本文選題：GmDof4 + GmDof11��； 參考：《揚州大學》2017年碩士論文

【摘要】：油菜是重要的油料作物,改善菜籽油的品質(zhì)和產(chǎn)量是油菜育種的一個重要方向。GmDof4和GmDof11基因是從大豆中克隆的與油脂合成相關(guān)的Dof(DNA binding with one finger)轉(zhuǎn)錄因子,參與大豆、擬南芥等種子油脂合成和積累,同時可能參與植物逆境脅迫應(yīng)答的調(diào)控。本研究以甘藍型油菜“揚油6號”為材料,利用農(nóng)桿菌介導的轉(zhuǎn)基因技術(shù),將GmDof4和GmDof11基因?qū)敫仕{型油菜中,分析GmDof4和GmDof11基因的導入對各株系種子品質(zhì)性狀的影響。主要研究結(jié)果如下:1.對以子葉為外植體的甘藍型油菜遺傳轉(zhuǎn)化方法進行了優(yōu)化。“揚油6號”子葉外植體的愈傷分化率達到94.9%,生苗率達到71.4%。以“揚油6號”子葉外植體進行遺傳轉(zhuǎn)化時,最適Kan篩選濃度為40 mg/L。使用含有GUS報告基因的pCambia-1303載體轉(zhuǎn)化子葉外植體,并利用潮霉素作為篩選劑進行轉(zhuǎn)基因苗的篩選。結(jié)果證明在侵染后第1周時各外植體的傷口以及子葉和子葉柄處已經(jīng)出現(xiàn)GUS的表達,而對生長4周的顏色正常且生長健壯的愈傷組織進行GUS染色后發(fā)現(xiàn)愈傷組織的大部分細胞為陽性轉(zhuǎn)化細胞。另外,我們獲得的11個35S:GUS轉(zhuǎn)基因苗中,6株為陽性,陽性率為54.5%。2.利用以子葉為外植體的轉(zhuǎn)基因方法進行了 2個GmDof基因過表達載體的遺傳轉(zhuǎn)化,對獲得的再生苗進行PCR及半定量RT-PCR鑒定。轉(zhuǎn)pBin438/GmDof4載體獲得的20株再生苗中PCR為陽性的有9株,其中6株檢測到表達;轉(zhuǎn)pBin438/GmDof11載體獲得的21株再生苗中有8株P(guān)CR呈陽性,其中有3株檢測到表達。3.利用近紅外光譜技術(shù)(NIRS)對獲得的陽性轉(zhuǎn)基因株系T2代種子進行營養(yǎng)成分的測定,與對照相比GmDof4和GmDof11轉(zhuǎn)基因株系種子中油脂、硫苷含量沒有顯著變化,其中GmDof4的一個過表達株系種子蛋白含量明顯降低。NIRS以及高效氣相色譜(GC)分析結(jié)果顯示各轉(zhuǎn)基因株系的脂肪酸比例發(fā)生了變化:與對照相比,轉(zhuǎn)基因株系中油酸含量均明顯升高,亞油酸和亞麻酸含量降低。4.利用qPCR分析了與蛋白和脂肪酸合成相關(guān)的基因表達,結(jié)果顯示accD基因的表達可以被GmDof4和GmDof11激活,但LACS表達無變化;過表達GmDof4可以誘導FAB2、FAD8的表達;而過表達GmDof11可以抑制FAD2、FAD6的表達;此外CRA1基因的表達可能也受到GmDof4的誘導。5.利用酵母單雜交技術(shù)驗證上述候選基因是否受GmDof蛋白的直接調(diào)控,結(jié)果發(fā)現(xiàn),GmDof4蛋白可以與aCCD、FAB2、FAD8基因上的啟動子元件互作;GmDof11蛋白可以與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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S565.4
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本文編號：1770207