不结球白菜金属硫蛋白基因的克隆及表达分析  

Cloning and Expression Analysis of Metallothionein Gene in Non-heading Chinese Cabbage

作　　者：刘东让 侯喜林[2] 张昌伟[2] 肖栋 LIU Dongrang;HOU Xilin;ZHANG Changwei;XIAO Dong(Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops, Ministry of Agriculture, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China;State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China;Jiangsu Engineering and Technology Center for Modern Horticulture, Nanjing 210095, China)

机构地区：[1]中国农业科学院蔬菜花卉研究所农业部园艺作物生物学与种质创制重点实验室,北京100081 [2]南京农业大学作物遗传与种质创新国家重点实验室,南京210095 [3]江苏现代园艺工程技术中心,南京210095

出　　处：《西北植物学报》2018年第12期2207-2214,共8页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家重点研发计划(2016YFD0101701);农业部园艺作物生物学与种质创制重点实验室开放课题(IVF201804).

摘　　要：通过RACE技术,从不结球白菜抗病品种‘苏州青’叶片克隆到金属硫蛋白(metallothionein)基因的全长cDNA序列(BcMT2)。序列分析结果表明,BcMT2基因的cDNA全长为528bp,其中开放阅读框长度为243bp,共编码80个氨基酸,相对分子质量为8.02×10^(3 )Da,理论等电点是4.61。氨基酸同源系统进化分析表明,不结球白菜BcMT2基因属于Ⅱ类植物MT2基因家族,且与同科植物的进化关系相近,其中与大白菜第5号染色体的基因(Bra029765)相似性最高(100%)。qRT-PCR分析表明,BcMT2基因在不结球白菜叶中表达最强;霜霉病菌诱导后,BcMT2基因在抗病品种‘苏州青’中的表达量于48h达到峰值,而在感病品种‘矮脚黄’中的表达量于72h达到峰值;盐处理条件下,BcMT2基因在抗病品种‘苏州青’中的表达量于12h达到峰值,而在感病品种‘矮脚黄’中的表达量于24h达到峰值;ABA处理下,‘苏州青’中其表达量于24h达到峰值,且在‘矮脚黄’中BcMT2基因的表达趋势与‘苏州青’类似;干旱处理条件下BcMT2基因的表达在两材料中均受到抑制。BcMT2原核表达分析发现,在37℃、1.0mmol·L^(-1)IPTG诱导2、4、6h后,均检测到了一条蛋白分子质量约为8×10^(3 )Da的表达特异条带,这与预期融合蛋白的相对分子质量的理论值8.02×10^(3 )Da相符。研究表明,不结球白菜BcMT2基因在受到生物胁迫和非生物胁迫等逆境条件下均发挥着重要作用,且BcMT2在大肠杆菌中成功实现融合表达,为进一步进行蛋白水平和转基因功能研究奠定了基础,也为选育高产、优质的不结球白菜新品种提供重要的理论依据。The full-length cDNA sequence of metallothionein gene ( BcMT 2) was cloned from the leaves of non-heading Chinese cabbage disease resistant cultivar ‘Suzhouqing’ by race technique. qRT-PCR was used to analyze the expression pattern of this gene in different tissues under biotic stress ( P. parasitica ) and abiotic stress (salt, drought, ABA) treatment conditions. SDS-PAGE technology was used for analysis of prokaryotic expression characteristics for this gene. Sequence analysis showed that the full-length cDNA of BcMT 2 gene was 528 bp. The length of the open reading frame was 243 bp and encoded a total of 80 amino acids. The relative molecular mass was 8.02×10^3 Da, and the theoretical isoelectric point was 4.61 . The phylogenetic analysis of amino acid homology showed that the non-heading Chinese cabbage BcMT 2 gene was belonged to the MT 2 gene family of class Ⅱ plants and had similar evolutionary relationship with other plants of the same family. Among of them, the gene had the highest homology (100%), comparing with the gene ( Bra029765 ) on A05 in Chinese cabbage. Quantitative real-time analysis showed that the expression of BcMT 2 gene was the strongest in leaves of non-heading Chinese cabbage. The expression of BcMT 2 gene was peaked at 48 h after infection by Pseudoperonospora cubensis in the disease-resistant variety ‘Suzhouqing’, while the expression level was peaked at 72 h in the susceptible variety ‘Aijiaohuang’. Under salt treatment condition, the expression level of BcMT 2 gene was peaked at 12 h in the resistant variety ‘Suzhouqing’, while the expression level was peaked at 24 h in the susceptible variety ‘Aijiaohuang’. Under ABA treatment, the expression level was peaked at 24 h in ‘Suzhouqing’, and the expression tendency of BcMT 2 gene in ‘Aijiaohuang’ was similar to that of ‘Suzhouqing’. The expression of BcMT 2 gene was inhibited in both materials under drought treatment conditions. Analysis of BcMT 2 prokaryotic expression revealed that a specific molecula

关 键 词：不结球白菜 金属硫蛋白基因 QRT-PCR 原核表达 

分 类 号：Q785[生物学—分子生物学] Q786