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  本文關(guān)鍵詞： 蜀恢818 Cry30Fa1基因 抗蟲性 拷貝數(shù) 農(nóng)藝性狀　出處：《四川農(nóng)業(yè)大學》2016年碩士論文　論文類型：學位論文

【摘要】：本研究以優(yōu)良的水稻恢復系蜀恢818(R818)為受體材料,采用農(nóng)桿菌介導的遺傳轉(zhuǎn)化方法,將本實驗室克隆的抗褐飛虱基因Cry30Fa1轉(zhuǎn)入其中,通過連續(xù)自交和DNA檢測在T6代獲得了目的基因純合的轉(zhuǎn)基因株系。通過目的基因PCR檢測和抗性表達鑒定,確定T5、T6代轉(zhuǎn)基因材料中的選擇標記基因Hyg(R)和抗蟲基因Cry30Fa1,最終篩選到無選擇標記基因的轉(zhuǎn)基因水稻株系。采用實時熒光定量PCR技術(shù)檢測Cry30Fa1基因的轉(zhuǎn)錄情況。同時,基于實時熒光定量PCR技術(shù)也檢測了轉(zhuǎn)基因材料中的Cry30Fa1基因拷貝數(shù)。采用Western Blot檢測Cry30Fa1基因蛋白表達情況。采用苗期集團抗蟲性鑒定法、苗期單株抗蟲性鑒定法和田間抗蟲性鑒定法,分別對3個轉(zhuǎn)基因株系進行了抗蟲性分析,篩選到中抗以上水平的抗褐飛虱轉(zhuǎn)基因水稻株系。此外,我們還對T6代轉(zhuǎn)基因水稻株系的主要農(nóng)藝性狀進行了調(diào)查。主要結(jié)果如下：1.對T5代的184個轉(zhuǎn)基因株系進行Cry30Fa1基因的PCR檢測,發(fā)現(xiàn)其中150個株系含有Cry30Fa1基因,而且T6代跟蹤檢測中篩選到29個目的基因純合的轉(zhuǎn)基因株系。通過對29個轉(zhuǎn)基因株系進行了Hyg(R)基因PCR檢測和浸泡表達檢測,最終獲得了15個無選擇標記基因的轉(zhuǎn)基因株系。2.通過實時熒光定量PCR技術(shù),本研究對29個T6代轉(zhuǎn)基因株系的Cry30Fa1基因在轉(zhuǎn)錄水平上的表達進行了檢測,結(jié)果顯示,所有轉(zhuǎn)基因株系的Cry30Fa1基因在轉(zhuǎn)錄水平上均有表達,但表達量差異較大,相對表達量在0.19-8.53之間。3.通過Western Blot技術(shù),本研究對29個T6代轉(zhuǎn)基因株系Cry30Fa1基因在蛋白質(zhì)水平上的表達進行了檢測,結(jié)果顯示,5個轉(zhuǎn)基因株系中沒有檢測到Bt蛋白質(zhì),余下的24個轉(zhuǎn)基因株系的Bt蛋白均有表達,但轉(zhuǎn)基因株系中Bt蛋白表達量存在較大差異,分布在0.13到11.6之間。4.通過分析了轉(zhuǎn)基因植株中Cry30Fa1基因拷貝數(shù),本研究發(fā)現(xiàn)29個轉(zhuǎn)基因株系中Cry30Fa1基因拷貝數(shù)存在較大差異,但有15個株系的拷貝數(shù)屬于低拷貝的株系。5.采用苗期集團抗蟲性鑒定法和田間抗蟲性鑒定法對其中3個轉(zhuǎn)基因株系進行了抗蟲性分析,結(jié)果發(fā)現(xiàn),苗期抗性均達到了7級,屬于中感材料；田間抗性達到3-5級,屬于中抗到抗的材料。同時,采用苗期單株抗蟲性鑒定法,對以上3個轉(zhuǎn)基因株系進行了殺蟲性分析,結(jié)果表明,3個轉(zhuǎn)基因株系中的褐飛虱死亡率顯著高于親本R818,這說明了轉(zhuǎn)基因株系增加了殺蟲能力。6.通過對29個轉(zhuǎn)基因株系主要農(nóng)藝性狀進行分析,結(jié)果表明,外源Bt基因?qū)λ局旮哂绊懽畲?有19個株系與親本之間存在顯著性差異；外源Bt基因?qū)ζ渌r(nóng)藝性狀如抽穗期、有效分蘗數(shù)、主穗長、結(jié)實率、千粒重等的影響較小。通過數(shù)據(jù)分析發(fā)現(xiàn)性狀得到改良的株系比性狀變差的株系多,這說明通過分子水平檢測和田間選育工作相結(jié)合,總會選到和親本相似農(nóng)藝性狀甚至是優(yōu)于親本的轉(zhuǎn)基因株系。目前,正在對余下的26個轉(zhuǎn)基因株系進行全面抗蟲性分析,以期獲得更多抗褐飛虱的轉(zhuǎn)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.

【學位授予單位】：四川農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S435.112.3

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1 王海鵬;轉(zhuǎn)Cry30Fa1基因抗褐飛虱水稻的研究[D];四川農(nóng)業(yè)大學;2016年

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本文編號：1452606