菠萝质膜水通道蛋白基因AcPIP2-4的克隆、重组载体构建

Gene Cloning and Recombinant Vector Construction of AcPIP2-4 in Ananas comosus(L.) Merrill Plasmalemma Aquaporin

本研究以菠萝(Ananas comosus (L.) Merrill)果实为材料提取总RNA并合成cDNA.根据菠萝质膜水通道蛋白基因(AcPIP2-4)序列(GenBank登录号:XM020240565.1)设计1对特异性引物,通过RT-PCR从果实cDNA中扩增AcPIP2-4的ORF序列,并将该序列亚克隆至原核表达载体pET32a(+).经酶切鉴定和序列分析,结果表明,原核表达重组载体pET32a(+)-AcPIP2-4构建成功.以重组载体pET32a(+)-AcPIP2-4质粒为模板,通过RT-PCR扩增AcPIP2-4的CDS序列,再将其亚克隆至载体pSP64 Poly(A)上.经酶切鉴定及序列分析,结果表明,重组载体pSP64 Poly(A)-AcPIP2-4构建成功.重组载体pET32a(+)-AcPIP2-4和pSP64 Poly(A)-AcPIP2-4的成功构建,为后续制备抗AcPIP2-4的抗体和AcPIP2-4 c的RNA,进而进行AcPIP2-4的组织表达和功能研究奠定基础.

Total RNA was extracted and synthesized into cDNA from pineapple(Ananas comosus(L.)Merrill)fruit.With a pair of specific primers,which were designed according to the full-length cDNA sequence of plasmalemma aquaporin gene AcPIP2-4 published in GenBank(Accession number:XM020240565.1),the open reading frame(ORF)sequence of AcPIP2-4 was amplified from fruit cDNA by RT-PCR. This sequence was subcloned into the prokaryotic expression vector pET32 a(+).The recombinant vector pET32 a(+)-AcPIP2-4 was successfully constructed by enzyme digestion identification and sequence analysis. Using the recombinant vector pET32 a(+)-AcPIP2-4 plasmid as template,the coding region sequence(CDS)of AcPIP2-4 was amplified by RT-PCR,and then cloned to the vector pSP64 Poly(A). The recombinant vector pSP64 Poly(A)-AcPIP2-4 was successfully constructed by enzyme digestion identification and sequence analysis. The successful construction of the recombinant vectors pET32 a(+)-AcPIP2-4 and p SP64 Poly(A)-AcPIP2-4 laid a foundation for the subsequent preparation of anti-AcPIP2-4 antibodies and cRNA,to study the tissue expression and function of AcPIP2-4.