Candidate Gene Association Analysis And Functional Validation Of ZmSAUR15,the Embryonic Callus Induction-Related Gene In Maize

Transgenic technology can shorten the breeding years,break the boundaries between species,and target the precise improvement of genes and other advantages.In the transgenic transformation system,excellent transformation receptors are the basis for successful transformation.The embryogenic callus induced by maize immature embryos is widely used because of its advantages of strong reproductive ability,not affected by seasons,and the ability to efficiently differentiate into complete plants.However,germplasm resources with strong embryogenic callus induction ability are scarce,and the commonly used transformation receptors at home and abroad mainly include American hybrid Hi II,maize inbred lines A188,B104,Zong 31,18-599 R,and Co1 cultivated by Zhongzhong Company.The few maize germplasm resources limit the rapid development and application of transgenic technology.In the early stage of our group,RNA sequencing of the callus of the maize inbred line 18-599R(18R)with the high ability to induce embryogenic callus by immature embryo from Sichuan Agricultural University was conducted,which revealed that the auxin signaling pathway responds to embryogenic is the most significant pathway.Auxin 2,4-D is an exogenous growth hormone commonly used in the culture of maize embryogenic callus.Previous have also reported the important role of 2,4-D in the formation of callus.We speculate that auxin metabolism pathway is an important metabolic pathway in the formation of maize embryogenic callus.This study focuses on the 16 response genes in the auxin signaling pathway in order to find the key genes that control the formation of maize embryogenic callus.Through the investigation of the ability of 309 maize inbred lines to generate embryogenic calli.Candidate gene association analysis methods,as well as Arabidopsis overexpression,maize mutants,maize overexpression,subcellular localization,histological observation and other means to verify the function of candidate genes.The conclusions are as follows:(1)Through the investigation of 309 embryonic callus traits of maize inbred lines under three environments,the results showed that the heritability of embryogenic callus induction rate(ECIR)reached 53.97%.Compared to the research of others,inbred lines with high induction rate and high differentiation rate,such as SCL155 and SCL250 were screened.Based on pedigree and population structure,it is speculated that the ECIR of inbred lines with American hybrid 78599 blood line is generally higher.(2)Among the 16 candidate genes,only ZmSAUR15 has significantly different polymorphisms in extreme materials with high and low induction rates,and there is a significant correlation between polymorphism and ECIR.Furthermore,the candidate gene association analysis was performed on the CDS and promoter regions of the ZmSAUR15 gene,and it was found that both the CDS and the promoter regions had excellent haplotypes represented by SCL155 and bad haplotypes represented by B73.(3)Subcellular localization indicates that ZmSAUR15 is localized in the nucleus.Two haplotypes in the CDS region were used to construct Arabidopsis overexpression vectors.The results showed that ZmSAUR15 both inhibited the formation of callus,and they had the same degree of inhibition,and the content of IAA in wild-type Arabidopsis callus was significantly higher than that of transgenic material,indicating that ZmSAUR15 negatively regulates the formation of embryogenic callus by affecting the synthesis of IAA in the auxin metabolism pathway.After the two CDS haplotypes were overexpressed in Arabidopsis thaliana,the callus phenotype was both inhibited,indicating that the CDS region variation is not the reason for the induction ability of embryogenic callus.Histological observations indicate that ZmSAUR15 may regulate callus formation by regulating cell size and cell expansion.(4)The GUS reporter gene was used to detect the effect of different haplotypes of ZmSAUR15 on the downstream gene driving activity.The results showed that the m RNA and GUS enzyme activities driven by the Pro ZmSAUR15B73 promoter were significantly higher than those driven by the Pro ZmSAUR15SCL155 promoter.And the expression of ZmSAUR15 during callus induction by haplotypes represented by B73 was higher than that by SCL155,and the ECIR of Pro ZmSAUR15B73 haplotype in GWAS population was significantly lower than that of Pro ZmSAUR15SCL155 Haplotype.The high induction rate line represented by SCL155 and SCL250 contains the auxin response original TGTCTC,while the low induction rate line B73 does not contain the auxin response element.It is speculated that ARF can effectively bind to the Pro ZmSAUR15SCL155 promoter to prevent the expression of ZmSAUR15 and make embryonic callus was successfully induced.(5)The callus tissue induced by the Mu mutant of maize ZmSAUR15 is dense,with meristematic regions,and the embryogenic callus are better than W22;overexpression of ZmSAUR15 in maize inbred line 31,compared with the control,the embryogenic callus induction rate of transgenic material was significantly reduced and the callus sprouting rate was reduced,knockout the ZmSAUR15 can improve the embryonic callus induction rate,which further verified that ZmSAUR15 negatively regulates the formation of embryogenic callus.Combined with previous studies and analysis of the previous transcriptome of our group,it was found that Zm ARF29 and Zm ARF20 transcription factors may be negative regulatory factors of ZmSAUR15.