Functional Analyses Of The Stress-inducible Promoter PZmCKS2、PZmCBF3and The Transcription Factor ZmZBED In Maize

Adverse environmental stresses such as drought, salinity, low temperature andpathogen attacks, have drastic effects on plant growth and development, whichsignificantly limit agricultural productivity. To solve the problem of severe multipleadversity stress in production and increase crop yield, the biological breedingtechnology which based on the genetic transformation is an important choice, inaddition to the conventional breeding technology. The cloning of key genes related toadverse stress is the core to solve the problem. However, cloning of adversitycross-inducible promoter is the important guarantee to realize molecular breedinggoals. Unfortunately, from the current application, the constitutive expressionpromoter is given priority, such as the Cauliflower mosaic virus35S promoter(CaMV35S), Ubiquitin promoter. The constitutive expression promoter, such asCaMV35S, has been used to assess the effects of transgene expression in various plantspecies. But the high expression of transgene in all tissues during all developmentalperiods, increases plant metabolism burden to a certain extent, makes a loss ofmaterial and energy, and then causes many negative effect on transgenic plants.Insteading of a constitutive promoter such as CaMV35S, an stress-inducible promoterto drive gene expression in transgenic plants can minimize the negative effects onplant.We have analyzed the genes expression of the corn seedling under low temperatureand drought stress by gene chip technology. On this experiment basis, we decided tostudy on protein kinase gene (ZmCKS2) and transcription factors (ZmCBF3) promoter.Their upstream sequences were obtained by alignment on maize B73whole-genomesequence web. The promoters of ZmCKS2and ZmCBF3were cloned by specificprimers, a series of5’-end deletion fragments were in fusion with GUS gene, theexpression vectors were transformed to Arabidopsis thaliana through theAgrobacterium and study the expression of GUS gene. At the same time, a transcription factor combined with ZmCKS2promoter was separated by yeastone-hybrid. The preliminary analysis has carried on the function of transcriptionfactor. The main results of this study as follows:1. we analyzed the gene mRNA levels by qRT–PCR after treating young maizeplants. These results indicated that ZmCKS2gene expression can be induced bydehydration, cold, and ABA stresses. To characterize the transcriptional control ofZmCKS2, the5’-upstream sequence of1853bp were cloned from maize genomic DNA.By the promoter sequence analysis on the PLACE and the PlantCARE databases,several core fragments were identified, such as MBS, CE3, TGA-element and ABREetc.2. Replacing35S promoter of binary expression vector pCAMBIA1301, theZmCKS2promoter was in fusion with GUS gene, which was transformed intoArabidopsis thaliana. The result showed that the promoter of ZmCKS2was inducedby abiotic stresses (drought, low temperature) and the different hormone (ABA,MeJA, SA) factors.3. Functional analyse of5’-end deletion fragments showed that the999bppromoter region was required for the highest basal expression of GUS, and the367bp sequence was the minimal promoter for ZmCKS2activation by low temperature,MeJA and SA. ABREs functional analysis showed that ABRE and CE3element wasthe positive regulatory elements of ZmCKS2gene. Two of them together was able tofeel ABA signal and play a role. This study also found that the effect of ABREs wasnot positive ratio with the number of ABREs.4. GUS expression assays indicated that the234-bp fragment upstream of theZmCBF3gene coding sequence confered a high level of GUS expression inArabidopsis. The PZmCBF3was activated by cold stress to regulate plantdevelopmental processes and adaptation to environment. The MYCCONSENSUSATelements (CANNTG) were responsible for the ability of PZmCBF3to be induced bycold. The ZmCBF3gene exhibited root-specific expression, which was relation withRAV1AAT and ROOTMOTIFPOX1elements 5. A protein combining with ZmCKS2promoter was separated by yeast one hybrid,the genetic code area of which coded174amino acids, the molecular weight of19.37KD, isoelectric point of8.99. The protein contains a zinc finger domain, composed of65amino acid, from the78th amino acid to142th amino acids. By homologoussequence comparison, It was found that the homology of the gene with other specieswas low, and the homology with the tobacco, tomato and Arabidopsis were14.47%,8.48%and4.66%, respectively. The protein localized in the nucleus and was no signalpeptide, which was named ZmZBED.6. By the activity analysis of GUS transient expression in tobacco, it showed thatZmZBED was a positive regulation factor of ZmCKS2promoter, could regulate theexpression of gene ZmCKS2.