棉花<i>WRKY7</i>基因克隆及其对黄萎病抗性分析

doi:10.7668/hbnxb.20193069

摘要/Abstract

摘要：

鉴定抗病基因并培育抗病棉花品种是目前根治棉花黄萎病的最好方法。利用MEGA 5.2等相关软件,构建棉花抗病相关基因WRKY7与拟南芥和水稻中抗病相关WRKY基因编码蛋白系统进化树。通过亚细胞定位技术、病毒诱导基因沉默技术、qPCR及GUS报告系统分析等技术,阐明GhWRKY7基因对大丽轮枝菌的诱导响应及其机制。结果表明:GhWRKY7和AtWKRY7高度同源,同属于Ⅱ类;GhWRKY7定位于植物细胞核内,其在棉花叶和根器官中的相对表达量显著高于茎;GhWRKY7基因表达受到大丽轮枝菌浸染诱导,在浸染24 h后呈显著上调。沉默GhWRKY7基因棉花对大丽轮枝菌抗性下降;和对照植株(表达TRV空载体植株)相比,接菌后沉默植株中GhPR1、GhPR3、GhPR4、GhPR5、GhPDF1.2、GhPAL1和GhCYP71B36等抗病相关基因的表达水平也显著下调,暗示GhWRKY7通过调控抗病相关基因的表达来提高植株的抗病性。构建GUS报告载体在烟草细胞中瞬时表达分析,揭示GhWRKY7基因能特异地结合GhCYP71B36启动子顺式元件,转录激活下游GhCYP71B36表达,从而提高棉花的抗病性。综上所述,GhWRKY7作为一个正调控棉花抗黄萎病的转录因子,参与如植保素Camalexin合成等下游抗病相关基因的表达,从而提高植株的抗病性。因此,GhWRKY7可以作为棉花抗病育种的候选基因,为棉花生产提供安全保障。

关键词: 陆地棉, 大丽轮枝菌, GhWRKY7, GhCYP71B36, 基因沉默

Abstract:

To eradicate Verticillium wilt of cotton,identification of resistant genes and breeding of resistant cotton varieties are the best methods at present.We used MEGA 5.2 and other related software to construct phylogenetic trees of the proteins encoded by WRKY7 genes in Gossypium hirsutum and Arabidopsis thaliana and Oryza sativa.The induction and response of GhWRKY7 gene to Verticillium dahliae and its mechanism were elucidate by subcellular localization,virus-induced gene silencing,qPCR and GUS reporting system analysis.The results showed that GhWRKY7 and AtWKRY7 were highly homologous and belonged to Group Ⅱ.GhWRKY7 was located in plant nucleus,and the relative expression of GhWRKY7 in cotton leaves and root organs was significantly higher than that in stems.GhWRKY7 gene expression was significantly upregulated after 24 h of inoculation.The GhWRKY7-silenced plants showed higher susceptibility to Verticillium dahliae infection compared to the control(Expression TRV empty vector plants),suggesting that GhWRKY7 gene positively regulated cotton disease resistance.Compared to the control,the expression levels of disease-resistance related genes,including GhPR1,GhPR3,GhPR4,GhPR5,GhPDF1.2,GhPAL1 and GhCYP71B36 in GhWRKY7-silenced plants significantly decreased after inoculation,indicating that GhWRKY7 improved the disease resistance of the plants due to increased the expression levels of disease-resistance related genes.Transient expression analysis of GhWRKY7 gene by constructing GUS reporter vector in tobacco cells revealed that GhCYP71B36 gene could specifically bound to cis-element of GhCYP71B36 promoter and transcriptionally activated downstream GhCYP71B36 expression,thus improved the disease resistance of cotton.In conclusion,GhWRKY7,as a transcription factor that positively regulates Verticillium wilt resistance in cotton,is involved in the expression of downstream disease-resistance related genes such as Camalexin synthesis,thereby improving plant disease resistance.Therefore,GhWRKY7 can be used as a candidate gene for cotton resistance breeding and provide security for cotton production.

