【作者】 李敏；

【导师】 周瑞阳；

【作者基本信息】 广西大学 ， 作物遗传育种， 2020， 博士

【摘要】 棉花（Gossypium hirsutum L.）是世界上最主要的天然纤维作物之一,也是重要的油料作物。棉花的杂种优势十分显著,但目前棉花细胞质雄性不育（Cytoplasmic male sterility,CMS）种质资源匮乏,利用的CMS系主要为哈克尼西棉细胞质。其细胞质供体为二倍体,细胞核供体为四倍体,因质核互作严重不协调,恢复系少,难以推广利用。为了解决远源杂交核置换回交所致的质核严重不协调问题,亟需选育质核同源CMS系。本研究基于陆地棉细胞核雄性不育两用系“洞A”的转录组测序结果,筛选到一个在不育株和可育株差异表达的基因GhbZIP1,并将其转入一个性状优良的陆地棉栽培种J4B中。在获得转基因雄性不育突变株后,将其与J4B进行饱和回交成功创制出了CMS系J4A。随后对J4A进行形态学和细胞学观察、线粒体基因组水平、全转录组水平和生化指标测定分析,得出以下主要结果:（1）形态学观察发现,与J4B相比,J4A花器官缩小、花柱突出、花药干瘪而不开裂,败育类型为无花粉型。（2）花粉败育特征观察发现,J4A的花药败育发生在减数分裂期,表现为在花药发育的整个过程中绒毡层细胞不发生降解,无四分体结构。亚细胞超微结构观察发现:J4A花药四分体时期绒毡层细胞的线粒体内嵴模糊。（3）线粒体重测序结果显示:3个CMS系（J4A-1、J4A-2和J4A-3）与其保持系J4B的线粒体基因组差异较小,同源性均高于99%;筛选到4个与CMS相关的ORFs（orf116b、orf186a-1、orf186a-2和orf305a）。（4）通过Illumina Hiseq 4000测序平台进行转录组测序,结果显示:在检测到的62,270个基因中,共筛选到4,461个差异表达基因（7.16%）,其中2,940个基因上调表达,1,521个基因下调表达。生物信息学分析发现,489个差异表达基因富集在氧化还原反应条目;47个差异表达基因参与糖酵解代谢途径,且下调基因数大于上调基因数。（5）通过Illumina Hiseq 4000测序平台进行miRNA测序,共获得1,293个miRNA,其中已知miRNA 734个,新miRNA 559个;预测到1,009个靶基因和1,054个靶基因位点;筛选到26差异表达miRNA。根据miRNA与其靶基因负调控关系,将m RNA测序结果与miRNA测序结果进行关联分析,筛选到一对可能与小孢子减数分裂异常有关的靶基因对:ghi-MIR7484-10/MAPKK6。（6）花药发育3个时期（花粉母细胞时期、减数分裂期和单核期）主要生化指标检测。活性氧（Reactive oxygen species,ROS）代谢相关生化指标（SOD、Mn-SOD、POD、CAT、H₂O₂和MDA）测定结果显示,在减数分裂期和单核期,J4A的SOD、Mn-SOD、POD和CAT酶活均显著低于J4B,H₂O₂和MDA含量均显著高于J4B。推测由于减数分裂期抗氧化酶（SOD、Mn-SOD、POD和CAT）活性降低,活性氧清除能力降低,导致活性氧（H₂O₂和MDA）积累。糖代谢相关生化指标（蔗糖、淀粉、可溶性糖和果糖）测定发现,在花药发育的减数分裂期和单核期,J4A花药中的蔗糖含量均高于J4B;而淀粉、可溶性糖和果糖含量均低于J4B。推测由于蔗糖发生积累,使葡萄糖和果糖的生成减少,进而导致淀粉含量降低和可溶性糖含量下降。更多还原

