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甘薯IbCAF1基因的克隆及耐盐性、抗旱性鉴定

本站小编 Free考研考试/2021-12-26
陈杉彬,, 孙思凡, 聂楠, 杜冰, 何绍贞, 刘庆昌, 翟红,*中国农业大学 / 农业农村部甘薯生物学与生物技术重点实验室 / 教育部作物杂种优势研究与利用重点实验室 / 北京市作物遗传改良重点实验室, 北京 100193

Cloning of IbCAF1 and identification on tolerance to salt and drought stress in sweetpotato

CHEN Shan-Bin,, SUN Si-Fan, NIE Nan, DU Bing, HE Shao-Zhen, LIU Qing-Chang, ZHAI Hong,*Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs / Laboratory of Crop Heterosis and Utilization, Ministry of Education / Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing 100193, China

通讯作者: *翟红, E-mail: zhaihong@cau.edu.cn

第一作者联系方式: E-mail: 757015572@qq.com
收稿日期:2020-02-27接受日期:2020-06-2网络出版日期:2020-07-02
基金资助:国家自然科学基金项目.31872878
国家重点研发计划项目.2018YFD1000700
国家重点研发计划项目.2018YFD1000704
国家现代农业产业技术体系建设专项.CARS-10


Received:2020-02-27Accepted:2020-06-2Online:2020-07-02
Fund supported: National Natural Science Foundation of China.31872878
National Key Research and Development Program of China.2018YFD1000700
National Key Research and Development Program of China.2018YFD1000704
China Agriculture Research System.CARS-10


摘要
CAF1 (CCR4-associated factor 1)基因在植物发育、抗病等方面发挥着重要的作用。本研究根据前期获得的差异表达EST序列, 克隆得到甘薯IbCAF1基因。IbCAF1基因的开放阅读框(open reading frame, ORF)长度为846 bp, 编码281个氨基酸, 分子量为32.13 kD, 等电点为4.83。氨基酸序列比对和系统进化树分析表明, IbCAF1与甘薯近缘野生种Ipomoea triloba (2x)同源蛋白ItlCAF1有较高的同源性, 序列一致性为96.8%。IbCAF1基因受到NaCl、PEG、ABA和H2O2的诱导表达。利用根癌农杆菌介导法将IbCAF1基因转入烟草, 过表达IbCAF1基因显著提高了转基因烟草植株的耐盐性和抗旱性。在200 mmol L -1 NaCl和10% PEG-6000的胁迫下, IbCAF1基因的过表达显著上调了转基因烟草植株中活性氧清除系统和脯氨酸合成相关基因的表达, 增加了SOD活性、POD活性、脯氨酸含量, 降低了H2O2含量和丙二醛含量。表明IbCAF1基因能够提高转基因烟草植株的耐盐性和抗旱性。本研究为后续甘薯IbCAF1基因耐盐抗旱基因工程研究奠定了基础。
关键词： 甘薯;IbCAF1;转基因烟草;耐盐;抗旱

Abstract
CAF1 (CCR4-associated factor 1) gene plays an important role in plant development and disease resistance. In this study, the IbCAF1 gene of sweetpotato was cloned according to the EST sequence. The ORF of IbCAF1 was 846 bp, encoding 281 amino acids, with a molecular weight of 32.13 kD and an isoelectric point of 4.83. The results of amino acid sequence alignment and phylogenetic tree analysis showed that IbCAF1 had higher homology with ItlCAF1, a homologous protein of Ipomoea triloba (2x), and the homology was 96.8%. IbCAF1 gene was induced and expressed by NaCl, PEG, ABA, and H2O2. The IbCAF1 gene was transferred into tobacco by Agrobacterium tumefaciens mediated transformation. The overexpression of IbCAF1 gene significantly improved the salt and drought tolerance of transgenic tobacco plants. After 200 mmol L -1NaCl and 10% PEG-6000 treatments, the transgenic tobacco plants showed significant upregulation of the genes involved in ROS scavenging system and proline biosynthesis related genes, significant increase of SOD activity, POD activity and proline content and significant decrease of H2O2 and malondialdehyde contents. These results demonstrate that the IbCAF1 gene could improve salt and drought tolerance in transgenic tobacco. This study will lay a foundation on salt and drought tolerance gene engineering of IbCAF1 gene in sweetpotato for the following research.
Keywords：sweetpotato;IbCAF1;transgenic tobacco;salt tolerance;drought tolerance


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本文引用格式
陈杉彬, 孙思凡, 聂楠, 杜冰, 何绍贞, 刘庆昌, 翟红. 甘薯IbCAF1基因的克隆及耐盐性、抗旱性鉴定[J]. 作物学报, 2020, 46(12): 1862-1869. doi:10.3724/SP.J.1006.2020.04045
CHEN Shan-Bin, SUN Si-Fan, NIE Nan, DU Bing, HE Shao-Zhen, LIU Qing-Chang, ZHAI Hong. Cloning of IbCAF1 and identification on tolerance to salt and drought stress in sweetpotato[J]. Acta Crops Sinica, 2020, 46(12): 1862-1869. doi:10.3724/SP.J.1006.2020.04045


土壤盐害和干旱严重影响了作物的产量, 成为限制农业生产的主要因素之一。据统计, 世界上存在着8亿公顷盐渍化土地, 约20%灌溉农业用地受到盐碱化的影响[1]。我国耕地中盐渍化面积达到920.9万公顷, 占全国耕地面积6.62%, 其中只有少部分被改良利用, 绝大部分仍未脱盐及不断遭受盐渍危害[2]。全球气候变化和人口增长引发的全球水资源短缺问题威胁着农业的可持续发展[3]。因此, 提高作物的耐盐性和抗旱性迫在眉睫。

Ccr4-Not复合物是一种多亚基的蛋白质复合物, 在真核生物中高度保守, 主要参与转录调控、mRNA降解、组蛋白修饰等重要生理过程[4,5]。CAF1 (CCR4-associated factor 1)是Ccr4-Not复合物中关键的亚基, 对复合物的形成及功能的行使起着重要作用[6]。CAF1属于DEDD家族, 是参与mRNA降解的主要脱腺苷酸酶之一, 在植物生长、胁迫响应和抵御微生物病原菌等方面发挥着重要作用[7,8]。Sarowar等[9]将辣椒CaCAF1基因转入番茄发现, CaCAF1基因的过量表达不但显著促进番茄的生长, 还增强了对番茄晚疫病病菌(Phytophthora infestans)的抗性。Liang等[10]研究发现, AtCAF1a基因的过量表达, 上调了PR1和PR2基因的表达量, 从而增强了拟南芥转基因植株对丁香假单胞菌(Pst DC3000)的抗性。AtCAF1基因的过表达增强了拟南芥转基因植株对坏死性真菌病原菌(Botrytis cinerea)的敏感性[11]。Shimo等[12]研究发现, 甜橙CsCAF1基因与甜橙溃疡病的抗性有关。有关CAF1基因提高植物的耐盐性报道较少。Walley等[13]研究发现, 在200 mmol L-1 NaCl胁迫下, 拟南芥Atcaf1a-1突变体种子的发芽率显著高于野生型对照。目前, 有关CAF1基因提高植物的抗旱性研究还未见报道。

甘薯是世界上第七大重要粮食作物, 同时也是饲料、工业原料、生物质能源作物[14]。然而, 其产量也受到盐和干旱胁迫的严重制约。因此, 培育耐盐抗旱品种是甘薯育种的重要目标之一。基因工程为定向改良甘薯耐盐抗旱性提供了可行的方法[15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]。本研究从甘薯中克隆得到IbCAF1基因, 其过量表达显著提高了转基因烟草植株的耐盐性和抗旱性。

