抗玉米螟、抗草甘膦、抗旱的轉(zhuǎn)基因復(fù)合性狀玉米種質(zhì)的創(chuàng)制
發(fā)布時間：2018-11-26 20:44
【摘要】：玉米是世界第一大糧食作物,被廣泛用于食品、飼料、工業(yè)加工、新能源開發(fā)等方面,玉米產(chǎn)量的波動會在世界范圍內(nèi)帶來一系列負(fù)面效應(yīng)。保證玉米高產(chǎn)優(yōu)質(zhì)和穩(wěn)產(chǎn),首先需要加強(qiáng)病蟲害和自然災(zāi)害的防治。近20年來,人們已經(jīng)利用基因工程手段成功培育出了具有抗蟲、抗病、抗逆以及耐除草劑等優(yōu)良性狀的玉米新種質(zhì),抗蟲和耐除草劑的轉(zhuǎn)基因玉米更是被廣泛種植,有效提高了玉米產(chǎn)量和降低了生產(chǎn)成本。當(dāng)前嚴(yán)重影響玉米產(chǎn)量的因素有蟲害、草害和干旱,培育具有抗蟲、耐除草劑、抗旱性狀的轉(zhuǎn)基因玉米品種是一項(xiàng)具有戰(zhàn)略意義的工作。利用多基因轉(zhuǎn)化技術(shù)培育具有復(fù)合性狀的轉(zhuǎn)基因玉米具有明顯競爭優(yōu)勢,是值得探索的課題。 本工作采用了對玉米螟具有強(qiáng)殺毒活性的crylAc-M基因以防治對我國玉米危害嚴(yán)重的亞洲玉米螟。crylAc-M基因是在保證cry1Ac氨基酸序列不變的前提下進(jìn)行密碼子及編碼框長度優(yōu)化的產(chǎn)物,使其在單子葉植物中能高效表達(dá)。為了提高玉米對除草劑草甘膦的耐受性,我們導(dǎo)入epsps和GAT兩個基因。epsps基因是從草甘膦極度污染的土壤中分離的可變鹽單胞菌(Halomonas Variabilis)中克隆的,擁有我國自主知識產(chǎn)權(quán)。GAT基因能使進(jìn)入植物體的草甘膦乙�；獬拘�。兩個耐除草劑基因同時發(fā)揮作用,希望能有效提高玉米植株抗草甘膦的能力。ZmPIS基因是本實(shí)驗(yàn)室從玉米中克隆得到的,該基因的表達(dá)強(qiáng)度受多種逆境脅迫誘導(dǎo),且過表達(dá)該基因可明顯提高玉米植株的抗旱能力。這4個基因構(gòu)建到同一植物表達(dá)載體中用于遺傳轉(zhuǎn)化。 本工作通過農(nóng)桿菌介導(dǎo)的玉米莖尖遺傳轉(zhuǎn)化法將上述4個基因(crylAc-M、 epsps、GAT、ZmPIS)分別轉(zhuǎn)入玉米自交系9801和齊319(Q319),獲得了轉(zhuǎn)基因植株。通過PCR檢測確定了目標(biāo)基因在轉(zhuǎn)化植株中存在,利用RT-PCR檢測了4個基因的轉(zhuǎn)錄強(qiáng)度,肯定它們在轉(zhuǎn)基因植株中穩(wěn)定表達(dá)。此外,利用Real-time RT-PCR和Western blot方法檢測了cryl Ac基因的表達(dá)豐度和cryl Ac蛋白的表達(dá)水平,肯定了cryl Ac基因在玉米植株中穩(wěn)定表達(dá)。通過逐代除草劑篩選、PCR檢測及抗蟲性鑒定,從大量轉(zhuǎn)基因株系中優(yōu)選出6個穩(wěn)定遺傳且抗玉米螟、抗除草劑草甘膦和耐旱性良好的轉(zhuǎn)基因玉米株系,其中株系L1-L3來自骨干自交系9801,Q1-Q3來自骨干自交系Q319。在此基礎(chǔ)上,以非轉(zhuǎn)基因自交系9801、Q319為對照,在嚴(yán)格控制條件下對6個轉(zhuǎn)基因株系進(jìn)行抗蟲、抗除草劑和抗旱檢測試驗(yàn)。 轉(zhuǎn)基因植株的抗蟲性分析 由于不同生長時期玉米對玉米螟危害的敏感程度有差異,本工作檢測了生長不同階段的轉(zhuǎn)基因玉米的抗蟲性,分別進(jìn)行了田間和室內(nèi)玉米螟接種試驗(yàn)。植株抗蟲性觀察結(jié)果顯示,這些高抗株系的營養(yǎng)生長期植株僅在最初接蟲的葉片上出現(xiàn)少量的幼蟲取食小孔,沒有受到進(jìn)一步危害的跡象,與對照植株相比差異達(dá)到極顯著差異水平。取灌漿期玉米的籽粒和苞葉進(jìn)行飼喂玉米螟幼蟲實(shí)驗(yàn),得出喂食轉(zhuǎn)基因玉米籽�；虬~的玉米螟死亡率顯著高于飼喂非轉(zhuǎn)基因的。這些結(jié)果表明轉(zhuǎn)基因玉米可有效防治玉米螟危害,達(dá)到實(shí)用標(biāo)準(zhǔn)。 轉(zhuǎn)基因植株的草甘膦抗性分析 在營養(yǎng)生長期,玉米葉片數(shù)與植株對草甘膦抗性成正相關(guān),即較大植株對草甘膦抗性也較強(qiáng)。在玉米三葉期噴灑1.05kg ae ha-1草甘膦的水溶液篩選抗草甘膦株系。在田間草甘膦抗性測試中,對六葉期玉米植株噴灑0.84kg ae ha-1(即大田除草應(yīng)用劑量)的草甘膦溶液,轉(zhuǎn)基因株系表現(xiàn)出了良好抗性,即達(dá)到生產(chǎn)中應(yīng)用水平。在進(jìn)行高濃度除草劑噴灑試驗(yàn)中,以兩倍應(yīng)用劑量的草甘膦溶液噴施植株,發(fā)現(xiàn)轉(zhuǎn)基因植株較未轉(zhuǎn)基因?qū)φ罩仓晔艹輨﹤Τ潭容p,受害癥狀出現(xiàn)晚,而對照植株幾乎全部死亡。 轉(zhuǎn)基因植株的抗旱性分析 對10葉期玉米植株的干旱脅迫試驗(yàn),觀察轉(zhuǎn)基因植株的抗旱能力。干旱處理期每天監(jiān)測并控制土壤相對含水量在17-18%左右,持續(xù)處理7天,然后恢復(fù)正常澆水。在干旱控水期間,相比于對照植株,轉(zhuǎn)基因株系葉片較伸展,萎焉程度較輕,恢復(fù)澆水后,也能夠較快恢復(fù)正常生長。光合作用和葉綠素?zé)晒鉁y定結(jié)果顯示：轉(zhuǎn)基因植株維持較強(qiáng)的光合能力和光系統(tǒng)II活性,對干旱脅迫的抗性顯著高于對照植株。 綜上所述,將crylAc-M、epsps、GAT、ZmPIS基因一同轉(zhuǎn)入玉米,賦予了植株抗玉米螟、抗除草劑草甘膦特性,提高了植株的抗旱性。本工作培育出了具有優(yōu)良復(fù)合性狀的轉(zhuǎn)基因玉米新材料,為培育多性狀疊加的轉(zhuǎn)基因新品種奠定了基礎(chǔ)。
[Abstract]:Corn is the first major food crop in the world, and is widely used in food, feed, industrial processing, new energy development and so on, and the fluctuation of corn output will bring a series of negative effects in the world. the high-yield and stable yield of the corn is ensured, and the prevention and control of the diseases and insect pests and the natural disasters are first needed. In the past 20 years, the genetic engineering method has been used to successfully cultivate new corn germplasm, anti-insect and herbicide resistant transgenic corn having excellent characters such as insect