家蚕黑卵与第二白卵差异表达基因的筛选与克隆
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摘要
家蚕(Bombyx mori)自古以来就是一种重要的经济昆虫,最近以来,家蚕又成了鳞翅目昆虫重要的模式生物。家蚕转基因技术在培育新品种、生物反应器的开发以及功能基因组学研究等方面具有重要应用价值,家蚕转基因育种技术的研究开发和利用已经成为蚕业科技界关注的焦点,而转基因阳性个体的高效检测技术是建立家蚕高效转基因技术的核心之一。
本课题研究的家蚕第二白卵(w-2:10-16.1)突变体的卵色和眼色与正常型十分容易区分,第二白卵对孵化、生命力、经济性状等方面的影响都很小,适合用作转基因个体筛选的标志基因。但之前对家蚕第二白卵形成的分子机制尚无了解,因此,我们利用野生型黑卵及其第二白卵近等基因系,联合运用全基因组芯片技术和实时荧光定量PCR技术,探索家蚕第二白卵性状相关的基因信息,以期为进一步阐明第二白卵性状形成的分子机制、开发新型转基因标记技术提供参考。主要研究结论如下:
一、家蚕全基因组芯片高通量检测家蚕第二白卵(w-2)性状相关的差异表达基因
采用由西南大学和博奥生物有限公司共同构建、含有23000个基因的家蚕全基因组寡聚核苷酸芯片进行检测。以表达差异≥2.0或≤0.5倍为标准,获得24h卵龄的第二白卵与野生型黑卵差异表达基因163个,获得48h卵龄的第二白卵与野生型黑卵差异表达基因186个。其中在24h和48h黑卵和白卵间表达差异最大的基因均为芯片探针sw04840对应的基因,差异倍数分别为32.4675和22.8311倍。二、差异表达基因的实时荧光定量PCR分析根据全基因组芯片检测分析获得的结果,挑选部分差异较大的基因以及w-2基因所在的第10连锁群上的差异表达基因,对芯片检测获得的差异表达候选基因在家蚕第
二白卵与野生型黑卵品系转色期(24h和48h)蚕卵的表达水平进行实时荧光定量
RT-PCR验证分析,结果表明大部分基因定量结果与芯片结果倾向一致,极少部分与芯片结果相反。其中基因芯片结果中表达差异最大的sw04840基因在real-time RT-PCR实验结果中也是差异最大的基因,在24h和48h的表达差异达到424.61和797.86倍,为正常型黑卵上调表达基因。通过进一步分析得知,该基因在是一个在黑卵中大量表达的基因,在24h和48h的黑卵中的表达量分别为内参基因Bmactin A3的1.92倍和0.73倍。另外,基因sw00132和基因sw04534在蚕卵发育的第48小时在黑白卵中的差异倍数也达到了数百倍,其中,基因sw00132为黑卵上调表达基因,sw04534则为白卵上调表达基因。在蚕卵发育的第24小时的白卵上调表达基因中,sw13775是差异最大的基因。
三、候选基因的电子克隆和RACE延伸
对于上述的4个基因,我们用它们各自探针序列,通过家蚕EST数据库对这四个基因进行电子延伸,并通过克隆测序验证拼接的正确性,结合3’RACE的测序结果拼接后得到了这四个基因的更多的序列信息。它们都包含了一个较长的开放阅读框的序列,sw13775,sw00132和sw04840这3个探针对应的基因获得了完整的3’端序列。
四、候选基因的序列分析和其编码蛋白分析
将这4个基因电子克隆和RACE延伸后的基因序列进行基因结构分析,发现sw13775和sw00132对应的基因是含有多个外显子的基因,但sw04840和sw04534对应的基因目前仅获得1个外显子。并且sw04840在家蚕基因组精细图数据库中也没有找到与之高度同源的序列,为家蚕中新发现的一个在黑白卵中表达差异很大的一个基因。
在NCBI等数据库中对4个候选基因进行BLASTX分析,发现sw13775对应的是家蚕乙醛还原酶(alcohol dehydrogenase)基因,sw00132与多种昆虫中的醛脱氢酶基因家族7中的一个基因高度同源,而sw04840和sw04534则没有找到与之高度同源的基因。通过所编码氨基酸序列的生物信息学分析,sw04534可能编码一种胞内酶,而sw04840则很可能不是一个编码酶蛋白的基因。
Silkworm, Bombyx mori is an important economic insect in China since the ancient times, Recently, it becomes an important organism model of lepidopteran insects. Its transgenic technology shows significant value in the fields of breeding new varieties, bioreactor and basic scientific development. So far breeding new variety by transgenic method will become the key point in the field of sericulture research, thus the development of detection method of positive transformation will be the core technology in this research field.
