水稻粒型基因DA3的克隆及功能分析

【作者】 秦进；

【导师】 曾晓芳；

【作者基本信息】 贵州大学 ， 生物学， 2025， 硕士

【摘要】 水稻(Oryza sativa L.)是世界重要的粮食作物之一,直接影响全球粮食供应安全。水稻粒型由粒长、粒宽和粒厚构成,是水稻产量的核心性状。水稻粒型是一个复杂的农艺性状,受多基因控制,其调控机制尚未完全明确。因此,进一步挖掘新的粒型控制基因并研究其调控机制,可以丰富水稻粒型调控网络,同时还可为水稻性状改良提供新的基因资源。课题组前期研究发现粒型调控基因DA3对水稻粒长和粒宽具有负调控作用,本研究在此基础上进一步对DA3基因的功能进行研究,获得如下结果:1.DA3基因的克隆及生物信息学分析DA3基因编码一个F-box蛋白,具有典型的F-box结构域,分子量33 k Da,理论等电点值5.44,脂肪系数89.05,具有疏水特性,在292-314位氨基酸处具有跨膜结构域,无信号肽,同时具有25个潜在磷酸化位点。系统发育分析表明,DA3蛋白与光稃稻F-box蛋白亲缘关系最近,与小麦F-box蛋白亲缘关系最远。2.DA3的表达模式及其亚细胞定位对DA3基因在水稻根、茎、叶、叶鞘、颖壳、5cm幼穗和10cm幼穗中的表达水平进行检测发现,DA3基因在水稻所检测的各组织中均有表达,在叶片中表达量最高,在根中最低。为进一步了解DA3基因的表达模式,我们克隆并构建了2041bp长度启动子驱动的DA3pro:RUBY启动子表达载体,利用农杆菌介导法遗传转化水稻,获得转基因植株。转基因植株的叶、叶鞘及茎秆、颖壳、根等部位均可观察到明显的红色,但在根中红色较浅,与q RT-PCR的结果一致。蛋白亚细胞定位结果表明DA3蛋白定位于细胞膜、细胞质与细胞核,但在细胞核中分布最多。3.DA3基因通过BR途径调控水稻株型及粒型利用CRISPR/Cas9基因编辑技术与转基因技术构建了DA3基因敲除突变体(da3)与超量表达转基因植株(OE),并对成熟期da3及OE植株表型进行观察。发现DA3基因负调控水稻株高、穗长、粒长及粒宽。同时还发现超量表达DA3基因植株表现出典型BR(油菜素内酯)缺陷的表型,表明DA3可能参与了BR调控途径。进一步研究发现da3突变体中BR相关基因表达量显著上调,而在OE植株中显著下调,表明DA3可能通过调控BR相关基因的表达来调控水稻株型及粒型。4.SCF复合体的预测及DA3下游靶蛋白的筛选F-box蛋白能与SKP1结合形成Skp1-Cullin-F-box(SCF)复合体,参与蛋白的泛素化降解过程。利用Alpha Fold对DA3与OsSKP20(SKP1同源蛋白)互作进行预测,结果显示ipTM和pTM值高达0.8,表明DA3可能与OsSKP20互作。da3突变体水稻籽粒变大,可能因DA3突变导致SCF的蛋白泛素化功能丧失,相关蛋白的表达量上升所导致。因此,我们对da3突变体及OE植株进行蛋白质组检测与分析,结果发现粒型调控基因DEP2的蛋白量在da3中显著上调,而在OE植株中显著下调。Alpha Fold预测结果表明DA3与DEP2可能存在互作关系。下一步将对DA3与DEP2进行互作验证。综上,我们发现了一个新的粒型调控基因DA3,DA3基因可能通过影响BR相关基因的表达量来调控水稻的株型与粒型。DA3蛋白可能与OsSKP20相互作用,参与形成SCF复合体,通过影响DEP2蛋白稳定性来参与调控水稻粒型的发育。更多还原

