Differential Genes Responsive To Tea Hooper(Ectropis Obliqua) Herbivore Were Screened By CDNA-AFLP And Molecular Cloning, Expression Of The SAMT In Tea Plant (Camellia Sinensis)

The insects of tea is always one of the major limit factors to the tea in quality and yield.and the molecular mechanism of defense responses induced by insects fed is not clear sofar.The study is still in the prelliminary stage. The study is about screening of the numberand type of different expression genes induced by Ectropis oblique feeding usingcDNA-AFLP in tea plant and qRT-PCR is used to verify the related different genes and itsexpression. Moreover, We mainly described the cDNA cloning, Prokaryotic expression ofCsSAMT as well as its expression characterisation in tea plant is induced by Ectropisoblique feeding and MeJA treatment. The Main results are as follows:(1) The different gene expression induced by Ectropis oblique feeding tea plant is analyzedusing cDNA-AFLP. The results showed that134EST(expressed sequence tags) wereobtained by sequencing and identifying from selected231TDFs(TDFs, Transcript-DerivedFragments), is58%of the total selected TDFs and the length range from80to700. thenup-regulated of TDFs is81and down-regulated is53, is60.4%and39.6%of the obtainedfragments, respectively.(2) According to blastx result of TDFs in GenBank, the functional classification ofobtained fragments are as follows, A main large group of these genes shared highhomology with genes involved in metabolism, is23.1%of the obtained TDFs, the secondlylarge groups to genes with function related to photosynthesis and energy as well as proteinsynthesis and storage, is9.0%and8.2%of the total TDFs, respectively, then6.7%to geneswith function related to signal transduction,4.5%to genes with function related todisease/defence, moreover, genes with functions related to transcription factor, transporter,cell growth and structure is1.5-3.0%; howover,6.7%to genes with unclear function and20.2%to genes with no similarity sequences. All TDFs were submitted to GenBank, theacession number is JK711037-JK711046, JK714343-JK714436and JK720971-JK721000.(3) The analysis of qRT-PCR showed that, the expression of TDFs which was identified toP450(21F1), ethylene response factor(22D), heat shock protein(27L3), MYB transcriptionfactor(28F) increased enormously after tea plant by Ectropis oblique feeding3h, and theincrease range of28F is maximum, is336.2times to control, the secondly is22D, then is 21F1and27L3; after feeding6h, the expression of all TDFs decrease quickly, but at48h,the all expression lower than that of control except27L3.(4) According to the TDF of26N, a full length cDNA of SAMT was obtained by3’/5’-RACE(rapid amplification of cDNA ends) techniques from leaves of tea plant. Thesequence length is1542bp, open reading frame (ORF) is1098bp, coding365amino acidsresidues and named as CsSAMT (GenBank: JQ406919), the theoretical molecular weight ofSAMT is41.7kD and PI is5.39; it presents53%highest similarity with the AmSA/BAMTof antirrhinum majus (GenBank: AAF98284) by multiple sequence alignment analysis,belongs to methyltransferase-7superfamily, and presents the same properties of SABATHmethyl transferase; CsSAMT contains conserved domain of methyltransferase-7superfamily by conserved domain analysis, it includes a conservative SAM binding sitesdomain Ⅰ [(V/I/L)(L/V)(D/E)(V/I)g(G/C)g(T/P)g], a conservative SA binding sitesdomain [SSYSVHWLS] and so on.(5) The result of prokaryotic expression showed that the fusion proteins of CsSAMT aremainly located in inclusion body(MW: about59kD), The fusion protein of CsSAMT is stillin inclusion body induced by different temperatures, different IPTG concentration andchanging use expression vector, and the activity of it was not measured.(6) the expression of CsSAMT increased to1.7times to control quickly after tea plant byEctropis oblique feeding3h, but the expression has been always down-regulating after12hand is30-62%to control; after treating with exogenous hormone of MeJA, the expressionof CsSAMT decreased a bit within6h, but at9h, the expression increased to3.0times tocontrol quickly, then the expression has been decreasing all the time.