【作者】 李霞；

【导师】 徐正君；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2018， 硕士

【摘要】 组蛋白是真核细胞生物染色体中的十分保守的一类小分子蛋白质,pI属于碱性,主要分为五种类型:H1、H2A、H2B、H3和H4。组蛋白的修饰作用主要包括了乙酰化、甲基化、磷酸化、腺苷酸化、泛素化等,组蛋白的修饰将会影响染色质的拼装,从而协助转录调控。组蛋白的修饰作用除了参与光反应和影响开花外,组蛋白的修饰作用被证明参与了其他刺激反应,包括激素,生物胁迫和各种非生物胁迫。我们实验室通过荧光差异显示和定量PCR技术,克隆了一个多逆境诱导的OsCaMBP水稻钙调素结合蛋白基因,通过酵母双杂交证明其能与CaM结合,热激能诱导其在水稻根、叶和叶鞘中的表达量变化,低温也能诱导该基因表达量的增加^[35]。本实验室而后再次利用酵母双杂交技术,将OsCaMBP作为诱饵蛋白从低温、干旱cDNA文库中筛选出了一个与其互作的基因OsSCL30b。文中所用OsH4a是本实验室通过酵母双杂交技术以OsSCL30b为诱饵蛋白从低温、干旱cDNA文库中筛选出的一个与其互作的蛋白,属于组蛋白H4超家族。主要研究结果如下:1、通过双分子荧光互补实验{BIFC)进一步验证两者互作。2、农杆菌介导瞬时转化烟草的亚细胞定位结果为OsH4a主要汇集在细胞核上。3、从水稻cDNA文库中克隆了OsH4a基因,生物信息学分析发现该基因等电点12,预测OsH4a分子量约11.4 KD,OsH4a开放阅读框为312个bp,编码了103个氨基酸残基,上游启动子区域有ABRE,G-box,CGTCA,TGACG和CCAAT等多个与逆境相关顺式作用元件。4、定量PCR分析OsH4a在各种非生物逆境下叶中的表达量,发现OsH4a基因在不同逆境下有不同程度的响应,而在高温下OsH4a的表达量被诱导的程度最高,在植物生长调节剂处理下OsH4a的表达量受ABA影响程度较高。5、我们构建了OsH4a基因的原核表达载体PET-32a:OsH4a,并通过体外诱导出OsH4a融合蛋白的表达,在此基础上探究了OsH4a蛋白在体外对逆境条件的响应,发现OsH4a蛋白在大肠杆菌中的表达能明显增强对高温、低温的耐受性。6、为了在真核生物中研究OsH4a基因对逆境的响应机制,从而应用到生产上,我们构建了OsH4a基因的正向超表达载体和反向抑制表达载体,通过农杆菌侵染水稻日本晴愈伤的方式,顺利得到转基因植株,共计六株正向超表达植株,五株反向抑制表达植株。并将转基因植株进行逆境处理的预实验,发现超表达植株在低温下的长势稍好于日本晴。更多还原

【Abstract】 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.更多还原