Construction Of Normalized Full-length CDNA Library Of Brown Planthopper Larvae And Analysis Of EST

The brown planthopper(Nilaparavata lugens, BPH)is a notorious long-distance migratory pest of rice plants in the worldwide include China. BPH attacks rice plant and sucks fluid from the vascular bundle and thus interferes with the translocation of assimilation products. In addition to sucking damage, rice disease cound be spread or induced by BPH infestation. Therefore, it affects growth and development of rice plants seriously and cause enormous yeild losses of rice.BPH was profoundly studied from 1970s in China and important progress had been made on characteristics of macrobiology and migration. To exploit inherent mechanism of disasters caused by BPH, the molecular mechanism of growth and development must be studied through molecular biological technology. cDNA library is the basis of gene expression profile and functional analysis, it can provide a platform for research of critical gene involved in physilogical process of BPH.Two methods of construction of full-length cDNA library for BPH larvae were compared. The results indicated that normalized full-length cDNA library which constructed using Creator SMART technology combined with DSN had better quality. Then 5000 clones were selected randomly from this library and sequenced from 5' end, the sequences were analyzed and some major results obtained were as follows:1. A full-length cDNA library of BPH larvae was constructed using SMART technology. The titer of original library was 1.06×10~6pfu/mL with 93% recombinant efficiency, the average size of inserted fragment was above 750bp. But the sequencing results indicated that redundancy of this cDNA library was too high, it was unsuitable for large-scale sequencing.2. A normalized full-length cDNA library of BPH larvae was constructed using Creator SMART technology combined with duplex-specific nuclease(DSN). The titer of original library was 1.75×10~6 cfu/mL, the recombinant efficiency was 93.33% and the average of insert size was above 1kb, these indicators showed this library was of high quality. Otherwise, 192 clones from it were selected randomly and sequenced, from the sequencing results, the redundancy of this library was 3.51% and the ratio of full- length cDNA was 54.8%. These results indicated it had significant effect of normalization. 3. 5063 clones from the normalized cDNA library were selected randomly and sequenced,4778 high quality sequences more than 100bp were produced after removing the vector sequences and contaminated sequences. The average length was 588.6bp.4. Using the Phrap software, the 4778 ESTs of high quality were analyzed by clustering and assemblying into 4302 unigene including 299 contigs and 4003 singlets, the number of unigene accounted for 90.04% of the total ESTs, which further validated the effect of normalization.5. The 4302 ESTs were compared with NCBI non-redundant nucleotide database using BLASTn and 1153 ESTs of unigenes matched, which accouted for 26.80% of all; they were compared with NCBI non-redundant protein database using BLASTx and 2679 ESTs of the unigenes matched, which accouted for 62.27% of all. After BLASTx analysis, gene information gained was as follows: 971 ESTs (22.57%) were known genes with function annotation or putative function annotation, 1708 ESTs (39.70%) shared homology but no function annotation, 1623 ESTs (37.73%) were novel genes that shared no homology in database. Additional, these ESTs were compared with EST database of brown planthopper and 2647 new genes(55.4%) were gained.6. The 971 genes with homology alignment were functionally classified into 9 different categories. A: replication, transcription and translation, B: substance metabolism, C: energy metabolism, D: defense mechanisms, E: cellular singal transduction, F: cellular biogenesis and cellular organization, G: cell growth and division, H: intracellular trafficking and secretion, I: general function predictin and unknown.7. Some genes involved in growth and development of BPH were identified, example for genes about transpoter, depense protein, wing polymorphism, metabolism and some other important genes. these genes will provide a reliable basis for the further study on biocontrolling of brown planthopper.