Bioinformatics Analysis Of Maize CKX Gene Family And Selection Of Cas9 Transgenic Maize Lines

Maize(Zea mays)is one of the three major food and feed crops in the world.It is also the source material for starch industry and green energy production.Therefore,maize is an important crop related to both food security and environmental protection.The demand for corn has increased year by year as the population has grown,livestock farming has developed and the demand for renewable energy has increased.However,due to the reduction of arable land area,climate change and other limitations,corn production has been unable to meet the demand.Cultivating maize varieties with high yield and high stress resistance can promote the increase of maize yield,so it is also the key to solve the imbalance of maize supply and demand.Bioinformatics and gene editing technology have become important techniques for gene function research,and can provide theoretical basis and technical methods for improving the yield trait and stress resistance of maize.Cytokinin oxidase/dehydrogenase regulates the balance of cytokinins in plants by degrading cytokinins,and then regulates the growth and development of plants.At the same time,it is closely related to the stress resistance and yield of plants.CKX is encoded by a multi-gene family,which contains 13 CKX genes in the maize genome.Previous studies mainly focused on Zm CKX1 and Zm CKX10,but lacked comprehensive analysis of the expression patterns of Zm CKX genes in different tissues,developmental stages and stress conditions.In this study,a variety of bioinformatic programs and softwares were used to systematically identify CKX gene in B73 genome.At the same time,the expression patterns of ZMCKX in different tissues,developmental times and conditions were systematically analyzed by using relevant data sets in GEO database.In addition,114Cas9 transgenic maize lines were verified to obtain single-copy,highly expressed transgenic lines for subsequent functional studies,genetic improvement or breeding applications of maize CKX gene and other genes.The main results are as follows:1.Using HMMER program to identify the CKX genes,we found that there are13 Zm CKX genes in the maize genome;Based on the gene ID,genome,GTF3 and CDS information,the 13 genes are distributed on the 7 chromosomes.Except Zm CKX11,the length of the other 12 genes is between 1.9 kb and 4.8 kb.The length of the CDS of the 13 Zm CKX genes ranged from 1.5 kb to 1.7 kb.2.The amino acid sequences of the Zm CKX genes were extracted according to their gene ID.The number of amino acids is between 494 and 582,and the relative molecular mass is between 53.89 and 62.15 k Da.The physical and chemical properties of the proteins were predicted using the online tool Ex PASy,the results showed that the most Zm CKXs are acidic protein.All Zm CKXs are hydrophilic proteins,with the theoretical isoelectric point between 5.31 and 6.61.Six Zm CKXs are stable proteins,other seven are unstable ones.The subcellular localization of Zm CKXs were predicted using Protcomp software,while Zm CKX1 and Zm CKX5 are outside the cell,all the others are located in vacuoles.The motifs of the Zm CKX were analyzed using MEME software.In the same branch of the phylogenetic tree,the type and numbers of motifs are relatively similar,while in different branches with less similarity.The analysis of gene structure had a similar conclusion.3.The MEGA-X software was used to investigate the evolutionary relationship among CKX genes in Arabidopsis,rice,sorghum and maize.The 43 CKX genes in the four species were clustered in 6 subgroups.The 13 Zm CKX genes were distributed in 5subgroups,with one subgroup contained 5 Zm CKX genes,and other subgroups contain1～3 Zm CKX genes.There are four pairs of Zm CKX(Zm CKX2/Zm CKX3,Zm CKX4/Zm CKX4 b,Zm CKX7/Zm CKX8,Zm CKX11/Zm CKX12)are closely related.Subsequent analysis proves that these 4 gene pairs are tandem repeat genes,with the Ka/Ks values less than 1.The JCVI program were used to analyze the collinearity between B73,Mo17,and Zea mexicana(Teo).Results showed that there were 6 pairs of collinear genes between B73 genome and Teo genome,and 8 pairs of collinear genes between B73 genome and Mo17 genome,indicating that there exist some chromosomal location variations between the CKX genes in the B73 and Mo17 genomes.4.The 2000 bp sequence upstream of Zm CKX were extracted and the plant CARE database was used to predict the cis-acting element in the promoter region.Promoter region of the Zm CKX gene mainly contains 7 kinds of cis-acting elements,including anaerobic response element ARE,light response element G-box,GATT-motif,I-box,the hypoxia-responsive original GC-motif,the zein metabolic regulation activation original O2-site,and the core promoter TATA-box.There are differences in the number and distribution of genes.5.The publicly available RNA-Seq data in the GEO database were carefully selected and analyzed,the results showed that the expression patterns of 13 Zm CKX genes were significantly different in different tissues,time and conditions.Under normal conditions,Zm CKX7 and Zm CKX8 were only expressed in male panicles,which showed tissue specificity.Zm CKX9 and Zm CKX11 were almost not expressed in all the tested tissues.Zm CKX2,Zm CKX4,Zm CKX6 and Zm CKX10 were highly expressed in multiple tissues.During seed development,Zm CKX2-6 and Zm CKX10 were highly expressed.Zm CKX6 and Zm CKX10 were highly expressed only at the early stage of seed development,while Zm CKX5 showed a pattern of high expression at the early and late stages and low expression at the middle stages of seed development.At the same time,the expression pattern of Zm CKX gene in embryo and endosperm is different from that in the whole seed.By looking at the expression pattern of embryo,endosperm and seed,it is found that Zm CKX5 and Zm CKX6 genes are highly expressed in the early stage of seed development,but do not occur in the early stage of embryo and endosperm development.Therefore,it is speculated that these two genes are highly expressed in other parts of seeds.Under different stress conditions,the expression pattern of Zm CKX gene changed differently.Under drought and submergation,the expression of Zm CKX1 gene was up-regulated in leaves and ovules,but down-regulated in roots.The expression of Zm CKX4 and Zm CKX4 b was up-regulated in roots,leaves,seeds,ovules and tassel under drought and submersion.The expression of Zm CKX2 gene was up-regulated in taproot,but down-regulated in crown root under 100 nm Na Cl treatment.6.114 T1 generation Cas9 transgenic lines was verified using Cas9 specific primers.For verified true transgenic lines,the Cas9 gene segregation ratio was investigated,and the chi-square test was used to verify whether the segregation ratio met the hypothetic segregation ratio.14 lines with a segregation ratio of 3:1 were determined,which were assumed to be single copy transgenic lines.Meanwhile,some transgenic lines met the segregation ratio of 15:1,and were presumed to be double copy transgenic lines.Because the insertion site of gene may affect its expression,q PCR assay was used to determine the level of Cas9 gene expression in single-copy lines.Real-time quantitative PCR results showed that the line 9T011 may be a singlecopy and highly expressed Cas9 transgenic line and can be used for follow-up research.