石榴花色苷相关基因(PgANS、PgCHS、PgACT)的克隆与序列分析

Cloning and Sequence Analysis of Anthocyanin Biosynthesis Related Genes(PgANS,PgCHS and PgACT) in Pomegranate(Punica granatum L.)

【目的】获得石榴果实PgANS、PgCHS和PgACT基因,并对这三个基因进行序列分析。【方法】以喀什甜石榴为试材,通过酚-氯仿法提取籽粒中的总RNA,用TIANScript cDNA试剂盒反转录合成第一链cDNA,并用特异引物对其RT-PCR扩增,将得到的cDNA片段连接到PMD19-T载体上,然后将该载体转到DH5α,并对阳性克隆进行序列分析。【结果】PgANS基因片段长度为474bp,Genbank登录号为KT779433,与NCBI数据库中KF841619、GU376749两种石榴核苷酸的同源性均为99%,与甘薯核苷酸(GU59821)、白桑核苷酸(JF499385)、草莓核苷酸(AY695817)的同源性分别为85%、79%、76%;PgCHS和PgACT基因片段长度分别为140bp和148bp,分别与NCBI数据库中JF747148、GU376750两种石榴核苷酸同源性为99%和93%。【结论】获得PgANS、PgCHS和PgACT基因片段,为进一步研究贮藏条件对石榴籽粒花色苷的影响奠定了基础。

[ Objective] To obtain PgANS, PgCHS and PgACT genes in Kashgar pomegranate kernels and analyze their sequence. [ Method ] Xinjiang pomegranate in Kashgar was selected as the materials. The total RNA of pomegranate Kernels was extracted with Phenol - chloroform. The first cDNA was synthesized with TIANScript kit,and amplified to cDNA by RT -PCR using special primer. The pMD19 -T vector linked with the eDNA fragment was transformed into Escherichia coli DH5α, and the positive clone was sequenced. [ Re- suit] The fragment of PgANS was 474bp, GenBank accession number was KT779433. Compared with Punica granatum L. which the accession numbers were KF841619, GU376749 on NCBI, the PgANS homology of nu- cleotide sequence was 99%. At the same time, the sequence analysis demonstrated that the deduced nucleo- tide sequence shared 85% , 79% and 76% homology with that of Ipomoea batatas （GU598212）, Morus alba （JF499385） and Fragariax ananassa （AY695817）; the fragment of PgCHS was 140bp and the PgACT was 148bp. Compared with Punica granatum L. （JF747148, GU376750） on NCBI, the PgCHS homology of nu- cleotide sequence was 99%, and the PgACT was 93%. [ Conclusion]The PgANS, PgCHS and PgACT genes have been obtained, which has laid the foundation for further studying the influence of storage conditions on the anthocyanins of Punica granatum L. kernels.