甘蓝型油菜蜡质相关基因的克隆与表达分析  

Cloning and Expression Analysis of Waxy-related Genes in Brassica napus

作　　者：周燕 黄小虎[1,2] 许代香 贾乐东 王瑞 李加纳[1,2] 曲存民[1,2] 

机构地区：[1]西南大学农学与生物科技学院/重庆市油菜工程技术研究中心,重庆400716 [2]西南大学农业科学研究院,重庆400716

出　　处：《农业生物技术学报》2017年第12期1918-1929,共12页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展计划(973)项目(No.2015CB150201);国家自然科学基金项目(No.31401412;No.U1302266和No.31571701);国家科技支撑计划项目(No.2013BAD01B03-12);中央高校基本科研业务费专项基金(No.XDJK2016B030);西南地区强优势杂种优势利用项目(No.2016YFD0101300)

摘　　要：植物叶片中,表皮蜡质对植物的生长发育和适应外界环境起着重要作用。本研究针对拟南芥(Arabidopsis thaliana)已发表的蜡质相关基因信息,基于生物信息学的方法获得以甘蓝型油菜(Brassica napus)品种中双11号为参考的10个蜡质相关基因的44个拷贝序列。然后,以在抽薹期前后叶片蜡质差异明显的3对自然突变的甘蓝型油菜(L1050,P144和W481)为材料,利用同源克隆的方法获得这些拷贝成员在3对甘蓝型油菜中的核苷酸和蛋白序列,通过多重比对分析发现,甘蓝型油菜蜡质基因(wax inducer 2,WIN2)家族中的Bn WIN2C01在核苷酸与氨基酸水平上均存在稳定的突变位点。利用q RT-PCR分析基因Bn WIN2C01在甘蓝型油菜突变体材料抽薹期前后叶片中的表达情况,结果表明该基因在表皮蜡质合成前后的叶片中达到极显著差异水平(P<0.01),说明Bn WIN2C01基因的特异性表达可能影响了叶片蜡质的合成。本研究将有助于阐明叶表皮蜡质形成过程的分子机理,为提高油菜抗逆性提供一定的参考依据。In plants, epicuticular wax plays important roles in plant growth and development, and it is indispensable for plants to adaptation to the external environment. Based on the known protein sequences of wax genes in Arabidopsis thaliana, 44 gene copies for 10 wax inducer 2（WIN2） gene families were identified and analyzed in the genome reference of rapeseed（Brassica napus） ZS11 by the bioinformatics methods. In this study, the c DNA were isolated from 3 oilseed（L1050, P144 and W481） with the differences on epicuticular waxy of leaves before bolting（BB） and after bolting（AB）, respectively. Then they were thoroughly cloned by the specific primers from them using the homology-based cloning strategy. However,homology comparison revealed that the nucleotide and protein had high sequence homology among them, butthe SNPs and amino acid mutations were stably detected in the sequences of Bn WIN2 C01. Meanwhile, the expression levels of Bn WIN2 C01 were detected using the q RT-PCR among the leaves before bolting（BB） and after bolting（AB） in different B. napus. The results showed that the highly differences were found among them, indicating that the specific expression of Bn WIN2 C01 might be involved in the biosynthesis of epicuticular waxy. Results will be helpful for deciphering the molecular mechanism of epicuticular waxy in B.napus, and provide a reference to improve the rapeseed stress resistance.

关 键 词：甘蓝型油菜 蜡质 蜡质基因(WIN2) 生物信息学分析 同源克隆 

分 类 号：S512.1[农业科学—作物学]