Preparation Of Expression Profile Chips And Research Of Gene Expression Differences Between Two Pacific Abalone Families

Pacific abalone, Haliotis discus hannai Ino is an important aquatic species in the north sea area of our country which distributed naturally in the Yellow sea and Bo sea. While to this day the function gene study of Haliotis discus hannai Ino. remains on its beginning. There are 92 sequences of nucleic acid which has been registered in the nucleic acid databank. Among these sequences there are 23 function genes which covered 8 kinds of genes. Furthermore the EST study of whole Haliotidae is very scarce. There are 26 EST which has been registered: 14 EST come from Haliotis asinine and 12 EST come from Haliotis asinine. The large scale sequencing technology of EST and gene profile chip plays an important role in the study of functional genome which can deal with a large amount of samples at one time. These two technologies have been used broadly in the clone of new genes,gene profile analysis of specific tissues and function annotation of the genome sequences as well as some other fields. As a quick and efficient method to get large amount function genes, EST sequencing method has accumulated a lot of successful experiences (Delseny et al., 1997; Ablett et al., 2000). So we can get a large quantity of expression information of function genes from Haliotis discus hannai Ino by building cDNA library and carrying out the analysis of EST sequences and get differently expressed genes of related characters with expression profile chip in a short time. This study has got an important progress by use of the technologies referred above. The results show as below. 1. This study had built a cDNA library from Haliotis discus hannai Ino.RwRh family of mutated series with red shell color. 2. cDNA library was used to carry out the large-scale EST sequencing experiment and 4494 EST which were longer than 150bp and the precision is better than 90% were found. 3205 EST without redundant sequences were found after the homology analysis through DNAtools software. The 3205 EST were used to do the BlastN and BlastX analysis against the nucleic acid database and protein database. Among the BlastN results, 178 EST had a significant homology with the function genes, 9 EST had a significant homology with the putative genes, 3 EST had a significant homology with the genes with unclear function and 10 EST had a significant homology with the sequences with no function. There were 3005 EST which had no significant homology with the nucleotide databases. 178 EST with high homology were classified into 7 categories according to the function: energy metabolism(34),cell structure and skeleton (36),digestive enzymes(18),regulatory and signal protein(17),DNA scaffold protein(2),translational and transcriptional protein(68),others(3). 912 EST had significant homology with function genes in the BlastX results. The 912 EST were classified into 9 categories according to the classification method of Adams: cell signal and relation (28),cell structure and motion(35),cell defense(39), gene expression and translation(122), metabolism(305), transportation(52), putative protein(110), protein with unclear function (119),protein unclassified(120).2293 EST were regarded as novel genes. There are 952 EST which has found high homologous function genes and structural sequences in both the results of BlastN and BlastX results and the remained 2253 EST were regarded as novel genes. 3. 4048 EST were selected from the HD-RwRh cDNA library to build the expression profile genechip of Pacific abalone which contains some EST which had been sequenced and analyzed and others had been not to carry out the BlastN and BlastX analysis. Each EST was repeated once. The chip contains 8192 dots:8096 EST and 64 positive control,32 negative control. The density of the chip is 1265 dots per cm2. 4. Pacific abalone with different growth speed were selected separately from RwRh and J1Rh families to do two groups of hybridization experiment with the gene chip and 436 differently expressed EST were found. 7 EST expressed differently remarkably in both hybridization experiments.