【作者】 李琦；

【导师】 徐辰武； 李鹏程；

【作者基本信息】 扬州大学 ， 作物栽培学与耕作学， 2023， 硕士

【摘要】 玉米是世界上重要的农作物之一,提高产量是玉米育种的重要目标。根系作为植物重要的器官之一,不仅可以吸收水分和养分,还可以对外界环境变化做出响应。生长素是植物生长发育所必需的激素,参与植物体内的细胞生长、根系伸长、维管组织的发育等多个过程。SAURs(Small Auxin-Up RNAs)基因家族是生长素早期响应的三大基因家族之一,解析SA URs基因家族成员的生物学功能将为玉米遗传育种提供重要的基因资源。利用课题组前期完成的357份玉米自交系的根系转录组数据,对37609个基因与24个根系性状进行相关分析,发现ZmSA UR21的表达量与多个根系性状显著相关,推测ZmSAUR21可能调控苗期玉米根系的生长发育。本文通过ZmSAUR21表达模式分析、遗传材料的创制、遗传材料的表型验证、转录组分析,初步解析了 ZmSA UR21在玉米根系发育过程中的生物学功能,主要研究结果如下:1.利用357份自交系根系转录组数据和苗期根系表型数据进行相关分析,发现ZmSAUR21的表达量与主胚根总根长(TPRL)、种子根总根长(TSRL)、总根长(TRL)等性状显著相关。对SAURs基因家族成员进行进化树分析和序列一致性分析,发现SAURs都有一个生长素诱导结构域,并且ZmSAUR21的生物学功能可能和OsSAUR4基因生物学功能相似。对ZmSAUR21的表达模式进行分析,发现在不同组织中,ZmSAUR21在玉米根系和胚乳中特异表达,并且在根系中表达较高,在种子萌发三天和七天后的主胚根分生区和伸长区中优势表达;在不同发育时间,ZmSAUR21在萌发后两天和四天后表达量较高,随后持续下降;在不同胁迫下,ZmSAUR21基因对温度的响应最大;在不同激素处理下,ZmSAUR21的表达水平对生长素的响应最明显;在不同空间上,在成熟区表达量最高。通过GUS染色证实ZmSAUR21在根系上特异表达,并且在根尖和侧根上表达量最高。亚细胞定位分析ZmSAUR21在细胞膜和细胞核上表达。2.为了验证ZmSAUR21的基因功能,我们构建ZmSAUR21的过表达株系和敲除突变体株系,对转基因材料进行表型分析发现:敲除突变体KO1、KO2的主胚根长、平均种子根长、总根长等性状显著低于野生型;过表达材料OE1的主胚根长,平均种子根长、总根长等性状显著高于野生型。对ZmSA UR21敲除突变体的根系进行半薄切片发现:相比于野生型,敲除突变体的根系分生区、伸长区和成熟区细胞体积均显著变小,细胞长度显著变短;过表达材料的细胞体积均显著变大,细胞长度显著变长;对转基因材料测定H+-ATPase活性,过表达材料的H+-ATPase活性显著升高,敲除突变体的H+-ATPase活性显著降低。3.为了解析ZmSA UR21调控苗期玉米根系发育可能的分子机制,对ZmSA UR21敲除突变体KO1、KO2和野生型的根系进行转录组测序。对比野生型和KO1、野生型和KO2的基因表达水平,共鉴定到989个差异表达基因;基因功能富集分析表明差异表达基因主要参与了激素介导的信号传导、生长素代谢、IAA-Ala结合水解酶活性等生物学过程;在生长素信号传导通路上鉴定到多个显著差异基因包括 ZmARF14/19、ZmAUX/IAA 7/23、ZmSAUR4/15、ZmPIN19等;鉴定到6个ZmPP2C基因和3个H+-ATP酶基因的表达存在显著差异。综上所述,本研究初步阐明ZmSAUR21可能通过激活H+-ATP酶活性,促进细胞膨大来调控玉米根系的伸长。研究结果为揭示玉米根系发育的分子机制和根系遗传改良奠定了基础。更多还原

