Cloning And Functional Analysis Of HbWRKY8 Transcription Factor In Hevea Brasiliensis

Rubber tree is a perennial tropical crop,belonging to the family Euphorbiaceae.It is the most economically important member of the genus Hevea because the milky latex extracted from the tree is the primary source of natural rubber.However,compared with the traditional rubber tree planting area,the yield of rubber tree in China is quite low,which might be caused by environmental restrictive factors:typhoon,low temperature,drought stress and disease.The natural rubber production is difficult to improve by expanding the scale of planting because of the narrow suitable environment.Improving the level of rubber tree per unit yield by breeding the high yield varieties is one of the effective ways.However,breeding new varieties of rubber tree is diffcult,due to a long cycle of sexuality of rubber tree.Therefore,the use of molecular biotechnology to explore the potential resistance of rubber trees is an effective method to breeding for abiotic stresses.The plant specific WRKY transcription factor is one of the largest TF families and WRKY Transcription factor are a class of DNA_binding proteins primarily found in plants and lower plants(with some exceptions),which is involved in diverse plant processes,such as growth,development and stress signaling through auto and cross regulation with different genes and transcription factors.In this study,we cloned a rubber tree WRKY transcription factor,named HbWRKY8,by RACE and investigated the expression patterns under specificity about abiotic stresses.Furthermore,the possible biological functions of HbWRKY8 were investigated by overexpression of HbWRKY8 in Arabidopsis thaliana plants.The main results were summarized as follows:1.The full length sequence of HbWRKY8 was 11241 bp containing a 915bp ORF,and encoded 304 amino acids.The deduced protein was 34.07kD with an isoelectric point of 5.86.A total of two introns were found in HbWRKY8,which 207bp and 128bp,respectively.2.Bioinformatic analysis showed that:HbWRKY8 had one conservative WRKY domain,belonging to the WRKY II c family members.,which can regulate gene expressions as a signal transduction factor in the rubber tree predicted.The deduced amino acid sequences located in the nucleus and contained no signal peptide,no transmembrane domain and no obvious hydrophobic region.3.The expression analysis of HbWRKY8 in rubber tree by fluorescence quantitative PCR showed that there were significant expression differences of HbWRKY8 among different 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 in bark was barely higher than that in latex,meanwhile,the HbWRKY8 gene almost did not express in the latex.These results indicated HbWRKY8 may be involved in the growth and development of the rabber tree.4.Under drought,salinity,PEG,and phytohormone(ABA,ETH,SA,JA,H2O2)stress treatments in the rubber tree,the up-regulation trend of HbWRKY8 in leaves is evident than in stem.The results showed that the HbWRKY8 gene could be used as a positive regulator in response to abiotic stress and participated in the stress in rubber tree.5.In order to research the function of HbWRKY8 on stress resistance of the plant,HbWRKY8 was cloned into a plant expression vector pCAMBIA2301 MSP-HbWRKY8 and overexpressed Arabidopsis.Based on phenotypic analysis research,various abiotic stress tolerances were investigated in transgenic Arabidopsis lines;the results indicated that HbWRKY8 may be a factor regulated to the signal transduction mechanism of plant salt stress,and its overexpression can enhance the sensitivity of plants under salt stress.