,, 魏梦园, 黎冬华, 周瑢, 张秀荣, 游均,中国农业科学院油料作物研究所/农业农村部油料作物生物学与遗传育种重点实验室,武汉 430062Cloning and Function Analysis of Sesame Galactinol Synthase Gene SiGolS6 in Arabidopsis
LIU AiLi,, WEI MengYuan, LI DongHua, ZHOU Rong, ZHANG XiuRong, YOU Jun,Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan 430062通讯作者:
责任编辑: 李莉
收稿日期:2019-12-30接受日期:2020-03-8网络出版日期:2020-09-01
| 基金资助: |
Received:2019-12-30Accepted:2020-03-8Online:2020-09-01
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刘爱丽,E-mail：
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刘爱丽, 魏梦园, 黎冬华, 周瑢, 张秀荣, 游均. 芝麻肌醇半乳糖苷合成酶基因SiGolS6的克隆及功能分析[J]. 中国农业科学, 2020, 53(17): 3432-3442 doi:10.3864/j.issn.0578-1752.2020.17.002
LIU AiLi, WEI MengYuan, LI DongHua, ZHOU Rong, ZHANG XiuRong, YOU Jun.
0 引言
【研究意义】芝麻（Sesamum indicum L.）是中国重要的传统油料作物之一,具有颇高的经济效益,在国内外都具有重要地位。干旱是限制各种农作物生产的主要逆境之一[1],近年来,中国芝麻产区干旱发生的范围呈现扩大的趋势,旱害造成芝麻减产20%以上,严重时甚至绝收[2]。因此,加强芝麻抗旱功能基因发掘,明确相关基因的调控机理,对培育抗旱芝麻新品种十分必要。棉子糖系列寡糖（raffinose family oligosaccharides,RFOs）是一种小分子渗透调节物[3],其代谢与植物的生长发育、抗逆反应等关系密切。肌醇半乳糖苷合成酶（galactinol synthase,GolS）是催化RFOs合成的关键酶[3],在植物非生物逆境抗性中发挥重要作用。【前人研究进展】植物在受到干旱、高盐和低温等多种逆境形成的渗透胁迫时,体内会积累渗透调节物使细胞液浓度升高,降低渗透势,使细胞吸水或保水能力得到提高,从而适应不利环境。RFOs就是其中一种小分子渗透调节物,它由一系列α-1,6-半乳糖连接到蔗糖上的6-葡萄糖基组成,主要包括棉子糖、水苏糖和毛蕊花糖[4]。RFOs广泛分布在植物的不同组织部位,在高等植物种子中含量相对较高[5,6]。有研究表明,一些体内不含RFOs或含量极低的植物,受到逆境胁迫后RFOs会迅速积累,植株抗逆性也随之增强[7,8],这一响应逆境的生理现象在番茄[9]、苜蓿[10]、辣椒[11]等植物中均存在。近年来,还发现棉子糖系列寡糖代谢与植物生长发育和种子活力建成关系密切[12]。GolS参与RFOs合成的第一步,该步骤是RFOs家族中各物质合成积累的关键步骤[13],因此,其在植物抗逆应答中的作用重大。大多数GolS基因受多种胁迫诱导表达,据报道,在拟南芥中鉴定了7个GolS基因（AtGolS1—AtGolS7）[8],其中,AtGolS1和AtGolS2的表达分别受干旱和高盐胁迫诱导,AtGolS3受冷胁迫诱导,说明它们均与抗逆应答相关。研究还发现AtGolS2超表达的拟南芥材料对干旱胁迫的耐受性显著增强[14],有报道还指出AtGolS2能够提高单子叶模式植物二穗短柄草的抗旱性[15]。另外,有研究发现木薯MeGolS5的表达量在干旱胁迫下会迅速上升,在大肠杆菌和拟南芥中过量表达该基因均能提高抗旱能力,说明GolS在植物抗旱调节中起着重要作用[3,16]。将牛耳草肌醇半乳糖苷合成酶基因（BhGolS1）在烟草中超表达,也显著提高了转基因植株的抗旱能力[17]。另外,玉米ZmGolS2受DREB2A转录因子调控,ZmGolS2与DREB2A超表达均可提高拟南芥抗逆性,并且对植株在正常条件下的生长没有任何负效应[18]。可见GolS在作物抗旱遗传改良中具有广阔的应用前景。前期工作中,在芝麻全基因组范围内鉴定了7个GolS基因（SiGolS1—SiGolS7）,表达分析显示,SiGolS2、SiGolS6和SiGolS7受干旱胁迫的强烈诱导[19]。【本研究切入点】虽然GolS基因在植物的干旱抗性中具有重要作用,芝麻SiGolS6的表达也受干旱胁迫诱导,但SiGolS6能否提高植物抗逆性,目前仍鲜见报道。【拟解决的关键问题】本研究从芝麻中分离SiGolS6,并利用农杆菌花序浸染法将其在拟南芥中超量表达,通过鉴定超量表达转SiGolS6拟南芥的抗旱性,分析其在干旱抗性中的功能,以期为芝麻抗旱遗传改良提供理论基础和基因资源。1 材料与方法
1.1 试验材料
所用芝麻品种为晋芝2号,菌株为大肠杆菌DH5α和农杆菌LBA4404,植物表达载体pCAMBIA1301S是在pCAMBIA1301基础上改建而成[20]。1.2 基因的克隆及序列分析
使用EASYspin Plus植物RNA提取试剂盒（北京艾德莱生物科技有限公司）提取干旱胁迫处理7 d的初花期抗旱芝麻品种晋芝2号叶片组织总RNA,用HiScipt II One Step RT-PCR Kit反转录试剂盒（南京诺唯赞生物科技有限公司）逆转录合成cDNA。根据前期对芝麻GolS基因家族的分析[19],通过登录号SIN_1022774在芝麻基因组数据库（
