转cry30Fal基因抗虫水稻研究

发布时间：2018-06-21 15:49

本文选题：水稻 + Bt杀虫晶体蛋白　；参考：《四川农业大学》2013年硕士论文

【摘要】：水稻是世界上重要的粮食作物,仅中国就有8亿人口以水稻为主食。同时,水稻也是虫害最严重的农作物,稻飞虱的危害每年造成巨大的损失,一般危害损失10-20%,有时甚至会更大。以化学防治为主的综合防治,对控制稻飞虱危害起了一定的作用。然而,长期、大量、广泛的使用化学农药,产生了如农药残留,环境污染,虫的抗药性增强等一系列问题,已经成为人们关注的焦点。随着生物技术的发展,利用基因工程手段培育抗虫能力强的新品种已成为发展的趋势和新的途径。目前农作物抗虫性改良的外源基因主要是从苏云金芽孢杆菌中分离的Bt毒蛋白基因。本研究的主要目的是利用基因工程手段培育具有自主知识产权的抗褐飞虱水稻品种。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cry30Fal基因转入其中,获得转基因植株。通过选择标记基因潮霉素抗性表达检测,目的基因的PCR检测,筛选到T0代阳性转基因植株。通过对T1代植株目的基因和选择标记基因的PCR检测,筛选到无选择标记基因的阳性植株。采用Western杂交检测T1代转基因阳性植株的蛋白表达情况。通过田问观察,收取农艺性状好的无选择标记的阳性转基因植株。通过T2代植株室内喂虫试验初步测定其抗虫性。本研究获得的主要结果如下： 1.构建了高效表达的转化双元载体pCDMAR-cry30Fal-Hyg,转化R818胚性愈伤,获得233株转基因再生植株,通过潮霉素抗性表达检测和目的基因的PCR检测,获得46株含cry30Fal基囚的阳性植株。 2.通过对转基因植株T1代39个株系共1680株的苗期叶片目的基因PCR检测,1680个单株中有1049株含有目的基因cry30Fal,631株不含目的基因cry30Fal。对含有cry30Fal目的基因的1049个单株进行了潮霉素抗性基因的PCR检测,检测到其中有353个单株含潮霉素抗性基因,696个单株不含潮霉素基因。检测结果表明,转基因T1代植株中,含cry30Fal目的基因并去掉潮霉素抗性基因的单株占总检测植株的41.43%。对T1代转基因植株进行遗传分离检测,经卡平方检验,T1代植株遗传分离检测中有64.10%符合3：1的分离。 3.蛋白含量测定结果表明：编号为1、2、3、4、10、11、12、13、17、25、29、30、31、32、33、36、37、38、39的T1代植株中没有目的蛋白表达或者表达量很少,在编号为5、6、7、8、9、14、15、16、18、19、20、21、22、23、24、26、27、28、34、35的T1代植株中目的蛋白有表达,并且目的蛋白条带的大小、颜色的深浅反应目的蛋白表达量的多少。 4.室内人工喂虫试验结果显示：对照高感TN1全部死亡,对照R818基本死亡,而转基因植株则部分死亡。经PCR检测,第一批次试验含目的基因植株的成活率为44.31%；不含目的基因植株的成活率为16.52%。含目的基因的植株成活率是不含目的基因植株的2.68倍。第二批次含目的基因植株的成活率为48.06%；不含目的基因植株的成活率为14.56%。含目的基因的植株成活率是不含目的基因植株的3.30倍。第三批次含目的基因的植株成活率为46.91%；不含目的基因植株的成活率为14.88%。含目的基因的植株成活率是不含目的基因植株的3.15倍。而三次合计的结果是含目的基因植株的成活率为46.41%,不含目的基因植株的成活率为15.34%,含目的基因的植株成活率是不含目的基因植株的3.03倍；因此,在一定范围内可以认为阳性转基因植株对褐飞虱是有一定抗性的。
[Abstract]:Rice is one of the most important food crops in the world. In China, only 800 million of the population is the main food of rice. At the same time, rice is the most harmful crop. The harm of rice planthopper causes huge losses every year. The damage is 10-20%, sometimes even bigger. The comprehensive control based on chemical control has played a certain role in controlling the harm of rice planthopper. However, a series of problems such as pesticide residues, environmental pollution, and insect resistance enhancement have become the focus of attention for a long time, extensive and extensive use of chemical pesticides, which have become the focus of attention. With the development of biotechnology, the development of new varieties with strong resistance to insect resistance by means of genetic engineering has become a trend and new way of development. The foreign gene for improving the insect resistance of the former crops is mainly the Bt toxin protein gene isolated from Bacillus thuringiensis. The main purpose of this study is to cultivate rice varieties with independent intellectual property by genetic engineering.
In this study, a good rice restorer line Shu Hui 818 (R818) was used as a receptor material and the genetic transformation method mediated by Agrobacterium tumefaciens was used to transfer the cry30Fal gene cloned in the laboratory to obtain transgenic plants. Through the selective detection of the resistance expression of the tagged gene hygromycin, the PCR detection of the target gene, and the screening of the positive transgenic plants of the T0 generation. The positive plants with no selection marker genes were screened by PCR detection of the target gene and selection marker gene of the T1 generation plant. The protein expression of the transgenic positive plants of the T1 generation was detected by Western hybridization. Through field observation, the positive transgenic plants with good agronomic characters were collected and the transgenic plants were fed in the T2 generation plant. The insect resistance was tested preliminarily.
