| Plants in the complex growth environment will be affected by biotic or abiotic stresses such as herbivores,pathogens and mechanical damage,resulting in local damage to plants.Plants have a defensive response to this damage,and the release of C6 green leaf volatiles is one of them[49].Green leaf volatiles include 6 carbon alcohols,aldehydes or esters,etc.,which are secondary metabolites of plant indirect defense against pests and have important biological functions and significance.The ADH gene is a key enzyme in the transformation pathway of green leaf volatiles.Alcohol dehydrogenase have catalytic activity,which can convert C6 aldehyde into C6 alcohol.However,there are few studies on the biological functions of ADH genes in tea plant.In our previous research,the expression level of alcohol dehydrogenase gene(CsADH)was significantly higher than that of the control group after the tea tree was fed.Based on the preliminary work,we selected and cloned two CsADH genes,and verified their functions and expression characteristics.The main findings are as follows:1.Based on the transcriptome data,the full length of the ORFs of the two CsADH genes were cloned from tea trees,which were named CsADH1 and CsADH2,respectively.Phylogenetic analysis showed that the two CsADH genes were highly homologous.2.The recombinant plasmids of the two genes were separately transferred into the E.coli expression strain BL21,and the expression was induced under optimal induction conditions to obtain a fusion protein.Prokaryotic expression results showed that the fusion proteins of the two genes were distributed in the supernatant after disruption,and the SDS-PAGE gel showed that their protein molecular weights were around 80 kDa.Subsequent qualitative analysis by GC-MS showed that the alcohol dehydrogenase has catalytic activity for catalyzing the conversion of C6 aldehyde to C6 alcohol.3.The results of qRT-PCR showed that the expression of CsADH1 was up-regulated at 6h after feeding,which was about 5 times of the control,and the other time points were down-regulated.CsADH2 had no significant change at all time points compared with the control;The expression of CsADH1 was not significantly induced in the treated leaves.CsADH2 was significantly up-regulated at 3h and 12h compared with the control,which was 3.5 times and 5 times higher than the control;The treatment showed significant up-regulation at 6h and 12h,respectively.ADH gene plays an important role in pest stress.In addition,the expression level of CsADH1 in flowers was significantly higher than that in other tissues,and CsADH2 was the highest in the third leaf.4.The function of CsADH1 and CsADH2 was verified in tea plants using gene suppression methods and verified by qRT-PCR.Quantitative results showed that the transcription level of CsADH2 was significantly decreased from 6 to 48 h during infusion incubation compared to control plants.The abundance of cis-3-hexenol in the treated tea leaves was also significantly reduced by GC-MS.This result indicates that CsADH2 may act as a C6 alcohol synthase in tea plants.5.The subcellular localization of CsADHl and CsADH2 was verified by constructing the fusion protein expression vector.The transient expression in Arabidopsis protoplasts was localized by laser confocal imaging,and it was found that CsADH1 and CsADH2 were diffused and localized in the cytoplasm. |
