小麦WDHN1基因的克隆、表达及功能分析

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摘要 YSK2型脱水素(dehydrins, DHNs)是植物中存在最多的DHNs形式，参与植物响应各种非生物逆境胁迫。为了研究YSK2型DHN的功能，从小麦(Triticum aestivum)中克隆了WDHN1基因(GenBank No. KR709259)，该基因编码区序列总长491 bp，含两个外显子和一个内含子，编码133个氨基酸；存在4个保守区域，即1个Y片段、1个S片段和2个K片段，与节节麦(Aegilops tauschii) DHN (EMT30992)亲缘关系最近。通过PCR扩增得到WDHN1启动子序列，该启动子存在2个脱落酸应答元件(abscisic acid (ABA) response element, ABRE)和3个MBS(MYB binding site)生物胁迫响应元件。通过qRT-PCR分析其非生物胁迫下的表达模式，结果表明，在低温、NaCl、ABA和PEG 6000胁迫下，小麦WDHN1基因表达均表现为先上升后下降的趋势，分别于6、60、12和48 h时表达量最高。组织特异性表达分析表明，WDHN1基因在小麦开花后22 d 的胚芽中表达量最高，具有明显的组织特异性。将WDHN1基因片段连接于原核表达载体pET28a中，转化大肠杆菌(Escherichia coli) BL21(DE3)，用异丙基硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside, IPTG) 诱导表达，获得20 kD的WDHN1蛋白。对重组大肠杆菌 WDHN1-pET28a-BL21(DE3)及其总蛋白进行非生物胁迫，结果表明，WDHN1蛋白还可以阻止蛋白聚合引起的蛋白变性，提高非生物胁迫下大肠杆菌的耐受性及乳酸脱氢酶(lactic dehydrogenase, LDH)的稳定性。研究结果为进一步利用该基因进行小麦抗性改良提供了理论依据。

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	刘浩
	杜娅
	李核
	王晓宇
	于正阳
	朱维宁
	张大鹏
	张林生

关键词 ：小麦, 脱水素(DHN), 非生物胁迫, 乳酸脱氢酶(LDH)

Abstract：YSK2 type dehydrins (DHNs), which are the most types of DHNs, have been shown to be involved in plant response and adaptation to various abiotic stresses. In order to determine the function of YSK2 DHNs, WDHN1 from wheat (Triticum aestivum) was cloned. The CDS of WDHN1 had a length of 491 bp, contained 2 exons and 1 intron, encoding a protein of 133 amino acids, containing one Y segment, one S segment and two K segments. A phylogenetic tree analysis with the related DHNs from different plant species indicated that WDHN1 shared homology with DNA from Aegilops tauschii(EMT30992). Based on PLACE and PlantCARE database analysis, the abiotic stress-related elements of WDHN1 promoter were determined, which contained 2 ABRE (abscisic acid (ABA) response element), and 3 MBS (MYB binding site). qRT-PCR analysis indicated that transcript accumulation occurred in response to low temperature, NaCl, ABA and PEG 6000 treatments. Tissue specific expression analysis showed that the transcript levels of WDHN1 reached the highest level at 22 d in embryo. The prokaryotic expression vector of WDHN1 was successfully constructed. The expression of fusion protein was obtained with isopropyl β-D-1-thiogalactopyranoside (IPTG) and its relative molecular weight was 20 kD. We investigated the anti-aggregation effects of protein WDHN1 on Escherichia coli viability and lactic dehydrogenase (LDH) activity during multiple abiotic stress treatments. WDHN1 protein enhanced tolerance of E. coli and LDH stabilization against diverse stresses via anti-aggregation effects. The results revealed that WDHN1 might serve as a potential stress response gene for the improvement of wheat inbred lines and cultivars under stress conditions in breeding activity.

Key words： Wheat Dehydrin (DHN) Abiotic stress Lactate dehydrogenase (LDH)

收稿日期: 2016-05-11 出版日期: 2016-10-01

基金资助:高等学校博士学科点专项科研基金

通讯作者: 张林生 E-mail: linszhang@nwsuaf.edu.cn

引用本文:

刘浩杜娅李核王晓宇于正阳朱维宁张大鹏张林生. 小麦WDHN1基因的克隆、表达及功能分析[J]. , 2016, 24(11): 1676-1687.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I11/1676

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