SiGolS6同源基因序列下载自NCBI（
Table 1
表1
表1本研究中所用引物序列
Table 1
| 引物名称 Primer name | 引物序列 Primer sequence (5′-3′) |
|---|---|
| SiGolS6q-F | CCAATCCCCGCAACCTATAAC |
| SiGolS6q-R | TGCTCTTTGTCGGTGTACTTC |
| Actinq-F | CCCGCTATGTATGTCGCCA |
| Actinq-R | AACCCTCGTAGATTGGCACAG |
| GolS6FL-F | GGGGTACCATGGTTCCTGAAATTATCATTCC |
| GolS6FL-R | CGGGATCCTCAACCGGAAACTTGATCAATTG |
新窗口打开|下载CSV
1.3 植物表达载体的构建
用BamHⅠ和KpnⅠ对pCAMBIA1301S表达载体进行双酶切获得线性化载体,将其通过琼脂糖凝胶电泳和琼脂糖凝DNA回收试剂盒（天根生化科技有限公司）纯化后获得高纯度线性化载体。将同样纯化后的SiGolS6片段与线性化pCAMBIA1301S载体通过同源重组的方法进行连接,获得植物表达载体pCAMBIA1301S-SiGolS6。1.4 转化拟南芥及阳性植株的鉴定
通过冻融法将pCAMBIA1301-SiGolS6植物表达载体转化LBA4404农杆菌感受态,挑取阳性克隆,菌落PCR鉴定后,将其接种到含有利福平（Rif）和卡那霉素（Kana）的LB液体培养基上,28℃振荡培养至OD600为0.4—0.6。然后采用花序浸染法浸染拟南芥花序,进行遗传转化[22]。浸染后的植株继续在室温22℃、12 h光照/12 h黑暗的培养间培养至收获种子（T0代）。将T0代种子消毒后播种于含潮霉素（30 mg·L-1）的MS培养基（添加25 mg·L-1头孢霉素抑菌）上筛选,将能正常生长的转基因植株移栽到含泥炭土、蛭石（体积比为1﹕3）的花钵中,同时以野生型拟南芥作为对照,继续生长收种,用于后续PCR试验鉴定筛选[23]。待上述初步筛选得到的阳性苗长至8片真叶后,用天根生化科技（北京）有限公司的植物DNA提取试剂盒提取叶片基因组DNA,以野生型拟南芥DNA为阴性对照,利用PCR方法鉴定是否获得T1代阳性转基因植株。
将T1代阳性转基因植株单株收获的T1代种子继续进行潮霉素筛选,获得T2代阳性植株,选取上述获得部分阳性株系将其移栽生长,再次提取叶片基因组DNA进行PCR分子鉴定,确定T2代阳性植株。
1.5 实时荧光定量PCR（qPCR）检测
取转基因T2代阳性植株和野生型植株幼嫩叶片,用EASYspin Plus植物RNA提取试剂盒（北京艾德莱生物科技有限公司）提取叶片总RNA,使用微量分光光度计测定RNA浓度和质量,反转录获得cDNA。利用Beacon Designer8.14设计SiGolS6荧光定量PCR引物SiGolS6q-F/R。拟南芥持家基因Actin2（At3g18780）作为内参基因,实时荧光定量PCR分析SiGolS6在转基因和野生型植株中的表达水平。实时荧光定量PCR试剂盒ChamQ Universal SYBR qPCR Master Mix购自南京诺唯赞生物科技有限公司,按照试剂盒说明书配置荧光定量PCR反应体系,实时荧光定量PCR分析平台为Roche LightCycler 480,基因特异性引物见表1。采用2-ΔΔCT法分析SiGolS6相对表达水平[24]。1.6 转基因拟南芥的耐旱性分析
在温室中,将不同株系的转基因拟南芥种子和野生型拟南芥种子直接点播于含3﹕1（v/v）蛭石和营养土的花钵（直径10 cm）中,先用保鲜膜保湿48 h,揭膜后置于室温22℃、12 h光照/12 h黑暗的培养间中继续培养,定期浇水。种子点播7 d后,间苗,每钵留苗3—4棵,再用保鲜膜保湿1 d。种子点播至土里第14 d,开始干旱处理。干旱处理前给所有植株浇足水,待吸水至饱和后,将放置花钵的大盘子里多余的水全部倒掉,以这一天计为干旱处理第1 d。干旱处理28 d后,复水生长5 d,观察表型并拍照（含复水5 d后表型）,并统计2种材料的存活数,计算存活率。选取正常生长、干旱胁迫28 d及复水5 d材料的地上部分,用清水冲洗其表面杂物,吸水纸吸干水分,在天平上称重,测定其鲜重变化情况。随机摘取15片大小相对一致的叶片,混合均匀后分成3份即时称量其鲜重,之后室温放置在滤纸上,每间隔0.5 h称重1次,计算其离体叶片失水率。取正常生长14 d的转基因和野生型植株,检测二者的棉子糖含量。棉子糖含量采用内标法由武汉迈特维尔生物科技有限公司（http://www.metware.cn/）基于Agilent7890B-7000D GC-MS/MS平台检测。取正常供水对照和干旱胁迫21 d后转基因与野生型植株的叶片,检测二者的相对电导率、过氧化物酶（peroxidase,POD）和超氧化物歧化酶（superoxide dismutase,SOD）活性,以及丙二醛（malondialdehyde,MDA）含量变化和O2-产生情况,每个指标每个株系取3—4个重复进行测定。使用浸泡法测定相对电导率[25]。POD、SOD活性测定试剂盒和MDA含量测定试剂盒购自南京建成生物工程研究所,具体试验流程及计算方法参照南京建成（www.njjcbio.com）丙二醛（MDA）测定试剂盒（TBA法）、总超氧化物歧化酶（T-SOD）测试盒（羟胺法）及过氧化物酶（POD）测定试剂盒（测植物）（比色法）说明书完成。
1.7 数据分析
试验数据用Microsoft Excel 2010和 SPSS11.5软件进行整理和统计分析。2 结果
2.1 SiGolS6序列分析
