过表达GmDof4和GmDof11基因对甘蓝型油菜种子脂肪酸组分的影响

发布时间：2018-04-18 21:41

  本文选题：GmDof4 + GmDof11　； 参考：《扬州大学》2017年硕士论文

【摘要】：油菜是重要的油料作物,改善菜籽油的品质和产量是油菜育种的一个重要方向。GmDof4和GmDof11基因是从大豆中克隆的与油脂合成相关的Dof(DNA binding with one finger)转录因子,参与大豆、拟南芥等种子油脂合成和积累,同时可能参与植物逆境胁迫应答的调控。本研究以甘蓝型油菜“扬油6号”为材料,利用农杆菌介导的转基因技术,将GmDof4和GmDof11基因导入甘蓝型油菜中,分析GmDof4和GmDof11基因的导入对各株系种子品质性状的影响。主要研究结果如下:1.对以子叶为外植体的甘蓝型油菜遗传转化方法进行了优化。“扬油6号”子叶外植体的愈伤分化率达到94.9%,生苗率达到71.4%。以“扬油6号”子叶外植体进行遗传转化时,最适Kan筛选浓度为40 mg/L。使用含有GUS报告基因的pCambia-1303载体转化子叶外植体,并利用潮霉素作为筛选剂进行转基因苗的筛选。结果证明在侵染后第1周时各外植体的伤口以及子叶和子叶柄处已经出现GUS的表达,而对生长4周的颜色正常且生长健壮的愈伤组织进行GUS染色后发现愈伤组织的大部分细胞为阳性转化细胞。另外,我们获得的11个35S:GUS转基因苗中,6株为阳性,阳性率为54.5%。2.利用以子叶为外植体的转基因方法进行了 2个GmDof基因过表达载体的遗传转化,对获得的再生苗进行PCR及半定量RT-PCR鉴定。转pBin438/GmDof4载体获得的20株再生苗中PCR为阳性的有9株,其中6株检测到表达;转pBin438/GmDof11载体获得的21株再生苗中有8株PCR呈阳性,其中有3株检测到表达。3.利用近红外光谱技术(NIRS)对获得的阳性转基因株系T2代种子进行营养成分的测定,与对照相比GmDof4和GmDof11转基因株系种子中油脂、硫苷含量没有显著变化,其中GmDof4的一个过表达株系种子蛋白含量明显降低。NIRS以及高效气相色谱(GC)分析结果显示各转基因株系的脂肪酸比例发生了变化:与对照相比,转基因株系中油酸含量均明显升高,亚油酸和亚麻酸含量降低。4.利用qPCR分析了与蛋白和脂肪酸合成相关的基因表达,结果显示accD基因的表达可以被GmDof4和GmDof11激活,但LACS表达无变化;过表达GmDof4可以诱导FAB2、FAD8的表达;而过表达GmDof11可以抑制FAD2、FAD6的表达;此外CRA1基因的表达可能也受到GmDof4的诱导。5.利用酵母单杂交技术验证上述候选基因是否受GmDof蛋白的直接调控,结果发现,GmDof4蛋白可以与aCCD、FAB2、FAD8基因上的启动子元件互作;GmDof11蛋白可以与CRA1、FAD2、FAD6基因互作。
[Abstract]:Rapeseed is an important oil crop. Improving the quality and yield of rapeseed oil is an important direction of rapeseed breeding. GmDof4 and GmDof11 genes are Dof(DNA binding with one fingerings related to oil synthesis cloned from soybean and participate in soybean.Oil synthesis and accumulation in Arabidopsis thaliana seeds may be involved in the regulation of plant stress response.In this study, GmDof4 and GmDof11 genes were introduced into Brassica napus by Agrobacterium tumefaciens-mediated transgenic technique, and the effects of GmDof4 and GmDof11 on seed quality were analyzed.The main results are as follows: 1.The genetic transformation method of Brassica napus with cotyledon as explant was optimized.The callus differentiation rate of "Yangyou 6" cotyledon explant reached 94.9%, and the seedling growth rate reached 71.4%.When the cotyledon explant "Yangyou 6" was used for genetic transformation, the optimal concentration of Kan was 40 mg / L.Cotyledon explants were transformed by pCambia-1303 vector containing GUS reporter gene and hygromycin was used as screening agent to screen transgenic seedlings.The results showed that the expression of GUS was found in the wound and cotyledon and cotyledon petiole of the explants at the first week after infection.GUS staining showed that most of the callus cells were positive transformed cells.In addition, 6 of the 11 35S:GUS transgenic plants were positive, and the positive rate was 54.5.The genetic transformation of two GmDof gene overexpression vectors was carried out by using cotyledons as explants. The regenerated seedlings were identified by PCR and semi-quantitative RT-PCR.Of the 20 regenerated seedlings obtained by pBin438/GmDof4 vector, 9 were positive for PCR, 6 were positive for PCR expression, and 8 were positive for PCR among 21 regenerated seedlings obtained by pBin438/GmDof11 vector, among which 3 were positive.Compared with the control, the content of glucosinolate in the seeds of GmDof4 and GmDof11 transgenic lines was not significantly changed by using near infrared spectroscopy (NIR) to determine the nutritional composition of the T2 generation seeds of the positive transgenic lines, and the results showed that there was no significant change in the content of glucosinolate in the seeds of GmDof4 and GmDof11 transgenic lines.The results of the analysis of seed protein content of one GmDof4 overexpression line and high performance gas chromatography (GC) showed that the fatty acid ratio of the transgenic lines had changed: compared with the control, the oleic acid content of the transgenic lines was significantly higher than that of the control lines, and the results showed that the content of oleic acid in the transgenic lines was significantly higher than that in the control lines.The contents of linoleic acid and linolenic acid decreased by .4.QPCR was used to analyze the expression of genes related to protein and fatty acid synthesis. The results showed that the expression of accD gene could be activated by GmDof4 and GmDof11, but the expression of LACS was not changed, overexpression of GmDof4 could induce the expression of FAB2nFAD8, and overexpression of GmDof11 could inhibit the expression of FAD2 and FAD6.In addition, the expression of CRA1 gene may also be induced by GmDof4.Yeast single hybrid technique was used to verify whether the candidate gene was directly regulated by GmDof protein. It was found that the GmDof4 protein could interact with the promoter element of the FAB2FAD8 gene. The GmDof11 protein could interact with the CRA1 / FAD2 / FAD6 gene.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.4
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本文编号：1770207