嫁接陆地棉查尔酮合成酶与查尔酮异构基因的克隆及r表达分析

摘要

Two new genes,GhCHS and GhCHI,were cloned from cotton (Gossypium hirsutum L.) cultivar HM-40,which were selected by iTRAQ of sea-land grafting cotton. The GhCHS open reading frame(ORF) was 1170 bp and encoded a protein of 389 amino acids. Many phosphorylation sites were found in GhCHS protein, which suggested that the function of GhCHS is related to phosphorylation of these sites. The cloned gene was most closely related to Theobroma cacao CHS protein by phylo-genetic analysis. The GhCHI open reading frame was 630 bp and encoded a protein of 209 amino acids. Many Phosphoryla-tion sites were found in GhCHI protein,which suggested that the function of GhCHI is related to phosphorylation of these sites. The cloned gene was most closely related to Hibiscus cannabinus CHI protein by phylogenetic analysis. Real-time quan-titative reverse transcription polymerase chain reaction showed that the two genes were rapidly activated after inoculation with Verticillium dahliae. When time prolonged,genes relative expression always increased. It was assumed that GhCHS and GhCHI might play an important role in the defense reaction against Verticillium wilt. Virus induced gene silencing was used to si-lence endogenous genes in resistant upland cotton by sea-land grafting cotton targeting a fragment of GhCHS or GhCHI. 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 GhCHS plants was 72.5,GhCHI plants was 67.5. These results confirmed that the GhCHS gene and GhCHI might relate to Verticillium wilt resistance in cotton.%从陆地棉(Gossypium hirsutum L.)HM-40中克隆了查尔酮合成酶GhCHS和查尔酮异构酶GhCHI基因.GhCHS基因的开放阅读框全长为1170 bp,编码389个氨基酸,查尔酮合成酶含有较多的磷酸化位点,推测激酶磷酸化可能与其功能的行使相关,进化分析表明查尔酮合成酶与可可(Theobroma cacao)的CHS蛋白相似性最高;GhCHI基因的开放阅读框全长为630 bp,编码209个氨基酸,查尔酮异构酶含有较多的磷酸化位点,推测激酶磷酸化可能与其功能的行使相关,进化分析表明查尔酮异构酶与红麻(Hibiscus cannabinus)的CHI蛋白相似性最高.qRT-PCR分析显示,在接种大丽轮枝菌VD07菌系后,这两个基因表达量逐渐增加,随着接菌处理时间的延长,相对表达量增加,推测它们在陆地棉抗黄萎病防卫反应中可能起重要作用.利用VIGS技术在嫁接棉中成功地沉默GhCHS基因和GhCHI基因,对沉默棉株接种大丽轮枝菌VD07,鉴定显示其病情指数分别为72.5和67.5,表现为感病;而转化空载体和未沉默的嫁接棉棉株的病情指数分别为30.0和31.2,表现为耐病,基因沉默后嫁接棉对黄萎病的抗性丧失.表明GhCHS基因和GhCHI基因在陆地棉抗黄萎病的过程中可能起重要作用.