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与低温相关的甜菜磷脂酰肌醇转运蛋白基因的克隆及表达分析

Cloning and Expression Analysis of Sugar Beet Phosphatidylinositol Transfer Genes Related to Low Temperature

  • 摘要: 真核细胞内膜系统间存在的磷脂酰肌醇转运蛋白PITP(phosphatidylinositol transfer proteins, PITP),能提高植物对逆境胁迫的耐受程度。本研究从甜菜(Beta vulgaris)基因组中分析获得甜菜PITP基因28个,通过低温胁迫,以RT-qPCR筛选出4个与低温胁迫相关最为显著的PITP基因,将其从甜菜中克隆出来,分别命名为Bvsec14、Bvsfh8、Bvsfh11和Bvpate4.2,基因长度分别为776、1 401、1 519、1 604 bp,均具有PITP基因家族所特有的SEC14保守结构域,分别定位在5号、9号、1号和8号染色体上。生物信息学分析结果显示,亚细胞定位预测表明其在细胞膜或细胞质上;4个基因编码蛋白都以无规则卷曲和α-螺旋为主,存在很多的磷酸化位点,以丝氨酸磷酸化数量最多;根据与其他物种的PITP的高度保守的SEC14区域比对,建立的4个基因的进化树表明,其与藜麦(Chenopodium quinoa)的亲缘关系最近;将拟南芥(Arabidopsis thaliana)中PITP蛋白与甜菜中与低温胁迫相关的PITP蛋白进行同源性分析,共分为4个亚组;4个基因编码蛋白之间没有直接互作关系且氨基酸序列间差异较大。以低温胁迫和RT-qPCR进行表达分析,结果表明,4个基因在表达量上无组织特异性,Bvsfh11、Bvsfh8、Bvpate4.2和Bvsec14受低温胁迫后的24 h之内都会出现过量表达,峰值出现在2 h和12 h,且峰值的表达量最低为对照组初始量的1.8倍,最高为25倍。上述结果表明,4个基因参与甜菜低温胁迫应答。本研究为研发新的甜菜抗性品种以及进一步探究PITP功能提供科学理论依据。

     

    Abstract: Phosphatidylinositol transfer proteins(PITP) exist in the inner membrane system of eukaryotic cells, which can improve the tolerance of plants in response to stress. In this study, 28 PITP genes were obtained from sugar beet(Beta vulgaris) genome. Under low temperature stress, four PITP genes most significantly related to low temperature stress were screened by RT-qPCR analysis and cloned from sugar beet. The four genes are named Bvsec14 with 776 bp, Bvsfh8 with 1 401 bp, Bvsfh11 with 1 519 bp and Bvpate4.2 with 1 604 bp and were located on chromosome 5, 9, 1 and 8 respectively, they all have a typical SEC14 conserved domain of PITP gene family. The subcellular localization prediction of four genes showed they were on cell membrane or cytoplasm by bioinformatics analysis, the structures of proteins encoded by four genes are mainly α-helix and irregular coil, many phosphorylation sites and the number of serine phosphorylation sites is the largest. According to the highly conserved SEC14 region of PITP compared with other species, the phylogenetic tree of four genes showed that they had the closest relative and PITP related to low temperature stress in sugar beet was analyzed and they were divided into four subgroups. There was no direct interaction between the proteins encoded by the four genes, and they had great differences in amino acid sequences. Under low temperature stress, RT-qPCR was used for expression analysis,analysis results showed that the four genes had inorganization specificity. Bvsfh11, Bvsfh8, Bvpate4.2 and Bvsec14 were overexpressed within 24 hours, the peak appeared at 2 h and 12 h, and the lowest expression was 1.8 times and the highest was 25 times of the control group. The above results showed that four genes were involved in the response of sugar beet under low temperature stress. This study provided a scientific theoretical basis for developing new sugar beet resistant varieties and further exploring the function of PITP.

     

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