采用基因芯片筛选棉花产量性状相关基因
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摘要
棉花作为一种重要的经济作物,在我国国民经济和社会发展中占有重要地位。由于我国人多地少,粮棉争地的矛盾十分突出,因此,培育高产优质棉花品种一直是棉花育种家长期追求的目标。
陆地棉以其产量高、适应性广的特点被广泛种植,海岛棉则以得天独厚的优良纤维品质著称于世,如何将陆地棉的高产与海岛棉的优质特点结合起来,从而培育出高产、优质的棉花新品种一直是育种家追求的目标,同时,也希望能够通过海陆杂交群体来研究得到高产优质的基因。本实验希望通过从海陆杂交群体中选择品质优良的基因型,利用其1ODPA (day past anthesis)的纤维cDNA与Affymetrix公司的棉花基因组芯片进行杂交,得到不同基因型的基因芯片数据。通过对基因芯片数据进行分析和实验验证,得到与棉花产量性状相关的基因。主要研究结果如下：
1、本实验室以陆地棉SG747为母本,海岛棉Giza75为父本,培育选择19个材料(含两个亲本),利用其开花后10天的纤维cDNA与Affymetrix公司的棉花基因组芯片进行杂交。根据2006、2007、2008三年17个回交自交系的产量相关性状(皮棉重量、铃重和衣分等)数据,用SAS软件进行ANOVO显著性分析,从17个系中选择皮棉、铃重、衣分等产量性状分别具有显著差异的几个材料。
2、将各性状中高低两组材料所对应的基因芯片数据进行比对,根据ratio>2.0或ratio<0.5的标准,得到关于皮棉性状的差异表达基因1508个,铃重性状的差异表达基因296个,衣分差异表达基因351个。其中仅在皮棉与铃重两个性状中共有的基因有185个,仅在皮棉与衣分两个性状中共有的基因有96个,仅在铃重与衣分两个性状中共有的基因有7个,三个性状共有的基因有51个。
3、用Blast2GO软件对三个性状的差异表达基因进行生物信息学分析,得到基因在生物学过程(biological process),细胞组分(cellular component),分子功能(molecular function)这三个方面的聚类结果。聚类结果显示,与细胞质膜发育相关的细胞组分、与过氧化物酶活性相关的分子功能、与抗逆相关和营养物质运输相关的生物学过程中所含的基因最多。
4、从皮棉、铃重、衣分三个性状差异表达基因中选择差异表达倍数大的基因各50个,共150个,选择的过程中避免了同一基因在不同性状间的重复选择。先进行RT-PCR分析,然后选择基因进行qRT-PCR分析。
5、通过RT-PCR分析,筛选到皮棉性状中在高产和低产材料中表达有差异的基因共8个。对这8个基因进行进一步的qRT-PCR分析,最终筛选到GhMKRP2和GhPOD两个基因,根据他们在不同时期不同材料中的表达情况,推测这两个基因对皮棉产量的形成有一定的影响。
Cotton is a major economic crop and an important variety for the Chinese textile industry. Since grain and cotton production compete for the same land resource, there is a conflict in China with increasing population and decreasing arable land, so how to breed varieties with high yield and good quality is always the breeder's target.
Land cotton is widely cultivated because of its high yield, and the sea island cotton is famous of its good fiber quality, so how to mix the high yield of land cotton and the good quality of sea island together to breed new varieties with high yield and good quality, and then do more research of the genes related to high yield and good quality by the cross of land cotton and sea island cotton, they are goals for breeders. In this research, the genes related to cotton yield were researched, the gene chip of Affymetrix and the backcross inbred lines of land cotton and sea island cotton are used. The main results are as follows:
1. The backcross inbred lines of BC2F5 were cultivated by the land cotton SG747 as the female parent and the sea island cotton Giza75 as the male parent. Totally 19 different genotypes were selected, and their fiber cDNAs of 10 days past anthesis were hybridized with the cotton gene chip of Affymetrix. Based on the above works, this research use the field data of the traits related to yield(lint yield、boll weight and lint percentage), and three years'data on 2006、2007、2008 were used. Then, do the ANOVO analysis with the SAS software, and select 3 genotypes separately of high lint yield and low lint yield、high boll size and low boll size and high lint percentage and low lint percentage, and every genotype in the high group is significant different with the low group of the single trait.
2. For the gene chip data comparasion,1508 differentially expressed (DE) genes were identified based on a two fold difference between the high lint yield and the low lint yield group,296 differentially expressed genes between the high boll size and the low boll size group,351 differentially expressed genes between the high lint percentage and low lint percentage group.185 differential expressed gene were common in lint yield and boll size,96 differential expressed genes were common in lint yield and lint percentage, 7 differential expressed genes in boll size and lint percentage, and 51 differential expressed genes in all three traits.
3. Bioinformatic analysis of these three traits were done by the Blast2GO software, and the results of clustering at biological process、cellular component and molecular function were grained. The results shows that, there are more genes related to the cellular component of plasma membrane, related to the molecular function of Oxidation reduction, related to the biological processes of response to the stress and transport.
4. Select 50 differential expressed genes of the three traits separately, totally 150 genes, and no duplicate in these 150 genes. First, do the RT-PCR analysis on these 150 differential expressed genes, then select some gene according to the RT-PCR results to do the qRT-PCR analysis.
5. According to the RT-PCR results, eight genes were preferentially expressed in the high yielding group or vice versa, in order to validate the results, the eight genes from the lint yield were selected to do the qRT-PCR, the results indicated that the two genes GhMKRP2 and GhPOD may be involved in the development of cotton fibers which in turn affect the cotton yield.