【Abstract】 Rice(Oryza sativa L.)is one of the most important cereal crops worldwide,playing a critical role in ensuring global food security.Grain shape,defined by grain length,width,and thickness,constitutes a key agronomic trait that directly influences yield potential.As a complex agronomic trait,the molecular mechanisms underlying rice grain shape regulation remain incompletely elucidated.Therefore,identifying novel grain shape-related genes and elucidating their regulatory networks will enrich our understanding of grain shape control and provide new genetic resources for rice improvement.Our previous studies identified DA3 as a negative regulator of grain length and width.Building on this foundation,the present study further investigates the function of DA3,with the following results:1.Cloning and Bioinformatics Analysis of DA3DA3 encodes a protein containing a typical F-box domain,with a molecular weight of 33 k Da.The DA3 encoded protein has a predicted molecular weight of 33 k Da,a theoretical isoelectric point(p I)of 5.44,and an aliphatic index of 89.05.Bioinformatic analyses indicate that DA3 is hydrophobic,contains a transmembrane domain spanning amino acid residues 292–314,lacks a signal peptide,and harbors 25 putative phosphorylation sites.Phylogenetic analysis revealed that the DA3 protein shares the highest sequence similarity with F-box proteins from Oryza glaberrima and exhibits the greatest evolutionary divergence from those in wheat.2.Expression Pattern and Subcellular Localization of DA3Quantitative expression analysis demonstrated that DA3 is constitutively expressed across multiple rice tissues,including roots,stems,leaves,leaf sheaths,glumes,and young panicles(5 cm and 10 cm in length),with peak expression observed in leaves and minimal expression in roots.To further characterize the expression pattern of DA3,a 2041-bp promoter was cloned to drive the DA3pro:RUBY reporter construct,which was introduced into rice via Agrobacterium-mediated transformation.Transgenic lines exhibited pronounced red pigmentation in leaves,leaf sheaths,stems,glumes,and roots,with the weakest signal in roots—consistent with q RT-PCR data.Subcellular localization assays revealed that DA3 is predominantly localized in the nucleus,with additional signals detected in the cytoplasm and cell membrane,suggesting potential multifunctional roles.3.DA3 Regulates Plant Architecture and Grain Shape via the BR PathwayUsing CRISPR/Cas9-mediated gene editing and transgenic overexpression approaches,DA3 knockout mutants(da3)and overexpression lines(OE)were generated.Phenotypic characterization revealed that DA3 acts as a negative regulator of plant height,panicle length,grain length,and grain width.Moreover,OE plants exhibited phenotypes typical of brassinosteroid(BR)-deficiency,suggesting that DA3may be involved in BR pathway.Further analysis revealed that the BR-related genes were significantly upregulated in da3 mutants but downregulated in OE plants,indicating that DA3 l modulates plant architecture and grain shape by regulating BR-related gene expression.4.Prediction of the SCF Complex and Identification downstream target proteins of DA3.F-box proteins typically associate with SKP1-like proteins to form Skp1-Cullin-F-box(SCF)E3 ubiquitin ligase complexes,which mediate targeted protein degradation via the ubiquitin-proteasome pathway.Alpha Fold3 prediction indicated a potential interaction between DA3 and OsSKP20(a SKP1 homolog),yielded high ipTM and pTM scores(0.8),supporting a potential physical interaction.The enlarged grain phenotype observed in da3 mutants may result from disrupted SCF complex function,leading to impaired ubiquitination and consequent accumulation of downstream target proteins.Comparative proteomic analysis identified DEP2 as significantly upregulated in da3 mutants and downregulated in OE lines.Alpha Fold modeling also predicted a potential interaction between DA3 and DEP2,although this requires experimental validation through biochemical assays.In conclusion,this study identified DA3 as a novel regulator of rice grain shape and plant architecture,likely functioning through modulation of the BR pathway.DA3may form an SCF-type E3 ubiquitin ligase complex with OsSKP20 to regulate the stability of key developmental proteins such as DEP2,thereby influencing grain development.These findings contribute to a deeper understanding of the molecular mechanisms controlling rice grain formation and provide new genetic tools for crop improvement.更多还原