| 摘要: |
| 【目的】研究脱水蛋白DHN14的功能及其在植物响应非生物胁迫中的潜在作用。【方法】从小麦品种“郑引1号”克隆获得脱水蛋白基因DHN14，对其进行生物信息学分析，利用实时定量PCR分析该基因在逆境胁迫下的表达水平，构建脱水蛋白DHN14基因的原核表达载体（重组菌pET28a-DHN14），经IPTG诱导表达后，在非生物胁迫下，研究该蛋白对大肠杆菌及乳酸脱氢酶（LDH）的保护作用。【结果】 克隆获得脱水蛋白基因DHN14的CDS为339 bp，编码112个氨基酸，该蛋白含有2个保守的K片段，属于高亲水性无序蛋白，其分子质量为24 ku，等电点(pI)约为6.28；多序列比对分析表明，DHN14蛋白与WCOR726（Triticum aestivum）脱水蛋白的亲缘关系最近；实时定量RT-PCR结果表明，脱水蛋白基因DHN14受干旱、低温和ABA诱导表达；在20 mmol/L金属离子（Co2+、Ni2+、Cu2+、Zn2+）胁迫下，表达DHN14重组蛋白的大肠杆菌的活力明显高于对照，表明该蛋白在大肠杆菌中对金属离子胁迫具有保护作用；用1.0 mmol/L过氧化氢进行胁迫处理，重组菌DHN14存活率显著增高，说明脱水蛋白DHN14能够提高大肠杆菌对过氧化氢胁迫的耐受性；在低温和脱水胁迫下，DHN14脱水蛋白对乳酸脱氢酶的活性具有保护作用。【结论】小麦脱水蛋白DHN14能够响应非生物胁迫，提高对低温、干旱、金属离子、过氧化氢的耐受性。 |
| 关键词: 脱水蛋白 非生物胁迫 小麦 乳酸脱氢酶 荧光实时定量PCR |
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| 基金项目:国家自然科学基金项目（31671608）；旱区作物逆境生物学国家重点实验室基金项目（CSBAA2017008） |
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| Functional analysis of K2 type wheat dehydrin DHN14 under abiotic stresses |
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SHI Xueying,TIAN Ye,LI He,et al
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| Abstract: |
| 【Objective】This paper studied the dehydrin function of DHN14 and its potential role in plant responses to abiotic stress.【Method】The dehydrin DHN14 was isolated from ‘Zhengyin 1’ cultivar of Triticum aestivum.Bioinformatics analysis of the dehydrin was conducted,and real-time quantitative PCR was used to demonstrate the expression of gene DHN14 under abiotic stresses.The recombinant plasmid pET28a-DHN14 was constructed and transformed into the Escherichia coli expression strain for inducible expression of the target protein by IPTG.The protective effect of the protein on E.coli and LDH under abiotic stress was also investigated.【Result】The dehydrin gene DHN14 was cloned and the full length of DHN14 CDS was 339 bp encoding 112 amino acids and containing 2 conserved K segments.DHN14 was a highly hydrophilic disorder protein and a 24 ku DHN14 protein induced by IPTG,and the theoretical isoelectric point (pI) value was 6.28.Multiple sequence alignment showed that it had the nearest relationship to a WCOR726 Triticum aestivum.The qRT-PCR assay indicated that the expression of DHN14 gene was induced by low temperature,PEG and ABA.In the E.coli transformants tolerance assays,the relative activity of transgenic E.coli was higher than control in 20 mmol/L (Co2+,Ni2+,Cu2+,and Zn2+) stress,which demonstrated that DHN14 protein could protect the activity of E.coli in metal ion stress.When the hydrogen peroxide concentration was 1.0 mmol/L,the viability of E.coli transformants was significantly increased.DHN14 can improve the tolerance of E.coli to heavy metal and hydrogen peroxide stress.The study on lactate dehydrogenase (LDH) showed that DHN14 dehydrin had protective effect on LDH activity under low temperature and dehydration stress.【Conclusion】The dehydrin DHN14 can respond to abiotic stress and improve the tolerance of crops in low temperature,drought,metal ion and hydrogen peroxide stresses. |
| Key words: dehydrin abiotic stress wheat enzyme activity protection Real-time PCR |