resistance, disease resistance, stress resistance and herbicide resistance, and is widely planted, thereby effectively improving the yield of the corn and reducing the production cost. The factors that have seriously affected the yield of the corn are insect, grass and drought, and the cultivation of the transgenic corn variety with the resistance to insects, herbicide and drought resistance is a strategic task. The use of multi-gene transformation technology to cultivate transgenic corn with compound character has obvious competitive advantage, and it is a topic to be explored. The invention adopts the cryl Ac-M gene with strong anti-virus activity on the corn silk to prevent and treat the Asian jade which has serious harm to the corn in China The m. crylAc-M gene is a product of codon and coding frame length optimization under the premise of ensuring that the amino acid sequence of the cry1Ac is unchanged, so that it can be highly effective in monocotyledonous plants. In order to improve the tolerance of the corn to the herbicide, we have introduced epsps and GAT The gene. epsps is cloned from the highly polluted soil of the grass and has the autonomous knowledge of China. Property rights. The GAT gene can be released from the plant, and can be released from the plant. The two herbicide-resistant genes play a role at the same time, hoping to effectively improve the resistance of the corn plants to the grass The ZmPIS gene is cloned from the corn, and the expression strength of the gene is induced by a plurality of adversity stress, and the overexpression of the gene can obviously improve the drought resistance of the maize plant. Ability. The four genes were built into the same plant expression vector for inheritance The above four genes (crylAc-M, epsps, GAT, ZmPIS) were transferred to the maize inbred lines 9801 and Q319 (Q319) by Agrobacterium-mediated maize stem-tip genetic transformation. The presence of the target gene in the transformed plant was determined by PCR, and the transcription level of the 4 genes was detected by RT-PCR, and they were positive in the transgenic plants. In addition, the expression level of crylAc gene and the expression level of crylAc were detected by Real-time RT-PCR and Western blot. 6 stable and anti-maize, anti-herbicide, and well-resistant transgenic corn lines are preferably selected from a large number of transgenic lines by screening, PCR and insect-resistant identification on a per-generation herbicide, wherein the lines L1-L3 are from the backbone inbred lines 9801, Q1-Q3 from the backbone selfing Based on the above, the non-transgenic inbred lines 9801 and Q319 were used as control, and 6 transgenic lines were anti-insect, anti-herbicide and anti-herbicide under the control of strict control. drought test The insect-resistance of the plants is different from the sensitivity of the corn to the corn in different growth stages, and the insect-resistance of the transgenic corn in different stages of growth is detected, and the field is respectively carried out. The results of the observation on the insect-resistance of the plants showed that the vegetative growth period of these high-resistant strains only showed a small number of larvae feeding the small holes on the leaves of the first worm, and there were no further