刚毛柽柳Th2CysPrx基因的互作蛋白及其表达模式分析

刘中原; 姜波; 吕佳欣; 李新苹; 高彩球

doi:10.3969/j.issn.1000-2006.201806018

刘中原,姜波,吕佳欣,李新苹,高彩球

南京林业大学学报（自然科学版） ›› 2019, Vol. 43 ›› Issue (02) : 86-92.

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南京林业大学学报（自然科学版） ›› 2019, Vol. 43 ›› Issue (02) : 86-92. DOI: 10.3969/j.issn.1000-2006.201806018

研究论文

刚毛柽柳Th2CysPrx基因的互作蛋白及其表达模式分析

刘中原, 姜波, 吕佳欣, 李新苹, 高彩球^*

作者信息 +

Interacting proteins of Tamarix hispida Th2CysPrx and their expression pattern analysis

LIU Zhongyuan, JIANG Bo, LÜ Jiaxin, LI Xinping, GAO Caiqiu^*

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文章历史 +

摘要

【目的】使用酵母双杂交系统研究刚毛柽柳中获得的2CysPrx(硫氧还蛋白过氧化物酶)基因(命名为Th2CysPrx)及编码蛋白的功能。【方法】通过酵母双杂交系统对该基因编码蛋白的互作蛋白进行筛选,进一步利用qRT-PCR 技术分析NaCl 和PEG₆₀₀₀胁迫下刚毛柽柳叶组织和根部组织中Th2CysPrx 基因与其互作蛋白基因的表达模式。【结果】酵母双杂交系统筛选获得了4 个可能与Th2CysPrx 互作的蛋白,分别为丙氨酸-乙醛酸转氨酶2(alanine-glyoxylate aminotransferase 2,ThAGT2)、苹果酸脱氢酶(malate dehydrogenase,ThMDH)、黄酮醇合酶(flavonol synthase,ThFLS)和扩展蛋白(expansin,ThEXP)。基因表达分析结果显示:盐胁迫下,柽柳叶和根中,Th2CysPrx和ThAGT2 基因的表达模式基本一致; 而干旱胁迫下,Th2CysPrx和ThAGT2 基因在叶和根中具有相同的表达模式。【结论】在盐和干旱胁迫下,ThAGT2 基因与Th2CysPrx 表达模式均趋于一致,表明Th2CysPrx 基因均可能通过与ThAGT2 基因的互作共同参与抗逆过程,为进一步研究Th2CysPrx 基因的抗逆机制,以及与其他抗逆基因的关系提供了依据。

Abstract

【Objective】In order to study the stress resistance mechanisms of the Th2CysPrx, the cDNA sequence of Th2CysPrx was cloned. And the interacting proteins were further identified in this study. 【Method】The CDS of Th2CysPrx gene was amplified and cloned into the GAL4 DNA binding domain of the pGEBKT7 vector, which served as the BD construct(pGBKT7-Th2CysPrx). The cDNA library of T. hispida was cloned into the pGADT7-Rec vector as the AD constructions(cDNA library/AD). The interacted proteins of Th2CysPrx were identified by using yeast two-hybrid assay. The recombinant BD vectors were transformed into the yeast Y2HGold cells including AD library, which were then grown on SD/-Trp-Leu-His-Ade/X-α-Gal/Aureobasidin A medium(QDO/X/A). The clones were sequenced and analyzed. And these clones may be the interacting proteins of Th2CysPrx. Furthermore, the expression patters of the Th2CysPrx and the 4 interaction proteins was analyzed by using the qRT-PCR. Total RNA was extracted from stems or leaves of T. hispida under different stress treatments. One thousand nanograms of total RNA treated with DNaseI was reverse-transcribed into cDNA in a reaction volume of 10 μL according to the manufacturer's protocol. The synthesized cDNAs were diluted 10-fold with sterile water and used as templates for qRT-PCR. The α-tubulin, β-tubulin, and β-actin genes were used as internal references. Each experiment was carried out by three technical and three biological replicates. The relative expression ratios calculated from the cycle threshold(Ct)according to the delta-delta Ct method. 【Result】The results showed that four different proteins which may interacte with Th2CysPrx gene were obtained. They were ThAGT2(alanine-glyoxylate aminotransferase 2), ThMDH(malate dehydrogenase), ThFLS(flavonol synthase)and ThEXP(expansin)proteins. Compared with the control, the expressions of Th2CysPrx gene showed difference under 0.4 mol/L NaCl and 20% PEG₆₀₀₀ stresses. The expression pattern of the interacting protein gene of ThAGT2 was basically the same expression patterns with Th2CysPrx under salt stress in T. hispida leaves and roots. However, under drought stress, the expression of Th2CysPrx gene was only consistent with ThAGT2 gene. 【Conclusion】Taken together, these results suggest that the Th2CysPrx gene may participate in the stress resistance process through interaction with ThAGT2 gene under salt and drought stress. This study laid the foundation to further investigate resistance mechanism of Th2CysPrx gene.

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刘中原,姜波,吕佳欣,李新苹,高彩球. 刚毛柽柳Th2CysPrx基因的互作蛋白及其表达模式分析[J]. 南京林业大学学报（自然科学版）. 2019, 43(02): 86-92 https://doi.org/10.3969/j.issn.1000-2006.201806018

LIU Zhongyuan, JIANG Bo, LÜ Jiaxin, LI Xinping, GAO Caiqiu. Interacting proteins of Tamarix hispida Th2CysPrx and their expression pattern analysis[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2019, 43(02): 86-92 https://doi.org/10.3969/j.issn.1000-2006.201806018

中图分类号： Q943.2

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基金

收稿日期:2018-06-26 修回日期:2018-09-08
基金项目:国家自然科学基金项目(31670679); 黑龙江省杰出青年科学基金(JC2017004)。
第一作者:刘中原(zhongyuanliu66@163.com)。
*通信作者:高彩球(gaocaiqiu@njfu.edu.cn),教授,ORCID(0000-0001-9953-9778)。