蒺藜苜蓿MtPUB基因克隆及进化分析

发布时间：2018-05-13 20:27

  本文选题：蒺藜苜蓿 + U-box　； 参考：《中国农业科学院》2016年博士论文

【摘要】：U-box蛋白在植物中广泛存在说明该类蛋白在植物体内发挥重要作用。很多U-box蛋白具有泛素连接酶E3活性以及在泛素化过程中特异地识别靶标蛋白等生理生化功能。AtPUB4蛋白是在拟南芥中最新发现的具有E3连接酶活性的PUB蛋白,研究表明AtPUB4基因能影响花粉绒毡层细胞的生长发育,利用其特性有可能创制温敏型雄性不育材料。蒺藜苜蓿(Medicago truncatula)是豆科苜蓿属的一年生草本植物。二倍体,具有生育期短、基因组小、自花授粉、易于转化、再生时间较短等特点,被作为豆科模式植物进行研究,2011年已完成全基因组测序。目前,有关蒺藜苜蓿U-box基因家族的研究尚未见报道。本试验克隆两个蒺藜苜蓿U-box基因MtPUB1和MtPUB2,通过生物信息学及生物技术手段分析了克隆基因结构、亚细胞定位、原核表达及基因的表达特性,成功构建了植物表达载体对拟南芥进行遗传转化。最后对MtPUB基因家族进化过程进行了分析。研究结果如下:1.利用同源克隆法获得蒺藜苜蓿MtPUB1和MtPUB2基因,该基因cDNA全长2448和2400bp,分别编码815和799个氨基酸,蛋白分子量为88.36 KDa和87.96 KDa,两个蛋白都是疏水性蛋白,无信号肽和跨膜结构。2.亚细胞定位预测MtPUB1基因定位在细胞质,MtPUB2基因定位在线粒体基质。构建原核表达载体,获得MtPUB1蛋白。3.利用实时荧光定量PCR分析MtPUB1和MtPUB2基因在蒺藜苜蓿不同组织的表达特异性,结果表明MtPUB1基因在花中表达量最高,MtPUB2在茎中表达量最高,两个基因在根部表达量最低。MtPUB1在NaCl、PEG、ABA诱导后,表达量上升,低温诱导表达量变化不明显。MtPUB2在NaCl诱导后表达量下降,在PEG、ABA及低温诱导下表达量都呈现出上升趋势。4.构建植物表达载体pBI121-MtPUB1和pBI121-MtPUB2,采用农杆菌介导法转化拟南芥Atpub4突变体,通过卡纳抗性筛选和PCR、RT-PCR检测后,获得MtPUB1的T1代转基因拟南芥种子。经过对转化拟南芥突变体和野生型拟南芥花粉扫描电镜观测,花粉改变不明显。5.通过对MtPUB基因的功能歧化分析,U-box和ARM两种功能结构域的主要歧化位点位于3个保守的基序上,在此基础上定位了9个歧化位点。6.对MtPUB基因序列选择压力的分析结果表明,MtPUB基因在强的负选择压力下经过复制后保留发挥基因功能,其中114个负选择位点所在序列与多序列联配获得保守序列相符,推测这一区域可使MtPUB蛋白与PREDEN发生特异性结合,因此产生不同功能。
[Abstract]:The widespread presence of U-box proteins in plants suggests that they play an important role in plants. Many U-box proteins have the activity of ubiquitin ligase E3 and the physiologic and biochemical functions such as target recognition protein. AtPUB4 protein is a newly discovered PUB protein with E3 ligase activity in Arabidopsis thaliana. The results showed that AtPUB4 gene could affect the growth and development of pollen tapetum cells, and the thermo-sensitive male sterile material could be created by using its characteristics. Medicago truncatula (Tribulus terrestris) is an annual herb of Alfalfa. Diploid, with the characteristics of short growth period, small genome, self-pollination, easy transformation and short regeneration time, has been studied as a legume model plant, and complete genome sequencing has been completed in 2011. At present, the study of U-box gene family of Tribulus terrestris has not been reported. In this study, two U-box genes of Tribulus terrestris, MtPUB1 and MtPUB2, were cloned. The structure, subcellular location, prokaryotic expression and expression characteristics of the cloned genes were analyzed by bioinformatics and biotechnology. Plant expression vector was successfully constructed for genetic transformation of Arabidopsis thaliana. Finally, the evolution process of MtPUB gene family was analyzed. The results are as follows: 1. The MtPUB1 and MtPUB2 genes of Tribulus terrestris were obtained by homologous cloning. The cDNA of the gene was 2448 and 2400bprespectively, encoding 815 amino acids and 799 amino acids, respectively. The molecular weight of the proteins was 88.36 KDa and 87.96 KDa. both proteins were hydrophobic proteins, no signal peptide and transmembrane structure. Subcellular localization predicted that the MtPUB1 gene was located in the cytoplasm of MtPUB2 gene in the mitochondrial matrix. Prokaryotic expression vector was constructed and MtPUB1 protein. 3. 3 was obtained. The expression specificity of MtPUB1 and MtPUB2 genes in different tissues of Medicago terrestris was analyzed by real-time fluorescence quantitative PCR. The results showed that the MtPUB1 gene expression was the highest in the flower and the lowest in the root. The expression of MtPUB2 decreased after induction of NaCl. The plant expression vectors pBI121-MtPUB1 and pBI121-MtPUB2 were constructed and transformed into Arabidopsis thaliana Atpub4 mutants by Agrobacterium tumefaciens. Transgenic Arabidopsis thaliana seeds of T1 generation of MtPUB1 were obtained by screening of Kana resistance and RT-PCR detection of pBI121-MtPUB2. The transformation of Arabidopsis mutants and wild type Arabidopsis pollen was observed by scanning electron microscope. Based on the analysis of the function of MtPUB gene, the main disambiguation sites of U-box and ARM were located on three conserved motifs, and the nine loci. 6 were located on the basis of this analysis. The results of sequence selection pressure of MtPUB gene showed that MtPUB gene remained functional after replication under strong negative selection pressure, and 114 negative selection sites were located at the same sequence as multiple sequences to obtain conserved sequences. It is speculated that this region can bind MtPUB protein to PREDEN specifically and thus produce different functions.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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