Key words: Gossypium hirsutum, Verticillium dahliae, GhWRKY7, GhCYP71B36, Gene silencing

曾焱明, 胡广, 贾培, 唐叶, 王炳婷, 武盼, 陆承哲, 陈爱民, 彭清忠, 吴家和. 棉花WRKY7基因克隆及其对黄萎病抗性分析[J]. 华北农学报, 2022, 37(6): 191-200. doi: 10.7668/hbnxb.20193069.

ZENG Yanming, HU Guang, JIA Pei, TANG Ye, WANG Bingting, WU Pan, LU Chengzhe, CHEN Aimin, PENG Qingzhong, WU Jiahe. Cotton WRKY7 Cloning and Analysis of Plant Resistance to Verticillium wilt[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 191-200. doi: 10.7668/hbnxb.20193069.

http://www.hbnxb.net/CN/Y2022/V37/I6/191

图/表 8

表1 引物序列

Tab.1 Primer sequence

引物名称 Primer name	正向引物(5'—3') Forward primer sequence (5'—3')	反向引物(5'—3') Reverse primer sequence (5' —3')
GhWRKY7	ATGACCGTGGAATTGAAGAT	AGATGATTCCAGGATCAAAG
GhCYP71B36_pro	CTAGACTGTCAATCATAATA	CCATCATAACTGAGTTTTGCAAA
GhWRKY7	GCAAAGATTGGTGTTTTGGAA	AATGACCTTCTTCAATC
qGhWRKY7	GGTTCAGCAGAGTCAGCCAT	GCAACGGCGAGAGAATGAAC
qGhCYP71B36	ACATGGTCATGCTTGGCTCA	TGACGGTTCCCATGTACAGC
qGhPR1	GGGGCAGTGCTGACCTATCG	TTAGCACAACCAAGATGGACAGAGT
qGhPR3	GATGACTCCACAATCACCGAAGC	GCGGTCTTCTACCTGGGCATT
qGhPR4	GCCTATTGCTCAACTTGGGA	CAATCTGCTGGAACAAGGCT
qGhPR5	CGACAACTCTGGCTCAGGAA	CCATCATCTGTAACTCGGCG
qGhPDF1.2	CTGTGGTAGCGGATGGTGATAAG	GTGCAGACGCATTTGCGAAGGAA
qGhPAL1	CTGCTAAGGGTGGTGTCGTCG	CCAATGACCTCCTGTACTCGGC
qGhUBQ7	GAAGGCATTCCACCTGACCAAC	CTTGACCTTCTTCTTCTTGTGCTTG

图1 GhWRKY7与拟南芥、水稻中抗病相关WRKY蛋白的系统进化树分析 Ⅰ类由2个WRKY结构域和C-X_4-5-C-X_22-23-H-X₁-H锌指结构构成;Ⅱ类由1个WRKY结构域和C-X_4-5-C-X_22-23-H-X₁-H锌指结构构成;Ⅲ类由1个WRKY结构域和C-X₇-C-X₂₃-H-X₁-C锌指结构构成。

Fig.1 Phylogenetic tree analysis of GhWRKY7 and WRKY proteins related to disease resistance in Arabidopsis thaliana and Oryza sativa Group Ⅰ consists of two WRKY domains and a C-X_4-5-C-X_22-23-H-X₁-H zinc finger structure;Group Ⅱ composed of a WRKY domain and a C-X_4-5-C-X_22-23-H-X₁-H zinc finger structure;Group Ⅲ consists of a WRKY domain and a C-X₇-C-X₂₃-H-X₁-C zinc finger structure.

图2 GhWRKY7亚细胞定位分析 A.35S∶GFP表达框示意图;B.35S∶ GhWRKY7-GFP表达框示意图;C.35S∶GhWRKY7-GFP和对照组35S∶GFP在激光共聚焦显微镜下细胞内分布。

Fig.2 Subcellular localization of GhWRKY7 A.Schematic diagram of 35S∶GFP expression frame;B.Schematic diagram of 35S∶GhWRKY7-GFP expression frame;C.The distribution of 35S∶GhWRKY7-GFP and control 35S∶GFP under laser confocal microscope.

