本文選題：甘藍(lán)型油菜 + NST3　； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：油菜是世界上分布最廣的油料作物之一,也是我國(guó)第一大油料作物。在我國(guó),菌核病和油菜倒伏是限制油菜產(chǎn)量最主要的因素。木質(zhì)素的含量的多少跟作物的抗病性、抗倒伏能力有關(guān)。植物細(xì)胞木質(zhì)化提高細(xì)胞壁的強(qiáng)度,不僅可以增強(qiáng)植物抗病性,也可以增強(qiáng)抗倒伏能力。油菜生長(zhǎng)后期是倒伏易發(fā)生的時(shí)期,也是菌核病的高發(fā)期,兩者變現(xiàn)為交互作用。前期研究發(fā)現(xiàn)在油菜莖稈中,S型木質(zhì)素單體含量越高,植物抗倒伏能力越強(qiáng),并且對(duì)菌核病的抗性也越強(qiáng)。本研究論文主要包括首先對(duì)甘藍(lán)型油菜不同器官的NST3基因的四個(gè)成員進(jìn)行表達(dá)分析,在線對(duì)四個(gè)成員進(jìn)行基因結(jié)構(gòu)分析,并對(duì)其編碼的蛋白進(jìn)行生物信息學(xué)分析,構(gòu)建NSR3家族的CRISPR-Cas9基因編輯系統(tǒng),構(gòu)建BnaA09g24190D成員的超表達(dá)載體。成功構(gòu)建載體后,將其轉(zhuǎn)化甘藍(lán)型油菜,并得到陽(yáng)性的轉(zhuǎn)基因苗。提取油菜轉(zhuǎn)基因苗DNA,設(shè)計(jì)特異引物擴(kuò)片段及測(cè)序。本實(shí)驗(yàn)的主要結(jié)果如下:1、油菜的NST3基因有四個(gè)成員,分別是BnaA05g17950D、BnaA09g24190D、BnaC05g24890D和BnaC05g28390D。四個(gè)成員中,BnaA09g24190D、BnaC05g24890D的表達(dá)量均高于其他兩個(gè)成員,在根、莖、花蕾花、角果和種子中BnaA09g24190D的表達(dá)量均比BnaC05g24890D高,在根中,BnaA09g24190D的表達(dá)量幾乎是BnaC05g24890D的5倍。在莖中,BnaA09g24190D的表達(dá)量幾乎是BnaC05g24890D的2.5倍。四個(gè)成員中,BnaA05g17950D、BnaC05g28390D的表達(dá)量較低,在子葉、葉和花蕾均不表達(dá),在其他部位的表達(dá)量也較低。通過基因的表達(dá)量分析,可以推測(cè),在油菜的這四個(gè)成員中,BnaA09g24190D、BnaC05g24890D在調(diào)節(jié)油菜次生細(xì)胞壁合成和調(diào)控木質(zhì)素單體組分起主要作用,尤其是BnaA09g24190D。2、對(duì)BnaA05g17950D、BnaA09g24190D、BnaC05g24890D和BnaC05g28390D在線進(jìn)行基因結(jié)構(gòu)分析。BnaA05g17950D全長(zhǎng)1819bp,CDS長(zhǎng)927bp,該基因有4個(gè)外顯子,3個(gè)內(nèi)含子;BnaA09g24190D全長(zhǎng)1776bp,CDS長(zhǎng)1095bp,該基因有3個(gè)外顯子,2個(gè)內(nèi)含子;BnaC05g24890D全長(zhǎng)1832bp,CDS長(zhǎng)1116bp,3個(gè)外顯子,2個(gè)內(nèi)含子;BnaC05g28390D全長(zhǎng)為2146bp,CDS長(zhǎng)978bp,5個(gè)外顯子,4個(gè)內(nèi)含子。對(duì)BnaA05g17950D、BnaA09g24190D、BnaC05g24890D和BnaC05g28390D編碼的蛋白質(zhì)進(jìn)行分析,分別含308,364,371,325個(gè)氨基酸,分子量在35.75~42.99KD之間,等電點(diǎn)均小于7,二級(jí)結(jié)構(gòu)中,α-螺旋占24.45%~29.54%,β-轉(zhuǎn)角占8.36%~8.77%,隨機(jī)卷曲占40.62%~48.35%,延伸鏈占17.25%~21.23%。它們都具有NAM典型特征的保守域,根據(jù)它們的保守域能夠預(yù)測(cè)出三級(jí)結(jié)構(gòu)。3、本實(shí)驗(yàn)共得到22株陽(yáng)性轉(zhuǎn)基因苗,在轉(zhuǎn)基因苗中,不同的基因成員的編輯情況也各不相同。在22株基因陽(yáng)性植物中,只有2株的BnaA05g17950D得到編輯,編輯的效率為9.09%。BnaA09g24190D在11株油菜中都得到編輯,編輯的效率為50%。BnaC05g24890D在8株轉(zhuǎn)基因油菜中都得到編輯,編輯的效率為36.4%。BnaC05g28390D在4株轉(zhuǎn)基因油菜中得到編輯,編輯的效率為18.2%。第3、22株油菜轉(zhuǎn)基因苗四個(gè)基因同時(shí)被編輯,第1、7株的BnaA09g24190D、BnaC05g24890D同時(shí)被編輯,第9株的油菜轉(zhuǎn)基因苗的BnaA09g24190D、BnaC05g24890D、BnaC05g28390D都被編輯,第12株轉(zhuǎn)基因油菜的BnaC05g24890D、BnaC05g28390D同時(shí)被編輯,第17株的轉(zhuǎn)基因油菜的BnaA09g24190D、BnaC05g24890D同時(shí)被編輯。CRISPR-Cas9編輯得到結(jié)果可分為三類:靶位點(diǎn)及其附近堿基缺失、插入、既有缺失又有插入。4、在四個(gè)成員中,BnaA09g24190D在調(diào)節(jié)油菜次生細(xì)胞壁合成和調(diào)控木質(zhì)素單體組分起最主要作用,因而本實(shí)驗(yàn)構(gòu)建了BnaA09g24190D的超量表達(dá)載體,并轉(zhuǎn)化油菜。本實(shí)驗(yàn)的后續(xù)工作,CRISPR-Cas9編輯和超表達(dá)的轉(zhuǎn)基因苗的木質(zhì)素單體含量變化情況、對(duì)抗菌核病的抗感情況和抗倒伏情況需要實(shí)驗(yàn)室的相關(guān)人員跟進(jìn)。
[Abstract]:Rape is one of the most widely distributed oil crops in the world, and it is also the largest oil crop in China. In China, sclerotius disease and rape lodging are the most important factors to limit the yield of rape. The content of lignin is related to the resistance and lodging resistance of the crops. The resistance to disease can also enhance the ability to resist lodging. The late growth of rape is a period of prone to lodging and also a period of high incidence of Sclerotinia. The earlier studies have found that the higher the content of S lignin monomers, the stronger the lodging resistance of plants and the stronger the resistance to Sclerotinia. To include the analysis of four members of the NST3 gene of different organs of Brassica napus (Brassica napus), four members were analyzed on line, and the encoded proteins were analyzed by bioinformatics. The CRISPR-Cas9 gene editing system of the NSR3 family was constructed, and the overexpression vector