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甜瓜属野生种Cucumis hystrix Chakr.胞质葡萄糖-6-磷酸脱氢酶基因的克隆与启动子结构分析

Cloning of cytoplasmic glucose-6-phosphate dehydrogenase gene and structure analysis of its promoter in Cucumis hystrix Chakr.
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摘要 根据植物葡萄糖-6-磷酸脱氢酶(G6PDH,EC1.1.1.49)氨基酸保守区域,设计简并引物,采用RT-PCR技术克隆得到甜瓜属野生种Cucumis hystrix Chakr.(2n=24)G6PDH基因cDNA片段;随后基于该序列设计特异引物,PCR方法筛选野生种BAC文库,获得2个BAC阳性单克隆。测序后获得了G6PDH基因全序列及其上游启动子序列,GenBank登录号:JQ771576。序列分析显示:G6PDH基因全长约6.5 kb,由15个外显子和14个内含子组成;内含子序列均符合5'-gt-ag-3'结构。外显子拼接后获得了G6PDH基因的开放阅读框(ORF)序列,序列全长1 551 bp,编码516个氨基酸,与黄瓜基因组网站公布的G6PDH基因核苷酸序列和氨基酸序列同源性分别为98.71%和99.03%。野生种G6PDH氨基酸序列N端缺少转运肽序列,确定为野生种胞质G6PDH。氨基酸序列与烟草、马铃薯、荷兰芹、猕猴桃、拟南芥、大豆、小麦、玉米、葡萄、杨树等植物胞质G6PDH同源性高达77%以上。系统发育树分析发现,甜瓜属胞质G6PDH与茄科植物最先聚类,植物胞质G6PDH在分子进化水平上与物种进化相符。启动子结构分析表明:序列含有启动子基本元件TATA-box、CAAT-box,还含有丰富的光响应元件,与环境胁迫响应相关的不同顺式作用元件,促进高水平转录的5UTR Py-rich stretch元件和提高表达的CAT-box元件等。 Based on the conserved sequences of glucose-6-phosphate dehydrogenase ( G6PDH ) amino acids of the other plants reported in GenBank, the degenerate primers were designed to isolate intermediate fragment of the G6PDH gene from Cucumis hystrix Chakr. (2n= 24), a wild species in Cucumis. According to the intermediate fragment of the C,6PDH gene we designed specific primers for PCR screening of BAC libraries of Cucumis hystrix Chakr. One of the two positive BAC clones obtained was sequenced. The sequence contained entire G6PDH gene, which was summited to GenBank, and the accession number was JQ771576. Sequence analysis indicated that the total length of G6PDH gene was 6.5 kb and consisted of 15 exons and 14 introns. The introns agreed with the structure 5'-gt-ag-3'. The ORF of G6PDH gene after splicing was 1 551 bp and encoded a protein containing 516 amino acid residues, The identities of nucleotides of ORF and amino acids sequence sharing with cucumber reported on the web of cucumber genome database were 98.71% and 99.03% ,respectively. We confirmed that the protein G6PDH was cytoplasmic because of the absence of N-end transferring peptide. Compared with Nicotiana benthamiana, Solanum tuberosum, Petroselinum crispum, Actinidia chinensis ,Arabidopsis thaliana, Glycine max, Triticum aestivum, Zea mays, Vitis vinifera, Populus suaveolens, et al, the cytoplasmic G6PDHs showed higher similarity than 77%. Phylogenetic analysis showed that cytoplasmic G6PDH clustered with Solanaceae family firstly and the molecular evolution of cytoplasmic G6PDH was corresponding to the evolution of species. The analysis of the promoter sequence indicated that the basic elements TATA-hox and CAAT-box of promoter were found. Moreover, there were abundant light responding elements,different elements involved in abiotic stress responses,two 5UTR Py-rich repeat elements increasing high tran- scription, and CAT-box activating higher expression.
出处 《南京农业大学学报》 CAS CSCD 北大核心 2013年第2期25-30,共6页 Journal of Nanjing Agricultural University
基金 国家自然科学基金重点项目(30830079) 国家自然科学基金项目(30972007 31071801) 国家重点基础研究发展计划项目(2009CB119001-01 2012CB113900) 国家863计划项目(2010AA10A108 2012AA100202) 江苏省科技支撑计划项目(BE2009310) 江苏省农业科技自主创新基金项目(CX(11)1002)
关键词 甜瓜属野生种 葡萄糖-6-磷酸脱氢酶基因 启动子 BAC文库 克隆 序列分析 Cucumis hystrix Chakr. G6PDH gene promoter BAC library clone sequence analysis
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