Cloning And Functional Identification Of HbWRKY7b From Hevea Brasiliensis

China is not the traditional area for rubber tree planting,and the rubber tree is affected by various abiotic stresses every year,which often results in the yield decline of rubber tree.The breeding of rubber tree with excellent characters is an important means to solve the problem.But the conventional breeding technology leads to a long period and low efficiency.Therefore,it is an important means to quickly obtain the rubber tree clones with high resistance and high yield by the modern molecular breeding technology.WRKY transcription factors are widely exists in many kinds of higher plants,and regulate various metabolic pathways in plants.So it is significant to study the WRKY transcription factors in the rubber tree.In this study,we cloned the HbWRKY7b gene from the rubber tree and analyzed its function about stress resistance through a series of experiments.The main results are as follows:1.The full length sequence of HbWRKY7b was 1173bp which contained a 792bp ORF,and encoded 263 amino acids.The predicted molecular weight of HbWRKY7b protein was 29.57kD,isoelectric point(PI)was 5.20,HbWRKY7b protein contained two introns,the length was 149bp and 232bp.2 Bioinformatics analysis shows HbWRKY7b protein contained one WRKY domain,which belongs to the class lie WRKY transcription factor.HbWRKY7b protein sequences without signal peptide,also does not belong to the secretory protein;HbWRKY7b protein has relatively strong hydrophilic property,which belongs to water-soluble protein;and there is no transmembrane structure in HbWRKY7b protein,so it is a typical non-transmembrane protein.Functional prediction of HbWRKY7b protein shows its role in transcriptional regulation in plants.3.Analysis of the expression of HbWRKY7b in tissues of rubber tree by using fluorescence quantitative PCR,the results showed that there were significant expression differences of HbWRKY7b between tissues of rubber tree,for example,the highest expression was in the flower,the expressions in leaves and callus took second place,and the expression of HbWRKY7b in bark was barely higher than that in latex,meanwhile,the HbWRKY7b gene almost did not express in the latex.4.With two kinds of cryogenic treatment of rubber tree,the quantitative results showed that HbWRKY7b was able to respond to the low temperature and was up-regulated in two modes;However,the expression of HbWRKY7b in the domesticated experimental group after low temperature acclimation at 10 l℃ was lower than that in the non domesticated experimental group.In addition,HbWRKY7b was up-regulated in response to abiotic stresses such as SA,H2O2,JA,ETH,ABA,drought,PEG and salt.The results showed that the HbWRKY7b gene could be used as a positive regulator in response to abiotic stress and participated in the stress resistance pathway of the plant5.In order to research the function of HbWRKY7b gene in stress resistance pathways of the plant,HbWRKY7b was cloned into plant expression vector pXCS-HAStrep and plant double gene co-expression vector pXCS-Dgene-HAStrep HbWRKY7b,and obtain the co-overexpressing Arabidopsis.Then,analysis of the expression patterns of the stress-resistance-related genes in over-expressing Arabidopsis plants under 1 ℃low-temperature,SA,JA and ABA by fluorescence quantitative PCR method.The results showed that the stress-resistance-related genes obviously upregulated in overexpressing Arabidopsis plants.Using phenotypic analysis research,various abiotic stress tolerance were investigated in transgenic Arabidopsis lines;The results indicated that HbWRKY7b may be a positive regulator of SA and JA signaling pathways in plants,and its overexpression could enhance the tolerance of plants to cold,high salinity,ABA,osmotic and drought stress.