转杜仲几丁质酶基因EuCHIT1烟草对真菌抗性增强的研究

Study on Transformed EuCHIT1 Gene in Tobacco Enhancing Resistance to Fungus

本研究从杜仲中分离并克隆了杜仲几丁质酶基因EuCHIT1,全长为1402 bp,包括序列长度为969 bp的开放阅读框,编码322个氨基酸。为验证EuCHIT1的抗菌能力,构建植物过表达载体pSH-35S-EuCHIT1后对烟草进行遗传转化获得转基因植株并开展了相关实验。结果表明,转基因烟草的几丁质酶含量显著高于野生型烟草,且添加了烟草粗蛋白EuCHIT1的培养基对尖孢镰刀菌的生长有着明显的抑制作用,同时,EuCHIT1基因显著增加了烟草叶片中过氧化氢酶(CAT)的活性。此外,为转EuCHIT1基因烟草对真菌病害的抗性机理进行研究,分析了转基因植株和野生型中水杨酸途径的关键基因PR-1a,与茉莉酸途径的关键基因COI1在接种灰霉菌之后的表达量。结果表明,转基因植株的COI1与PR-1a在接种病原菌前的表达量均显著高于野生型植株。但接种病原菌后,COI1的表达受到抑制,而PR-1a基因在接种病原菌后的最大表达量是野生型烟草的33.8倍。说明EuCHIT1基因的过表达增强了烟草对真菌的抗性,提高烟草的CAT活性及植株中病程相关蛋白基因的表达,从而增强植株对灰霉菌的抑制作用,提高植株的抗真菌病害能力。

In this study,a chitinase gene EuCHIT1 from Eucommia ulmoides with a total length of 1402 bp,including an open reading frame with a sequence length of 969 bp,encoding 322 amino acids.In order to verify the antifungal ability of EuCHIT1,constructed plant overexpression vector pSH-35S-EuCHIT1 was constructed and genetically transformed tobacco to obtain transgenic plants and related experiments were carried out.The results showed that the content of chitinase in transgenic tobacco was significantly higher than that in wild-type tobacco,and the medium supplemented with tobacco crude protein EuCHIT1 had a significant inhibitory effect on the growth of Fusarium oxysporum.EuCHIT1 gene significantly increased the activity of catalase(CAT)in tobacco leaves.In addition,in order to explore the resistance mechanism of EuCHIT1-transgenic tobacco against fungi mold,the expression levels of PR-1a and COI1 were analysed,the key genes in the salicylic acid pathway and jasmonic acid pathway,in transgenic plants and wild types after inoculation with Botrytis cinerea.The results showed that the expression levels of COI1 and PR-1a in transgenic plants were significantly higher than those in wild type plants before inoculation with pathogens.However,the expression of COI1 was inhibited after inoculation with pathogens,and the maximum expression of PR-1a gene in transgenic tobacco after inoculation with pathogens was 33.8 times that of wild-type tobacco.In conclusion,the overexpression of EuCHIT1 gene enhanced the resistance of tobacco to fungi,increased the activity of CAT and the expression of protein genes related to disease course in tobacco plants,and thus enhanced the inhibition effect of plants on Botrytis cinerea and the ability of plants to resist fungus diseases.