本文選題：水稻 + Bt殺蟲晶體蛋白　； 參考：《四川農(nóng)業(yè)大學(xué)》2013年碩士論文

【摘要】：水稻是世界上重要的糧食作物,僅中國就有8億人口以水稻為主食。同時(shí),水稻也是蟲害最嚴(yán)重的農(nóng)作物,稻飛虱的危害每年造成巨大的損失,一般危害損失10-20%,有時(shí)甚至?xí)�。以化學(xué)防治為主的綜合防治,對(duì)控制稻飛虱危害起了一定的作用。然而,長(zhǎng)期、大量、廣泛的使用化學(xué)農(nóng)藥,產(chǎn)生了如農(nóng)藥殘留,環(huán)境污染,蟲的抗藥性增強(qiáng)等一系列問題,已經(jīng)成為人們關(guān)注的焦點(diǎn)。隨著生物技術(shù)的發(fā)展,利用基因工程手段培育抗蟲能力強(qiáng)的新品種已成為發(fā)展的趨勢(shì)和新的途徑。目前農(nóng)作物抗蟲性改良的外源基因主要是從蘇云金芽孢桿菌中分離的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識(shí)產(chǎn)權(quán)的抗褐飛虱水稻品種。 本研究以優(yōu)良的水稻恢復(fù)系蜀恢818(R818)為受體材料,采用農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化方法,將本實(shí)驗(yàn)室克隆的cry30Fal基因轉(zhuǎn)入其中,獲得轉(zhuǎn)基因植株。通過選擇標(biāo)記基因潮霉素抗性表達(dá)檢測(cè),目的基因的PCR檢測(cè),篩選到T0代陽性轉(zhuǎn)基因植株。通過對(duì)T1代植株目的基因和選擇標(biāo)記基因的PCR檢測(cè),篩選到無選擇標(biāo)記基因的陽性植株。采用Western雜交檢測(cè)T1代轉(zhuǎn)基因陽性植株的蛋白表達(dá)情況。通過田問觀察,收取農(nóng)藝性狀好的無選擇標(biāo)記的陽性轉(zhuǎn)基因植株。通過T2代植株室內(nèi)喂蟲試驗(yàn)初步測(cè)定其抗蟲性。 本研究獲得的主要結(jié)果如下： 1.構(gòu)建了高效表達(dá)的轉(zhuǎn)化雙元載體pCDMAR-cry30Fal-Hyg,轉(zhuǎn)化R818胚性愈傷,獲得233株轉(zhuǎn)基因再生植株,通過潮霉素抗性表達(dá)檢測(cè)和目的基因的PCR檢測(cè),獲得46株含cry30Fal基囚的陽性植株。 2.通過對(duì)轉(zhuǎn)基因植株T1代39個(gè)株系共1680株的苗期葉片目的基因PCR檢測(cè),1680個(gè)單株中有1049株含有目的基因cry30Fal,631株不含目的基因cry30Fal。對(duì)含有cry30Fal目的基因的1049個(gè)單株進(jìn)行了潮霉素抗性基因的PCR檢測(cè),檢測(cè)到其中有353個(gè)單株含潮霉素抗性基因,696個(gè)單株不含潮霉素基因。檢測(cè)結(jié)果表明,轉(zhuǎn)基因T1代植株中,含cry30Fal目的基因并去掉潮霉素抗性基因的單株占總檢測(cè)植株的41.43%。對(duì)T1代轉(zhuǎn)基因植株進(jìn)行遺傳分離檢測(cè),經(jīng)卡平方檢驗(yàn),T1代植株遺傳分離檢測(cè)中有64.10%符合3：1的分離。 3.蛋白含量測(cè)定結(jié)果表明：編號(hào)為1、2、3、4、10、11、12、13、17、25、29、30、31、32、33、36、37、38、39的T1代植株中沒有目的蛋白表達(dá)或者表達(dá)量很少,在編號(hào)為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達(dá),并且目的蛋白條帶的大小、顏色的深淺反應(yīng)目的蛋白表達(dá)量的多少。 4.室內(nèi)人工喂蟲試驗(yàn)結(jié)果顯示：對(duì)照高感TN1全部死亡,對(duì)照R818基本死亡,而轉(zhuǎn)基因植株則部分死亡。經(jīng)PCR檢測(cè),第一批次試驗(yàn)含目的基因植株的成活率為44.31%；不含目的基因植株的成活率為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為48.06%；不含目的基因植株的成活率為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為46.91%；不含目的基因植株的成活率為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。而三次合計(jì)的結(jié)果是含目的基因植株的成活率為46.41%,不含目的基因植株的成活率為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,在一定范圍內(nèi)可以認(rèn)為陽性轉(zhuǎn)基因植株對(duì)褐飛虱是有一定抗性的。
[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.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：S511

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