家蚕第二隐性赤蚁基因(ch-2)的图位克隆
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摘要
着色是动物最具多样化的特征之一,体色艳丽,形状奇特的蝴蝶和部分蛾类,具有很高的观赏价值。体色的研究对探讨昆虫遗传多态性、适应机制及生物进化等具有重要的意义。家蚕的色彩突变涉及家蚕的各个生长时期,胚胎期,幼虫期、蛹期、成虫期,包括蚕茧也具有丰富的颜色变化。不同的基因变异导致控制家蚕体内的各种黑色素、蝶啶、眼黄素等在表皮细胞和真皮细胞中的积累和相互作用,可以形成不同体色突变。目前已发现100多种突变都与着色模式异常相关,因此,研究家蚕体色形成机制,有助于为色素模式的遗传学基础研究提供丰富资源,使人们深层次认识色素功能,同时为家蚕品种资源的多样性分析提供了依据。
第二隐性赤蚁基因(ch-2)是少数只在蚁蚕时期显现出可辨别表型的体色突变型之一,作为标志性基因,本研究利用SSR、STS和CAPs分子标记技术和siRNA干扰技术,对其进行图位克隆和功能补偿验证,其主要内容及结果如下: 1.对ch-2基因的初步定位和连锁分析
供试家蚕品种正常黑蚁P50和突变体赤蚁ch-2品种k04均由中国农业科学院蚕业研究所提供。用k04和P50作为亲本组建F1代及BC1回交群体(k04×p50)×k04和k04×(k04×P50),分别记作BC1F和BC1M。基于雌性家蚕的W与Z染色体不发生交换的特点,采用此两种回交群体通过已经构建的家蚕SSR分子标记连锁图谱用BC1F群体进行连锁分析,用BC1M群体构建遗传连锁图,寻找与ch-2基因连锁的标记,结果显示在第18连锁群上发现7个多态SSR标记(S1804、S1807、S1808、S1809、S1812、S1814 and S1819),重新设计微卫星位点的引物,结果在S1804和S1807附近找到2个新的多态SSR标记ZR1807和ZR1817,这9个标记在BC1F群中的电泳谱中所有黑色个体均表现出与F1相同的杂合带型;而所有赤蚁带型与k04一致,为纯合型;用BC1M群体采用作图软件Mapmaker 3.0,根据整个数据阵计算重组值(LOD取5.0),绘制出ch-2基因的分子连锁图。绘制的遗传连锁图的遗传距离为70.7cM,ch-2基因位于中间69.6cM处,S1814和S1819与ch-2基因距离最近,与ch-2基因的距离分别是7.9cM和1.1cM。
2对第二隐性赤蚁基因ch-2的精细定位及ch-2基因全长的获得
经与家蚕基因组数据库进行比对,S1814位于家蚕基因组精细图的nscaf2902的1.526Mb处,而S1819不能在基因组精细图中比对到序列,说明S1819的序列位于已知的家蚕基因组精细图中的scaffold外部,这与其位于遗传连锁图的尾端是符合的,说明S1819所在区域的序列还没有能够被测通或者没有能够被正确拼装。于是我们在nscaf2902和nscaf2901上用Primer.primer5.0设计更多的STS和CAPS引物的标记,通过测序酶切PCR产物将ch-2锁定在300kb以内的区域,通过Blast程序搜索,经同源序列比对,发现预测基因BGIBMGA008245-TA表皮蛋白(CPG6 )基因和BGIBMGA008268-TA基因,可能与着色相关。查询数据库,并了解该基因的功能,将目标锁定为BGIBMGA008245-TA表皮蛋白(CPG6)基因。通过对P50表皮进行5’和3’RACE-cDNA扩增,利用DNAMAN拼接得到了ch-2基因的全长。
3.候选基因功能验证用家蚕品种正常黑蚁P50和突变体赤蚁ch-2品种k04的不同催青天数的蚕卵,液氮速冻后提取RNA,反转录为cDNA后进行普通PCR扩增,凝胶回收PCR产物,克隆测序,比较亲本间的序列差异。结果发现表皮蛋白(CPG6)的表达在催青第一天和第四天均无差异,而第九天在突变体中存在156bp左右的缺失表达,根据表皮蛋白(CPG6)在突变体中的表达差异性,对差异序列进行SiRNA设计,注射到经过不同时间催青处理的蚕卵中进行催青,并对孵化的卵数进行统计,对未孵化的卵进行解剖,发现了赤色的蚁蚕。
Coloration is one of the variegated characteristics for insects.It is quite a value to view and admire for radiantly beautifμL and odd butterflies and some moth. it is interesting and impotant to explore the genetic polymorphisms, adaptive mechanism and biological evolution for insects. Colour mutation are involved in various growth period, including embryonic phase, larval phase, pupa stage, adult stage and cocoon. Different mutants caused the accumulation and interaction of various kinds of melanin, pteridine, xanthommatin in epidermal cells and corium cells, which are resulted in forming different body color. It has been more than 100 mutants which have been found to correlate with coloring model. Based on the study and research on the mechanism of the body color, it can provide knowledge for insect pigmentation and understand the modle of genetic basis, at the same time it can provide the evidence for the analysis of genetic diversity of silkworm.