克隆获得SiGolS6全长CDS,序列长度为921 bp,编码306个氨基酸,蛋白分子量为35.07 kD,等电点为4.75。多序列比对和保守结构域分析发现芝麻SiGolS6与其他植物中的同源基因都含有一个糖基转移酶保守结构域（glycosyl transferase,family 8,IPR002495）,属于糖基转移酶超家族（图1-A）。图1
新窗口打开|下载原图ZIP|生成PPT图1SiGolS6和其他物种GolS的多序列比对（A）和系统进化分析（B）
SiGolS6：芝麻Sesamum indicum,XP_011087512.1;CsGolS：甜橙Citrus sinensis,XP_006444560.1;PtGolS：毛果杨Populus trichocarpa,XP_011087512.1;AtGolS1：拟南芥Arabidopsis thaliana,NP_182240.1;GrGolS：棉花Gossypium raimondii,XP_012490292.1;VvGolS：葡萄Vitis vinifera,XP_002281369.1;SlGolS：番茄Solanum lycopersicum,NP_001234668.2;StGolS：马铃薯Solanum tuberosum,ADW78838.1;SbGolS：高粱Sorghum bicolor,XP_002467954.1;BdGolS：二穗短柄草Brachypodium distachyon,XP_003558046.1;SitGolS：谷子Setaria italica,XP_004984579.1;OsGolS：水稻Oryza sativa,XP_015628833.1;ZmGolS：玉米Zea mays,NP_001105748.2。划线处为Glycosyl transferase, family 8（IPR002495）保守结构域The line marks the Glycosyl transferase, family 8 (IPR002495) conserved domain
Fig. 1Multiple sequence alignment (A) and phylogenetic analysis (B) of SiGolS6 and its homologs
为了进一步研究芝麻SiGolS6蛋白在物种中的进化位置及亲缘关系,应用MEGA5.2程序构建了多物种的系统进化树（图1-B）。结果表明,芝麻SiGolS6和马铃薯中的同源基因亲缘关系较近。
2.2 转基因拟南芥阳性植株鉴定
将pCAMBIA1301S-SiGolS6转化拟南芥,T1转基因材料经潮霉素筛选,共获得6个独立的转基因植株。22℃光照培养箱培养7—10 d,T2转基因株系在含潮霉素的MS筛选培养基上生长状况良好（图2-A）,而野生型（wild type,WT）拟南芥根系较短,均黄化死去（图2-B）。提取WT和T2转基因植株的DNA,进行目的基因的分子验证,除WT外,均能扩增出目的条带（图2-C）。随机选择3个独立的转基因株系OE-2、OE-3、OE-4进行qPCR检测,结果显示,3个转基因株系中SiGolS6的表达显著高于野生型（图2-D）。GolS是催化棉子糖合成的关键酶[3]。进一步检测了SiGolS6超表达T3转基因株系及野生型中的棉子糖含量,结果表明,转基因拟南芥的棉子糖含量显著高于野生型植株（图3）。图2
新窗口打开|下载原图ZIP|生成PPT图2转基因拟南芥的获得和鉴定
A、B：含潮霉素的MS筛选培养基上生长的T2转基因（A）和野生型（B）植株;C：T2阳性转基因植株鉴定,1—6：转基因株系;WT：野生型对照;D：转基因株系中SiGolS6的表达量检测。*表示在P<0.05水平上差异显著;**表示在P<0.01水平上差异显著。下同
Fig. 2Acquisition and identification of transgenic Arabidopsis
A, B: Transgenic (A) and wild type (B) plants growing on selective medium containing hygromycin; C: Identification of positive T2 generation transgenic Arabidopsis plants; 1-6: SiGolS6 transgenic lines; WT: Wild type; D: Transcript level of SiGolS6 in transgenic Arabidopsis plants. * indicate significant difference at P<0.05 level; **indicates significant difference at P<0.01 level. The same as below
图3
新窗口打开|下载原图ZIP|生成PPT图3转基因株系中棉子糖含量检测
Fig. 3Raffinose content in transgenic Arabidopsis plants
2.3 转基因拟南芥的抗旱性分析
为了研究转SiGolS6拟南芥的抗旱性,对正常培养14 d的T3转基因植株和野生型植株进行干旱胁迫处理（不浇水）。结果表明,正常供水条件下,转基因株系和野生型植株均正常生长,二者长势无明显区别;干旱胁迫处理28 d,野生型植株萎蔫程度较转基因株系严重（图4-A）,复水处理（恢复正常供水）5 d后,50%的转基因植株基本恢复,而野生型植株存活率不到10%（图4-A和图4-C）。图4
新窗口打开|下载原图ZIP|生成PPT图4转基因拟南芥耐旱性鉴定
A：干旱胁迫下野生型和转基因植株的表型;B：野生型和转基因植株离体叶片失水速率,3次重复;C：干旱胁迫复水后野生型和转基因植株的存活率;D—E：干旱胁迫下野生型和转基因植株鲜重（D）和相对电导率（E）
