Cloning And Stress Resistance Analysis Of OsH4a Gene In Rice

Histone is a type of very conservative small-molecule basic protein in the chromosome of eukaryotes,mainly including five types:H1,H2A,H2B,H3 and H4.The modification of histones mainly includes acetylation,methylation,adenylation,phosphorylation,ubiquitination and etc.The modification of histones will affect chromatin assembly and assist transcriptional regulation.Modifications of histones in addition to participating in photoreactions and affecting flowering,it has been shown to be involved in other stimuli including hormones,biotic stress,and various abiotic stresses.In rice a multi-stress induced gene of calmodulin-binding protein was named OsCaMBP that was cloned in our laboratory by fluorescence differential display and quantitative PCR.It was demonstrated by yeast two-hybrid that it could bind to CaM,and heat shock induced its expression in rice root,leaf and leaf sheath,low temperature can induce an increase in the expression of this gene^[35].In this laboratory,we used yeast two-hybrid technology again and used OsCaMBP as a bait protein from a low-temperature,drought cDNA library to screen a OsSCL30b gene that interacted with OsCaMBP.The histone OsH4a was selected from a low-temperature,drought cDNA library using OsSCL30b as the bait protein with the yeast two-hybrid technique,which belongs to the histone H4 superfamily.The main findings are as follows:1?The bimolecular fluorescence complementation?BIFC?was used to further validates that they interact with each.2?Agrobacterium-mediated transient transformation of tobacco subcellular localization indicates that OsH4a mainly accumulates in the nucleus.3?The OsH4a gene was cloned from the rice cDNA library.Bioinformatics analysis revealed that the gene has an isoelectric point of 12,a predicted molecular weight of 11.4 KD,an open reading frame of 312 bp,encoding 103 amino acid residues,and the upstream promoter region includes multiple stress-related cis-acting elements such as ABRE,G-box,CGTCA,TGACG,and CCAAT.4?Quantitative PCR was used to analyze the expression level of OsH4a gene in leaves under different stress conditions.It was found that OsH4a gene was expressed under different stress conditions.The expression level of OsH4a under high temperature is the highest level,under the treatment of plant growth regulators,the expression of OsH4a was highly affected by ABA..5?We constructed the prokaryotic expression vector PET-32a:OsH4a and induced the expression of the OsH4a fusion protein in vitro.Based on this,we explored the response of OsH4a protein to adversity conditions in vitro,it was found that the expression of OsH4a protein in E.coli can significantly enhance the tolerance to high temperature and low temperature.6?In order to study the response mechanism of the OsH4a gene to adversity in eukaryotes and apply it to production,we constructed the positive overexpression vector and reverse suppression expression vector of the OsH4a gene and then transformed vector into rice callus by Agrobacterium infection.Transgenic plants were successfully obtained.A total of six positively overexpressing plants and five reverse suppression expressing plants.The pre-experiment of adversity treatment of the transgenic plants revealed that the over-expressed plants were slightly better than Nipponbare at the low temperature.