中国野生葡萄抗白粉病新基因遗传转化及检测方法研究
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摘要
本研究以长穗无核白葡萄(Vitis vinifera cv. Long Thompson Seedless)为材料,对农杆菌侵染后叶片从愈伤组织到不定芽发生阶段的绿色荧关蛋白分布进行了观察;以拟南芥(Arabidopsis thaliana)为材料,通过农杆菌介导的花序浸蘸法,进行了中国野生葡萄抗白粉病新基因(GLOXrg,登录号:DQ201181)的遗传转化研究;以无核白葡萄(Vitis vinifera cv. Thompson Seedless)为材料,以脱水素基因为工具基因,进行了适于内、外源基因检测的方法研究。取得的主要结果如下:
1.通过长穗无核白葡萄叶片农杆菌侵染后从愈伤化阶段到不定芽发生阶段绿色荧光蛋白的观察,认为在愈伤化阶段,在培养基中可只加入脱菌抗生素(头孢霉素,Cef) 500 mg/L,而不加入选择抗生素(潮霉素,HygB) 9 mg/L;在不定芽发生阶段,应当同时加入脱菌抗生素和选择抗生素。
2.通过农杆菌介导的花序浸蘸法向拟南芥转化GLOXrg,经过花序浸蘸、种子采收、种子氯气消毒、潮霉素抗性筛选(MS + Hyg 50 mg/L),获得潮霉素抗性株系2株。
3.建立了一种适于内、外源基因拷贝数鉴定及获得不同拷贝序列的方法(基于梯状回收的PCR法)。该方法步骤为:首先,基因组DNA被限制性内切酶充分酶切;其次,酶切产物通过琼脂糖凝胶电泳分离,分离产物被梯状回收;再次,通过PCR鉴定基因拷贝数;最后,测定PCR产物序列,获得每个拷贝核苷酸序列。
4.应用“基于梯状回收的PCR法”法鉴定了无核白葡萄脱水素基因拷贝数,并获得了不同拷贝核苷酸序列。结果表明无核白葡萄脱水素基因拷贝数为2个,两者高度同源,在GenBank登录号分别为EF371881和EF371882。
GFP analysis of the infected explants was applied, and the phases covered were from callus formation to adventitious shoot formation; Also, we did the genetic transformation of the novel gene resistant to powdery mildew(GLOXrg, accession number: DQ201181) from Chinese wild grapevine on Arabidopsis thaliana by the Agrobacterium tumefaciens-mediated floral-dip method; Besides, we did some research on identification method available for endogenous and exogenous genes, and the reference gene used was dehydrin gene in Vitis vinifera cv. Thompson Seedless
1. Based on GFP analysis from callus formation phase to adventitious shoot formation phase of the explant after infection with Agrobacterium tumefaciens, we obtained the following conclusion: it was available to just add cefotaxime (cef) 500 mg/L into the medium during the callus formation phase, and during the adventitious shoot formation phase, the cef and hygromycin B (hygB) 9 mg/L should were both added into the medium.
2. Through Agrobacterium tumefaciens-mediated floral-dip transformation of the novel gene resistant to powdery mildew from Chinese wild grapevine on Arabidopsis thaliana, and after floral-dip、harvest of seeds、chlorine sterilization of seeds、selection on hygB added medium (MS + HygB 50 mg/L), two lines resistant to hygB were obtained.
3. One method (ladderlike purification-based PCR method) available for estimating copy number and obtaining sequence of each copy was developed. The procedures of this method were as follows: First, DNA was digested with restriction endonuclease; Second, DNA fragments were separated on agarose gel, and separated fragments were purified ladderlikely; Third, copy number was estimated by PCR approach; Last, PCR products were sequenced for obtaining sequence of each copy.
4. Copy number of dehydrin gene was estimated and sequence of each copy was obtained by ladderlike purification-based PCR method. It was shown that there were two copies of dehydrin gene in Vitis vinifera cv. Thompson Seedless, and these two copies were highly homologous to each other in nucleotide lever and the accession numbers were EF371881and EF371882 respectively.
1.通过长穗无核白葡萄叶片农杆菌侵染后从愈伤化阶段到不定芽发生阶段绿色荧光蛋白的观察,认为在愈伤化阶段,在培养基中可只加入脱菌抗生素(头孢霉素,Cef) 500 mg/L,而不加入选择抗生素(潮霉素,HygB) 9 mg/L;在不定芽发生阶段,应当同时加入脱菌抗生素和选择抗生素。
2.通过农杆菌介导的花序浸蘸法向拟南芥转化GLOXrg,经过花序浸蘸、种子采收、种子氯气消毒、潮霉素抗性筛选(MS + Hyg 50 mg/L),获得潮霉素抗性株系2株。
3.建立了一种适于内、外源基因拷贝数鉴定及获得不同拷贝序列的方法(基于梯状回收的PCR法)。该方法步骤为:首先,基因组DNA被限制性内切酶充分酶切;其次,酶切产物通过琼脂糖凝胶电泳分离,分离产物被梯状回收;再次,通过PCR鉴定基因拷贝数;最后,测定PCR产物序列,获得每个拷贝核苷酸序列。
4.应用“基于梯状回收的PCR法”法鉴定了无核白葡萄脱水素基因拷贝数,并获得了不同拷贝核苷酸序列。结果表明无核白葡萄脱水素基因拷贝数为2个,两者高度同源,在GenBank登录号分别为EF371881和EF371882。
GFP analysis of the infected explants was applied, and the phases covered were from callus formation to adventitious shoot formation; Also, we did the genetic transformation of the novel gene resistant to powdery mildew(GLOXrg, accession number: DQ201181) from Chinese wild grapevine on Arabidopsis thaliana by the Agrobacterium tumefaciens-mediated floral-dip method; Besides, we did some research on identification method available for endogenous and exogenous genes, and the reference gene used was dehydrin gene in Vitis vinifera cv. Thompson Seedless
1. Based on GFP analysis from callus formation phase to adventitious shoot formation phase of the explant after infection with Agrobacterium tumefaciens, we obtained the following conclusion: it was available to just add cefotaxime (cef) 500 mg/L into the medium during the callus formation phase, and during the adventitious shoot formation phase, the cef and hygromycin B (hygB) 9 mg/L should were both added into the medium.
2. Through Agrobacterium tumefaciens-mediated floral-dip transformation of the novel gene resistant to powdery mildew from Chinese wild grapevine on Arabidopsis thaliana, and after floral-dip、harvest of seeds、chlorine sterilization of seeds、selection on hygB added medium (MS + HygB 50 mg/L), two lines resistant to hygB were obtained.
3. One method (ladderlike purification-based PCR method) available for estimating copy number and obtaining sequence of each copy was developed. The procedures of this method were as follows: First, DNA was digested with restriction endonuclease; Second, DNA fragments were separated on agarose gel, and separated fragments were purified ladderlikely; Third, copy number was estimated by PCR approach; Last, PCR products were sequenced for obtaining sequence of each copy.
4. Copy number of dehydrin gene was estimated and sequence of each copy was obtained by ladderlike purification-based PCR method. It was shown that there were two copies of dehydrin gene in Vitis vinifera cv. Thompson Seedless, and these two copies were highly homologous to each other in nucleotide lever and the accession numbers were EF371881and EF371882 respectively.
引文
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