signs of further harm, compared with the control plants. The difference reached a very significant level of difference. The grain and bract leaf of the corn in the grouting period were fed with the experiment of the corn and larvae, and the mortality rate of the corn in the grain or bract of the transgenic corn was obtained. The results show that the transgenic corn can effectively prevent and treat the jade. The hazard of the rice has reached the practical standard. The resistance of the plant of the transgenic plant was analyzed in the vegetative period, the number of the leaves of the corn and the resistance of the plant to the plant were positive. The results showed that the resistance of the larger plants to the plant was stronger than that of the larger plants.-1. The anti-plant strain was selected from the aqueous solution of the grass and the root of the plant. In the field of the resistance test of the plant in the field, a total of 0. 84kg ae ha-1 (i.e., the application dose of the field weeding) was sprayed on the maize plants in the six-leaf period, and the expression of the transgenic lines In the high-concentration herbicide spraying test, the plant was sprayed with a two-fold application dose of the grass-calamine solution, and the transgenic plants were found to be less harm to the herbicide than the non-transgenic control plants. The symptoms were late, and the control plants Almost all of the deaths. The drought resistance of the transgenic plants is about 10 leaf. The drought stress test of the rice plant was carried out to observe the drought resistance of the transgenic plants. The relative water content of the soil was monitored and controlled every day in the drought treatment period. 7-18%, continuous treatment for 7 days, and then normal watering. During drought-controlled water, the transgenic line leaves were spread, withering, compared to the control plants. The results of photosynthesis and chlorophyll fluorescence showed that the transgenic plants maintained strong photosynthetic capacity and light. In conclusion, cryl Ac-M, epsps, GAT and ZmPIS gene were transferred to the corn, and the resistance to drought stress was significantly higher than that of control plants. The plant has the characteristics of resistance to the corn and the herbicide, and the drought resistance of the plant is improved.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：S513
本文編號：2359578
[Abstract]:Corn is the first major food crop in the world, and is widely used in food, feed, industrial processing, new energy development and so on, and the fluctuation of corn output will bring a series of negative effects in the world. the high-yield and stable yield of the corn is ensured, and the prevention and control of the diseases and insect pests and the natural disasters are first needed. In the past 20 years, the genetic engineering method has been used to successfully cultivate new corn germplasm, anti-insect and herbicide resistant transgenic corn having excellent characters such as insect resistance, disease resistance, stress resistance and herbicide resistance, and is widely planted, thereby effectively improving the yield of the corn and reducing the production cost. The factors that have seriously affected the yield of the corn are insect, grass