基因枪法介导幼胚转NAC-43基因小麦的获得及插入位点的初步研究
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摘要
小麦是我国重要的粮食作物之一,利用植物基因工程技术对小麦进行遗传改良,是改善小麦品质的一个重要的方法之一。小麦遗传改良的重要目标有：增强小麦抗病抗虫和耐逆境的能力,提高小麦产量,改善小麦品质。本研究以8种普通西南小麦品种作为材料,以这些小麦的幼胚作为受体诱导出愈伤组织并且研究愈伤组织的分化及再生,比较各品种间愈伤组织的诱导能力以及愈伤组织的分化再生能力,筛选小麦幼胚适宜组织培养的基因型。经过筛选之后,以小麦品种绵阳11和川农23的幼胚愈伤组织为实验材料,用基因枪法对幼胚诱导的胚性愈伤组织进行遗传转化,将重新构建好的PBI121-NAC-43表达载体转入小麦中,获得转基因植株。并且用TAIL—PCR技术对转基因阳性植株中目的基因的插入位点进行研究并对该技术进行一定的优化。获得的实验结果为：
1.通过对8种小麦材料进行幼胚愈伤组织的诱导以及分化再生的研究,最后明确了大多数基因型小麦幼胚的愈伤组织诱导能力差别不是很大。而对分化再生及成苗的影响很大。利用剥胚法诱导小麦幼胚诱导出愈伤组织,并且对其进行分化和再生的培养,最终筛选出了出愈率比较高,愈伤组织的质量好,分化的能力比较强以及成苗率最高的绵阳11和川农23两个优良的西南小麦基因型材料。
2.以PMD19-T-NAC-43为模板,克隆得到NAC-43基因,并将其构建到PBI121表达载体中。利用建立的小麦幼胚遗传转化体系将新构建的插入有NAC-43基因的PBI121表达载体与含有bar基因的PAHC20表达载体共转化入小麦品种绵阳11和川农23中,获得了转基因植株。并对转基因植株进行分子检测,经PCR初步鉴定,以及及回收连接转化测序后对所测序列进行比对证实目的基因NAC-43已转入其中。
3.根据载体PBI121-NAC-43中左右边界的已知序列(npt和gus基因)分别设汁了3个与其边界距离不等的嵌套的特异性引物,共计6个特异性引物,并按照小麦物种普遍存在的蛋白质保守氮基酸序列设计了三个简并引物,将简并引物与特异引物相互组合,进行TAIL—PCR反应,同时做了这两种引物之间加入量比例的对比研究以及二三次反应中模板稀释倍数的对比研究,从而初步优化了TAIL—PCR技术的操作并对转基因阳性植株中目的基因的插入位点进行了载体的序列分析。
Wheat is one of the important food crops, It is one of the important methods to improve the quality of wheat by Using plant genetic engineering to geneticly improve the wheat.The Important goal of genetic improvement of wheat are enhancing the ability of wheat to insect resistance and stress tolerance, increasing wheat production, improving wheat quality. In this study, using eight kinds of Common Southwest Wheat as materials induce The immature embryo into and different the callus and Finally regenerat into plants. We also Compare to the ability of the induction and differentiation and regeneration between species,in order to screen the bast genotypes what have the bast ability of The Tissue culture. On this basis,using the Callus which come from Improved strains of southwestern wheat-Mianyang 11 and chuannong23-as experimental material, Using particle bombardment to Transduce PBI121-NAC-43 into the wheat and get the transgenic plants. After getting the transgenic plants, we also study the insertion sites which has the target genes in transgenic plants by using TAIL-PCR technique and certainly optimize this technology. The main results obtained are as follows:
1.Through studing 8 different genotypes of wheat about immature embryos of callus induction,differentiation and regeneration, We find that it is not differentbetween the majority of immature embryos of wheat genotypes callus induction capacity but The Regeneration and Sprouting are On the contrary. using eembryo-isolated method(EI),we induce callus from immature wheat embryos and culture for differentiation and regeneration, Finally,we selecte Mianyang 11 and chuanong 23 as the excellent southwest of wheat genotypes which have the high rate of callus, good callus quality, high capacity of differentiation and high rate of seedling.
2. Using PMD19-T-NAC-43 as template, we clone NAC-43 gene and constructe NAC-43 gene to a new expression vector-PBI121. Then we transforme the PBI121-NAC-43 and PAHC20 which contains the bar gene into mianyangll and chuanong23 to get transgenic wheat.
