農(nóng)桿菌介導(dǎo)的抗草甘膦基因?qū)τ衩鬃越幌?8-599幼胚的遺傳轉(zhuǎn)化
發(fā)布時(shí)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(chǎn)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(zhǎng)、人民生活水平的提高,以及畜牧業(yè)的極速發(fā)展,對(duì)玉米需求量顯著增加。田間的雜草與玉米競(jìng)爭(zhēng)陽(yáng)光和肥料,形成草害,引起減產(chǎn),嚴(yán)重制約了玉米產(chǎn)量的增加。人工撥草和機(jī)械除草費(fèi)時(shí)費(fèi)力且效果不理想；化學(xué)除草方便、經(jīng)濟(jì)有效,業(yè)已成為現(xiàn)代農(nóng)業(yè)技術(shù)重要組成部分。目前,全世界農(nóng)業(yè)生產(chǎn)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(duì)玉米也具有滅生性作用,這就使在玉米生產(chǎn)中使用草甘膦受到限制。因此,培育出具有自主知識(shí)產(chǎn)權(quán)的抗草甘膦的玉米新品種,進(jìn)行試驗(yàn)、示范、推廣,能降低除草成本和勞動(dòng)強(qiáng)度、提高工作效率、增強(qiáng)我國(guó)玉米的國(guó)際市場(chǎng)競(jìng)爭(zhēng)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(dá)載體pCAMBIA3301中,以農(nóng)桿菌菌株EHA105攜帶含有目的基因的表達(dá)載體進(jìn)行介導(dǎo)轉(zhuǎn)化,遺傳轉(zhuǎn)化受體材料為自交系18-599R的幼胚；實(shí)驗(yàn)首先探討了熱激與離心對(duì)幼胚浸染的影響,并對(duì)農(nóng)桿菌轉(zhuǎn)化玉米幼胚過(guò)程中幼胚大小、篩選劑濃度,頭孢霉素濃度等因素進(jìn)行優(yōu)化,為建立良好的玉米幼胚轉(zhuǎn)化體系奠定基礎(chǔ)；獲得抗草甘膦轉(zhuǎn)基因玉米植株,并進(jìn)行的PCR檢測(cè)與產(chǎn)物測(cè)序,結(jié)果表明目的基因已整合到玉米基因組中；對(duì)陽(yáng)性植株進(jìn)行葉面噴施草甘膦,結(jié)果表明轉(zhuǎn)基因玉米后代具有草甘膦抗性。主要研究結(jié)果如下： 1目的基因的獲得。以表達(dá)載體P35S-2301-EPSPS為模板,用高保真酶擴(kuò)增獲得目的基因片段并測(cè)序,利用NCBI的在線軟件Blast、ORF Finder分析,結(jié)果表明,擴(kuò)增的序列全長(zhǎng)為1563bp,編碼464個(gè)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(dá)載體構(gòu)建。將構(gòu)建好pMD19T-2mg2-epsps載體和pCAMBIA3301表達(dá)載體同時(shí)進(jìn)行雙酶切,回收目的片段并進(jìn)行連接轉(zhuǎn)化；經(jīng)酶切、測(cè)序驗(yàn)證結(jié)果表明所連接確實(shí)為目的片段,沒(méi)有發(fā)生突變。 3玉米幼胚遺傳轉(zhuǎn)化系統(tǒng)的優(yōu)化。首先探討了熱激與離心對(duì)幼胚浸染的影響,并對(duì)農(nóng)桿菌轉(zhuǎn)化玉米幼胚過(guò)程中幼胚大小、頭孢霉素濃度、篩選劑濃度等因素設(shè)置不同水平處理,結(jié)果表明：最適合的處理?xiàng)l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(shí),可以得到質(zhì)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(nóng)桿菌生長(zhǎng),還可以保持幼胚良好的生長(zhǎng)狀態(tài)；利用不同濃度的草甘膦對(duì)浸染的幼胚進(jìn)行篩選,比較理想的篩選濃度為1.5-2.0mmol/L。 4轉(zhuǎn)基因苗的PCR鑒定。對(duì)96株To代再生植株進(jìn)行多次重復(fù)的PCR鑒定,15株再生植株的基因組DNA擴(kuò)增出與陽(yáng)性對(duì)照大小一致的的片段,表明目的基因已經(jīng)成功轉(zhuǎn)入玉米基因組中；T1代株系進(jìn)行PCR檢測(cè),獲得四株轉(zhuǎn)基因株系,這些陽(yáng)性的株系經(jīng)RT-PCR檢測(cè),表明抗草甘膦基因在mRNA水平上得到了有效轉(zhuǎn)錄；對(duì)陽(yáng)性植株進(jìn)行葉面噴施草甘膦,結(jié)果表明轉(zhuǎn)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(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：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(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：S513
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