Cloning And Functional Analysis Of HbWRKY7 And HbWRKY11 In Hevea Brasiliensis

Stress-resistance breeding is one of the important objectives in rubber tree breeding. Because of the long breeding cycle of rubber tree and the lack of early prediction means, it is useful to improve the stress resistance of rubber tree by modern molecular biological techniques, so the process of stress-resistance breeding can be accelerated in rubber tree. WRKY is a kind of plant specific transcription factors, which can activate or inhibit the expression of defense genes, and play a crucial role in signal transduction between different signaling pathways. In this study, we cloned MWRKY7 and HbWRKYll by RACE and investigated the function about stress resistance. The main results were summarized as follows:1. The full length sequence of HbWRKY7 was 1196bp containing a 804bp ORF, and encoded 267 amino acid. The deduced protein was 30.11kD with an isoelectric point of 5.79. HbWRKY7 had two intron including 114bp and 197bp sequense. The full length sequence of HbWRKY11 was 1104bp containing a 780bp ORF, and encoded 259 amino acid. The deduced protein was 29.39kD with an isoelectric point of 5.04. HbWRKY11 had two intron including 187bp and 228bp sequense.2. Bioinformatic analysis shows that:HbWRKY7 and HbWRKY11 have one conservative WRKY domain by each, belonging to the WRKY Ⅱd and WRKY Ⅱc family members.The deduced amino acid sequences located in the nucleus and contained no signal peptide, no transmembrane domain and no obvious hydrophobic region. HbWRKY7 and HbWRK11 can regulate gene expression, and HbWRKY11 also as a growth factor in the growth and development of the rubber tree by prodicted.3. The expression of HbWRKY7 and HbWRKY11 in different tissues were assayed by real time fluorescent PCR. The results showed that the tissue-specific expressions of HbWRKY7 were flowers>callus>leaves>stem>latex; HbWRKYll were leaves>flowers=stem>callus> latex; HbWRKY7 and HbWRKY11 were not expressed in latex almost. These showed HbWRKY7 and HbWRKY11 may be involved in the growth and development of the rubber tree.4. In Cold Stress, HbWRKY7 and HbWRKY11 were upregulated significantly, and the increasing trend of HbWRKY7 was more obvious. HbWRKY7 was significantly increased, HbWRKY11 had not obvious change with cold acclimation by 10℃. In response to biotic stress such as ABA、ETH、SA、JA、H2O2、NaCL、PEG and Dry, HbWRKY7 was upregulated, HbWRKY11 also was upregulated except ETH, the upregulated trends of HbWRKY7 was evident than HbWRKY11, leaves was evident than stem. These showed HbWRKY7 and HbWRKYll were induced by adversity and participated in different defense response, Which were stress resistance related genes.5. We successfully constructed an co-expression vector named pXCS-HbWRKY 11-HbWRKY7-HAStrep, which overexpressed in Arabidopsis thaliana. Among transgenic lines, the highest expression levels of HbWRKYll and HbWRKY7 were detected in two single copy lines, S2 and S5. The results showed that HbWRKY11 and HbWRKY7 can express simultaneously, and the expression level of HbWRKY7 was more than that of HbWRKY11, but the difference was not significant. Under 4℃ treatment, the cold related genes such as COR15a、COR47 and RD29A were upregulated in all transgenic Arabidopsis. The trends was HbWRKY7>HbWRKY11> pXCS-HbWRKY11-HbWRKY7-HAStrep relative to WT. In transgenic Arabidopsis, SA related genes like AtPR1 and AtNPR1, JA related genes like AtLOX2 and AtCO11 were upregulated in response to SA and JA. The data further indicated that HbWRKY7 and HbWRKY11 involved in the signaling pathway of SA and JA, and play an active role in the disease resistance in plants.