多年生黑麦草P5CS基因的cDNA克隆、表达及亚细胞定位

以多年生黑麦草(Lolium perenne)‘Derby'为试材,采用RT-PCR结合RACE技术,克隆获得1个Δ1–吡咯啉–5–羧酸合成酶基因(P5CS)的cDNA序列,全长2528bp,推断其编码716个氨基酸,命名为LpP5CS。序列分析表明:LpP5CS基因与小麦TaP5CS和水稻OsP5CS基因核苷酸序列的相似性分别为92.05%和85.82%,氨基酸序列的相似性分别为93.99%和87.99%。半定量PCR结果表明,LpP5CS基因在多年生黑麦草根、茎,叶均有表达。盐处理下,表达量高于对照,其中叶片中表达量最高,根中最低。200mmol·L-1NaCl处理下,LpP5CS基因表达量随处理时间延长,有先升高后降低的趋势。洋葱鳞茎表皮细胞的瞬时表达显示,LpP5CS蛋白定位于细胞膜和细胞核。

Cloning,Expression and Subcellular Localization of P5CS Gene from Perennial Ryegrass(Lolium perenne L.)

In this study，full length cDNA sequence of a putative Δ1-pyrroline-5-carboxylate synthase gene（LpP5CS）was cloned from perennial ryegrass（Lolium perenne L.‘Derby’）leaves using RT-PCR and rapid amplification of cDNA ends（RACE）. Sequence analysis showed that the nucleotide sequence of this gene is 2 528 bp，containing a complete open reading frame and encoding 716 amino acids. Nucleotide and amino acid sequence analysis revealed that LpP5CS shares high identity with the orthologs from Triticum aestivum（92.05%，93.99%）and Oryza sativa（85.82%，87.99%），respectively. Semi-quantitative RT-PCR analysis showed that LpP5CS expressed in different tissues. Various elevated levels of LpP5CS expression have been detected when the seedling roots exposed to high salinity. Tested with salt solution，its expression was higher than control，and the highest in leaves，the lowest in roots，The contents of LpP5CS increased first then decreased with the processing time is extended after 200 mmol · L-1 NaCl treatment. Finally，subcellular localzation assays showed that the LpP5CS protein was present in the nucleus and at the plasmalemma.