• 论文与研究报告 •

### 刚毛柽柳ThP5CR基因的克隆及抗逆功能分析

1. 东北林业大学 林木遗传育种国家重点实验室 哈尔滨 150040
• 收稿日期:2016-08-15 修回日期:2016-12-27 出版日期:2017-07-25 发布日期:2017-08-23
• 通讯作者: 高彩球
• 基金资助:
国家高技术研究发展计划（863计划）（2013AA102701）。

### Cloning and Stress Tolerance Analysis of ThP5CR from Tamarix hispida

Tang Feifei, Zhao Yulin, Wang Peilong, Feng Deming, Song Yi, Gao Caiqiu

1. State Key Laboratory of Tree Genetics and Breeding Northeast Forestry University Harbin 150040
• Received:2016-08-15 Revised:2016-12-27 Online:2017-07-25 Published:2017-08-23

Abstract: [Objective] Proline plays an important osmotic regulatory role in plant responses to salt and drought stress. Therefore, the tolerance to stress of plants can be improved by increasing the proline content in the plants. Pyrroline-5-carboxylic acid reductase (P5CR) is an important reductase for the synthesis of proline. In this study, the ThP5CR gene was cloned by screening the Tamarix hispida transcriptome libraries. And its stress resistance function was further studied. The study laid a theoretical foundation for using the gene in tree breeding through genetic engineering.[Method] The ThP5CR cDNA sequence was obtained by searching with T. hispida transcriptome libraries using "Pyrroline 5 Carboxylate Reductase" as a keyword. Further, RT-PCR cloning and sequencing of the gene were used to verify the sequence of ThP5CR. And the sequence was analyzed using bioinformatics tools. The Real time RT-PCR was used to analyze the expression of ThP5CR genes in the roots and leaves of T. hispida under different stress treatments. For further analysis of stress resistance function of ThP5CR gene, the plant overexpression vector pROKⅡ-ThP5CR was constructed and transient transformed into T. hispida. At the same time, the vector pROKⅡ was also transient infected into T. hispida as a control. The proline content, MDA content, H2O2, and DAB, NBT and Evans blue staining under NaCl and mannitol stress were measured and compared between the pROKⅡ-ThP5CR transient transformed T. hispida and the control.[Result] The result showed that the cDNA length of ThP5CR was 1 432 bp, containing a length of 822 bp open reading frame encoding 273 amino acids. The relative molecular weight of ThP5CR protein was 28.29 kDa, with isoelectric point 9.22. And typical NADP+ domain and L-Proline domain were found in the ThP5CR sequence. Compared to the control, the expressions of ThP5CR gene showed difference under two abiotic stresses and three hormone treatments. Furthermore, the expressions were significantly different at least at a stress point time for each treatment. In addition, the expressions of ThP5CR gene were more significantly influenced under NaCl stress, PEG6000stress and ABA hormone treatment than the other treatments. The expressions of ThP5CR in the pROKⅡ-ThP5CR transient transformed T. hispida were obviously higher than it in the control, indicating that the overexpression lines were successfully obtained. Further, the result of physiological indicators and physiological staining showed that proline content was higher in the overexpression lines than in the control under non-stress conditions. However, the proline content was more significantly higher than that in the control plants under 150 mmol·L-1 NaCl and 200 mmol·L-1 mannitol stress. The NBT and DAB staining and H2O2 content also showed that the accumulation of O2· and H2O2 in the overexpression lines were significantly lower than those in the control. The Evans blue staining and MDA content showed that the stain of the overexpression lines were lesser intensive and the MDA content was lower compared with those of control.[Conclusion] ThP5CR gene can respond to NaCl, PEG stress, ABA and other hormones, which may be involved in salt and drought stress in T. hispida. All results indicated that transient overexpression of ThP5CR improved the salt and drought stresses tolerance by increasing proline content and enhancing the ability to remove reactive oxygen species within the cell to reduce the accumulation of H2O2 and O2·, thereby reducing cell damage or cell death and enhancing plant resistance. ThP5CR was proved to be a candidate gene of responding to stresses.