本文選題：甘蔗 + 煙草　； 參考：《廣西大學(xué)》2016年碩士論文

【摘要】：甘蔗是世界上最重要的糖料和能源作物。在我國,甘蔗的產(chǎn)糖量占全國糖總產(chǎn)量的92%-94%。培育高產(chǎn)高糖抗逆性強(qiáng)的甘蔗新品種是確保我國甘蔗糖業(yè)可持續(xù)發(fā)展的重要基礎(chǔ)。在甘蔗品種遺傳改良中,常規(guī)育種仍然是培育甘蔗優(yōu)良新品種的主要手段,但基因工程育種正表現(xiàn)出明顯的優(yōu)勢,其運(yùn)用也愈加廣泛。本研究以甘蔗蔗糖轉(zhuǎn)運(yùn)蛋白基因SoSUT5基因作為外源基因構(gòu)建了植物表達(dá)載體,進(jìn)行了煙草和甘蔗的遺傳轉(zhuǎn)化。主要研究結(jié)果如下：1.煙草的遺傳轉(zhuǎn)化。構(gòu)建了雙子葉植物表達(dá)載體pRI101-ON-SoSUT5,通過農(nóng)桿菌介導(dǎo)轉(zhuǎn)化法侵染煙草的葉盤,將載體上的T-DNA區(qū)域轉(zhuǎn)入煙草品種K 346中,經(jīng)過組織培養(yǎng)獲得煙草再生植株31株。通過PCR檢測,擴(kuò)增npt Ⅱ基因和目的基因,分別得到轉(zhuǎn)化效率為100%(npt Ⅱ+)和93.55%(SoSUT5+)。測定了野生型、轉(zhuǎn)空載體pRI 101-ON型和轉(zhuǎn)重組載體pRI 101-ON-SoSUT5型煙草的可溶性糖含量，，以轉(zhuǎn)pRI101-ON-SoSUT5型轉(zhuǎn)空載體pRI 101-ON型野生型。2. RNAi載體的構(gòu)建。利用SiRNA Target Finder預(yù)測軟件,分析SoSUT5基因的siRNA干擾片段,最終選擇一段550 bp的序列作為干擾片段。將該片段分別正向和反向插入干擾載體pTCK 303中,構(gòu)建了RNAi載體pTCK 303-SoSUT5。同時(shí)以甘蔗品種B8的愈傷組織為材料,進(jìn)行了Hyg的濃度篩選實(shí)驗(yàn)。結(jié)果表明,甘蔗愈傷誘導(dǎo)階段和分化階段的Hyg的最佳篩選濃度為50 mg/L,生根階段的Hyg的最佳篩選濃度為40 mg/L3.甘蔗的遺傳轉(zhuǎn)化。構(gòu)建了單子葉植物表達(dá)載體pUBTC-SoSUT5,通過農(nóng)桿菌介導(dǎo)轉(zhuǎn)化法侵染甘蔗愈傷組織,將載體的T-DNA區(qū)域轉(zhuǎn)入甘蔗品種B8中。經(jīng)過組織培養(yǎng),在PPT 2.5 mg/L壓力下篩選48 d獲得甘蔗再生植株128株。移栽后進(jìn)行PCR檢測標(biāo)記基因bar,其中陽性植株有91株,轉(zhuǎn)化效率71.09%。
[Abstract]:Sugar cane is the most important sugar and energy crop in the world. In our country, sugar yield of sugarcane accounts for 92-94 percent of total sugar production in China. Cultivating new sugarcane varieties with high yield and high sugar resistance is an important basis to ensure the sustainable development of sugarcane industry in China. In the genetic improvement of sugarcane varieties, conventional breeding is still the main means of breeding sugarcane fine new varieties, but genetic engineering breeding is showing obvious advantages, and its application is becoming more and more extensive. In this study, the sugarcane sucrose transporter gene SoSUT5 gene was used as the exogenous gene to construct the plant expression vector, and the genetic transformation of tobacco and sugarcane was carried out. The main results are as follows: 1. Genetic transformation of tobacco. A dicotyledonous plant expression vector pRI101-ON-SoSUT5 was constructed. The T-DNA region of the vector was transferred into tobacco variety K346 by Agrobacterium tumefaciens-mediated transformation, and 31 tobacco regenerated plants were obtained by tissue culture. Npt 鈪

本文編號(hào)：1926179