1 材料与方法

1.1 植物材料

以甘薯品种鲁薯3号为材料克隆IbCAF1基因。利用烟草品种Wisconsin 38 (W38)分析IbCAF1基因的功能。

1.2 IbCAF1基因的克隆与序列分析

根据前期逆境胁迫获得的IbCAF1基因EST序列, 在甘薯近缘野生种Ipomoea trifida (2n=2x=30)数据库(http://sweetpotato-garden.kazusa.or.jp/blast.html)中进行BLAST分析, 获得IbCAF1的ORF序列。参照杨元军等[30]的方法提取甘薯总RNA。用TaKaRa公司的PrimeSript RT Kit合成cDNA。使用Primer Premier 5软件, 根据IbCAF1基因ORF序列设计扩增引物(表1), 以cDNA为模板, 进行PCR扩增。PCR扩增体系为25 μL, 包含模板1.0 μL、10×Taq Reaction buffer 2.5 μL、Taq DNA Polymerase (5 U μL-1) 0.2 μL、dNTP (10 mmol L-1) 0.5 μL、IbCAF1-F和IbCAF1-R (10 μmol L-1) 各0.5 μL、ddH2O 19.8 μL。PCR扩增程序为94℃ 5 min; 95℃ 30 s, 57℃ 30 s, 72℃ 1 min, 35个循环; 72℃ 5 min; 4℃保温。

Table 1
表1
表1本研究所用引物
Table 1Primers used in this study
引物名称
Primer name		引物序列
Primer sequence (5°-3°)
IbCAF1扩增引物
Primers for IbCAF1 amplification		IbCAF1-F: ATGGGTGTACAAGAAGATGTTTTG
IbCAF1-R: CTAAAAAACTTCTAGTCCGTACAATACT
鉴定转基因引物
Primers for identifying transformants		IbCAF1-OPF: GAGGCTTACGCAGCAGGTC
IbCAF1-OPR: TTATAATTCATACTCGTGGAAGCGC
实时定量PCR引物
Primers for real-time quantitative PCR		IbActin-F: AGCAGCATGAAGATTAAGGTTGTAGCAC
IbActin-R: TGGAAAATTAGAAGCACTTCCTGTGAAC
Actin-F: GAGGAATGCAGATCTTCGTG
Actin-R: TCCTTGTCCTGGATCTTAGC
IbCAF1RT-F: TCAGCTACCTCATCGACGAC
IbCAF1RT-R: AGTCAACCCGAGCTGAATCA
SOD-F: CTATTACCGACAAGCAGATTCCTC
SOD-R: TACCACAAGCAACCCTTCCAC
APX-F: GATGTTCCCTTTCACCCTGG
APX-R: CAGATAGACCCATTTGCTTCACA
POD-F: TCCGGGAGCCACACCATTGG
POD-R: TGGTCGGAATTCAACAG
P5CS-F: TTGTGACACGGACTGATGGAA
P5CS-R: TATCTAAGCCGCTGACGACCA

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利用ProtParam (https://web.expasy.org/protparam/) 分析蛋白质的理化性质, 利用SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) 在线预测蛋白质二级结构, 利用CDS (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) 预测保守结构域, 利用NCBI数据库进行Blastp分析(http://blast.ncbi.nlm.nih.gov/Blast.cgi), 寻找该基因的同源序列, 并利用MEGA7.0 (http://www.megasoftware.net/)构建同源进化树。

1.3 IbCAF1基因的表达分析

选取长势良好的甘薯试管苗, 在MS固体培养基中继代培养30 d, 在1/2 Hoagland溶液中培养3 d, 然后将试管苗分别浸入含有200 mmol L-1 NaCl、20% PEG-6000、100 μmol L-1 ABA和10 mmol L-1 H2O2的1/2 Hoagland溶液中处理0、0.5、1、3、6、12、24、48 h, 处理后的试管苗存于-80℃冰箱待用。以甘薯肌动蛋白基因(β-Actin)作为内参基因, 用Primer Premier 5软件根据IbCAF1基因的ORF非保守区间设计实时定量PCR引物(表1)。使用SYBR Premix Ex Taq (Tli RNaseH Plus)荧光定量试剂盒(宝生物工程(大连)有限公司, RR420)进行qRT-PCR分析。

1.4 烟草遗传转化

将构建的pCAMBIA1300-IbCAF1-GFP质粒通过冻融法转入根癌农杆菌EHA105中。采用叶盘法对烟草品种W38进行转化[31]。将侵染后的烟草叶盘接种于含有15 mg L-1潮霉素、400 mg L-1头孢氨苄、1.0 mg L-1 6-BA和0.1 mg L-1 NAA的MS培养基上, 每天13 h、54 μmol m-2 s-1光照, (27±1)℃培养30 d, 然后将再生芽培养于含有15 mg L-1潮霉素、400 mg L-1头孢氨苄、1.0 mg L-1 6-BA和0.1 mg L-1 NAA的1/2 MS培养基上, 直至长成完整植株。对再生植株进行PCR检测, 所用引物序列见表1。

1.5 耐盐性、抗旱性鉴定

参照Jiang等[32]和喻娜等[33]的方法(略有改动), 对转基因烟草植株进行耐盐和抗旱性离体鉴定。将转基因植株和野生型对照植株分别培养于含有200 mmol L-1 NaCl和10% PEG-6000的MS培养基上, 培养条件为(27±1)℃, 每天13 h、54 μmol m-2 s-1光照, 胁迫培养4周后, 观察植株生长状态, 并测定相关生理生化指标。参考Zhai等[26]的方法测定脯氨酸、MDA、H2O2含量、SOD和POD活性。

1.6 抗逆相关基因的表达分析

对0、200 mmol L-1NaCl或10% PEG-6000处理4周的转基因烟草植株的抗逆相关基因的表达量进行分析。用Primer Premier 5软件设计基因的特异扩增引物。抗逆相关基因包括活性氧清除相关基因SOD、APX、POD, 以及脯氨酸合成相关基因P5CS。参照Huo等[34]设计内标基因烟草肌动蛋白基因(actin)的检测引物。引物序列见表1。

1.7 统计分析

每个试验设置3次重复, 通过P<0.01或P<0.05的t检验分析差异显著性。

2 结果与分析

2.1 IbCAF1基因的克隆与序列分析

从甘薯品种鲁薯3号中克隆了IbCAF1基因, 该基因的ORF长度为846 bp, 编码281个氨基酸, 分子量为32.13 kD, 理论等电点pI为4.83, 是酸性蛋白, 不稳定系数为36.36, 亲水性平均系数为-0.167, 说明该蛋白是一个稳定的亲水蛋白。IbCAF1基因编码蛋白是由45.55%的α螺旋、36.30%的不规则卷曲、13.52%的延伸链和4.63%的β折叠组成。IbCAF1属于CAF1超家族成员(图1-A)。IbCAF1基因与甘薯近缘野生种Ipomoea triloba (2n=2x=30)亲缘关系最近, 序列一致性为96.8% (图1-B, C)。

图1

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图1IbCAF1基因序列分析

A: IbCAF1蛋白序列分析; B: 甘薯CAF1蛋白和其他植物中的CAF1蛋白序列比对; C: 甘薯CAF1蛋白和其他植物中的CAF1蛋白的同源进化树分析。ItlCAF1: 三裂叶野牵牛(XP_031110715.1); InCAF1: 牵牛(XP_019199562.1); NtCAF1: 烟草(XP_016511744.1); SpCAF1: 番茄近缘野生种(XP_015079674.1); CaCAF1: 辣椒(NP_001312000.1); SlCAF1: 番茄(XP_004241342.1); StCAF1: 马铃薯(XP_006361099.1)。
Fig. 1Sequence analysis of IbCAF1

A: sequence analysis of IbCAF1 protein. B: multiple sequence alignment of IbCAF1 and CAF1 proteins from other plants. C: phylogenetic analysis of IbCAF1 and CAF1 proteins from other plants. ItlCAF1: Ipomoea triloba (XP_031110715.1); InCAF1: Ipomoea nil (XP_019199562.1), NtCAF1: Nicotiana tabacum (XP_016511744.1); SpCAF1: Solanum pennellii (XP_015079674.1), CaCAF1: Capsicum annuum (NP_001312000.1), SlCAF1: Solanum lycopersicum (XP_004241342.1), StCAF1: Solanum tuberosum (XP_006361099.1).