Fig. 4Characteristics of transgenic plants under drought stress
A: Phenotype of WT and transgenic plants under drought stress; B: Water loss from detached leaves of WT and transgenic plants at indicated time points; C: Survival rate of WT and transgenic plants after drought stress; D-E: Fresh weight (D) and relative electrical conductivity (E) of WT and transgenic plants after drought stress
离体叶片失水率试验显示,野生型植株的失水率始终高于转基因株系（图4-B）。选取正常生长、干旱胁迫28 d及复水5 d的材料,分别测量其鲜重。结果显示,正常供水条件下,转基因材料的鲜重与野生型无明显差异。干旱胁迫及复水处理后,转基因植株的鲜重则显著高于野生型（图4-D）。电导率测定结果显示,干旱处理21 d后,转基因株系的相对电导率也显著低于野生型植株（图4-E）。
活性氧（ROS）和MDA含量、SOD及POD活性是衡量植物抗逆性状的重要生理指标。氮蓝四唑（nitrobluetetrazolium,NBT）染色结果显示,正常供水条件下,转基因材料染色程度和野生型材料基本一致,说明二者在正常条件下产生的超氧自由基差异不大。而干旱胁迫下,转基因材料较野生型材料染色面积小,说明胁迫条件下,转基因株系积累的活性氧明显低于野生型材料（图5-A）。取干旱胁迫21 d的拟南芥植株,分别检测其MDA含量、SOD及POD活性,发现转基因株系的相对MDA含量明显低于野生型植株（图5-B）,相对SOD活性和POD活性则明显高于野生型拟南芥植株（图5-C和图5-D）。
图5
新窗口打开|下载原图ZIP|生成PPT图5转基因拟南芥氧化相关生理指标测定
A：正常供水和干旱胁迫下离体叶片的氮蓝四唑（NBT）染色;B—D：干旱胁迫下野生型和转基因植株相对MDA含量（B）、相对SOD活性（C）和相对POD活性（D）
Fig. 5Determination of oxidation-related physiological indexes in transgenic Arabidopsis plants
A: Nitro blue tetrazolium staining of leaves from WT and transgenic plants under normal or drought treatments; B-D: Relative content of MDA (B), relative activity of SOD (C) and relative activity of POD (D) of WT and transgenic plants after drought stress
3 讨论
3.1 芝麻SiGolS6序列分析
SiGolS6是芝麻糖基转移酶超家族中的一员,通过对芝麻SiGolS6和其他物种GolS进行多序列比对并构建系统进化树,表明SiGolS6与马铃薯同源基因的亲缘关系较近。有研究发现,RFOs对马铃薯在胁迫条件下的耐受能力起着关键作用[26],而GolS是马铃薯RFOs合成过程中的关键酶,可见GolS对马铃薯在逆境下的抗性也至关重要。同源性分析发现芝麻SiGolS6与马铃薯亲缘关系较近,说明SiGolS6可能也参与植物逆境胁迫抗性。前期研究工作中,我们在芝麻基因组中鉴定了7个GolS基因（SiGolS1—SiGolS 7）,表达分析发现,SiGolS6受干旱胁迫的强烈诱导[19]。而前人研究表明,拟南芥、水稻、玉米、白杨和番茄中GolS基因的表达在干旱、高盐等非生物胁迫条件下上升[14,27-29]。通过进一步研究证明了上述一些基因在逆境胁迫中发挥了重要作用,如拟南芥AtGolS2的表达受干旱胁迫诱导,而超表达AtGols2不仅能够增强拟南芥的抗旱、抗盐以及抗氧化胁迫的能力[14,30],而且能够提高单子叶模式植物二穗短柄草以及水稻的抗旱性[15,31]。以上结果表明芝麻SiGolS6可能参与植物抗逆反应。3.2 芝麻SiGolS6对转基因拟南芥耐旱性的影响
植物在正常条件下RFOs含量很少,但在逆境胁迫下,RFOs的积累会被诱导,从而增强植株的抗逆性[7-8,32]。GolS是催化RFOs合成的关键酶[3],其活性会影响RFOs的积累水平[33]。检测转基因和野生型植株的棉子糖含量发现,转基因拟南芥的棉子糖含量显著高于野生型植株,说明GolS表达量的升高确实导致了RFOs的积累。通过对转基因拟南芥材料在正常及干旱胁迫条件下的表型鉴定发现,在正常条件下, 转基因植株与野生型植株的生长（鲜重）无显著差异,但在干旱胁迫处理及复水后,转基因植株的鲜重显著高于野生型植株。干旱胁迫过程中,转基因植株的萎蔫程度较野生型轻;复水5 d后,大约50%的转基因植株基本恢复正常生长状态,而野生型植株死亡率则达到90%以上。进一步分析发现,转基因植株的离体叶片失水速率低于野生型;干旱胁迫下,转基因植株的相对电导率也显著低于野生型。以上结果都说明在干旱胁迫下,SiGolS6提高了转基因植株的耐旱能力。此外,转基因拟南芥植株的抗旱性可能与SiGolS6表达量水平存在一定的关系。如表达量较高的OE-2家系在胁迫复水后,长势较OE-3和OE-4更好。存活率、鲜重和相对电导率等指标也显示OE-2家系的耐旱性强于OE-3和OE-4。3.3 芝麻SiGolS6对转基因拟南芥抗氧化能力的影响
