本试验旨在测定和比较5个绿壳蛋鸡群体中EAV-HP在溶质载体有机阴离子转运蛋白家族成员1B3（solute carrier organic anion transporter family member 1B3,SLCO1B3）基因的5′-非编码区插入整合的频率和类型。所选5个绿壳蛋鸡群体包括3...本试验旨在测定和比较5个绿壳蛋鸡群体中EAV-HP在溶质载体有机阴离子转运蛋白家族成员1B3（solute carrier organic anion transporter family member 1B3,SLCO1B3）基因的5′-非编码区插入整合的频率和类型。所选5个绿壳蛋鸡群体包括3个北京地区的商业群体（北京-LK、北京-LF和北京-YM）、贵州长顺绿壳蛋鸡群体和四川省地方品种旧院黑鸡;采用双重PCR对EAV-HP的插入整合进行检测,并利用DNA测序的方法判断EAV-HP的主要突变位点。结果表明,5个绿壳蛋鸡群体均拥有一定数量的EAV-HP插入整合的个体,表现为杂合（LC/N）和纯合（LC/LC）基因型,均可产绿壳蛋,其中旧院黑鸡的绿壳蛋频率最低,仅为28.90%,其次是北京-LF群体,为46.94%,其他3个群体的绿壳蛋频率为83.33%~84.62%;经卡方检验,北京-LF和旧院黑鸡群体符合Hardy-Weinberg平衡（P〉0.05）,而其他3个群体则极显著偏离Hardy-Weinberg平衡状态（P〈0.01）。DNA测序结果显示这5个绿壳蛋鸡群体中EAV-HP插入整合的序列与之前报道的东乡绿壳蛋鸡的KC632577.1完全一致。地方品种旧院黑鸡的绿壳蛋频率较低,建议利用双重PCR检测的分子标记辅助选择方法加以提高。展开更多
To construct the suicide vector of hhd B gene marker-free mutant in Haemophilus parasuis( HPS),two pairs of specific primers were designed and synthesized according to the hhd B gene upstream and downstream sequences ...To construct the suicide vector of hhd B gene marker-free mutant in Haemophilus parasuis( HPS),two pairs of specific primers were designed and synthesized according to the hhd B gene upstream and downstream sequences of HPS published in Gen Bank. The hhd B gene upstream and downstream sequences were amplified by PCR,which were further ligated( hhd B-up + down) through overlapping PCR method. NotⅠand SalⅠrestriction enzyme sites were introduced on both ends of the ligated sequence. After the corresponding digestion,the hhd B-up + down sequence was directionally cloned to the suicide plasmid vector p EMOC2. Results showed that the suicide vector of hhd B gene marker-free deleted( p EMOC2Δhhd B) with stable inheritance in E. coli β2155 strain was successfully obtained,thereby laying the foundation for construction of HPS-hhd B gene marker-free mutant strain.展开更多
文摘本试验旨在测定和比较5个绿壳蛋鸡群体中EAV-HP在溶质载体有机阴离子转运蛋白家族成员1B3（solute carrier organic anion transporter family member 1B3,SLCO1B3）基因的5′-非编码区插入整合的频率和类型。所选5个绿壳蛋鸡群体包括3个北京地区的商业群体（北京-LK、北京-LF和北京-YM）、贵州长顺绿壳蛋鸡群体和四川省地方品种旧院黑鸡;采用双重PCR对EAV-HP的插入整合进行检测,并利用DNA测序的方法判断EAV-HP的主要突变位点。结果表明,5个绿壳蛋鸡群体均拥有一定数量的EAV-HP插入整合的个体,表现为杂合（LC/N）和纯合（LC/LC）基因型,均可产绿壳蛋,其中旧院黑鸡的绿壳蛋频率最低,仅为28.90%,其次是北京-LF群体,为46.94%,其他3个群体的绿壳蛋频率为83.33%~84.62%;经卡方检验,北京-LF和旧院黑鸡群体符合Hardy-Weinberg平衡（P〉0.05）,而其他3个群体则极显著偏离Hardy-Weinberg平衡状态（P〈0.01）。DNA测序结果显示这5个绿壳蛋鸡群体中EAV-HP插入整合的序列与之前报道的东乡绿壳蛋鸡的KC632577.1完全一致。地方品种旧院黑鸡的绿壳蛋频率较低,建议利用双重PCR检测的分子标记辅助选择方法加以提高。
基金Supported by Science and Technology Project of Guangdong Province(2013B020307002)
文摘To construct the suicide vector of hhd B gene marker-free mutant in Haemophilus parasuis( HPS),two pairs of specific primers were designed and synthesized according to the hhd B gene upstream and downstream sequences of HPS published in Gen Bank. The hhd B gene upstream and downstream sequences were amplified by PCR,which were further ligated( hhd B-up + down) through overlapping PCR method. NotⅠand SalⅠrestriction enzyme sites were introduced on both ends of the ligated sequence. After the corresponding digestion,the hhd B-up + down sequence was directionally cloned to the suicide plasmid vector p EMOC2. Results showed that the suicide vector of hhd B gene marker-free deleted( p EMOC2Δhhd B) with stable inheritance in E. coli β2155 strain was successfully obtained,thereby laying the foundation for construction of HPS-hhd B gene marker-free mutant strain.
