【作者】 易斌；

【导师】 傅廷栋； 涂金星；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 杂种优势利用是提高油菜产量、品质和抗性的有效途径。雄性不育是油菜杂种优势利用主要的途径,但是对于雄性不育机理的研究却远远滞后。甘蓝型油菜细胞核雄性不育(GMS)败育彻底,不育性表现稳定,细胞质来源丰富,恢复源广泛,在国内已成为油菜杂种优势利用的重要途径之一。S45AB来源于四川大学,由潘涛等(1988)在Oro中发现的自然突变体衍生而来,经过多代的兄妹交(25代以上)保存,通过杂交试验证明S45A的不育性受两对重叠隐性基因控制(潘涛等,1988)。在近等基因系S45AB中,一个不育位点处于杂合状态,另外一个不育位点处于隐性纯合状态。本研究以S45AB作为基础材料,对处于杂合状态的隐性核不育基因进行了遗传定位和克隆的研究,主要结果如下:1.3年的育性调查结果表明:兄妹交后代的育性分离比稳定在1:1,可育株自交后代的育性比例符合3:1。这一结果进一步证实了在两型系S45AB中控制育性的两个位点中只有一个处于分离状态,我们将这个位点的不育基因命名为Bnms1;另外一个位点处于隐性纯合状态,不育基因命名为Bnms2。2.半薄切片观察发现可育花药和不育花药在减数分裂前没有明显差异。四分体时期四分体没有明显差异,不育花药的绒毡层比可育花药的稍厚。在单核花粉期小孢子差异明显,不育小孢子没有花粉外壁的合成,发育长时间停留在单核花粉期并最终解体;不育花药的绒毡层表现为径向扩大,液泡化明显。S45A败育发生的关键时期在四分体至单核花粉期,绒毡层异常、花粉外壁的缺失是导致败育的主要原因。3.应用AFLP与BSA结合的方法分析近等基因系S45AB,共筛选了2560对AFLP引物组合,获得7个与目的基因(Bnms1)连锁的AFLP标记AF1-AF7。用包含310个单株的近等基因系群体将Bnms1基因定位在标记AF3和AF7之间,标记与基因间的遗传距离分别为1.6cM和0.3cM,标记AF1和AF2与基因共分离。4.回收、克隆了以上7个AFLP特异片段,测序结果表明AF1-AF7的精确长度分别为203bp、241bp、211bp、186bp、187bp、284bp和362bp。通过PCR-walking分离AFLP标记的侧翼序列,将AF1、AF3、AF6和AF7分别延长至7,499bp、997bp、1,203bp和656bp,并转化为4个SCAR标记SC1、SC3和SC6和SC7。用1,974个单株的S45AB近等基因系将Bnms1基因定位在标记SC1和SC7之间,标记与Bnms1基因间的遗传距离分别为0.1cM和0.3cM。5.利用DH群体(Zhongyou821×Bao604)为作图群体,构建了一张包含237个标记的遗传图谱,图谱总长1,625.6cM,包括2个RFLP标记,65个RAPD标记,86个SSR标记,84个SRAP标记。利用两个DH群体(Tapidor×Ningyou7、Zhongyou821×Ba0604)的遗传图谱将不育基因Bnms1定位于N7连锁群,并在N7连锁群上找到一个与Bnms1基因连锁的共显性标记Na12A02,Na12A02与基因的距离为2.6cM。6.以分子标记SC7、SC1和SC6的特异片段为探针筛选Tapidor BAC文库,得到41个阳性克隆。利用标记SC1和SC7将Bnms1基因定位在BAC克隆BAC1上,对克隆BAC1进行shotgun测序。基于BAC序列开发了6个新标记SCS-SC13,在4132个单株群体中Bnms1基因被定位在标记SC8和SC11之间,标记与Bnms1基因间的遗传距离分别为0.05cM和0.15cM,SC8和SC11在BAC克隆BAC1上的物理距离为21.2-kb。7.用21.2-kb的序列检索GenBank的EST数据库和拟南芥数据库(AGIGenes),结果表明该区域存在四个候选基因,四个候选基因在甘蓝型油菜上的排列顺序与拟南芥基因的排列顺序一致。第一个和第四个候选基因是功能未知的基因,第二个候选基因属于NAP类型基因,第三个候选基因是CYP450基因家族成员,分析认为第二和第三个候选基因可能与花粉发育相关,以下用G14、G15表示。8.Northern blot分析表明G14和G15的表达量在S45A和S45B之间没有明显差异。在小于1mm、1mm～2mm、2mm～3mm和3mm～4mm四种大小的花蕾之间G14的表达量没有变化,表达水平很低;G15在1mm～2mm、2mm～3mm大小的的花蕾中特异表达。9.比较测序发现,G14在S45A和S45B之间存在2个错义突变,导致第79位的缬氨酸突变为丙氨酸;第226位的丙氨酸突变为丝氨酸。G15在S45A和S45B之间也有2个错义突变,导致第179位的甘氨酸突变为精氨酸;第297位的缬氨酸突变为丙氨酸。10.构建了转基因载体pBnG15、pAtG15RNAi-1和pAtG15RNAi-2,获得了5株甘蓝型油菜的阳性转化植株和2株表现为雄性不育的拟南芥植株。更多还原

【Abstract】 Utilization of heterosis in rapeseed is an efficient path to enhance yield,improve quality,and to strengthen resistance.The genie male sterile(GMS) system has been applied to this crop as an effective and economical pollination control system in China for its stable and complete sterility,rich sources of cytoplasm and widely spread of restorers.S45AB,a recessive GMS two-type line in Brassica napus,was derived from male sterile mutant of the Brassica napus canola variety Oro,genetic analysis indicated that two duplicate recessive genes controlled the male sterility in S45 populations(Pan et al.1988).In this study,S45AB was used to fine map and clone the recessive GMS gene,Bnms1.Main results are as follows:1.The NIL population displayed a ratio of ms to mf plants that did not differ significantly from 1:1.The segregation of fertile to sterile plants in the selfing generation from the male fertile plant of NIL did not differ significantly from 3:1. This result indicated that the recessive allele at the first locus(designed as Bnms1) was heterozygous,while the recessive allele at the second locus(designed as Bnms2) was homozygous.2.The observation on anther microscopical structure showed that it was no difference between S45A and S45B before the stage of meiosis.The tapetum of S45A was a little thicker than that of S45B at the stage of tetrad,but the tetrad in S45A was normal.Soon after the microspore released from tetrad,there was distinct difference between the microspore of S45A and S45B,the surface of microspore was smooth in S45A without the exine formation,the mierospore stopped developping and disaggregated;The tapetum of male sterile anther enlarged radial