棉花S-腺苷甲硫氨酸合成酶基因的克隆及表达分析

S-adenosylmethionine Synthetase Gene Coloning and Its Expression Analysis on Gossypium hirsutum L.

利用i TRAQ技术从陆地棉(Gossypium hirsutum L.)HM-40中筛选并克隆到S-腺苷甲硫氨酸合成酶Gh SAMS基因。Gh SAMS基因的开放阅读框全长为1 182 bp,编码393个氨基酸,预测Gh SAMS蛋白质含有15个磷酸化位点,推测其功能的行使可能与激酶磷酸化相关,进化分析表明S-腺苷甲硫氨酸合成酶与茶树(Camellia sinensis)的SAMS蛋白质相似性最高。q RT-PCR分析表明,在接种黄萎病菌VD07菌系后,Gh SAMS表达量逐渐增加,随着接种时间的延长,其相对表达量随之增加,推测其在陆地棉抗黄萎病防卫反应中可能起重要作用。在嫁接棉株中,利用VIGS技术成功地沉默Gh SAMS基因,然后对沉默棉株接种黄萎病菌VD07,鉴定其病情指数为62.5,抗性级别为感病;而转化空载体和未接种载体处理的嫁接棉株病情指数分别为30.0和31.2,抗性级别为耐病,表明Gh SAMS基因沉默后嫁接棉对黄萎病的抗性丧失。推测Gh SAMS基因在陆地棉抗黄萎病的过程中可能起重要作用。

The Gh SAMS gene was cloned from cotton（Gossypium hirsutum L.） cultivar HM-40, which was selected by i TRAQ of sea-land grafting cotton. The Gh SAMS open reading frame（ORF） was 1 182 bp and encoded a protein of 393 amino acids.Many Phosphorylation sites were found in Gh SAMS protein, which suggested that the function of Gh SAMS is related to phosphorylation of these sites. The cloned gene was most closely related to Camellia sinensis CHS protein by phylogenetic analysis. Real-time quantitative reverse transcription polymerase chain reaction showed that Gh SAMS gene was rapidly activated after inoculation with Verticillium dahliae. When time prolonged,the expression of related genes always inc reased. It was assumed that the Gh SAMS gene might play an important role in the defense reaction against Verticillium wilt. Virus induced gene silencing was used to silence endogenous genes in resistant upland cotton by sea-land grafting cotton targeting a fragment of Gh SAMS. VD07 was used to inoculate silenced cotton plants to identify disease resistance. The results showed that the disease indices of wild type vector control and sea-land grafting cotton plants were 30.0 and 31.2,respectively. The disease index of silenced Gh SAMS plants was 62.5. These results confirmed that the Gh SAMS gene might relate to Verticillium wilt resistance in cotton.