2.2 IbCAF1基因的表达分析

IbCAF1基因在鲁薯3号茎中的表达水平显著高于在叶和根中(图2-A)。其在离体培养的鲁薯3号植株中的表达受到NaCl、PEG-6000、ABA和H2O2的强烈诱导, 在200 mmol L-1 NaCl处理24 h时达到高峰(1.4倍), 在20% PEG-6000处理12 h时达到高峰(15.4倍), 在100 μmol L-1 ABA处理12 h时达到高峰(4.4倍), 在10 mmol L-1 H2O2处理0.5 h时达到高峰(1.7倍)(图2-B)。

图2

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图2IbCAF1基因在鲁薯3号中的表达分析

A: IbCAF1基因在鲁薯3号不同组织中的表达; B: 200 mmol L-1 NaCl、20% PEG-6000、100 μmol L-1 ABA和10 mmol L-1 H2O2分别处理不同时间后, 鲁薯3号中IbCAF1基因的表达分析。*与**分别表示在0.05和0.01水平下差异显著。
Fig. 2Expression analysis of IbCAF1 in Lushu 3

A: expression analysis of IbCAF1 gene in different tissues of Lushu 3; B: expression analysis of the IbCAF1 gene in Lushu 3 after different times (h) in response to 200 mmol L-1 NaCl, 20% PEG-6000, 100 μmol L-1 ABA and 10 mmol L-1 H2O2, respectively. * and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively.


2.3 转基因烟草植株的获得

利用Wang等[31]的方法获得12株转基因烟草植株, 即L1, L2, …, L12。qRT-PCR结果显示, 转基因植株中IbCAF1的表达量均显著高于WT (图3)。选取表达量较高的L1、L5和L9株系进行后续的分析。

图3

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图3转基因烟草植株的IbCAF1基因的qRT-PCR分析

**表示在0.01水平下差异显著。
Fig. 3Expression analysis of IbCAF1 gene in transgenic tobacco plants by qRT-PCR

**: significantly different at the 0.01 probability level.


2.4 过表达IbCAF1基因能够提高烟草的耐盐性和抗旱性

将3个转基因株系(L1、L5和L9)和野生型植株分别在200 mmol L-1 NaCl和10% PEG-6000的MS培养基上和无胁迫条件下培养4周。转基因植株和野生型植株在无胁迫条件下生长无明显差异。在盐胁迫和干旱胁迫下, 转基因植株生长良好(图4-A), 叶片中积累了较少的H2O2 (图4-B, D)和O2- (图4-C), SOD活性、POD活性和脯氨酸含量均显著高于WT, MDA含量在盐和干旱胁迫下显著低于WT (图4-E~H)。

图4

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图4IbCAF1增强了转基因烟草植株的耐盐性和抗旱性

A: 转IbCAF1基因烟草植株和WT烟草植株在无胁迫或添加200 mmol L-1 NaCl或10% PEG-6000的1/2 MS培养基上培养4周; B~H: 在无胁迫、200 mmol L-1 NaCl或10% PEG-6000的1/2 MS培养基上培养4周的IbCAF1转基因烟草和WT烟草叶片的DAB染色(B)、NBT染色(C)、H2O2含量(D)、SOD活性(E)、POD活性(F)、脯氨酸含量(G)、丙二醛含量(H)。*与**分别表示在0.05和0.01水平下差异显著。
Fig. 4IbCAF1 enhances salt and drought tolerance in transgenic tobacco plants

A: responses of IbCAF1-transgenic and WT tobacco plants cultured for 4 weeks on half-MS medium supplemented without stress or with 200 mmol L-1 NaCl or 10% PEG-6000; B-H: DAB staining (B), NBT staining (C), H2O2 content (D), SOD activity (E), POD activity (F), proline content (G), and MDA content (H) in the leaves of IbCAF1 transgenic and WT tobacco plants cultured for 4w on half-MS medium supplemented with no stress, 200 mmol L-1 NaCl or 10% PEG-6000. * and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively.


2.5 转基因植株抗性相关基因的表达分析

在200 mmol L-1 NaCl和10% PEG-6000的胁迫下, 过表达株系与WT相比, ROS清除相关基因SOD、APX和POD上调表达, 脯氨酸合成相关基因P5CS上调表达(图5)。

图5

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图5转基因植株及WT植株的抗逆相关基因的表达分析

*与**分别表示在0.05和0.01水平下差异显著。
Fig. 5Relative expression of abiotic stress-responsive genes in the transgenic and WT plants

* and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively.


3 讨论

CAF1是参与mRNA降解的主要脱腺苷酸酶之一, 在调控基因表达和影响生物学性状方面起着重要作用[7]。目前, CAF1在酵母和动物中被广泛研究,在植物中的作用还不清楚。只有少数植物, 如拟南芥[10-11,13]、辣椒[9]、甜橙[12]等有CAF1基因功能的研究报道, 表明该基因在植物的生长发育及生物和非生物胁迫的应答过程中发挥重要作用。到目前为止, CAF1基因在植物中的耐盐作用还不清楚, 抗旱性研究还未见报道。本研究发现IbCAF1基因能够被NaCl、PEG、ABA和H2O2诱导上调表达(图2-B), 并且IbCAF1基因的过量表达增强了转基因烟草植株的耐盐抗旱性(图4)。

在盐或干旱胁迫下, 植物体内常常会产生大量的活性氧(ROS), 如超氧阴离子(O2?)和过氧化氢(H2O2)等。ROS的大量累积会造成细胞氧化损伤, 对植物有很大的毒害作用。超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)和过氧化物酶(POD)等ROS清除系统, 可以解毒ROS, 以减少植物细胞中的氧化损伤而增强抗逆性[19,35]。在盐或干旱胁迫下, 脯氨酸水平的升高增强了植物的耐盐性和抗旱性[28,35]。脯氨酸可以调节植物细胞质pH防止其酸化, 保护膜完整性, 同时还具有清除活性氧的功能[36]。丙二醛(MDA)的含量变高时会致使细胞发生膜损伤, 植物的耐盐抗旱性也会因此减弱[37,38]。在本研究中, 盐或干旱胁迫条件下, 在IbCAF1过量表达的烟草植株中, 活性氧清除相关基因SOD、APX和POD和脯氨酸合成相关基因P5CS被显著上调表达, 增加了SOD活性, POD活性, 脯氨酸含量, 降低了H2O2含量和MDA含量, 从而增强了转基因烟草植株的耐盐性和抗旱性(图4和图5)。

4 结论

IbCAF1基因的过表达通过上调活性氧清除和脯氨酸合成相关基因的表达, 增加SOD活性、POD活性、脯氨酸含量, 降低H2O2含量和丙二醛含量, 从而增强转基因烟草植株的耐盐性和抗旱性。IbCAF1基因将在提高甘薯等植物耐盐抗旱性方面具有一定的应用潜力。

参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

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The physiological and molecular mechanisms of tolerance to osmotic and ionic components of salinity stress are reviewed at the cellular, organ, and whole-plant level. Plant growth responds to salinity in two phases: a rapid, osmotic phase that inhibits growth of young leaves, and a slower, ionic phase that accelerates senescence of mature leaves. Plant adaptations to salinity are of three distinct types: osmotic stress tolerance, Na(+) or Cl() exclusion, and the tolerance of tissue to accumulated Na(+) or Cl(). Our understanding of the role of the HKT gene family in Na(+) exclusion from leaves is increasing, as is the understanding of the molecular bases for many other transport processes at the cellular level. However, we have a limited molecular understanding of the overall control of Na(+) accumulation and of osmotic stress tolerance at the whole-plant level. Molecular genetics and functional genomics provide a new opportunity to synthesize molecular and physiological knowledge to improve the salinity tolerance of plants relevant to food production and environmental sustainability.