【Abstract】 Cotton（Gossypium hirsutum L.）is one of the main natural fiber crops in the world,and also an significant oil crop.The heterosis of cotton is very significant,but cotton cytoplasmic male sterility（CMS）germplasm resources are scarce at present.The CMS lines used are mainly cotton cytoplasmic.Because the cytoplasmic donor is diploid,and the nuclear donor is tetraploid donor,the cytoplasmic nuclear interaction is seriously discordant,and there are few restorer lines.In order to solve the problem of serious cytoplasmic nucleus disharmony caused by nucleus replacement backcross in distant hybridization,it is necessary to breed CMS lines with homologous cytoplasm and nucleus.In this study,based on the transcriptome sequencing results of the genic male sterile line"Dong A"in upland cotton,a GhbZIP1 with differential expression in sterile and fertile plants was screened and transferred into an upland cotton cultivar J4B with excellent characters.After obtaining the transgenic male sterile mutant,the CMS line J4A was successfully developmented by saturated backcrossing with J4B.After that,the morphological and cytological observation,mitochondrial genome,whole transcriptome analysis and biochemical characteristics of J4A were carried out,the main results are as follows:（1）Morphological observation showed that the flowers organ of J4A were smaller,the style was prominent and the anthers were shriveled without dehiscence,and the abortion type of J4A belonged to non-pollen type.（2）Observation of pollen abortion characteristics showed that the anther abortion stage of J4A was from microspore pollen mother cell stage to tetrad stage（meiosis）,which showed that tapetum cells did not degrade and no tetrad structure appeared during the whole period of anther development.The ultrastructural observation of anther cells showed that the inner ridges of mitochondria in tapetal cells of J4A were fuzzy at tetrad stage.（3）The mitochondrial genome of three CMS lines（J4A-1,J4A-2 and J4A-3）and their maintainer line J4B were dwarfed,and their similarity was beyond 99%.Four ORFs（orf116b,orf186a-1,orf186a-2 and orf305a）with CMS related gene characteristics were found in the CMS lines J4A-1,J4A-2 and J4A-3,which were identified as CMS related candidate genes.（4）The anthers at abortion stage were sequenced by Illumina Hiseq 4000 sequencing platform.The results of m RNA transcriptome sequencing showed that 4,461 differentially expressed genes（7.16%）were screened out of62,270 genes detected,among which 2,940 genes were up-regulated and 1,521genes were down-regulated.Bioinformatics analysis showed that 489differentially expressed genes were enriched in redox reaction,47 differentially expressed genes were involved in glycolysis pathway,and more down regulated genes were found.By analyzing the regulatory network of the selected differentially expressed genes,the screening scope of cotton CMS key genes was narrowed.（5）Mi RNAs were sequenced by Illumina Hiseq 4000 sequencingplatform,a total of 1,293 miRNAs were obtained,including 734 known miRNAs and 59 new miRNAs,and there were 26 differentially expressed miRNAs.A pair of target genes（ghi-MIR7484-10/MAPKK6）that may be related to the abnormal meiosis of microspore were screened by miRNA and m RNA association analysis.According to the regulatory relationship between miRNA and its target genes,the correlation analysis of m RNA sequencing results and miRNA sequencing results can more accurately screen the key genes involving the regulation of CMS more accurately.（6）Biochemical indexes were determined.The biochemical indexes （SOD,Mn-SOD,POD,CAT,H₂O₂and MDA）related to the metabolism of reactive oxygen species（ROS）were measured at three important stages of anther development:pollen mother cell stage,meiosis stage and monocyte stage.The results showed that the activities of SOD,Mn-SOD,POD and CAT of J4A were lower than those of J4B,and the contents of H₂O₂and MDA were significantly higher than those of J4B at meiosis stage and monocyte stage.It is speculated that the decrease of antioxidant enzyme activities（SOD,Mn-SOD,POD and CAT）during meiosis leads to the accumulation of reactive oxygen species（H₂O₂and MDA）in J4A anthers.Sugar metabolism related biochemical indexes（sucrose,starch,soluble sugar and fructose）were measured at three important stages of anther development:pollen mother cell stage,meiosis stage and mononuclear stage.The results showed that the sucrose content of J4A was higher than that of J4B at meiosis stage and mononuclear stage,and the contents of starch,soluble sugar and fructose were lower than that of J4B at the three important stages of anther development.It was speculated that the abnormal accumulation of sucrose led to the decrease of glucose and fructose,which resulted in the decrease of starch content and soluble sugar content.更多还原