魏丽娟^,**, 申树林^**, 黄小虎, 马国强, 王曦彤, 杨怡玲, 李洹东, 王书贤, 朱美晨, 唐章林, 卢坤, 李加纳^,*, 曲存民^,*西南大学农学与生物科技学院 / 油菜工程研究中心 / 西南大学现代农业科学研究院, 重庆 400715

Genome-wide association analysis reveals zinc-tolerant loci of rapeseed at germination stage

WEI Li-Juan^,**, SHEN Shu-Lin^**, HUANG Xiao-Hu, MA Guo-Qiang, WANG Xi-Tong, YANG Yi-Ling, LI Huan-Dong, WANG Shu-Xian, ZHU Mei-Chen, TANG Zhang-Lin, LU Kun, LI Jia-Na^,*, QU Cun-Min^,*College of Agronomy and Biotechnology, Southwest University / Chongqing Engineering Research Center for Rapeseed / Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China

通讯作者: * 李加纳, E-mail: ljn1950@swu.edu.cn, Tel: 023-68250701; 曲存民, E-mail: drqucunmin@swu.edu.cn, Tel: 023-68250701

** 同等贡献(Contributed equally to this work)
收稿日期:2020-02-18接受日期:2020-04-15网络出版日期:2020-05-08

基金资助:

国家自然科学基金项目.31701460 中央高校基本科研业务费.XDJK2019C041 中央高校基本科研业务费.XDJK2020D023 重庆市基础科学与创新研究项目.cstc2016shms-ztzx80010 重庆市基础科学与创新研究项目.cstc2017jcyjAX0321 国家现代农业产业技术体系建设专项.CARS-12 高等学校学科创新引智基地111项目.B12006

Received:2020-02-18Accepted:2020-04-15Online:2020-05-08

Fund supported:

National Natural Science Foundation of China.31701460 Fundamental Research Funds for the Central Universities.XDJK2019C041 Fundamental Research Funds for the Central Universities.XDJK2020D023 Chongqing Basic Scientific and Advanced Technology Research.cstc2016shms-ztzx80010 Chongqing Basic Scientific and Advanced Technology Research.cstc2017jcyjAX0321 China Agricultural Research System.CARS-12 Intellectual Base for Discipline Innovation in Colleges and Universities.B12006

作者简介 About authors
魏丽娟, E-mail: lijuan525888@163.com















摘要
锌(Zn)是重要的微量元素之一, 但土壤中过量的锌累积会影响植物的生长发育。本研究以不同遗传来源的140份甘蓝型油菜为材料, 利用芸薹属60K SNP芯片对锌胁迫下(30 mg L^-1)甘蓝型油菜发芽期相对下胚轴长(RHL)进行全基因组关联分析, 筛选与甘蓝型油菜发芽期下胚轴长度显著关联的SNP位点及候选基因。群体结构分析表明, 供试的140份甘蓝型油菜被分为2个亚群, 其中89%材料间亲缘关系小于0.1, 说明供试群体材料亲缘关系比较远。GWAS分析共检测到8个与RHL显著关联的SNP位点, 单个SNP位点分别可解释22.0%~33.2%的表型变异。转录组分析获得的差异基因GO富集分析结果表明, 上调表达基因主要参与氧化还原反应、离子转运、胁迫反应、防御反应和硫化合物转运。综合全基因组关联分析和转录组测序结果, 共鉴定到19个与锌胁迫相关的候选基因, 包括编码锌指蛋白家族成员(B-box型和ZFP1)、谷胱甘肽转移酶GSTU21、过氧化物酶家族蛋白、ABC和MFS转运蛋白及细胞壁相关激酶蛋白和一些重要的转录因子(BnaA07g27330D、BnaA02g30270D、BnaA07g27840D、BnaA07g31860D和BnaA07g28000), 为深入解析油菜锌胁迫分子机制提供了参考。
关键词： 甘蓝型油菜;重金属;锌胁迫;全基因组关联分析;发芽期

Abstract
Zinc (Zn) is one of the important mircroelements, but the excessive amount application would affect plant growth and development. Genome-wide association analysis (GWAS) was performed on the relative hypocotyl length (RHL) using the 140 B. napus genotyped under zinc stress treatment (30 mg L^-1) at germination stage by Illumina 60K SNP array, and then significant SNP locus and candidate genes were detected. In the study, the population structure analysis revealed that the 140 B. napus were classified into two subgroups, and the kinship coefficients of the 89% materials were less than 0.1, indicating the tested population had a distant relationship. GWAS analysis indicated that there were significantly 8 SNP locus correlated to RHL, and single SNP loci could give an explanation on the 22.0%-33.2% phenotype variation. The differential expressed gene (DEGs) were detected by RNA-Seq. GO enrichment analysis indicated that the up-regulated genes mainly participated in redox reaction, ion transport, stress response, defense response and sulfur compound transport. Nineteen candidate genes response to zinc stress were identified by GWAS analysis and RNA-seq, including the genes encoding zinc finger protein (B-box type and ZFP1), glutathione transferase GSTU21, peroxidase family protein, ABC and MFS transporters, cell wall-related kinase protein, and genes encoding transcription factors (TF), such as BnaA07g27330D (MYB), BnaA02g30270D (bHLH), BnaA07g27840D (WRKY57), BnaA07g31860D (ORA47), and BnaA07g28000 (NAC). This study laid the foundation for understanding the molecular mechanism of zinc stress in B. napus.
Keywords：Brassica napus;heavy metal;zinc stress;GWAS;germination stage


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本文引用格式
魏丽娟, 申树林, 黄小虎, 马国强, 王曦彤, 杨怡玲, 李洹东, 王书贤, 朱美晨, 唐章林, 卢坤, 李加纳, 曲存民. 锌胁迫下甘蓝型油菜发芽期下胚轴长的全基因组关联分析[J]. 作物学报, 2021, 47(2): 262-274. doi:10.3724/SP.J.1006.2021.04037
WEI Li-Juan, SHEN Shu-Lin, HUANG Xiao-Hu, MA Guo-Qiang, WANG Xi-Tong, YANG Yi-Ling, LI Huan-Dong, WANG Shu-Xian, ZHU Mei-Chen, TANG Zhang-Lin, LU Kun, LI Jia-Na, QU Cun-Min. Genome-wide association analysis reveals zinc-tolerant loci of rapeseed at germination stage[J]. Acta Agronomica Sinica, 2021, 47(2): 262-274. doi:10.3724/SP.J.1006.2021.04037


近年来, 土壤重金属污染问题愈发严重, 对人类身体健康和生态系统产生重大影响^[1]。锌是植物生长发育所必需的微量元素之一, 适量锌可促进植物生长发育, 增强植物对不良环境的适应性^[2,3]。然而过度使用不仅会造成严重的环境污染, 还会降低土壤微生物的活性, 减缓土壤有机质的分解, 进而影响植物根的生长和光合作用^[4,5,6]。油菜被认为是修复重金属(如Cu、Cd、Zn)污染土壤的理想植物之一^[7,8,9,10], 对锌具有高耐受性, 在锌胁迫下仍可获得较高的生物产量^[7,11]。曹春信等^[12]通过研究油菜地上部器官对锌富集的特征发现, 锌在茎秆中的含量大于籽粒。挖掘耐锌性关键位点和候选基因对耐锌性油菜品种的培育具有重要意义, 全基因组关联分析被广泛应用于作物重金属抗性位点的检测。Zhang等^[13]利用全基因组关联分析方法, 在水稻中共检测到31个耐锌QTL位点。Chen等^[14]利用Brassica Illumina 60K SNP芯片进行全基因组关联分析, 检测出25个甘蓝型油菜耐镉QTL位点, 并找到NRAMP6、IRT1、CAD1和PCS2四个候选基因。Zhang等^[15]检测到4个甘蓝型油菜耐镉位点, 并鉴定出一些候选基因, 如HIPP27、EXPB4、EMB1793和CDSP3。曲存民等^[16]检测到70个甘蓝型油菜耐砷性的显著关联位点, 并筛选出PHT3;3和PHT1;9等与甘蓝型油菜砷离子吸收转运相关的候选基因。超富集植物对重金属的积累和耐性机制研究也取得了一定进展, 许多蛋白参与锌的吸收、转运和贮存, 包括CDF (Cation Diffusion Facilitator)蛋白家族^[17]、NRAMP蛋白家族^[18]、ZIP蛋白家族^[19]、HMA蛋白家族(P-type ATPases)^[20]、YSL (yellow stripe-like)蛋白家族^[21]、ABC (ATP-binding cassette)转运蛋白家族^[2,22]。但关于甘蓝型油菜耐锌性QTL定位和候选基因鉴定并未见相关报道。

