毛竹非组培转基因及转DhNiR基因水稻后代性状分析

发布时间：2019-06-10 20:22

【摘要】：竹子传统杂交与选择育种困难,造成育种水平滞后,成为竹产业进一步提升的限制因素。转基因是有效育种手段,但是竹子再生比较困难,遗传转化成功的报道很少。本研究主要从两方面入手,一方面通过非组培转基因研究,避开竹子组培再生的难题;另一方面是对能够促进植物再生的基因如DhNiR(Dendrocalamus hamiltonii,Dh)进行研究,通过将促进再生基因整合入双元表达载体,以提高竹子再生与转化效率。本试验的目的是分别将这两种方法应用到竹子中,使竹子的转化效率得到有效的提高。本项目首先以毛竹种子为实验材料,用农杆菌介导植物萌动种子基因转化方法将CP4-EPSPS基因成功转化到毛竹种子中,成功获得了转基因植株。试验结果如下:在共培养时,菌液浓度以OD600=0.07为最适浓度,太低或过高的浓度都不利于苗的转化和萌发;且共培养液中加入400μmol·L-1乙酰丁香酮,可使出苗率提高8%左右;经PCR分子验证获得1株毛竹转基因植株。其次是对转DhNiR基因水稻进行后代性状分析,试验结果如下:以转DhNiR的T2代水稻成熟种子和野生型水稻成熟种子为试验材料,经愈伤组织诱导及增殖后,选取生长良好的致密愈伤组织进行分化培养,愈伤诱导率和苗分化时间上均表现出差异,转DhNiR的水稻愈伤组织在转入分化培养基第25天时绿点愈伤率达到了100%,而野生型水稻绿点愈伤率仅为56.37%,且苗分化时间比野生型水稻早5天左右,表明DhNiR在水稻再生能力提高方面有促进作用;以转DhNiR基因的水稻和野生型水稻的叶片、茎、根、愈伤组织为样本,测定各个样本中的亚硝酸还原酶活性,转DhNiR基因水稻各部分组织的亚硝酸还原酶活性均高于野生型水稻,且达到了极显著差异;对转DhNiR水稻和野生型水稻的种子、叶片及根分别进行硝酸盐和亚硝酸盐含量测定,结果显示转DhNiR基因水稻各部分组织中的硝酸盐和亚硝酸盐含量也普遍高于野生型水稻。
[Abstract]:The difficulty of traditional hybrid and selection breeding of bamboo, which leads to the lag of breeding level, has become a limiting factor for the further promotion of bamboo industry. Transgenic is an effective breeding method, but bamboo regeneration is difficult, and there are few reports of successful genetic transformation. This study mainly starts from two aspects, on the one hand, through non-tissue culture transgenic research, to avoid the problem of bamboo tissue culture regeneration; On the other hand, genes that can promote plant regeneration, such as DhNiR (Dendrocalamus hamiltonii,Dh, are studied in order to improve the efficiency of bamboo regeneration and transformation by integrating the promoting genes into binary expression vectors. The purpose of this experiment is to apply these two methods to bamboo respectively, so that the transformation efficiency of bamboo can be effectively improved. In this project, using Phyllostachys pubescens seeds as experimental materials, CP4-EPSPS gene was successfully transformed into Phyllostachys pubescens seeds by Agrobacterium tumefaciens mediated seed gene transformation, and transgenic plants were successfully obtained. The results were as follows: in co-culture, OD600=0.07 was the optimum concentration, too low or too high concentration was not conducive to seedling transformation and germination; The emergence rate of Phyllostachys pubescens was increased by about 8% when 400 渭 mol 路L-1 acetyleugenone was added to the co-culture medium, and a transgenic plant of Phyllostachys pubescens was obtained by PCR molecular verification. Secondly, the characters of the progenies of transgenic rice with DhNiR gene were analyzed. The results were as follows: the mature seeds of T2 generation rice and wild type rice mature seeds transformed with DhNiR were used as experimental materials, and after Callus induction and proliferation, When the dense calli with good growth were selected for differentiation and culture, the calli induction rate and seedling differentiation time were different. The green spot healing rate of rice calli transferred to DhNiR reached 100% on the 25th day after being transferred to the differentiation medium. However, the green spot healing rate of wild type rice was only 56.37%, and the seedling differentiation time was about 5 days earlier than that of wild type rice, which indicated that DhNiR could promote the improvement of rice regeneration ability. Taking the leaves, stems, roots and calli of transgenic rice with DhNiR gene as samples, the activity of nitrate reductase in each sample was determined. The activity of nitrate reductase in each part of transgenic rice with DhNiR gene was higher than that in wild type rice. And reached a very significant difference; The contents of nitrate and nitrite in seeds, leaves and roots of transgenic DhNiR rice and wild type rice were determined respectively. The results showed that the contents of nitrate and nitrite in the tissues of transgenic rice with DhNiR gene were also generally higher than those in wild type rice.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S795.7;S511

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