| In recent years,low temperature and drought stress occured frequently in tea gardens,which have adverse effects on tea production.Therefore,it is necessary to study how tea plant responds to low temperature and drought stress.Phenylpropane synthesis pathway is a very important secondary metabolic pathway in plants.Many secondary metabolites such as anthocyanins,lignin,hormones,flavonoids and plant antitoxins are derived from phenylpropane synthesis pathway.They play an important role in the process of plant stress resistance.Phenylalanine ammonia-lyase is the entrance enzyme and rate-limiting enzyme of this pathway.Therefore,it is of great significance to study what role it plays in the response process of low temperature and drought stress in tea plant.Based on the results of the tea plant genome,we cloned the cDNA and promoter sequences of the CsPAL-a and CsPAL-d members of phenylalanine ammonia-lyase gene family,predicted the encoded amino acid sequences,and conducted bioinformatics analysis on them.The tea seedlings were treated with low temperature(4℃)and drought.The effects of the above two stresses on tea leaves and the expression patterns of CsPAL-a and CsPAL-d were analyzed by measuring the soluble sugar,soluble protein,proline,malondialdehyde,relative conductivity and antioxidant enzyme activity.The specific expressions of CsPAL-a and CsPAL-d were analyzed by Real-time fluorescence quantification and histochemical staining.The function of CsPAL-a and CsPAL-d in model plants(Arabidopsis thaliana)was verified.The main results are as follows: 1.Cloning and sequence analysis of CsPAL-a and CsPAL-dTwo CsPAL genes,named CsPAL-a and CsPAL-d were cloned from tea plant.CsPAL-a contains a 2121 bp ORF,encoding 706 amino acid residues.CsPAL-d contains a 2145 bp ORF,encoding 714 amino acid residues.Sequence analysis showed that both CsPAL-a and CsPAL-d were hydrophilic proteins without any transmembrane domain.The amino acid sequences of CsPAL-a and CsPAL-d have conserved domains of Phenylalanine synthetase.Homology analysis showed that the two CsPALs were highly homologous to PALs in Vitis vinifera,,PopuludL,Durio zibethinus,Daucus carota L..Phylogenetic analysis showed the two CsPALs are closer to Vitis vinifera.2.CsPAL-a and CsPAL-d respond to low temperature and drought stressThe qPCR test results showed that the expression level of CsPAL-a in tea plants was about 7 times that of CsPAL-d under normal growth conditions.After artificially treating the tea trees with low temperature and drought,the expression levels of CsPAL-a and CsPAL-d were significantly increased.The expression of CsPAL-a was up-regulated by about 3 times,and the expression of CsPAL-d was up-regulated by up to 10-fold.3.Expression analysis of CsPAL-a and CsPAL-dTissue-specific expression results showed that CsPAL-a and CsPAL-d were expressed in all tissue parts of the tea tree,with the highest relative expression in the stem,followed by the young bud and the first leaf,and the lowest in the mature leaf.These results showed that CsPAL-a and CsPAL-d had different expression patterns.4.The expression level of CsPAL-a and CsPAL-d increased the resistance to cold and drought stressSensitivity analysis of ABA and JA on overexpressed materials showed that the germination rate of transgenic Arabidopsis seeds decreased compared with wild type,and the sensitivity to ABA increased during germination.Under different degrees(4℃ and-20℃)of low temperature treatment and artificial simulated drought treatment,the results showed that transgenic Arabidopsis lines have stronger cold tolerance and drought tolerance.5.Cloning and functional characterization of CsPAL-a and CsPAL-d promoterStudies on promoter function can clarify the regulatory mechanism of gene expression,which is helpful to analyze gene function.Therefore,this study cloned the regulatory sequences of 1232 bp upstream of CsPAL-a and 1643 bp upstream of CsPAL-d containing common cis-acting elements such as TATA-box,CAAT-box and a large number of typical plant light response elements.In addition,these sequences also contain many specific response elements,such as cold response element,dehydration response element,ethylene response element,auxin response element,stress response element etc.GUS gene was fused into the cloned promoters for genetic transformation and GUS staining analysis was carried out in transgentic arabidopsis.The results showed that all of the two promoters could drive GUS gene in mature leaf,seedings,root,flower expect ilique,indicating that these promoters have tissue expression specificity. |
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