【作者】 王新宇；

【导师】 郑志民；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2024， 博士

【摘要】 蓖麻（Ricinus communis L.）是大戟科蓖麻属植物,广泛种植于全球各地。在东非,蓖麻主要以多年生乔木的形态生长。而西亚的蓖麻主要是一种多年生的灌木。在印度和中国,蓖麻多以栽培的灌木形态出现。蓖麻种子含有40%-55%蓖麻油,这使得蓖麻已经成为我国最主要的经济林树种之一。蓖麻油富含羟基化的蓖麻油酸,其含量高达90%以上,这种高纯度和高羟基化特性使得蓖麻油在工业上得到了广泛应用。近年来国内对蓖麻需求大幅度上升,但其产量改善有限,供需缺口逐步加大。为了进一步提高蓖麻产量,一个行之有效的途径是将现代分子生物学结合至传统品种选育的过程中进行遗传改良。近年来,一些研究已经揭示了与蓖麻种子大小相关的数量性状位点,但是调控蓖麻种子大小的分子机制仍不清楚。本研究旨在解析蓖麻种子大小的遗传分子机制,深入剖析蓖麻种子形成的调控机理,并为蓖麻的遗传改良提供重要的指导和理论依据。本研究主要实验结果如下:（1）定位影响蓖麻种子大小的主效QTL（qSS3）。以内蒙古大种子栽培品系DL01和埃塞俄比亚小种子野生品系WH11作为亲本进行杂交,构建遗传分离群体。通过扫描电镜和石蜡切片对亲本种子进行细胞学分析后发现,DL01的种皮细胞和胚乳细胞展现出更大面积和更多数量,这表明蓖麻种子大小变化的细胞学机制是种皮和胚乳细胞数量和大小变化导致的。随后选取包含240个单株的F₂代群体作为用于数量性状位点（QTL,Quantitative Trait Locus）定位的F₂小群体,基于亲本的差异筛选多态性的标记并构建连锁图谱,最终定位到Chr03染色体上Rc-1-3和Rc1-1分子标记之间存在一个同时调控粒长、粒宽以及百粒重的主效QTL（qSS3）。BSA（Bulked Segregant Analysis）混池测序结果进一步证明了QTL定位的准确性并缩短了区间。随后对定位区间进行加密标记并利用F₂代群体487株极端表型植株将目的基因定位到Rc-1-30.97和Rc-1-31.15之间的180 kb区间内。（2）qSS3编码miRNA396b。在图位克隆区间内进行基因注释时,共鉴定出20个基因,其中6个基因在亲本之间存在非同义突变。进一步的遗传分析结果显示已注释基因的突变在资源品种中并不保守。进而通过亲本候选区间的序列比对发现一个17.6kb结构变异存在于亲本之间,PCR鉴定分析结果显示大种子表型的蓖麻资源品种均丢失了该17.6 kb的片段。对此区间进行基因注释鉴定到非编码RNA的一个成员miR396b,RT-q PCR结果显示在DL01中并未检测到miR396b的前体和成熟序列的表达量。（3）miR396b-RcGRF4/RcGIF2模块参与蓖麻种子发育过程。在蓖麻全基因组中鉴定miR396的保守靶基因GRF（Growth Regulation Factor）以及GRF的辅助因子GIF（GRF-Interacting Factor）),共鉴定到11个RcGRFs基因和2个RcGIFs基因。RcGRFs和RcGIFs基因组织表达特异性分析结果表明,RcGRF4和RcGIF2在DL01中呈现出更高的相对表达量并在种子中特异高表达。3’RACE实验和双荧光素酶报告实验表明了miR396b可以特异的识别并靶向切割RcGRF4,酵母双杂交实验也证明了RcGRF4和RcGIF2可以形成功能复合体共同行使功能。在拟南芥中异源超表达RcGRF4显著增加了拟南芥种子的大小,一定程度上证明了miR396b-RcGRF4/RcGIF2模块在种子发育中的重要作用。（4）RcGRF4靶向生长素合成限速酶RcYUC6并激活其表达。对DL01和WH11种子进行转录组测序分析,筛选差异表达基因。生长素合成通路的限速酶RcYUC6在DL01中显著的上调表达,酵母单杂交、双荧光素酶报告实验以及电泳迁移率实验验证了RcGRF4与RcYUC6之间的靶向关系。实验结果表明,RcGRF4可以结合在RcYUC6的启动子区域并激活其表达。（5）生长素影响蓖麻种子发育。通过检测种子中生长素含量,来验证内源生长素的合成不足与WH11的小种子表型之间的关系。结果表明,WH11种子中的生长素含量显著低于DL01种子中的生长素含量。蓖麻生殖生长时期对其花序施加外源生长素,在一定程度上挽救了WH11的小种子表型,扫描电镜和石蜡切片结果也说明了外源生长素促进了细胞的增殖与增大。综上所述,本研究应用经典图位克隆的方式定位到影响蓖麻种子大小的主效位点qSS3,并证明了该位点编码miR396b。此外,miR396b-RcGRF4/RcGIF2-RcYUC6调控通路被首次发现并证明参与调控蓖麻种子的发育,该结果为蓖麻的分子辅助高产育种提供了一个新的思路。更多还原

