辣椒<i>CaWRKY30</i>转录因子的克隆、亚细胞定位及番茄斑萎病毒侵染下的表达分析

doi:10.7668/hbnxb.20192715

摘要/Abstract

摘要：

为研究辣椒CaWRKY30转录因子与番茄斑萎病毒互作的响应机制,以辣椒湘研11为试验材料,通过RNA提取、RT-PCR、切胶纯化及克隆等试验获得CaWRKY30编码序列。生物信息学分析结果表明,CaWRKY30全长1 122 bp,编码373个氨基酸,该基因编码的蛋白含有1个WRKY保守结构域和1个C₂H₂结构域,属于典型的Ⅱ(e)亚家族成员。系统发育分析表明,与马铃薯StWRKY22氨基酸序列的亲缘关系最近。本氏烟叶片亚细胞定位试验发现,CaWRKY30在本氏烟幼苗中定位于细胞核和细胞膜,并且导致细胞膜加厚。实时荧光定量PCR分析结果表明,观察番茄斑萎病毒机械摩擦接种辣椒后的病毒积累量,发现病毒积累量在接种1~14 d逐渐上升,在接种后14 d处于最大值,接种14 d后病毒积累量逐渐下降;同时,CaWRKY30受番茄斑萎病毒接种后诱导,在接种病毒1~14 d CaWRKY30表达量上调,14 d达到峰值,14 d以后表达量逐渐下降。综上,获得了CaWKRY30转录因子基因序列,该转录因子定位于细胞核和细胞膜,并初步阐述了辣椒CaWRKY30转录因子在番茄斑萎病毒胁迫下的表达趋势。

关键词: 辣椒, CaWRKY30基因克隆, 生物信息学, 番茄斑萎病毒, 表达分析

Abstract:

In order to study the response mechanism of pepper CaWRKY30 transcription factor and Tomato spotted wilt orthotospovirus,it was experimental materials with pepper Xiangyan 11.The CaWRKY30 coding sequence was obtained by RNA extraction,RT-PCR,split gel and cloning.Biological information analysis results showed that CaWRKY30 full length was 1 122 bp,encoding 373 amino acids,the gene encoded protein contains 1 WRKY conservative domain and 1 C₂H₂ domain,belonged to a typical Ⅱ(e)subfamily member.System evolution analysis showed that the relative relationship with the potato StWRKY22 amino acid sequence was recently.It was found that CaWRKY30 was positioned in the nucleus and cell membranes in its cigarette seedlings,and leads to cell membranes.The results of Real-time fluorescence quantitative PCR analysis showed that the viral accumulation of Tomato spotted wilt orthotospovirus mechanical friction-vaccination was found that the viral accumulation was gradually increased from 1 to 14 days after inoculation,and virus accumulation reached its maximum in 14 days,after inoculation 14 days,viral accumulation gradually declined.At the same time,CaWRKY30 was induced by Tomato spotted wilt orthotospovirus,when the inoculation 1-14 days,the CaWRKY30 expression was raised,and the peak was reached in 14 days,the expression in 14 days gradually decreased.In summary,it obtained the CaWRKY30 transcription factor gene sequence,which was located in the nucleus and cell membrane,and preliminarily explained the expression trend of CaWRKY30 transcription factors under the stress of Tomato spotted wilt orthotospovirus.

Key words: Pepper, CaWRKY30 gene cloning, Bioinformatics, Tomato spotted wilt orthotospovirus, Expression analysis

贾志强, 许云玉, 高雪, 陶宏征, 陈增敏, 刘雅婷, 李永忠. 辣椒CaWRKY30转录因子的克隆、亚细胞定位及番茄斑萎病毒侵染下的表达分析[J]. 华北农学报, 2022, 37(3): 186-192. doi: 10.7668/hbnxb.20192715.

JIA Zhiqiang, XU Yunyu, GAO Xue, TAO Hongzheng, CHEN Zengmin, LIU Yating, LI Yongzhong. Cloning,Subcellular Positioning of Pepper CaWRKY30 Transcription Factor, and Its Expression Analysis by Tomato spotted wilt orthotospovirus Infected[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(3): 186-192. doi: 10.7668/hbnxb.20192715.

http://www.hbnxb.net/CN/Y2022/V37/I3/186

图/表 6

表1 试验所用引物信息

Tab.1 Experiment primer information

引物 Primer	引物序列(5'-3') Primer sequences(5'-3')	功能 Function
CaWRKY30-F	ATGGAGGAAGATTGGGAT	克隆引物
CaWRKY30-R	TCAAACACCGCCAGCTGC
CaW30-BamHⅠ-F	CGCggatccATGCAGTGGTCAGAGGCTGC	亚细胞定位
CaW30-KpnⅠ-R	CGGggtaccAAACACCGCCAGCTGCGGTG
qTSWV-F	CTGTGAGGCTTGCCATAAT	qRT-PCR
qTSWV-R	CTAAGGCTTCCTTGGTGTC
qCaW30-F	TTTGAAGGGTTAGATGAA
qCaW30-R	GTTGTGGCATTATTTGAC
qUBI3-F	TGTCCATCTGCTCTCTGTTG
qUBI3-R	CACCCCAAGCACAATAAGAC

图1 辣椒 CaWRKY30基因的PCR扩增

Fig.1 PCR amplification of pepper CaWRKY30 gene

图2 辣椒CaWRKY30的系统发育分析

Fig.2 Systematic development analysis of pepper CaWRKY30

图3 辣椒CaWRKY30的多序列比对分析

Fig.3 Multi-sequence comparison analysis of pepper CaWRKY30

图4 CaWRKY30亚细胞定位

Fig.4 CaWRKY30 subcellular positioning

图5 TSWV侵染下CaWRKY30的表达分析 A.CaWRKY30变化;B.TSWV侵染植物后的变化;使用SPSS 26.0软件采用Duncan's模型进行ANOVA统计学分析(P< 0.05)。

Fig.5 Expression analysis of CaWRKY30 under TSWV infection A.CaWRKY30 changes;B.Changes after TSWV infects plants;SPSS 26.0 software was used for ANOVA statistical analysis using Duncan's model(P<0.05).

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辣椒CaWRKY30转录因子的克隆、亚细胞定位及番茄斑萎病毒侵染下的表达分析

Cloning,Subcellular Positioning of Pepper CaWRKY30 Transcription Factor, and Its Expression Analysis by Tomato spotted wilt orthotospovirus Infected

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辣椒CaWRKY30转录因子的克隆、亚细胞定位及番茄斑萎病毒侵染下的表达分析

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