为挖掘甘蓝型油菜与耐锌性状相关联的关键位点及候选基因, 本研究采用GWAS方法获得与下胚轴长显著关联的SNP标记, 结合转录组数据获得锌胁迫前后差异表达基因, 进一步确定影响下胚轴长的关键候选基因。本研究不仅为甘蓝型油菜耐锌性种质资源的鉴定提供分子标记, 而且可以为耐锌性油菜资源利用和新品种培育提供理论依据。

1 材料与方法

1.1 试验材料

供试材料为国内外收集的甘蓝型油菜自交系品种140份, 其中国内材料123份, 主要来自重庆、四川、湖北、湖南等地, 其余17份材料来自加拿大和德国等国家, 所有材料由西南大学重庆市油菜工程技术研究中心收集并提供。

1.2 试验处理与性状分析

从140份研究材料中随机抽取10份材料(附表1), 分别用0、5、10、15、20、30和50 mg L^-1浓度的Zn(NO₃)₂进行处理, 比较分析发现, 处理浓度为30 mg L^-1时材料间长势差异较大, 确定为最佳处理浓度(表1)。试验分为对照组和处理组, 重复3次, 挑选大小均匀、饱满的种子各100粒, 分别播种于对照纯水和30 mg L^-1的Zn(NO₃)₂溶液处理的培养盘 (长37.0 cm × 宽24.5 cm × 高6.5 cm, 体积为4.5 L), 覆盖保鲜膜。生长条件为昼夜温度为25℃, 光照/黑暗时间为16 h/8 h, 光照强度为100 μmol m^-2 s^-1, 相对湿度为60%^[23]。播种7 d后, 选取长势一致的5株幼苗, 分别测量对照和锌胁迫下的下胚轴长(hypocotyl length, HL)。将处理和对照的相对值作为衡量锌胁迫对甘蓝型油菜生长的影响^[24], 记为相对下胚轴长(relative hypocotyl length, RHL)。计算公式: 相对性状值(%)=处理组测定值/对照组测定值×100%^[16]。利用Microsoft Excel 2013绘制正态分布图, 利用SPSS统计分析软件进行偏度和峰度分析。

Table S1
附表1
附表1140份甘蓝型油菜材料信息
Table S1Detailed information of the 140 B. napus accessions

