水稻金属硫蛋白基因(MT2bL)启动子的克隆与表达分析  

Cloning and Expression Analysis of MT2bL Promoter in Rice(Oryza sativa L.)

作　　者：李伟[1] 吴超[1] 吕永刚[2] 张小玲[1] 郭效琼 张慧鑫[1] 董陈文华 陈丽娟[1] 李东宣 

机构地区：[1]云南农业大学稻作研究所,昆明650201 [2]云南省农业科学院粮食与作物研究所,昆明650205 [3]省部共建云南生物资源保护与利用国家重点实验室,昆明650201

出　　处：《分子植物育种》2017年第10期3856-3869,共14页Molecular Plant Breeding

基　　金：“十三五”国家重点研发计划项目(2016YFD0101101-5);NSFC-云南联合基金重点项目(U11-36604);省部共建云南生物资源保护与利用国家重点实验室开放课题基金;国家自然科学基金(31560115)共同资助

摘　　要：金属硫蛋白(metallothionein,MT)富含半胱氨酸,而且能够结合多种金属离子,调控细胞内金属代谢的平衡。本研究根据水稻在减数分裂期、开花期和开花后5 d的基因芯片数据结果,从水稻品种黎榆B(O.sativa L.ssp.japonica C.V.Liyu B)基因组中克隆得到了一个植物MT-2类基因Metallothionein2b-Like(Os MT2b L)的启动子序列,序列全长2 063 bp。利用植物启动子区域顺式作用元件数据库(PLACE)分析表明该启动子序列含有5个CURE(copper-response element)元件(GTAC)以及多个其它顺式作用元件。构建了多个启动子融合表达载体(p Ca MV35S::GUS,p MT2b L::GUS,p Ubi1::GUS,p CYC::GUS和p ACT1::GUS),并通过根癌农杆菌介导转化水稻愈伤组织获得了转基因植株。GUS组织染色结果分析表明,Os MT2b L基因启动子在水稻幼苗期的胚根、胚芽和胚芽鞘中具有高水平的GUS表达活性,特别是在胚根根尖和胚芽顶端的GUS表达活性较高;在减数分裂期的植株叶片、颖壳和开花10 d后的未成熟种子中GUS表达活性也较高;GUS蛋白荧光定量分析结果表明,Os MT2b L基因启动子在水稻叶片中驱动GUS基因表达的活性比Ca MV35S基因启动子高出3倍以上。Metallothionein（MT） is rich in cysteine, and can combine a variety of metalions to regulate the balance of intracellular metal metabolism. In this study, we cloned Oryza sativa Metallothionein2 b-Like（Os MT2 b L）promoter sequence with the length of 2 063 bp based on microarray results at meiotic, flowering and 5 days after pollination（DAP） stages from Liyu B（O. sativa L. ssp. japonica C.V.）. We used plant promoter region cis-element database PLACE to analyze and found that these regulatory cis-elements include 5 copper-response elements（CURE） and other cis-elements. We constructed a number of promoter fusion expression vectors（p Ca MV35 S：：GUS, pMT2bL：：GUS, p Ubi1：：GUS, p CYC：：GUS and p ACT1：：GUS） and obtained transgenic plants by Agrobacterium tumefaciens-mediated transformation metho d. The results of GUS histochemical assays showed that Os MT2bL gene promoter had high level of GUS expression in radicle, embryo and coleoptile at rice seedling stage, especially in the tip of radicle and the top of embryo. The level of GUS expression was also high in the leaf, panicles, and immature seeds of 10 days after pollination at the meiosis stage. The results of GUS protein fluorescence quantitative analysis showed that the expression level of GUS gene drove by Os MT2bL gene promoter was three times higher than Ca MV35 S gene promoter in rice leaves.

关 键 词：水稻(Oryza SATIVA L.) OsMT2bL基因 启动子 GUS基因表达 

分 类 号：Q943.2[生物学—植物学] S511[农业科学—作物学]