香蕉果实SMART cDNA文库的构建及利用PCR方法筛选香蕉Actin2基因

The Construction of SMART cDNA Library from Banana Fruit and the Screening of Actin2 Gene by Using PCR Method

利用SMART(switching mechanismat5’end of RNA transcript)技术,提取果实少量总RNA,经15-25轮LD-PCR扩增获得全长ds-cDNA,构建了海南主栽的食用香蕉巴西蕉(Musa AAA Group Cavendish)果实的cDNA文库。所构建的文库容量为5×106Pfuml-1,重组率93%。利用此cDNA文库,采用96孔板PCR法筛选香蕉Actin2基因,测序结果显示,序列全长1723bp,编码区长1134bp,编码378个氨基酸,与蝴蝶兰Actin2基因序列同源率达83%,已递交GenBank,接受号692696。

In this paper, SMART （switching mechanism at 5＇ end of RNA transcript）was used in the construction of cDNA library of postharvested banana fruit. Total RNA was isolated from banana fruit after harvested for 2 days and cDNA was synthesized by using 15-25 round LD-PCR （Long-distance PCR）. Full length of amplified ds-cDNA （Double-strained complement DNA） was obtained and used in the construction of cDNA library afterward. The capacity of the library was measured to be 5×10^6 Pfu ml^-1, with 93% recombinant. A cDNA was amplified by using PCR with this library diluted in 96-well-plate as templates. The sequence of this cDNA shows 83% similarity to A ctin2 gene in the NCBI database （Accession No. 692696）.