本文關(guān)鍵詞： 雙單倍體DM 農(nóng)桿菌轉(zhuǎn)化 增強(qiáng)子捕獲 側(cè)翼序列擴(kuò)增　出處：《青海大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：馬鈴薯作為世界上第四大糧食作物,同時也是中國的第四大糧食作物,在人民生活中占著越來越重要的地位。馬鈴薯全基因組測序的完成,幫助人們加深對馬鈴薯遺傳規(guī)律的了解,加強(qiáng)對其豐富的遺傳資源的開發(fā),同時也加快分子育種的運(yùn)用。雙單倍體馬鈴薯DM(DM13-516-R44)為測序材料之一,本研究以它為材料,通過農(nóng)桿菌介導(dǎo)將構(gòu)建好的增強(qiáng)子捕獲T-DNA載體導(dǎo)入馬鈴薯的外植體中,獲得轉(zhuǎn)基因植株,得到轉(zhuǎn)基因植株后,利用潮霉素設(shè)計(jì)引物對生根較好的轉(zhuǎn)化苗進(jìn)行PCR鑒定,初步構(gòu)建了馬鈴薯增強(qiáng)子捕獲系群體。此外,利用PCR-walking技術(shù)擴(kuò)增了突變體植株的側(cè)翼序列,并結(jié)合已經(jīng)公布的馬鈴薯序列對部分側(cè)翼序列進(jìn)行了分析。主要的研究結(jié)果如下:1.初步建立了馬鈴薯增強(qiáng)子捕獲系群體,獲得了12000株左右的轉(zhuǎn)化苗,對1500株轉(zhuǎn)化苗進(jìn)行陽性鑒定,有978個陽性株系。同時,優(yōu)化了馬鈴薯DM的轉(zhuǎn)化體系,主要的實(shí)驗(yàn)條件為:誘導(dǎo)分化培養(yǎng)基中頭孢霉素(cef)的濃度為600mg/L、潮霉素(hyg)為8mg/L,外植體預(yù)培養(yǎng)13天、侵染時間8min、農(nóng)桿菌濃度OD600=0.4、共培養(yǎng)3天。2.利用PCR-walking技術(shù)擴(kuò)增了馬鈴薯突變體的側(cè)翼序列,對60個陽性突變體進(jìn)行了擴(kuò)增,其中28個突變體擴(kuò)增出單一條帶并測序,其測序結(jié)果與馬鈴薯全基因組序列比對后,發(fā)現(xiàn)有6個突變體是有效插入,對插入位點(diǎn)的信息做了初步分析。3.將鑒定過的30個陽性突變體植株種植于溫室,獲得了30個突變體植株的微型種薯,觀察到部分突變體的表型變化。將460個已鑒定的陽性突變體植株種植,觀察其表型變化。
[Abstract]:Potato, as the world's 4th largest food crop, is also the 4th largest food crop in China, and plays an increasingly important role in people's lives. To help people to understand the genetic law of potato, to strengthen the development of its abundant genetic resources, and to accelerate the application of molecular breeding. DM13-516-R44, a dihaploid potato, is one of the sequencing materials. The constructed enhancer was introduced into potato explants by Agrobacterium tumefaciens to obtain transgenic plants. The transformed plants were identified by PCR with hygromycin primer design. In addition, the flanking sequence of the mutant plant was amplified by PCR-walking technique. Some flanking sequences were analyzed based on the published potato sequences. The main results were as follows: 1. A population of potato enhancer captors was established, and about 12000 transformed seedlings were obtained. There were 978 positive lines in 1500 transformed seedlings. Meanwhile, the transformation system of DM in potato was optimized. The main experimental conditions were as follows: the concentration of cefocefin was 600 mg / L, hygm hyg was 8 mg / L in induced differentiation medium, and the explant was precultured for 13 days. The infection time was 8 min, the concentration of Agrobacterium tumefaciens was OD600,0.4, and co-cultured for 3 days. 2. The flanking sequence of potato mutants was amplified by PCR-walking technique, and 60 positive mutants were amplified, of which 28 mutants were amplified by a single band and sequenced. The results of sequencing were compared with the whole genome sequence of potato. It was found that 6 mutants were effectively inserted. The information of insertion site was analyzed preliminarily. The 30 identified positive mutants were planted in greenhouse. The phenotypic changes of some mutants were observed, and 460 identified positive mutants were planted to observe the phenotypic changes.
【學(xué)位授予單位】：青海大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S532

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