类番茄茄CBF1基因转化烟草及生理检测

发布时间：2018-06-01 18:40

  本文选题：CBF1 + 类番茄茄　； 参考：《牡丹江师范学院》2016年硕士论文

【摘要】：类番茄茄(Solanum lycopersicoides)是茄属(Solanum)中番茄(Lycopersicon esculentum Mill)近缘野生种,在-1.25~5.3℃的条件下能正常开花结果。烟草(Nicotiana tabacum L.)为茄科(Solanleae)烟草属(Nicotiana)一年生或有限多年生草本植物,作为中国主要的经济作物之一,低温冻害一直制约着优质烟草的生产发展。CBF1基因是CBF家族的一种抗冷转录因子,它首先在拟南芥中被克隆出来。它能够诱导低温调节蛋白(COR蛋白)的表达,使未经过低温驯化的植株具备一定的抗冻力,因此通过转化CBF基因,可以提高烟草的抗冻性,从而提高烟草产量和改善其品质。当植物处于低温胁迫的状态下,较低的温度会使植物脱水并对植物细胞膜造成严重伤害,而抗冷转录因子对保护细胞膜结构起到了重要的作用,促使植株在某种程度上减缓或抵制低温。本试验以无菌烟草叶片为受体材料,采用农杆菌介导的叶盘法转化烟草。研究结果如下:1、以烟草(中烟100)叶片为外植体,通过筛选各阶段的最适培养基建立了烟草的再生体系。其中,培养基MS+6-BA 2.0 mg/L+NAA 0.1 mg/L是烟草叶片不定芽分化的最佳培养基;MS+6-BA 2.0 mg/L是烟草不定芽增殖培养的最佳培养基;MS+NAA 0.1 mg/L是烟草不定芽生根培养的最佳培养基。2、将农杆菌侵染后的烟草叶片放入含有不同Kan浓度的筛选培养基中,筛选出抗性芽对Kan的抗性临界值。该临界值为50 mg/L的Kan溶液。3、确立了烟草的最佳转化体系:外植体预培养48 h,再用OD600为0.4左右的农杆菌菌液侵染外植体5 min,接种于含乙酰丁香酮(AS)的共培养培养基中,共培养48 h后除菌,头孢霉素(Cef)的除菌浓度为500mg/L。4、通过叶盘法对烟草进行遗传转化,获得5株转基因植株。采用CTAB法提取转基因植株DNA并进行PCR检测,得到大约750 bp的清晰条带,表明CBF1基因成功转入烟草中。5、对转基因烟草进行生理指标检测,经过4℃低温处理24 h和22℃恢复处理72 h后,SOD、POD、游离脯氨酸、CAT活性均增加,并高于未经转化烟草活性,而MDA活性降低且始终低于未转基因烟草活性,说明在一定程度上提高了烟草的抗冷性。
[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，

本文编号：1965227