陆地棉以其产量高、适应性广的特点被广泛种植,海岛棉则以得天独厚的优良纤维品质著称于世,如何将陆地棉的高产与海岛棉的优质特点结合起来,从而培育出高产、优质的棉花新品种一直是育种家追求的目标,同时,也希望能够通过海陆杂交群体来研究得到高产优质的基因。本实验希望通过从海陆杂交群体中选择品质优良的基因型,利用其1ODPA (day past anthesis)的纤维cDNA与Affymetrix公司的棉花基因组芯片进行杂交,得到不同基因型的基因芯片数据。通过对基因芯片数据进行分析和实验验证,得到与棉花产量性状相关的基因。主要研究结果如下：
1、本实验室以陆地棉SG747为母本,海岛棉Giza75为父本,培育选择19个材料(含两个亲本),利用其开花后10天的纤维cDNA与Affymetrix公司的棉花基因组芯片进行杂交。根据2006、2007、2008三年17个回交自交系的产量相关性状(皮棉重量、铃重和衣分等)数据,用SAS软件进行ANOVO显著性分析,从17个系中选择皮棉、铃重、衣分等产量性状分别具有显著差异的几个材料。
2、将各性状中高低两组材料所对应的基因芯片数据进行比对,根据ratio>2.0或ratio<0.5的标准,得到关于皮棉性状的差异表达基因1508个,铃重性状的差异表达基因296个,衣分差异表达基因351个。其中仅在皮棉与铃重两个性状中共有的基因有185个,仅在皮棉与衣分两个性状中共有的基因有96个,仅在铃重与衣分两个性状中共有的基因有7个,三个性状共有的基因有51个。
3、用Blast2GO软件对三个性状的差异表达基因进行生物信息学分析,得到基因在生物学过程(biological process),细胞组分(cellular component),分子功能(molecular function)这三个方面的聚类结果。聚类结果显示,与细胞质膜发育相关的细胞组分、与过氧化物酶活性相关的分子功能、与抗逆相关和营养物质运输相关的生物学过程中所含的基因最多。
4、从皮棉、铃重、衣分三个性状差异表达基因中选择差异表达倍数大的基因各50个,共150个,选择的过程中避免了同一基因在不同性状间的重复选择。先进行RT-PCR分析,然后选择基因进行qRT-PCR分析。
5、通过RT-PCR分析,筛选到皮棉性状中在高产和低产材料中表达有差异的基因共8个。对这8个基因进行进一步的qRT-PCR分析,最终筛选到GhMKRP2和GhPOD两个基因,根据他们在不同时期不同材料中的表达情况,推测这两个基因对皮棉产量的形成有一定的影响。
Cotton is a major economic crop and an important variety for the Chinese textile industry. Since grain and cotton production compete for the same land resource, there is a conflict in China with increasing population and decreasing arable land, so how to breed varieties with high yield and good quality is always the breeder's target.
Land cotton is widely cultivated because of its high yield, and the sea island cotton is famous of its good fiber quality, so how to mix the high yield of land cotton and the good quality of sea island together to breed new varieties with high yield and good quality, and then do more research of the genes related to high yield and good quality by the cross of land cotton and sea island cotton, they are goals for breeders. In this research, the genes related to cotton yield were researched, the gene chip of Affymetrix and the backcross inbred lines of land cotton and sea island cotton are used. The main results are as follows:
1. The backcross inbred lines of BC2F5 were cultivated by the land cotton SG747 as the female parent and the sea island cotton Giza75 as the male parent. Totally 19 different genotypes were selected, and their fiber cDNAs of 10 days past anthesis were hybridized with the cotton gene chip of Affymetrix. Based on the above works, this research use the field data of the traits related to yield(lint yield、boll weight and lint percentage), and three years'data on 2006、2007、2008 were used. Then, do the ANOVO analysis with the SAS software, and select 3 genotypes separately of high lint yield and low lint yield、high boll size and low boll size and high lint percentage and low lint percentage, and every genotype in the high group is significant different with the low group of the single trait.
2. For the gene chip data comparasion,1508 differentially expressed (DE) genes were identified based on a two fold difference between the high lint yield and the low lint yield group,296 differentially expressed genes between the high boll size and the low boll size group,351 differentially expressed genes between the high lint percentage and low lint percentage group.185 differential expressed gene were common in lint yield and boll size,96 differential expressed genes were common in lint yield and lint percentage, 7 differential expressed genes in boll size and lint percentage, and 51 differential expressed genes in all three traits.
3. Bioinformatic analysis of these three traits were done by the Blast2GO software, and the results of clustering at biological process、cellular component and molecular function were grained. The results shows that, there are more genes related to the cellular component of plasma membrane, related to the molecular function of Oxidation reduction, related to the biological processes of response to the stress and transport.
4. Select 50 differential expressed genes of the three traits separately, totally 150 genes, and no duplicate in these 150 genes. First, do the RT-PCR analysis on these 150 differential expressed genes, then select some gene according to the RT-PCR results to do the qRT-PCR analysis.
5. According to the RT-PCR results, eight genes were preferentially expressed in the high yielding group or vice versa, in order to validate the results, the eight genes from the lint yield were selected to do the qRT-PCR, the results indicated that the two genes GhMKRP2 and GhPOD may be involved in the development of cotton fibers which in turn affect the cotton yield.
引文
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