Cloning And Stress-resistance Study Of Heat Shock Transcription Factor ZmHsf04 From Zea Mays

Maize as a major cash crop worldwide,is not only a primary food resource,but also an significant raw material.Abiotic stresses,especially high temperature,salinity and drought,have serious effects on maize growth and development,and are important factors limiting maize yield and quality.Therefore,screening stress-resistant gene and breeding new crop cultivars with better stress resistance,has become a main approach in the genetic improvement of crop resistance breeding.In the long-term evolution process,plants have formed a variety of mechanisms to adapt and survive under various environmental stresses.Studies on transcription factor showed that they play important roles in plant growth and development and improving plant tolerance to adversity stresses.Thereinto,heat shock transcription factor(Hsf)is a kind of highly conservative and important members,which widely involved in plants response to abiotic stresses.However,there is less study on its function and action mechanism in maize.In this study,we isolated a abiotic stresses strongly induced gene,ZmHsf04,from maize inbred line B73 and then preliminary analyzed the functions and mechanisms response to high temperature and salinity.The main results as follows:1.The results of RT-PCR showed that Zmhsf04 was induced by high temperature,salinity,PEG,low temperature and exogenous ABA.Tissue-specific expression pattern indicated that Zmhsf04 was expressed in roots,stems,leaves,tassel,filaments,ear and embryo,however,the higher expression level was in stems and leaves.2.ZmHsf04 was cloned from maize inbred line B73 by PCR.Sequence analysis showed that the entire coding region of ZmHsf04 is 1074 bp and encodes a protein of 357 amino acids that its relative molecular mass is 40.50 kD,and isoelectric point is 4.99.Amino acid sequence analysis indicated that the protein includes a highly conservative DNA-binding domain(DBD),and contains the typical structural domains of subfamily A2.3.A fusion expression vector p1305-ZmHsf04:GFP was constructe.The instantaneous expression of tobacco showed that ZmHsf04 is localized into the nucleus.Regional truncation screening experiment indicated that ZmHsf04 could transcribe in yeast cells,and C-terminal AHA2 motif is the smallest active region,but the point mutations experiment showed that tryptophan(W)located in AHA2 motif is not the main functional site.4.We acquiried ZmHsf04 overexpression transgenic Arabidopsis by an Agrobacterium infection method.Under normal conditions,transgenic lines had no obvious difference compare with wild type.After stresses treatments,ZmHsf04 overexpression transgenic Arabidopsis exhibited enhanced basal and acquired thermotolerance and salt stress tolerance.In addition,the expression of stress related genes in transgenic Arabidopsis was apparently higher than WT plants,suggesting that expression of these genes were actived by ZmHsf04 gene.5.ZmHsf04 overexpression transgenic Arabidopsis also showed improved sensitivity to ABA,which suggested that ZmHsf04 gene may be involved in the ABA signal transduction pathway,consequently improved the tolerance to high-temperature and salinity.