| Maize(Zea mays L.)is one of the most important food,feed,chemical raw materials and energy crops in the world.Cultivating new maize varieties with high yield,high quality and strong stress resistance is an important way to promote the development of maize industry.With the rapid development of transgenic and gene editing techniques,genetic engineering techniques which depend on genetic transformation,are playing a more and more important role in maize breeding.Maize immature embryos are the most commonly used receptors for maize genetic transformation,because of their strong ability of regeneration in tissue culture.The genetic transformation mediated by Agrobacterium tumefaciens has been widely used in the genetic transformation of maize immature embryos because of its low copy number of exogenous genes and strong genetic stability of progeny lines.However,the transformation efficiency of Agrobacterium tumefaciens was significantly different among different maize genotypes.At present,only a few maize inbred lines such as A188,B104,C01 and Zong31 showed high transformation efficiency on being infected by Agrobacterium tumefaciens.Most of maize elite lines are not sensitive to be infected by Agrobacterium tumefaciens and thereby can not be used as transgenic receptor materials.Therefore,revealing the molecular mechanism underlying Agrobacterium tumefaciens infecting maize immature embryos,identifying the major genes regulating this process and applying these to the improvement of elite inbred lines can accelerate the efficiency of maize genetic engineering breeding.The main results are as follows:1.With Agrobacterium tumefaciens infection efficiency of extremely low inbred line18-599 R and extremely high inbred line A188 as the research objects,transcriptome sequencing(RNA-seq)analysis showed that 18-599 R and A188 responded to Agrobacterium tumefaciens infection gene(CLvs CK)12529 and 5626,respectively.Among them,the specific response genes of 18-599 R and A188 were 8483 and 1580,respectively,and the common response genes were 4046.The common response genes of the two materials are mainly involved in response to stress,response to biotic stimulus,response to endogenous stimulus,response to biotic stimulus,cell communication and other biological processes related to plant-pathogen interaction.2.According to the differential gene analysis of Agrobacterium tumefaciens infection process between the two materials(A188vs18-599R),a total of 8291 genes(including 848 transcription factors)were significantly up-regulated and 3450 genes(including 203 transcription factors)were significantly down-regulated.The number of up-regulated genes was significantly more than that of down-regulated genes,indicating that the high expression of a large number of genes in A188 promoted the infection of Agrobacterium tumefaciens.The enrichment of KEGG pathway of differentially expressed genes between the two materials identified the rich biological processes in which they participated.3.18-599 R and A188 identified 11 and 14 gene co-expression modules of the genes in response to Agrobacterium tumefaciens infected,respectively.The significant enrichment biological process of genes in each module were identified by KEGG analysis.Through the correlation analysis between module eigenvalues and samples,seven stage-specific co-expression modules were identified by the two materials.The gene regulatory networks of these stage-specific co-expression modules were constructed,and 70 stage-specific key genes were identified.4.The phenotypic values of immature embryo infection efficiency(e GFP relative expression level)of 340 maize inbred lines in GWAS population were collected,and 186 inbred lines were successfully infected by Agrobacterium tumefaciens,and the phenotypic values were used for GWAS analysis.A total of 30 SNPs significantly associated with infection efficiency phenotype were identified in the three infection stages of the two environments,and a total of 315 candidate genes were identified in the upstream and downstream 220kb(LD)regions.In addition,combined with the materials with high infection efficiency of Agrobacterium tumefaciens identified in this study and 144 materials with strong callus induction and differentiation ability identified in the previous study,41 inbred lines could be directly used as excellent transgenic receptor materials.The germplasm resources of transgenic receptors in maize were broadened.5.Based on the results of GWAS and RNA-Seq analysis,specific responses of 73 and17 GWAS candidate genes were identified in transcriptase 18-599 R and A188,respectively.These 90 genes were identified as key candidate genes regulating agrobacterium infection efficiency phenotype.Six genes were randomly selected for q RT-PCR validation in common transgenic maize receptor materials 18-599 R,A188,C01,B104 and B73.The results showed that these key candidate genes regulated agrobacterium infection in young maize embryos.Combining the results of GWAS and RNA-Seq analysis,among the GWAS candidate genes,there are 73 and 17 genes were identified to respond specifically in 18-599 R and A188 in transcriptome analysis,respectively.These 90 genes were identified as key candidate genes regulating the phenotype of Agrobacterium tumefaciens infection efficiency.Six of these genes were randomly selected for q RT-PCR verification in maize transgenic receptor materials 18-599 R,A188,C01,B104 and B73.The results showed that these key candidate genes regulated the infection of maize immature embryos by Agrobacterium tumefaciens.6.The functional verification results of ZmHRGP,a key candidate gene identified by GWAS and RNA-Seq,showed that the deletion of ZmHRGP reduced the content of lignin in the cell wall and reduced the defense function of plants,thus improving the efficiency of Agrobacterium tumefaciens transformation of maize immature embryos. |
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