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

doi:10.7668/hbnxb.20192715

Abstract

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

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.

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Figures/Tables 6

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

Fig.1 PCR amplification of pepper CaWRKY30 gene

Fig.2 Systematic development analysis of pepper CaWRKY30

Fig.3 Multi-sequence comparison analysis of pepper CaWRKY30

Fig.4 CaWRKY30 subcellular positioning

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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