Functional Analysis Of Maize ZmSKIP In Response To Drought Stress

Maize is not only the world’s main food and feed,but also an important industrial raw material.Drought is the most important abiotic stress factor affecting maize production,so it is of great significance to mine drought-tolerant genes and apply them to molecular breeding.In the early stage of the research group,a drought-related QTL was located through genetic linkage,and the drought response gene ZmSKIP was selected by combining gene expression data.Studies in Arabidopsis have shown that the SKIP gene,as a component of the spliceosome,plays an important role in the response to plant stress response by participating in pre-mRNA alternative splicing and gene expression regulation.In order to study the role of SKIP in maize response to drought stress,this study first analyzed the spatial and temporal specificity of ZmSKIP expression,and found that its expression in drought-tolerant inbred lines and drought-sensitive inbred lines was significantly different.Therefore,the promoter sequences of the ZmSKIP gene in the corresponding materials were cloned separately,and transformed tobacco and Arabidopsis thaliana were analyzed by GUS histochemical staining and fluorescent quantitative PCR to initially verify the function of their specific promoters.To study the function of ZmSKIP,the full-length cDNA sequence of ZmSKIP gene was cloned and transformed into tobacco leaves for subcellular localization of the target gene.Construct ZmSKIP overexpression vector,transform Arabidopsis thaliana and skip-1mutants at the same time,and carry out genetic transformation in maize,compare the phenotype difference between transgenic positive plants and wild type under different water conditions,and preliminary analyze the function of ZmSKIP,transcriptome sequencing analysis of transgenic overexpressed maize.The main results are as follows:1.ZmSKIP gene qRT-PCR tissue expression analysis showed that: ZmSKIP is expressed in various tissues of maize,with the highest expression level in the ear of the ear.The drought-tolerant and drought-sensitive maize inbred lines treated with20% PEG and the expression level of ZmSKIP gene were found.In drought-tolerant materials,the expression of ZmSKIP was significantly increased after stress-induced,and in different tissues The expression level in inbred lines was significantly higher than drought sensitive inbred lines.Analysis of the response of ZmSKIP gene under different hormone treatments showed that ZmSKIP can respond to the treatment of ABA and ETH,but does not respond to the processing of GA and IAA.2.The ZmSKIP promoter sequence(p-ZmSKIP-P1: 1393 bp,p-ZmSKIP-P2:1233bp)was cloned from the drought-tolerant material and drought-sensitive material,respectively.MYB and many other cis-acting elements related to stress response and light response.3.The promoter of ZmSKIP can respond to abiotic stress.The promoter of ZmSKIP was fused with the GUS reporter gene.Using the tobacco leaf transient expression system,GUS staining results showed that the promoter sequence of ZmSKIP had transcriptional activity,and the promoter activity of drought-tolerant material was significantly higher than that of drought-sensitive material.The results of GUS staining of transgenic Arabidopsis showed that the leaves,stems,flowers,and pods could be stained,which was consistent with the results of ZmSKIP that can be expressed in multiple tissues.The GUS quantification results of transgenic Arabidopsis thaliana under mannitol and NaCl stress treatment conditions showed that the expression level of GUS increased under stress treatment conditions.4.The 160 bp difference in the ZmSKIP gene promoter may be related to the drought tolerance of the material.Combined with co-expression network analysis,this study screened out a MYB transcription factor MYBR63 that may be related to the insertion.It can be speculated that MYBR63 can be combined with the ZmSKIP promoter to regulate the expression of ZmSKIP,thereby affecting the drought tolerance of different corn inbred lines.5.The full-length cDNA sequence of ZmSKIP gene was cloned,a total of1830 bp,a fusion expression vector of CaMV35S::ZmSKIP:e GFP was constructed,and the tobacco leaf was transiently transformed by Agrobacterium-mediated method.The results showed that the ZmSKIP protein was located in the nucleus and nucleus.6.Overexpression of ZmSKIP can increase Arabidopsis resistance to abiotic stress.The transgenic Arabidopsis plants overexpressing ZmSKIP had a significantly higher germination rate under mannitol and NaCl stress than wild type,and their drought tolerance and survival rate under drought stress were higher than those of wild type plants.Further using qRT-PCR and RT-PCR to analyze the expression and splicing forms of plants related to stress under drought stress,it was found that the expression levels of AtDREB and AtRD29 B genes in drought stress overexpression materials were significantly increased compared with the wild type and splicing of AtRD29 B When the form changed,the expression levels of AtLEA and AtPOD genes decreased significantly,while the expression levels of AtAPX did not differ significantly.These results indicate that ZmSKIP may improve the drought tolerance of plants by regulating the expression of AtDREB,AtRD29 B and other genes.7.ZmSKIP can complementary the phenotype of Arabidopsis skip-1 mutant.Using the CaMV35S::ZmSKIP overexpression vector to complement the Arabidopsis mutants through Agrobacterium-mediated method,the results showed that the germination rate of skip-1/35S::ZmSKIP,the growth of underground and aerial parts are similar to the wild type.ZmSKIP can restore all aspects of the mutant phenotype to a certain extent,indicating that it has a similar function to AtSKIP gene..8.Overexpression of ZmSKIP in maize can improve drought tolerance of plants,but does not affect yield-related traits.In the three-leaf one-core stage,the growth potential of the overexpressed material and the wild type was observed,and it was found that the plant root length and total root area were significantly higher than that of CO1 after ZmSKIP overexpression compared with the overexpressed receptor material CO1.In the five-leaf one-core stage,the drought resistance was identified in the soil,and the results showed that the over-expressed material was more drought-resistant than CO1.In terms of ears,compared with CO1,there was no significant difference in the indexes of ear weight,ear thickness,ear rows,100-grain weight,grain length,and grain width.9.ZmSKIP overexpression transgenic corn transcriptome sequencing results show that the expression of a large number of genes has changed.A total of 44842 genes were counted,of which 889 were significant responses,320 were up-regulated,569 were down-regulated,46161 alternative splicing events were detected,and 10360 AS genes had 1360 significant AS events involving 961 gene,but up-regulated genes and differential splicing genes overlapped only 10 genes.