王佳丽, 黄贤金, 钟太洋, 陈志刚 . 盐碱地可持续利用研究综述
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盐碱地资源的开发利用对粮食生产具有重大贡献。但水资源的缺乏、气候变化使盐碱地开发利用工作更具挑战性。文章系统总结了近年来盐碱地可持续利用研究取得的重要进展：(1)盐碱地可持续利用的技术研发、排盐水环境安全处理及技术配套管理研究；(2) 盐碱地可持续利用的农户技术选择行为研究；(3) 盐碱地可持续利用的科学研究方法。提出盐碱地可持续利用进一步研究的建议：加强多学科交叉融合的研究；强调农户技术选择行为研究；增强研究方法的科学性。
Wang J L, Huang X J, Zhong T Y, Chen Z G . Review on sustainable utilization of salt-affected land
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Yang S J, Vanderbeld B, Wan J X, Huang Y F . Narrowing down the targets: towards successful genetic engineering of drought tolerant crops
Mol Plant, 2010,3:469-490.
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Drought is the most important environmental stress affecting agriculture worldwide. Exploiting yield potential and maintaining yield stability of crops in water-limited environments are urgent tasks that must be undertaken in order to guarantee food supply for the increasing world population. Tremendous efforts have been devoted to identifying key regulators in plant drought response through genetic, molecular, and biochemical studies using, in most cases, the model species Arabidopsis thaliana. However, only a small portion of these regulators have been explored as potential candidate genes for their application in the improvement of drought tolerance in crops. Based on biological functions, these genes can be classified into the following three categories: (1) stress-responsive transcriptional regulation (e.g. DREB1, AREB, NF-YB); (2) post-transcriptional RNA or protein modifications such as phosphorylation/dephosphorylation (e.g. SnRK2, ABI1) and farnesylation (e.g. ERA1); and (3) osomoprotectant metabolism or molecular chaperones (e.g. CspB). While continuing down the path to discovery of new target genes, serious efforts are also focused on fine-tuning the expression of the known candidate genes for stress tolerance in specific temporal and spatial patterns to avoid negative effects in plant growth and development. These efforts are starting to bear fruit by showing yield improvements in several crops under a variety of water-deprivation conditions. As most such evaluations have been performed under controlled growth environments, a gap still remains between early success in the laboratory and the application of these techniques to the elite cultivars of staple crops in the field. Nevertheless, significant progress has been made in the identification of signaling pathways and master regulators for drought tolerance. The knowledge acquired will facilitate the genetic engineering of single or multiple targets and quantitative trait loci in key crops to create commercial-grade cultivars with high-yielding potential under both optimal and suboptimal conditions.

Molin L, Puisieux A . C. elegans homologue of the Caf1 gene, which encodes a subunit of the CCR4-NOT complex, is essential for embryonic and larval development and for meiotic progression
Gene, 2005,358:73-81.
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The evolutionary conserved CCR4-NOT multi subunit complex is involved in different aspects of mRNA metabolism, including mRNA synthesis initiation and mRNA deadenylation (shortening of the poly(A) tail) in yeast and higher eukaryotes. Here we report the characterization of the gene encoding the Caf1 subunit of this complex in Caenorhabditis elegans, ccf-1, and the phenotypes associated with its inactivation. Use of staged populations and of mutants strains with altered germline showed that ccf-1 is predominantly expressed in embryos and adults. Loss of ccf-1 function, by both RNAi and a deletion allele, caused early embryonic and larval lethality. It also resulted in sterility in both males and hermaphrodites by blocking germ cell development at the pachytene stage of meiosis I. These results reveal that ccf-1 is an essential factor for both somatic and germline development in C. elegans. Functional analysis of ccf-1 may contribute to the understanding of the molecular role of the CCR4-NOT complex.

Collart M A . The CCR4-NOT complex is a key regulator of eukaryotic gene expression
WIREs RNA, 2016,7:438-454.
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The Ccr4-Not complex is a multisubunit complex present in all eukaryotes that contributes to regulate gene expression at all steps, from production of messenger RNAs (mRNAs) in the nucleus to their degradation in the cytoplasm. In the nucleus it influences the post-translational modifications of the chromatin template that has to be remodeled for transcription, it is present at sites of transcription and associates with transcription factors as well as with the elongating polymerase, it interacts with the factors that prepare the new transcript for export to the cytoplasm and finally is important for nuclear quality control and influences mRNA export. In the cytoplasm it is present in polysomes where mRNAs are translated and in RNA granules where mRNAs will be redirected upon inhibition of translation. It influences mRNA translatability, and is needed during translation, on one hand for co-translational protein interactions and on the other hand to preserve translation that stalls. It is one of the relevant players during co-translational quality control. It also interacts with factors that will repress translation or induce mRNA decapping when recruited to the translating template. Finally, Ccr4-Not carries deadenylating enzymes and is a key player in mRNA decay, generic mRNA decay that follows normal translation termination, co-translational mRNA decay of transcripts on which the ribosomes stall durably or which carry a non-sense mutation and finally mRNA decay that is induced by external signaling for a change in genetic programming. Ccr4-Not is a master regulator of eukaryotic gene expression. WIREs RNA 2016, 7:438-454. doi: 10.1002/wrna.1332 For further resources related to this article, please visit the WIREs website.

Berthet C, Morera A M, Asensio M J, Chauvin M A, Morel A P, Dijoud F, Magaud J P, Durand P, Rouault J P . CCR4-associated factor CAF1 is an essential factor for spermatogenesis
Mol Cell Biol, 2004,24:5808-5820.
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The CCR4-associated protein CAF1 has been demonstrated to play several roles in the control of transcription and of mRNA decay. To gain further insight into its physiological function, we generated CAF1-deficient mice. They are viable, healthy, and normal in appearance; however, mCAF1(-/-) male mice are sterile. The crossing of mCAF1(+/-) mice gave a Mendelian ratio of mCAF1(+/+), mCAF1(+/-), and mCAF1(-/-) pups, indicating that haploid mCAF1-deficient germ cells differentiate normally. The onset of the defect occurs during the first wave of spermatogenesis at 19 to 20 days after birth, during progression of pachytene spermatocytes to haploid spermatids and spermatozoa. Early disruption of spermatogenesis was evidenced by Sertoli cell vacuolization and tubular disorganization. The most mature germ cells were the most severely depleted, but progressively all germ cells were affected, giving Sertoli cell-only tubes, large interstitial spaces, and small testes. This phenotype could be linked to a defect(s) in germ cells and/or to inadequate Sertoli cell function, leading to seminiferous tubule disorganization and finally to a total disappearance of germ cells. The mCAF1-deficient mouse provides a new model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

Cui Y J, Ramnarain D B, Chiang Y C, Ding L H, McMahon J S, Denis C L . Genome wide expression analysis of the CCR4-NOT complex indicates that it consists of three modules with the NOT module controlling SAGA-responsive genes
Mol Genet Genomics, 2008,279:323-337.
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Of the nine known members of the CCR4-NOT complex, CCR4/CAF1 are most important in mRNA deadenylation whereas the NOT1-5 proteins are most critical for transcriptional repression. Whole genome microarray analysis using deletions in seven of the CCR4-NOT genes was used to determine the overall mRNA expression patterns that are affected by members of the yeast CCR4-NOT complex. Under glucose conditions, ccr4 and caf1 displayed a high degree of similarity in the manner that they affected gene expression. In contrast, the not deletions were similar in the way they affected genes, but showed no correlation with that of ccr4/caf1. A number of groups of functionally related proteins were specifically controlled by the CCR4/CAF1 or NOT modules. Importantly, the NOT proteins preferentially affected SAGA-controlled gene expression. Also, both the CCR4/CAF1 and NOT group of proteins shared much greater similarities in their effects on gene expression during the stress of glucose deprivation. BTT1, a member of the nascent polypeptide association complex that binds the ribosome, was shown to be a tenth member of the CCR4-NOT complex, interacting through CAF130. Microarray analysis indicated that BTT1 and CAF130 correlate very highly in their control of gene expression and preferentially repress genes involved in ribosome biogenesis. These results indicate that distinct portions of the CCR4-NOT complex control a number of different cellular processes.

Feng L K, Yan Y B . The N-terminus modulates human Caf1 activity, structural stability and aggregation
Int J Biol Macromol, 2012,51:497-503.
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Caf1 is a deadenylase component of the CCR4-Not complex. Here we found that the removal of the N-terminus resulted in a 30% decrease in human Caf1 (hCaf1) activity, but had no significant influence on main domain structure. The removal of the N-terminus led to a decrease in the thermal stability, while the existence of the N-terminus promoted hCaf1 thermal aggregation. Homology modeling indicated that the N-terminus had a potency to form a short alpha-helix interacted with the main domain. Thus the N-terminus played a role in modulating hCaf1 activity, stability and aggregation.

