盐地碱蓬(Suaeda salsa)中SsINPS基因的分子克隆及表达特性分析

摘要

肌醇-1-磷酸 (I-1-P)合成酶 (EC5.5.1.4,INPS)是肌醇生物合成中的关键酶,催化葡萄糖-6-磷酸(G-6-P)到I-1-P的反应.从该实验室已构建的 NaCl 400 mmol/L处理的盐地碱蓬(Suaeda salsa)cDNA文库中克隆了肌醇-1-磷酸合成酶的全长cDNA (S.salsa myo-inositol-1-phosphate synthase,SsINPS),基因注册号为AF433879.SsINPS全长约1 986 bp,含有开放式阅读框架1 530 bp,3′和5′的非翻译区分别为139 bp和317 bp;推导的氨基酸序列全长510个氨基酸残基,分子量约为56.7 kD,pI值为5.35.BLAST同源性分析表明,该cDNA与已报告的冰叶日中花 (Mesembryanthemum crystallinum) 的INPS基因同源性最高,其中,核苷酸水平的同源性为91%,氨基酸水平上的同源性为 84%.以SsINPS全长cDNA为探针进行的Southern杂交结果表明,SsINPS基因在盐地碱蓬基因组中只有一个拷贝;Northern结果表明,在盐处理(400 mmol/L的NaCl)下,SsINPS在叶中的表达量有显著的增加.从而说明SsINPS在盐胁迫下是上升调节的.%Myo-inositol-1-phosphate (I-1-P) synthase (INPS,EC5.5.1.4) catalyzes the reaction from glucose-6-phosphate (G-6-P) to I-1-P,the first step of myo-inositol (Ins) biosynthesis.A full-length cDNA clone,named SsINPS (Genbank accession number:AF433879),which showed highest homology to the INPS from the common ice plant (Mesembryanthemum crystallinum) (84% identity in nucleotide sequence and 91% identity in deduced amino acid sequence),was isolated from a λZap-cDNA library constructed from NaCl-treated Suaeda salsa aerial tissue.The total size of SsINPS is 1 986 bp with an open reading frame of 1 530 bp.Of it,the 5′ and 3′non-coding region is 139 bp and 317 bp respectively.The deduced structure of INPS contains 510 amino acids with a calculated molecular weight of 56.7 kD,and an estimated pI of 5.35.Southern blot analysis showed that there is only one copy of this gene in S.salsa genome.Northern blot analysis indicated that the expression level of SsINPS in S.salsa leaves was significantly increased after being treated with NaCl 400 mmol/L.The results show that SsINPS is upregulated by salt stress.