编号 Number	材料名称 Accessions		生态型 Ecotype	来源 Source
1	中双3号Zhongshuang 3		半冬性Semi-winter	中国浙江Zhejiang, China
2	镇油5号Zhenyou 5		半冬性Semi-winter	中国江苏Jiangsu, China
3	SWU46		半冬性Semi-winter	中国重庆Chongqing, China
4	WH-127		半冬性Semi-winter	中国湖北Hubei, China
5	WH-41		半冬性Semi-winter	中国湖北Hubei, China
6	WX10329		半冬性Semi-winter	中国湖南Hunan, China
7	JY-16		半冬性Semi-winter	中国湖北Hubei, China
8	黔油331 Qianyou 331		半冬性Semi-winter	中国贵州Guizhou, China
编号 Number	材料名称 Accessions		生态型 Ecotype	来源 Source
9	SWU47		半冬性Semi-winter	中国重庆Chongqing, China
10	浙双72 Zheshuang 72		半冬性Semi-winter	中国浙江Zhejiang, China
11	沪油17 Huyou 17		半冬性Semi-winter	中国上海Shanghai, China
12	中双7号Zhongshuang 7		半冬性Semi-winter	中国武汉Wuhan, China
13	WH-26		半冬性Semi-winter	中国湖北Hubei, China
14	Taisetsu		冬性Winter	日本Japan
15	M114		半冬性 Semi-winter	中国China
16	中油589 Zhongyou 589		半冬性Semi-winter	中国武汉Wuhan, China
17	农林43 Nonglin 43		冬性Winter	日本Japan
18	广德8104 Guangde 8104		半冬性Semi-winter	中国江苏Jiangsu, China
19	至尊Zhizun		半冬性Semi-winter	中国湖北Hubei, China
20	中双2号Zhongshuang 2		半冬性Semi-winter	中国湖北Hubei, China
21	中双4号Zhongshuang 4		半冬性Semi-winter	中国湖北Hubei, China
22	华油3号Huayou 3		半冬性Semi-winter	中国湖北Hubei, China
23	宁油14 Ningyou 14		半冬性Semi-winter	中国江苏Jiangsu, China
24	华油12 Huayou 12		半冬性Semi-winter	中国湖北Hubei, China
25	湖北白花油菜Hubei white flower rape		半冬性Semi-winter	中国湖北Hubei, China
26	WH-58		半冬性Semi-winter	中国湖北Hubei, China
27	699		半冬性Semi-winter	中国湖北Hubei, China
28	SWU63		半冬性Semi-winter	中国重庆Chongqing, China
29	WH-57		半冬性Semi-winter	中国湖北Hubei, China
30	苏油1号Shuyou 1		半冬性Semi-winter	中国江苏Jiangsu, China
31	WH-19		半冬性Semi-winter	中国湖北Hubei, China
32	中双4号Zhongshuang 4		半冬性Semi-winter	中国湖北Hubei, China
33	Nakate Chousen		春性Spring	朝鲜DPRK
34	广德761 Gaungde 761		半冬性Semi-winter	中国江苏Jiangsu, China
35	SWU110		半冬性Semi-winter	中国重庆Chongqing, China
36	7094		半冬性Semi-winter	中国湖北Hubei, China
37	SWU71		半冬性Semi-winter	中国重庆Chongqing, China
38	1111		半冬性Semi-winter	中国湖北Hubei, China
39	Erake		半冬性Semi-winter	波兰Poland
40	Campino		春性Spring	德国Germany
41	宁油10号 Ningyou 10		半冬性Semi-winter	中国江苏Jiangsu, China
42	中双11DH Zhongshuang 11DH		半冬性Semi-winter	中国湖北Hubei, China
43	浙双8号 Zheshuang 8		半冬性Semi-winter	中国浙江Zhejiang, China
44	cresor		春性Spring	法国France
45	浙油17 Zheyou 17		半冬性Semi-winter	中国浙江Zhejiang, China
46	华航901 Huahang 901		半冬性Semi-winter	中国湖北Hubei, China
47	Wesreo		春性Spring	澳大利亚Australia
48	南川长角 Nanchuansiliqua		半冬性Semi-winter	中国重庆Chongqing, China
49	J-917		半冬性Semi-winter	中国湖北Hubei, China
50	浙双6号 Zheshuang 6		半冬性Semi-winter	中国浙江Zhejiang, China
51	荣选 Rongxuan		半冬性Semi-winter	中国江苏Jiangsu, China
52	SWU106		半冬性Semi-winter	中国重庆Chongqing, China
编号 Number	材料名称 Accessions		生态型 Ecotype	来源 Source
53	D2		春性Spring	丹麦Denmark
54	豫油1号 Yuyou 1		半冬性Semi-winter	中国重庆Chongqing, China
55	WH-37		半冬性Semi-winter	中国湖北Hubei, China
56	J-951		半冬性Semi-winter	中国湖北Hubei, China
57	全紫油菜 Quanzi rape		春性Spring	中国江苏Jiangsu, China
58	SWU69		半冬性Semi-winter	中国重庆Chongqing, China
59	SWU60		半冬性Semi-winter	中国重庆Chongqing, China
60	丰油9号 Fengyou 9		半冬性Semi-winter	中国河南Henan, China
61	宁油1号 Ningyou 1		半冬性Semi-winter	中国湖北Hubei, China
62	SWU80		半冬性Semi-winter	中国重庆Chongqing, China
63	10-C29		半冬性Semi-winter	中国湖北Hubei, China
64	秦油5号 Qinyou 5		半冬性Semi-winter	中国陕西Shaanxi, China
65	Sophia		春性Spring	德国Germany
66	WaseChousen		春性Spring	朝鲜DPRK
67	Korina		春性Spring	德国Germany
68	J-915		半冬性Semi-winter	中国湖北Hubei, China
69	Bienvenu		半冬性Semi-winter	法国France
70	SWU102		半冬性Semi-winter	中国重庆Chongqing, China
71	漕泾胜利 Caojingshengli		半冬性Semi-winter	中国上海Shanghai, China
72	563		半冬性Semi-winter	中国湖南Hunan, China
73	皖油15号 Wanyou 15		半冬性Semi-winter	中国安徽Anhui, China
74	阳光198 Yangguang 198		半冬性Semi-winter	中国湖北Hubei, China
75	湘油13号 Xiangyou 13		半冬性Semi-winter	中国湖南Hunan, China
76	皖油16号 Wanyou 16		半冬性Semi-winter	中国安徽Anhui, China
77	华油13号 Huayou 13		半冬性Semi-winter	中国湖北Hubei, China
78	湘油15号 Xiangyou 15		半冬性Semi-winter	中国湖南Hunan, China
79	Tapidor		冬性Winter	法国France
80	10-C24		半冬性Semi-winter	中国湖北Hubei, China
81	圣光77 Shengguang 77		半冬性Semi-winter	中国湖北Hubei, China
82	史力丰 Shilifeng		半冬性Semi-winter	中国江苏Jiangsu, China
83	沪油3号 Huyou 3		半冬性Semi-winter	中国上海Shanghai, China
84	Santana		半冬性Semi-winter	中国China
85	10-C34		半冬性Semi-winter	中国湖北Hubei, China
86	11-Y7-117		半冬性Semi-winter	中国湖北Hubei, China
87	SWU99		半冬性Semi-winter	中国重庆Chongqing, China
88	宁油12 Ningyou 12		半冬性Semi-winter	中国江苏Jiangsu, China
89	Comet		春性Spring	瑞典Sweden
90	WH-20		半冬性Semi-winter	中国湖北Hubei, China
91	扬J6711 Yang J6711		半冬性Semi-winter	中国江苏Jiangsu, China
92	7191		半冬性Semi-winter	中国湖北Hubei, China
93	中双9号 Zhongshuang 9		半冬性Semi-winter	中国湖北Hubei, China
94	Daechosen		半冬性Semi-winter	朝鲜DPRK
95	Monty		半冬性Semi-winter	澳大利亚Australia
96	纬隆88 Weilong 88		半冬性Semi-winter	中国陕西Shanxi, China
编号 Number	材料名称 Accessions		生态型 Ecotype	来源 Source
97	沪油14 Huyou 14		半冬性Semi-winter	中国上海Shanghai, China
98	A117		半冬性Semi-winter	中国陕西Shaanxi, China
99	WH-15		半冬性Semi-winter	中国湖北Hubei, China
100	2011-7103		半冬性Semi-winter	中国湖北Hubei, China
101	SWU68		半冬性Semi-winter	中国重庆Chongqing, China
102	浙油18 Zheyou 18		半冬性Semi-winter	中国四川Zhejiang, China
103	Yan 81-2		半冬性Semi-winter	中国重庆Chongqing, China
104	2012-K8053		半冬性Semi-winter	中国湖北Hubei, China
105	淮油6号 Huaiyou 6		半冬性Semi-winter	中国江苏Jiangsu, China
106	2012-3546		半冬性Semi-winter	中国湖北Hubei, China
107	wx 10296		半冬性Semi-winter	中国湖南Hunan, China
108	2012-8380		半冬性Semi-winter	中国湖北Hubei, China
109	SWU92		半冬性Semi-winter	中国重庆Chongqing, China
110	农林42 Nonglin 42		半冬性Semi-winter	日本Japan
111	大花球 Dahuaqiu		半冬性Semi-winter	中国江苏Jiangsu, China
112	华油6号 Huayou 6		半冬性Semi-winter	中国湖北Hubei, China
113	CY12Q95406		半冬性Semi-winter	中国四川Sichuan, China
114	SWU49		半冬性Semi-winter	中国重庆Chongqing, China
115	SWU70		半冬性Semi-winter	中国重庆Chongqing, China
116	华双128 Huashuang 128		半冬性Semi-winter	中国湖北Hubei, China
117	皖油早 Wanyouzao		半冬性Semi-winter	中国安徽Anhui, China
118	宁油12 Ningyou 12		半冬性Semi-winter	中国江苏Jiangsu, China
119	滁油1号 Chuyou 1		半冬性Semi-winter	中国安徽Anhui, China
120	10-JP3		半冬性Semi-winter	中国湖北Hubei, China
121	2359		半冬性Semi-winter	中国湖北Hubei, China
122	宁油8号 Ningyou 8		半冬性Semi-winter	中国江苏Jiangsu, China
123	CY21PXW-84		半冬性Semi-winter	中国四川Sichuan, China
124	SWU88		半冬性Semi-winter	中国重庆Chongqing, China
125	Sida		春性Spring	加拿大Canada
126	川油20 Chuanyou 20		半冬性Semi-winter	中国四川Sichuan, China
127	沪油19 Huyou 19		半冬性Semi-winter	中国上海Shanghai, China
128	WH-62		半冬性Semi-winter	中国湖北Hubei, China
129	华油14 Huayou 14		半冬性Semi-winter	中国湖北Hubei, China
130	CY12GJ-1		半冬性Semi-winter	中国四川Sichuan, China
131	湘油11号 Xiangyou 11		半冬性Semi-winter	中国湖南Hunan, China
132	浙油601 Zheyou 601		半冬性Semi-winter	中国浙江Zhejiang, China
133	SWU108		半冬性Semi-winter	中国重庆Chongqing, China
134	CY14PXW-18		半冬性Semi-winter	中国四川Sichuan, China
135	Nca		半冬性Semi-winter	中国湖北Hubei, China
136	甘油5号 Ganyou 5		半冬性Semi-winter	中国湖北Hubei, China
137	SWU94		半冬性Semi-winter	中国重庆Chongqing, China
138	中油821 Zhongyou 821		半冬性Semi-winter	中国湖北Hubei, China
139	川油18 Chuanyou 18		半冬性Semi-winter	中国四川Sichuan, China
140	wx 10213		半冬性Semi-winter	中国湖北Hunan, China

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Table 1
表1
表1不同Zn(NO₃)₂溶液浓度下油菜下胚轴长度
Table 1Hypocotyl length with different Zn(NO₃)₂ concentration solutions in B. napus

品种 Accessions	Zn(NO3)2溶液浓度 Concentration of Zn(NO3)2 solutions (mg L-1)
	0	5	10	15	20	30	50	70
1	5.76	4.46	4.74	5.82	3.78	2.54	2.54	1.68
2	4.38	4.38	4.02	3.16	2.38	2.54	2.52	2.08
3	6.80	6.64	5.02	6.02	4.34	2.62	2.24	2.34
4	7.60	7.66	6.64	5.20	4.20	3.14	2.76	2.14
5	6.70	6.28	5.24	4.32	3.24	3.20	2.84	1.80
6	6.25	5.88	5.13	4.90	3.59	2.81	2.58	2.01
7	7.90	6.30	8.40	6.52	5.88	5.46	4.84	2.60
8	5.90	7.98	8.64	4.50	4.08	4.58	4.64	2.40
9	6.30	7.90	7.10	5.60	6.54	4.50	3.74	2.40
10	6.96	7.08	8.06	5.73	6.01	4.86	4.94	2.91
平均值 Mean (cm)	6.45	6.46	6.30	3.78	3.58	2.48	3.36	2.24
标准差SD (cm)	1.00	1.29	1.68	1.17	1.25	0.87	1.07	0.37
变异系数 CV (%)	0.155	0.200	0.267	0.309	0.349	0.352	0.319	0.166

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1.3 基因型分析及SNP标记定位

利用芸薹属60K SNP芯片, 参照Qu等^[25,26]方法, 对140份材料进行基因型分析, 去掉最小基因型频率(minor allele frequency, MAF)低于0.05和SNP得率(call frequency)小于80%的标记。为确定SNP在染色体上的位置, 利用探针序列与基因组进行BLASTN比对, E-value设定为1e^-5。去掉Bit-Score相同的SNP, 最终获得30,288个SNP标记用于群体性状的关联分析。

1.4 群体结构分析与亲缘关系

利用STRUCTURE软件进行甘蓝型油菜群体结构分析^[27], 组群数目(K)设定为1~10, K值运行5次(number of iterations), 将MCMC (Markov Chain Monte Carlo)不作数迭代(length of burn-in period)和不作数迭代后的MCMC全部设为100,000次, 在混合模型下进行运算。最后用运算得到的后验概率值和2个连续的后验概率值的变化速率(ΔK)来确定群体中存在的亚群数目^[28]。利用SPAGeDi v1.4软件进行亲缘关系(relative kinship)分析, 并计算亲缘关系的矩阵^[29]。

