【作者】 李敏；

【导师】 颜康； 宋健民；

【作者基本信息】 山东农业大学 ， 生物技术与工程（专业学位）， 2024， 硕士

【摘要】 富含丝氨酸/精氨酸的蛋白(Serine/arginine-rich,SR),是真核生物的一种高度保守的剪切因子,主要负责前体mRNA的剪接加工。可变剪切是由剪接因子调控的真核细胞转录后调控过程中关键加工步骤。超过90%的动物含内含子的基因和40-70%的植物含内含子的基因能够发生可变剪切。SR45a蛋白属于特异SR蛋白,在植物的高温胁迫应答调控过程及其生长发育中起到关键的作用。高温胁迫是严重影响小麦(Triticum aestivum L.)产量、品质和生长的重要因素。在本研究中,我们成功地从小麦中克隆了拟南芥AtSR45a的同源基因,并将其命名为TaSR45a,并对小麦SR蛋白结构、染色体定位、基因结构及进化关系等进行分析;之后运用RT-qPCR技术、Western Blot、亚细胞定位和酵母筛库分析,通过过表达获得TaSR45a转基因的小麦,并在此基础上对TaSR45a基因的功能进行探究,其结果如下:(1)利用BLASTP方法对拟南芥和水稻SR家族基因进行搜索,然后进行HMM搜索和结构域验证,在小麦基因组中共鉴定出60个SR基因。进一步分析了60个TaSRs基因的理化性质;小麦SR蛋白的氨基酸数量在182-428之间,平均为280,SR蛋白的分子量在20.56-47.2 kDa之间。进一步对小麦SR蛋白结构,启动子序列,氨基酸序列以及组织表达模式进行了初步分析。(2)通过RT-qPCR表达分析表明,经过高温处理TaSR45a的表达量随着处理时间的延长而增加,说明TaSR45a受高温诱导表达。(3)通过RT-qPCR验证经过干旱处理后TaSR45a的表达情况,发现TaSR45a基因在干旱中受诱导,以及组织表达模式分析表明TaSR45a表达量存在组织差异性,在籽粒中的表达量最高,在叶中表达量最低。(4)Western Blot实验表明在高温胁迫下显著增加TaSR45a蛋白的积累。(5)构建pMDC83-Ubi::TaSR45a-GFP载体,观察TaSR45a的亚细胞定位,结果显示TaSR45定位于细胞核。(6)同源克隆获得Fielder小麦TaSR45a基因,构建过表达载体p3300-Ubi::TaSR45a转小麦,并进行转基因小麦筛选和高温表型验证,发现TaSR45a基因的过量表达可以增加小麦的高温抗性。(7)酵母筛库实验进行TaSR45a互作蛋白的筛选,共筛选到18个互作蛋白。更多还原

【Abstract】 Serine/arginine-rich protein(SR),a highly conserved eukaryotic splicing factor,function mainly in splicing of precursor mRNA.Alternative splicing is a key processing step in the post-transcriptional regulation controlled by splicing factors.More than 90%intron-containing genes in animal and 40-70%intron-containing genes in plant undergo alternative splicing events.Among the SR family proteins,SR45a is specific splicing factor in plants which plays a key role in the stress responses and development in plants.Heat stress is an important factor that seriously affects the yield,quality and growth of wheat.In this study,we successfully cloned the homologous gene AtSR45a from wheat and named it TaSR45a,and analyzed the structure,chromosome localization,gene structure,amino acid sequence and evolutionary relationship of wheat SR protein.Then RT-qPCR technology,Western Blot,subcellular localization and yeast sieve bank analysis were used to obtain TaSR45a gene from wheat through overexpression and artificial gene knockout.On this basis,the function of TaSR45a gene was explored,and the results were as follows:(1)Based on BLASTP search with Arabidopsis and rice SR family genes,followed by performing HMM search and domain verification,a total of 60 SR homologs were identified in wheat genome.Further analysis of the physical and chemical properties of the 60 TaSRs genes;The amino acid length of the wheat SR protein ranged from 182 to 428,with an average of 280.The molecular weight of the protein was 20.56-47.2 kDa,and further preliminary analysis of the structure of the wheat SR protein,promoter sequence,amino acid sequence and tissue expression pattern was carried out.(2)The expression analysis of TaSR45a by RT-qPCR showed that,in the seedling stage,the expression of TaSR45a increased with the increase of the treatment time after high temperature treatment,indicating that TaSR45a was induced at high temperature.(3)The expression of TaSR45a after drought treatment was verified by RT-qPCR,and it was found that TaSR45a gene was induced in drought,and the analysis of tissue expression pattern showed that the expression of TaSR45a was tissue-specific,with the highest expression in grains and the lowest expression in leaves.(4)The Western Blot experiment showed that TaSR45a protein was significantly increased under high temperature stress.(5)The pMDC83-Ubi::TaSR45a-GFP vector was constructed to observe the subcellular localization of TaSR45,and the results showed that TaSR45a was localized in the nucleus.(6)Fielder wheat TaSR45a gene was cloned,and the overexpression vector p3300-Ubi::TaSR45a was constructed to transfer wheat to Fielder.The transgenic wheat was screened and verified by heat stress phenotype.It was found that the transgenic wheat was resistant to heat stress.(7)The yeast sieve library experiment was conducted to screen TaSR45a interaction proteins,a total of 18 interacting proteins were screened.更多还原