Functional Analysis Of ZmHsfA4? Gene To Drought Stress In Maize

Maize is one of the three major cereal crops in China,it can be used as both food and industrial raw material,which plays important role in ensuring food production and food security.During the process of growth,maize often suffers from many environmental stresses,of which the abiotic stresses are major factors,such as high temperature,drought and high salt,which leads to a decline of maize yield and quality.Therefore,it's of great significance to cultivate new resistance varieties to ensure the production and quality of crop.At present,transgenic technology is one of the important means for crop modification and improving plant stress resistance.Transcription factors have been identified to play an important role in the process of plant stress response,which can simultaneously activate or inhibit the expression of multiple downstream genes,therefore,their effects are more significant.Up to now,many plant transcription factor genes were identified to participate in the response to stress response.HSF is one of the most important families of transcription factors in plants,which is widely involved in the process of plant growth and abiotic stress response,and the mechanism of HSF genes has been well studied in Arabidopsis thaliana and tomato.However,the real roles of maize HSF genes are still unknown.In this study,we cloned the ZmHsfA4 a gene from maize,and then study the gene characterization and expression patterns of ZmHsfA4 a,as well as its role under drought stress.We further identified the interacted protein of ZmHsfA4 a to elucidate its functional mechanism.The main results are as follows:1.The full-length CDS sequence of ZmHsfA4 a was cloned from maize B73 and the sequencing result was consistent with the published sequence in B73 genome database.The gene ZmHsfA4 a was 1302 bp in length and encoded 433 amino acids.Gene microarray data showed that ZmHsfA4 a was expressed in all the tissues of maize,and the expression level of ZmHsfA4 a was high in the leaf.The result of q-PCR was consistent with the chip data that ZmHsfA4 a was expressed in all six tested tissues,including roots,stems,leaf,tassel,cornsilk,and stegophyll,and the expression level in the leaves was high.The induced expression pattern analysis showed that ZmHsfA4 a was up-regulated by drought and high temperature,while was down-regulated under ABA and NaCl treatments.2.The result of subcellular localization showed that ZmHsfA4a-p1305 fusion protein was expressed only in the nucleus,indicating that ZmHsfA4 a was a nuclear protein.The results of transcriptional activity analysis indicated that ZmHsfA4 a had transcription activity in yeast strain AH109 and the key active region was located at 230-280 Amino acids.3.Overexpression vector ZmHsfA4a-p1301 a was constructed and transformed into Arabidopsis thaliana by Agrobacterium tumefaciens.The germination rate of transgenic Arabidopsis thaliana was increased on MS medium containing 300 mM mannitol,and under drought conditions,the tolerance of transgenic plants was also enhanced.Expression analysis showed that the expression level of stress-related genes in transgenic Arabidopsis increased after drought treatment,and were higher than that in wild type plants.4.Overexpression of ZmHsfA4 a in rice zhonghua11 enhanced transgenic rice resistance to drought stress.The relative conductivity and malondialdehyde(MDA)content of transgenic rice plants were significantly lower than those of wild plants after drought treatment,and the expression level of stress-related genes in transgenic rice was higher than that of wild plants.These results further indicated that The ZmHsfA4 a enhanced transgenic rice tolerance to drought stress.In addition,the transgenic rice were more sensitive to exogenous ABA,indicating that ZmHsfA4 a was involved in ABAmediated signaling pathway in rice.5.Overexpression of ZmHsfA4 a increased transgenic maize tolerance to drought stress.The relative water loss rate of transgenic plants was lower than that of wild type plants,and the relative conductivity,malondialdehyde(MDA)content of transgenic maize were significantly lower than that of wild type plants,while the free proline content was significantly higher than that of wild type plants,which further indicated that ZmHsfA4 a enhanced maize drought resistance.In addition,The ABA content of the transgenic maize was significantly higher than that of the wild type plant after drought treatment,and the expression of ZmVP14,an ABA synthesis-related gene,was consistent with the trend of ABA content,indicating that ZmHsfA4 a was involved in ABA signal transduction pathway in maize.Moreover,BIFC and pulldown experiments showed that ZmHsfA4 a could interact with itself to form homologous multimers,meanwhile,it could interact with EMP2.These results indicate that ZmHsfA4 a is involved in the regulation of drought stress response through ABA signal transduction pathway,and EMP2 participates in the process of regulation of ZmHsfA4?.