Analysis Of Abiotic Stress Related Functions Of Genes ZmWRKY50 And ZmWRKY44 In Maize (Zea Mays L.)

Maize is an important food crop, forage crop and commercial crop in our country. The growth and development of maize is inhibited by biotic stress (insect pest infection) and abiotic stress (drought stress, salt stress, cold stress, heat stress and other disadvantageous factors) during growth process. Based on application of modern molecular biology techniques, excavation and genetic transformation of important stress resistance gene conbined with other excellent characters through marker-assisted selectionis one of the effective methods to get the corn varieties with strong stress tolerance and increase maize yield. WRKY gene family has been suggested to play important roles in regulation of transcriptional reprogramming associated with plant stress responses and the plant growth and specifically bind to the W-box in the promoter regions of the target genes. WRKY transcript factor contains a highly conserved WRKY domain, which can experimental evidences that WRKY transcription factors might be involved in several different processes and display functional diversity. Thus, WRKY genes and its target genes are potentially important stress resistance gene.In our study, we mainly make a preliminary function study of the ZmWRKY50 and ZmWRKY44 genes in maize. Firstly, we obtained the cDNA sequences of ZmWRKY50 and ZmWRKY44 from maizesequence database. And phylogenetic tree analysis was performed followed by cDNA sequences of ZmWRKY50 and ZmWRKY44 gene. Secondly, the temporal and spatial expression patterns of ZmWRKY50 and ZmWRKY44 genes were detected by qRT-PCR under different abiotic stress conditions. Thirdly, the cDNA fragments of ZmWRKY50 and ZmWRKY44 gene were isolated from the maize inbred line 178 and the overexpression vectors pCBP-ZmWRKY50 and pCBP-ZmWRKY44 were transformed into the Arabidopsis mediated by Agrobacterium.Phenotype analysis for homozygous 35S::ZmWRKY44 plant was performed accordingly. Fourthly, the transient vectors 35S::ZmWRKY50-GFP and 35S::ZmWRKY44-GFP were constructed and transformed into the leaf of Nictotiana benthamiana mediated by Agrobacterium to determine the subcellular location of ZmWRKY50 and ZmWRKY44. Finally, we performed the yeast transcriptional activation experiment of pGBKT7-ZmWRKY50 and pGBKT7-ZmWRKY44 to verify the transcriptional activation function. The main results obtained are listed as follows:1. Phylogenetic tree analysis showed that the highest homology of ZmWRKY50 is OsWRKY68 in rice andthat of ZmWRKY44 is HvWRKY31 in barley.2. RT-qPCR detection showed that ZmWRKY44 expression was influenced by salt stress, high temperature, H2O2 and ABA level.3. pCBP-ZmWRKY50 and pCBP-ZmWRKY44 vectors were transformed into the Arabidopsis mediated by Agrobacteriumand positive homozygous transgenic plants were obtained after PCR detection and sequencing.4. Phenotype analysis of salt tolerance for homozygous 35S::ZmWRKY44 demonstrated that 35S::ZmWRKY44 plantwas sensitive to salt stress to some extent.5. Subcellular localization showed that ZmWRKY50 and ZmWRKY44 were all located in the nucleus.6. Yeast transcriptional activation test showed that ZmWRKY50 and ZmWRKY44 have the function of self-ranscriptional activaty.