【作者】 周倩；

【导师】 李绅崇； 刘芳；

【作者基本信息】 云南大学 ， 农艺与种业（专业学位）， 2024， 硕士

【摘要】 非洲菊(Gerbera hybrida)是世界五大鲜切花之一,在全球花卉市场上具有很高的商业价值,也是云南花卉产业的主打产品。随着消费升级和需求的多样化,传统花型的非洲菊已难以满足市场和人们的审美需要,而拉丝系列的非洲菊越来越受消费者的欢迎。因此,挖掘调控非洲菊丝状花瓣的关键基因,分析非洲菊丝状花瓣的形成机理,建立非洲菊高效的遗传转化体系,有助于为非洲菊花型分子设计育种提供理论基础和技术支持。为此,本研究结果如下:通过WOX蛋白家族保守结构域分析,在非洲菊高质量基因组数据中比对获得了 16个非洲菊WOX蛋白家族成员序列。与拟南芥和水稻的WOX家族成员进行多重序列比对后,绘制出了非洲菊、拟南芥和水稻WOX家族成员的系统进化树。非洲菊WOX家族成员在进化中可分为古老(Ancient)分支、中间(Intermediate)和现代(Modern)分支。多数(12/16)非洲菊WOX家族基因属于现代分支,中间分支包含了3个成员,而古老分支只有一个WOX家族成员。拟南芥和矮牵牛的研究证明WOX1和WOX3共同调控花瓣融合的生长发育,其功能缺失导致丝状花瓣的形成。在非洲菊基因组中,我们鉴定了3个相关基因:Ger011293为AtWOX1的同源基因,而AtWOX3在非洲菊中有两个同源基因,分别为 Ger006225(GhWOX3a)和Ger045807(GhWOX3b)。通过在拟南芥wox1 wox3双突变体中分别过表达非洲菊WOX1、WOX3a和WOX3b基因,均不同程度地恢复了拟南芥突变体的花瓣宽度,弥补拟南芥中花瓣的表型缺陷。通过qPCR表达量验证,在上述三种转基因拟南芥株系中,GhWOX1、GhWOX3a、GhWOX3b的表达量显著高于纯合双突变体woxlwox3的表达量,并且随着表达量的升高,花瓣宽度恢复的也越好。上述研究证实了GhWOX1、GhWOX3a、GhWOX3b在花瓣融合调控中具有较为保守的基因功能,是非洲菊丝状花瓣形成的关键基因。另外,本研究还初步探索了非洲菊中与不定芽再生相关的关键基因。在愈伤组织形成过程中筛选出特异表达的基因,如GhWOX4、GhWOX8、GhWOX11、GhWUS1、Gh WUS2、GhWUS3、GhWUS4。通过基因表达模式分析,上述基因与愈伤组织形成呈正相关,是非洲菊不定芽形成的关键调控候选基因。更多还原

【Abstract】 Gerbera hybrida,ranking among the top five cut flowers globally,holds significant commercial value in the global flower market and is a major product in the flower industry of Yunnan,China.With the upgrading of consumption and the diversification of demands,the traditional flower shape of G.hybrida has become increasingly difficult to satisfy the market and people’s aesthetic needs,and the lace-like series of G.hybrida has gained increasing popularity among consumers.Therefore,identifying the key genes regulating the filamentous petals of G.hybrida,analyzing the formation mechanism of its filamentous petals,and establishing an efficient genetic transformation system for G.hybrida will contribute to providing theoretical basis and technical support for the molecular design breeding of G.hybrida flower types.The main findings of this study are as follows:Through the analysis of conserved domains of the WOX protein family,16 member sequences of the G.hybrida WOX protein family were obtained by aligning with high-quality genome data of G.hybrida.Multiple sequence alignments were conducted with the WOX family members of Arabidopsis thaliana and Oryza sativa,and a phylogenetic tree of the WOX family members of G.hybrida,Arabidopsis thaliana,and Oryza sativa was constructed.The WOX family members of G.hybrida can be divided into ancient,intermediate,and modern branches during evolution.Most(12/16)of the G.hybrida WOX family genes belong to the modern branch,while the intermediate branch contains three members,and the ancient branch has only one WOX family member.Studies on Arabidopsis thaliana and Petunia hybrida have demonstrated that WOX1 and WOX3 jointly regulate the growth and development of petal fusion,and their functional loss leads to the formation of filamentous petals.In the G.hybrida genome,we identified three related genes:Ger011293 as the homologous gene of AtWOX1,and AtWOX3 has two homologous genes in G.hybrida,namely Ger006225(GhWOX3a)and Ger045807(GhWOX3b).By overexpressing the GhWOX1,GhWOX3a,and GhWOX3b genes in the Arabidopsis thaliana woxl wox3 double mutant,the petal width of the mutant was restored to varying degrees,compensating for the phenotypic defects in the petals of Arabidopsis thaliana.qPCR expression verification showed that the expression levels of GhWOX1,GhWOX3a,and GhWOX3b in the above three transgenic Arabidopsis thaliana lines were significantly higher than those in the homozygous double mutant wox1 wox3,and as the expression levels increased,the petal width also recovered better.The above research confirms that GhWOX1,GhWOX3a,and GhWOX3b have conserved gene functions in petal fusion regulation and are key genes in the formation of filamentous petals in G.hybrida.In addition,this study also initially explored the key genes related to adventitious bud regeneration in G.hybrida.Genes specifically expressed during callus formation were screened out,such as GhWOX4,Gh WOX8,GhWOX11,GhWUS1,GhWUS2,GhWUS3,and GhWUS4.Through the analysis of gene expression patterns,the above genes were positively correlated with callus formation and is a key regulatory candidate gene for adventitious bud formation in G.hybrida.更多还原