Isolation And Functional Characterization Of HbWRKY1 Gene From Hevea Brasiliensis

WRKYs are a kind of plant specific transcription factor family, which are involved in the physiological and biochemical processe related to various stresses, aging, et al. Pathogen infection, mechanical wounding and plant hormones (such as ethylene, jasmonic acid, salicylic acid) can induce the expression of WRKY genes. The rubber tree (Hevea brasiliensis) is the main source of natural rubber. To clone the WRKY gene and investigate its potential functions will be very important for our understanding of the stress-tolerance and tapping-related molecular characteristisc in H.brasiliensis. The results are as follows:1. Based on the screening of an ethephon-induced differentially expressed cDNA library from latex in H.brasiliensis, an expressed sequence tag (EST) with high homology to WRKY genes, HbSSH125 was identified. Employing RACE and RT-PCR techniques, a novel member of the WRKY gene family, designated as HbWRKYl (GenBank Accession Number:GU372969), was isolated from H. brasiliensis.2. Bioinformatis analisis showed that the full cDNA of HbWRKYl has 1234-bp and contains a 912-bp open reading frame encoding a putative protein of 303 amino acids. The deduced amino acid sequence shared high identities of 79%,73%and 58% to the WRKY proteins from Ricinus communis (GenBank accession number EEF47235), Populus tremula x Populus alba (GenBank accession number ABK41486) and Glycine max (GenBank accession number ABC26920), in which one highly conserved WRKY domain, one FLYWCH zinc finger domain with two cysteins and two histidines in the C-terminal region were identified. The HbWRKY1 is one of the group-Ⅱmember of WRKY family.3. The 1377-bp 5’regulatory sequence of HbWRKY1 was cloned from leaf genomic DNA of H.brasiliensis by Genome Walking method. Sequence analysis showed that it consists of a typical core promoter region of eukaryotic, several promoter elements such as TATA-box and CAAT-box, as well as many cis-regulatory elements related to hormone and stress responses. It is suggested that HbWRKY1 transcription factor should be involved in the regulation of stress-and hormone-responses in H.brasiliensis.4. The expression patterns of HbWRKYl gene were investigated in H.brasiliensis by semi-quantitative RT-PCR analysis. The results demonstrated that the transcripts of HbWRKY1 were present in all tested organs, but its level was significantly different in different organs. The HbWRKY1 gene was more strongly expressed in latex than in flower, whereas it was weakly expressed in barks, roots and leaves. ET, JA and tapping induced the expression of HbWRKYl in the latex while its expression in the leaves was also strongly induced by drought, ABA and Oidium heveae infection.5. The HbWRKY1-overexpressing plant expression vector, pCAMBIA1304-HbWRKYl, was constructed and 65 transgenic tobacco plants were obtained by agrobacterium-mediated leaf disc transformation method. Drought-stress tests confirmed that the overexpression of HbWRKYl could obviously increase the drought-tolerance of trangenic tobacoo plants, but no obvious differences in morphology were detected between transgenic and nontransgenic plants after tapping or heat stress.Conclusion:The expression of HbWRKYl was constitutively expressed in all tested organs but at different levels. The HbWRKY1 gene was more strongly expressed in latex than in flower, whereas it was weakly expressed in barks, roots and leaves. ET, JA and tapping induced the expression of HbWRKY1 in the latex while its expression in the leaves was also strongly induced by drought, ABA and Oidium heveae infection. The HbWRKYl in H.brasiliensis was a multi-function transcription factor. Drought stress tests showed that the overexpression of HbWRKY1 significantly improved the tolerance of transgenic plants to drought, and the HbWRKYl functions in improving the plant tolerance to drought.