1.5 全基因组关联分析及候选基因鉴定

利用TASSEL 5.0软件^[30]中的一般线性模型(General Linear Model, GLM)和混合线性模型(Mixed Linear Model, MLM), 结合SNP芯片基因型数据, 以Q值和亲缘关系K值作为协变量, 进行全基因组关联分析, 确定关联位点和标记对表型变异的解释率(R²)。本研究采用了6种模型进行关联分析, 包括GLM中的naive、Q和PCA模型, MLM中的K、Q+K和PCA+K模型。根据每个SNP位点的-log₁₀(P)观察值和期望值, 利用R软件(https://www.r-project. org/)绘制Quantile-Quantile散点图(QQ plot)。曼哈顿图(Manhattan plot)利用R语言程序包的mrMLM (Multi-Locus Random-SNP-Effect Mixed Linear Model)进行绘制^[31]。与性状显著关联的SNP位点的阈值为P < 3.30×10^-5, 计算方法为: 1/N (N = 30,288)^[16]。利用TASSEL 5.0软件^[30]计算linkage disequilibrium (LD)在油菜不同染色上的分布情况, 确定不同染色体的LD衰减距离, 根据LD确定候选基因区间。

1.6 甘蓝型油菜锌胁迫转录组测序

取甘蓝型油菜自交系07191锌胁迫处理前和处理7 d后的幼苗, 于液氮中保存, 参照曲存民等^[16]方法提取总RNA, 利用Illumina Hiseq 2500进行转录组测序, 以甘蓝型油菜为参考基因组^[32], 利用Cuffdiff筛选差异表达基因, 筛选标准为FDR < 0.01, log₂|(fold change)|>1。上调和下调的差异表达基因GO富集分析采用基迪奥云平台进行分析(https://www.omicshare.com/)。

2 结果与分析

2.1 甘蓝型油菜发芽期锌胁迫最佳浓度确定

为了确定油菜发芽期锌胁迫的最佳处理浓度, 本研究随机挑选10个材料, 分别利用不同浓度的Zn(NO₃)₂溶液和纯水为对照进行处理, 发现处理浓度为30 mg L^-1时, 这些材料下胚轴长度明显受到抑制, 且材料间变异较大(表1)。因此, 在发芽期筛选耐锌油菜的最佳浓度为30 mg L^-1。

2.2 甘蓝型油菜锌胁迫性状表型变异分析

对140份油菜正常和锌胁迫条件下的下胚轴长度进行统计发现, 在正常条件下, 甘蓝型油菜下胚轴长度为3.66~7.99 cm, 变异系数为15.8%; 锌胁迫下, 下胚轴长度为2.28~6.47 cm, 变异系数为18.8%; 相对下胚轴长的变异范围为0.38~1.08, 均值为0.73, 变异系数为17.6%, 表现出广泛的表型变异(表2)。锌胁迫下, 大部分材料的下胚轴长度都较正常生长状态的下胚轴短, 只有5个半冬性材料例外(甘油5号、SWU94、中油821、川油18和wx10213), 表明30 mg L^-1锌胁迫严重抑制了甘蓝型油菜下胚轴伸长。相对下胚轴长呈连续性正态分布, 说明其受多基因控制, 表型为典型的数量性状遗传特点(图1)。

Table 2
表2
表2锌胁迫下甘蓝型油菜发芽期性状统计分析
Table 2Statistical analysis of the traits at germination stage under zinc stress

性状	最小值	最大值	均值±标准差	变异系数	偏度	峰度
Trait	Min.(cm)	Max.(cm)	Mean±SD	CV (%)	Skewness	Kurtosis
CHL	3.66	7.99	5.77±0.92	15.85	-0.13	0.83
THL	2.28	6.47	4.24±0.80	18.84	0.41	0.02
RHL	0.38	1.08	0.73±0.13	17.57	0.28	0.15

CHL: 正常条件下的下胚轴长; THL: 锌胁迫下的下胚轴长; RHL: 正常与锌胁迫下相对下胚轴长。
CHL: the hypocotyl length under normal condition; THL: the hypocotyl length under zinc stress; RHL: the relative hypocotyl length under normal and zinc stress treatments.

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图1

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图1锌胁迫下甘蓝型油菜相对下胚轴长频率分布图

Fig. 1Frequency distribution of RHL under zinc stress in B. napus

2.3 群体结构和亲缘关系分析

利用30,288个SNP标记进行关联群体的群体结构和亲缘关系分析, 当K=2时, ΔK值最大(图2-A), 因此, 该群体的亚群数为2, 生成的最优群体结构的Q矩阵见图2-B。140份甘蓝型材料中, 89%材料间亲缘关系小于0.1 (图2-C), 表明该群体内材料间的亲缘关系较远。

图2

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图2基于SNP标记估算群体结构关系图和亲缘关系图

A: lnP(D)和ΔK变化关系图; B: K = 2时群体结构图; C: 140份甘蓝型油菜亲缘关系图。
Fig. 2Population structure and relative kinship diagram based on SNP markers

A: plot of lnP(D) and ΔK; B: population structure profile (K = 2), the areas of the two colors (green and red) indicate the proportion of each subgroup, respectively; C: relative kinship diagram of 140 B. napus.

2.4 耐锌胁迫下相对下胚轴长度的全基因组关联分析

对相对下胚轴长度的QQ图进行分析(图3), 结果表明, K+Q模型检测到的标记的P值较其他模型更接近期望值, 其降低了假阳性, 关联分析结果更可靠。全基因组关联分析共检测到8个显著关联的SNP位点(图3和表3), 分别位于A02、A07、C04和C06染色体上, 单个SNP解释的表型变异为22.0%~33.2%。为了确定显著关联SNP位点的候选基因区间, 本研究对这些关联位点的染色体进行LD分析, 利用R²值估计LD, A02和A07染色体的衰减距离为300 kb, C04染色体衰减距离为700 kb, C06染色体的衰减距离为1 Mb (图4-A)。另外, A07染色体上22.2 Mb~23.9 Mb与C06染色体34.9 Mb~37.2 Mb位于共线性区间内(图4-B), 说明找到的显著关联的SNP位点位于这2个共线性区间内。

图3

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图3锌胁迫下甘蓝型油菜相对下胚轴长QQ图和曼哈顿图

Fig. 3QQ and Manhattan plots on RHL under zinc stress treatment in B. napus



Table 3
表3
表3锌胁迫下甘蓝型油菜发芽期相对下胚轴长显著关联的SNP位点
Table 3Significant SNP locus associated with the relative hypocotyl length at germination stage under zinc stress in B. napus

染色体 Chr.	位置 Position (bp)	P值 P-value	显著性 -log10 (P)	贡献率 R2 (%)
A02	22,075,111	2.97E-06	5.53	24.62
A07	20,094,111	3.97E-06	5.40	22.11
A07	22,001,673	3.49E-06	5.46	21.99
A07	23,402,490	7.70E-06	5.11	19.90
A07	23,555,825	2.92E-06	5.53	22.06
A07	23,580,640	7.50E-08	7.13	33.18
C04	4,456,230	3.12E-06	5.51	24.66
C06	36,835,784	2.55E-06	5.59	24.45

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图4

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图4甘蓝型油菜染色体不同物理距离的LD分布(A)和共线性分析(B)

Fig. 4LD distribution (A) and synteny analysis (B) in different distances in B. napus

2.5 锌胁迫下油菜幼苗差异表达基因

转录组分析结果表明, 锌处理油菜幼苗7 d后, 与对照相比, 上调基因2733个, 下调基因3652个, 其中差异转录因子/基因616个, 主要包括bHLH (77)、ERF (73)、NAC (65)、MYB (60)、WRKY (55)和C2H2 (33)家族(图5)。ERF、NAC和WRKY家族基因上调数目较多, 而bHLH、MYB和C2H2家族基因下调基因数目较多。本研究对上调和下调基因分别进行GO功能富集分析, 上调基因主要富集在细胞外区域和细胞膜, 分子功能是硫酸盐跨膜转运功能, 主要参与氧化还原反应、离子转运、胁迫反应、防御反应和硫化合物转运(图6-A)。下调基因主要富集在细胞外区域、质外体和细胞膜区域, 主要的分子功能是抗氧化活性和过氧化物酶活性, 主要参与抗氧化反应、氧化还原反应和细胞壁生物合成(图6-B)。

