【作者】 刘斌美；

【导师】 吴跃进； 童继平；

【作者基本信息】 安徽农业大学 ， 生物物理学， 2005， 硕士

【摘要】 粳型水稻 Y98149 是从离子束诱变后代中获得的显性半矮秆突变体。本研究利用RAPD 方法在水稻显性半矮秆突变体 Y98149 和其野生型 Y98148 之间进行了 RAPD标记筛选工作,从基因组水平上揭示了两者的差异程度,为突变的基因提供分子生物学证据。对稳定的多态性 RAPD 标记进行了 SCAR 标记转化,成功地获得了与 3 个与水稻显性半矮秆基因连锁的 SCAR 标记,并对克隆的 RAPD 标记进行了相关序列分析,结果表明:1、利用 1100 个 10 碱基寡核苷酸随机引物筛选 Y98148 和 Y98149,共获得 4 个稳定的多态性引物:S1041、S1075、S1076 和 S1272;2、通过计算得到突变体 Y98149 与其野生型 Y98148 的相似系数为 0.9995,遗传距离为 0.0005,表明 Y98148 与 Y98149 遗传背景具有极高的相似性,为证明二者是近等基因系提供了分子水平证据;3、通过克隆、测序,成功地得到 3 个 RAPD 标记的序列,分别命名为 S1041₅₂₅、S1076₅₄₉和 S1272₄₀₃,提交到 DDBJ 数据库,接受号为 AB207912、AB207913 和 AB207914;根据所测的序列设计特异性引物,并在近等基因系间进行了 PCR 分析,成功地将RAPD 标记 S1041₅₂₅、 S1076₅₄₉、S1272₄₀₃转化为 SCAR 标记 SCS1041₄₉₈、 SCS1076₅₁₀ 、SCS1272₃₈₈;4、利用 Y98148×Y98149 的 F₂ 分离群体 384 个单株对 SCAR 标记进行了验证和连锁分析,SCAR 标记 SCS1041₄₉₈ 、SCS1076₅₁₀ 和 SCS1272₃₈₈PCR 扩增结果与田间农艺性状吻合率分别为 88.8%、93.2%和 85.2%,与显性半矮秆基因的遗传距离分别为12.6cM、7.5cM 和 16.3cM;5、通过序列分析和同源性搜索,片段 S1041₅₂₅ 和 S1076₅₄₉ 分别编码 111 和 90 个氨基酸序列的多肽,其中 S1076₅₄₉ 的多肽序列与原核生物的蔗糖磷酸转移酶基因有 94%的同源性,与一些植物中的膜蛋白也有相当高的同源性。片段 S1272₄₀₃的核苷酸序列与水稻第七染色体上的两个 BAC 克隆 B1249D05（AP006451）和 OJ1212_C12（AP005604） 同源性为 99%,B1249D05 和 OJ1212_C12 是重叠克隆,S1272₄₀₃ 位于二者重叠区域中,进而初步将水稻显性半矮秆基因定位于第七染色体上。 本研究通过分子生物学方法鉴定了新的水稻显性半矮秆基因,获得了研究该基因的有利的分子标记,为该基因的利用提供了有用的信息。水稻显性半矮秆基因初步定位的完成,为下一步精细定位和图位克隆该基因奠定了坚实的基础。更多还原

【Abstract】 Through ion beam mutagenesis, a new dominant semi-dawrfism mutant was obtained.In this study, DNA frigerprints of Y98149 and its wild type Y98148 were obtained withRAPD technology, which has revealed the discrepancy degree of genome and offeredmolecular evidence of mutanted gene. Four stable molypophic RAPD primers were found,which produced four special RAPD bands. Three special RAPD bands wererecovered ,purified and inserted pUCm-T vector, and then transferred into E.coli. JM109.Positive colonies were identified for sequencing. According to the obtainedsequences,18²4-mer specific primers were designed as SCAR primers, and theSCAR-PCR reaction conditions were optimized. Consequently, RAPD markers weresuccessfully converted into SCAR markers and the sequences of the RAPD fragments wereanalyzed.The results indicated:1. A totle of 1100 arbitrary 10-mer oligonucleotide primers were screened on the genomicDNA of Y98149 and Y98148 using RAPD technology. Among which, four polymorphicprimers were selected: S1041、S1075、S1076 and S1272;2. Through caculating, the similar co and genetic distance of Y98148 and Y98149 are0.9995 and 0.0005, respectively, which indicates that Y98148 and Y98149 have the highlyhomology genetic background and offer molercular evidences to confirm they are NILs;3. The three special RAPD bands were cloned and sequenced and three sequences wereobtained, which were named S1041₅₂₅、S1076₅₄₉ and S1272₄₀₃, respectively. The sequenceswere submitted to DDBJ and the accession numbers were AB207912、AB207913 andAB207914.Three sets of SCAR primers were designed according to the obtainedsequences. The SCAR primers were successfully amplified by PCR method betweenY98148 and Y98149. Consequently, RAPD markers S1041₅₂₅、S1076₅₄₉ and S1272₄₀₃ weresuccessfully converted into SCAR markers SCS1041498、SCS1076510 and SCS1272388.4. F₂ progeny with 384 individuals of Y98148×Y98149 was analyzed to map SCARmarkers in relationship to this gene and verify SCAR markers. The result indicated that thegenetic distances of SCS1041498、SCS1076510 and SCS1272388 to this gene were 12.6cM、7.5cM and 16.3cM and the selected accuracy were 88.8%、93.2%和 85.2%, respectively.5. S1041₅₂₅ and S1076₅₄₉ each has one ORF（open reading fragment）,which encoded 111and 90 AA resdues, respectively. The sequence of S1076₅₄₉ has high homologous with themRNA sequence and amino acid sequence of sugar phosphotransferase component II B , in94% homologenity of amino acid sequence in the region between 245th and 517thnucleotide sequence of it. It also had high homologenity of amino acid sequence with somemembrane protein of plants. The blast result indicated the nucleotide sequence of markerS1272403 was 99% identical with a part of two BAC clones B1249D05（AP006451）andOJ1212_C12（AP005604）on chromosome 7 and single copy in rice genomic DNA.B1249D05 and OJ1212_C12 has overlay area of 23Kb, marker S1272403 lies in this area.So this new semi-dwarf gene was located on chromosome 7 preliminarily. To summarize, this study maked a great progress in molecular markers linked to thenew dominant semi-dwarf gene and identified the mutant gene with molecular biologymethods, which has offered useful information for the use of this gene. The preliminarilygene location was completed, which established the solid foundation of positional cloningof this gene.更多还原