番茄AP2a和MYB12转录因子的克隆及CYRSV侵染下的表达分析

Cloning of tomato SlAP2a and SlMYB12 transcription factor genes, and their expression analysis infected by chilli yellow ringspot virus（CYRSV）

关键词:  番茄，AP2a，MYB12，辣椒黄化环斑病毒，基因克隆，表达模式

Key words:  tomato, AP2a, MYB12, chilli yellow ringspot virus（CYRSV）, gene clone, expression pattern

摘要:

番茄SlAP2a和SlMYB12是重要的转录因子，但其在辣椒黄化环斑病毒（chilli yellow ringspot orthotospovirus, CYRSV）侵染下的表达模式还未见研究报道。为探究SlAP2a和SlMYB12的功能及其在CYRSV侵染下的表达响应模式，该研究以番茄品种‘靓思’为试验材料，通过RNA提取、逆转录、克隆等试验获得SlAP2a和SlMYB12编码序列，并采用生物信息学方法分析SlAP2a和SlMYB12蛋白的功能结构域及理化性质，同时分析蛋白的亚细胞定位，再运用实时荧光定量PCR检测CYRSV侵染后SlAP2a和SlMYB12在叶片中的表达情况。结果表明：（1）SlAP2a全长1 206 bp，编码401个氨基酸，SlMYB12全长1 017 bp，编码338个氨基酸。番茄AP2a与咖啡AP2a核苷酸序列的亲缘关系最近，而番茄MYB12与马铃薯MYB12核苷酸序列的亲缘关系最近。（2）亚细胞定位实验发现，SlAP2a和SlMYB12蛋白定位于细胞核和细胞膜。（3）SlAP2a和SlMYB12的表达受CYRSV接种后诱导，在接种病毒后4、7、9 d，SlAP2a和SlMYB12的表达量上调，而在14 d时表达量降低，但仍高于对照组。该研究成功克隆了转录因子SlAP2a和SlMYB12基因，并初步阐明了番茄SlAP2a和SlMYB12转录因子在CYRSV侵染下的表达模式。

Abstract:

TThe tomato transcription factors SlAP2a and SlMYB12 are considered to play siginificant roles in regulatory processes; however, their expression patterns in response to chilli yellow ringspot virus (CYRSV) infection have not yet been documented. To investigate the functional roles of SlAP2a and SlMYB12 and their expression response patterns during CYRSV infection, this study utilized the ‘Liangsi’ tomato as the experimental material. The coding sequences of SlAP2a and SlMYB12 were obtained through experimental procedures including RNA extraction, reverse transcription, and cloning. The functional domains and physicochemical characteristics of the SlAP2a and SlMYB12 proteins were analyzed using bioinformatics approaches, and their subcellular localization was also examined. Real-time fluorescence quantitative PCR was employed to quantify the expression levels of SlAP2a and SlMYB12 in leaves following CYRSV infection. The results were as follows: (1) The full-length SlAP2a sequence was 1 206 bp, encoding 401 amino acids, whereas the full-length SlMYB12 was 1 017 bp, encoding 338 amino acids. Phylogenetic analysis of tomato SlAP2a indicated a close evolutionary relationship with the coffee AP2a gene. Similarly, phylogenetic analysis of tomato SlMYB12 revealed relative close relationship with the potato MYB12 gene. (2) Subcellular localization analysis revealed that the SlAP2a and SlMYB12 proteins were localized in the nucleus and cell membrane. (3) The expression levels of SlAP2a and SlMYB12 were up-regulated at 4, 7, and 9 days post-inoculation (dpi), and its expression level decreased at 14 dpi, although it remained significantly higher than that in the control (CK) group. In summary, the transcription factor genes SlAP2a and SlMYB12 have been successfully cloned, and this study preliminarily elucidates the expression patterns of the tomato SlAP2a and SlMYB12 transcription factors during CYRSV infection.