枸杞LmP5CS基因的克隆及表达分析

目的:为进一步研究枸杞抗逆境胁迫的机制,并为转基因育种,提供理论依据。提高农作物的抗逆性提供优质的基因资源。方法:提选取盐胁迫后脯氨酸含量变化较大的耐盐植物枸杞为材料,用1.5%NaCl处理后,提取枸杞叶片总RNA,利用 RT-PCR 及3' RACE方法克隆获得吡咯啉-5-羧酸合成酶(delta 1-pyrroline-5-carboxylate synthetase,P5CS)基因的全长cDNA,命名为LmP5CS,构建pH7m24GW,3rc-LmP5CS植物表达载体。结果:LmP5CS基因的ORF长2 154 bp,编码1个等电点为6.07、分子量为 77.5kDa、由717个氨基酸组成的蛋白。枸杞在200 mmol/L NaCl 盐胁迫下, LmP5CS基因表达量随处理时间,有先升高后降低的趋势,9h基因表达量最高,脯氨酸含量变化与之一致。结论:LmP5CS基因在盐胁迫下脯氨酸含量的变化中起关键作用。

Cloning and Expression Analysis of LmP5CS Gene from Lycium chinense Miller

Proline is the important osmotic regulation substances in plants and plays a critical role in improving the stress tolerance of plants. The materials is Lycium chinense Miller,which proline content changes significantly after salt stress. After 1.5% NaCl stress, full length cDNA sequence of a putative Δ1-pyrroline-5-carboxylate synthase gene(P5CS)was cloned from Lycium chinense Miller leaves using RT-PCR and 3’rapid amplification of cDNA ends(RACE),named LmP5CS, and construct expression vector pH7m24GW,3rc -LmP5CS. Sequence analysis showed that the complete open reading frame (ORF) of this gene is 2154 bp, encoding for a protein of 717 amino acids with an isoelectric point of 6.07 and a molecular weight of 77.5 kDa.After 200 mmol/L NaCl stress, protein expression level increased at first and decreased subsequently, proline content changed in accordance with that. The semi-quantitative PCR result suggests that LmP5CS plays an important role in proline content change responses to salt stress.