【作者】 王海凤；

【导师】 赵惠贤；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 小麦是世界和我国最主要粮食作物之一,但由于小麦是六倍体,基因组很大,遗产背景复杂,所以小麦是最后一个获得转化成功的大宗粮食作物。在小麦的遗传转化中,幼胚是应用最多的外植体,但幼胚的再生率受基因型和培养基的激素配比的影响很大;目前小麦的遗传转化研究主要集中在少数几种再生能力较高的品种上,而生产上大面积推广和主栽的品种由于再生能力较低难以应用于转基因实践。因此,选取优良的受体基因型,优化小麦幼胚组织培养再生体系显得极为重要。本研究以黄淮麦区或陕西省近年来审定和大面积推广的优良小麦品种郑麦9023、小偃22、西农2000、西农928等为试材,对其小麦幼胚组织培养再生性能进行比较;在此基础上进行以下转基因研究:①通过基因枪共转化法将玉米调控花青素合成的转录因子基因Zm-c1或Zm-r(s)与筛选标记基因Bar同时导入小麦中,试图通过后代分离获得不含标记基因的转基因植株;②以安全标记基因GFP作为筛选基因,利用基因枪介导法将玉米中能增大种子体积的基因ZmAL1转入小麦中,以获得安全的转基因小麦。本研究旨在获得含目标基因的转基因小麦材料,为进一步研究这些基因对转基因小麦目标性状的影响及其遗传稳定性奠定基础工作;为利用基因工程改良小麦品质和产量性状提供基础资料。取得了以下主要研究结果:(1)不同品种小麦幼胚组织培养再生性能比较通过不同小麦品种幼胚组织培养表明,不同基因型的小麦品种的愈伤组织的诱导率都较高,都达到95%以上,但愈伤组织的质量有所差异;不同小麦品种的分化率差异很大。供试的五个品种的分化能力依次为西农928>郑麦9023>西农2000>小偃22>西农979。结合诱导率和分化率来说,西农928和西农2000诱导出的愈伤组织状态较好,分化率也相对较高,可以作为转基因的良好受体材料。(2)基因枪介导的玉米Zm-c1或Zm-r(s)基因和Bar基因共转化研究采用基因枪共转化法将玉米调控花青素合成的Zm-c1或Zm-r(s)基因和Bar基因转入小麦品种西农2000、西农928和小偃22中。经过多次重复检测,转Zm-c1基因的西农2000和西农928的再生植株中分别有3株和1株扩增到与阳性质粒大小一致的617 bp目标片段,目标基因的转化率分别为0.23%和0.08%;Bar基因分别有13株和5株扩增到与质粒一致的400 bp左右的片段,转化率分别为0.98%和0.42%。转Zm-r(s)基因的西农2000的再生植株中有4株PCR扩增到与阳性质粒大小一致的628bp目标片段,目标基因转化率为0.13%;Bar基因有15株扩增到与质粒一致的400 bp左右的片段,转化率为0.5%。在所有含目的基因的转基因植株中都含有Bar基因。(3)基因枪介导的转ZmalI基因小麦植株的获得和转基因后代材料的鉴定利用基因枪介导法,将由谷蛋白胚乳特异表达启动子驱动的ZmalI+ GFP融合蛋白基因导入陕农138小麦中,并对转基因小麦进行分子检测。基因枪共轰击3 000个幼胚,获得再生植株59株;利用载体上安全标记基因GFP的特异引物对所获得59株T0代再生植株进行PCR检测,发现有16株转基因阳性植株,转化率为0.53 %。16个T0代转基因植株中有10株收到种子,按单株收获后将其中6株收获的种子分别全部种植后获48株T1代植株,PCR检测结果发现有35株T1代转基因阳性植株,说明T1代植株目标基因发生了分离;从这35株T1代阳性植株中随机挑取7株,每株取一粒种子进行目标基因表达的RT-PCR检测,结果7粒种子中有6粒检测到目标基因已经表达,说明在绝大部分T2代种子中目标基因已得到了表达。更多还原

【Abstract】 Wheat is one of the most important food crops in China and the world.But common wheat is hexapliod, and the background of wheat is very complicated. So,wheat is the last one of the food crop which was transformed successfully.In wheat genetic transformation, immature embryo is the most application explant,but the regeneration rate was influenced by genotypes and the hormone ratio medium.Only a few highly regenerated varieties were used in wheat transformation,while a large quantity of elite cultivars can not be transformed successfully due to their poor regeneration property.This resulted in the transformation of wheat developing very slowly.Therefore selection of excellent receptor genotypes and optimization of wheat regeneration culture system from immature embryo is urgent in wheat transgenic work.The excellent wheat varieties planted in HuangHuai areas or shaanxi province in recent years,incluging zhengmai 9023,xiaoyan 22,xinong 2000,xinong 928 and so on, were selected as materials in this study to compare their regeneration properties system of wheat immature embryo tissue culture.On these basis,we do the following transgenic research: (1) Through the co-transformation by gene gun, we introduce the Zm - c1 or Zm - r (s) gene which control the corn anthocyanins synthesis and Bar gene into wheats, trying to get plants that excluding marker genes by offspring separation of transgenic plants; (2) Using