The color of wild type silkworm egg is yellow-white at the beginning, about 20h later, gradually become darker and darker,and dark brown finally. The color of the white egg 2 (w-2) mutant of silkworm is yellow-white with a little light red, and eyes of moth are white. So it’s easy to distinguish w-2 silkworm from wild type by egg color and ommateum,also the white egg 2 trait is regular hereditary. w-2 has no negative influences on its hatching, vitality and economic characters,thus it is suitable for marker gene for transgenic silkworm research. Now the understanding of molecular mechanism of the w-2 of silkworm eggs is still not clear.
In this study, we use the established near-isogenic line of w-2 of silkworm,whole genomic gene chip and Real-time Quantitative RT-PCR to explore the functional genes for characteristics of w-2 of silkworm. The result of the experiment will be helpful to elucidate the molecular mechanism of w-2, and be useful for developing new marker for the transgenic silkworm.
The main conclusions we made in this study are as follows: 1. The differentially expressed genes high-throughput explored by silkworm whole genomic gene chip
The silkworm whole genomic gene chip which contains 23000 genes is constructed by CapitalBio corporation and southwest university jointly, is used to find the differrentially expressed genes between white egg 2 and normal eggs of silkworm.Using an criterion of ratio≥2.0 or ratio≤0.5, We obtained 163 genes, in expression of multiple between normal eggs and white eggs that develop to the 24th h, and when silkworm eggs develop to the and 48th h, we obtained 186. The biggest difference expression level gene is sw04840.When silkworm eggs develop to the 24th h and 48th h, the expression level of sw04840 gene in wild type silkworm is 32.4675 and 22.8311 folds respectively comparing to that in white egg 2 mutant.
2. Analysis of differentially expressed genes by Real-time fluorescence quantitative RT-PCR.
According to the results obtained by the whole genomic gene chip, we use Real-time fluorescence quantitative RT-PCR to identify some larger differrentially expressed genes and some differrentially expressed genes located on the 10th linkage group. The results show that the degree of concordance between data generated by the two methods is high. According to the results obtained by Real-time fluorescence quantitative RT-PCR, gene sw04840 is also the biggest difference expression level gene. When silkworm eggs develop to the 24th h and 48th h, the expression level of sw04840 gene in wild type silkworm is 424.61 and 797.86 folds respectively comparing to that in white egg 2 mutant.Further analysis showed that this gene is an abundantly expressed gene in wild type strain eggs, when wild type strain eggs develop to the 24h and 48h, the expression level of sw04840 gene relative to Bmactin A3 is about 1.92times and 0.73 times respectively. In addition, when silkworm eggs develop to the 48th h, the expression level of sw00132 and sw04534 between white egg 2 and normal eggs of silkworm is also reached several hundred times, and when silkworm eggs develop to the 24th h, the biggest difference expression level gene is sw13775 in up-regulate genes in white egg 2.We select this four gene as candidate genes.
3. In silico cloning and RACE elongation of the candidate genes.
For the above four candidate genes, we use in silico extention technology with their probe sequence as seed sequence to extend their sequence, and with assembly with the sequencing results of 3’RACE elongation,we obtained more sequence information of them.. Sequence analysis revealed that all of them contains a long open reading frame(ORF), and gene sw13775, sw00132 and sw04840 contains full 3’end sequence.
4. Analysis the sequence of the candidate genes and the protein that encoded by them
We analysis the gene structure of the four candidate genes,the results show that the gene sw13775 and gene sw00132 contain more than one exons,but the gene sw04840 and the gene sw04534 contains only one.and in database of silkworm genome assembly we did not find any sequence highly homologous with sw04840, indicate that sw04840 is a new gene which is significant difference expressed between white egg 2 and normal eggs.
