本文選題：甘藍(lán)型油菜 + BnaSOT基因�。� 參考：《湖南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：磺酸基轉(zhuǎn)移酶(SOT, EC 2.8.2.-)是在動(dòng)植物都廣泛存在的一類重要的蛋白家族,能夠催化磺酸基團(tuán)從供體到底物的轉(zhuǎn)移反應(yīng),使底物發(fā)生磺酸化。研究表明,擬南芥中有22個(gè)編碼磺酸基轉(zhuǎn)移酶(SOT)的基因(AtSOT),其中,AtSOT12基因的表達(dá)能夠被鹽、滲透壓力和激素誘導(dǎo)而大大加強(qiáng)。并且AtSOT12基因的T-DNA插入突變體(sot12)在發(fā)芽率上表現(xiàn)出對(duì)NaCl和脫落酸(ABA)的高度敏感性。本研究對(duì)SOT蛋白家族在白菜、甘藍(lán)和甘藍(lán)型油菜中的分布和分類情況進(jìn)行了系統(tǒng)的生物信息學(xué)分析。在此基礎(chǔ)上結(jié)合蛋白和基因結(jié)構(gòu)的分析,選取了3個(gè)甘藍(lán)型油菜BnaSOT12基因作為試驗(yàn)研究對(duì)象。構(gòu)建了BnaSOT12的過表達(dá)載體,并對(duì)擬南芥和甘藍(lán)型油菜進(jìn)行了遺傳轉(zhuǎn)化實(shí)驗(yàn)。獲得了如下試驗(yàn)結(jié)果：1.白菜基因組的分析表明：白菜中存在56個(gè)編碼BraSOT蛋白的基因,其中53個(gè)基因能夠分別被定位在白菜的染色體上�；蛟谌旧w上的分布并不均勻：A07號(hào)和A09號(hào)染色體含BraSOT基因最多,A05號(hào)染色體不包含BraSOT基因。56個(gè)基因能夠較好的與擬南芥一致被分類為9個(gè)亞家族。多數(shù)BraSOT基因表現(xiàn)出沒有內(nèi)含子的特征。2.甘藍(lán)基因組的分析表明：甘藍(lán)基因組存在33個(gè)編碼BolSOT蛋白的基因,分別定位到甘藍(lán)的9條染色體上,并且在C03號(hào)和C06號(hào)染色體上分別最為密集。33個(gè)BolSOT蛋白的分類很不均勻,第7類亞家族成員最多,沒有與擬南芥第4和第9類亞家族蛋白對(duì)應(yīng)的同源蛋白。多數(shù)BolSOT基因不含有內(nèi)含子,且各基因長度相差較大。3.甘藍(lán)型油菜基因組的分析表明：甘藍(lán)型油菜基因組存在有115個(gè)編碼BnaSOT蛋白的基因。這些基因在A07、A09、C06和C09號(hào)染色體上的分布最多。其中AtSOT12基因的同源基因有13個(gè),表明該基因在甘藍(lán)型油菜中進(jìn)行了復(fù)制和倍增,暗示其具有重要的生物學(xué)功能。進(jìn)而通過基因結(jié)構(gòu)和蛋白結(jié)構(gòu)的對(duì)比,確定了在結(jié)構(gòu)上與AtSOT12相似度最高的三個(gè)基因：BnaC02g35150D、BnaA09g53490D、BnaA09g19440D,分別命名為BnaSOT12a、BnaSOT12b、BnaSOT12c,作為試驗(yàn)研究對(duì)象。4.分別構(gòu)建了BnaSOT12a、BnaSOT12b、BnaSOT12c基因的過表達(dá)載體,獲得了BnaSOT12a的過表達(dá)植株。對(duì)突變體的抗鹽性實(shí)驗(yàn)表明,甘藍(lán)型油菜BnaSOT12a基因在擬南芥中的過表達(dá),能提高轉(zhuǎn)基因植株對(duì)NaCl脅迫的耐受能力。5.利用pFGC5941：:35S:BnaSOT12a載體對(duì)甘藍(lán)型油菜進(jìn)行了遺傳轉(zhuǎn)化,分別應(yīng)用了子葉柄農(nóng)桿菌浸染法和下胚軸農(nóng)桿菌浸染法兩種方法。子葉柄農(nóng)桿菌浸染法轉(zhuǎn)化湘油-15甘藍(lán)型油菜獲得9株抗性植株,轉(zhuǎn)化率為0.77%；下胚軸農(nóng)桿菌浸染法中使用了湘油-15甘藍(lán)型油菜和中雙-6甘藍(lán)型油菜兩種材料,總共獲得92株分化的抗性苗,在產(chǎn)生愈傷組織和分化出不定芽的過程中,中雙-6相對(duì)于湘油-15甘藍(lán)型油菜能表現(xiàn)出更強(qiáng)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.
【學(xué)位授予單位】：湖南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S565.4

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