【Abstract】 Maize is one of the most important crops in the world,and improving yield is an important goal of maize breeding.As one of the important organs of plants,roots can not only absorb water and nutrients,but also respond to external environmental changes.Auxin is an essential hormone for plant growth and development.It is involved in many processes in plants,such as cell growth,root elongation,and vascular tissue development.The Small Auxin Up RNAs(SAURs)gene family is one of the three early auxin responsive gene families.Understanding the biological functions of SAURs genefamily members will provide important genetic resources for maize breeding.In our previous study,RNA-seq of root was conducted in 357 maize inbred lines,and the correlation analysis was conducted between expression level of 37,609 genes and 24 root traits.It was found that the expression of ZmSA UR21 was significantly correlated with multiple root traits,suggesting that ZmSA UR21 may regulate the growth and development of maize root at seedling stage.In this study,the biological function of ZmSAUR21 in maize root development was preliminarily analyzed through the expression pattern analysis,genetic material creation,phenotype verification of genetic material,and transcriptome analysis.The main research results are as follows:1.Correlation analysis of root transcriptome and root traits of 357 inbred lines at seedling stage found that the expression of ZmSAUR21 was significantly correlated with the total length of primary root(TPRL),total length of seminal root(TSRL),and total root length(TRL)using the.The phylogenetic tree analysis and sequence identity analysis of SA URs gene family members showed that SAURs all have an auxin-induced domain,and the biological function of ZmSA UR21 may be similar to that of OsSA UR4 gene.Analysis of the expression pattern of ZmSAUR21 showed that ZmSAUR21 was specifically expressed in the root and endosperm of maize,and dominant expression in the main radicle meristematic zone and elongation zone after three and seven days of seed germination.At different developmental times,the expression of ZmSAUR21 was higher after two and four days of germination.Under different stress,ZmSA UR21 gene showed the greatest response to temperature.Under different hormone treatments,the expression level of ZmSAUR21 showed the most obvious response to auxin.In different root region,the expression level was highest in the mature zone.GUS staining confirmed that ZmSAUR21 was specifically expressed in roots,with the highest expression in apical and lateral roots.The subcellular localization analysis showed that ZmSAUR21 was expressed in the cell membrane and nucleus.2.In order to verify the function of ZmSAUR21 gene,we constructed the ZmSAUR21 overexpression line and knockout mutant line.Phenotypic analysis of transgenic lines showed that the primary root length,average length of seminal root,and total root length of knockout mutant KO1 and KO2 were significantly lower than those of the wild type.The primary root length,average length of seminal root,and total root length of the overexpressed material OE1 were significantly higher than those of the wild type.Compared with the wild type,the cell volume of the root meristem,elongation,and maturation zone was significantly smaller,and the cell length was significantly shorter in mutant.The cell volume was significantly larger and the cell length was significantly longer in the overexpressed lines.The H+-ATPase activity was measured in the transgenic material,and the H+-ATPase activity was significantly increased in the overexpression material and decreased in the knockout mutant.3.To understand the possible molecular mechanism of ZmSAUR21 regulating maize root development at seedling stage,transcriptome sequencing was performed in roots of ZmSA UR21 knockout mutants KO1,KO2 and wild type.Comparing the gene expression levels between wild type and KO1,wild type and KO2,989 differentially expressed genes were identified totally.Gene function enrichment analysis showed that the differentially expressed genes were mainly involved in biological processes such as hormone-mediated signal transduction,auxin metabolism,and IAA-Ala binding hydrolase activity.In the auxin signaling pathway,a number of significant differential genes were identified,including ZmARF14/19,ZmAUX/IAA7/23,ZmSAUR4/15,ZmPIN19,etc.Significant differences in the expression of six ZmPP2C genes and three H+-ATPase were identified.Taken together,this study initially clarified that ZmSAUR21 may regulate maize root elongation by activating H+-ATPase activity and promoting cell enlargement.Our results lay the foundation for revealing the molecular mechanism of maize root development and root genetic improvement.更多还原