The second recessive chocolate gene (ch-2) is one of the few appeared red color only in newly hatached larvae period, which can distinguish to normal black ones. We used SSR, STS to make map-based cloning this gene and use and siRNA methods to analyze the functional compensation verification, the contents are as follows:
1. Initial Linkage Analysis of Gene ch-2 and MolecμLar Mapping
We used silkworm strain P50 as wild type and k04 (ch-2/ch-2) variety as parents, both of which are preserved in SericμLture Research Institute, Chinese Academy of AgricμLtural Sciences. Owing to a lack of crossing over in females, reciprocal Backcrossed BC1F progeny were used for Linkage analysis and mapping of the ch-2 gene in chr18 using silkworm stains P50 and k04, which are classified as being black and red larvae. We found seven SSR markers including S1804、S1807、S1808、S1809、S1812、S1814 and S1819, which were identified to be linked to the ch-2 gene. Redesigned microsatellite loci primers, we found 2 makers near S1804 and S1807, all the 9 primers have the same homozygous profile as the parental ch-2 in the chocolate individuals of BC1F, and heterozygous in the wild type individuals as in F1.The recombination fractions were then calculated from the whole dataset using MAPMAKER 3.0 at a LOD score of 5.0 for further confirmation using BC1M, we constructed a linkage map of 70.7cM, with ch-2 mapped at 69.6cM and the genetic distance between ch-2 gene and the nearest marker S1814 and S1819 is 7.9cM and 1.1cM.The order of the SSR markers was ch-2 and S1819. 2. Fine mapping of ch-2 gene and the full sequence of ch-2
S1814 located at the site of 1.526Mb in nscaf2902 after blasting the Bombyx mori genome database, but the sequence of S1819 can’t be blast in the genome database, which meant that the sequence of S1819 is located outside the scaffolds of genome, and this is coincident to the result that the ch-2 gene is located in the end of the established linkage map. We screening nscaf2901 and nscaf2902, design new STS makers and CAPs makers using Primer.primer5.0, to map ch-2 gene precisely, at last we had narrowed the ch-2 gene in a region of 300kb. We blasted all of the candidate genes in NCBI and silk worm genome database, and found that the predicted genes BGIBMGA008245-TA (skin protein gene CPG6) and gene BGIBMGA008268-TA might be related to body color. We blast the Bombyx mori genome database to get the function and at last we consider BGIBMGA008245-TA(CPG6)as the important candidate gene. We get the full sequence of ch-2 using DNAMAN after we had a 5’and 3’RACE-cDNA amplification of P50epidermis.
