菠萝AcPIP1∶2基因的克隆和重组载体的构建

Cloning and Constructing Expression Vector of AcPIP 1∶2 Gene from Pineapple

构建菠萝(Ananas comosus(Linn.) Merr.)质膜水通道蛋白基因(AcPIP1∶2)原核表达载体和体外转录载体,为制备AcPIP1∶2抗体和含有Poly(A)+的AcPIP1∶2转录子奠定基础.以菠萝果实为试验材料,提取总RNA并反转录成cDNA.以cDNA为模板,根据GenBank中公布的AcPIP1∶2全长cDNA序列(登录号:XM020229679.1)设计合成特异性引物进行PCR扩增,获得AcPIP1∶2开放阅读框(ORF)序列.序列分析表明,获得的AcPIP1∶2序列与GenBank中公布的序列一致,序列长度为867 bp.该序列经Hind III和Xho I双酶切后亚克隆至原核表达载体pET32a(+),酶切、测序表明,成功构建了重组载体pET32a(+)-AcPIP1∶2.将pET32a(+)-AcPIP1∶2转到大肠杆菌表达菌株BL21(DE3)中表达,结果表明,异丙基硫代半乳糖苷(IPTG)诱导产生分子量为50 ku的融合蛋白.该序列经Hind III和Xba I双酶切后亚克隆至载体pSP64 Poly(A)中,酶切、测序表明,成功构建了重组载体pSP64 Poly(A)-AcPIP1∶2.

The study aims to construct prokaryotic expression vector and transcription vector in vitro of plasma membrane aquaporin protein gene(AcPIP1∶2) from pineapple(Ananas comosus(Linn.) Merr.),laying a foundation for the preparation of AcPIP1∶2 antibody and AcPIP1∶2 transcriptor containing Poly(A)+.Total RNA was extracted from pineapple fruit and the cDNA was synthesized by reverse transcription.Using cDNA as template,AcPIP1∶2 open reading frame(ORF) sequence was amplified by RT-PCR with specific primers designed and synthesized according to the full-length cDNA sequence of AcPIP1∶2 published in GenBank(Accession number:XM020229679.1).Sequence analysis showed that the obtained AcPIP1∶2 sequence was identical to the original sequence and its sequence length was 867 bp. AcPIP1∶2 was sub-cloned into the prokaryotic expression vector pET32 a(+) after the digestion with the enzyme Hind III and Xho I.The recombinant vectors pET32 a(+)-AcPIP1∶2 was successfully constructed,which was identified by endonuclease digestion and sequence analysis.pET32 a(+)-AcPIP1∶2 was transferred into the Escherichia coli expression strain BL21(DE3) for expression,and the results showed that IPTG induced the fusion protein with a molecular weight of 50 ku.AcPIP1∶2 was sub-cloned into vector pSP64 Poly(A) after the digestion with the enzyme Hind III and Xba I.The recombinant vectors pET32 a(+)-AcPIP1∶2 was successfully constructed, which was identified by endonuclease digestion and sequence analysis.