Sarowar S, Oh H W, Cho H S, Baek K H, Seong E S, Joung Y H, Choi G J, Lee S, Choi D . Capsicum annuum CCR4-associated factor CaCAF1 is necessary for plant development and defence response
Plant J, 2007,51:792-802.
DOI:10.1111/j.1365-313X.2007.03174.xURLPMID:17587232 [本文引用: 2]
The CCR4-associated factor 1 (CAF1) protein belongs to the CCR4-NOT complex, which is an evolutionary conserved protein complex and plays an important role in the control of transcription and mRNA decay in yeast and mammals. To investigate the function of CAF1 in plants, we performed gain- and loss-of-function studies by overexpression of the pepper CAF1 (CaCAF1) in tomato and virus-induced gene silencing (VIGS) of the gene in pepper plants. Overexpression of CaCAF1 in tomato resulted in significant growth enhancement, with increasing leaf thickness, and enlarged cell size by more than twofold when compared with the control plants. A transmission electron microscopic analysis revealed that the CaCAF1-transgenic tomato plants had thicker cell walls and cuticle layers than the control plants. In addition to developmental changes, overexpression of CaCAF1 in tomato plants resulted in enhanced resistance against the oomycete pathogen Phytophthora infestans. Additionally, microarray, northern and real-time polymerase chain reaction analyses of CaCAF1-transgenic tomato plants revealed that multiple genes were constitutively upregulated, including genes involved in polyamine biosynthesis, defence reactions and cell-wall organogenesis. In contrast, VIGS of CaCAF1 in pepper plants caused significant growth retardation and enhanced susceptibility to the pepper bacterial spot pathogen Xanthomonas axonopodis pv. vesicatoria. Our results suggest roles for plant CAF1 in normal growth and development, as well as in defence against pathogens.

Liang W X, Li C B, Liu F, Jiang H L, Li S Y, Sun J Q, Wu X Y, Li C Y . The Arabidopsis homologs of CCR4-associated factor 1 show mRNA deadenylation activity and play a role in plant defence responses
Cell Res, 2009,19:307-316.
DOI:10.1038/cr.2008.317URLPMID:19065152 [本文引用: 2]
Messenger RNA (mRNA) turnover in eukaryotic cells begins with shortening of the poly (A) tail at the 3' end, a process called deadenylation. In yeast, the deadenylation reaction is predominantly mediated by CCR4 and CCR4-associated factor 1 (CAF1), two components of the well-characterised protein complex named CCR4-NOT. We report here that AtCAF1a and AtCAF1b, putative Arabidopsis homologs of the yeast CAF1 gene, partially complement the growth defect of the yeast caf1 mutant in the presence of caffeine or at high temperatures. The expression of AtCAF1a and AtCAF1b is induced by multiple stress-related hormones and stimuli. Both AtCAF1a and AtCAF1b show deadenylation activity in vitro and point mutations in the predicted active sites disrupt this activity. T-DNA insertion mutants disrupting the expression of AtCAF1a and/or AtCAF1b are defective in deadenylation of stress-related mRNAs, indicating that the two AtCAF1 proteins are involved in regulated mRNA deadenylation in vivo. Interestingly, the single and double mutants of AtCAF1a and AtCAF1b show reduced expression of pathogenesis-related (PR) genes PR1 and PR2 and are more susceptible to Pseudomonas syringae pv tomato DC3000 (Pst DC3000) infection, whereas transgenic plants over-expressing AtCAF1a show elevated expression of PR1 and PR2 and increased resistance to the same pathogen. Our results suggest roles of the AtCAF1 proteins in regulated mRNA deadenylation and defence responses to pathogen infections.

Kwon T, Yi Y B, Nam J . Overexpression of AtCAF1, CCR4-associated factor 1 homologue in Arabidopsis thaliana, negatively regulates wounding-mediated disease resistance
J Plant Biotechnol, 2011,38:278-284.
DOI:10.5010/JPB.2011.38.4.278URL [本文引用: 2]

Shimo H M, Terassi C, Lima Silva C C, de Lima Zanella J, Mercaldi G F, Rocco S A, Benedetti C E . Role of the Citrus sinensis RNA deadenylase CsCAF1 in citrus canker resistance
Mol Plant Pathol, 2019,20:1105-1118.
DOI:10.1111/mpp.12815URLPMID:31115151 [本文引用: 2]
Poly(A) tail shortening is a critical step in messenger RNA (mRNA) decay and control of gene expression. The carbon catabolite repressor 4 (CCR4)-associated factor 1 (CAF1) component of the CCR4-NOT deadenylase complex plays an essential role in mRNA deadenylation in most eukaryotes. However, while CAF1 has been extensively investigated in yeast and animals, its role in plants remains largely unknown. Here, we show that the Citrus sinensis CAF1 (CsCAF1) is a magnesium-dependent deadenylase implicated in resistance against the citrus canker bacteria Xanthomonas citri. CsCAF1 interacted with proteins of the CCR4-NOT complex, including CsVIP2, a NOT2 homologue, translin-associated factor X (CsTRAX) and the poly(A)-binding proteins CsPABPN and CsPABPC. CsCAF1 also interacted with PthA4, the main X. citri effector required for citrus canker elicitation. We also present evidence suggesting that PthA4 inhibits CsCAF1 deadenylase activity in vitro and stabilizes the mRNA encoded by the citrus canker susceptibility gene CsLOB1, which is transcriptionally activated by PthA4 during canker formation. Moreover, we show that an inhibitor of CsCAF1 deadenylase activity significantly enhanced canker development, despite causing a reduction in PthA4-dependent CsLOB1 transcription. These results thus link CsCAF1 with canker development and PthA4-dependent transcription in citrus plants.

Walley J W, Kelley D R, Nestorova G, Hirschberg D L, Dehesh K . Arabidopsis deadenylases AtCAF1a and AtCAF1b play overlapping and distinct roles in mediating environmental stress responses
Plant Physiol, 2010,152:866-875.
DOI:10.1104/pp.109.149005URLPMID:19955262 [本文引用: 2]
To maintain homeostasis in an ever-changing environment organisms have evolved mechanisms to reprogram gene expression. One central mechanism regulating gene expression is messenger RNA (mRNA) degradation, which is initiated by poly(A) tail shortening (deadenylation). The carbon catabolite repressor 4-CCR4 associated factor1 (CCR4-CAF1) complex is the major enzyme complex that catalyzes mRNA deadenylation and is conserved among eukaryotes. However, the components and functions of this global regulatory complex have not been well characterized in plants. Here we investigate the CAF1 family in Arabidopsis (Arabidopsis thaliana). We identify 11 AtCAF1 homologs and show that a subset of these genes are responsive to mechanical wounding, among them are AtCAF1a and AtCAF1b whose expression levels are rapidly and transiently induced by wounding. The differential expression profiles of the various AtCAF1s suggest that not all AtCAF1 genes are involved in stress-responsive regulation of transcript levels. Comparison of misexpressed genes identified via transcript profiling of Atcaf1a and Atcaf1b mutants at different time points before and after wounding suggests that AtCAF1a and AtCAF1b target shared and unique transcripts for deadenylation with temporal specificity. Consistent with the AtPI4Kgamma3 transcript exhibiting the largest increase in abundance in Atcaf1b, AtCAF1b targets AtPI4Kgamma3 mRNA for deadenylation. Stress-tolerance assays demonstrate that AtCAF1a and AtCAF1b are involved in mediating abiotic stress responses. However, AtCAF1a and AtCAF1b are not functionally redundant in all cases, nor are they essential for all environmental stresses. These findings demonstrate that these closely related proteins exhibit overlapping and distinct roles with respect to mRNA deadenylation and mediation of stress responses.