3. The candidate gene and function analysis
RT-PCR primers were designed based on the EST of these genes, and we found there were 156bp deletions in BGIBMGA008245 -TA in k04’s embryo after 9 days incubation, but there was no distinguish between ch-2 and P50 in the embryo after 1day and 4days’incubation. We designed siRNA according to the CPG6, and microinjected into the Nistari eggs which were dealed with different days’incubation. In the end, we had a statistics about the eggs incubation and dissected the eggs which didn’t hatch, and ch-2 phenotype newly-hatched larvae had been observed after RNA knock down of CPG6 gene.
第二隐性赤蚁基因(ch-2)是少数只在蚁蚕时期显现出可辨别表型的体色突变型之一,作为标志性基因,本研究利用SSR、STS和CAPs分子标记技术和siRNA干扰技术,对其进行图位克隆和功能补偿验证,其主要内容及结果如下: 1.对ch-2基因的初步定位和连锁分析
供试家蚕品种正常黑蚁P50和突变体赤蚁ch-2品种k04均由中国农业科学院蚕业研究所提供。用k04和P50作为亲本组建F1代及BC1回交群体(k04×p50)×k04和k04×(k04×P50),分别记作BC1F和BC1M。基于雌性家蚕的W与Z染色体不发生交换的特点,采用此两种回交群体通过已经构建的家蚕SSR分子标记连锁图谱用BC1F群体进行连锁分析,用BC1M群体构建遗传连锁图,寻找与ch-2基因连锁的标记,结果显示在第18连锁群上发现7个多态SSR标记(S1804、S1807、S1808、S1809、S1812、S1814 and S1819),重新设计微卫星位点的引物,结果在S1804和S1807附近找到2个新的多态SSR标记ZR1807和ZR1817,这9个标记在BC1F群中的电泳谱中所有黑色个体均表现出与F1相同的杂合带型;而所有赤蚁带型与k04一致,为纯合型;用BC1M群体采用作图软件Mapmaker 3.0,根据整个数据阵计算重组值(LOD取5.0),绘制出ch-2基因的分子连锁图。绘制的遗传连锁图的遗传距离为70.7cM,ch-2基因位于中间69.6cM处,S1814和S1819与ch-2基因距离最近,与ch-2基因的距离分别是7.9cM和1.1cM。
2对第二隐性赤蚁基因ch-2的精细定位及ch-2基因全长的获得
经与家蚕基因组数据库进行比对,S1814位于家蚕基因组精细图的nscaf2902的1.526Mb处,而S1819不能在基因组精细图中比对到序列,说明S1819的序列位于已知的家蚕基因组精细图中的scaffold外部,这与其位于遗传连锁图的尾端是符合的,说明S1819所在区域的序列还没有能够被测通或者没有能够被正确拼装。于是我们在nscaf2902和nscaf2901上用Primer.primer5.0设计更多的STS和CAPS引物的标记,通过测序酶切PCR产物将ch-2锁定在300kb以内的区域,通过Blast程序搜索,经同源序列比对,发现预测基因BGIBMGA008245-TA表皮蛋白(CPG6 )基因和BGIBMGA008268-TA基因,可能与着色相关。查询数据库,并了解该基因的功能,将目标锁定为BGIBMGA008245-TA表皮蛋白(CPG6)基因。通过对P50表皮进行5’和3’RACE-cDNA扩增,利用DNAMAN拼接得到了ch-2基因的全长。
3.候选基因功能验证用家蚕品种正常黑蚁P50和突变体赤蚁ch-2品种k04的不同催青天数的蚕卵,液氮速冻后提取RNA,反转录为cDNA后进行普通PCR扩增,凝胶回收PCR产物,克隆测序,比较亲本间的序列差异。结果发现表皮蛋白(CPG6)的表达在催青第一天和第四天均无差异,而第九天在突变体中存在156bp左右的缺失表达,根据表皮蛋白(CPG6)在突变体中的表达差异性,对差异序列进行SiRNA设计,注射到经过不同时间催青处理的蚕卵中进行催青,并对孵化的卵数进行统计,对未孵化的卵进行解剖,发现了赤色的蚁蚕。
Coloration is one of the variegated characteristics for insects.It is quite a value to view and admire for radiantly beautifμL and odd butterflies and some moth. it is interesting and impotant to explore the genetic polymorphisms, adaptive mechanism and biological evolution for insects. Colour mutation are involved in various growth period, including embryonic phase, larval phase, pupa stage, adult stage and cocoon. Different mutants caused the accumulation and interaction of various kinds of melanin, pteridine, xanthommatin in epidermal cells and corium cells, which are resulted in forming different body color. It has been more than 100 mutants which have been found to correlate with coloring model. Based on the study and research on the mechanism of the body color, it can provide knowledge for insect pigmentation and understand the modle of genetic basis, at the same time it can provide the evidence for the analysis of genetic diversity of silkworm.
