通过沉默GA20-ox基因获矮化苹果植株的研究

发布时间：2018-06-10 13:36

本文选题：苹果 + GA20-ox基因　；参考：《沈阳农业大学》2016年博士论文

【摘要】：苹果(Malus domestica Borkh.)是世界范围内栽培最为广泛、经济价值较高的果树树种之一。本研究以RNAi为沉默目的基因的关键技术,将编码GA20-ox基因的沉默信号用农杆菌介导法导入苹果当中,用以阻断GA的生物合成从而获得转基因矮化砧木,探讨基于系统获得性沉默原理利用转基因苹果砧木改良非转基因苹果接穗的可行性。具体研究结果如下：1.以平邑甜茶种胚为外植体,在筛选出适合的胚培养培养基的情况下建立了继代增殖体系、组培苗生根体系和组培微嫁接体系。较适宜的增殖培养基为MS+1 mg·L_(-1) 6-BA +0.1 mg·L_(-1) NAA+0.2 mg.L_(-1)GA,在此培养基上增殖系数为3.37。适于平邑甜茶生根的培养基配方为1/2MS+0.6 mg·L_(-1) IBA+0.5 g·L_(-1) AC,生根率为69.67%。最适于微嫁接的培养基是MS+0.3 mg·L_(-1) IAA+1.0 mg.L_(-1)BA,接穗和砧木均选用继代培养40 d的试管苗,并且接穗具有2片真叶时嫁接成活率最高,用平邑甜茶作为砧木的嫁接成活率高,嫁接口愈合最好。2.建立了平邑甜茶组培苗叶片为外植体离体再生不定芽体系。最适宜的培养基是MS+4.0 mg·L_(-1)6-BA+0.40 mg·L_(-1)NAA;近轴面与培养基接触的叶片再生不定芽频率高于远轴面与培养基接触的叶片再生不定芽频率；暗培养对叶片再生不定芽是必需的,暗培养21 d左右能明显提高叶片的再生效率；选择继代培养四周的叶色嫩绿有光泽、长势较强的组培苗叶片为外植体,可以显著提高苹果离体叶片的再生频率；再生植株在添加25mg·L_(-1)Kan培养30 d时,再生植株叶片出现白化,40 d叶片枯萎死亡；培养基中添加0.5mg·L_(-1)GA时,能显著提高组培苗的伸长生长趋势。3.根据GenBank中登陆的苹果GA20-ox基因的序列,依照基因的同源性设计引物扩增寒富苹果基因组DNA,获得一条466 bp的片段,并命名为MdGA20-ox;经分析此片在不同基因型苹果中序列一致,以pKANNNBAL载体为中间载体,利用Xho Ⅰ/EcoR Ⅰ和Hind Ⅲ/Xba Ⅰ分别进行双酶切,将MdGA20-ox插入连接到pKANNNBAL上,然后利用Not I单酶切,并与pART27载体连接,最终构建成RNA干扰载体pRNAi-GA20ox。4.利用农杆菌介导法将pRNAi-GA20ox导入寒富和平邑甜茶中来沉默GA20-ox基因,转化试验共获得寒富转基因系5株,平邑甜茶转基因系3株。由于GA20-ox基因表达受到抑制,转基因系的的茎和节间长度变短,植株高度低于非转基因对照,其中植株高度最矮的是T-HF_2,相当于非转基因对照的57%,节数是对照的1.3倍 (4.3/3.4)节间长度0.44 cm；另外转基因系的增殖率都有所提高,这些结果表明GA20-ox在控制植株营养生长中具有重要作用。试验也尝试用GA3外源喷施田间苗,结果转基因系都恢了普通生长型。用非转基因系嘎啦为接穗(GL),植株高度最矮、节间最短的转基因系寒富(T-HF_2)和平邑甜茶(T-PT_3)为砧木的微嫁接研究结果表明转基因砧木是可以影响接穗生长的,如GL/T-HF_2 (1.50 cm)伸长长度虽接近于GL/HF (1.44 cm),但节间数(4.53)和节间长度(0.63 cm)均显著高于GL/HF (3.00,1.00 cm),而对照GL/T-PT_3为植株节间数4.83,节间长度0.61 cm。从表型变化来看,接穗高度GL/HFGL/T-HF_2GL/T-PT_3。
[Abstract]:Malus domestica Borkh. is one of the most widely cultivated and economically valuable fruit tree species in the world. This study takes RNAi as the key technique of the silent target gene, and introduces the silent signal of the GA20-ox gene into Apple by Agrobacterium mediated method to block the biosynthesis of GA and obtain the transgenic dwarf rootstock. The feasibility of Using Transgenic Apple Rootstock to improve non transgenic apple scion based on the principle of system acquired silence was discussed. The specific results are as follows: 1. the subculture system, the rooting system of tissue culture seedlings and the tissue culture micro grafting system have been established with the suitable embryo culture medium as the explants in the case of the suitable embryo culture medium. 1. The more suitable proliferation medium is MS+1 mg. L_ (-1) 6-BA +0.1 mg. L_ (-1) NAA+0.2 mg.L_ (-1). BA, scion and rootstock were all subculture 40 d test tube seedlings, and when the scion had 2 true leaves, the grafting survival rate was the highest, the grafting survival rate was high with Pingyi sweet tea as the rootstock, and the best.2. of the marrying interface was established to build the regeneration adventitious bud system of the tissue cultured leaves of Pingyi sweet tea. The most suitable medium was MS+4.0 mg. L_ (-1). 