Analysis Of Expression Pathways Regulated By OsWRKY28 And Its Role In Bacterial Blight Resistance In Rice

Bacterial blight(BB),which is caused by Xanthomonas oryzae pv.oryzae(Xoo),has been regarded as one of the three Bacterial diseases of rice and it was harmful to the quality and yield of rice when it occurs seriously.Combining the disease resistance mechanism of rice and using new rice breeding technology to develop resistant varieties is the most effective way to control rice blight and other diseases.On the one hand,this paper selects the resist-related genes with negative regulation,and USES CRISPR/Cas9 gene editing technology to carry out gene knockout in rice varieties with good traits,so as to obtain new materials similar to spontaneous mutation.On the other hand,based on the previous research in the laboratory,the function of OsWRKY28 gene in rice resistance to bacterial blight and brown planthopper was preliminarily analyzed by combining transcriptome data.The main results are listed below:1.Using CRISPR/Cas9 gene editing CRISPR/Cas9 gene editing vectors targeting OsPi21 and OsBON1 genes was constructed.Transgenic plants were obtained through agrobacterium-mediated rice genetic transformation.2.The promoter element analysis of OsWRKY28 gene was carried out.According to the analysis on Plant CARE website,it was found that OsWRKY28 contains multiple elements of response hormones such as MeJA,ABA,GA and IAA,among which 4 elements respond to MeJA.In addition,there are TC repeat regions responding to biological stress,low-temperature response elements responding to abiotic stress,and flavonoid synthesis regulatory elements,etc.3.Quantitative PCR results showed that the expression of OsWRKY28 was induced by MeJA,GA and IAA.OsWRKY28 is capable of sensing changes in these hormones and its expression is regulated by these hormones,so it is speculated that it may be involved in MeJA and other mediated resistance signaling pathways.4.Transcriptome results showed that compared with wild-type plants,1,287 genes of overexpressed OsWRKY28 plants were more than twice as differentially expressed,with 807 up-regulated and 480 down-regulated.There were 2,293 differentially expressed genes in oswrky28-cas9 transgenic plants,including 1,594 up-regulated genes and 699 downregulated genes.These genes include disease-resistant and insect-related regulatory genes NPRs and OsLecRKs,key genes related to hormone synthesis,OsICS,OsLOXs,OsEILs and other genes,as well as transcription factor family genes such as OsWRKYs,OsbZIPs and OsNACs.The differential expression of these genes indicates that OsWRKY28 may be involved in hormone-mediated pest resistance signal regulation pathway.5.The inoculation experiment was carried out on the rice plants of the homozygous mutant with T-DNA insertion in the background of Dongjin(Oryza sativa japonica cv.Dongjin,DJ).The results showed that the average disease spot of ordinary DJ rice was longer than that of the rice strains of T-DNA insertion mutant after 14 days of inoculation.For ordinary Japanese fine rice and on the background of Japanese fine OsWRKY28 transgenic expression of material and CRISPR-Cas9 knockout genetically modified materials to bacterial leaf blight fungus inoculation experiment,after 14 days of disease spot statistics,statistical results as follows: disease spot OsWRKY28 over-expression material length,slightly higher than Japan’s shine and knock out after OsWRKY28 material disease spot length is slightly lower than the super express.The results of the inoculation resistance identification of the two materials were consistent,which suggested that OsWRKY28 transcription factor had a negative regulatory role in the resistance of rice to bacterial blight.6.Identification of brown planthopper resistance by DJ and DJ background T-DNA insertion mutants.The preliminary results showed that the T-DNA insertion mutant of OSWRKY28 showed leaf wilting and weak phenotype in the leaves of DJ(control group)and OSWRKY28(experimental group)at the seedling age of 14 days.It was speculated that the resistance to brown planthopper was slightly weaker than that of wild-type DJ material.