with a number of large vacuoles.The key stage taking place male sterility was from microsporocyte to mierospores.Extine development defective caused male sterlity.3.AFLP technology combined with bulked segregant analysis(BSA) was used to identify the genetic markers for Bnms1 gene in the NIL population.2560 pairs of AFLP primers were screened,and seven genetic markers AF1-AF7 linked to the Bnms1 gene were obtained.An NIL population including 310 individuals was used to map the Bnms1 gene,two nearest markers AF3 and AF7 bracketed Bnms1 at distances of 1.6cM and 0.3cM,respectively,AF1 and AF2 were co-segregated with Bnms1 gene.4.The fragments were cloned and sequenced,which indicated that AF1-AF7 were 203bp,241bp,211bp,186bp,187bp,284bp and 362bp in length,respectively. The four AFLP markers(AF1,AF3,AF6 and AF7) were successfully extended by PCR-walking.The four extended sequences were 7,499bp,997bp,1,203bp and 656bp, respectively,and they were converted into SCAR markers(designated as SC1,SC3, SC6 and SC7).Another population in the same NIL including 1,974 individuals was used for fine mapping of the Bnms1 gene.As a result,the Bnms1 gene was flanked by two SCAR markers,SC1 and SC7,with genetic distance of 0.1cM and 0.3cM, respectively.5.Using a DH population derived from the cross Zhongyou821×Bao604,a genetic linkage map spanning 1,625.6cM was constructed,which including 2 RFLP markers,65 RAPD markers,86 SSR markers and 84 SRAP markers.Based on two linakge map constructed using DH populations(Tapidor×Ningyou7 and Zhongyou821×Bao604),the Bnms1 gene was mapped on linkage group N7.One co-dominant SSR marker located on linkage group N7,Na12A02,was confirmed to link to the Bnms1 gene.The distance between the marker and Bnms1 gene was 2.6cM.6.Three probes(SC7,SC1 and SC6) screened the Tapidor BAC library,and 41 positive BAC clone were identified.One BAC clone BAC1 which might contain the Bnms1 gene was identified by two blacketing SCAR markers,SC1 and SC7.The BAC clone was sequenced using the shotgun sequencing strategy.Two and six recombinants with the Bnms1 gene were detected by two SCAR markers(SC8 and SC11) based on the sequence of BAC1,respectively.The physical distance between SC8 and SC11 was 21.2-kb.That meaned the Bnms1 gene was narrowed into a 21.2-kb DNA fragment.7.Searches for putative candidate gene were performed using TBLASTN searches in the plant EST database at http://www.ncbi.nlm.nih.gov/and AGI Genes database at http://www.arabidopsis.org/using 21.2-kb sequence as the query sequence. The result indicated that the 21.2-kb region included four candidate genes,and the gene order was the same between Brassica napus and Arabidopsis,it indicated that the Bnms1 gene located in a collinearity region of Brassica napus and Arabidopsis. The functions of the first and the fourth candidate were unknow,the second candidate gene was a NAP like type,the third candidated gene was a member of CPY450 family, and both the second and the third candidate gene were related with pollen development,which were designed as G14 and G15,respectively.8.Northern blot analysis showed that the expressions of G14 and G15 were neither distinctly different between S45A and S45B.The expression of G14 was not different among four stages tested and expression was low in bud,while G15 specially expressed from the microsporocyte stage to microsorpe stage.9.Comparative sequence analysis identified 2 missense mutations between S45A and S45B at both candidate genes,and those caused two amino acid conversion,V to A and A to S in G14,G to R and V to A in G15,respectively. 10.Plant expression vectors pBnGA15 and RNA interference expression vectors, pAtG15RNAi-1 and pAtG15RNAi-2,were constructed.Five transgenic Brassica napus plants and two male sterile Arabidopsis plants were obtained,respectively.更多还原