图5

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图5油菜锌胁迫后上调和下调转录因子基因数目

Fig. 5Number of up-regulated and down-regulated transcription factors under zinc stress treatment in B. napus

图6

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图6油菜锌胁迫后差异表达上调(A)和下调基因(B) GO富集分析

Fig. 6Differentially up-regulated (A) and down-regulated (B) genes by GO enrichment analysis under zinc stress treatment in B. napus

2.6 GWAS与转录组结合鉴定共有基因

根据各染色体的LD确定候选区间, 参照油菜基因组注释信息^[32], 共找到19个与锌胁迫相关的候选基因(表4), 这些基因主要包括编码锌指蛋白家族成员(B-box型和ZFP1)、谷胱甘肽转移酶GSTU21、过氧化物酶家族蛋白、ABC和MFS转运蛋白及细胞壁相关激酶蛋白, 并找到一些重要的转录因子, 如MYB、bHLH、NAC、ERF和WRKY, 大部分基因(除GSTU21、ZFP1)。

Table 4
表4
表4甘蓝型油菜锌胁迫下相关性状候选基因
Table 4Candidate genes of the traits correlated with zinc stress treatment in B. napus

候选基因 Candidate genes	物理位置 Physical position	拟南芥同源基因 Homologs in Arabidopsis	基因注释 Gene annotation	差异倍数 log2 (Fold change)
BnaA02g30040D	Chr.A02: 21813781-21815282	AT5G48250	B-box型锌指蛋白 B-box type zinc finger protein with CCT domain	2.18
BnaA02g30270D	Chr.A02: 21934212-21936672	AT5G48560	bHLH转录因子 Basic helix-loop-helix (bHLH) DNA-binding protein	-6.62
BnaA07g27330D	ChrA07: 19877781-19879164	AT1G68670	MYB转录因子 MYB-like transcription factor family protein	2.15
BnaA07g27340D	Chr.A07: 19884616-19887579	AT1G68690	蛋白激酶家族 Protein kinase superfamily protein	4.55
BnaA07g27500D	Chr.A07: 19996402-19997781	AT1G68850	过氧化物酶家族蛋白 Peroxidase superfamily protein	-2.06
BnaA07g27840D	Chr.A07: 20196357-20198149	AT1G69310	WRKY57转录因子 WRKY DNA-binding protein 57	2.64
BnaA07g28000D	Chr.A07: 20266703-20267687	AT1G69490	NAC转录因子 NAC-like, activated by AP3/PI (NAP)	2.01
BnaA07g31860D	Chr.A07: 22136736-22137327	AT1G74930	ERF转录因子 ORA47	3.71
BnaA07g34210D	Chr.A07: 23311591-23312410	AT1G78360	谷胱甘肽转移酶 (GSTU21) Glutathione S-transferase TAU 21 (GSTU21)	-3.13
BnaA07g35030D	Chr.A07: 23695006-23698790	AT1G79610	Na+/H+逆向转运蛋白 Na+/H+ antiporter 6 (NHX6)	2.29
BnaA07g35090D	Chr.A07: 23714116-23714860	AT1G79680	类细胞壁相关激酶WALL ASSOCIATED KINASE (WAK)-LIKE 10 (WAKL10)	6.52
BnaA07g35350D	Chr.A07: 23800896-23801571	AT1G80730	锌指蛋白 Zinc-finger protein 1 (ZFP1)	-7.08
BnaC04g06210D	Chr.C04: 4439046-4439940	AT3G55090	ABC转运蛋白 ABC-2 type transporter family protein	2.10
BnaC04g06300D	Chr.C04: 4503767-4505873	AT2G39210	主要协助转运蛋白超家族 Major facilitator superfamily protein (MFS)	3.11
BnaC06g38840D	Chr.C06: 36254140-36254958	AT1G78360	谷胱甘肽转移酶(GSTU21) Glutathione S-transferase TAU 21 (GSTU21)	-4.29
BnaC06g38850D	Chr.C06: 36255467-36256984	AT1G78360	谷胱甘肽转移酶(GSTU21) Glutathione S-transferase TAU 21 (GSTU21)	-2.35
BnaC06g39970D	Chr.C06: 36849441-36853390	AT1G79610	Na+/H+逆向转运蛋白 Na+/H+ antiporter 6 (NHX6)	4.01
BnaC06g40020D	Chr.C06: 36867031-36869575	AT1G69730	细胞壁相关激酶蛋白 Wall-associated kinase family protein	4.43
BnaC06g40250D	Chr.C06: 36958965-36959628	AT1G80730	锌指蛋白 Zinc-finger protein 1 (ZFP1)	-2.49

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3 讨论

重金属污染可归纳为2类, 一类是一些具有显著生物毒性的重金属物, 如镉(Cd)、砷(As)等, 另一类为超过一定量后具有生物毒性的一些重金属, 如锌(Zn)、铜(Cu)等^[33]。近年来, 土壤重金属污染越来越引起人们关注^[34]。在土壤中过量的锌富积会抑制植物生长, 导致营养失衡、萎黄叶片, 以及光合作用受损^[5,6]。研究表明, 甘蓝型油菜对重金属锌具有较强的耐受性, 在较高浓度下仍可获得较高的生物产量^[2,3,4,5]。然而关于油菜响应锌胁迫的分子机制尚未见报道。在甘蓝型油菜中, 通过GWAS分析与基因组信息相结合已成为挖掘油菜数量性状基因座的常规手段, 并获得了一些与非生物胁迫相关的候选基因^{[14-16,35-37]}。因此, 本研究通过对锌胁迫下140份甘蓝型油菜发芽期的相对下胚轴长进行了全基因组关联分析, 共检测到8个显著关联的SNP位点, A02、C04和C06染色体上显著关联的SNP位点分别位于22.0、4.4和36.8 Mb, A07染色体上显著关联的SNP位点位于22.0 Mb和23.5 Mb。曲存民等^[16]将砷胁迫下甘蓝型油菜的RHL定位在A02: 19.2 Mb~22.0 Mb、A07: 19.2 Mb~22.5 Mb、A07: 23.1 Mb~23.5 Mb、C04: 4.3 Mb~4.5 Mb和C06: 31.6 Mb~36.8 Mb, 与本研究定位结果一致, 这些位点具有一因多效性, 可能在不同重金属胁迫过程中都起着重要作用。

在植物中, 重金属可以引起许多代谢过程发生变化, 这些变化主要是通过氧化还原反应引起的, 同时造成抗氧化酶的活化、细胞膜通透性改变和离子的置换等反应^[38]。本研究发现锌胁迫后, 上调的差异表达基因主要富集在细胞膜, 参与氧化还原反应、离子转运、防御反应和硫化合物转运。本研究结合全基因组关联分析和转录组测序, 找到19个与锌胁迫相关的候选基因, 包括转录因子/基因, 如WRKY57、ORA47和BnaA07g28000D (NAC), WRKY57可以通过清除活性氧提高植物抗旱和抗盐能力^[39,40]; 植物受到伤害和水分胁迫时, ORA47可通过调控脱落酸和茉莉酸信号途径来提高抗性^[41]。本研究中WRKY57和ORA47在锌胁迫后显著上调表达, 可能在锌胁迫过程中起着重要作用, 但具体的调控机制还需进一步研究。

本研究还找到了B-box型和锌指蛋白ZFP1, 锌指蛋白中的锌指结构可以结合锌离子形成稳定的指状结构, 编码锌指蛋白的众多基因已经被证明在生物和非生物胁迫过程中起着重要作用^[42,43]。Sun等^[44]研究表明, 菜豆MTF通过激活色氨酸生物合成调控烟草耐镉性, 锌指基序是其重要的结合金属反应的元件。拟南芥锌指蛋白ZAT6通过谷胱甘肽途径提高镉抗性^[45], 而锌指蛋白ZAT11负调控拟南芥镍抗性^[46]。本研究找到的B-box型锌指蛋白基因BnaA02g30040D在锌胁迫后表达量上调, 而锌指蛋白ZFP1基因表达量下调, 它们可能通过不同的机制来响应胁迫。另外, 本研究找到2个编码Na⁺/H⁺逆向转运蛋白的基因NHX6 (BnaA07g35030D和BnaC06g39970D), 锌胁迫后其表达量上调, Yang等^[47]研究表明, 浮萍NHX1基因通过减少Cd²⁺流入和增加Cd²⁺外排提高重金属镉抗性。本研究中NHX6表达上调, 可能增加了Zn²⁺的外排, 从而提高锌抗性。

