Identification Of The ADH Gene Family In Tea Plant And Analysis Of The Expression Pattern Of CsADH17 Gen

Alcohol dehydrogenase(ADH,EC 1.1.1.1)is an oxidoreductase with a highly conserved domain and is a member of the dehydrogenase/reductase(MDR)protein gene superfamily.Human Chase(1999)divided the plant ADH family into several subfamilies such as ADH0,ADH1,BAD1,CAD1,CAD2,FDH1,KELa,TRR1,and TRR2.ADH exists widely in organisms,and generally uses NAD~+and NADP~+as coenzymes,and plays an important role in plant growth and development,resistance to abiotic stresses such as drought,waterlogging,chilling,hypoxia and salt damage.Tea tree(Camellia sinensis(L.)O.Ktze)is an important economic crop in my country.A good ecological environment is the main factor affecting the normal growth of tea trees and the quality of tea leaves.This kind of adversity stress greatly affects its yield and quality.However,there are few studies on the tea tree ADH family and its family genes related to stress in tea tree.This study focuses on the tea tree ADH gene family,and conducts bioinformatics analysis of the basic biological information(including phylogenetic evolution)of the tea tree ADH gene family members.tree,conserved domains,protein motifs,chromosomal localization,abiotic stress expression pattern analysis,etc.).By selecting genes with high expression levels in the gene family,designing gene primers,and performing real-time fluorescence quantitative analysis,it is verified whether the results are related to the predicted results of the genome database.The CsADH17 gene was cloned and genetically transformed into tobacco,and the expression level and related physiological indicators of transgenic tobacco under abiotic stress were measured and analyzed to understand the way the related genes of the tea tree ADH gene family were regulated in tea tree.The main research results are as follows:1.Identification and analysis of CsADH gene familyUsing the BLAST function on TBtool,51 tea tree ADH gene family members were screened and their physicochemical properties were predicted.The tea tree ADH gene family was analyzed by bioinformatics method,and the 51 tea tree CsADH genes were divided into two groups according to their structural domains.The second group had the most members,including 41 CsADH members,and the first group had 10 CsADH members.Gene structure and conserved motif analysis found that most genes were highly conserved and had great similarity to each other.From the analysis of expression patterns in eight tissues of tea plant,it was found that most CsADHs were expressed in various tissues,and the expression level in young leaves was higher than that in other tissues.The expression patterns of different abiotic stresses indicated that the CsADH genes of tea plant were all involved in the regulation of tea plants under abiotic stress,and had different degrees of response under different abiotic stresses.2.Quantitative expression analysis of five CsADH genesThe expression patterns of five genes(CsADH17,CsADH23,CsADH26,CsADH35,CsADH44)in tea plants under drought and low temperature stress were analyzed by real-time fluorescence quantitative analysis.Under low temperature treatment from 0to 24,the expression of CsADH17 gradually increased over time,and the expression of CsADH23,CsADH26 and CsADH35 first decreased and then increased.It is predicted that the expression of these two genes will gradually increase if the low temperature stress is continued.,while the expression of CsADH44 was the highest at the beginning and then decreased;in the 0-72 h of drought treatment,the expression of CsADH17 was the highest at 48 h,and the expression of CsADH23,CsADH26 and CsADH35 first decreased and then increased,and then increased at 72 h.When the expression level was the highest,the expression level of CsADH44 generally trended from high to low.Based on the expression patterns in tea plants under drought and low temperature stress,the functional verification of CsADH17 gene in tobacco was selected.3.Functional verification of CsADH17 gene in transgenic tobaccoIn order to identify the function of CsADH17 gene in plants,the overexpression vector p BWA(V)KS-CsADH17 of tea CsADH17 gene was constructed.GUS histochemical staining and DNA extraction were performed for PCR identification.The results showed that GUS activity was not detected in wild-type tobacco plants,but GUS activity was detected in transgenic tobacco plants,and the target band was not detected in wild-type plants,while the expected target band was detected in transgenic plants.Finally,CsADH17 transgenic tobacco was obtained.plant.Drought stress treatment of wild-type tobacco plants and CsADH17 transgenic tobacco plants showed that the relative expression level of CsADH17 transgenic tobacco plants was higher under drought stress treatment than wild-type.The results of the determination of antioxidant enzyme activities in wild-type tobacco plants and CsADH17 transgenic tobacco plants after 21 d of drought stress treatment showed that the activities of CAT,POD,SOD and APX in leaves of CsADH17 transgenic tobacco plants were all high after 21 d of drought stress treatment.in wild-type tobacco plants.At the same time,the MDA content of CsADH17 transgenic tobacco leaves was significantly lower than that of wild-type tobacco,indicating that the drought stress resistance of CsADH17 transgenic tobacco plants was significantly improved.By GC-MS determination,the CsADH17 gene increased the content of six aroma components in transgenic tobacco,including nicotine,neolipadiene,benzyl alcohol,benzaldehyde,dihydrokiwifruit lactone and diene nicotine,and enhanced the consumption of tobacco leaves.Taste and aroma,while reducing the irritation and bitterness of tobacco leaves,making the aroma in tobacco leaves softer.