【作者】 张征锋；

【导师】 郑用琏；

【作者基本信息】 华中农业大学 ， 遗传学， 2006， 博士

【摘要】 植物杂种优势的利用推动了农作物产量、品质的大幅度提高。玉米是世界上最早将CMS及育性恢复系统应用于杂种优势育种的作物之一。CMS及育性恢复（restorer of fertility，Rf）系统为实践中杂交种的生产提供了巨大的便利，不仅能节省大量人工去雄，而且可减少因去雄不净所造成的混杂，充分发挥杂种优势的作用；同时CMS是研究核质互作分子机理的重要模式性状。玉米S-CMS系的育性由线粒体上育性相关基因和核基因（Rf3／rf3）共同决定。S组CMS是玉米三个主要细胞质雄性不育组群中最大的一个组，其育性表现较不稳定，影响了其在育种实践中的应用。为了进一步发挥玉米S-CMS在杂优利用中的作用，克隆育性恢复基因Rf3并阐明育性恢复机理显得尤其重要。图位克隆是一种有效的基因克隆方法，其成功的基础需要有目标基因区域高分辨率的遗传图谱，前人对核育性恢复基因Rf3已进行了定位，但所定位的遗传图距较远，远不能满足图位克隆的需要。本研究拟在本室前期对玉米S-CMS研究的基础上，以Rf3／rf3近等基因系S-MO17^Rf3Rf3／S-MO17^rf3rf3为材料，利用近等基因系间Rf3区段的分子标记信息，结合AFLP分子标记开发技术对Rf3进行精细定位，筛选与其紧密连锁或共分离的分子标记。同时利用cDNA-AFLP技术，在转录水平上研究近等基因系S-MO17^Rf3Rf3／S-MO17^rf3rf3不同发育时期基因的差异表达谱，以期鉴定玉米S-CMS育性恢复相关基因。主要研究结果如下：1．为开展玉米S-型细胞质雄性不育核恢复基因的精细定位，选用恢复基因近等基因系（NIL）材料构建回交一代群体(S-Mo17^rf3rf3×S-Mo17^Rf3Rf3)×N-Mo17^rf3rf3。对所有单株进行田间花粉观察与室内镜检，二者结果一致，BC₁F₁群体中178株被划分为可育株；165株为不育株，符合1：1分离比，在本研究材料中该育性恢复性状受一对基因控制。2．选用Rf3所在的2.09bin区域的13对SSR标记进行双亲间多态性筛选，结果表明，仅标记UMC2184在双亲的2.09bin区间存在多态性，该标记被用于鉴定分析群体中的基因型并作为其它标记的锚定位点。采用EcoRI+MseI、PstI+MseI及EcoRI+PstI三种酶切方式共528对选择性扩增引物组合在双亲及DNA池中进行AFLP分析，共产生大约29，000条带纹。选择差异性引物组合进一步对可育／不育两DNA池的单株DNA进行检测，最终发现E3P1、E7P6、E10P7及E12M7共4个标记在个体中的带型与相应亲本一致，其序列相互不重复，代表4个不同的片段，作为候选的紧密连锁标记进一步在BC₁F₁群体中检测基因型。3．回收标记E3P1、E7P6、E10P7及E12M7对应的差异扩增片段，最终将AFLP标记E3P1及E12M7分别转化为共显性标记CAPSE3P1及显性标记SCARE12M7。4．用筛选、验证所得到的一个SSR标记UMC2184、2个AFLP标记E7P6、E10P7及2个基于PCR的标记SCARE12M7、CAPSE3P1构建了恢复基因Rf3区域的遗传连锁图，图谱全长11.1cM，平均间距为2.22cM。将Rf3定位于AFLP标记E7P6与SCAR标记SCARE12M7之间，遗传图距分别为0.9cM和1.8cM。为恢复基因Rf3的图位克隆奠定了基础。5．对分子标记SCARE12M7序列进行BLASTx分析及相关图谱比较，发现其与已定位的玉米抗丝黑穗病QTL紧密连锁或共分离。讨论了该标记用于玉米S-CMS育性恢复及丝黑穗抗性双性状聚合选择的潜力的可能性。6．利用64对引物组合对S-Mo17^rf3rf3和S-Mo17^Rf3Rf3苗期叶片及抽雄早期花粉进行cDNA-AFLP分析。共显示TDFs约2500条，片段大小介于80-800bp之间。其中差异表达片段分为质的表达差异和量的表达差异两种类型。质的差异表达带纹共122条，分为7大类型。对在恢复系叶片和花粉中表达但在不育系中沉默和仅在恢复系花粉中表达的33条片段进行序列分析和同源性比较，发现其分别代表：蛋白质降解与贮存相关基因3个，约占9％；转录调控相关基因5个，约占15％；信号转导相关基因4个，约占12％；能量代谢相关基因3个，约占9％；细胞生长与分裂相关基因3个，约占9％；转座子相关基因1个，约占3％；未知蛋白编码基因9个，约占28％；新发现的序列5个，约占15％。其中26S proteasome regulatory particle与本室前期利用cDNA芯片的研究结果表现相同的表达趋势。用电子拼接及在基因组cDNA中扩增、测序验证：该基因全长1329bp，编码443个氨基酸组成的多肽。经Clustal W比较分析，玉米26S proteasome regulatory subunit与水稻、拟南芥、人类中同源蛋白质序列相似性分别达91％、77％、41％。7．在已测序的差异表达片段中，与转录调控相关的基因占15％；对序列E1同源性搜索发现：其代表基因编码蛋白包含PPR模体。对该基因在恢复系及不育系苗期叶片、抽雄期叶片、抽雄期花粉、苗期根4个材料进行RT-real-time PCR分析，结果表明：该基因在不育系和恢复系两种材料的不同组织部位存在明显的表达模式差异，在可育材料的抽雄期花粉中，该基因表达量最高，为幼苗叶片及抽雄期叶片表达量的4倍，在根部表达量达到最低；而在不育材料抽雄期花粉中表达量比抽雄期叶片略低，为幼苗叶片表达量的2倍，根部表达量为最低水平。研究结果暗示该PPR相关基因可能与育性恢复有关。更多还原