3. According to the left and right borders'known sequence of vector PBI121-NAC-43 (npt and gus gene), we severally design 3 nested primers which are varying distances from the border, totaly of 6 specific Primers, and design 3 degenerate primers according to the conserved amino acid sequences of wheat protein. Then we Combinate the degenerate primers and specific primers in various and make the TAIL-PCR reaction. Also we make the Comparative study of the dosage-ratio of the two primers and the template dilution of two or three times in the reaction. Thus we initially optimized TAIL-PCR technology, and study the target gene insertion sites of transgenic plants.
1.通过对8种小麦材料进行幼胚愈伤组织的诱导以及分化再生的研究,最后明确了大多数基因型小麦幼胚的愈伤组织诱导能力差别不是很大。而对分化再生及成苗的影响很大。利用剥胚法诱导小麦幼胚诱导出愈伤组织,并且对其进行分化和再生的培养,最终筛选出了出愈率比较高,愈伤组织的质量好,分化的能力比较强以及成苗率最高的绵阳11和川农23两个优良的西南小麦基因型材料。
2.以PMD19-T-NAC-43为模板,克隆得到NAC-43基因,并将其构建到PBI121表达载体中。利用建立的小麦幼胚遗传转化体系将新构建的插入有NAC-43基因的PBI121表达载体与含有bar基因的PAHC20表达载体共转化入小麦品种绵阳11和川农23中,获得了转基因植株。并对转基因植株进行分子检测,经PCR初步鉴定,以及及回收连接转化测序后对所测序列进行比对证实目的基因NAC-43已转入其中。
3.根据载体PBI121-NAC-43中左右边界的已知序列(npt和gus基因)分别设汁了3个与其边界距离不等的嵌套的特异性引物,共计6个特异性引物,并按照小麦物种普遍存在的蛋白质保守氮基酸序列设计了三个简并引物,将简并引物与特异引物相互组合,进行TAIL—PCR反应,同时做了这两种引物之间加入量比例的对比研究以及二三次反应中模板稀释倍数的对比研究,从而初步优化了TAIL—PCR技术的操作并对转基因阳性植株中目的基因的插入位点进行了载体的序列分析。
Wheat is one of the important food crops, It is one of the important methods to improve the quality of wheat by Using plant genetic engineering to geneticly improve the wheat.The Important goal of genetic improvement of wheat are enhancing the ability of wheat to insect resistance and stress tolerance, increasing wheat production, improving wheat quality. In this study, using eight kinds of Common Southwest Wheat as materials induce The immature embryo into and different the callus and Finally regenerat into plants. We also Compare to the ability of the induction and differentiation and regeneration between species,in order to screen the bast genotypes what have the bast ability of The Tissue culture. On this basis,using the Callus which come from Improved strains of southwestern wheat-Mianyang 11 and chuannong23-as experimental material, Using particle bombardment to Transduce PBI121-NAC-43 into the wheat and get the transgenic plants. After getting the transgenic plants, we also study the insertion sites which has the target genes in transgenic plants by using TAIL-PCR technique and certainly optimize this technology. The main results obtained are as follows:
1.Through studing 8 different genotypes of wheat about immature embryos of callus induction,differentiation and regeneration, We find that it is not differentbetween the majority of immature embryos of wheat genotypes callus induction capacity but The Regeneration and Sprouting are On the contrary. using eembryo-isolated method(EI),we induce callus from immature wheat embryos and culture for differentiation and regeneration, Finally,we selecte Mianyang 11 and chuanong 23 as the excellent southwest of wheat genotypes which have the high rate of callus, good callus quality, high capacity of differentiation and high rate of seedling.
2. Using PMD19-T-NAC-43 as template, we clone NAC-43 gene and constructe NAC-43 gene to a new expression vector-PBI121. Then we transforme the PBI121-NAC-43 and PAHC20 which contains the bar gene into mianyangll and chuanong23 to get transgenic wheat.
3. According to the left and right borders'known sequence of vector PBI121-NAC-43 (npt and gus gene), we severally design 3 nested primers which are varying distances from the border, totaly of 6 specific Primers, and design 3 degenerate primers according to the conserved amino acid sequences of wheat protein. Then we Combinate the degenerate primers and specific primers in various and make the TAIL-PCR reaction. Also we make the Comparative study of the dosage-ratio of the two primers and the template dilution of two or three times in the reaction. Thus we initially optimized TAIL-PCR technology, and study the target gene insertion sites of transgenic plants.
引文
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