二穗短柄草钙依赖型蛋白激酶基因BdCDPK14克隆及表达分析

发布时间：2018-01-23 07:55

本文关键词： 二穗短柄草 BdCDPK基因克隆生物信息学分析干旱胁迫表达　出处：《南方农业学报》2017年05期 　论文类型：期刊论文

【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。
[Abstract]:[objective] to clone and analyze CDPK14 gene of Calcium-dependent protein Kinase (CDPK14) and detect its expression under drought stress. [methods] A specific primer was designed according to the results of NCBI search and cDNA was used as template. BdCDPK14, a member of CDPK gene family, was amplified by PCR and bioinformatics analysis of BdCDPK14 gene encoding protein was carried out by on-line analysis software. RT-PCR was used to detect the expression of BdCDPK14 gene under drought stress of PEG-6000. [results] the BdCDPK14 gene (. The length of the Gen Bank fragment was 1750bp. The open reading frame ORFN 1545 BP encodes 514 amino acids, the molecular weight of the encoded protein is 56.78 KD, the theoretical isoelectric point is 5.45, and the fat index is 78.21. The instability index was 38.66, belonging to the close relationship between the stable protein BdCDPK14 and wheat TaCPK13 protein ABY59018. There are four EF-hands structures, protein tyrosine kinase domain, lipopolysaccharide kinase family, serine / threonine protein kinase activation region and predicted transmembrane domain. It is mainly composed of random coils and 伪 -helix, located on chloroplast and cytoplasmic membrane, under PEG-6000 drought stress. The relative expression of BdCDPK14 gene did not change after 3 h stress, but the relative expression level began to increase after 6 h stress. [conclusion] the cloned BdCDPK14 gene is one of the members of CDPK gene family, which is involved in drought stress resistance. It can be used as a candidate gene to study the drought resistance mechanism of two-spike short stalk grass.
【作者单位】：武汉职业技术学院生物工程学院;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;
【基金】：湖北省自然科学研究计划项目(B2016581) 教育部高校博士学科点专项科研基金项目(20120142110075) 湖北省高等学校优秀中青年科技创新团队计划项目(T201535) 武汉职业技术学院博士科研基金项目(2016BS001)
【分类号】：Q943.2
【正文快照】： 0引言【研究意义】二穗短柄草(Brachypodium distachy-on)是一种温带禾本科植物,是用于研究小麦、水稻作物新型模式植物的理想植株,具有重要的经济价值。钙依赖型蛋白激酶(CDPK)为单肽链,在结构上具有明显的特征,从N端到C端存在4个功能区(可变区、催化区、连接区和调控区),是

【相似文献】