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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/LNaCl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。
OBJECTIVE: To provide a theoretical basis for further research on the mechanism of Lycium barbarum in anti-stress and for genetically modified breeding. Improve the resistance of crops to provide high quality genetic resources. Methods: The salt-tolerant plant Lycium chinense L., whose content of proline changed greatly after salt stress, was selected as the material. After treated with 1.5% NaCl, the total RNA of Lycium barbarum L. leaves was extracted and cloned by RT-PCR and 3’RACE The full-length cDNA of delta-pyrroline-5-carboxylate synthetase (P5CS) gene was named as LmP5CS, and the pH7m24GW and 3rc-LmP5CS plant expression vectors were constructed. Results: The ORF of LmP5CS gene was 2 154 bp in length and encoded a protein of 717 amino acids with an isoelectric point of 6.07 and a molecular weight of 77.5 kDa. Under 200 mmol / L NaCl stress, the expression level of LmP5CS gene in Lycium barbarum increased first and then decreased with the time of treatment. The expression level of LmP5CS gene was the highest at 9h, and the content of proline was the same. Conclusion: LmP5CS gene plays a key role in the change of proline content under salt stress.