本文關(guān)鍵詞： 旱稻 農(nóng)桿菌介導法 除草劑 PCR Southern雜交　出處：《河北科技大學》2016年碩士論文　論文類型：學位論文

【摘要】：由于水資源短缺,導致水稻的生產(chǎn)受到限制,旱稻作為一個需水量少耐干旱的品種,具有良好的發(fā)展及應用前景。由于蟲害是導致水稻減產(chǎn)的一個重要因素,利用轉(zhuǎn)基因培育具有抗蟲性的旱稻品種,具有經(jīng)濟效益和生態(tài)效益。PPA基因具有殺蟲的生理活性,在抗蟲轉(zhuǎn)基因作物育種方面應用前景廣闊。本研究選用“旱稻2號”作為實驗材料,以Bar基因作為篩選標記基因,通過農(nóng)桿菌介導法將抗蟲基因PPA導入旱稻的基因組中,對轉(zhuǎn)化體系進行中的各種因素進行了優(yōu)化,培育出了有抗除草劑、抗蟲活性的轉(zhuǎn)基因旱稻植株。通過PCR對轉(zhuǎn)化旱稻植株進行了鑒定,PCR結(jié)果證明PPA基因已經(jīng)成功整合到旱稻2號基因組中。大田噴灑0.5 mg/mL的草銨膦,篩選得到有除草劑抗性的T_1代植株。在實驗室對T_2代轉(zhuǎn)化株系進行純合體篩選,對全部攜帶除草劑抗性的株系進行PCR鑒定,篩選純合株系。本實驗利用地高辛標記的Southern雜交技術(shù)對篩選得到的轉(zhuǎn)PPA的旱稻2號株系進行檢測。Southern雜交有許多影響因素,例如:酶切體系、樣品DNA的量、探針濃度、雜交條件等。實驗結(jié)果表明:在60μL的酶切體系中適宜加入15μg的DNA,90U的內(nèi)切酶,酶切15-16 h。在雜交體系中5 mL雜交液中適宜加入的探針的量為150 ng。Southern雜交結(jié)果表明:PPA基因以多拷貝的形式整合到旱稻2號基因組中。
[Abstract]:Due to the shortage of water resources, rice production is restricted. As a variety with less water requirement and drought tolerance, Upland rice has a good prospect of development and application. Transgenic upland rice varieties with insecticidal resistance have economic and ecological benefits. PPA gene has insecticidal physiological activity. In this study, "Upland Rice 2" was selected as experimental material and Bar gene as screening marker gene. The insect-resistant gene PPA was introduced into the genome of Upland rice by Agrobacterium tumefaciens mediated method. The factors in the transformation system were optimized, and the herbicide resistance was developed. Transgenic upland rice plants with insecticidal activity. The transgenic upland rice plants were identified by PCR. The results showed that the PPA gene had been successfully integrated into the genome of Upland Rice 2. The plants with herbicide resistance were screened by homozygous screening in the laboratory, and all the lines with herbicide resistance were identified by PCR. In this experiment, digoxigenin labeled Southern hybridization was used to detect the PPA transgenic Upland Rice strain 2. Southern blot analysis showed that there were many factors affecting the selection, such as enzyme digestion system, sample DNA quantity and probe concentration. The results showed that 15 渭 g of DNA-90U endonuclease could be added into 60 渭 L enzyme system. The suitable amount of probes in 5 mL hybridization solution was 150 ng.Southern hybridization. The results showed that the gene was integrated into the genome of Upland Rice 2 in the form of multiple copies.
【學位授予單位】：河北科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S511.6;Q943.2

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