The second recessive chocolate gene (ch-2) is one of the few appeared red color only in newly hatached larvae period, which can distinguish to normal black ones. We used SSR, STS to make map-based cloning this gene and use and siRNA methods to analyze the functional compensation verification, the contents are as follows:
1. Initial Linkage Analysis of Gene ch-2 and MolecμLar Mapping
We used silkworm strain P50 as wild type and k04 (ch-2/ch-2) variety as parents, both of which are preserved in SericμLture Research Institute, Chinese Academy of AgricμLtural Sciences. Owing to a lack of crossing over in females, reciprocal Backcrossed BC1F progeny were used for Linkage analysis and mapping of the ch-2 gene in chr18 using silkworm stains P50 and k04, which are classified as being black and red larvae. We found seven SSR markers including S1804、S1807、S1808、S1809、S1812、S1814 and S1819, which were identified to be linked to the ch-2 gene. Redesigned microsatellite loci primers, we found 2 makers near S1804 and S1807, all the 9 primers have the same homozygous profile as the parental ch-2 in the chocolate individuals of BC1F, and heterozygous in the wild type individuals as in F1.The recombination fractions were then calculated from the whole dataset using MAPMAKER 3.0 at a LOD score of 5.0 for further confirmation using BC1M, we constructed a linkage map of 70.7cM, with ch-2 mapped at 69.6cM and the genetic distance between ch-2 gene and the nearest marker S1814 and S1819 is 7.9cM and 1.1cM.The order of the SSR markers was ch-2 and S1819. 2. Fine mapping of ch-2 gene and the full sequence of ch-2
S1814 located at the site of 1.526Mb in nscaf2902 after blasting the Bombyx mori genome database, but the sequence of S1819 can’t be blast in the genome database, which meant that the sequence of S1819 is located outside the scaffolds of genome, and this is coincident to the result that the ch-2 gene is located in the end of the established linkage map. We screening nscaf2901 and nscaf2902, design new STS makers and CAPs makers using Primer.primer5.0, to map ch-2 gene precisely, at last we had narrowed the ch-2 gene in a region of 300kb. We blasted all of the candidate genes in NCBI and silk worm genome database, and found that the predicted genes BGIBMGA008245-TA (skin protein gene CPG6) and gene BGIBMGA008268-TA might be related to body color. We blast the Bombyx mori genome database to get the function and at last we consider BGIBMGA008245-TA(CPG6)as the important candidate gene. We get the full sequence of ch-2 using DNAMAN after we had a 5’and 3’RACE-cDNA amplification of P50epidermis.
3. The candidate gene and function analysis
RT-PCR primers were designed based on the EST of these genes, and we found there were 156bp deletions in BGIBMGA008245 -TA in k04’s embryo after 9 days incubation, but there was no distinguish between ch-2 and P50 in the embryo after 1day and 4days’incubation. We designed siRNA according to the CPG6, and microinjected into the Nistari eggs which were dealed with different days’incubation. In the end, we had a statistics about the eggs incubation and dissected the eggs which didn’t hatch, and ch-2 phenotype newly-hatched larvae had been observed after RNA knock down of CPG6 gene.
引文
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