| 摘要: |
| 【目的】克隆青杄类伸展蛋白（extensin-like proteins,ELPs）基因PwELP1并分析该基因的表达特性，为研究ELP基因家族的功能奠定基础。【方法】以青杄cDNA文库为模板，用RACE-PCR方法克隆青杄类伸展蛋白编码基因PwELP1的cDNA全长,利用DNAMAN确定PwELP1的编码框及蛋白氨基酸的翻译，运用clustalx软件与Espript工具进行多序列比对，利用MEGA 5软件的邻位相连法构建系统树,并对编码蛋白进行生物信息学分析；利用RT-qPCR检测PwELP1基因在青杄不同组织中表达的特异性、花粉和种子不同萌发时期及幼苗受到不同逆境处理时的表达差异，分析该基因的组织发育及逆境响应表达特点。【结果】PwELP1基因全长832 bp，其编码一个由159个氨基酸组成的蛋白，该蛋白的N端与C端均有比较保守的结构域；此蛋白二级结构由1个α-螺旋、5个β-折叠与3个β-转角结构构成，对其三级结构的预测印证了这一结果，同时发现该蛋白中还存在部分无规则卷曲。其分子质量为17.03 ku，理论等电点为5.81。PwELP1在青杄花粉中的表达量较高，且在花粉萌发后期（30～36 h）以及种子萌发初期（第2天）表达量高；PwELP1的表达不同程度地受低温、高温、茉莉酸甲酯（MeJA）、H2O2、脱水以及脱落酸（ABA）等非生物逆境胁迫处理的诱导，尤其是H2O2、高温和ABA，处理初期表达量就显著升高。【结论】PwELP1在青杄花粉和种子萌发过程中可能发挥重要功能，并可参与青杄对非生物逆境胁迫的响应过程。 |
| 关键词: 青杄 类伸展蛋白 组织表达 非生物胁迫 |
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| 基金项目:国家自然科学基金面上项目（31270663） |
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| Cloning and expression analysis of PwELP1 gene in Picea wilsonii |
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GAO Yintao,ZHOU Yanni,LUO Chaobing,et al
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| Abstract: |
| 【Objective】This study cloned and conducted molecular characterization for PwELP1 gene from Picea wilsonii to provide basis for investigating functions of ELP gene family.【Method】The full length cDNA of PwELP1 was obtained by RACE-PCR assays based on the cDNA library of grown Picea wilsonii.The ORF finding and the protein translation of PwELP1 were done by DNAMAN.The multiple sequences alignment was done by clustalx and Espript.Phylogenetic tree was obtained by Neighbor-joining in MEGA 5.The calculation of theoretical isoelectric point and molecular weight of PwELP1 protein were completed by Compute pI/Mw tool in Expasy.The secondary and tertiary structure prediction of PwELP1 protein was completed by Jpred and swiss-model,respectively.The tissue-specific expressions,the differential expressions in different germination periods of pollen and seed,and the differential expressions of seedling under different abiotic stresses were investigated by RT-qPCR assays.【Result】PwELP1 encoded a protein with 159 amino acids,which had relatively conserved domain in both N-terminal and C-terminal.As the prediction of secondary structure,the protein consisted of one α-helix,five β-sheets,three β-turns,which was prove by protein tertiary structure prediction Furthermore,a few coils exsisted in the tertiary structure.The theoretical molecular weight of PwELP1 was 17.03 ku and the isoelectric point was 5.81.Tissue specific expression experiments revealed that the expression level in pollen was the highest among all tissues of Picea wilsonii.During pollen germination,PwELP1 was highly expressed at the end of germination process (30-36 h).The expression of PwELP1 was up-regulated in inhibition period (2 days) in seed germination.Seedling tolerance treatment experiments suggested that the expression level of PwELP1 was regulated under chilling,heat,methyl jasmonate,hydrogen peroxide,dehydration and abscisic acid (ABA) treatments,especially under hydrogen peroxide,heat and ABA treatments.【Conclusion】PwELP1 might have a potential function in pollen germination,seed germination and responding to a serious of abiotic stress processes. |
| Key words: Picea wilsonii extension-like protein tissue expression abiotic stresses |