重金属处理下植物细胞产生活性氧造成细胞氧化损伤, 为了抵抗活性氧对细胞的损伤, 植物会产生抗氧化酶及非酶抗氧化剂清除自由基^[48]。锌处理下小麦过氧化氢含量、过氧化物酶及过氧化氢酶活性均显著提高, 增强了小麦对锌的耐受性^[49]。本研究找到一些与氧化还原反应相关的基因, 如编码过氧化物酶家族蛋白和谷胱甘肽转移酶基因GSTU21。GST可使GSH与内源电子化合物结合而产生脱毒^[50], 重金属(Cu和Al)胁迫下, 拟南芥GST基因表达上调, 超表达水稻GSTU30基因提高了拟南芥重金属耐性^[51,52]。但是本研究中, GSTU21基因在锌胁迫后表达量下调, 可能是由于锌处理植株后, 重金属造成的氧化胁迫反应超过了油菜自身的协调能力, 抗氧化能力受到明显抑制。

另外, 本研究还定位到一些编码ABC和MSF转运蛋白的基因, 其功能是将重金属转运到液泡内, 在重金属转运过程中起着重要作用^[53,54], 这些转运蛋白基因表达量上调, 可能通过相似的途径增强植株对锌的抗性。拟南芥铝胁迫后, 可以诱导WAK1基因的表达^[55]; WALK4基因启动子的突变导致水稻对钾、钠、铜和锌超敏感, WALK4突变导致水稻茎中锌含量下降^[56]; WAKL10基因表达量上调, 其可能在锌胁迫过程中起着重要作用。本研究为深入研究甘蓝型油菜响应锌胁迫的分子机制奠定了基础。

4 结论

全基因组关联分析共鉴定出8个与锌胁迫显著关联的SNP位点, 结合转录组测序结果, 共找到19个候选基因, 可能在锌胁迫过程中起着重要作用。

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An extensive soil survey was conducted to study pollution sources and delineate contamination of heavy metals in one of the metalliferous industrial bases, in the karst areas of southwest China. A total of 597 topsoil samples were collected and the concentrations of five heavy metals, namely Cd, As (metalloid), Pb, Hg and Cr were analyzed. Stochastic models including a conditional inference tree (CIT) and a finite mixture distribution model (FMDM) were applied to identify the sources and partition the contribution from natural and anthropogenic sources for heavy metal in topsoils of the study area. Regression trees for Cd, As, Pb and Hg were proved to depend mostly on indicators of anthropogenic activities such as industrial type and distance from urban area, while the regression tree for Cr was found to be mainly influenced by the geogenic characteristics. The FMDM analysis showed that the geometric means of modeled background values for Cd, As, Pb, Hg and Cr were close to their background values previously reported in the study area, while the contamination of Cd and Hg were widespread in the study area, imposing potentially detrimental effects on organisms through the food chain. Finally, the probabilities of single and multiple heavy metals exceeding the threshold values derived from the FMDM were estimated using indicator kriging (IK) and multivariate indicator kriging (MVIK). The high probabilities exceeding the thresholds of heavy metals were associated with metalliferous production and atmospheric deposition of heavy metals transported from the urban and industrial areas. Geostatistics coupled with stochastic models provide an effective way to delineate multiple heavy metal pollution to facilitate improved environmental management. (C) 2014 Elsevier B.V.

[35] Zhang F, Xiao X, Xu K, Cheng X, Xie T, Hu J, Wu X. Genome-wide association study (GWAS) reveals genetic loci of lead (Pb) tolerance during seedling establishment in rapeseed (Brassica napus L.)
BMC Genomics, 2020,21:139.
[本文引用: 1]

[36] Zhang J, Mason A S, Wu J, Liu S, Zhang X, Luo T, Redden R, Batley J, Hu L, Yan G. Identification of putative candidate genes for water stress tolerance in canola (Brassica napus)
Front Plant Sci, 2015,6:1058.
DOI:10.3389/fpls.2015.01058URLPMID:26640475
Drought stress can directly inhibit seedling establishment in canola (Brassica napus), resulting in lower plant densities and reduced yields. To dissect this complex trait, 140 B. napus accessions were phenotyped under normal (0.0 MPa, S0) and water-stressed conditions simulated by polyethylene glycol (PEG) 6000 (-0.5 MPa, S5) in a hydroponic system. Phenotypic variation and heritability indicated that the root to shoot length ratio was a reliable indicator for water stress tolerance. Thereafter, 66 accessions (16 water stress tolerant, 34 moderate and 16 sensitive lines) were genotyped using 25,495 Brassica single nucleotide polymorphisms (SNPs). Genome-wide association studies (GWAS) identified 16 loci significantly associated with water stress response. Two B. napus accessions were used for RNA sequencing, with differentially-expressed genes under normal and water-stressed conditions examined. By combining differentially-expressed genes detected by RNA sequencing with significantly associated loci from GWAS, 79 candidate genes were identified, of which eight were putatively associated with drought tolerance based on gene ontology of Arabidopsis. Functional validation of these genes may confirm key drought-related genes for selection and breeding in B. napus. Our results provide insight into the genetic basis of water stress tolerance in canola.

[37] Ke L, Lei W, Yang W, Wang J, Gao J, Cheng J, Sun Y, Fan Z, Yu D. Genome-wide identification of cold responsive transcription factors in Brassica napus L
BMC Plant Biol, 2020,20:62.
DOI:10.1186/s12870-020-2253-5URLPMID:32028890 [本文引用: 1]
BACKGROUND: Cold stress is one of the primary environmental factors that affect plant growth and productivity, especially for crops like Brassica napus that live through cold seasons. Till recently, although a number of genes and pathways involved in B. napus cold response have been revealed by independent studies, a genome-wide identification of the key regulators and the regulatory networks is still lack. In this study, we investigated the transcriptomes of cold stressed semi-winter and winter type rapeseeds in short day condition, mainly with the purpose to systematically identify the functional conserved transcription factors (TFs) in cold response of B. napus. RESULTS: Global modulation of gene expression was observed in both the semi-winter type line (158A) and the winter type line (SGDH284) rapeseeds, in response to a seven-day chilling stress in short-day condition. Function analysis of differentially expressed genes (DEGs) revealed enhanced stresses response mechanisms and inhibited photosynthesis in both lines, as well as a more extensive inhibition of some primary biological processes in the semi-winter type line. Over 400 TFs were differentially expressed in response to cold stress, including 56 of them showed high similarity to the known cold response TFs and were consistently regulated in 158A and SGDH284, as well as 25 TFs which targets were over-represented in the total DEGs. A further investigation based on their interactions indicated the critical roles of several TFs in cold response of B. napus. CONCLUSION: In summary, our results revealed the alteration of gene expression in cold stressed semi-winter and winter ecotype B. napus lines and provided a valuable collection of candidate key regulators involved in B. napus response to cold stress, which could expand our understanding of plant stress response and benefit the future improvement of the breed of rapeseeds.

[38] Shahid M, Natasha , Khalid S, Abbas G, Niazi N K, Murtaza B, Rashid M I, Bibi I. Redox mechanisms and plant tolerance under heavy metal stress: genes and regulatory networks
In: Sablok G, eds. Plant Metallomics and Functional Omics. Cham: Springer, 2019. pp 71-105.
[本文引用: 1]

[39] Jiang Y, Qiu Y, Hu Y, Yu D. Heterologous expression of AtWRKY57 confers drought tolerance in Oryza sativa
Front Plant Sci, 2016,7:145.
DOI:10.3389/fpls.2016.00145URLPMID:26904091 [本文引用: 1]
Drought stress is a severe environmental factor that greatly restricts plant distribution and crop production. Recently, we have found that overexpressing AtWRKY57 enhanced drought tolerance in Arabidopsis thaliana. In this study, we further reported that the Arabidopsis WRKY57 transcription factor was able to confer drought tolerance to transgenic rice (Oryza sativa) plants. The enhanced drought tolerance of transgenic rice was resulted from the lower water loss rates, cell death, malondialdehyde contents and relative electrolyte leakage while a higher proline content and reactive oxygen species-scavenging enzyme activities was observed during stress conditions. Moreover, further investigation revealed that the expression levels of several stress-responsive genes were up-regulated in drought-tolerant transgenic rice plants, compared with those in wild-type plants. In addition to the drought tolerance, the AtWRKY57 over-expressing plants also had enhanced salt and PEG stress tolerances. Taken together, our study indicates that over-expressing AtWRKY57 in rice improved not only drought tolerance but also salt and PEG tolerance, demonstrating its potential role in crop improvement.

