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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。
In this study, total RNA of Chinese narcissus petals was used as a template for reverse transcription PCR. PCR products with the same size as expected were amplified and reverse transcribed PCR products were recovered. The products were ligated into pEASY-T1 cloning vector and introduced into E. coli DH5α After cloning, identified by colony PCR and double digestion, the recombinant plasmid with the target gene was screened and sequenced. The sequencing results showed that 1 131 bp of the ORF fragment of Chinese narcissus fragrant gene encoded 376 amino acid residues, which was 99.2% homologous with the reported Chinese narcissus SAMT (JX273470). The target gene fragment was linked to the PBI121 expression vector and the Nt SAMT gene was transformed into Asarina procumbens by Agrobacterium tumefaciens-mediated transformation. Twelve transformed plants were obtained. The 12 suspected transgenic plants were tested by PCR, of which 8 were positive. Positive plants were detected by RT-PCR, of which 5 were positive. The results showed that the study successfully cloned Nt SAMT Narcissus gene and expressed in the goldfish vine plants.