本文選題：玉米 + CP4-EPSPS基因�。� 參考：《山西大學(xué)》2016年碩士論文

【摘要】：玉米(Zea mays L.)作為世界三大糧食作物之一,是重要的糧食作物、飼料以及工業(yè)加工原料。而雜草是玉米減產(chǎn)的主要危害之一,在玉米生長(zhǎng)過程中會(huì)爭(zhēng)奪水分,養(yǎng)分和陽光,并且引起病蟲害的發(fā)生。因而研究如何有效經(jīng)濟(jì)的控制玉米田間雜草具有重要意義。隨著生物技術(shù)的發(fā)展,利用基因工程的手段獲得抗草甘膦作物新品種已成為最理想的手段。自1998年,抗草甘膦轉(zhuǎn)基因玉米品種GA21獲得批準(zhǔn)商業(yè)化種植以來,各種抗草甘膦、抗蟲、抗病等優(yōu)良性狀的轉(zhuǎn)基因玉米品種相繼推出,轉(zhuǎn)基因玉米是世界上種植面積最廣的轉(zhuǎn)基因糧食作物。本研究中利用超聲波輔助花粉介導(dǎo)轉(zhuǎn)化法和超聲波輔助農(nóng)桿菌介導(dǎo)萌發(fā)種子轉(zhuǎn)化法兩種方法將攜帶目的基因——抗草甘膦基因CP4-EPSPS的載體轉(zhuǎn)入到玉米自交系昌7-2中。其中pM3301UbiSpCP4載體選用了組成型強(qiáng)啟動(dòng)子——玉米泛素啟動(dòng)子pZmUbi-1啟動(dòng)表達(dá),同時(shí)該載體攜帶了葉綠體信號(hào)肽(Signal Peptide),有助于目的基因成功定位到葉綠體內(nèi),使得CP4基因在玉米葉綠體中更好的表達(dá)。另外這兩種新型的轉(zhuǎn)基因方法是以花粉和萌發(fā)的種子作為受體,避開了組織培養(yǎng)再生植株的過程,且無基因型依賴性,為廣大育種工作者提供了簡(jiǎn)便經(jīng)濟(jì)且易于操作的兩種種質(zhì)創(chuàng)新途徑。另外玉米自交系昌7-2作為目前生產(chǎn)中廣泛使用的優(yōu)良自交系,農(nóng)藝性狀良好,獲得轉(zhuǎn)基因株系后能最快應(yīng)用于生產(chǎn)。主要研究結(jié)果如下:將構(gòu)建好的載體pM3301UbiSpCP4利用超聲波輔助花粉介導(dǎo)的轉(zhuǎn)基因方法對(duì)1552株(T0代)玉米進(jìn)行遺傳轉(zhuǎn)化,成功獲得了943粒結(jié)實(shí)種子。對(duì)T1代出苗的648個(gè)轉(zhuǎn)化株涂抹2%濃度的草甘膦進(jìn)行田間鑒定篩選,有29株長(zhǎng)勢(shì)健康,表現(xiàn)出抗草甘膦的性狀,通過PCR對(duì)這些株系進(jìn)行目的基因檢測(cè),其中有16個(gè)株系擴(kuò)增出陽性條帶,RT-PCR顯示16個(gè)陽性株系中外源基因CP4-EPSPS正常轉(zhuǎn)錄。同時(shí)進(jìn)行了PCR-Southern blot檢測(cè),進(jìn)一步證明了外源基因CP4-EPSPS穩(wěn)定遺傳到了T1代。T1代的16個(gè)株系自交后收獲種子(T2代),對(duì)T2代部分轉(zhuǎn)基因株系涂抹2%濃度的草甘膦,篩選出了5個(gè)抗性良好的轉(zhuǎn)基因株系A(chǔ)nti-3,11,12,23,29。經(jīng)PCR、Southern、ELISA檢測(cè)結(jié)合田間抗草甘膦篩選試驗(yàn),最終獲得了3個(gè)高抗草甘膦、單拷貝且抗除草劑蛋白表達(dá)量較高的轉(zhuǎn)基因玉米株系A(chǔ)nti-3,23,29。本研究為抗除草劑轉(zhuǎn)基因玉米的研究奠定了基礎(chǔ),并為育種工作提供有價(jià)值的新種質(zhì)。
[Abstract]:Zea mays L.As one of the three major food crops in the world, it is an important food crop, fodder and raw material for industrial processing.Weed is one of the main harms of maize yield reduction. It will compete for water, nutrients and sunlight in the process of maize growth, and cause the occurrence of diseases and insect pests.Therefore, it is of great significance to study how to control weeds in maize field economically.With the development of biotechnology, it has become the most ideal method to obtain new varieties of glyphosate resistant crops by genetic engineering.Since 1998, when glyphosate resistant transgenic maize (GA21) was approved for commercial planting, a variety of transgenic maize varieties with excellent characters, such as glyphosate resistance, insect resistance and disease resistance, have been introduced one after another.Transgenic corn is the most widely grown genetically modified food crop in the world.In this study, ultrasonic assisted pollen mediated transformation and ultrasonic assisted Agrobacterium tumefaciens seed transformation were used to transfer the vector carrying the target gene, glyphosate resistant gene CP4-EPSPS, into maize inbred line Chang7-2.Among them, the pM3301UbiSpCP4 vector selected the component strong promoter-maize ubiquitin promoter pZmUbi-1 to initiate expression, and the vector carried the chloroplast signal peptide Signal Peptidea, which was helpful for the target gene to be successfully located in the chloroplast.CP4 gene is better expressed in maize chloroplast.In addition, the two new transgenic methods used pollen and germinated seeds as receptors, avoiding the process of tissue culture and regeneration, and without genotypic dependence.It provides a simple, economical and easy to operate ways for breeders to innovate germplasm.In addition, the maize inbred line Chang7-2 is widely used in production at present. It has good agronomic characters and can be used in production as soon as transgenic lines are obtained.The main results are as follows: 943 seed bearing seeds were successfully obtained by genetic transformation of 1552 maize plants in T0 generation by using the constructed vector pM3301UbiSpCP4.Field identification and screening of 648 transgenic plants with 2% concentration of glyphosate were carried out in T1 generation. 29 of them had healthy growth and showed resistance to glyphosate. The target genes of these lines were detected by PCR.16 of them amplified positive bands and RT-PCR showed normal transcription of foreign gene CP4-EPSPS in 16 positive lines.At the same time, PCR-Southern blot analysis showed that the exogenous gene CP4-EPSPS was stably inherited to 16 lines in T1 generation and T1 generation, and then harvested the seeds of T 2 generation, and applied 2% glyphosate to some transgenic lines in T2 generation.Five transgenic lines with good resistance were screened out.Three transgenic maize lines with high glyphosate resistance, single copy and high herbicide protein expression were obtained by PCR Southern Elisa combined with field glyphosate resistant screening test.This study laid a foundation for the study of herbicide resistant transgenic maize and provided valuable new germplasm for breeding.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S513

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 宋紅霞;惠國(guó)強(qiáng);楊海鵬;溫琳;孫毅;劉小紅;張紅梅;;不同保存方法和研磨方式對(duì)玉米總DNA提取效果的影響[J];山西農(nóng)業(yè)科學(xué);2015年09期

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本文編號(hào)：1735176