Expression Pattern Analysis Of Three Maize WRKY Transcription Factor Genes

As a significant indicator, to improve maize disease resistance through modern molecular biology techniques is an effective way. Compared to traditional breeding methods of disease-resistant variety, transgenic means is with shorter production term and stronger purpose. Transcription factor (TF) is a kind of DNA-binding protein which can bind to cis-acting elements in gene promoter to ensure mRNA copies of target genes in one particular time and space spot. A large number of studies showed that WRKY transcription factor is involved in multiple metabolic pathways as well as disease resistance response. The three WRKY proteins related to disease-resistant process are cloned through bioinformatic analysis and molecular biology methods, whose change of expression under treatment of Rhizoclonia solani AG1-1A, SA and MeJA was obtained through qRT-PCR. Prokaryotic expression vectors in E.coli BL21were constructed and three fusion proteins were induced successfully by1PTG. A overexpression vector for the WRKY gene of significant differential expression was constructed and introduced into maize through Agrobaclerium tumefaciens EHA105to lay the foundation for identification of gene function. The main results are as follows:1) Primers were designed by Primer Premier5.0according to nucleotide sequences published on NCBI, cDNA of maize inbred line R15leaf sheath treated with Rhizoclonia solani AG1-IA for1h was used as template for PCR. The sequcncing result of WRKY62, WRKY71and WRKY76indicated that nucleotide sequence of the three genes was in accord with sequence in database for98%、93%、100%separately and kept96%,89%,100%similarity on amino acid sequence with no change of protein domain.2) According to the sequencing result and restriction enzyme cutting sits in prokaryotic expression vector pET32a(+), primers were designed to separate CDS using preserved bacterium liquid as template. And then recombinant vectors were constructed after a series of restriction endonuclease reactions, ligation reactions and sequencing validation. In order to find out how concentration of IPTG impact the result, four concentration gradients of0.2,0.5,0.8and1.0mmol/L were set, surprisingly, the three fusion proteins could be induced between45KD and66.2KD compared to control group after the recombinant plasmids were transformed into E.coli BL21, the size of which coincided well with expected value, however, there was almost no difference on quantity of protein between the four IPTG concentrations, concentration of0.2mmol/L was used in follow-on experiments. Solubility analysis of the three fusion protein was operated under two situations, one was to be induced for8h at30℃and the other was for4h at37℃, nevertheless, all of them were in the form of inclusion bodies.3) Maize inbred lines R15and478which are resistant and susceptible to Rhizoctonia solani are experimental materials, the plants without inoculation are control, the samples inoculated with Rhizoctonia solani for1h,2h,4h,6h,12h and24h are experimental treatment. The qRT-PCR result showed that expression of the three genes after inoculation rose considerably at1h, especially that of WRKY71and WRKY76peaked at1h and2h after stress. The expression demonstrated a great difference for WRKY62in R15and478, which rose to peak at24h with a decrease at2h in R15and peaked at12h with almost no change among other time in478. Consequently, we speculated the three genes maybe are positive factors involved in resistance reaction process under the stress of Rhizoctonia solani. In addition, with the treatment of SA, the plants without spraying of herbicides are control, the samples sprayed of herbicides for1h,2h,4h,6h,12h and24h are experimental treatment. The expression of WRKY62and WRKY76were up-regulated while that of WRKY71was up-regulated first and then down-regulated in both maize inbred lines. Under the treatment of MeJA, the expression of WRKY62was up-regulated in478while it had the same trend as WRKY71with the treatment of SA. Both WRKY71and WRKY76were suppressed after stress in478, the expression in R15for WRKY71was up-regulated with a suppression at2h and the latter appeared to be down-regulated first and then up-regulated.4) Overexpression of WRKY76was operated to valid its function. CDS with specific restriction enzyme cutting sites was acquired based on the sequencing result and expression vector pCAMBIA3301. Recombinant vector was constructed and named by pCAMBIA3301-WRKY76which was transformed into maize shoot apical meristems transformation system established with inbred line18-599W by Agrobacterium tumefaciens-mediated method.121resistant seedlings of To generation were obtained after screening with herbicide Basta and3positive transgenic plants were detected by PCR with gene-specific primer designed based on the sequence of35S promoter and WRKY76.