甘蓝型油菜BnaSOT12基因在植物抗逆中的作用研究

发布时间：2018-04-21 00:30

本文选题：甘蓝型油菜 + BnaSOT基因　；参考：《湖南农业大学》2016年硕士论文

【摘要】：磺酸基转移酶(SOT, EC 2.8.2.-)是在动植物都广泛存在的一类重要的蛋白家族,能够催化磺酸基团从供体到底物的转移反应,使底物发生磺酸化。研究表明,拟南芥中有22个编码磺酸基转移酶(SOT)的基因(AtSOT),其中,AtSOT12基因的表达能够被盐、渗透压力和激素诱导而大大加强。并且AtSOT12基因的T-DNA插入突变体(sot12)在发芽率上表现出对NaCl和脱落酸(ABA)的高度敏感性。本研究对SOT蛋白家族在白菜、甘蓝和甘蓝型油菜中的分布和分类情况进行了系统的生物信息学分析。在此基础上结合蛋白和基因结构的分析,选取了3个甘蓝型油菜BnaSOT12基因作为试验研究对象。构建了BnaSOT12的过表达载体,并对拟南芥和甘蓝型油菜进行了遗传转化实验。获得了如下试验结果：1.白菜基因组的分析表明：白菜中存在56个编码BraSOT蛋白的基因,其中53个基因能够分别被定位在白菜的染色体上。基因在染色体上的分布并不均匀：A07号和A09号染色体含BraSOT基因最多,A05号染色体不包含BraSOT基因。56个基因能够较好的与拟南芥一致被分类为9个亚家族。多数BraSOT基因表现出没有内含子的特征。2.甘蓝基因组的分析表明：甘蓝基因组存在33个编码BolSOT蛋白的基因,分别定位到甘蓝的9条染色体上,并且在C03号和C06号染色体上分别最为密集。33个BolSOT蛋白的分类很不均匀,第7类亚家族成员最多,没有与拟南芥第4和第9类亚家族蛋白对应的同源蛋白。多数BolSOT基因不含有内含子,且各基因长度相差较大。3.甘蓝型油菜基因组的分析表明：甘蓝型油菜基因组存在有115个编码BnaSOT蛋白的基因。这些基因在A07、A09、C06和C09号染色体上的分布最多。其中AtSOT12基因的同源基因有13个,表明该基因在甘蓝型油菜中进行了复制和倍增,暗示其具有重要的生物学功能。进而通过基因结构和蛋白结构的对比,确定了在结构上与AtSOT12相似度最高的三个基因：BnaC02g35150D、BnaA09g53490D、BnaA09g19440D,分别命名为BnaSOT12a、BnaSOT12b、BnaSOT12c,作为试验研究对象。4.分别构建了BnaSOT12a、BnaSOT12b、BnaSOT12c基因的过表达载体,获得了BnaSOT12a的过表达植株。对突变体的抗盐性实验表明,甘蓝型油菜BnaSOT12a基因在拟南芥中的过表达,能提高转基因植株对NaCl胁迫的耐受能力。5.利用pFGC5941：:35S:BnaSOT12a载体对甘蓝型油菜进行了遗传转化,分别应用了子叶柄农杆菌浸染法和下胚轴农杆菌浸染法两种方法。子叶柄农杆菌浸染法转化湘油-15甘蓝型油菜获得9株抗性植株,转化率为0.77%；下胚轴农杆菌浸染法中使用了湘油-15甘蓝型油菜和中双-6甘蓝型油菜两种材料,总共获得92株分化的抗性苗,在产生愈伤组织和分化出不定芽的过程中,中双-6相对于湘油-15甘蓝型油菜能表现出更强的分化能力。
[Abstract]:Sulfonic acid transferase (EC 2.8.2.-) is an important protein family which exists widely in animals and plants. It can catalyze the transfer reaction of sulfonic groups from the donor to the end and make the substrates sulfonate. In Arabidopsis thaliana, there are 22 genes encoding SOT, in which the expression of AtSOT12 gene can be greatly enhanced by salt, osmotic pressure and hormone induction. Moreover, the T-DNA insertion mutant of AtSOT12 gene was highly sensitive to NaCl and abscisic acid (Aba) in germination rate. The distribution and classification of SOT protein family in Chinese cabbage, cabbage and Brassica napus were systematically analyzed by bioinformatics. Based on the analysis of protein and gene structure, three BnaSOT12 genes of Brassica napus were selected. The overexpression vector of BnaSOT12 was constructed and genetic transformation of Arabidopsis thaliana and Brassica napus was carried out. The following results are obtained: 1: 1. The genome analysis of Chinese cabbage showed that 56 genes encoding BraSOT protein existed in Chinese cabbage, 53 of which could be located on the chromosomes of Chinese cabbage. The distribution of the genes on chromosomes was not uniform. Chromosome A05 contained the most BraSOT genes, and chromosome A05 did not contain BraSOT genes. 56 genes could be classified into 9 subfamilies in good agreement with Arabidopsis thaliana (Arabidopsis thaliana). Most BraSOT genes showed no intron characteristics. 2. The analysis of cabbage genome showed that there were 33 genes encoding BolSOT protein in cabbage genome, which were located on 9 chromosomes of cabbage, and were the most dense on chromosome C03 and C06, respectively. The classification of 33 BolSOT proteins was very uneven. The 7 th subfamily had the most members and had no homologous proteins corresponding to the 4 and 9 subfamily proteins of Arabidopsis thaliana. Most BolSOT genes do not contain introns, and the length of each gene varies by 3. 3. The genome analysis of Brassica napus showed that there were 115 genes encoding BnaSOT protein in Brassica napus genome. These genes were most distributed on chromosomes A 07, A 09, C 06 and C 09. There were 13 homologous genes of AtSOT12 gene, which indicated that the gene was duplicated and multiplied in Brassica napus, suggesting that it had important biological function. Through the comparison of gene structure and protein structure, three genes, BnaC02g35150D, BnaA09g53490Dand BnaA09g19440D, were identified as BnaSOT12bnBnaSOT12BnaSOT12c. The overexpression vectors of BnaSOT12b BnaSOT12b BnaSOT12c gene were constructed, and the overexpression plants of BnaSOT12a were obtained. The salt-tolerance experiment of mutant showed that the overexpression of BnaSOT12a gene in Arabidopsis thaliana could improve the tolerance of transgenic plants to NaCl stress. The genetic transformation of Brassica napus was carried out by pFGC5941::35S:BnaSOT12a vector. Two methods, Agrobacterium tumefaciens soaking in cotyledon petiole and Agrobacterium tumefaciens in hypocotyls, were used. Nine resistant plants were obtained by Agrobacterium tumefaciens soaking method, and the transformation rate was 0.77. Two kinds of materials, Xiangyou -15 Brassica napus and medium double -6 Brassica napus were used in Hypocotyl Agrobacterium tumefaciens soaking method. A total of 92 differentiated resistant seedlings were obtained. In the process of callus formation and adventitious bud differentiation, medium and double -6 showed stronger differentiation ability than Xiangyou-15 Brassica napus.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S565.4

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