雏鸡肠组织β1基因的鉴定及时空表达特性

Identification and temporal and spatial expression characteristics of β1 in chicken intestine

设计1对引物，建立RT—PCR方法对β1基因mRNA进行鉴定；以GAPDH基因为内参，建立检测鸡β1基因表达水平的sYBRGreenI实时荧光定量PCR方法，并用此方法测定1～20日龄雏鸡不同肠段组织81基因表达水平。结果显示，从鸡肠组织成功鉴定出β1基因mRNA；检测内参基因GAPDH和目的基因β1的实时荧光定量PCR方法扩增效率一致（扩增曲线斜率差〈0．1），满足使用比较Ct值法对B1基因mRNA进行相对定量分析的前提；扩增曲线、熔解曲线及凝胶电泳结果提示该方法特异性好。实时荧光定量PCR检测结果表明，1日龄雏鸡十二指肠和盲肠、10日龄雏鸡空肠和直肠、5日龄雏鸡回肠组织中β1基因相对表达量最高。结果表明，鸡肠组织中存在β1基因表达，并在不同日龄不同肠段表达水平存在差异。

To establish a method of detecting the level of β1 expression using real-time fluorescence quantitative PCR,and test the temporal and spatial expression characteristics of β1 in chicken in- testine. A pair of primers was designed for proving the presence of β1 gene mRNA in chicken in- testine. Another pair of primers for real-time fluorescence quantitative PCR was designed to es- tablish a rapid and specific SYBR Green I real-time fluorescence quantitative PCR assay, β1 gene expression levels of 1 to 20-day-old chickens intestinal tissue were detected using the established assay. β1 gene mRNA was identified in the chicken intestinal tissue. The reference gene GAPDH and the target gene β1 had the same amplification efficiency （slope difference of the amplification curve 〈0.1） ,which meets the prerequisite of analyzing the expression level of β1 gene by comparative Ct method. The amplification curve, melting curve and gel electrophoresis results demonstrated that the established method was very specific. The expression levels of β1 gene mRNA differed in different day-old chicken intestinal tissue, the expression levels of β1 gene mRNA were the highest in jejunum and rectum of 10-day-old chicken,duodenum and cecum of 1-day-old chicken, ileum of 5-day-old chicken. The expression of β1 gene existes in chicken intestine,and the expression levels of β1 gene mRNA differ in different day-old chicken intestinal tissue. The research provides a basis for further research on the invasion mechanism of chicken infected with Shigella.