枳抗逆基因PtrZPT2-3克隆与表达分析

Cloning and expression analysis of PtrZPT2-3 from trifoliate orange(Poncirus trifoliata)

【目的】分离和克隆枳双锌指蛋白基因PtrZPT2-3(GenBank登录号为KF279693),了解其在枳响应环境胁迫和逆境相关激素诱导的表达情况。【方法】以枳[Poncirus trifoliata(L.)Raf]实生苗为试验材料,对植株进行低温、干旱、高盐、ABA处理,使用电子克隆和RT-PCR的方法从枳中克隆到一个新的C2H2型锌指蛋白基因,命名为PtrZPT2-3,并研究该基因在4种胁迫处理下的表达模式。【结果】PtrZPT2-3的cDNA序列长930 bp,含有534 bp的开放阅读框(ORF),编码178个氨基酸残基的蛋白,蛋白推测分子质量为19.54 kDa,蛋白的等电点是8.91。PtrZPT2-3包含2个C2H2型锌指结构域,在其C-末端含有一个转录抑制结构域(DLN)。系统发生分析结果表明,PtrZPT2-3与植物中已经发现的双锌指蛋白ZPT2-14、JrZFP2、PtaZFP2、ZPT2-12、ZPT2-13亲缘关系较近。半定量表达分析结果表明,枳PtrZPT2-3基因在干旱和ABA胁迫下的根、茎、叶中表达上调。【结论】枳PtrZPT2-3是C2H2型锌指蛋白家族的新成员,可能在柑橘响应干旱和ABA胁迫的过程中起了重要的作用。

[Objective]The aim of this study was to isolate a novel ZPT2-related protein gene from trifoliate orange（PtrZPT2-3, GenBank accession KF279693 ） and analyze its expression patterns in different tissues and in response to environmental stresses and stress-related hormones. [Method]Under low temperature, drought, high salt, plant ABA treatment,PtrZPT2-3 was cloned from trifoliate orange by in silico and RT-PCR approach,and the gene expression patterns under stress was studied. Using in silico and RT- PCR approach, PtrZPT2-3 was cloned from trifoliate orange. The PtrZPT2-3 expression patterns under cold, salt, drought and ABA stresses in leaves, stems and roots of trifoliate orange seedlings were detect- ed by semi-quantitative RT-PCR. [Result]The PtrZPT2-3 cDNA is 930bp in length with a 534 bp ORF（open reading frame）, encoding a deduced protein of 178 amino acids with a predicted molecular mass of 19.54 kDa and an isoelectric point of 8.91. The PtrZPT2-3 protein contains two C2H2-type zinc finger domains, a B-box at its N-terminus functioning as a putative nuclear localization signal （NLS）, a putative transcription repression domain （DLN-box） at the C-terminus. Phylogenetic tree showed that PtrZPT2-2 was clustered closely with ZPT2-14, JrZFP2, PtaZFP2, ZPT2 - 12, ZPT2 - 13. [ Conclusion ] Our data suggest that PtrZPT2-3 is a new member of the ZPT2-related protein genes and may play an important role in citrus response to drought and ABA stresses.