在逆境胁迫条件下,植物体内的ROS会大量积累,导致细胞受到损害,并对植物生长发育产生不利影响[34]。有研究表明,RFOs可以作为抗氧化剂,限制ROS过量积累,保护细胞免受氧化损伤[35,36]。超量表达拟南芥和鹰嘴豆GolS基因的转基因材料均表现出RFOs含量上升,在逆境胁迫条件下ROS积累较少,氧化损伤较小,对逆境胁迫抗性显著增强[35,37]。本研究中,NBT染色结果表明,干旱胁迫下转基因株系积累的O2-低于野生型材料,转基因株系的MDA含量也显著低于野生型,说明转基因植株在干旱胁迫下ROS积累较少,受到的氧化损伤相对较低。SOD和POD是植物酶促抗氧化系统的重要组分,在活性氧清除和非生物逆境抗性中发挥着重要作用[34]。超量表达南极发草（Deschampsia antarctica）DaGolS2和水稻OsGolS2都可以提高转基因水稻中ROS清除相关酶基因的表达,降低胁迫条件下ROS积累水平,显著增强转基因材料对干旱和低温的抗性[38]。本研究也发现SiGolS6转基因拟南芥株系中SOD和POD活性显著高于野生型。综上所述,SiGolS6转基因植株通过提高抗氧化能力增强了对干旱胁迫的抗性。4 结论
在拟南芥中超量表达芝麻肌醇半乳糖苷合成酶基因SiGolS6,提高了转基因植株棉子糖含量和抗氧化酶活性,增强了转基因植株干旱胁迫耐受性。推测SiGolS6可能参与植物对干旱胁迫的应答过程。参考文献 原文顺序
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被引期刊影响因子
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Galactinol synthase (UDP-galactose:inositol galactosyltransferase) is the first unique enzyme in the biosynthetic pathway of raffinose saccharides. Its role as a regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean (Glycine max L. Merrill) seeds was examined. Galactinol synthase activity and concentrations of sucrose, stachyose, and raffinose were compared during seed development between two genotypes that were high and two genotypes that were low in mature seed raffinose saccharide concentration. In all genotypes, sucrose concentration increased as seed development progressed, but in both low raffinose saccharide genotypes, greater increases in sucrose concentration were observed late in seed development. Sucrose to stachyose ratios in mature seeds were 2.3-fold greater in low raffinose saccharide genotypes than in the high raffinose saccharide genotypes. During seed development, higher levels of galactinol synthase activity were observed in the high raffinose saccharide genotypes than in the low raffinose saccharide genotypes. A common linear relationship for all four soybean genotypes was shown to exist between galactinol formed estimated from galactinol synthase activity data and the concentration of galactose present in raffinose saccharides. Results of this study implied that galactinol synthase is an important regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean seeds.
