農桿菌介導的抗草甘膦基因對玉米自交系18-599幼胚的遺傳轉化
發(fā)布時間：2018-09-07 09:05
【摘要】：玉米作為三大糧食作物之一,在飼料生產中扮演重要的角色,也為生物能源制造提供良好原材料；伴隨人口的增長、人民生活水平的提高,以及畜牧業(yè)的極速發(fā)展,對玉米需求量顯著增加。田間的雜草與玉米競爭陽光和肥料,形成草害,引起減產,嚴重制約了玉米產量的增加。人工撥草和機械除草費時費力且效果不理想；化學除草方便、經濟有效,業(yè)已成為現(xiàn)代農業(yè)技術重要組成部分。目前,全世界農業(yè)生產中使用量最大最廣泛的除草劑是草甘膦。由于草甘膦殺草譜比較寬,對玉米也具有滅生性作用,這就使在玉米生產中使用草甘膦受到限制。因此,培育出具有自主知識產權的抗草甘膦的玉米新品種,進行試驗、示范、推廣,能降低除草成本和勞動強度、提高工作效率、增強我國玉米的國際市場競爭力。 本研究主要是將抗草甘膦的抗性基因2mg2-epsps插入到植物表達載體pCAMBIA3301中,以農桿菌菌株EHA105攜帶含有目的基因的表達載體進行介導轉化,遺傳轉化受體材料為自交系18-599R的幼胚；實驗首先探討了熱激與離心對幼胚浸染的影響,并對農桿菌轉化玉米幼胚過程中幼胚大小、篩選劑濃度,頭孢霉素濃度等因素進行優(yōu)化,為建立良好的玉米幼胚轉化體系奠定基礎；獲得抗草甘膦轉基因玉米植株,并進行的PCR檢測與產物測序,結果表明目的基因已整合到玉米基因組中；對陽性植株進行葉面噴施草甘膦,結果表明轉基因玉米后代具有草甘膦抗性。主要研究結果如下： 1目的基因的獲得。以表達載體P35S-2301-EPSPS為模板,用高保真酶擴增獲得目的基因片段并測序,利用NCBI的在線軟件Blast、ORF Finder分析,結果表明,擴增的序列全長為1563bp,編碼464個氨基酸；與原始載體基因堿基序列同源性為99%,氨基酸序列匹配度為100%。 2表達載體構建。將構建好pMD19T-2mg2-epsps載體和pCAMBIA3301表達載體同時進行雙酶切,回收目的片段并進行連接轉化；經酶切、測序驗證結果表明所連接確實為目的片段,沒有發(fā)生突變。 3玉米幼胚遺傳轉化系統(tǒng)的優(yōu)化。首先探討了熱激與離心對幼胚浸染的影響,并對農桿菌轉化玉米幼胚過程中幼胚大小、頭孢霉素濃度、篩選劑濃度等因素設置不同水平處理,結果表明：最適合的處理條件為熱激40℃,3min,冷處理25℃,2min,不離心,此處理得到最高的抗性愈傷得率為52%；浸染的幼胚大小為0.8-1.3mm時,可以得到質地較好的愈傷組織；頭孢霉素為400mg/L的濃度的不僅可以抑制農桿菌生長,還可以保持幼胚良好的生長狀態(tài)；利用不同濃度的草甘膦對浸染的幼胚進行篩選,比較理想的篩選濃度為1.5-2.0mmol/L。 4轉基因苗的PCR鑒定。對96株To代再生植株進行多次重復的PCR鑒定,15株再生植株的基因組DNA擴增出與陽性對照大小一致的的片段,表明目的基因已經成功轉入玉米基因組中；T1代株系進行PCR檢測,獲得四株轉基因株系,這些陽性的株系經RT-PCR檢測,表明抗草甘膦基因在mRNA水平上得到了有效轉錄；對陽性植株進行葉面噴施草甘膦,結果表明轉基因玉米后代具有草甘膦抗性。。
[Abstract]:As one of the three major grain crops, maize plays an important role in feed production and provides good raw materials for bioenergy production. With the growth of population, the improvement of people's living standards and the rapid development of animal husbandry, the demand for maize has increased significantly. At present, glyphosate is the most widely used herbicide in agricultural production in the world. Because of the comparison of glyphosate herbicidal spectrum, it has become an important part of modern agricultural technology. Wide, also has a sterile effect on corn, which limits the use of glyphosate in Maize production. Therefore, cultivating new varieties of corn with independent intellectual property rights of glyphosate resistance, testing, demonstration, promotion, can reduce the cost of weeding and labor intensity, improve work efficiency, enhance the competitiveness of China's Maize in the international market.
In this study, the glyphosate-resistant gene 2mg 2-epsps was inserted into the plant expression vector pCAMBIA 3301. Agrobacterium strain EHA105 carrying the expression vector containing the target gene was used to mediate the transformation, and the genetic transformation receptor material was the immature embryo of inbred line 18-599R. The size of immature embryo, the concentration of screening agent and cephalosporin in the process of transforming maize immature embryo by Agrobacterium tumefaciens were optimized to lay a foundation for establishing a good system of transforming maize immature embryo. Glyphosate was sprayed on the leaves of the positive plants. The results showed that the progeny of transgenic maize had glyphosate resistance.