of BnaA09g24190D members was constructed. The main results of this experiment are as follows: 1, the NST3 gene of rapeseed has four members, which are four members of BnaA05g17950D, BnaA09g24190D, BnaC05g24890D and BnaC05g28390D., BnaA09g24190D, Bna, respectively. The expression of C05g24890D is higher than that of the other two members. The expression of BnaA09g24190D in root, stem, bud flower, pod and seed is higher than that of BnaC05g24890D. In the root, the expression of BnaA09g24190D is almost 5 times as much as BnaC05g24890D. In the stem, the expression of BnaA09g24190D is almost 2.5 times that of BnaC05g24890D. Among four members, BnaA05g17950D, The expression of BnaC05g28390D is low, not expressed in cotyledon, leaves and buds, and is also low in other parts. Through the analysis of gene expression, it is possible to speculate that in these four members of rape, BnaA09g24190D and BnaC05g24890D play a major role in regulating the secondary cell wall of rape and regulating the lignin monomer components, especially Bn. AA09g24190D.2, BnaA05g17950D, BnaA09g24190D, BnaC05g24890D and BnaC05g28390D are on line gene structural analysis.BnaA05g17950D full length 1819bp, CDS long 927bp, the gene has 4 exons and 3 introns, BnaA09g24190D full length 1776bp, CDS long, this gene has 3 exons and 2 introns. P, 3 exons and 2 introns, the full length of BnaC05g28390D is 2146bp, CDS long 978bp, 5 exons and 4 introns. BnaA05g17950D, BnaA09g24190D, BnaC05g24890D and BnaC05g28390D encoded proteins are analyzed with 308364371325 amino acids, the molecular weight is between 35.75~42.99KD, and the isoelectric point is less than 7, and the two structure, alpha - The spiral accounts for 24.45%~29.54%, the beta angle is 8.36%~8.77%, the random curls account for 40.62%~48.35%, the extension chain accounts for the conservative domain with the typical NAM characteristics of the 17.25%~21.23%., and the three stage structure.3 can be predicted according to their conservative domain. This experiment results in a total of 22 positive transgenic seedlings, and the editing of different gene members in the transgenic seedlings is also the same. Of the 22 gene positive plants, only 2 of the BnaA05g17950D were edited, and the efficiency of the editing was 9.09%.BnaA09g24190D in 11 rapeseed. The efficiency of editors was edited in 8 transgenic rapeseed. The efficiency of the editing was that the efficiency of the editing was to be compiled in 4 transgenic rapeseed. Editing, the efficiency of editing is that four genes of 18.2%. 3,22 plant transgenic seedlings were edited simultaneously, the BnaA09g24190D of the 1,7 strain, BnaC05g24890D were edited simultaneously, the BnaA09g24190D, BnaC05g24890D, BnaC05g28390D of the ninth transgenic rape seedlings were edited, the BnaC05g24890D of twelfth transgenic rapeseed, BnaC05g28390D were edited at the same time, seventeenth The results of BnaA09g24190D and BnaC05g24890D edited.CRISPR-Cas9 in transgenic rapeseed can be divided into three categories: target site and its adjacent base deletion, insertion, both deletion and insertion of.4, and in the four members, BnaA09g24190D plays the most important role in regulating the secondary cell wall synthesis and regulating the lignin component of the rape. In this experiment, the overexpression vector of BnaA09g24190D was constructed and rapeseed was transformed. The follow-up work of this experiment, the changes of lignin content in the CRISPR-Cas9 edited and overexpressed transgenic seedlings, the resistance to the Sclerotinia and the resistance to lodging need to be followed up by the related personnel in the laboratory.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2;S565.4

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