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Brachypodium distachyon (purple false brome) is a herbaceous species belonging to the grass subfamily Pooideae, which also includes major crops like wheat, barley, oat and rye. The species has been established as experimental model organism for understanding and improving cereal crops and temperate grasses. The complete genome of Bd21, the community standard line of B. distachyon, has been sequenced and protocols for Agrobacterium-mediated transformation have been published. Further improvements to the experimental platform including better evaluation systems for transgenic plants are still needed. Here we describe the growth conditions for Bd21 plants yielding highly responsive immature embryos that can generate embryogenic calli for transformation. A prolonged 20-h photoperiod produced seeds with superior immature embryos. In addition, osmotic treatment of embryogenic calli enhanced the efficiency of transfection by particle bombardment. We generated transgenic plants expressing Arabidopsis thaliana galactinol synthase 2 (AtGolS2) in these experiments. AtGolS2-expressing transgenics displayed significantly improved drought tolerance, increasing with increased expression of AtGolS2. These results demonstrate that AtGolS2 can confer drought tolerance to monocots and confirm that Brachypodium is a useful model to further explore ways to understand and improve major monocot crop species.
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Accumulation of compatible osmolytes, such as soluble sugars, in plants is an important osmoprotective mechanism. Sugars play a role in osmotic adjustment and are associated with stabilization of proteins and cell structures, reactive oxygen species scavenging, signaling functions or induction of adaptive pathways. Galactinol is the galactosyl donor for the synthesis of raffinose family oligosaccharides (RFOs) and its synthesis by galactinol synthase (GolS) is the first committed step of the RFOs biosynthetic pathway. GolS genes are induced by a variety of stresses in both stress-sensitive and tolerant-plant species; however, the mechanism of transcriptional regulation is not fully established. In this paper, we characterized a GolS gene (BhGolS1) that was dehydration and ABA-inducible in the resurrection plant Boea hygrometrica and conferred dehydration tolerance in a transgenic tobacco system. Four W-box cis-elements were identified in the BhGolS1 promoter and shown to be bound by an early dehydration and ABA-inducible WRKY gene (BhWRKY1). These data suggest a mechanism where BhWRKY1 is likely to function in an ABA-dependent signal pathway to regulate BhGolS1 expression, which leads to the accumulation of RFOs in desiccation-tolerant B. hygrometrica leaves.