【Abstract】 Utilization of heterosis has facilitated the improvement of yield and quality of crop.Cytoplasmic male sterility （CMS） has been used extensively in breeding for theproduction of a range of F₁ hybrid crops because of its advantages on decreasing the costof labor and improving seed quality. Moreover, the system of CMS/Rf is a perfect modelfor studying molecular mechanism of the interaction between nucleolus and cytoplasm.Maize is one of the pioneer crops whose heterosis is enhanced through this system. Theunstanable fertility of maize S-CMS limits its utility in practice. For using CMS in maizeproduction by the greatest extent, it is necessary to theoretically elucidate the mechanismsof steility and restoration of fertility. Map-based cloning is an effective method of genecloning and the genetic linkage map in the vicinity of target gene with high density is thebasis to map-based cloning. Several attemps have been made to tag Rf3 with molecularmarkers, however, the distances between them and Rf3 are not yet near enough to clonethis gene.In the present study, we screened AFLP and SSR markers polymorphic between nearisogenic lines （NILs）. A BC₁F₁ population from a pair of NILs with different Rf3 locus wasconstructed for linkage analysis. Additionally, the differentially expressed profile indifferent tissues and times of S-MO17^Rf3Rf3/S-MO17^rf3rf3 was analyzed throughcDNA-AFLP technology. And some primary results are listed as follows:1. The BC₁F₁ generation yielded 178 semi-fertile （having 50% or more stainablepollen） and 165 sterile plants. This segregation ratio fits a monogenic Mendelianinheritance model of 1 fertile （Rf3rf3）: 1 sterile （rf3rf3）, it confirmed that fertilityrestoration was conditioned by one dominant restorer gene in these maize materials.2. One of 13 SSRs on chromosome 2.09 bin, UMC2184, was detected to showcodominant polymorphism between parents and bulks. Thus UMC2184 were employed toanalyze linkage relationship between itself and rf3 and perform anchor point to othermarkers. The total 528 AFLP primer combinations （PC） were screened for theirpolymorphism between parents and bulks. Only 4 polymorphic fragments from E3P1,E7P6, E10P7 and E12M7 were confirmed as candidate markers tightly linked to Rf3 andanalyzed subsequently using the BC₁F₁ population.3. Through designing primer and detecting in the parents and bulks, AFLP markersE3P1 and E12M7 are converted into codominant CAPS and dominant SCAR marker,respectively.4. A linkage map was constructed around the Rf3 locus, which was mapped on the distal region of chromosome 2 long arm with the help of SSR marker UMC2184. Thetotal size of this map is 11.1 cM and E7P6 and SCARE12M7 delimited a 2.7cM windowwith 0.9 cM and 1.8 cM from Rf3, respectively5. BLASTx analysis showed that the marker SCARE12M7 was high homologouswith resistance protein. Through comparing and assembling the mapped results of thisstudy and others, we assumed that marker SCARE12M7 was tightly linked orcosegregant with gene resistant to head smut. The potential of SCARE12M7 in assessingand selecting both Rf3 gene and gene resistant to head smut was discussed.6. Differential expression of genes in young leaves and pollen between S-Mo17^rf3rf3and S-Mo17^Rf3Rf3 was investigated via cDNA-AFLP. 2500 fragments distributing80-800bp were displayed by 64 primer combinationa. 33 TDFs which expressed only infertile pollen or expressed in fertile materials specifically were sequenced successfully.By BLAST analysis, these sequences were assigned into 8 categories based on theirbiology function: protein synthesis and fate （9%）; regulation on the transcript level （15%）;signal transduction （12%）; energy metabolism （9%）; cell developing and dividing （9%）;transposable element （3%）; unknown function protein （28%）; novel ESTs （15%）.7. Through comparing functions of 33 differentially expressed genes with thosederived from analysis of cDNA-microarray （Zhang, 2004）, protein synthesis and faterelative genes account for 9% and 13%, respectively. Among them, 26S proteasomeregulatory particle expressed high in fertile pollen in two studies above mentioned.Through in silico cloning strategy, the candidate cDNA sequence of maize 26Sproteasome regulatory particle was obtained, it covered 1451bp in which coding sequencewas 1329bp and coded 443 amino acids. Clustering analysis using the software of ClustalW shows that the homologic rate between 26S proteasome regulatory subunit5 in maizeand its homology in rice, arabidopsis, human is 91%, 77% and 41%, respectively.8. Regulation on the transcript level relative genes occupy 15% in the sequenceddifferentially expressed genes. BLASTx analysis for sequence E1 indicates therepresented gene coding protein containing PPR model. Through expressed patternanalysis in different tissues and times by RT-real-time PCR, this gene expressed in thepollen on the highest level for S-Mo17^Rf3Rf3 but on the highest level in the leaves oftasseling time for S-Mo17^rf3rf3.Differentially expressed patter of this gene indicates the itsrelationship with between it and fertility restoring in maize S-CMS.更多还原