Preliminary Study On The Function Of ZmMPK16 And ZmERF9 In Response To Low Phosphorus Stress In Maize

Maize is a very important food,economic and feed crop in the world.Its yield and quality directly affect the food security and life quality of human beings.Low phosphorus abiotic stress has a very significant impact on the whole growth period of maize,which restricts the growth and development of maize seedlings and the yield and quality of maize plants.In the early stage,the laboratory carried out the collection of low phosphorus stress and related phenotypic traits in the maize field seedling stage in the natural population composed of 356 maize inbred lines.In this study,two candidate genes of two traits were further studied.The main results are as follows:1.The candidate gene ZmMPK16(Mitogen-activated Protein Kinase 16)is located at the end of chromosome 2,which is significantly related to the plant height phenotype change(PH-R: Plant-High Pi-deficient/sufficient Ratio)under low phosphorus stress,and the explanation degree of phenotype variation is more than13%.The candidate gene ZmERF9(Ethylene Responsive Factor 9)is located at the end of chromosome 8,which is significantly related to the change of root dry weight under low phosphorus stress(RDW-R: Root-Dry-Weight Pi-deficient/sufficient Ratio),and the explanation degree of phenotypic variation is higher than 8%.2.In order to verify the association sites,the SNP region of the candidate gene ZmMPK16 was amplified and sequenced by PCR using phenotype extreme materials.The results showed that there was an ideal type separation between phenotype extreme materials.The single base variation of A-G in Phosphorus Deficient sensitive materials and transitory materials results in serine proline substitution.The SNP region of ZmERF9 was amplified by PCR and sequenced.The results showed that in addition to the two highly correlated SNPs in the coding region,there were three adjacent small fragment insertion deletions(Indel)in the promoter region,which were significantly correlated with the phenotype.3.Two materials were selected to analyze the expression of ZmERF9 under the stress of phosphorus deficiency according to the difference of indel.The expression of ZmERF9 in the leaves of deletion material was down regulated by the treatment of phosphorus deficiency,but the expression of ZmERF9 in the roots did not change significantly.The expression of ZmERF9 in the leaves of the insertion material increased first and then decreased under the treatment of phosphorus deficiency,and the expression of ZmERF9 in the roots showed a slight upward trend.The indel of the ZmERF9 promoter related site can lead to the change of potential AtGRF7 binding elements,suggesting that ZmERF9 may be related to the expression of zmgrf.Therefore,the correlation between the expression of three GRF genes and ZmERF9 in maize was identified and detected by homology comparison.The results showed that the correlation between the expression of ZmERF9 and GRFs was significantly different in indel.The results showed that the regulation of ZmERF9 gene was different in two different promoter genotypes under low phosphorus stress.The expression of ZmERF9 gene was slightly up regulated by abscisic acid(ABA),ethylene(ETH)and gibberellin(GA3),but not by indole 3 acetic acid(IAA).The results of Maize Tissue expression experiment showed that the expression of leaves was higher than that of roots.Atflowering stage,the expression of supporting root was the highest,and the expression of male flower organ was also relatively high.4.The results of subcellular localization showed that ZmMPK16 was located in the cytoplasm,and there was no difference between the two transcripts.ZmERF9 was located in the nucleus.5.Amino acid sequence homology showed that the similarity between AtMPK16 and ZmMPK16 was the highest in Arabidopsis,and the similarity between AtERF9 and ZmERF9 was the highest.Compared with wild type,the changes of root system related indexes of mpk16 mutants were not significant before and after low phosphorus treatment,while the dry weight of the above ground part of the mutants was significantly reduced under low phosphorus stress,indicating that the deletion of AtMPK16 gene in Arabidopsis had a significant impact on the growth and development of Arabidopsis,Especially the part of the aboveground.The above ground dry weight of erf9 mutant was not significantly changed before and after low phosphorus treatment.However,the ratio of the root length,root surface area,root volume and root tip number of erf9 under low phosphorus stress was lower than that of wild type,which indicated that AtERF9 in Arabidopsis was important for root development,especially under phosphorus deficiency stress.6.The overexpression vectors of ZmMPK16 and ZmERF9 were constructed.The overexpression of maize CO1(KN5585)was mediated by Agrobacterium tumefaciens and identified by PCR.In the next step,we will use transgenic maize to carry out a series of subsequent biological function verification.