中国水仙NtSAMT基因的克隆及转化金鱼藤  

Clone of the NtSAMT Gene in Narcissus tazetta L.Var. chinensis Roem and Transformation of Asarina procumbens

作　　者：郑清波[1] 赵明明[1] 郑鑫[1] 刘绪明[1] 樊金萍[1] 

机构地区：[1]东北农业大学园艺学院,哈尔滨150030

出　　处：《基因组学与应用生物学》2016年第2期420-428,共9页Genomics and Applied Biology

基　　金：中国黑龙江省省教育厅项目(12531014)资助

摘　　要：本研究以中国水仙花瓣总RNA为模板进行反转录PCR,扩增得到与预期大小相同的PCR产物带,回收反转录PCR产物,将其与p EASY-T1克隆载体连接并导入大肠杆菌DH5α进行克隆,经菌落PCR和双酶切鉴定,筛选带有目的基因的重组质粒并进行测序。测序结果表明中国水仙花香基因SAMT c DNA的ORF片段1 131 bp编码376个氨基酸残基,与已报导的中国水仙SAMT(JX273470)的同源性为99.2%。将目的基因片段与PBI121表达载体连接,并利用农杆菌介导法将Nt SAMT基因转化到金鱼藤(Asarina procumbens)中,以金鱼藤无菌苗茎段为遗传转化外植体,通过抗性筛选获得12株转化植株,对获得的12株疑似转基因植株进行PCR检测,其中有8株呈阳性;通过RT-PCR检测得到的阳性植株,其中有5株呈阳性。检测结果表明,该研究成功克隆水仙花Nt SAMT基因并在金鱼藤植物中得到表达。A novel cDNA was cloned by PT-PCR using the total RNA as template, and amplified to the desirable size. The RT-PCR products were reclaimed and transformed into E. coli DH5a together with the pEASY-T1 vector. Identified by colony PCR and digestion of BamH I and EcoR V, the recombinant plasmid with target gene was screened out and conducted the sequence analysis. Results of the sequence analysis showed that the ORF fragment of the SAMT cDNA was successfully cloned form Narcissus tazetta L. var. chinensis roem gene, with the length of 1 131 bp and encoded 376 amino acids fragment which shared 99.2% homology to that of previously reported SAMT cDNA from Narcissus tazetta L. var. chinensis roem （JX273470）. Connecting the purpose gene fragments and PBI121 expression vector, and using the method of agrobacterium mediated NtSAMT genes into A sarina procumbens, with sections of stems for genetic transformation receptor, twelve putative transgenic plants were obtained, and 8 were identified as positive transgenic plants by PCR assay; and 5 positive plants were obtained by RT-PCR test. This study successfully cloned the SAMT gene of Narcissus tazetta L. var. chinensis roem and expressed in A sarina procumbens.

关 键 词：NtSAMT 基因克隆 金鱼藤 表达载体构建 转化 

分 类 号：S682.21[农业科学—观赏园艺]