the GFP gene as the safety marker genes, we transfer the ZmAL1 gene which can increase the volume of corn seed into wheat, in order to obtain safety transgenic wheat.The purpose of study is to obtain transgenic wheat with target genes,lay the foundation for functional identification of these genes in transgenic wheat.The main results obtained as follows:(1) Comparison of tissue culture regeneration properties of the immature embryo from different wheat.cultivarsThrough the immature embryo tissue culture of different wheat varieties ,we indicated that the induction rate of different genotypes of wheat varieties is higher, all above 95 percent, but callus quality is differences;The differentiation rate of different varieties of wheat genotypes is very different.The order of differentiation capacity of selected five varieties is: Xinong 928 > Zhengmai 9023 > Xinong 2000 > Xiaoyan 22 > Xinong 979.Considering both induction rate and the differentiation rate,Xinong 928 and Xinong 2000 are much better for plant regeneration.(2) Co-transformation of the corn Zm-c1 or Zm-r (s) and Bar gene by Biolistic Particle The genes transcription factors,Zm-c1 or Zm-r(s) which control the anthocyanins synthesis in corn gene was transformed into wheat varieties of xinong 2000, xinong 928 and xiaoyan 22 with bar as marker gene. there were 3 and 1 positive transgenic plants with the Zm-c1 gene in Xinong 2000 and Xinong 928, respectively .The transfermation ratio is respectively 0.23% and 0.08%;The respectively got there were thirteen and five transfegenic plants with Bar gene in Xinong 2000 and Xinong 928,respectively.The transfermation ratio is 0.98% and 0.42% ,respectively.With regards to transformation with the Zm-r(s) gene,we got 4 positive transgenic plants in Xinong 2000.The transfermation ratio is 0.13%.Fifteen transgenic plants with Bar gene were obtained in Xinong 2000.The transfermation ratio is 0.5%. All the transfer plants with the target gene contain the Bar gene.(3) Transformation of Wheat with ZmalI gene by Biolistic Particle and Molecular IdentificationThe over expressed vector pGlu-exbessa::ZmalI,which contained the fusion protein gene ZmalI and GFP controlled by the special glutenin promoter,was introduced into wheat by Biolistic Particle.Then 3 000 embryos of shaannong 138 were bombarded by Biolistic Particle, and 59 regenerated plants were obtained. 16 out of 59 were identified to be transgenic positive plants by PCR assay in the T0 plants.The transformation frequency was 0.53 %.10 of the 16 T0 transgenic plants generated seeds.We get 48 plants of T1 progency.PCR analysis indicated that 35 of the 48 T1 plants have the target gene.This implied that the target gene segragated in T1 plants.Seven of the 35 T1 plants were randomly selected to check the expression of the target in the kernels by performing the RT-PCR,The results showed that 6 of the 7 grains had the gene expression, indicating that the target genes in most seeds of T2 generation plant has been expressed.更多还原