高羊茅逆境胁迫蛋白基因FaUSP的克隆、表达及生物学功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0744

摘要/Abstract

摘要：

逆境胁迫蛋白（Universal stress protein,USP）参与干旱、高温、氮胁迫和高盐等多种逆境胁迫反应。为研究逆境胁迫蛋白USP在高羊茅中的表达模式,采用cDNA末端快速扩增技术从高羊茅叶片中克隆USP基因,命名为FaUSP。结果表明,FaUSP全长为844 bp,含501 bp的开放阅读框,共编码166个氨基酸,具有USP家族典型的USPA结构域。经系统进化树分析,高羊茅FaUSP蛋白与禾本科植物小麦、大麦、山羊草的USP蛋白亲缘关系较近。荧光定量PCR表明,高羊茅FaUSP受干旱、高温、氮胁迫及高盐胁迫的诱导表达上调,构建FaUSP超量表达载体,利用农杆菌侵染法转化菊苣,干旱胁迫下过量表达FaUSP菊苣可溶性蛋白、GR、SOD、POD、CAT的合成显著高于野生型,表明FaUSP的超量表达可以增强菊苣的抗旱能力,推测该基因与抗旱性相关。

关键词: 高羊茅, FaUSP, 克隆, 遗传转化

Abstract:

Universal stress proteins（USP）involve in multiple abiotic responses including drought stress,heat stress,nitrogen deficiency and salt stress. To explore the expression pattern of USP gene in Festuca arundinacea,a USP was cloned through rapid amplification of cDNA ends from the leaves of F. arundinacea,and designated as FaUSP. The sequence analysis results showed that the full-length cDNA（844 bp）was obtained with a predicated 501 bp open reading frame,which encoded 166 amino acids,and it had the typical USPA domain of USP family. Phylogenetic analysis revealed that FaUSP in the F. arundinacea was most closely related to USP from other Gramineae plants such as Hordeum vulgare,Triticum aestivum and Aegilops tauschii. The qRT-PCR results show that expression of FaUSP gene was up-regulated after induced via drought stress,heat stress,nitrogen deficiency and salt stress. A plant overexpression vector was constructed and transformed into chicory by Agrobacterium infection. The soluble protein,glutathione reductase（GR）,superoxide dismutase（SOD）,peroxidase（POD）,and catalase（CAT）in the FaUSP overexpression strains were significantly higher than that of wild-type under drought stress. These results indicate that overexpression of FaUSP gene enhances the drought resistance of chicory,thus it is speculated that USP gene is associated with drought resistance.

Key words: Festuca arundinacea, FaUSP, cloning, genetic transformation

陈莹, 陈锡, 王茜, 王小利. 高羊茅逆境胁迫蛋白基因FaUSP的克隆、表达及生物学功能分析[J]. 生物技术通报, 2021, 37(2): 32-39.

CHEN Ying, CHEN Xi, WANG Qian, WANG Xiao-li. Cloning,Expression and Biological Function Analysis of Universal Stress Protein in Festuca arundinacea[J]. Biotechnology Bulletin, 2021, 37(2): 32-39.

图/表 8

表1 高羊茅FaUSP克隆及荧光定量PCR的引物序列

引物名称	引物序列（5'-3'）	扩增类别
FU1	CAATGGAAGGCTCAGA	5' RACE
FU2	AGAGAGGGGGATCAAAGG
FU3	GGATGGTCTCCCTCTGCT
FU4	CATCCCCATGAGCTGCCTGGTTATC	3' RACE
FU5	AAAGGGTGCTCTTAGGCAGCGTCAG
FU-fwd1	AGAGGTGGGTTGGCCTGGC	中间片段PCR扩增
FU-rev1	TCGCTGGCTTGACAACTGTG
UBI-fwd1	CACCTCGATCACCCACCTCT	UBI内参引物
UBI-rev1	AGGGTCTCCGATAACCTCCA
FU-fwd2	CCTTTGATCCCCCTCTCTGAA	荧光定量PCR引物
FU-rev2	AAACCACCGTGACCTCCTTCT

图1 FaUSP基因的RACE扩增 1：5' RACE;2：3' RACE;3：FaUSP基因核心片段;M：DL2000

图2 FaUSP核苷酸及推导的氨基酸序列

图3 植物USP同源基因氨基酸序列的系统进化树分析

图4 非生物胁迫下FaUSP基因在高羊茅中的转录水平

图5 转基因菊苣的表型

图6 转基因菊苣FaUSP的表达分析

图7 转基因菊苣在干旱胁迫下的生理应答差异

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