Isolation And Function Study Of WRKY Transcription Factors Related To Stress In Hevea Brasiliensis

Hevea brasiliensis is the main source of natural rubber, which is planted in the non-traditional area in China. Low temperature and drought are the main restricting factors of rubber yield and geographic distribution. For Chinese rubber industry, it is important to breed rubber clones with high yield, cold-and drought tolerance. Rubber tree is the perennial woody tree, so it is difficult to breed rubber clones in a few years through conventional breeding way because of its long growth cycle. The rapid development of molecular biology, tissue culture technology and gene engineering technique certainly provide a more promising method for rubber improvement.WRKY transcription factors family, found in the recent twenty years, is one of the largest families of transcriptional regulators. The WRKY proteins play important roles in regulating plant growth and development, response to biotic and abiotic stresses, and they can be induced by pathogenic bacteria, wounding, various abiotic stresses and plant signal material such as SA, JA, ET and ABA. In this study, four HbWRKY genes, named HbWRKY1, HbWRKY2, HbWRKY3, HbWRKY4, respectively, were isolated from rubber tree by RACE technology, on the base of WRKY ESTs from a rubber cDNA library founded in our lab. Their functions were studied by qRT-PCR; plant expression vectors were built and transformed into Arabidopsis thaliana and H. brasiliensis mediated by Agrobacterium tumefaciens. The main results are as followings:1. Isolation and analysis of HbWRKY genes from H. brasiliensisFour HbWRKY genes were isolated from rubber tree by RACE technology. The full DNA of HbWRKY1is3780bp with the composition of four exons and three introns. It contains a2211-bp open reading frame encoding a putative protein of737amino acids. HbWRKY1protein, containing two highly conserved WRKYGQK domains and one C2H2zinc finger domain, belongs to the group I of WRKY family. Interestingly, another sequence, found in leaves, latex and barks, also encoded a protein of737amino acids. We named them HbWRKY1a and HbWRKY lb respectively, and only75amino acids were different between them. HbWRKY1a is79.70389kD and HbWRKYlb is79.6026kD.The full DNA of HbWRKY2is2273bp with the composition of six exons and five introns. It contains a1773-bp open reading frame encoding a putative protein of590amino acids, and belongs to group IIb of WRKY family. HbWRKY2protein is63.9419kD, in which one highly conserved WRKYGQK domain and one C2H2zinc finger domain in the C-terminal region were identified.The full DNA of HbWRKY3is1255bp with the composition of three exons and two introns. It contains a963-bp open reading frame encoding a putative protein of320amino acids, and belongs to group IIe of WRKY family. HbWRKY3protein is36.152kD, in which one highly conserved WRKYGQK domain and one C2H2zinc finger domain in the C-terminal region were identified.The full DNA of HbWRKY4is1009bp with the composition of three exons and two introns. It contains a786-bp open reading frame encoding a putative protein of262amino acids, and belongs to group IIa of WRKY family. HbWRKY2protein is28.8086kD, in which one highly conserved WRKYGQK domain and one C2H2zinc finger domain in the C-terminal region were identified.2. Cloning and analysis of promotersThe promoter length of HbWRKY1was2319bp, in which there were W-boxs, responsed to heat stress element (HSE), TGA-element and other cis-elements. The promoter region of HbWRKY2was1276bp, in which there were W-boxs,. HSE, TCA-element and other cis-elements. The promoter region of HbWRKY3was1068bp containing W-boxs, wound stress motif (WUN-motif), ABRE, LTRE and other cis-elements. The promoter region of HbWRKY4was708bp, in which there were W-boxs, ABRE, LTRE and other cis-elements. It was indicated that HbWRKY transcription factor should be involved in the regulation of stress-and hormone-responses in H.brasiliensis.3. Functions of four HbWRKY genesTissue specific analysis showed that the highest relative expression level of HbWRKY genes was in rubber flower. The expression of HbWRKY1in rubber tree was induced by low temperature and ABA. HbWRKY2was in response to drought, PEG and cold stresses, and was induced by SA at early stage in rubber leaves; the expression of HbWRKY2was induced by high salt, PEG, cold, ET in rubber tree barks. HbWRKY3in rubber leaves was in response to PEG, high salt and low temperature stresses, and was rapidly induced by MeJA, SA, ABA and H2O2at early stage; the expression of HbWRKY3was induced by drought, PEG, low temperature, MeJA, SA and ET t in rubber tree barks. The expression of HbWRKY4in rubber tree leaves was in response to drought, PEG and low temperature stresses, and rapidly induced by MeJA, SA, ABA, H2O2and ET at early stage; the expression of HbWRKY4was induced by high salt, ET and cold, and sharply increased by cold treatment in rubber tree barks.HbWRKY2over-expression enhanced the ability of drought and salt tolerance in A. thaliana; In HbWRKY2over-expressed Arabidopsis, the expression of AtRD22and AtRD29A were increased in high salt treatment; the expression of AtNPRl and AtPR1was highly increased in SA and MeJA treatment; the expression of AtLOX2was highly increased in MeJA treatment. It indicated that HbWRKY2played a role in SA and MeJA signal pathway, maybe involved in resisting to pathogenic bacteria. The expression of cold-related genes AtRD29A, AtCOR15A and AtCOR47was induced in cold treatment, on the other side, over-expression of HbWRKY2increased the ability of Arabidopsis to resist cold stress by increasing proline and soluble sugar content in physiological.HbWRKY3over-expression enhanced the ability to resist to drought in A. thaliana. The expression of AtERS1and AtETR1was rapidly induced by ET in HbWRKY3over-expressed Arabidopsis.HbWRKY4over-expression enhanced the ability of drought and salt tolerance in A. thaliana. The expression levels of AtRD22and AtRD29A were increased in high salt treatment in HbWRKY4over-expressed plants. HbWRKY4over-expression also increased the expression of cold-related gene AtCOR15A and AtCOR47under low temperature stress.4. Genetic transformation of H. brasiliensispCAMBIA2301was as a plant expression vector, and HbWRKY2gene was transformed into embryogenic calli of CATAS7-33-97and CATAS8-79medited by Agrobacterium strain EHA105. To investigate the effect of co-cultured time on efficiency of genetic transformation, the frequency of stable expression of GUS gene (uidA) was as an index. The results showed that5d and6d were the optional co-cultured time for CATAS7-33-97and CATAS8-79, respectively. Four resistance calli lines from CATAS7-33-97and CATAS8-79were obtained and identified by PCR and GUS staining.The bar gene was used as a selecting marker, and bialaphos was as screening reagent, HbWRKY4was transformed into embryogenic calli of CATAS8-79mediated by Agrobacterium strain GV3101::pMP90RK. The results showed that the optimal screening concentration of bialaphos was3mg/L. In the study, three resistance embryogenic callus lines,234somatic embryos were acquired and22regeneration plantlets were obtained. The PCR analysis showed that all the regeneration plantlets were positive individuals, the exougenous HbWRKY4was indentified in all plantlets. Southern blotting analysis of13positive plantlets showed that the bar gene with low copies as a foreign gene had been successfully integrated into the genomic DNA of the transgenic plantlets of rubber tree. It was demonstrated that bar gene with bialaphos can be used as an effective screening system in Hevea genetic transformation. The Agrobacterium-mediated genetic transformation method with bar gene as a selecting marker was preliminarily established.