Molecular Cloning, Functional Identification And Expression Characteristic Of The HDR, HDS Gene From MEP Pathway In Camellia Sinensis

Terpenoids are a class of compounds which constructed from their basicstructural unit-isoprene. Terpenoids is an important part of secondary metabolitesin tea plant, and their also plays an important role in indirect defense.4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS),4-hydroxy-3-methyl-but-2-enyl diphosphate reductase (HDR), the two above-mentioned participate inthe last two steps of the MEP pathways which is upstream biosynthesis pathway interpenes metabolism. The HDR is also a critical rate-limiting enzyme of MEPpathway. However, the research around HDS and HDR and its function in tea plantis still unknown. In this paper, we mainly described the cDNA cloning,identification and expression analysis of the HDS, HDR in different organs ofCamellis sinensis. The preliminary results are as follows:1. First, the two EST sequences which have the high homology with the HDS,HDR were isolated from SSH library after Ectropic oblique attack in the leaves ofCamellis sinensis. Then we get HDS, HDR full-length cDNA sequences by usingthe RACE and named for CsHDS, CsHDR. The full length of CsHDS is2603bpwith a open reading frame of2226bp, encoding741amino acids; The full length ofCsHDR is1716bp with a open reading frame of1380bp, encoding459aminoacids. The two gene have been submitted to GenBank and the accession numbersare JQ014629, JQ014628.2. Bioinformatics analysis revealed CsHDS had two conservative GcpEfunctional domains and three active site of conserved cysteine residues like otherHDS in plant; The CsHDR had a conservative LYTB functional domains and fouractive site of conserved cysteine residues The two genes all contained chloroplasttransit peptides.3. Tissue specific expression analysis showed that the CsHDS had the highestexpression in the mature leaves, the expression was3.09folds than the bud; But theCsHDR gene had a high expression in mature leaves, roots, petals, theseexpressions were2.24-2.56folds than the bud; All the two genes had the lowestexpression in young stems, were0.62and0.57fold than the bud.4. The CsHDS, CsHDR expressions were significantly up-regulated and hadthe same up-regulated trend after the Ectropic oblique attack,24h after the feedingthe expression of the two genes get to the highest expression, the expressions were 10.31folds and7.13folds than the control group, the expression of two geneschanged non-significant in48h after feeding; However, under the mechanicalinjury, The CsHDS and CsHDR expression were significantly up-regulated，but theup-regulated folds were less than the changes after after the Ectropic oblique attack;Use Ectropic oblique mouth secretions in5time dilution treated the tea leaves,CsHDS and CsHDR expression were significantly up-regulated in6h after thetreatment, and the values were2.01folds and3.46folds than the control group,respectively.5. We found6h after the treatment which sprayed the tea plant with methyljasmonate (MeJA), The expressions of CsHDS were up-regulated to2.31folds thanthe control group, but later, the expression down-regulated to a similar level thanthe control group; The expressions of CsHDR were significantly higher than thecontrol group;12h after the salicylic acid (SA) treatment can induce the CsHDSand CsHDR expression up-regulated, and the expressions were1.92folds and1.71folds than the control group, respectively, but in other time points after the spraying,the expressions of CsHDS were significantly lower than the control group, and theexpressions of CsHDR had non significantly changes than the control group;Abscisic acid (ABA) induced CsHDS significantly down-regulated, and the theexpression of CsHDR had the same changes except the6h after the treatment;4days After sprayed with recommend dose of imidacloprid, the expressions ofCsHDS were significant up-regulated but CsHDR expressions showed the reveresvariations than the control group.