紫茎泽兰处理鸡柔嫩艾美耳球虫后差异基因筛选和分析

Screening and Analysis of Differential Expression Genes in the Eupatorium adenophorum Treatment of Chicken Eimeria tenella

为获得紫茎泽兰处理鸡柔嫩艾美耳球虫后差异基因,将0.5%紫茎泽兰提取液作用于鸡柔嫩艾美耳球虫卵囊孢子化过程,采用抑制消减杂交技术(SSH)筛选处理后卵囊的差异表达基因,通过GO和COG功能预测分析,并采用实时荧光定量PCR验证差异表达的基因。结果显示,采用SSH成功构建了紫茎泽兰处理前后鸡柔嫩艾美耳球虫卵囊的cDNA消减文库,获得86条ESTs序列,经拼接和聚类后得到31条独立基因(Unigenes),其中有23个基因有功能注释,8个基因没有功能注释,另外有4个基因没有同源性匹配。为进一步验证文库的特异性,从中随机选取3个差异表达基因,运用实时荧光定量PCR技术验证表面抗原13、3-羟酰辅酶A脱氢酶和细胞色素P450基因在紫茎泽兰处理前后的表达差异,结果显示,3个基因在经紫茎泽兰处理的鸡球虫孢子化卵囊中的表达量明显低于未处理组,说明紫茎泽兰对柔嫩艾美耳球虫卵囊具有一定的活性抑制作用。本试验获取了紫茎泽兰作用柔嫩艾美耳球虫卵囊的主要调控基因,为将紫茎泽兰研发成环境杀虫剂奠定了良好的理论基础,也可为球虫致弱疫苗或基因缺失疫苗的靶标筛选研究提供参考。

To obtain the biological information of differentially expressed genes of Eimeria tenella oocysts treated with Eupatorium adenophorum,0.5%Eupatorium adenophorum were adopted to treat oocyst sporulation process of coccidiosis,suppression subtractive hybridization(SSH)was used to screen differential expression genes of chicken coccidian oocysts after treatment,GO and COG function prediction were used to analyze those genes,and Real-time PCR was used to verify the results of SSH.The results showed that a subtractive cDNA library of chicken Eimeria tenella before and after treatment of Eupatorium adenophorum was successfully constructed,86 ESTs sequences and 31 Unigenes were obtained after splicing and clustering,of which 23 Unigenes were with function annotation,8 Unigenes with no functional annotation,and 4 Unigenes had no homology matching.3 differentially expressed genes were randomly selected to further verify the specificity of the cDNA library by Real-time PCR,and the results showed that the expression of surface antigen 13,3-hydroxyacyl coenzyme A dehydrogenase and cytochrome P450 genes in Eimeria tenella sporulated oocysts in Eupatorium adenophorum treatment group was obviously lower than that in untreated group,indicating that Eupatorium adenophorum had an inhibitory effect on the activity of oocysts.The main regulation effect of Eupatorium adenophorum on genes were obtained in this study,which laid a theoretical foundation for the development of Eupatorium adenophorum environmental pesticides,and could provide references for the screening of coccidiosis attenuated vaccine or gene deletion vaccine targets.