[40] Jiang Y, Liang G, Yu D. Activated expression of WRKY57 confers drought tolerance in Arabidopsis
Mol Plant, 2012,5:1375-1388.
URLPMID:22930734 [本文引用: 1]

[41] Chen H Y, Hsieh E J, Cheng M C, Chen C Y, Hwang S Y, Lin T P. ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) regulates jasmonic acid and abscisic acid biosynthesis and signaling through binding to a novel cis-element
New Phytol, 2016,211:599-613.
[本文引用: 1]

[42] Zang D D, Li H Y, Xu H Y, Zhang W H, Zhang Y M, Shi X X, Wang Y C. An arabidopsis zinc finger protein increases abiotic stress tolerance by regulating sodium and potassium homeostasis, reactive oxygen species scavenging and osmotic potential
Front Plant Sci, 2016,7:1272.
DOI:10.3389/fpls.2016.01272URLPMID:27605931 [本文引用: 1]
Plant zinc finger proteins (ZFPs) comprise a large protein family and they are mainly involved in abiotic stress tolerance. Although Arabidopsis RING/FYVE/PHD ZFP At5g62460 (AtRZFP) is found to bind to zinc, whether it is involved in abiotic stress tolerance is still unknown. In the present study, we characterized the roles of AtRZFP in response to abiotic stresses. The expression of AtRZFP was induced significantly by salt and osmotic stress. AtRZFP positively mediates tolerance to salt and osmotic stress. Additionally, compared with wild-type Arabidopsis plants, plants overexpressing AtRZFP showed reduced reactive oxygen species (ROSs) accumulation, enhanced superoxide dismutase and peroxidase activity, increased soluble sugars and proline contents, reduced K(+) loss, decreased Na(+) accumulation, stomatal aperture and the water loss rate. Conversely, AtRZFP knockout plants displayed the opposite physiological changes when exposed to salt or osmotic stress conditions. These data suggested that AtRZFP enhances salt and osmotic tolerance through a series of physiological processes, including enhanced ROSs scavenging, maintaining Na(+) and K(+) homeostasis, controlling the stomatal aperture to reduce the water loss rate, and accumulating soluble sugars and proline to adjust the osmotic potential.

[43] Cao H J, Huang P Y, Zhang L L, Shi Y K, Sun D D, Yan Y X, Liu X H, Dong B, Chen G Q, Snyder J H, Lin F C, Lu J P. Characterization of 47 Cys₂-His₂ zinc finger proteins required for the development and pathogenicity of the rice blast fungus Magnaporthe oryzae
New Phytol, 2016,211:1035-1051.
URLPMID:27041000 [本文引用: 1]

[44] Sun N, Liu M, Zhang W, Yang W, Bei X, Ma H, Qiao F, Qi X. Bean metal-responsive element-binding transcription factor confers cadmium resistance in tobacco
Plant Physiol, 2015,167:1136-1148.
DOI:10.1104/pp.114.253096URLPMID:25624396 [本文引用: 1]
Cadmium (Cd) is highly toxic to plants. Modulation of Cd-responsive transcription is an important way for Cd detoxification in plants. Metal-responsive element (MRE) is originally described in animal metallothionein genes. Although functional MREs also exist in Cd-regulated plant genes, specific transcription factors that bind MRE to regulate Cd tolerance have not been identified. Previously, we showed that Cd-inducible bean (Phaseolus vulgaris) stress-related gene2 (PvSR2) produces a short (S) PvSR2 transcript (S-PvSR2) driven by an intronic promoter. Here, we demonstrate that S-PvSR2 encodes a bean MRE-binding transcription factor1 (PvMTF-1) that confers Cd tolerance in tobacco (Nicotiana tabacum). PvMTF-1 expression was up-regulated by Cd at the levels of RNA and protein. Importantly, expression of PvMTF-1 in tobacco enhanced Cd tolerance, indicating its role in regulating Cd resistance in planta. This was achieved through direct regulation of a feedback-insensitive Anthranilate Synthase alpha-2 chain gene (ASA2), which catalyzes the first step for tryptophan biosynthesis. In vitro and in vivo DNA-protein interaction studies further revealed that PvMTF-1 directly binds to the MRE in the ASA2 promoter, and this binding depends on the zinc finger-like motif of PvMTF-1. Through modulating ASA2 up-regulation by Cd, PvMTF-1 increased free tryptophan level and subsequently reduced Cd accumulation, thereby enhancing Cd tolerance of transgenic tobacco plants. Consistent with this observation, tobacco transiently overexpressing ASA2 also exhibited increased tolerance to Cd. We conclude that PvMTF-1 is a zinc finger-like transcription factor that links MRE to Cd resistance in transgenic tobacco through activation of tryptophan biosynthesis.

[45] Chen J, Yang L, Yan X, Liu Y, Wang R, Fan T, Ren Y, Tang X, Xiao F, Liu Y, Cao S. Zinc-finger transcription factor ZAT6 positively regulates cadmium tolerance through the glutathione- dependent pathway in Arabidopsis
Plant Physiol, 2016,171:707-719.
URLPMID:26983992 [本文引用: 1]

[46] Liu X M, An J, Han H J, Kim S H, Lim C O, Yun D J, Chung W S. ZAT11, a zinc finger transcription factor, is a negative regulator of nickel ion tolerance in Arabidopsis
Plant Cell Rep, 2014,33:2015-2021.
DOI:10.1007/s00299-014-1675-7URLPMID:25163803 [本文引用: 1]
KEY MESSAGE: ZAT11, a Zinc Finger of Arabidopsis Thaliana 11, is a dual-function transcriptional regulator that positively regulates primary root growth but negatively regulates Ni (2+) tolerance. Zinc Finger of Arabidopsis Thaliana 11 (ZAT11) is a C2H2-type zinc finger protein that has been reported to function as an active transcriptional repressor. However, the biological function of ZAT11 remains unknown. Here we show that GFP-tagged ZAT11 is targeted to the nucleus. Analysis of plants expressing ZAT11 promoter-GUS showed that ZAT11 is highly expressed in roots and particularly in root tips. To identify the biological function of ZAT11, we constructed three independent lines of ZAT11 overexpressing transgenic plant (ZAT11 OE). ZAT11 OE enhanced the elongation of primary root but reduced the metal tolerance against nickel ion (Ni(2+)). The reduced Ni(2+) tolerance of ZAT11 OE was correlated with decreased accumulation of Ni(2+) in plants. The decreased accumulation of Ni(2+) in ZAT11 OE was caused by the reduced transcription of a vacuolar Ni(2+) transporter gene. Taken together, our results suggest that ZAT11 is a dual function transcriptional regulator that positively regulates primary root growth but negatively regulates Ni(2+) tolerance.

[47] Yang L, Wei Y, Na L, Zeng J Y, Han Y J, Zuo Z J, Wang S T, Zhu Y R, Zhang Y, Sun J S, Yong W. Declined cadmium accumulation in Na⁺/H⁺ antiporter (NHX1) transgenic duckweed under cadmium stress
Ecotoxicol Environ Saf, 2019,182:109397.
DOI:10.1016/j.ecoenv.2019.109397URLPMID:31299476 [本文引用: 1]
Cadmium (Cd) is a serious threat to plants health. Though some genes have been reported to get involved in the regulation of tolerance to Cd, the mechanisms underlying this process are not fully understood. Na(+)/H(+) antiporter (NHX1) plays an important role in Na(+)/H(+) trafficking. The salt and cadmium stress tolerance were found to be enhanced by NHX1 in duckweed according to our previous study, however, its function in Cd(2+) flux under Cd stress has not been studied. Here we explored the Cd(2+) flux in wild type (WT) and NHX1 transgenic duckweed (NHX1) under Cd stress. We found that the Cd(2+) influx in NHX1 duckweed was significantly declined, followed by an increased Cd(2+) efflux after 20min treatment of Cd, which resulted a less accumulation of Cd in NHX1. Reversely, inhibition of NHX1 by amiloride treatment, enhanced Cd(2+) influx in NHX1 duckweed, subsequently delayed Cd(2+) efflux in both genotypes of duckweed under Cd(2+) shock. H(+) efflux in NHX1 duckweed was lower compare with that in WT with 20min Cd(2+) shock. NHX1 also increased the pH value with Cd(2+) stress in the transgenic rhizoid. These finding suggested a new function of NHX1 in regulation of Cd(2+) and H(+) flow during short-term Cd(2+) shock.

