大白菜抗霜霉病同源基因的克隆与分析
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摘要
大白菜(Brassica campestris ssp.pekinensis)原产我国,其栽培面积及产量均居我国蔬菜之首。霜霉病是大白菜三大病害之一,严重影响大白菜的产量和品质,因此,抗霜霉病育种一直是大白菜的重要育种目标。随着分子生物学技术的发展,克隆并利用植物的抗性基因,研究这些基因的结构和功能已成为生物技术领域的热点。本研究以大白菜自交系‘85-1’的BAC文库为材料,应用同源序列法筛选抗病同源基因的BAC克隆,对获得的10个阳性克隆进行酶切分析,构建了亚克隆文库,并对筛选的阳性亚克隆进行了测序分析。本试验的研究结果如下:
1.根据植物抗霜霉病基因保守区设计简并引物,利用三步PCR方法对大白菜‘85-1’BAC文库进行筛选,从19200个BAC克隆中筛选到含有目的基因的10个阳性克隆。对10个阳性克隆进行NotⅠ酶切,经脉冲场电泳检测,10个克隆的插入片段在80-100kb之间。将10个阳性克隆分别用EcoRⅠ、BamHⅠ及HindⅡ酶切,电泳检测酶切结果,由酶切图谱显示,选择阳性克隆94-G-17和限制性内切酶HindⅡ建立亚克隆文库。
2.含抗霜霉病同源基因的亚克隆文库含有6200个克隆,覆盖BAC克隆的180倍左右。随机挑取85个亚克隆进行EcoRI,PstI双酶切,经琼脂糖凝胶电泳检测,其中78个克隆酶切后均有插入片段,其大小基本分布在1-5kb之间,平均插入片段大小为3kp左右。7个克隆酶切后只有载体片段,无插入片段,空载率为8.36%。
3.利用上述简并引物,采用三步PCR方法对亚克隆文库进行筛选,获得抗霜霉病同源基因全长,编码区长度为3063bp,含有抗病同源基因具有的TIR结构域、NB-AR结构域和NB-ARC结构域。
Chinese cabbage (Brassica campestris L.ssp. pekinensis) originating from China, is one of the most important vegetable crops in China,with the largest cultivated area and yield. Downy mildew is one of three most important diseases in Chinese cabbage, impacting on its yield and quality seriously. Therefore, resistant to downy mildew has been an important objective in Chinese cabbage breeding. With the development of molecular biology techniques, cloning and using of plant resistance genes, studying on the structure and function have become a hot point in biotechnology field. In this study, the resistance gene analogs analysis was used to screen resistance gene from the BAC library of Chinese cabbage inbred line‘85-1’and 10 positive clones were obtained and analyzed by restriction enzyme digestion. A sub-clone library was constructed and the screened positive sub-clone was sequenced. The main results are as follows:
1. The degenerated primers were designed according to the conserved domain of downy mildew resistant genes in plants. The Chinese cabbage‘85-1’BAC library was screened through three steps of PCR amplification, and 10 positive clones contained target gene from 19200 BAC clones were achieved. Ten positive clones were digested by NotⅠ, in which the inserted fragments ranged from 80 to 100kb revealed by pulsed-field gel electrophoresis. The positive clone 94-G-17 and restriction enzyme HindⅡwere selected to construct a sub-clone library based on the result of pulsed-field gel electrophoresis, digesting the 10 positive clones by EcoRⅠ, BamHⅠand HindⅡ.
2. The sub-clone library with downy mildew resistant genes consisted of 6 200 clones, covering 180-fold of the BAC clone in size. Eighty-five sub-clones picked at random were double digested by restriction enzyme EcoRI and PstI, of which 78 clones were digested into fragments by agrose gel electrophoresis, ranging from 1 to 5kb, with an average insert size 3kb .Seven clones had vector fragments without inserts, with a empty clones rate of 8.36%.
3. Using above degenerated primers, the sub-clone library was screened through three steps of PCR and achieved the whole length of Downy Mildew Resistance Gene Analogs,the effective coding length 3063 bp, containing TIR domain, NB-AR domain and NB-ARG domain within disease resistant genes.
1.根据植物抗霜霉病基因保守区设计简并引物,利用三步PCR方法对大白菜‘85-1’BAC文库进行筛选,从19200个BAC克隆中筛选到含有目的基因的10个阳性克隆。对10个阳性克隆进行NotⅠ酶切,经脉冲场电泳检测,10个克隆的插入片段在80-100kb之间。将10个阳性克隆分别用EcoRⅠ、BamHⅠ及HindⅡ酶切,电泳检测酶切结果,由酶切图谱显示,选择阳性克隆94-G-17和限制性内切酶HindⅡ建立亚克隆文库。
2.含抗霜霉病同源基因的亚克隆文库含有6200个克隆,覆盖BAC克隆的180倍左右。随机挑取85个亚克隆进行EcoRI,PstI双酶切,经琼脂糖凝胶电泳检测,其中78个克隆酶切后均有插入片段,其大小基本分布在1-5kb之间,平均插入片段大小为3kp左右。7个克隆酶切后只有载体片段,无插入片段,空载率为8.36%。
3.利用上述简并引物,采用三步PCR方法对亚克隆文库进行筛选,获得抗霜霉病同源基因全长,编码区长度为3063bp,含有抗病同源基因具有的TIR结构域、NB-AR结构域和NB-ARC结构域。
Chinese cabbage (Brassica campestris L.ssp. pekinensis) originating from China, is one of the most important vegetable crops in China,with the largest cultivated area and yield. Downy mildew is one of three most important diseases in Chinese cabbage, impacting on its yield and quality seriously. Therefore, resistant to downy mildew has been an important objective in Chinese cabbage breeding. With the development of molecular biology techniques, cloning and using of plant resistance genes, studying on the structure and function have become a hot point in biotechnology field. In this study, the resistance gene analogs analysis was used to screen resistance gene from the BAC library of Chinese cabbage inbred line‘85-1’and 10 positive clones were obtained and analyzed by restriction enzyme digestion. A sub-clone library was constructed and the screened positive sub-clone was sequenced. The main results are as follows:
1. The degenerated primers were designed according to the conserved domain of downy mildew resistant genes in plants. The Chinese cabbage‘85-1’BAC library was screened through three steps of PCR amplification, and 10 positive clones contained target gene from 19200 BAC clones were achieved. Ten positive clones were digested by NotⅠ, in which the inserted fragments ranged from 80 to 100kb revealed by pulsed-field gel electrophoresis. The positive clone 94-G-17 and restriction enzyme HindⅡwere selected to construct a sub-clone library based on the result of pulsed-field gel electrophoresis, digesting the 10 positive clones by EcoRⅠ, BamHⅠand HindⅡ.
2. The sub-clone library with downy mildew resistant genes consisted of 6 200 clones, covering 180-fold of the BAC clone in size. Eighty-five sub-clones picked at random were double digested by restriction enzyme EcoRI and PstI, of which 78 clones were digested into fragments by agrose gel electrophoresis, ranging from 1 to 5kb, with an average insert size 3kb .Seven clones had vector fragments without inserts, with a empty clones rate of 8.36%.
3. Using above degenerated primers, the sub-clone library was screened through three steps of PCR and achieved the whole length of Downy Mildew Resistance Gene Analogs,the effective coding length 3063 bp, containing TIR domain, NB-AR domain and NB-ARG domain within disease resistant genes.
引文
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