Establishment Of CDNA-AFLP System And Its Application In The Differential Expression Of Cyprinus Carpio

cDNA-AFLP (cDNA-amplified fragment length polymorphism, cDNA-AFLP) is a molecular technique used to detect cDNA polymorphism,which developed from the differential display reverse transcriptase PCR(DDRT-PCR) and amplified fragment length polymorphism (AFLP) . The technology has the advantages of no need sequence information, high sensitivity and reproducibility , and can give a comprehensive analysis of transcription. we established a cDNA-AFLP reaction system of carp for the first time through the optimization and improvement of the key steps, and screening 69 polymorphic cDNA-AFLP markers. The study include the following:1.Establishment of cDNA-AFLP System for carpcDNA-amplified fragment length polymorphism technology was used for mirro carp and an appropriate reaction system was established in this study。It was proved that good result could be achieved by the digestion with MseI and BstYI of two and three hours separately, the ligation reaction system consisted mainly of 0.2 pmol/ul adapterⅠ,2 pmol/ul adaperⅡ,100 ng cDNA and 0.12 U/ul T4 ligate enzyme and the BstYI and MseI selective primer ratio of 1:4。The establishment of cDNA-AFLP reaction system provided a foundation for transcriptomic research of carp.2.Screening cDNA-AFLP markers of carpcDNA-AFLP markers of carp were isolated based on the established system. 69 primer combinations were screened from 96 pairs of primers combined with 12 MseI-NN and 8 BstYI-T/C-N. These primers may provide basic materials for the study of differential expression and transcription of carp.3.Preliminary analysis of differential gene expression in immune-related organizations of carp.The infected and normal mirro carp were collected from groups of high-density cage culture in the Yalu River during the outbreak of fish diseases. Fresh brain and spleen were collected for the following study and treated by liquid nitrogen immediately . Informations on differential expression were obtained by differences screening. The results as follows:( 1 ) In brain and spleen, between normal and infected states,the population genetic distance was 0.2434 and 0.2437,and genetic differentiation index (Gst) was 0.2560 and 0.2678 respectively. The Neighbor-Jioning dendrogram showed that all individuals were mainly clustered into two broad clusters . All results indicate that there were differential expression between normal and disease populations( 2 )Slightly higher expression of the spleen than the brain, whether in normal or infection period. Differentially expressed genes were 45 and 55 in brain and spleen respectively. Differences not only in the expression level, but also qualitatively different. Under normal conditions, significant differences in the two tissue are mainly based on the expression abundance. In infection stage, the two organizations, changes in the expression level was not obvious, but the number of genes expressed significantly increased, and the dominant factor in significant differences changed into a qualitative difference from expression level. Types of difference include three categories.①in the same organization, expresstion only in one of the infected or normal stage.②in the same organization, expression in both of infected and normal stages, but there is significant difference in expression level .③the same organization, genes expressed in part of the individuals of the infected or normal group.