| WRKY transcription factor family,one of the largest families of transcriptional regulators,is newly discovered in the recent twenty years,and they play important roles in plant growth and development and responses to abiotic and biotic stresses.In this study,two HbWRKY genes,designated HbWRKY27 and HbWRKY57,were cloned from Hevea brasiliensis(renyan7-33-97),and their expression characteristics were analysed.Furthermore,the possible biological functions of HbWRKY57 were investigated through transformation of HbWRKY57 gene into Arabidopsis thaliana.The main results are as followings:1.The full length of the cDNA sequences of two WRKY transcription factor genes,HbWRKY27 and HbWRKY57,was obtained from the latex of rubber trees according to the latex transcriptome database.2.Bioinformatics analysis showed that the HbWRKY27 gene contained an open reading frame(ORF)of 1,266 bp encoding a protein with 422 amino acid residues and the proportion of G and C was 48.82%;and that the HbWRKY57 gene contained an ORF of 921 bp encoding a protein with 306 amino acid residues and the proportion of G and C was 49.84%.Both the HbWRKY27 and the HbWRKY57 proteins had a typical WRKY domain and a C2H2 zinc finger motif,and belonged to the class II protein of the WRKY transcription factor family.3.The expression patterns of the HbWRKY27 and HbWRKY57 genes were investigated using real-time quantitative PCR(RT-qPCR).In different tissues of rubber trees including root,bud,bark,xylem,petiole,leaf and latex,the expression level of HbWRKY27 gene was the highest in the bark but the lowest in the bud;while the expression level of HbWRKY57 was the highest in the xylem but the lowest in the leaf.In different development stages of leaf including leaf bud,bronze,discoloration,light green,stable,early senescence,mid-term senescence and late senescence stages,the expression level of HbWRKY27 gene was the highest in the bronze period but the lowest in the discoloration period while the expression level of HbWRKY57 gene was the highest in the late senescence period but the lowest in the discoloration period.Ethrel,methyl jasmonic acid(MeJA)and bark tapping(a mechanical damage)can significantly up-regulate the expression of both HbWRKY27 and HbWRKY57 genes in the latex of rubber trees.However,in the latex and the bark tissue of rubber trees suffered from tapping panel dryness(TPD),different expression patterns were found between the HbWRKY27 and HbWRKY57 genes.In the latex of rubber trees with TPD,the HbWRKY27 gene was remarkably induced while the HbWRKY27 gene was lowered in mRNA abundance;whereas in the bark tissue of rubber trees with TPD,the HbWRKY27 gene was significantly down-regulated while the HbWRKY27 gene was greatly up-regulated in their mRNA levels.4.The subcellular localization of HbWRKY27 and HbWRKY57 was performed by transgenic Tabacoo using the plant expression vectors pCAMBIA1302-HbWRKY27 and pCAMBIA1302-HbWRKY57.Tabacco plants were transformed by agrobacterium,and the results demonstrated that both HbWRKY27 and HbWRKY57 were located in the nucleus of the leaf cells.5.Yeast two-hybrid assay was conducted to detect the activities of transcriptional activation using the fusion expression proteins pGBKT7-HbWRKY27 and pGBKT7-HbWRKY57.The results showed that both HbWRKY27 and HbWRKY57 had transcription activating domain and could activate the expression of report gene through combining with the GAL4 DNA-binding domain.6.The biological functions of HbWRKY57 were further studied using the transgenic A.thaliana plants,which overexpressed HbWRKY57 gene in T3 plants.Compared with the wild type Arabidopsis plants,the transgenic plants had not only stronger vitality and ability to resist stresses,but had a longer lifetime with an earlier blooming. |
