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刚毛柽柳Th2CysPrx基因的互作蛋白及其表达模式分析(PDF)

《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

Issue:
2019年02期
Page:
86-92
Column:
研究论文
publishdate:
2019-03-30

Article Info:/Info

Title:
Interacting proteins of Tamarix hispida Th2CysPrx and their expression pattern analysis
Article ID:
1000-2006(2019)02-0086-07
Author(s):
LIU Zhongyuan JIANG Bo LÜ Jiaxin LI Xinping GAO Caiqiu*
(State Key Laboratory of Tree Genetics and Breeding(Northeast Forestry University), Harbin 150040, China)
Keywords:
Tamarix hispida yeast two-hybrid interacting protein 2CysPrx
Classification number :
Q943.2
DOI:
10.3969/j.issn.1000-2006.201806018
Document Code:
A
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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Last Update: 2019-03-30