为进一步丰富番鸭羽色性状遗传机制的研究,运用Illumina高通量测序技术对不同性别的黑羽及白羽番鸭皮肤组织进行转录组测序,经GO富集分析和KEGG通路注释对差异表达基因功能进行解析,利用qRT-PCR对部分RNA-Seq数据进行验证。结果显示,公...为进一步丰富番鸭羽色性状遗传机制的研究,运用Illumina高通量测序技术对不同性别的黑羽及白羽番鸭皮肤组织进行转录组测序,经GO富集分析和KEGG通路注释对差异表达基因功能进行解析,利用qRT-PCR对部分RNA-Seq数据进行验证。结果显示,公黑羽番鸭皮肤(HFPF.G)vs母黑羽番鸭皮肤(HFPF.M)获得差异基因2 131个,公白羽番鸭皮肤(BFPF.G)vs母白羽番鸭皮肤(BFPF.M)获得差异基因780个,黑羽番鸭皮肤(HFPF)vs白羽番鸭皮肤(BFPF)获得差异基因684个,通过上述三组韦恩分析,以黑、白羽番鸭皮肤对比组(HFPF vs BFPF)504个差异基因为番鸭羽色候选基因,进一步筛选获得番鸭羽色性状相关前10个差异表达,其溶质转运蛋白(Solute carrier,SLC)家族成员SLC7A11和SLC25A4可能对番鸭黑、白羽色性状遗传起重要调控作用。同时发现细胞色素P450对外源物质的代谢作用信号通路、谷胱甘肽代谢信号通路、cAMP信号通路等可能参与番鸭黑白羽色性状遗传的调控过程。研究表明,SLC7A11和SLC25A4可用于黑羽番鸭的羽毛分子辅助选育。展开更多
The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as te...The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.展开更多
文摘为进一步丰富番鸭羽色性状遗传机制的研究,运用Illumina高通量测序技术对不同性别的黑羽及白羽番鸭皮肤组织进行转录组测序,经GO富集分析和KEGG通路注释对差异表达基因功能进行解析,利用qRT-PCR对部分RNA-Seq数据进行验证。结果显示,公黑羽番鸭皮肤(HFPF.G)vs母黑羽番鸭皮肤(HFPF.M)获得差异基因2 131个,公白羽番鸭皮肤(BFPF.G)vs母白羽番鸭皮肤(BFPF.M)获得差异基因780个,黑羽番鸭皮肤(HFPF)vs白羽番鸭皮肤(BFPF)获得差异基因684个,通过上述三组韦恩分析,以黑、白羽番鸭皮肤对比组(HFPF vs BFPF)504个差异基因为番鸭羽色候选基因,进一步筛选获得番鸭羽色性状相关前10个差异表达,其溶质转运蛋白(Solute carrier,SLC)家族成员SLC7A11和SLC25A4可能对番鸭黑、白羽色性状遗传起重要调控作用。同时发现细胞色素P450对外源物质的代谢作用信号通路、谷胱甘肽代谢信号通路、cAMP信号通路等可能参与番鸭黑白羽色性状遗传的调控过程。研究表明,SLC7A11和SLC25A4可用于黑羽番鸭的羽毛分子辅助选育。
基金Supported by the National Natural Science Foundation of China(Grant No.30430030)Specialized Research Fund for the Doctoral Program of Higher Education(Grant No.20061117004)
文摘The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.
