| 二穗短柄草钙依赖型蛋白激酶基因BdCDPK14克隆及表达分析 |
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| 引用本文： | 韦淑亚,赵旭东,杨广笑,何光源.二穗短柄草钙依赖型蛋白激酶基因BdCDPK14克隆及表达分析[J].南方农业学报,2017,48(5). |
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| 作者姓名： | 韦淑亚 赵旭东 杨广笑 何光源 |
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| 作者单位： | 1. 武汉职业技术学院 生物工程学院,武汉,430074;2. 科技部国际科技合作基地(基因工程)/分子生物物理学教育部重点实验室/华中科技大学 生命科学与技术学院,武汉,430074 |
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| 基金项目： | 湖北省自然科学研究计划项目，教育部高校博士学科点专项科研基金项目，湖北省高等学校优秀中青年科技创新团队计划项目，武汉职业技术学院博士科研基金项目 |
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| 摘 要： | 【目的】克隆分析二穗短柄草钙依赖型蛋白激酶(CDPK)基因BdCDPK14,并检测其在干旱胁迫下的表达量,为揭示钙依赖型蛋白激酶的抗旱调控机制打下基础。【方法】根据NCBI检索结果设计特异引物,以二穗短柄草cDNA为模板,采用PCR扩增二穗短柄草CDPK基因家族成员BdCDPK14,利用在线分析软件对BdCDPK14基因编码蛋白进行生物信息学分析,并采用RT-PCR检测PEG-6000干旱胁迫下的BdCDPK14基因表达量。【结果】从二穗短柄草叶片中克隆获得的BdCDPK14基因(Gen Bank登录号XM_003564390)片段长度1750 bp,其开放阅读框(ORF)1545 bp,编码514个氨基酸,其编码蛋白分子量56.78 kD,理论等电点5.45,脂肪指数78.21,不稳定指数38.66,属于稳定蛋白。BdCDPK14蛋白与小麦TaCPK13蛋白(ABY59018)的亲缘关系最近,含有4个EF-hands结构、蛋白酪氨酸激酶结构域、脂多糖激酶家族、ATP结合区域、丝氨酸/苏氨酸蛋白激酶激活区和预测跨膜区等结构域,主要由无规卷曲和α-螺旋构成,位于叶绿体和细胞质膜上。在PEG-6000干旱胁迫下,BdCDPK14基因在胁迫3 h内的相对表达量无明显变化,胁迫6 h后相对表达量开始升高,至胁迫12 h时的相对表达量最高。【结论】克隆获得的BdCDPK14基因为二穗短柄草CDPK基因家族成员之一,参与其抗干旱胁迫反应,可作为候选基因用于二穗短柄草抗旱机制研究。
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| 关 键 词： | 二穗短柄草 BdCDPK14基因 克隆 生物信息学分析 干旱胁迫 表达 |
Clone and expression of calcium-dependent protein kinase BdCDPK14 gene from Brachypodium distachyon |
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| Abstract: | Objective]The present experiment was conducted to clone calcium dependent protein kinase (CDPK) Bd-CDPK14 gene from Brachypodium distachyon and detect its expression level under drought stress, in order to provide a foundation for further research on regulation mechanism of BdCDPK14 gene in response to drought. Method]Specific primers were designed based on NCBI retrieval results, and cDNA of B. distachyon was used as template. BdCDPK14, one of B. distachyon CDPK genes was cloned by PCR amplification. Encoded protein of BdCDPK14 gene was analyzed by online software on its bioinformatics. Its expression level under PEG-6000 drought stress was detected by RT-PCR. Result]Bd-CDPK14 gene(GenBank accession number: XM_003564390) cloned from B. distachyon was 1750 bp in length, open reading frame(ORF) was 1545 bp CDS, coding 514 amino acids. The molecular weight of encoded protein was 56.78 kD, isoelectric point 5.45, fat index 78.21 and instability index 38.66, belonging to stable protein. BdCDPK14 protein con-tained several domains including four EF-hands, protein tyrosine kinase domain, lipopolysaccharide kinase family, ATP-binding region, serine/threonine protein kinase active-site region and predicted transmembrane region. It shared the closest genetic relationship with TaCPK13 protain(ABY59018) of wheat. BdCDPK14 protein was a protein with random coil andα-helix located in chloroplast and plasma membrane. Conclusion]The cloned BdCDPK14 gene from B. distachyon leaf is a member of CDPK genes, and is involved in drought-resistant reaction in B. distachyon. So it can be served as a candi-date gene to further study its role in drought-resistant in B. distachyon. |
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| Keywords: | Brachypodium distachyon BdCDPK14 gene clone bioinformatics analysis drought stress expression |
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