Differential Gene Expression Profiles Analysis Of Tea Plant Induced By Tea Looper (Ectropic Oblique) Attack Using DDRT-PCR

The particularity of tea which is one kind of drinks brings a very major difficulty in harmful insect preventing and controlling, because Chemistry agricultural chemicals and the use of anti-insect genes are restricted. In recent years, the technology of pest preventing and controlling has obtained very greatly progressive, but the pest of tea still repeatedly caused disaster, the situation of pesticide residue exceeded the allowed figure still be stern. Therefore, it must have a new theoretical breakthrough in synthesis controlling in the anti-insect breeding technology of tea, the anti-insect gene resources of tea, the preparation of anti-insect medicine of tea.The gene expression spectrum difference demonstrated technology (GESDDT) was one effective method which recently emerged and research homogeneous cell under the different condition of the gene expression difference. At present ,it has demonstrated the unique superiority and succeeded applies in the food induction defense to the plant to avoid the harmful insect, the molecular mechanism, excavates the plant endogene anti-insect gene the research area.This research faces the stern question about our country tea productionreality and take nongkangzao as the research object to research the gene ex pression spectrum difference performance characteristic which caused by ident ical tea leaves blade by the ectropis obliqua through the DDRT-PCR method,and use the biological information method to research the structure and the function the difference expression gene carried on. With the resistant related unknown function sequence, RACE technology is used to clone its span DN A sequence and the sequence analysis.We hope this research could obtain thefollowing result through related molecular mechanism and the induction defense to the ectropisobliqua taken by tea:(1) Ten pair of directing was used in the difference demonstration analys is processes between the processing group and the comparison group ,and theexpression difference has found out between the processing group and the comparison group.Techniques of differential display for gene expression profile, recently developed, are effective and practicable to explore gene expression and used in the fields of plant-insect interactions and searching insect-resistant genes successfully. DDRT-PCR was used to explore the differential gene expression profiles analysis of tea plant induced by Ectropic obliqua attack. The results were showed that 222 differential expression fragments were obtained, which was 32.8% of total fragments, by amplified in anchor primers A3, A4, A6, A7, A8 and random primers R1-R8, R12, respectively. 20 fragments of which were re-amplified by RT-PCR. Based on the BLASTx search in GenBank, it were found that eight of the fragments shared no homology, five of the fragments shared homology with the unknown proteins, and seven of the fragments shared higher score homology with the known proteins, which can be divided into six groups related to photosynthetic pigment protein in photosystem II (C15) , abiotic resistance induced protein(D22), indicant metabolism enzyme(A45), nucleic acid and protein transposon regulated factors(A12, D63), auxin-regulated protein(E94) and VIP2 protein(D73). The fragments of C15, A45, D22, D73 and E94 were firstly found in the studies on the molecular mechanisms of plant-insect interactions, up to day.The results were showed that the indican metabolized enzyme and the un-biology resistant-induced protein are correlated with ressistance to pests. The complete cDNA sequence of A45 and D22 were cloned by Rapide amplification of cDNA ends (RACE) technique. Analysis of A45 cloned complete cDNA showed that it encompassed an open reading frame (ORF) with 2340bp enconding 767 amino acids. The 5' untranslated regions contains 72bp and 3' untranslated regions contains 120bp, Analysis of D22 cloned complete cDNA showed that it encompassed an open reading frame (ORF) with 2034bp enconding 677amino acids. The 5' untranslated regions contains 128bp and 3' untranslated regions contains 157bp, Homology searches with the deduced 767 amino acids residues reverled A45 shares 85% similarity with to the straw berry.β-xyloseglucoside may participate in hydrolyzingβ-primeverosidase,changing into volatility xyloseglucoside-residues, Accordingly tea leaf comesinto being resistant to insect-pests and disease. And then the volatility xyloseglucoside-residues are the chemical message matters among in tea plant ,pest and natural enemy,the aromatic of indican ofen tempt the insects of natural enemy ,thereby, tea plant produce resistance to pests indirectly.So xyloseglucoside may take important part in induction defense to the plant to avoid the harmful insect.