1. The target gene was obtained by using P35S-2301-EPSPS as the template and amplified by high-fidelity enzymes. The sequence was analyzed by NCBI on-line software Blast and ORF Finder. The results showed that the amplified sequence was 1 563 BP in length and encoded 464 amino acids. The homology with the base sequence of the original vector gene was 99%, and the amino acid sequence was 99%. The matching degree is 100%.
The pMD19T-2mg 2-epsps vector and the pCAMBIA3301 expression vector were digested by double enzyme digestion, and the target fragment was recovered and transformed. The results of enzyme digestion and sequencing showed that the fragment was indeed the target fragment, and no mutation occurred.
3. Optimization of genetic transformation system of immature embryo of maize. Firstly, the effect of heat shock and centrifugation on immature embryo dissemination was discussed, and different levels of treatment were set for immature embryo size, cephalosporin concentration, screening agent concentration and other factors in the process of transformation of immature embryo of Maize by Agrobacterium tumefaciens. The results showed that the most suitable treatment conditions were heat shock 40 C, 3 min, cold treatment 25 C Without centrifugation, the highest yield of resistant callus was 52%; when the immature embryos were 0.8-1.3 mm in size, the callus with better texture could be obtained; the concentration of cephalosporin 400 mg/L could not only inhibit the growth of Agrobacterium tumefaciens, but also keep the immature embryos in good growth state; different concentrations of glyphosate could be used to immerse the immature embryos. The embryos were screened with an ideal screening concentration of 1.5-2.0mmol/L..
4. PCR identification of transgenic seedlings. Repeated PCR identification of 96 Togeneration regenerated plants showed that the genomic DNA of 15 regenerated plants produced fragments of the same size as the positive control, indicating that the target gene had been successfully transferred into the maize genome. Four transgenic lines were obtained from T1 generation by PCR detection. RT-PCR showed that glyphosate-resistant genes were effectively transcribed at the mRNA level, and glyphosate was sprayed on the leaves of the positive plants. The results showed that the progenies of transgenic maize had glyphosate resistance.
【學位授予單位】：四川農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：S513
[Abstract]:As one of the three major grain crops, maize plays an important role in feed production and provides good raw materials for bioenergy production. With the growth of population, the improvement of people's living standards and the rapid development of animal husbandry, the demand for maize has increased significantly. At present, glyphosate is the most widely used herbicide in agricultural production in the world. Because of the comparison of glyphosate herbicidal spectrum, it has become an important part of modern agricultural technology. Wide, also has a sterile effect on corn, which limits the use of glyphosate in Maize production. Therefore, cultivating new varieties of corn with independent intellectual property rights of glyphosate resistance, testing, demonstration, promotion, can reduce the cost of weeding and labor intensity, improve work efficiency, enhance the competitiveness of China's Maize in the international market.
In this study, the glyphosate-resistant gene 2mg 2-epsps was inserted into the plant expression vector pCAMBIA 3301. Agrobacterium strain EHA105 carrying the expression vector containing the target gene was used to mediate the transformation, and the genetic transformation receptor material was the immature embryo of inbred line 18-599R. The size of immature embryo, the concentration of screening agent and cephalosporin in the process of transforming maize immature embryo by Agrobacterium tumefaciens were optimized to lay a foundation for establishing a good system of transforming maize immature embryo. Glyphosate was sprayed on the leaves of the positive plants. The results showed that the progeny of transgenic maize had glyphosate resistance.
1. The target gene was obtained by using P35S-2301-EPSPS as the template and amplified by high-fidelity enzymes. The sequence was analyzed by NCBI on-line software Blast and ORF Finder. The results showed that the amplified sequence was 1 563 BP in length and encoded 464 amino acids. The homology with the base sequence of the original vector gene was 99%, and the amino acid sequence was 99%. The matching degree is 100%.
The pMD19T-2mg 2-epsps vector and the pCAMBIA3301 expression vector were digested by double enzyme digestion, and the target fragment was recovered and transformed. The results of enzyme digestion and sequencing showed that the fragment was indeed the target fragment, and no mutation occurred.
3. Optimization of genetic transformation system of immature embryo of maize. Firstly, the effect of heat shock and centrifugation on immature embryo dissemination was discussed, and different levels of treatment were set for immature embryo size, cephalosporin concentration, screening agent concentration and other factors in the process of transformation of immature embryo of Maize by Agrobacterium tumefaciens. The results showed that the most suitable treatment conditions were heat shock 40 C, 3 min, cold treatment 25 C Without centrifugation, the highest yield of resistant callus was 52%; when the immature embryos were 0.8-1.3 mm in size, the callus with better texture could be obtained; the concentration of cephalosporin 400 mg/L could not only inhibit the growth of Agrobacterium tumefaciens, but also keep the immature embryos in good growth state; different concentrations of glyphosate could be used to immerse the immature embryos. The embryos were screened with an ideal screening concentration of 1.5-2.0mmol/L..
4. PCR identification of transgenic seedlings. Repeated PCR identification of 96 Togeneration regenerated plants showed that the genomic DNA of 15 regenerated plants produced fragments of the same size as the positive control, indicating that the target gene had been successfully transferred into the maize genome. Four transgenic lines were obtained from T1 generation by PCR detection. RT-PCR showed that glyphosate-resistant genes were effectively transcribed at the mRNA level, and glyphosate was sprayed on the leaves of the positive plants. The results showed that the progenies of transgenic maize had glyphosate resistance.
【學位授予單位】：四川農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：S513
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