Screening Of Differential Expressed Gene From Channa Argus Challenged With A.Veronii And Study On The Expression Pattern Of The Representative Genes

Channa argus, one of valuable fishes which is loved by the customers, is been food and medicine. In recent years, more kinds of diseases occur with the incremental farming area and the intensive aquaculture, especially the bacterial disease. This has become a significant barrier to the aquaculture development of Channa argus. Currently, to feed antibiotics is the general method to cure the bacterial disease. However, the drug residue and resistance is increasing. To develop the new no-toxic drug and disease resistance breeding is the tendency of the sustainable development of Channa argus aquaculture. Hence, it requires further study to antibacterial immunity-related genes and the immunologic mechanism of Channa argus.In this research, the head-kidney SSH cDNA library from Channa argus infected with Aeromonas veronii was constructed using Suppression Subtractive Hybridization. And by screening and BLASTn,202expressed sequence tags(ESTs) were acquired.73ESTs were unknown sequence, and the other129ESTs were grouped under6clusters based on their function. The immunity-related genes consist of25ESTs and represented13%of the whole ESTs.From the head-kidney SSH library, the gene segment of Cu/Zn-SOD, Rac2, and MPO were picked, and the full length were acquired with RACE. By the bioinformatics, the chemicophysical properties, protein structure and amino acid homology were analyzed. The expression and distribution of the three genes in health Channa argus and the expression change after challenge with Aeromonas veronii were detected by real-time quantitative PCR (RT-PCR).These results showed that the full length of Cu/Zn-SOD gene contained810bases, whose ORF contained465bases and encoded154amino acids. The multiple sequence alignment showed the amino acid sequence of Cu/Zn-SOD shared95%homologies with Tilapia Mossambica. The RT-PCR results showed that the SOD was expressed in head-kidney, kidney, gill, heart, spleen, liver, muscle, and intestines, and the relative expression quality of liver was the highest. After challenge with Aeromonas veronii, the average trend of expression quality was after rising to decline and maintained for a period of time, and then rose.The full length of Rac2gene contained1179bases, whose ORF contained579bases and encoded192amino acids. The amino acid sequence showed that the Rac2from most species all shared high homologies. The RT-PCR results showed that the Rac2was expressed in head-kidney, kidney, gill, heart, spleen, liver, and muscle, but not in intestines, and the relative expression quality of head-kidney was the highest. After challenge with Aeromonas veronii, the expression quality changed with time and all of them rose at36hour post-infection and exceeded it at0hr P.I.The full length of MPO gene contained3181bases, whose ORF contained2301bases and encoded766amino acids. The amino acid sequence showed that the MPO from most species was different from one another. The RT-PCR results showed that the Rac2was expressed in head-kidney, kidney, gill, heart, spleen, liver, and muscle, but not in intestines, and the relative expression quality of head-kidney was the highest. After challenge with Aeromonas veronii, the expression quality was low in most tissue and all rose at36hr P.I..In this work, we successfully constructed the head-kidney SSH cDNA library from Channa argus, and a number of ESTs related to antibacterial immunity was acquired. The full length of three genes was amplified and systematic analyzed, and then their patterns of expression were explored. These results can lay the foundation for further study about the molecular mechanism of immunity and the basic theoretical data for resistance breeding and disease control.