[48] 李洋, 于丽杰, 金晓霞. 植物重金属胁迫耐受机制
中国生物工程杂志, 2015,35(9):94-104.
[本文引用: 1]

Li Y, Yu L J, Jin X X. The mechanism of heavy metal stress in plants
China Biotechnol, 2015,35(9):94-104 (in Chinese with English abstract).
[本文引用: 1]

[49] 李小宁. 锌胁迫对小麦种子萌发及幼苗生理生化特性的影响
西北师范大学硕士毕业论文, 甘肃兰州, 2013.
[本文引用: 1]

Li X N. Effects of Zinc Stress on Seed Germination, Physiological and Biochemical Characteristics in Wheat Seedling
MS Thesis of Northwest Normal University, Lanzhou, Gansu, China, 2013 (in Chinese with English abstract).
[本文引用: 1]

[50] Kumar S, Trivedi P K. Glutathione S-transferases: role in combating abiotic stresses including arsenic detoxification in plants
Front Plant Sci, 2018,9:751.
URLPMID:29930563 [本文引用: 1]

[51] Ezaki B, Gardner R C, Ezaki Y, Matsumoto H. Expression of aluminum-induced genes in transgenic Arabidopsis plants can ameliorate aluminum stress and/or oxidative stress
Plant Physiol, 2000,122:657-665.
DOI:10.1104/pp.122.3.657URLPMID:10712528 [本文引用: 1]
To examine the biological role of Al-stress-induced genes, nine genes derived from Arabidopsis, tobacco (Nicotiana tabacum L.), wheat (Triticum aestivum L.), and yeast (Saccharomyces cerevisiae) were expressed in Arabidopsis ecotype Landsberg. Lines containing eight of these genes were phenotypically normal and were tested in root elongation assays for their sensitivity to Al, Cd, Cu, Na, Zn, and to oxidative stresses. An Arabidopsis blue-copper-binding protein gene (AtBCB), a tobacco glutathione S-transferase gene (parB), a tobacco peroxidase gene (NtPox), and a tobacco GDP-dissociation inhibitor gene (NtGDI1) conferred a degree of resistance to Al. Two of these genes, AtBCB and parB, and a peroxidase gene from Arabidopsis (AtPox) also showed increased resistance to oxidative stress induced by diamide, while parB conferred resistance to Cu and Na. Al content of Al-treated root tips was reduced in the four Al-resistant plant lines compared with wild-type Ler-0, as judged by morin staining. All four Al-resistant lines also showed reduced staining of roots with 2',7'-dichloro fluorescein diacetate (H(2)DCFDA), an indicator of oxidative stress. We conclude that Al-induced genes can serve to protect against Al toxicity, and also provide genetic evidence for a link between Al stress and oxidative stress in plants.

[52] Srivastava D, Verma G, Chauhan A S, Pande V, Chakrabarty D. Rice (Oryza sativa L.) tau class glutathione S-transferase (OsGSTU30) overexpression in Arabidopsis thaliana modulates a regulatory network leading to heavy metal and drought stress tolerance
Metallomics, 2019,11:375-389.
DOI:10.1039/c8mt00204eURLPMID:30516767 [本文引用: 1]
Glutathione S-transferases (GSTs) are well-known enzymes due to their role in detoxification of xenobiotic compounds. However, their biochemical action is still not so clear in imparting tolerance to several abiotic stresses in crop plants. In our previous study, we observed that rice tau class OsGSTU30 plays a significant role in the detoxification of Cr(vi). Interestingly, q-RT PCR analysis also revealed higher expression of OsGSTU30 under drought conditions. In this study, we characterize OsGSTU30 in response to drought as well as heavy metal [Cr(vi)] stresses through overexpression in Arabidopsis thaliana. Biochemical and physiological analyses revealed that OsGSTU30 overexpression lines have improved tolerance against both stresses as compared to wild-type plants. Kinetic analysis and molecular docking confirmed that OsGSTU30 enzyme possesses both GST as well as glutathione peroxidase (GPx) like activity. Differentially expressed stress-responsive genes were also identified by transcriptome analysis, involved in different biological pathways during abiotic stresses. These results suggest the signaling functions of OsGSTU30 apart from its catalytic activity during abiotic stress responses and can be further exploited for improving the stress tolerance in crops.

[53] Zhang J, Martinoia E, Lee Y. Vacuolar transporters for cadmium and arsenic in plants and their applications in phytoremediation and crop development
Plant Cell Physiol, 2018,59:1317-1325.
DOI:10.1093/pcp/pcy006URLPMID:29361141 [本文引用: 1]
Soil contamination by heavy metals and metalloids such as cadmium (Cd) and arsenic (As) poses a major threat to the environment and to human health. Vacuolar sequestration is one of the main mechanisms by which plants control toxic materials including Cd and As. Understanding the mechanisms of heavy metal tolerance and accumulation can be useful for both phytoremediation and safe crop development. In this review, we summarize recent advances in deciphering the molecular mechanisms underlying vacuolar sequestration of Cd and As, and discuss potential biotechnological applications of this knowledge and efforts towards attaining these goals.

[54] Kim D Y, Bovet L, Maeshima M, Martinoia E, Lee Y. The ABC transporter AtPDR8 is a cadmium extrusion pump conferring heavy metal resistance
Plant J, 2007,50:207-218.
URLPMID:17355438 [本文引用: 1]

[55] Korenkov V, King B, Hirschi K, Wagner G J. Root-selective expression of AtCAX4 and AtCAX2 results in reduced lamina cadmium in field-grown Nicotiana tabacum L
Plant Biotechnol J, 2009,7:219-226.
URLPMID:19175521 [本文引用: 1]

[56] Hou X W, Tong H Y, Selby J, DeWitt J, Peng X X, He Z H. Involvement of a cell wall-associated kinase, WAKL4, in Arabidopsis mineral responses
Plant Physiol, 2005,139:1704-1716.
DOI:10.1104/pp.105.066910URLPMID:16286448 [本文引用: 1]
The cell wall-associated receptor kinase (WAK) and WAK-like kinase (WAKL) gene family members are good candidates for physical linkers that signal between the cell wall and the cytoplasmic compartment. Previous studies have suggested that while some WAK/WAKL members play a role in bacterial pathogen and heavy-metal aluminum responses, others are involved in cell elongation and plant development. Here, we report a functional role for the WAKL4 gene in Arabidopsis (Arabidopsis thaliana) mineral responses. Confocal microscopic studies localized WAKL4-green fluorescent protein fusion proteins on the cell surfaces suggesting that, like other WAK/WAKL proteins, WAKL4 protein is associated with the cell wall. Histochemical analyses of the WAKL4 promoter fused with the -glucuronidase reporter gene have shown that WAKL4 expression is induced by Na+, K+, Cu2+, Ni2+, and Zn2+. A transgenic line with a T-DNA insertion at 40-bp upstream of the WAKL4 start codon was characterized. While the T-DNA insertion had little effect on the WAKL4 transcript levels under normal growth conditions, it significantly altered the expression patterns of WAKL4 under various conditions of mineral nutrients. Semiquantitative and quantitative reverse transcription-PCR analyses showed that the promoter impairment abolished WAKL4-induced expression by Na+, K+, Cu2+, and Zn2+, but not by Ni2+. Whereas the WAKL4 promoter impairment resulted in hypersensitivity to K+, Na+, Cu2+, and Zn2+, it conferred a better tolerance to toxic levels of the Ni2+ heavy metal. WAKL4 was required for the up-regulation of zinc transporter genes during zinc deficiency, and the WAKL4 T-DNA insertion resulted in a reduction of Zn2+ accumulation in shoots. A WAKL4-green fluorescent protein fusion gene driven by either the WAKL4 native promoter or the 35S constitutive promoter complemented the phenotypes. Our results suggest versatile roles for WAKL4 in Arabidopsis mineral nutrition responses.