转Cry30Fa1基因抗褐飞虱水稻的研究

发布时间：2018-01-21 21:42

本文关键词： 蜀恢818 Cry30Fa1基因抗虫性拷贝数农艺性状　出处：《四川农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：本研究以优良的水稻恢复系蜀恢818(R818)为受体材料,采用农杆菌介导的遗传转化方法,将本实验室克隆的抗褐飞虱基因Cry30Fa1转入其中,通过连续自交和DNA检测在T6代获得了目的基因纯合的转基因株系。通过目的基因PCR检测和抗性表达鉴定,确定T5、T6代转基因材料中的选择标记基因Hyg(R)和抗虫基因Cry30Fa1,最终筛选到无选择标记基因的转基因水稻株系。采用实时荧光定量PCR技术检测Cry30Fa1基因的转录情况。同时,基于实时荧光定量PCR技术也检测了转基因材料中的Cry30Fa1基因拷贝数。采用Western Blot检测Cry30Fa1基因蛋白表达情况。采用苗期集团抗虫性鉴定法、苗期单株抗虫性鉴定法和田间抗虫性鉴定法,分别对3个转基因株系进行了抗虫性分析,筛选到中抗以上水平的抗褐飞虱转基因水稻株系。此外,我们还对T6代转基因水稻株系的主要农艺性状进行了调查。主要结果如下：1.对T5代的184个转基因株系进行Cry30Fa1基因的PCR检测,发现其中150个株系含有Cry30Fa1基因,而且T6代跟踪检测中筛选到29个目的基因纯合的转基因株系。通过对29个转基因株系进行了Hyg(R)基因PCR检测和浸泡表达检测,最终获得了15个无选择标记基因的转基因株系。2.通过实时荧光定量PCR技术,本研究对29个T6代转基因株系的Cry30Fa1基因在转录水平上的表达进行了检测,结果显示,所有转基因株系的Cry30Fa1基因在转录水平上均有表达,但表达量差异较大,相对表达量在0.19-8.53之间。3.通过Western Blot技术,本研究对29个T6代转基因株系Cry30Fa1基因在蛋白质水平上的表达进行了检测,结果显示,5个转基因株系中没有检测到Bt蛋白质,余下的24个转基因株系的Bt蛋白均有表达,但转基因株系中Bt蛋白表达量存在较大差异,分布在0.13到11.6之间。4.通过分析了转基因植株中Cry30Fa1基因拷贝数,本研究发现29个转基因株系中Cry30Fa1基因拷贝数存在较大差异,但有15个株系的拷贝数属于低拷贝的株系。5.采用苗期集团抗虫性鉴定法和田间抗虫性鉴定法对其中3个转基因株系进行了抗虫性分析,结果发现,苗期抗性均达到了7级,属于中感材料；田间抗性达到3-5级,属于中抗到抗的材料。同时,采用苗期单株抗虫性鉴定法,对以上3个转基因株系进行了杀虫性分析,结果表明,3个转基因株系中的褐飞虱死亡率显著高于亲本R818,这说明了转基因株系增加了杀虫能力。6.通过对29个转基因株系主要农艺性状进行分析,结果表明,外源Bt基因对水稻株高影响最大,有19个株系与亲本之间存在显著性差异；外源Bt基因对其它农艺性状如抽穗期、有效分蘖数、主穗长、结实率、千粒重等的影响较小。通过数据分析发现性状得到改良的株系比性状变差的株系多,这说明通过分子水平检测和田间选育工作相结合,总会选到和亲本相似农艺性状甚至是优于亲本的转基因株系。目前,正在对余下的26个转基因株系进行全面抗虫性分析,以期获得更多抗褐飞虱的转基因株系。
[Abstract]:Based on the excellent rice restorer line Shuhui 818 (R818) receptor material, using the method of Agrobacterium mediated genetic transformation, the cloned BPH resistance gene Cry30Fa1 into which, through the detection of continuous selfing and DNA obtained the target gene in homozygous T6 transgenic lines. The expression and identification the PCR gene, detection and determination of T5 resistance, Hyg marker gene T6 in transgenic materials (R) and insect resistant gene Cry30Fa1, finally selected marker free transgenic rice lines. The expression of real-time fluorescence quantitative PCR detection of Cry30Fa1 gene. At the same time, real-time fluorescence quantitative PCR detection the Cry30Fa1 gene copy number in transgenic materials. Based on the detection of Western by Cry30Fa1 gene protein expression of Blot. By Seedbox insect resistance identification method, seedling resistance identification method and field Insect resistance identification method, each of the 3 transgenic lines were screened in insect resistance analysis, resistance above the level of brown planthopper resistance in transgenic rice. In addition, we are also on the T6 transgenic rice lines of the main agronomic traits were investigated. The main results are as follows: 1. of the 184 transgenic T5 generation strains were detected Cry30Fa1 gene PCR, found that 150 strains contain Cry30Fa1 gene, and T6 detection and tracking screened 29 genes homozygous transgenic lines. The 29 transgenic lines of Hyg (R) PCR gene expression detection and immersion detection, finally obtained 15 marker free transgenic lines.2. by real-time fluorescent quantitative PCR, the expression of Cry30Fa1 gene in 29 T6 transgenic lines at the transcriptional level was detected, results showed that all the transgenic lines Cry30Fa1 At the transcriptional level was expressed, but the expression difference in relative expression between 0.19-8.53.3. through Western Blot technology, this study was tested on the expression of Cry30Fa1 gene of 29 T6 transgenic plant at the protein level showed that 5 transgenic lines were not detected in Bt protein the expression, the remaining 24 transgenic lines had Bt protein, but the transgenic lines in Bt protein expression differences, distribution between 0.13 to 11.6.4. through the analysis of the Cry30Fa1 gene copy number in transgenic plants, the study found 29 Cry30Fa1 transgenic lines in gene copy number differences, copy but the number of 15 strains belonging to low copy strain.5. resistance analysis of the 3 transgenic lines were used in group identification of insect resistance and field resistance identification method, the results show that at the seedling stage The resistance reached 7, which belongs to the sense of material; field resistance reached 3-5, belonging to the anti anti material. At the same time, the seedling bioassay method of insecticidal plant, analysis of more than 3 transgenic lines. The results showed that 3 transgenic lines was significantly higher than in brown planthopper this shows that the parent R818, transgenic lines increased insecticidal ability of.6. through the main agronomic traits of 29 transgenic lines were analyzed, the results show that the exogenous Bt gene on rice plant height, between 19 lines and their parents have significant differences; exogenous Bt gene on other agronomic traits such as heading, effective tiller number, panicle length, 1000 grain weight and seed setting rate, little effect. Through the analysis of the data found that traits of improved strains than traitvariation strains, indicating that through the combination of molecular detection and field breeding work, The transgenic lines which are similar to their parents and even better than their parents are always selected. At present, the 26 remaining transgenic lines are being analyzed comprehensively for the purpose of obtaining more transgenic lines resistant to brown planthopper.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.112.3

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