Analysis of differentially expressed genes in the precocious line of Eimeria maxima and its parent strain using suppression subtractive hybridization and cDNA microarrays
Analysis of differentially expressed genes in the precocious line of Eimeria maxima and its parent strain using suppression subtractive hybridization and cDNA microarrays
复制标题
抑制消减杂交和cDNA微阵列分析艾美球虫早熟系及其亲本差异表达基因
DOI：
10.1007/s00436-010-2149-7
复制
发表时间：
2011-04-01
影响因子：
2
通讯作者：
Huang, Bing
中科院分区：
文献类型：
--
作者：
Dong, Hui;Lin, Jiaojiao;Huang, Bing
艾美耳球虫早熟系是通过从先前感染鸡的粪便中收集的卵囊重复传代获得的，具有独特的表型，在免疫鸡对抗球虫病方面发挥着重要作用。然而，艾美耳球虫早熟表型的遗传基础仍然知之甚少。为了研究早熟系巨型艾美球菌及其亲本菌株孢子化卵囊中基因表达的变化，通过抑制消减杂交（SSH）构建了消减cDNA文库。从SSH cDNA文库中筛选出总共3,164个cDNA片段来构建cDNA微阵列并进一步鉴定差异表达基因。通过实时PCR验证了微阵列数据的可信度。总共获得了 360 个有效的表达序列标签 (EST)，代表 32 个独特的序列。在早熟品系中，21 个基因被验证为下调，11 个基因被上调。对公共序列数据库进行同源检索发现，有6个基因编码的蛋白与已报道的蛋白同源，包括柔嫩弓形虫的菱形样蛋白和转氢酶、弯形虫的丝氨酸蛋白酶抑制剂和阳离子转运ATP酶、巨型艾美球虫的热休克蛋白以及弓形虫的保守假设蛋白。因此，其余 26 个 EST 之前尚未报道过。这些差异表达基因的进一步表征将有助于理解艾美耳球虫早熟表型的遗传基础。
The precocious line of Eimeria spp., obtained by repeated passages of oocysts initially collected from feces of previously infected chickens, has unique phenotypes and plays an important role in immunizing chickens against coccidiosis. However, the genetic basis of precocious phenotype in Eimeria is still poorly understood. To investigate gene expression changes in sporulated oocysts between the precocious line of E. maxima and its parent strain, subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH). A total of 3,164 cDNA fragments were selected from the SSH cDNA libraries to fabricate cDNA microarrays and further identify the differentially expressed genes. The credibility of the microarray data was verified by real-time PCR. A total of 360 valid expressed sequence tags (ESTs) were obtained, which represented 32 unique sequences. Twenty-one genes were validated as downregulated and 11 genes as upregulated in the precocious line. Homology searching of the public sequence database showed that six genes encoded proteins homologous with previously reported proteins, including rhomboid-like protein and transhydrogenase of E. tenella, serpin, and cation-transporting ATPase of E. acervulina, a heat-shock protein of E. maxima, and a conserved hypothetical protein of Toxoplasma gondii. Thus, the remaining 26 ESTs have not been previously reported. Further characterization of these differentially expressed genes will be useful in understanding the genetic basis for the precocious phenotype in Eimeria spp.