本文選題：CBF1 + 類番茄茄　； 參考：《牡丹江師范學(xué)院》2016年碩士論文

【摘要】：類番茄茄(Solanum lycopersicoides)是茄屬(Solanum)中番茄(Lycopersicon esculentum Mill)近緣野生種,在-1.25~5.3℃的條件下能正常開花結(jié)果。煙草(Nicotiana tabacum L.)為茄科(Solanleae)煙草屬(Nicotiana)一年生或有限多年生草本植物,作為中國主要的經(jīng)濟(jì)作物之一,低溫凍害一直制約著優(yōu)質(zhì)煙草的生產(chǎn)發(fā)展。CBF1基因是CBF家族的一種抗冷轉(zhuǎn)錄因子,它首先在擬南芥中被克隆出來。它能夠誘導(dǎo)低溫調(diào)節(jié)蛋白(COR蛋白)的表達(dá),使未經(jīng)過低溫馴化的植株具備一定的抗凍力,因此通過轉(zhuǎn)化CBF基因,可以提高煙草的抗凍性,從而提高煙草產(chǎn)量和改善其品質(zhì)。當(dāng)植物處于低溫脅迫的狀態(tài)下,較低的溫度會(huì)使植物脫水并對(duì)植物細(xì)胞膜造成嚴(yán)重傷害,而抗冷轉(zhuǎn)錄因子對(duì)保護(hù)細(xì)胞膜結(jié)構(gòu)起到了重要的作用,促使植株在某種程度上減緩或抵制低溫。本試驗(yàn)以無菌煙草葉片為受體材料,采用農(nóng)桿菌介導(dǎo)的葉盤法轉(zhuǎn)化煙草。研究結(jié)果如下:1、以煙草(中煙100)葉片為外植體,通過篩選各階段的最適培養(yǎng)基建立了煙草的再生體系。其中,培養(yǎng)基MS+6-BA 2.0 mg/L+NAA 0.1 mg/L是煙草葉片不定芽分化的最佳培養(yǎng)基;MS+6-BA 2.0 mg/L是煙草不定芽增殖培養(yǎng)的最佳培養(yǎng)基;MS+NAA 0.1 mg/L是煙草不定芽生根培養(yǎng)的最佳培養(yǎng)基。2、將農(nóng)桿菌侵染后的煙草葉片放入含有不同Kan濃度的篩選培養(yǎng)基中,篩選出抗性芽對(duì)Kan的抗性臨界值。該臨界值為50 mg/L的Kan溶液。3、確立了煙草的最佳轉(zhuǎn)化體系:外植體預(yù)培養(yǎng)48 h,再用OD600為0.4左右的農(nóng)桿菌菌液侵染外植體5 min,接種于含乙酰丁香酮(AS)的共培養(yǎng)培養(yǎng)基中,共培養(yǎng)48 h后除菌,頭孢霉素(Cef)的除菌濃度為500mg/L。4、通過葉盤法對(duì)煙草進(jìn)行遺傳轉(zhuǎn)化,獲得5株轉(zhuǎn)基因植株。采用CTAB法提取轉(zhuǎn)基因植株DNA并進(jìn)行PCR檢測,得到大約750 bp的清晰條帶,表明CBF1基因成功轉(zhuǎn)入煙草中。5、對(duì)轉(zhuǎn)基因煙草進(jìn)行生理指標(biāo)檢測,經(jīng)過4℃低溫處理24 h和22℃恢復(fù)處理72 h后,SOD、POD、游離脯氨酸、CAT活性均增加,并高于未經(jīng)轉(zhuǎn)化煙草活性,而MDA活性降低且始終低于未轉(zhuǎn)基因煙草活性,說明在一定程度上提高了煙草的抗冷性。
[Abstract]:Solanum lycopersicoides (Solanum lycopersicoides) is a wild species of Lycopersicon esculentum Milli in Solanum. Nicotiana tabacum L. As an annual or finite perennial herbaceous plant of Solanleaeana (Solanleaeana), as one of the main cash crops in China, low temperature freezing injury has been restricting the development of high-quality tobacco production. CBF1 gene is a cold resistant transcription factor of CBF family. It was first cloned in Arabidopsis thaliana. It can induce the expression of low temperature regulated protein (cor), and make the plants without low temperature acclimation have a certain freezing resistance. Therefore, the transformation of CBF gene can improve the frost resistance of tobacco, thus increase the yield and improve the quality of tobacco. When plants are under low temperature stress, lower temperatures can dehydrate plants and cause serious damage to cell membranes, and cold resistant transcription factors play an important role in protecting cell membrane structures. Urge plants to slow down or resist low temperatures to some extent. In this study, aseptic tobacco leaves were used as receptor materials and Agrobacterium tumefaciens mediated leaf disc method was used to transform tobacco. The results are as follows: 1. The regeneration system of tobacco was established by screening the optimum medium for each stage with the leaves of tobacco (Zhongyan 100) as explants. Of which, MS 6-BA 2.0 mg/L NAA 0.1 mg/L is the best medium for adventitious bud differentiation of tobacco leaves. MS 6-BA 2.0 mg/L is the best medium for tobacco adventitious bud proliferation and culture. MS NAA 0.1 mg/L is the best medium for rooting of tobacco adventitious buds. The infected tobacco leaves were placed in the screening medium containing different concentrations of Kan. The critical value of resistance of resistant buds to Kan was screened out. The critical value was 50 mg/L Kan solution. 3. The optimal transformation system of tobacco was established. The explants were precultured for 48 hours, then infected with Agrobacterium tumefaciens solution with OD600 about 0.4 for 5 minutes, and inoculated in co-culture medium containing acetyl eugenone. After 48 hours of co-culture, the concentration of cefcefin was 500 mg / L. 5 transgenic plants were obtained by genetic transformation of tobacco by leaf disk method. The DNA of transgenic plants was extracted by CTAB method and detected by PCR, and the clear bands of 750bp were obtained, which indicated that the CBF1 gene was successfully transferred into tobacco, and the physiological indexes of transgenic tobacco were detected. After 24 h and 22 鈩，

本文編號(hào)：1965227