| Zizania latifolia is a perennial shallow water herb of the Gramineae,which is the second largest aquatic vegetable in China after lotus root.The formation of the fungus gall is closely related to the infection of its endophytic fungus.At present,the mechanism of Z.latifolia in response to the infection of Ustilago esculenta is unclear.Mitogen-activated protein kinase is located at the lowest downstream of the MAPK cascade pathway,and when receiving the signal transmitted upstream,it can phosphorylate the corresponding downstream substrates such as protein kinases,cytoskeletal proteins and transcription factors,so as to realize the regulation of plant growth,development and stress.The research group had completed the inoculation of male Z.latifolia with U.esculenta in the early stage,and RNA-seq sequencing analysis found that “MAPK signaling pathway-plant” is an important pathway for male Z.latifolia to respond to U.esculenta infection,and MPK3 is an important candidate gene.In order to further study the characteristics of the MAPK family gene and its role in responding to biological stress,this study screened and identified the members of the MAPK gene family,and analyzed the structure,conserved domain,cis-acting element of the promoter region,collinearity,etc.of the family members,as well as the expression pattern of U.esculenta infection.In addition,the ZlMPK3 gene(a candidate gene in response to the infection of U.esculenta)was cloned,and transformed into Arabidopsis thaliana mediated by Agrobacterium.The ZlMPK3 overexpression plants were identified,and its response to Botrytis cinerea infection was studied.The main findings are as follows:1.Through homologous alignment and domain validation,25 ZlMPKs(ZlMPK1-ZlMPK25)were identified from the genomic data.Phylogenetic tree analysis showed that25 ZlMPK proteins belonged to four subgroups A,B,C,and D.There were 10 ZlMPK proteins in the three subgroups A,B and C,all of which had TEY conserved motifs.The D subgroup had 15 ZlMPK proteins with TDY conserved motifs.The 25 ZlMPKs were unevenly distributed on 11 chromosomes,and many ZlMPKs were localized on the cytoplasm.The cis-acting elements prediction showed that ZlMPKs were involved in hormone signal transduction and stress.Collinear analysis showed that gene fragment duplication and gene tandem replication events were prevalent in these genes.2.Inoculation of 5-leaf male Z.latifolia with U.esculenta,and the leaf RNA at different time points after inoculation were extracted.Expression of ZlMPKs were detected by quantitative real-time PCR.The results showed that the expression level of most ZlMPKs changed,among which,21 ZlMPKs showed up-regulated expression,while the other 4 genes were down-regulated expression.Additionally,9 out of 11 MAPK-Pathogen infection pathway related genes were up-regulated 3 h after inoculation with U.esculenta.3.The ZlMPK3 gene was cloned,and p CAMBIA1300-ZlMPK3 overexpression vector was constructed,and transformed into A.thaliana mediated by Agrobacterium.The results showed that the c DNA length of the ZlMPK3 gene was 1110 bp.PCR detection and sequencing analysis showed that the p CAMBIA1300-ZlMPK3 overexpression vector was successfully constructed.Resistance screening and molecular detection showed two overexpressed plants were obtained.4.The overexpressed A.thaliana were inoculated with Botrytis cinerea.Phenotypic observation revealed significant leaf damage at the inoculation site of overexpressed plants,while the leaves of wild-type plants were normal.The total RNA of leaves was extracted at24 h after inoculation,and the expression of MAPK-pathogen infection pathway related genes were detected by quantitative real-time PCR.The results showed that the relative expression of At ACS6 and 5 CAP genes were significantly up-regulated in overexpression plants,while At PR1 and At FRK1 were significantly down-regulated.The above results showed that the resistance of overexpressing plants was weakened to Botrytis cinerea infection. |
PDF Full Text Request