and drought, and the cultivation of the transgenic corn variety with the resistance to insects, herbicide and drought resistance is a strategic task. The use of multi-gene transformation technology to cultivate transgenic corn with compound character has obvious competitive advantage, and it is a topic to be explored. The invention adopts the cryl Ac-M gene with strong anti-virus activity on the corn silk to prevent and treat the Asian jade which has serious harm to the corn in China The m. crylAc-M gene is a product of codon and coding frame length optimization under the premise of ensuring that the amino acid sequence of the cry1Ac is unchanged, so that it can be highly effective in monocotyledonous plants. In order to improve the tolerance of the corn to the herbicide, we have introduced epsps and GAT The gene. epsps is cloned from the highly polluted soil of the grass and has the autonomous knowledge of China. Property rights. The GAT gene can be released from the plant, and can be released from the plant. The two herbicide-resistant genes play a role at the same time, hoping to effectively improve the resistance of the corn plants to the grass The ZmPIS gene is cloned from the corn, and the expression strength of the gene is induced by a plurality of adversity stress, and the overexpression of the gene can obviously improve the drought resistance of the maize plant. Ability. The four genes were built into the same plant expression vector for inheritance The above four genes (crylAc-M, epsps, GAT, ZmPIS) were transferred to the maize inbred lines 9801 and Q319 (Q319) by Agrobacterium-mediated maize stem-tip genetic transformation. The presence of the target gene in the transformed plant was determined by PCR, and the transcription level of the 4 genes was detected by RT-PCR, and they were positive in the transgenic plants. In addition, the expression level of crylAc gene and the expression level of crylAc were detected by Real-time RT-PCR and Western blot. 6 stable and anti-maize, anti-herbicide, and well-resistant transgenic corn lines are preferably selected from a large number of transgenic lines by screening, PCR and insect-resistant identification on a per-generation herbicide, wherein the lines L1-L3 are from the backbone inbred lines 9801, Q1-Q3 from the backbone selfing Based on the above, the non-transgenic inbred lines 9801 and Q319 were used as control, and 6 transgenic lines were anti-insect, anti-herbicide and anti-herbicide under the control of strict control. drought test The insect-resistance of the plants is different from the sensitivity of the corn to the corn in different growth stages, and the insect-resistance of the transgenic corn in different stages of growth is detected, and the field is respectively carried out. The results of the observation on the insect-resistance of the plants showed that the vegetative growth period of these high-resistant strains only showed a small number of larvae feeding the small holes on the leaves of the first worm, and there were no further signs of further harm, compared with the control plants. The difference reached a very significant level of difference. The grain and bract leaf of the corn in the grouting period were fed with the experiment of the corn and larvae, and the mortality rate of the corn