6-BA+0.40 mg. L_ (-1) NAA; the frequency of adventitious buds regenerated from the leaves in contact with the medium is higher than that of the leaves from the culture medium. The dark culture is necessary for the regenerated adventitious buds of the leaves. The regeneration efficiency of the leaves can be obviously improved by the dark culture of 21 d, and the green and glossy leaf color of the selected subculture leaves is bright and glossy. The regeneration frequency of the leaves could be significantly increased by the leaves of strong growing tissue culture seedlings. When the regenerated plants were adding 25mg. L_ (-1) Kan for 30 d, the leaves of the regenerated plants were whitened and 40 d leaves withered and died. When 0.5mg. L_ (-1) GA was added to the medium, the elongation trend of the tissue culture seedlings could be significantly increased according to GenB. The sequence of the apple GA20-ox gene which was landed in ank was designed to amplify the genomic DNA of cold rich apple according to the homology of the gene. A fragment of 466 BP was obtained and named MdGA20-ox. After analyzing the sequence in different genotype apples, the pKANNNBAL vector was used as the intermediate carrier, and Xho I /EcoR I and Hind III /Xba I were used respectively. Double enzyme cut, insert MdGA20-ox into pKANNNBAL, then use Not I single enzyme to cut, and connect with pART27 carrier, and finally construct RNA interference carrier pRNAi-GA20ox.4. using Agrobacterium mediated method to transfer pRNAi-GA20ox into cold rich and Pingyi sweet tea to silence GA20-ox gene. Transformation test obtained 5 transgenic lines of cold rich, Pingyi sweet 3 strains of tea transgenic lines. Because of the inhibition of GA20-ox gene expression, the stem and internode length of the transgenic lines were shorter and the height of the plant was lower than that of the non transgenic control. The height of the plant was T-HF_2, equivalent to 57% of the non transgenic control, and the number of nodes was 1.3 times (4.3/3.4) of the internode length of 0.44 cm, and the proliferation rate of the transgenic lines was both. These results suggest that GA20-ox plays an important role in controlling the growth of plant nutrition. The experiment also tried to spray the field seedlings with GA3, and the transgenic lines were restored to the common growth type. The transgenic line was GL, the height of the plant was the shortest, the shortest internode was T-HF_2 and T-PT_3 (T-PT_3). The results of the rootstock micro grafting showed that the transgenic rootstock could affect the growth of the scion, for example, the length of GL/T-HF_2 (1.50 cm) was close to GL/HF (1.44 cm), but the number of internodes (4.53) and internode length (0.63 cm) were significantly higher than GL/HF (3.00,1.00 cm), while the number of plants was 4.83, and the length of internode 0.61 cm. changed from phenotypic variation. Look, the height of the scion is GL/HFGL/T-HF_2GL/T-PT_3.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S661.1

【相似文献】