The main results obtained in this study are as follows:
1. a highly expressed transformation dual vector pCDMAR-cry30Fal-Hyg was constructed, and R818 embryogenic callus was transformed. 233 transgenic plants were obtained. Through the detection of hygromycin resistance expression and the PCR detection of the target gene, 46 positive plants containing cry30Fal were obtained.
2. through the detection of the leaf target gene PCR of 1680 strains of T1 generation of transgenic plants, 1049 of the 1680 single plants contained the target gene cry30Fal and 631 without the target gene cry30Fal. for the PCR detection of the hygromycin resistance gene of 1049 single plants containing the cry30Fal target gene, and 353 single strains were detected. The mycomycin resistance gene and 696 single plants did not contain the hygromycin gene. The results showed that in the transgenic T1 generation, the single plant containing the cry30Fal target gene and the hygromycin resistant gene accounted for the genetic separation of the T1 generation transgenic plants with the 41.43%. of the total detected plant, and there were 64.10% characters in the genetic separation test of the T1 generation by the square test. The separation of 3:1.
The determination of 3. protein content showed that there was no expression or expression of the target protein in T1 plants numbered 1,2,3,4,10,11,12,13,17,25,29,30,31,32,33,36,37,38,39, and the target egg white was expressed in the T1 generation plant numbered 5,6,7,8,9,14,15,16,18,19,20,21,22,23,24,26,27,28,34,35, and the size of the target protein band, The depth of color reflects the amount of target protein expressed.
4. the results of indoor artificial feeding test showed that the control of high sensitivity TN1 was all dead, the control R818 died, and the transgenic plants were partially dead. The survival rate of the plant containing the target gene was 44.31% by the PCR test, and the survival rate of the plant without the target gene was 16.52%. containing the target gene without the target base. The survival rate of second batches of target genes was 48.06% for 2.68 times. The survival rate of the plant without target gene was 3.30 times that of the target gene without the target gene. The survival rate of the plant containing the target gene was 46.91%, and the survival rate of the plant without the target gene was 14.88%., and the survival rate of the plant without the target gene was 2.68. The survival rate of the plant containing the target gene is 3.15 times as high as that of the plant without the target gene. The result of the three combination is that the survival rate of the plant containing the target gene is 46.41%, the survival rate of the plant without the target gene is 15.34%, the survival rate of the plant containing the target gene is 3.03 times that of the plant without the target gene; therefore, it can be considered in a certain range. The positive transgenic plants were 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】：2013
【分类号】：S511

【参考文献】