甘蓝型油菜—新疆野生油菜二体异附加系BAC文库的构建
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摘要
目前作物生产上的杂交育种主要是利用细胞质雄性不育技术,形成不育系、恢复系和保持系三系或不育系、恢复系两系育种,其中油菜品种中约70%为细胞质雄性不育杂种,恢复系是应用此项技术必不可少的条件,如果能够得到恢复基因,就有可能利用此基因建立新的恢复系。而得到恢复基因并深入的研究其功能的有效途径之一就是构建BAC文库。
新疆野生油菜(Sinapis arvensis L.)生长势强,抗病虫性好,芥酸及硫甙含量低,具有雄性不育胞质特征,是植物育种中优良的种质资源。本文以含有Nsa细胞质雄性不育恢复基因甘蓝型油菜——新疆野生油菜二体异附加系Nsa恢1的成熟叶片为材料,以CopyControl~(TM) pCC1BAC~(TM)(HindⅢCloning-Readyl)Vector为载体,在Zhang和Peterson等人的BAC文库构建方法的基础上改进,建立了一套高效的油菜BAC文库构建技术体系,得到了浓度适当、降解较少的核DNA琼脂糖包埋块,并通过不同浓度梯度酶量酶切实验得到最适合构建文库的酶切条件(1.2U HindⅢ,37℃酶切10min)。通过回收得到浓度、片段大小合适的核DNA酶切片段并建立了高效的连接体系,共得到69120个阳性克隆,94%的插入片段分布在80—230kb之间,76%的插入片段超过100kb,平均插入片段达110kb,相当于甘蓝型油菜基因组大小的6.3倍;筛选到任意基因的概率为99.82%;随机挑选3个大于100kb的克隆进行稳定性继代实验验证。结果表明,所挑选克隆的各代之间的外源片段大小一致,说明BAC克隆在培养的过程中是很稳定的,没有重排现象。并构建完成了BAC一级混合池、二级混合池各一份。
该文库的建成,为油菜基因的克隆及基因组学研究提供了技术平台。
Cytoplasmic male sterility(CMS) is widely used in cross breeding,which consists of sterile line,restoring line and/or maintainer line.About 70%of Brassica variaties are Cytoplasmic male sterility(CMS) hybrids.Restoring line is one of the most important parts in the CMS technology.If we can get the restore gene,we could study its structure and use it to develop some more restoring lines,for this purpose we constructed a BAC library.
The Sinapis arvensis Xinjiang is crucifer,and a wild breed of Sinapis arvensis L.It is good at resist diseases and insects,low content of erucic acid and glucosinolates.We used the disomic alien addition line which consists two chromosomes of Sinapis arvensis Xinjiang wild rape and the Nsa Cytoplasmic male sterility restore gene,and CopyControl~(TM) pCC1BACTM(HindⅢCloning-Ready) Vector followed the manuals of Zhang and Perterson to construct a BAC library.We got plugs with good quality and tested enzyme units at 1.2 U Hind/Ⅲ,and then we reclaimed the big DNA segments to link with the vector.The library consisting of 69120 clones from the analysis of 100 randomly selected BAC clones,the average insert size is estimated at 110kb,94%are 80-230kb,76%are more than 100kb.As reported that the Brassica napus haploid genome size is 1.2×10~9bp,the library represents about 6.3×haploid genome equivalents. This provides a 99.82%probability of finding any specific gene.At last we make a primary pool and a secondary pool of this library.
The results indicate that this BAC library has high quality and high coverage,and is sufficient for target gene isolation.
新疆野生油菜(Sinapis arvensis L.)生长势强,抗病虫性好,芥酸及硫甙含量低,具有雄性不育胞质特征,是植物育种中优良的种质资源。本文以含有Nsa细胞质雄性不育恢复基因甘蓝型油菜——新疆野生油菜二体异附加系Nsa恢1的成熟叶片为材料,以CopyControl~(TM) pCC1BAC~(TM)(HindⅢCloning-Readyl)Vector为载体,在Zhang和Peterson等人的BAC文库构建方法的基础上改进,建立了一套高效的油菜BAC文库构建技术体系,得到了浓度适当、降解较少的核DNA琼脂糖包埋块,并通过不同浓度梯度酶量酶切实验得到最适合构建文库的酶切条件(1.2U HindⅢ,37℃酶切10min)。通过回收得到浓度、片段大小合适的核DNA酶切片段并建立了高效的连接体系,共得到69120个阳性克隆,94%的插入片段分布在80—230kb之间,76%的插入片段超过100kb,平均插入片段达110kb,相当于甘蓝型油菜基因组大小的6.3倍;筛选到任意基因的概率为99.82%;随机挑选3个大于100kb的克隆进行稳定性继代实验验证。结果表明,所挑选克隆的各代之间的外源片段大小一致,说明BAC克隆在培养的过程中是很稳定的,没有重排现象。并构建完成了BAC一级混合池、二级混合池各一份。
该文库的建成,为油菜基因的克隆及基因组学研究提供了技术平台。
Cytoplasmic male sterility(CMS) is widely used in cross breeding,which consists of sterile line,restoring line and/or maintainer line.About 70%of Brassica variaties are Cytoplasmic male sterility(CMS) hybrids.Restoring line is one of the most important parts in the CMS technology.If we can get the restore gene,we could study its structure and use it to develop some more restoring lines,for this purpose we constructed a BAC library.
The Sinapis arvensis Xinjiang is crucifer,and a wild breed of Sinapis arvensis L.It is good at resist diseases and insects,low content of erucic acid and glucosinolates.We used the disomic alien addition line which consists two chromosomes of Sinapis arvensis Xinjiang wild rape and the Nsa Cytoplasmic male sterility restore gene,and CopyControl~(TM) pCC1BACTM(HindⅢCloning-Ready) Vector followed the manuals of Zhang and Perterson to construct a BAC library.We got plugs with good quality and tested enzyme units at 1.2 U Hind/Ⅲ,and then we reclaimed the big DNA segments to link with the vector.The library consisting of 69120 clones from the analysis of 100 randomly selected BAC clones,the average insert size is estimated at 110kb,94%are 80-230kb,76%are more than 100kb.As reported that the Brassica napus haploid genome size is 1.2×10~9bp,the library represents about 6.3×haploid genome equivalents. This provides a 99.82%probability of finding any specific gene.At last we make a primary pool and a secondary pool of this library.
The results indicate that this BAC library has high quality and high coverage,and is sufficient for target gene isolation.
引文
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3.陈玉卿,徐希龙.新疆、云南野生油菜病毒病菌核病的抗性鉴定[J].中国油料,1988,10(专辑):49-52.
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5.方玉达,刘大钧.植物可转化基因组文库的发展和应用[J].生物技术通报.1999(5):12-15.
6.官春云,李少骞.新疆野生油菜细胞遗传学研究[J].遗传学报,1995,22(6):470-477.
7.官春云.新疆野生油菜与野芥(Sinapis arvensis L.)遗传性状的比较研究[J].作物学报,1996,22(2):214-219.
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