The analysis of deduced amino acid of the above four candidate genes with blastx shows that gene sw13775 corresponds to the alcohol dehydrogenase of silkworm; sw00132 highly homology with the aldehyde dehydrogenase 7 family, member A1. But we can’t find protein homology with protein encoded by sw04534 and sw04840. Bioinformatics analysis show that sw04534 is likely to be an endocellular enzyme and protein encoded by sw04840 probably not a enzyme.
本课题研究的家蚕第二白卵(w-2:10-16.1)突变体的卵色和眼色与正常型十分容易区分,第二白卵对孵化、生命力、经济性状等方面的影响都很小,适合用作转基因个体筛选的标志基因。但之前对家蚕第二白卵形成的分子机制尚无了解,因此,我们利用野生型黑卵及其第二白卵近等基因系,联合运用全基因组芯片技术和实时荧光定量PCR技术,探索家蚕第二白卵性状相关的基因信息,以期为进一步阐明第二白卵性状形成的分子机制、开发新型转基因标记技术提供参考。主要研究结论如下:
一、家蚕全基因组芯片高通量检测家蚕第二白卵(w-2)性状相关的差异表达基因
采用由西南大学和博奥生物有限公司共同构建、含有23000个基因的家蚕全基因组寡聚核苷酸芯片进行检测。以表达差异≥2.0或≤0.5倍为标准,获得24h卵龄的第二白卵与野生型黑卵差异表达基因163个,获得48h卵龄的第二白卵与野生型黑卵差异表达基因186个。其中在24h和48h黑卵和白卵间表达差异最大的基因均为芯片探针sw04840对应的基因,差异倍数分别为32.4675和22.8311倍。二、差异表达基因的实时荧光定量PCR分析根据全基因组芯片检测分析获得的结果,挑选部分差异较大的基因以及w-2基因所在的第10连锁群上的差异表达基因,对芯片检测获得的差异表达候选基因在家蚕第
二白卵与野生型黑卵品系转色期(24h和48h)蚕卵的表达水平进行实时荧光定量
RT-PCR验证分析,结果表明大部分基因定量结果与芯片结果倾向一致,极少部分与芯片结果相反。其中基因芯片结果中表达差异最大的sw04840基因在real-time RT-PCR实验结果中也是差异最大的基因,在24h和48h的表达差异达到424.61和797.86倍,为正常型黑卵上调表达基因。通过进一步分析得知,该基因在是一个在黑卵中大量表达的基因,在24h和48h的黑卵中的表达量分别为内参基因Bmactin A3的1.92倍和0.73倍。另外,基因sw00132和基因sw04534在蚕卵发育的第48小时在黑白卵中的差异倍数也达到了数百倍,其中,基因sw00132为黑卵上调表达基因,sw04534则为白卵上调表达基因。在蚕卵发育的第24小时的白卵上调表达基因中,sw13775是差异最大的基因。
三、候选基因的电子克隆和RACE延伸
对于上述的4个基因,我们用它们各自探针序列,通过家蚕EST数据库对这四个基因进行电子延伸,并通过克隆测序验证拼接的正确性,结合3’RACE的测序结果拼接后得到了这四个基因的更多的序列信息。它们都包含了一个较长的开放阅读框的序列,sw13775,sw00132和sw04840这3个探针对应的基因获得了完整的3’端序列。
四、候选基因的序列分析和其编码蛋白分析
将这4个基因电子克隆和RACE延伸后的基因序列进行基因结构分析,发现sw13775和sw00132对应的基因是含有多个外显子的基因,但sw04840和sw04534对应的基因目前仅获得1个外显子。并且sw04840在家蚕基因组精细图数据库中也没有找到与之高度同源的序列,为家蚕中新发现的一个在黑白卵中表达差异很大的一个基因。
在NCBI等数据库中对4个候选基因进行BLASTX分析,发现sw13775对应的是家蚕乙醛还原酶(alcohol dehydrogenase)基因,sw00132与多种昆虫中的醛脱氢酶基因家族7中的一个基因高度同源,而sw04840和sw04534则没有找到与之高度同源的基因。通过所编码氨基酸序列的生物信息学分析,sw04534可能编码一种胞内酶,而sw04840则很可能不是一个编码酶蛋白的基因。
Silkworm, Bombyx mori is an important economic insect in China since the ancient times, Recently, it becomes an important organism model of lepidopteran insects. Its transgenic technology shows significant value in the fields of breeding new varieties, bioreactor and basic scientific development. So far breeding new variety by transgenic method will become the key point in the field of sericulture research, thus the development of detection method of positive transformation will be the core technology in this research field.