【Abstract】 Castor bean(Ricinus communis L.)is a plant belonging to the Euphorbiaceae family and is widely cultivated around the world.In East Africa,castor beans primarily grow as perennial trees,while they are mostly found as perennial shrubs in Western Asia.In India and China,castor beans are commonly seen as cultivated or wild shrubs.The seeds of castor beans contain40%to 55%castor oil,making it one of the most important economic tree species in China.Castor oil is rich in hydroxylated ricinoleic acid,with content exceeding 90%.This high purity and hydroxylated characteristic have led to its extensive industrial applications.However,demand for castor beans has significantly increased in recent years in China,while production improvements have been limited,resulting in a growing supply-demand gap.To boost castor bean production,an effective approach is to integrate modern molecular biology into the traditional breeding process for genetic improvement.Recent studies have identified quantitative trait loci associated with seed size in castor beans,but the molecular mechanisms regulating seed size remain unclear.This research aims to elucidate the genetic and molecular mechanisms underlying castor bean seed size,delve into the regulatory mechanisms of seed formation,and provide important guidance and theoretical basis for the genetic improvement of castor beans.The main experimental results of this study are as follows.(1)Mapping the main QTL(qSS3)affecting the size of castor seeds.A genetic segregating population was constructed by hybridizing the large-seed cultivar DL01 from Inner Mongolia with the small-seed wild type WH11 from Ethiopia.Cytological analysis via scanning electron microscopy and paraffin sections revealed that the seed coat and endosperm cells of DL01 were larger and more numerous,indicating that seed size variation is due to changes in cell number and size.A subset of 240 F2 individuals was selected for QTL mapping,leading to the identification of a major QTL(qSS3)regulating seed length,width,and weight on Chr03 between Rc-1-3 and Rc1-1 markers.BSA(Bulked segregant analysis)confirmed the QTL mapping accuracy and further narrowed its interval.Furthermore,fine mapping using 487 extreme phenotypes individuals localized the target gene to a 180 kb region between Rc-1-30.97 and Rc-1-31.15.(2)qSS3 encodes miRNA396b.Genetic analysis of the mapped region revealed that mutations in annotated genes were not conserved among resource varieties.Sequence alignment showed a 17.6 kb structural variant between the parents,with PCR analysis indicating that larger-seed accessions lacked this 17.6 kb fragment.Gene annotation identified a non-coding RNA member,miR396b,and RT-q PCR results showed no expression of miR396b precursor or mature sequence in DL01.(3)miR396b-RcGRF4/RcGIF2 module participate in the development process of castor seeds.11 conserved target genes of miR396,specifically Growth Regulation Factors(GRFs),and 2 auxiliary factors,GRF-Interacting Factors(GIFs),were identified in the castor genome.Relative expression analysis of RcGRFs and RcGIFs across various tissues revealed that RcGRF4 and RcGIF2 had higher expression levels in DL01,particularly high expression in seeds.3’RACE and dual-luciferase assays demonstrated that miR396b specifically recognizes and cleaves RcGRF4.Yeast two-hybrid experiments further confirmed that RcGRF4 and RcGIF2,as an auxiliary factor,could form a functional complex to jointly exert their effects.Overexpression of RcGRF4 in Arabidopsis significantly increased seed size,providing evidence for the important role of the miR396b-RcGRF4/RcGIF2 module in seed development.(4)RcGRF4 targets the growth hormone synthesis rate limiting enzyme RcYUC6 to activate its expression.To further elucidate how RcGRF4 influences seed development,transcriptome sequencing was performed on seeds from DL01 and WH11 to identify differentially expressed genes.The rate-limiting enzyme in the auxin biosynthesis pathway,RcYUC6,was significantly upregulated in DL01.Yeast one-hybrid,dual-luciferase assays,and electrophoretic mobility shift assays validated the targeting relationship between RcGRF4 and RcYUC6.The results indicated that GRF4 binds to the promoter region of RcYUC6 and activates its expression.(5)Auxin affects the development of castor seeds.Additionally,the content of auxin in the seeds was measured to investigate the relationship between insufficient endogenous auxin synthesis and the small seed phenotype of WH11.The results indicated that the auxin levels in WH11 seeds were significantly lower than those in DL01 seeds.During the reproductive growth phase of the castor bean,the application of exogenous auxin to the inflorescences partially rescued the small seed phenotype of WH11.Scanning electron microscopy and paraffin sectioning results also demonstrated that exogenous auxin promoted cell proliferation and enlargement.In summary,this study employs classical map-based cloning to identify the major QTL qSS3 affecting castor seed size,encoding miR396b.Additionally,the miR396b-RcGRF4/RcGIF2-RcYUC6 regulatory pathway was discovered and demonstrated to play a role in seed development,offering new insights for molecular-assisted breeding strategies to improve castor yield.更多还原