酿酒酵母耐铜基因CUP1的克隆及其植物表达载体的构建

Cloning of yeast CUP1 gene and construction its plant expression vector

酿酒酵母铜金属硫蛋白 ( copper metallothionein,Cu MT) ,属于第 II型金属硫蛋白 ,Cu MT由酿酒酵母耐铜基因 CUP1编码 ,由 61个氨基酸残基组成 ,其中半胱氨酸残基 1 3个 ,占 Cu MT总氨基酸数的 2 1 .3%。 Cu MT与铜离子有较强的结合力 ,在酵母细胞中主要参与过量铜的解毒作用。本研究通过PCR方法 ,从酵母基因组 DNA中扩增出了 CUP1基因 ,克隆于 p UC1 9的多克隆位点。扩增片段长 4 86bp,包括 ORF及其部分下游序列。所得克隆与发表序列的 ORF一致 ,推导出的氨基酸序列与发表序列完全一样。用于植物转 CUP1基因用的表达载体已构建完毕 ,分别命名为 p BI CUP1和 p CM CUP1。对植物 (如苎麻、水稻、水生植物等 )

The baker yeast copper metallothionein (CuMT), the class II metallothionein, consists of 61 amino acid residues, including 13 cysteine residues. CuMT was encoded by yeast CUP1 gene. Possessing special affinity to copper ions, CuMT plays a key role in copper detoxification in yeast cells. In this study, the CUP1 gene was amplified from baker yeast genomic DNA by PCR and cloned into the MCS of pUC19. The amplified fragment was 486 bp, including ORF of CUP1 and part of its downstream sequence. DNA sequence analysis indicated that the ORF of the amplified fragment was same as the published ORF of CUP1 and the amino acid sequence of the putative polypeptide was the same as that of CUP1 .The plant expression vectors, named pBICUP1 and pCMCUP1 respectively, have been constructed for transformation of rice, ramie and hydrophytes.