in the grain or bract of the transgenic corn was obtained. The results show that the transgenic corn can effectively prevent and treat the jade. The hazard of the rice has reached the practical standard. The resistance of the plant of the transgenic plant was analyzed in the vegetative period, the number of the leaves of the corn and the resistance of the plant to the plant were positive. The results showed that the resistance of the larger plants to the plant was stronger than that of the larger plants.-1. The anti-plant strain was selected from the aqueous solution of the grass and the root of the plant. In the field of the resistance test of the plant in the field, a total of 0. 84kg ae ha-1 (i.e., the application dose of the field weeding) was sprayed on the maize plants in the six-leaf period, and the expression of the transgenic lines In the high-concentration herbicide spraying test, the plant was sprayed with a two-fold application dose of the grass-calamine solution, and the transgenic plants were found to be less harm to the herbicide than the non-transgenic control plants. The symptoms were late, and the control plants Almost all of the deaths. The drought resistance of the transgenic plants is about 10 leaf. The drought stress test of the rice plant was carried out to observe the drought resistance of the transgenic plants. The relative water content of the soil was monitored and controlled every day in the drought treatment period. 7-18%, continuous treatment for 7 days, and then normal watering. During drought-controlled water, the transgenic line leaves were spread, withering, compared to the control plants. The results of photosynthesis and chlorophyll fluorescence showed that the transgenic plants maintained strong photosynthetic capacity and light. In conclusion, cryl Ac-M, epsps, GAT and ZmPIS gene were transferred to the corn, and the resistance to drought stress was significantly higher than that of control plants. The plant has the characteristics of resistance to the corn and the herbicide, and the drought resistance of the plant is improved.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：S513
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 俞慧;江城梅;趙文紅;;草甘膦毒性作用研究進(jìn)展[J];蚌埠醫(yī)學(xué)院學(xué)報(bào);2012年06期
2 余桂容;劉艷;杜文平;宋軍;張蓮;陸偉;徐利遠(yuǎn);;農(nóng)桿菌介導(dǎo)的轉(zhuǎn)雙價基因耐草甘膦玉米研究[J];分子植物育種;2013年03期
3 白萍;生物農(nóng)藥Bt殺蟲劑研究進(jìn)展[J];河南化工;1998年12期
4 朱華晨;許新萍;肖國櫻;袁隆平;李寶健;;利用四價抗病基因提高超級雜交稻的抗性[J];中國科學(xué)C輯:生命科學(xué);2006年04期
5 吳才君,范淑英;植物轉(zhuǎn)基因沉默[J];江西農(nóng)業(yè)大學(xué)學(xué)報(bào);2004年01期
6 金丹;陳明;馬瑞強(qiáng);楊志榮;陳建;;利用DNA shuffling技術(shù)鑒定草甘膦N-乙酰轉(zhuǎn)移酶抗性相關(guān)位點(diǎn)[J];中國農(nóng)業(yè)科技導(dǎo)報(bào);2007年05期
7 李圣彥;郎志宏;朱莉;李秀影;張杰;何康來;黃大f ;;利用密碼子優(yōu)化提高Bt cry1Ah基因在轉(zhuǎn)基因玉米(Zea mays L.)中的表達(dá)[J];中國農(nóng)業(yè)科技導(dǎo)報(bào);2011年06期
8 雷凱健;盧維維;洪玉枝;劉子鐸;;土壤總DNA中草甘膦N-乙酰轉(zhuǎn)移酶基因的克隆及酶學(xué)特性分析[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2007年01期
9 林長福;玉米田化學(xué)除草現(xiàn)狀及發(fā)展趨勢[J];農(nóng)藥;1999年09期
10 蘇少泉;草甘膦述評[J];農(nóng)藥;2005年04期
相關(guān)博士學(xué)位論文 前3條
1 張淑娟;利用轉(zhuǎn)基因技術(shù)創(chuàng)造適合纖維素乙醇生產(chǎn)的玉米新材料[D];山東大學(xué);2011年
2 李朝霞;磷匱乏影響玉米根系發(fā)育機(jī)制的研究[D];山東大學(xué);2011年
3 王延鋒;轉(zhuǎn)Bt基因抗蟲玉米田間試驗(yàn)與遺傳穩(wěn)定性分析[D];東北農(nóng)業(yè)大學(xué);2010年
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