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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 binds to multiple metal ions to regulate the balance of intracellular metal metabolism. In this study, a plant MT was cloned from the genome of rice cultivar “O.sativa L.ssp. Japonica CVLiyu B” according to the data of the gene chip at the stage of meiosis, flowering and 5 days after flowering. -2 gene of Metallothionein2b-Like (Os MT2b L) promoter sequence, the full length of 2 063 bp. Analysis of plant promoter promoter cis-acting element database (PLACE) indicated that the promoter sequence contained five CURE (GTAC) elements as well as many other cis-acting elements. A number of promoter fusion expression vectors (p CaMV35S :: GUS, pMT2b L :: GUS, pUbi1 :: GUS, pCYC :: GUS and pACT1 :: GUS) were constructed and introduced into Agrobacterium tumefaciens Transformed rice callus obtained transgenic plants. The results of GUS staining showed that the promoter of Os MT2b L gene had high GUS expression activity in radicle, germ and coleoptile at rice seedling stage, especially at the apical and embryo tips GUS expression activity was also higher in plant leaves, glume shells and immature seeds after 10 days of flowering at the meiosis stage. Fluorescence quantitative analysis of GUS protein showed that the promoter of Os MT2b L gene drives GUS gene in rice leaves The expression activity is more than 3 times higher than that of CaMV35S gene promoter.