Liu Q C . Sweet potato omics and biotechnology in China
Plant OMICS: J Plant Mol Biol Omics, 2011,4:295.
[本文引用: 1]

Park S C, Kim Y H, Jeong J C, Kim C Y, Lee H S, Bang J W, Kwak S S . Sweetpotato late embryogenesis abundant 14 ( IbLEA14) gene in?uences lignification and increases osmotic- and salt stress-tolerance of transgenic calli
Planta, 2011,233:621-634.
DOI:10.1007/s00425-010-1326-3URL [本文引用: 1]
Late embryogenesis abundant 14 (LEA14) cDNA was isolated from an EST library prepared from dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas). Quantitative RT-PCR revealed a variety of different IbLEA14 expression patterns under various abiotic stress conditions. IbLEA14 expression was strongly induced by dehydration, NaCl and abscisic acid treatments in sweetpotato plants. Transgenic sweetpotato non-embryogenic calli harboring IbLEA14 overexpression or RNAi vectors under the control of CaMV 35S promoter were generated. Transgenic calli overexpressing IbLEA14 showed enhanced tolerance to drought and salt stress, whereas RNAi calli exhibited increased stress sensitivity. Under normal culture conditions, lignin contents increased in IbLEA14-overexpressing calli because of the increased expression of a variety of monolignol biosynthesis-related genes. Stress treatments elicited higher expression levels of the gene encoding cinnamyl alcohol dehydrogenase in IbLEA14-overexpressing lines than in control or RNAi lines. These results suggest that IbLEA14 might positively regulate the response to various stresses by enhancing lignification.

Kim S H, Ahn Y O, Ahn M J, Lee H S, Kwak S S . Down-regulation of β-carotene hydroxylase increases β-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato
Phytochemistry, 2012,74:69-78.
DOI:10.1016/j.phytochem.2011.11.003URL [本文引用: 1]
Sweetpotato (Ipomoea batatas Lam.) is an important industrial crop and source of food that contains useful components, including antioxidants such as carotenoids. beta-Carotene hydroxylase (CHY-beta) is a key regulatory enzyme in the beta-beta-branch of carotenoid biosynthesis and it catalyzes hydroxylation into both beta-carotene to p-cryptoxanthin and beta-cryptoxanthin to zeaxanthin. To increase the beta-carotene content of sweetpotato through the inhibition of further hydroxylation of beta-carotene, the effects of silencing CHY-beta in the carotenoid biosynthetic pathway were evaluated. A partial cDNA encoding CHY-beta was cloned from the storage roots of orange-fleshed sweetpotato (cv. Shinhwangmi) to generate an RNA interference-IbCHY-beta construct. This construct was introduced into cultured cells of white-fleshed sweetpotato (cv. Yulmi). Reverse transcription-polymerase chain reaction analysis confirmed the successful suppression of IbCHY-beta gene expression in transgenic cultured cells. The expression level of phytoene synthase and lycopene p-cyclase increased, whereas the expression of other genes showed no detectable change. Down-regulation of IbCHY-beta gene expression changed the composition and levels of carotenoids between non-transgenic (NT) and transgenic cells. In transgenic line #7, the total carotenoid content reached a maximum of 117 mu g/g dry weight, of which beta-carotene measured 34.43 mu g/g dry weight. In addition, IOCHY-beta-silenced calli showed elevated p-cryptoxanthin and zeaxanthin contents as well as high transcript level P450 gene. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) in transgenic cells was more than twice that in NT cells. RNA-IbCHY-beta calli increased abscisic acid (ABA) content, which was accompanied by enhanced tolerance to salt stress. In addition, the production of reactive oxygen species measured by 3,3'-diaminobenzidine (DAB) staining was significantly decreased in transgenic cultured cells under salt stress. Taken together, the present results indicate that down-regulation of IbCHY-beta increased beta-carotene contents and total carotenoids in transgenic plant cells and enhanced their antioxidant capacity. (C) 2011 Elsevier Ltd.

Kim S H, Kim Y H, Ahn Y O, Ahn M J, Jeong J C, Lee H S, Kwak S S . Downregulation of the lycopene ε-cyclase gene increases carotenoid synthesis via the β-branch-specific pathway and enhances salt-stress tolerance in sweetpotato transgenic calli
Physiol Plant, 2013,147:432-442.
DOI:10.1111/j.1399-3054.2012.01688.xURLPMID:22938023 [本文引用: 1]
Lycopene epsilon-cyclase (LCY-epsilon) is involved in the first step of the alpha-branch synthesis pathway of carotenoids from lycopene in plants. In this study, to enhance carotenoid synthesis via the beta-branch-specific pathway [which yields beta-carotene and abscisic acid (ABA)] in sweet potato, the expression of IbLCY-epsilon was downregulated by RNAi (RNA interference) technology. The RNAi-IbLCY-epsilon vector was constructed using a partial cDNA of sweet potato LCY-epsilon isolated from the storage root and introduced into cultured sweet potato cells by Agrobacterium-mediated transformation. Both semi-quantitative Reverse transcription polymerase chain reaction (RT-PCR) of carotenoid biosynthesis genes and high-performance liquid chromatography (HPLC) analysis of the metabolites in transgenic calli, in which the LCY- epsilongene was silenced, showed the activation of beta-branch carotenoids and its related genes. In the transgenic calli, the beta-carotene content was approximately 21-fold higher than in control calli, whereas the lutein content of the transgenic calli was reduced to levels undetectable by HPLC. Similarly, expression of the RNAi-IbLCY-epsilon transgene resulted in a twofold increase in ABA content compared to control calli. The transgenic calli showed significant tolerance of 200 mM NaCl. Furthermore, both the beta-branch carotenoids content and the expression levels of various branch-specific genes were higher under salt stress than in control calli. These results suggest that, in sweet potato, downregulation of the epsilon-cyclization of lycopene increases carotenoid synthesis via the beta-branch-specific pathway and may positively regulate cellular defenses against salt-mediated oxidative stress.

Kim S H, Jeong J C, Park S, Bae J Y, Ahn M J, Lee H S, Kwak S S . Down-regulation of sweetpotato lycopene β-cyclase gene enhances tolerance to abiotic stress in transgenic calli
Mol Biol Rep, 2014,41:8137-8148.
DOI:10.1007/s11033-014-3714-4URL [本文引用: 1]
Lycopene beta-cyclase (LCY-beta) is a key enzyme involved in the synthesis of alpha- and beta-branch carotenoids such as alpha-carotene and beta-carotene through the cyclization of lycopene. IbLCY-beta had a length of 1,506 bp and approximately 80 % nucleotide sequence identity with that of tomato LCY-beta. IbLCY-beta was strongly expressed in leaves, and expression was enhanced by salt-stress and osmotic-stress conditions. To characterize the LCY-beta gene (IbLCY-beta) of sweetpotato (Ipomoea batatas), it was isolated and transformed into calli of white-fleshed sweetpotato using an IbLCY-beta-RNAi vector. Transgenic IbLCY-beta-RNAi calli had yellow to orange color and higher antioxidant activity compared to that of white, nontransgenic (NT) calli. Transgenic cells had significantly higher contents of total carotenoids, although lycopene was not detected in transgenic or NT cells. All transgenic calli had strongly activated expression of carotenoid biosynthetic genes such as beta-carotene hydroxylases (CHY-beta), cytochrome P450 monooxygenases (P450), and carotenoid cleavage dioxigenase 1 (CCD1). Transgenic cells exhibited less salt-induced oxidative-stress damage compared to that of NT cells, and also had greater tolerance for polyethylene glycol (PEG)-mediated drought compared to that of NT cells, due to the higher water content and reduced malondialdehyde (MDA) content. The abscisic acid content was also higher in transgenic cells. These results show that a study of IbLCY-beta can facilitate understanding of the carotenoid biosynthetic pathway in sweetpotato. IbLCY-beta could be useful for developing transgenic sweetpotato enriched with nutritional carotenoids and with greater tolerance to abiotic stresses.

Liu D G, He S Z, Zhai H, Wang L J, Zhao Y, Wang B, Li R J, Liu Q C . Overexpression of IbP5CR enhances salt tolerance in transgenic sweetpotato
Plant Cell Tiss Org Cult, 2014,117:1-16.
DOI:10.1007/s11240-013-0415-yURL [本文引用: 2]

Liu D G, Wang L J, Liu C L, Song X J, He S Z, Zhai H, Liu Q C . An Ipomoea batatas iron-sulfur cluster scafold protein gene, IbNFU1, is involved in salt tolerance
PLoS One, 2014,9:e93935.
DOI:10.1371/journal.pone.0093935URLPMID:24695556 [本文引用: 1]
Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. An iron-sulfur cluster scaffold protein gene, IbNFU1, was isolated from a salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line LM79 in our previous study, but its role in sweetpotato stress tolerance was not investigated. In the present study, the IbNFU1 gene was introduced into a salt-sensitive sweetpotato cv. Lizixiang to characterize its function in salt tolerance. The IbNFU1-overexpressing sweetpotato plants exhibited significantly higher salt tolerance compared with the wild-type. Proline and reduced ascorbate content were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbNFU1 up-regulated pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that IbNFU1gene is involved in sweetpotato salt tolerance and enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system.