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Sesame (Sesamum indicum L.) is an important oilseed crop. However, multiple abiotic stresses severely affect sesame growth and production. Raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, play an important role in desiccation tolerance of plants and developing seeds. In the present study, three types of key enzymes, galactinol synthase (GolS), raffinose synthase (RafS) and stachyose synthase (StaS), responsible for the biosynthesis of RFOs were identified at the genome-wide scale in sesame. A total of 7 SiGolS and 15 SiRS genes were identified in the sesame genome. Transcriptome analyses showed that SiGolS and SiRS genes exhibited distinct expression profiles in different tissues and seed developmental stages. Comparative expression analyses under various abiotic stresses indicated that most of SiGolS and SiRS genes were significantly regulated by drought, osmotic, salt, and waterlogging stresses, but slightly affected by cold stress. The up-regulation of several SiGolS and SiRS genes by multiple abiotic stresses suggested their active implication in sesame abiotic stress responses. Taken together, these results shed light on the RFOs-mediated abiotic stress resistance in sesame and provide a useful framework for improving abiotic stress resistance of sesame through genetic engineering.
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Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.
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The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
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Apical shoot tips were dissected from donor plants (cultured in several conditions) and cryopreserved using the droplet-vitrification technique. The effect of two preculture treatments (sucrose pretreatment medium or cold-culturing during two weeks) on donor plants of four potato species (Solanum commersonii, S. juzepcukii, S. ajanhuiri, and Solanum tuberosum) was studied. Post-cryopreservation meristem growth and plant recovery were influenced by the treatments, but the effect on the regeneration was strongly genotype-dependent. The highest post-rewarming plant recovery percentage was obtained using meristems dissected from donor plants of S. commersonii cultured on sucrose pretreatment medium or cold-cultured. Both preculture conditions also enhanced plant recovery in S. juzepcukii compared to control cultures. Cold preculture, however, proved to be undesirable for S. tuberosum whereas sucrose pretreatment had a positive impact on the plant regeneration of this species. The determination of changes in the concentration of soluble sugars revealed sugar accumulation, especially of sucrose and the raffinose family of oligosaccharides (RFOs), which can be linked to tolerance towards the cryopreservation. Additionally, a study of the proteome of the donor plantlets after the pretreatments by 2D-fluorescence difference gel electrophoresis (DIGE) was carried out to identify differentially abundant proteins. Carbon metabolism-related proteins, together with stress-response and oxidative-homeostasis related proteins were the main class of proteins that changed in abundance after the pretreatments. Our results suggest that oxidative homeostasis-related proteins and sugars may be associated with the improved tolerance to cryopreservation and the ability to cold acclimate by S. commersonii in contrast to the other genotypes. The increased accumulation of sucrose and RFOs play a fundamental role in the response to stress in potato and may help to acquire tolerance to cryopreservation.
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A considerable increase in the concentration of raffinose was detected in rice seedlings exposed to chilling for more than 4 days. The content of raffinose in leaf blades increased from hardly detectable levels (0-1 days) to approximately 9 mg/g FW after 11 days of chilling treatment. Accumulation occurred in leaf blades but not in sheaths. Analysis of the expression of candidate genes related to galactinol and raffinose synthesis revealed that transcript levels of two galactinol synthase and four raffinose synthase genes increased in leaf blades before the accumulation of raffinose became detectable.
DOI:10.1093/glycob/cws121URLPMID:22879458
The raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, are synthesized by a set of distinct galactosyltransferases, which sequentially add galactose units to sucrose. The accumulation of RFOs in plant cells are closely associated with the responses to environmental factors, such as cold, heat and drought stresses. Systematic analysis of genes involved in the raffinose metabolism has not been reported to date. Searching the recently available working draft of the maize genome, six kinds of enzyme genes were speculated, which should encode all the enzymes involved in the raffinose metabolism in maize. Expression patterns of some related putative genes were analyzed. The conserved domains and phylogenetic relationships among the deduced maize proteins and their homologs isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as galactinol synthase (ZmGolS5, ZmGolS45 and ZmGolS37), raffinose synthase (ZmRS1, ZmRS2, ZmRS3 and ZmRS10), stachyose synthase (ZmRS8) and beta-fructofuranosidase, are encoded by multiple gene members with different expression patterns. These results reveal the complexity of the raffinose metabolism and the existence of metabolic channels for diverse RFOs in maize and provide useful information for improving maize stress tolerance through genetic engineering.