图3 GhWRKY7表达分析 A.GhWRKY7组织表达分析;B.大丽轮枝菌对GhWRKY7诱导表达分析;C、D.SA和JA对GhWRKY7诱导表达分析。不同字母表示差异显著(P<0.05)。图4—6同。

Fig.3 Expression analysis of GhWRKY7 A.Tissue expression analysis of GhWRKY7;B.Induced expression analysis of GhWRKY7 by V.dahliae;C,D.Expression analysis of GhWRKY7 induced by SA and JA.The different alphabets stand for significant difference(P<0.05).The same as Fig.4—6.

图4 GhWRKY7沉默载体构建及沉默植株获得 A.pYL-156-GhWRKY7沉默载体构建示意图,其中包含强启动子(35S_pro)、NOS_ter终止子序列和用于各种目的基因片段构建的多克隆位点(Multiple clone sites,MCS),GhWRKY7基因特异性片段大小为300 bp;B.阳性植株叶片白化表型;C.阴性对照与沉默植株中GhWRKY7基因相对表达量分析。

Fig.4 Construction of GhWRKY7-silencing vector and development of silencing plants A.Construction schematic of pYL-156-GhWRKY7-silencing vector,which contains duplicated enhancers(35S_pro),NOS terminator(NOS_ter)and Multiple clone sites(MCS)for construction of various target gene fragments,the GhWRKY7 gene specific fragment size was 300 bp;B.Leaf albino phenotype of positive plants;C.Analysis of GhWRKY7 gene relative expression levels in negative control and silent plants.

图5 GhWRKY7沉默植株抗病性分析 A.GhWRKY7沉默植株与对照组发病表型;B.植株发病率;C.病情指数;D.接菌棉花主茎秆切面维管束表型:WT为未接菌正常发育棉花植株;TRV∶00和TRV∶GhWRKY7为对照和沉默植株接菌21 d后,发病植株的主茎秆切面表型;E.接菌植株茎段恢复培养后,大丽轮枝菌生长情况。

Fig.5 The disease-resistant analysis of GhWRKY7-silenced plants A.Phenotype of GhWRKY7-silenced plants and control group;B.Rates of diseased plant;C.Disease index;D.Vascular bundle phenotypes on the stem section of inoculated plant and WT:WT is the normal plants without inoculation;TRV:00 and TRV:GhWRKY7 are the main stem section phenotypes of the infected plants after inoculation for 21 days;E.The recovery culture of V.dahliae from the stem segment of the inoculated plants.

图6 GhWRKY7沉默降低抗病标志基因的表达 A.GhWRKY7沉默植株与对照组中SA抗病标志基因相对表达量;B.JA抗病标志基因相对表达量;C.GhCYP71B36相对表达量。GhWRKY7沉默植株与对照组的表达量测定于大丽轮枝菌浸染后21 d。

Fig.6 Expression analysis of disease resistance marker genes in GhWRKY7-silenced plants and control plants A.Relative expression levels of three SA marker genes in GhWRKY7-silenced plants and control plants; B. Relative expression levels of three JA marker genes;C.Relative expression levels of GhCYP71B36. GhWRKY7-silenced plants and control group infection with V.dahliae at 21 days were sampled.

图7 GhWRKY7基因表达载体和GhCYP71B36_pro基因报告载体构建示意图及GUS染色 A.pBI121-GhWRKY7表达载体与pBI121-GhCYP71B36_pro-GUS报告载体构建示意图;B.左为烟草瞬时表达的GUS染色结果,右为烟草叶片各部分注射情况。阳性对照pBI121-GUS菌液。

Fig.7 Schematic diagram of GhWRKY7 gene effector vector and GhCYP71B36_pro gene reporter vector construction and GUS staining results A.Schematic diagram of construction of pBI121-GhWRKY7 expression vector and pBI121-GhCYP71B36_pro-GUS reporting vector;B.GUS staining results of transient expression of tobacco on the left,on the right is the injection of each part of tobacco leaf.Positive control pBI121-GUS bacterial solution.

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