Liu D G, Wang L J, Zhai H, Song X J, He S Z, Liu Q C . A novel ɑ/β-hydrolase gene IbMas enhances salt tolerance in transgenic sweetpotato
PLoS One, 2014,9:e115128.
DOI:10.1371/journal.pone.0115128URLPMID:25501819 [本文引用: 1]
Salt stress is one of the major environmental stresses in agriculture worldwide and affects crop productivity and quality. The development of crops with elevated levels of salt tolerance is therefore highly desirable. In the present study, a novel maspardin gene, named IbMas, was isolated from salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line ND98. IbMas contains maspardin domain and belongs to alpha/beta-hydrolase superfamily. Expression of IbMas was up-regulated in sweetpotato under salt stress and ABA treatment. The IbMas-overexpressing sweetpotato (cv. Shangshu 19) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content was significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbMas up-regulated the salt stress responsive genes, including pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductase, SOD, psbA and phosphoribulokinase genes, under salt stress. These findings suggest that overexpression of IbMas enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and increasing reactive oxygen species scavenging capacity.

Liu D G, He S Z, Song X J, Zhai H, Liu N, Zhang D D, Ren Z T, Liu Q C . IbSIMT1, a novel salt-induced methyltransferase gene from Ipomoea batatas, is involved in salt tolerance
Plant Cell Tissue Organ Cult, 2015,120:701-715.
DOI:10.1007/s11240-014-0638-6URL [本文引用: 1]

Wang B, Zhai H, He S Z, Zhang H, Ren Z T, Zhang D D, Liu Q C . A vacuolar Na+/H+ antiporter gene, IbNHX2, enhances salt and drought tolerance in transgenic sweetpotato
Sci Hortic, 2016,201:153-166.
DOI:10.1016/j.scienta.2016.01.027URL [本文引用: 1]

Wang F B, Tong W J, Zhu H, Kong W L, Peng R H, Liu Q C, Yao Q H . A novel Cys2/His2 zinc fnger protein gene from sweetpotato, IbZFP1, is involved in salt and drought tolerance in transgenic Arabidopsis
Planta, 2016,243:783-797.
DOI:10.1007/s00425-015-2443-9URLPMID:26691387 [本文引用: 1]
MAIN CONCLUSION: IbZFP1, encoding a Cys 2/His 2 zinc finger protein gene from sweetpotato, enhances salt and drought tolerance in transgenic Arabidopsis by regulating ABA signaling pathway, proline biosynthesis, stress responses and ROS scavenging. In plants, Cys2/His2 zinc finger proteins play important roles in regulating the growth and development or responses to abiotic stresses. In this study, a novel Cys2/His2 zinc finger protein gene, named IbZFP1, was isolated from drought-tolerant sweetpotato [Ipomoea batatas (L.) Lam.] line Xu55-2. Subcellular localization analysis in onion epidermal cells indicated that IbZFP1 was localized to the nucleus. Expression analysis in yeast showed that the full length of IbZFP1 exhibited transcriptional activation. Expression of IbZFP1 was induced by NaCl, polyethylene glycol and abscisic acid (ABA). Overexpression of IbZFP1 significantly enhanced salt and drought tolerance in transgenic Arabidopsis plants. Real-time quantitative PCR (qRT-PCR) analysis showed that overexpression of IbZFP1 up-regulated the genes involved in ABA signaling pathway, proline biosynthesis, stress responses, and ROS scavenging under salt and drought stresses. Meanwhile, Western blot and enzymatic analyses showed that the activities of 9-cis-epoxycarotenoid dioxygenase, pyrroline-5-carboxylate synthase, superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase were also increased. Further component analyses indicated that the significant increase of ABA, proline, soluble sugar and total chlorophyll content and the significant reduction of H2O2 and malonaldehyde content were observed under salt and drought stresses. In addition, the rates of electrolyte leakage and water loss were reduced in transgenic plants. The overall results demonstrate the explicit role of IbZFP1 in conferring salt and drought tolerance in transgenic Arabidopsis plants. The IbZFP1 gene has the potential to be used to enhance the tolerance to abiotic stresses in plants.

Wang F B, Zhai H, An Y Y, Si Z Z, He S Z, Liu Q C . Overexpression of IbMIPS1 gene enhances salt tolerance in transgenic sweetpotato
J Integr Agric, 2016,15:271-281.
DOI:10.1016/S2095-3119(14)60973-4URL [本文引用: 1]

Zhai H, Wang F B, Si Z Z, Huo J X, Xing L, An Y Y, He S Z, Liu Q C . A myo-inositol-1-phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and stem nematode resistance in transgenic sweet potato
Plant Biotechnol J, 2016,14:592-602.
DOI:10.1111/pbi.2016.14.issue-2URL [本文引用: 2]

Li R J, Kang C, Song X J, Yu L, Liu D G, He S Z, Zhai H, Liu Q C . A ζ-carotene desaturase gene, IbZDS, increases β-carotene and lutein contents and enhances salt tolerance in transgenic sweetpotato
Plant Sci, 2017,262:39-51.
DOI:10.1016/j.plantsci.2017.05.014URLPMID:28716419 [本文引用: 1]
zeta-Carotene desaturase (ZDS) is one of the key enzymes in carotenoid biosynthesis pathway. However, the ZDS gene has not been applied to carotenoid improvement of plants. Its roles in tolerance to abiotic stresses have not been reported. In this study, the IbZDS gene was isolated from storage roots of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Nongdafu 14. Its overexpression significantly increased beta-carotene and lutein contents and enhanced salt tolerance in transgenic sweetpotato (cv. Kokei No. 14) plants. Significant up-regulation of lycopene beta-cyclase (beta-LCY) and beta-carotene hydroxylase (beta-CHY) genes and significant down-regulation of lycopene epsilon-cyclase (epsilon-LCY) and epsilon-carotene hydroxylase (epsilon-CHY) genes were found in the transgenic plants. Abscisic acid (ABA) and proline contents and superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants under salt stress. The salt stress-responsive genes encoding pyrroline-5-carboxylate reductase (P5CR), SOD, CAT, ascorbate peroxidase (APX) and POD were found to be significantly up-regulated in the transgenic plants under salt stress. This study indicates that the IbZDS gene has the potential to be applied for improving beta-carotene and lutein contents and salt tolerance in sweetpotato and other plants.

Kang C, Zhai H, Xue L Y, Zhao N, He S Z, Liu Q C . A lycopene β-cyclase gene, IbLCYB2, enhances carotenoid contents and abiotic stress tolerance in transgenic sweetpotato
Plant Sci, 2018,272:243-254.
DOI:10.1016/j.plantsci.2018.05.005URLPMID:29807598 [本文引用: 2]
Lycopene beta-cyclase (LCYB) is an essential enzyme that catalyzes the conversion of lycopene into alpha-carotene and beta-carotene in carotenoid biosynthesis pathway. However, the roles and underlying mechanisms of the LCYB gene in plant responses to abiotic stresses are rarely known. This gene has not been used to improve carotenoid contents of sweetpotato, Ipomoea batatas (L.) Lam.. In the present study, a new allele of the LCYB gene, named IbLCYB2, was isolated from the storage roots of sweetpotato line HVB-3. Its overexpression significantly increased the contents of alpha-carotene, beta-carotene, lutein, beta-cryptoxanthin and zeaxanthin and enhanced the tolerance to salt, drought and oxidative stresses in the transgenic sweetpotato (cv. Shangshu 19) plants. The genes involved in carotenoid and abscisic acid (ABA) biosynthesis pathways and abiotic stress responses were up-regulated in the transgenic plants. The ABA and proline contents and superoxide dismutase (SOD) activity were significantly increased, whereas malonaldehyde (MDA) and H2O2 contents were significantly decreased in the transgenic plants under abiotic stresses. The overall results indicate that the IbLCYB2 gene enhances carotenoid contents and abiotic stress tolerance through positive regulation of carotenoid and ABA biosynthesis pathways in sweetpotato. This gene has the potential to improve carotenoid contents and abiotic stress tolerance in sweetpotato and other plants.

