刚毛柽柳ThP5CR基因的克隆及抗逆功能分析

Cloning and Stress Tolerance Analysis of ThP5CR from Tamarix hispida

【目的】脯氨酸在植物对盐和干旱胁迫应答中起重要的渗透调节作用,增加植物体内脯氨酸含量可以提高植物抗逆性。吡咯啉-5-羧酸还原酶(P5CR)是合成脯氨酸的重要还原酶,本研究拟从抗旱耐盐植物刚毛柽柳中克隆获得1个ThP5CR基因,研究该基因的抗逆功能,为该基因用于林木基因工程育种奠定理论基础。【方法】以"Pyrroline 5 Carboxylate Reductase"作为关键词,对实验室前期构建的刚毛柽柳转录组序列进行比对查找获得ThP5CR基因c DNA序列,并通过RT-PCR和测序验证克隆获得的ThP5CR基因序列。利用生物信息学工具进行序列分析。利用Real time RT-PCR分析不同逆境处理下ThP5CR基因在刚毛柽柳根和叶中的表达情况。为了进一步分析ThP5CR基因的抗逆功能,构建了植物过表达载体pROKⅡ-ThP5CR,并进行刚毛柽柳瞬时转化,同时以pROKⅡ瞬时侵染刚毛柽柳作为对照。分析比较Na Cl和甘露醇胁迫后2种瞬时转化刚毛柽柳的脯氨酸、MDA、H2O2含量和二氨基联苯胺(DAB)、氯化硝基四氮唑蓝(NBT)及伊文思蓝(Evans blue)染色情况,以综合评定ThP5CR基因的抗逆功能。【结果】ThP5CR基因编码273个氨基酸,编码蛋白分子量为28.29 k Da,理论等电点为9.22。含有典型的NADP结构域和P5CR二聚体结构域。2种胁迫(Na Cl和PEG6000)和3种激素(ABA、GA3和JA)处理后,刚毛柽柳ThP5CR基因的表达都发生了不同程度的改变,且每种处理后至少有1个时间点发生了明显改变。此外,Na Cl胁迫、PEG6000胁迫和ABA激素处理对ThP5CR基因的表达产生的影响更为显著。对2种瞬时表达刚毛柽柳植株中ThP5CR基因的表达分析显示成功获得瞬时过表达转基因株系(标记为OX)。进一步的生理指标和生理染色结果显示,非胁迫条件下,过表达植株脯氨酸含量高于对照植株(标记为Con);150 mmol·L-1Na Cl和200 mmol·L-1甘露醇胁迫后,2种转基因株系脯氨酸含量差异则更显著;NBT、DAB染色和H2O2含量测定结果显示,OX植株的O-·2和H2O2含量明显低于对照;而Evans blue染色结果和MDA含量测定表明,与对照相比,过表达ThP5CR植株着色更浅、MDA含量更低。【结论】ThP5CR基因能对Na Cl、PEG胁迫和ABA等激素处理做出应答,可能参与了刚毛柽柳抗旱耐盐胁迫应答。在150 mmol·L-1Na Cl和200 mmol·L-1甘露醇胁迫下瞬时过表达ThP5CR植株可通过增加脯氨酸含量增强细胞内清除活性氧能力,使O-·2和H2O2的积累减少,从而减少细胞受损或细胞死亡,增强植物的抗逆性。本研究初步证实ThP5CR基因是一个逆境胁迫应答候选基因。

【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 ThP5 CR 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 ThP5 CR c DNA 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 ThP5 CR. And the sequence was analyzed using bioinformatics tools. The Real time RT-PCR was used to analyze the expression of ThP5 CR genes in the roots and leaves of T. hispida under different stress treatments. For further analysis of stress resistance functionof ThP5 CR gene,the plant overexpression vector pROKⅡ-ThP5 CR 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 Na Cl and mannitol stress were measured and compared between the pROKⅡ-ThP5 CR transient transformed T. hispida and the control.【Result】 The result showed that the c DNA length of ThP5 CR was 1 432 bp,containing a length of 822 bp open reading frame encoding 273 amino acids. The relative molecular weight of ThP5 CR protein was 28. 29 k Da,with isoelectric point 9. 22. And typical NADP＋domain and L-Proline domain were found in the ThP5 CR sequence. Compared to the control,the expressions of ThP5 CR 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 ThP5 CR gene were more significantly influenced under Na Cl stress,PEG6000 stress and ABA hormone treatment than the other treatments. The expressions of ThP5 CR in the pROKⅡ-ThP5 CR 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-1Na Cl and 200 mmol·L-1mannitol stress. The NBT and DAB staining and H2O2 content also showed that the accumulation of O-·2and 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】ThP5 CR gene can respond to Na Cl,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 ThP5 CR 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 O-·2,thereby reducing cell damage or cell death and enhancing plant resistance.ThP5 CR was proved to be a candidate gene of responding to stresses.