Further confirmation of second- and third-generation Eimeria necatrix merozoite DEGs using suppression subtractive hybridization
In our previous study, we obtained a large number of differentially expressed genes (DEGs) between second-generation merozoites (MZ-2) and third-generation merozoites (MZ-3) of Eimeria necatrix using RNA sequencing (RNA-seq). Here, we report two subtractive cDNA libraries for MZ2 (forward library) and MZ3 (reverse library) that were constructed using suppression subtractive hybridization (SSH). PCR amplification revealed that the MZ2 and MZ3 libraries contained approximately 96.7% and 95% recombinant clones, respectively, and the length of the inserted fragments ranged from 0.5 to 1.5 kb. A total of 106 and 111 unique sequences were obtained from the MZ2 and MZ3 libraries, respectively, and were assembled into 13 specific consensus sequences (contigs or genes) (5 from MZ2 and 8 from MZ3). The qRT-PCR results revealed that 11 out of 13 genes were differentially expressed between MZ-2 and MZ-3. Of 13 genes, 11 genes were found in both SSH and our RNA-seq data and displayed a similar expression trend between SSH and RNA-seq data, and the remaining 2 genes have not been reported in both E. necatrix genome and our RNA-seq data. Among the 11 genes, the expression trends of 8 genes were highly consistent between SSH and our RNA-seq data. These DEGs may provide specialized functions related to the life-cycle transitions of Eimeria species.
KeywordsEimeria Merozoite Suppression subtractive hybridization Differentially expressed genes
We would like to thank all organizations which funded this work and all the teachers who cooperated in technical assistance.
Conceived and designed the experiments: JP, SJ. Performed the experiments: SJ, ZF, LL, DD. Analyzed the data: SJ, JJ, HJ, JP. Wrote the paper: SJ and JP. All authors read and approved the final manuscript.
This work was supported by “the National Key R&D Program of China” (2017YFD0500400), the National Natural Science Foundation of China (grant number 31472181 to JPT, grant number 31602039 to DDL), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Compliance with ethical standards
The study was approved by the Animal Care and Use Committee of the College of Veterinary Medicine, Yangzhou University (Approval ID: SYXK [Su] 2012-0029)
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
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