糙皮侧耳Pleurotus ostreatus子实体凝集素基因polectin2及其启动子的克隆及功能鉴定  

Cloning and function analysis of fruiting body lectin gene from Pleurotus ostreatus

作　　者：苏文英[1] 盛立柱 代月婷 宋冰[1] 付永平[1] 李玉[1] 

机构地区：[1]吉林农业大学农学院食药用菌教育部工程中心,吉林长春130118 [2]龙泉市食药用菌产业办公室,浙江龙泉323700

出　　处：《菌物学报》2017年第8期1111-1120,共10页Mycosystema

基　　金：公益性行业(农业)科研专项(201503137);973项目(2014CB138305);吉林省秸秆综合利用技术创新平台(吉高平合字[2014]B-1);吉林农业大学校内启动基金(2015007);吉林省科技发展项目(20140101148JC)~~

摘　　要：凝集素在食用菌的生长发育、抗病虫等逆境及抗肿瘤等方面发挥着重要的作用,但是关于糙皮侧耳凝集素基因的抗逆功能研究还比较薄弱。因此,本文利用PCR技术克隆得到糙皮侧耳子实体凝集素polectin2基因及其启动子序列,测序和生物信息学结果显示该基因不含内含子,完整开放阅读框为408bp,编码136个氨基酸,预测蛋白分子量15.3k Da;启动子polectinpro序列长度为927bp,生物数据库PLACE分析结果表明polectinpro含有低温、干旱胁迫诱导和病程相关等抗逆元件。然后,构建含有GFP融合蛋白的重组植物表达载体pCAMBIA1302-polectinpro-gfp,并进行烟草遗传转化,结果证明该启动子能够在低温诱导下驱动gfp基因表达。同时成功构建了含有polectin2的重组植物表达载体pCAMBIA1301-polectin2并进行了烟草遗传转化;对阳性转化植株T1代和野生型种子进行低温胁迫实验,结果表明转基因种子萌发率明显高于野生型。综上所述,推测polectin2基因具有耐低温功能,且其启动子具有低温诱导活性。上述结果为进一步利用子实体凝集素polectin2基因提高食用菌的耐低温等抗逆功能提供了理论依据和技术支持。The fungal lectins have many biological functions, such as regulating the growth and development, stress tolerance,antitumor, etc. Yet, the functions of lectin genes in response to various biotic and abiotic stress in Pleurotus ostreatus are poorly understood. In this study, cloning and functional identification of polectin2 gene and its promoter polectinpro from the fruiting body of P. ostreatus are carried out. The sequencing and bioinformatics analysis indicate that the promoter and cDNA of polectin2 are 927bp and 408bp respectively, encoding 136 amino acids without intron. The molecular weight of lectin2 is 15.3kDa using the prediction by PLACE. The results obtained by PLACE database analysis indicated that the nucleotide sequence of polectinpro contained multi regulatory elements, such as regulating the functional genes responding to low temperature, drought and pathogens. GFP fusion expression vector of pCAMBIA1302-polectinpro-gfp and the recombinant expression vector of pCAMBIA1301-lectin2 were constructed, and transformation to tobacco was conducted. The results showed that the gfp gene was fusion-expressed in the transgenic plants, indicating this promoter could induce by cold stress. It was revealed that To and T1 transgenic plants contained the exogenous gene polectin2, and the germination rate of T1 transgenic seeds under cold stress was higher than that of the wild type of tobacco. The results suggest the lectin2 gene from the fruiting body of P. ostreetus could resist cold stress. Our study will provide the new gene resource for further breeding edible mushroom against abiotic stress.

关 键 词：糙皮侧耳 凝集素基因 低温 功能验证 转化植株 

分 类 号：S646[农业科学—蔬菜学]