The color of wild type silkworm egg is yellow-white at the beginning, about 20h later, gradually become darker and darker,and dark brown finally. The color of the white egg 2 (w-2) mutant of silkworm is yellow-white with a little light red, and eyes of moth are white. So it’s easy to distinguish w-2 silkworm from wild type by egg color and ommateum,also the white egg 2 trait is regular hereditary. w-2 has no negative influences on its hatching, vitality and economic characters,thus it is suitable for marker gene for transgenic silkworm research. Now the understanding of molecular mechanism of the w-2 of silkworm eggs is still not clear.
In this study, we use the established near-isogenic line of w-2 of silkworm,whole genomic gene chip and Real-time Quantitative RT-PCR to explore the functional genes for characteristics of w-2 of silkworm. The result of the experiment will be helpful to elucidate the molecular mechanism of w-2, and be useful for developing new marker for the transgenic silkworm.
The main conclusions we made in this study are as follows: 1. The differentially expressed genes high-throughput explored by silkworm whole genomic gene chip
The silkworm whole genomic gene chip which contains 23000 genes is constructed by CapitalBio corporation and southwest university jointly, is used to find the differrentially expressed genes between white egg 2 and normal eggs of silkworm.Using an criterion of ratio≥2.0 or ratio≤0.5, We obtained 163 genes, in expression of multiple between normal eggs and white eggs that develop to the 24th h, and when silkworm eggs develop to the and 48th h, we obtained 186. The biggest difference expression level gene is sw04840.When silkworm eggs develop to the 24th h and 48th h, the expression level of sw04840 gene in wild type silkworm is 32.4675 and 22.8311 folds respectively comparing to that in white egg 2 mutant.
2. Analysis of differentially expressed genes by Real-time fluorescence quantitative RT-PCR.
According to the results obtained by the whole genomic gene chip, we use Real-time fluorescence quantitative RT-PCR to identify some larger differrentially expressed genes and some differrentially expressed genes located on the 10th linkage group. The results show that the degree of concordance between data generated by the two methods is high. According to the results obtained by Real-time fluorescence quantitative RT-PCR, gene sw04840 is also the biggest difference expression level gene. When silkworm eggs develop to the 24th h and 48th h, the expression level of sw04840 gene in wild type silkworm is 424.61 and 797.86 folds respectively comparing to that in white egg 2 mutant.Further analysis showed that this gene is an abundantly expressed gene in wild type strain eggs, when wild type strain eggs develop to the 24h and 48h, the expression level of sw04840 gene relative to Bmactin A3 is about 1.92times and 0.73 times respectively. In addition, when silkworm eggs develop to the 48th h, the expression level of sw00132 and sw04534 between white egg 2 and normal eggs of silkworm is also reached several hundred times, and when silkworm eggs develop to the 24th h, the biggest difference expression level gene is sw13775 in up-regulate genes in white egg 2.We select this four gene as candidate genes.
3. In silico cloning and RACE elongation of the candidate genes.
For the above four candidate genes, we use in silico extention technology with their probe sequence as seed sequence to extend their sequence, and with assembly with the sequencing results of 3’RACE elongation,we obtained more sequence information of them.. Sequence analysis revealed that all of them contains a long open reading frame(ORF), and gene sw13775, sw00132 and sw04840 contains full 3’end sequence.
4. Analysis the sequence of the candidate genes and the protein that encoded by them
We analysis the gene structure of the four candidate genes,the results show that the gene sw13775 and gene sw00132 contain more than one exons,but the gene sw04840 and the gene sw04534 contains only one.and in database of silkworm genome assembly we did not find any sequence highly homologous with sw04840, indicate that sw04840 is a new gene which is significant difference expressed between white egg 2 and normal eggs.
The analysis of deduced amino acid of the above four candidate genes with blastx shows that gene sw13775 corresponds to the alcohol dehydrogenase of silkworm; sw00132 highly homology with the aldehyde dehydrogenase 7 family, member A1. But we can’t find protein homology with protein encoded by sw04534 and sw04840. Bioinformatics analysis show that sw04534 is likely to be an endocellular enzyme and protein encoded by sw04840 probably not a enzyme.
引文
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