| Maize(Zea mays L)with the largest planting area is one of the main food crops in China,which occupies an important position in China's food security.The occurrence and prevalence of maize gray leaf spot disease limit the production of maize in China.At present,the genetic mechanism of resistance to gray leaf spot in maize is still completely clear,and maize's resistance genes to to gray leaf spot still needs to be further explored and identified.In view of this,This study selected the high resistance to gray spot disease inbred line T32 and the high-sensitivity gray spot disease inbred line Jiao51 as test materials,Through high-throughput sequencing technology,transcriptome analysis of infection and uninfection anti-sensing materials was carried out to systematically study different expression genes(DEGs)between the materials and the related biological processes and metabolic pathways involved before and after being stressed by maize gray leaf spot disease pathogens Combining with the results of fine-mapping results in the previous study,we screened and cloned important candidate genes for resistance to gray leaf spot in maize,and analyzed the expression of candidate genes,functional site identification and bioinformatics analysis on the candidate genes,Hopefully,the present research will lay the solid foundation for the molecular marker-assisted breeding and genetic improvement of corn gray spot disease resistance.The main results are as follows:1.With self-intersecting T32 and Jiao51 as test materials for field natural bacteria and non-bacteria treatment,set up three biological repetitions,in the sampling period of spiked leaf sampling and sequencing,A total of 12 c DNA libraries were established to obtained 533.2 Million Clean Reads.Compared with the control,the number of genes was increased by 3187 and reduced by 1658.Compared with the control,the number of genes was lowered by 1608 because there were 2923.GO functional annotation analysis of differential expression genes was found to be mainly involved in biological processes such as metabolic processes,cellular processes,catalytic activity,bio-modulation and stimulating reactions.KEGG analysis of the metabolic pathways involved in differential genes showed that differential expression genes were mainly concentrated in biosynthesis,metabolic pathways,phenyl-propane biosynthesis,amino acid biosynthesis,carbon metabolism,microbial metabolism in different environments,biosynthesis of antibiotics,plant-pathogen interactions,etc.Through further analysis of plant hormone signal transduction and plant-pathogenic bacteria interoperability pathology,it was found that there are many different expression genes associated with the four hormone signaling pathogens,pathogens of shedding acid,jasmine acid,growth hormone and cytosin,and many related protein genes involved in plant-pathogen interoperability,all of which may be involved in the regulation of maize response to gray spot disease.2.In the early stage of the research group,the main QTL for gray spot disease resistance,q GLS8,was located in the physical range of about 124 kb on the eighth chromosome of maize,and the expression patterns of 7 genes in the finely mapped range were analyzed by using transcriptome sequencing data and found that there are two This gene is different between T32 and Jiao51.The expression level of Zm00001d008808 in T32 increased by 4.21 times,and the difference multiple was1.579.After inoculation in T32,the expression level of Zm00001d008812 decreased by 7 times,and the difference in expression level was 3.3,The other genes were not expressed or expressed very low,combined with gene annotation,the gene Zm00001d008808 was predicted as a candidate gene for resistance to maize gray leaf spot disease.3.Through cloning the gene Zm00001d008808 were obtained a total length of2235 bp of the gene sequence,the gene sequence of the resistance material is 99.6%and a total of 7 base mutations.Amino acid sequence prediction found that the gene encoded a total of 375 amino acids,amino acid sequence esotericity was 99.2% in the exon region of 5 mutated bases,Three amino acid mutation sites were found,,the first was in the 68 th place in T32.In T32,the line(L)Jiao51 was proline(P),the second was in the 250 th bit,T32 suline(T)Jiao51 was alanine(A),the third was in 362 bits,T32 proline(P),Jiao51 serine(S).4.Through the expression of Zm00001d008808 gene in different tissues of fluorescence quantitative analysis,it was concluded that the candidate gene was mainly induced by a large number of resistant material blades after inoculation,followed by the Tassel,where the relative expression of the resistance material leaves was 3.3 times the relative expression of the disease-sensing material leaves,showing a significant difference.The prediction of subcellular location shows that the gene was mainly located in the nucleus and the Chloroplast matrix,and the physical and chemical properties prediction indicates that the gene belongs to an unstable protein with an isoelectrine(p I)value of 10.12 and a protein hydro hydro-hydrogenic average of-0.343.5.Candidate genes were cloned and sequenced in six materials susceptible to maize gray leaf spot,and two important functional sites were found through amino acid multi-sequence alignment.The first was at position 68,and T32 was leucine(L).The other seven materials were all proline(P).The second was at position 362,and T32 was proline(P).The other seven materials were all serine(S).it can be speculated that these two amino acid mutation points may be causing Zm00001d08808 to produce resistance to the important point. |
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