Zhang H, Gao X R, Zhi Y H, Li X, Zhang Q, Niu J B, Wang J, Zhai H, Zhao N, Li J G, Liu Q C, He S Z . A non-tandem CCCH-type zinc-finger protein, IbC3H18, functions as a nuclear transcriptional activator and enhances abiotic stress tolerance in sweet potato
New Phytol, 2019,223:1918-1936.
DOI:10.1111/nph.15925URLPMID:31091337 [本文引用: 1]
CCCH-type zinc-finger proteins play essential roles in regulating plant development and stress responses. However, the molecular and functional properties of non-tandem CCCH-type zinc-finger (non-TZF) proteins have been rarely characterized in plants. Here, we report the biological and molecular characterization of a sweet potato non-TZF gene, IbC3H18. We show that IbC3H18 exhibits tissue- and abiotic stress-specific expression, and could be effectively induced by abiotic stresses, including NaCl, polyethylene glycol (PEG) 6000, H2 O2 and abscisic acid (ABA) in sweet potato. Accordingly, overexpression of IbC3H18 led to increased, whereas knock-down of IbC3H18 resulted in decreased tolerance of sweet potato to salt, drought and oxidation stresses. In addition, IbC3H18 functions as a nuclear transcriptional activator and regulates the expression of a range of abiotic stress-responsive genes involved in reactive oxygen species (ROS) scavenging, ABA signaling, photosynthesis and ion transport pathways. Moreover, our data demonstrate that IbC3H18 physically interacts with IbPR5, and that overexpression of IbPR5 enhances salt and drought tolerance in transgenic tobacco plants. Collectively, our data indicate that IbC3H18 functions in enhancing abiotic stress tolerance in sweet potato, which may serve as a candidate gene for use in improving abiotic stress resistance in crops.

杨元军, 王玉萍, 翟红, 刘庆昌 . 甘薯块根总RNA的高效快速提取方法
分子植物育种, 2008,6:193-196.
[本文引用: 1]

Yang Y J, Wang Y P, Zhai H, Liu Q C . A simple and rapid procedure for RNA isolation from storage roots of sweetpotato ( Ipomoea batatas)
Mol Plant Breed, 2008,6:193-196 (in Chinese with English abstract).
[本文引用: 1]

Wang L J, He S Z, Zhai H, Liu D G, Wang Y N, Liu Q C . Molecular cloning and functional characterization of a salt tolerance-associated gene IbNFU1 from sweetpotato
J Integr Agric, 2013,12:27-35.
DOI:10.1016/S2095-3119(13)60202-6URL [本文引用: 2]

Jiang T, Zhai H, Wang F B, Zhou H N, Si Z Z, He S Z, Liu Q C . Cloning and characterization of a salt tolerance-associated gene encoding trehalose-6-phosphate synthase in sweetpotato
J Integr Agric, 2014,13:1651-1661.
DOI:10.1016/S2095-3119(13)60534-1URL [本文引用: 1]

喻娜, 郭新勇, 焦天奇, 祝建波 . 转小拟南芥ApHRD基因烟草获得及其抗旱性鉴定
西北植物学报, 2010,30:2385-2393.
[本文引用: 1]

Yu N, Guo X Y, Jiao T Q, Zhu J B . Transformation of ApHRD gene and drought-tolerance identification of transgenic plants in tobacco
Acta Bot Boreali-Occident Sin, 2010,30:2385-2393 (in Chinese with English abstract).
[本文引用: 1]

Huo J X, Du B, Sun S F, He S Z, Zhao N, Liu Q C, Zhai H . A novel aldo-keto reductase gene, IbAKR, from sweet potato confers higher tolerance to cadmium stress in tobacco
Front Agric Sci Eng, 2018,5:206-213.
[本文引用: 1]

Gill S S, Tuteja N . Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants
Plant Physiol Biochem, 2010,48:909-930.
DOI:10.1016/j.plaphy.2010.08.016URLPMID:20870416 [本文引用: 2]
Various abiotic stresses lead to the overproduction of reactive oxygen species (ROS) in plants which are highly reactive and toxic and cause damage to proteins, lipids, carbohydrates and DNA which ultimately results in oxidative stress. The ROS comprises both free radical (O(2)(-), superoxide radicals; OH, hydroxyl radical; HO(2), perhydroxy radical and RO, alkoxy radicals) and non-radical (molecular) forms (H(2)O(2), hydrogen peroxide and (1)O(2), singlet oxygen). In chloroplasts, photosystem I and II (PSI and PSII) are the major sites for the production of (1)O(2) and O(2)(-). In mitochondria, complex I, ubiquinone and complex III of electron transport chain (ETC) are the major sites for the generation of O(2)(-). The antioxidant defense machinery protects plants against oxidative stress damages. Plants possess very efficient enzymatic (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; glutathione reductase, GR; monodehydroascorbate reductase, MDHAR; dehydroascorbate reductase, DHAR; glutathione peroxidase, GPX; guaicol peroxidase, GOPX and glutathione-S- transferase, GST) and non-enzymatic (ascorbic acid, ASH; glutathione, GSH; phenolic compounds, alkaloids, non-protein amino acids and alpha-tocopherols) antioxidant defense systems which work in concert to control the cascades of uncontrolled oxidation and protect plant cells from oxidative damage by scavenging of ROS. ROS also influence the expression of a number of genes and therefore control the many processes like growth, cell cycle, programmed cell death (PCD), abiotic stress responses, pathogen defense, systemic signaling and development. In this review, we describe the biochemistry of ROS and their production sites, and ROS scavenging antioxidant defense machinery.

Smirnoff N, Cumbes Q J . Hydroxyl radical scavenging activity of compatible solutes
Phytochemistry, 1989,28:1057-1060.
DOI:10.1016/0031-9422(89)80182-7URL [本文引用: 1]

Bao A K, Wang S M, Wu G Q, Xi J J, Zhang J L, Wang C M . Overexpression of the Arabidopsis H+-PPase enhanced resistance to salt and drought stress in transgenic alfalfa (Medicago sativa L.)
Plant Sci, 2009,176:232-240.
DOI:10.1016/j.plantsci.2008.10.009URL [本文引用: 1]

Kumar V, Shriram V, Kishor P B K, Jawali N, Shitole M G . Enhanced proline accumulation and salt stress tolerance of transgenic indica rice by over-expressing P5CSF129A gene
Plant Biotechnol Rep, 2010,4:37-48.
DOI:10.1007/s11816-009-0118-3URL [本文引用: 1]
Δ1-pyrroline-5-carboxylate synthetase (P5CS) is a proline biosynthetic pathway enzyme and is known for conferring enhanced salt and drought stress in transgenics carrying this gene in a variety of plant species; however, the wild-type P5CS is subjected to feedback control. Therefore, in the present study, we used a mutagenized version of this osmoregulatory gene-P5CSF129A, which is not subjected to feedback control, for producing transgenic indica rice plants of cultivar Karjat-3 via Agrobacterium tumefaciens. We have used two types of explants for this purpose, namely mature embryo-derived callus and shoot apices. Various parameters for transformation were optimized including antibiotic concentration for selection, duration of cocultivation, addition of phenolic compound, and bacterial culture density. The resultant primary transgenic plants showed more enhanced proline accumulation than their non-transformed counterparts. This proline level was particularly enhanced in the transgenic plants of next generation (T1) under 150mM NaCl stress. The higher proline level shown by transgenic plants was associated with better biomass production and growth performance under salt stress and lower extent of lipid peroxidation, indicating that overproduction of proline may have a role in counteracting the negative effect of salt stress and higher maintenance of cellular integrity and basic physiological processes under stress.]]>
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