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Galactinol synthase (GolS; EC 2.4.1.123) is a member of the glycosyltransferase eight family that catalyzes the first step in the biosynthesis pathway of the raffinose family of oligosaccharides (RFOs). The accumulation of RFOs in response to abiotic stress indicates a role for RFOs in stress adaptation. To obtain information on the roles of RFOs in abiotic stress adaptation in trees, we investigated the expression patterns of nine Populus trichocarpa GolS (PtrGolS) genes with special reference to stress responses. PtrGolS genes were differentially expressed in different organs, and the expressions of PtrGolS4 and PtrGolS6 were relatively high in all tested organs. The expression levels of all PtrGolS genes, except PtrGolS9, changed in response to abiotic stress in gene- and stress-type-specific manners. Moreover, short- and long-term stress treatments revealed that induction of PtrGolS by salt stress is obvious only in the early period of treatment (within 24 h), whereas water-deficit stress treatments continued to upregulate PtrGolS gene expression after two days of treatment, in addition to induction within 24 h of treatment. Consistent with these expression patterns, the galactinol content in leaves increased after four days of drought stress, but not under salt stress. Our findings suggest divergent roles for PtrGolS genes in abiotic stress responses in poplars.
DOI:10.1104/pp.108.122465URLPMID:18502973 [本文引用: 1]
Galactinol synthase (GolS) is a key enzyme in the synthesis of raffinose family oligosaccharides that function as osmoprotectants in plant cells. In leaves of Arabidopsis (Arabidopsis thaliana) plants overexpressing heat shock transcription factor A2 (HsfA2), the transcription of GolS1, -2, and -4 and raffinose synthase 2 (RS2) was highly induced; thus, levels of galactinol and raffinose increased compared with those in wild-type plants under control growth conditions. In leaves of the wild-type plants, treatment with 50 mum methylviologen (MV) increased the transcript levels of not only HsfA2, but also GolS1, -2, -3, -4, and -8 and RS2, -4, -5, and -6, the total activities of GolS isoenzymes, and the levels of galactinol and raffinose. GolS1- or GolS2-overexpressing Arabidopsis plants (Ox-GolS1-11, Ox-GolS2-8, and Ox-GolS2-29) had increased levels of galactinol and raffinose in the leaves compared with wild-type plants under control growth conditions. High intracellular levels of galactinol and raffinose in the transgenic plants were correlated with increased tolerance to MV treatment and salinity or chilling stress. Galactinol and raffinose effectively protected salicylate from attack by hydroxyl radicals in vitro. These findings suggest the possibility that galactinol and raffinose scavenge hydroxyl radicals as a novel function to protect plant cells from oxidative damage caused by MV treatment, salinity, or chilling.
DOI:10.1111/pbi.12731URLPMID:28378532 [本文引用: 1]
Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non-transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.
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DOI:10.3389/fpls.2015.01092URLPMID:26697045 [本文引用: 2]
Abiotic stresses such as drought, cold, salt and heat cause reduction of plant growth and loss of crop yield worldwide. Reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide anions (O2 (*-)), hydroxyl radical (OH*) and singlet oxygen ((1)O2) are by-products of physiological metabolisms, and are precisely controlled by enzymatic and non-enzymatic antioxidant defense systems. ROS are significantly accumulated under abiotic stress conditions, which cause oxidative damage and eventually resulting in cell death. Recently, ROS have been also recognized as key players in the complex signaling network of plants stress responses. The involvement of ROS in signal transduction implies that there must be coordinated function of regulation networks to maintain ROS at non-toxic levels in a delicate balancing act between ROS production, involving ROS generating enzymes and the unavoidable production of ROS during basic cellular metabolism, and ROS-scavenging pathways. Increasing evidence showed that ROS play crucial roles in abiotic stress responses of crop plants for the activation of stress-response and defense pathways. More importantly, manipulating ROS levels provides an opportunity to enhance stress tolerances of crop plants under a variety of unfavorable environmental conditions. This review presents an overview of current knowledge about homeostasis regulation of ROS in crop plants. In particular, we summarize the essential proteins that are involved in abiotic stress tolerance of crop plants through ROS regulation. Finally, the challenges toward the improvement of abiotic stress tolerance through ROS regulation in crops are discussed.
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Fructans and raffinose family oligosaccharides (RFOs) are the two most important classes of water-soluble carbohydrates in plants. Recent progress is summarized on their metabolism (and regulation) and on their functions in plants and in food (prebiotics, antioxidants). Interest has shifted from the classic inulin-type fructans to more complex fructans. Similarly, alternative RFOs were discovered next to the classic RFOs. Considerable progress has been made in the understanding of structure-function relationships among different kinds of plant fructan metabolizing enzymes. This helps to understand their evolution from (invertase) ancestors, and the evolution and role of so-called
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