一种新型甜菜胞质雄性不育系的分子生物学鉴定

对通过种间杂交获得的两种胞质雄性不育系甜菜(命名为Cl和M,其细胞质来源分别为Beta cicla Turkey和Beta maritima)和通常的Owen型胞质不育系甜菜(简称作S)以及这3种不育系通用的保持系(简称N)进行了线粒体基因组的RFLP和RAPD对比分析.结果表明Cl与S相比线粒体组织结构具有明显的差别.用6种异源线粒体基因作探针对6种限制性内切酶片段进行Southern杂交分析,发现Cl和S线粒体基因组之间存在明显多态性.但S和M之间却异乎寻常地表现一致.RAPD分析验证了Cl和 S之间的差异,此外,在Cl中发现 3种 4.2 kb大小的特异存在的RNA分子.根据结果认为Cl是一种不同于S的新型细胞质不育系.用RAPD方法也找到了S与M之间差异.酶切结果表明N与3个不育系的mtDNA非常相似.但在杂交和RAPD分析中均找到了N系的特异片段.这些片段是否与育性相关尚有待分析.     

水稻细胞质雄性不育系和保持系的线粒体COⅠ、COⅡ基因组织结构差异的分析

我们研究了水稻珍汕97不育系和保持系的线粒体DNA的PstⅠ、HindⅢ、BamHⅠ酶切电泳带型,发现了不育系和保持系的线粒体DNA分子结构的显著不同,而不育系和保持系的叶绿体DNA的HindⅢ酶切片段没有差异。以线粒体的细胞色素C氧化酶亚基Ⅰ、Ⅱ(COⅠ、COⅡ)基因为探针,与珍汕97不育系、保持系的叶绿体、线粒体DNA酶切片段进行分子杂交,证明叶绿体DNA没有COⅠ、COⅡ基因的同源顺序,发现不育系和保持系COⅠ、COⅡ基因有组织结构上的差异,但不能确证这些差异是否与细胞质雄性不育有关。     

K、V、T型小麦细胞质雄性不育系叶绿体DNA的SSR分析及RuBP羧化酶活性比较

为了探讨小麦细胞质雄性不育(CMS)与叶绿体DNA(cpDNA)的关系,揭示CMS机理。以2套K、V、T型同核异质不育系(A)及其保持系(B)‘太911289’和‘冀5418’、育性恢复的F_1和各自的质供体粘果山羊草(Aegilops kotschyi)、偏凸山羊草(Aegilops ventricosa)、提莫菲维小麦(Triticum timopheevii)为试验材料,利用cpSSR引物对小麦cpDNA进行比较分析,筛选出代表不同胞质不育系的引物,探讨CMS与叶绿体的关系;同时测定由叶绿体DNA与核DNA共同编码的RuBP羧化酶的活性,为小麦K、V、T雄性不育类型的应用提供理论依据。结果表明:(1)K型、V型、T型不育系与保持系的cpDNA之间均呈现多态性,且不同的细胞质之间存在特异片段,这从DNA水平上提供了3类不育系胞质来源不同的证据,并分别找到5对和7对可以鉴定V型和T型不育系的特异引物。(2)除K型‘冀5418’与其胞质供体的cpDNA出现差异外,不育系与其胞质供体的cpDNA没有差异,而且不育系与其育性恢复的F_1代cpSSR扩增片段之间也没有差异,很好地遗传了其母本性状。(3)在起身期和开花期,不仅2套不育系的RuBP羧化酶活性显著高于保持系,且在不育系与恢复系杂交的F_1中,随着育性的恢复其RuBP羧化酶活性高于保持系而低于各自不育系;而在拔节期的不育系与保持系之间、不育系之间、以及F_1代与不育系和保持系之间的RuBP酶活性大小没有显著差异。这可能与拔节期主要是营养生长有关系。     

CCACA碱基序列是玉米CMS-S线粒体orf355-orf77转录本剪切识别位点

玉米S型细胞质雄性不育系（CMS-S）及其近等基因恢复系是研究核 质互作机制的重要遗传资源和理想模式体系．目前认为,CMS-S花粉败育是由其线粒体内细胞质不育基因orf355-orf77表达的毒性蛋白引起,而核育性恢复基因Rf3可通过引发orf355-orf77转录本的降解而解除其毒性作用,使花粉育性得以恢复．本研究采用Northern杂交和3′-RACE技术确定了orf355-orf77转录本的剪切位点,并发现在育性恢复的花粉中,orf355-orf77转录本被剪切成小片段之后聚合了poly(A)序列,推测这一过程加速了mRNA分子的降解,是育性恢复的关键环节．利用生物信息学方法分析了orf355-orf77转录本6个剪切位点的侧翼序列,发现在剪切位点下游10个碱基的位置均含有5′-CCACA-3′序列,推测该序列受到特定功能蛋白的识别,然后募集核酸内切酶对其进行剪切．研究结果可为揭示玉米CMS S育性恢复机理提供重要的理论依据．     

水稻线粒体atpA基因的克隆及其与细胞质雄性不育的关系

本研究以水稻BT型细胞质雄性不育系秋光和相应的保持系秋光为材料,提取线粒体DNA,用限制性内切酶完全酶解,以玉米线粒体atpA基因和波菜叶绿体atpA基因作为探针,进行分子杂交,将保持系线粒体atpA基因定位在3.5kb的Bam HI酶切片段上,并且以pBR322为载体,克隆了这一片段,另外,在Bam HI完全酶解普带的杂交结果中,不育系线粒体基因组中有两条阳性杂交带,分别是3.5kb和2.9kb,而保持系线粒体基因组中只有3.5kb一条阳性杂交带,因而认为水稻不育系线粒体基因组中可能有两个atpA基因拷贝,而相应的保持系线粒体基因组中只有一个atpA基因拷贝。     

水稻线粒体atpA基因的克隆及其与细胞质雄性不育的关系

本研究以水稻BT型细胞质雄性不育系秋光和相应的保持系秋光为材料，提取线粒体DNA，用限制性内切酶完全酶解，以玉米线粒体atpA基因和波菜叶绿体atpA基因作为探针，进行分子杂交，将保持系线粒体atpA基因定位在3.5kb的Bam HI酶切片段上，并且以pBR322为载体，克隆了这一片段，另外，在Bam HI完全酶解普带的杂交结果中，不育系线粒体基因组中有两条阳性杂交带，分别是3.5kb和2.9kb，而保持系线粒体基因组中只有3.5kb一条阳性杂交带，因而认为水稻不育系线粒体基因组中可能有两个atpA基因拷贝，而相应的保持系线粒体基因组中只有一个atpA基因拷贝。     

水稻线粒体基因组翻译产物与细胞质雄性不育性

通过线粒体离体翻译产物的电泳和放射性自显影分析,发现水稻BT型不育细胞质(农虎26A和丰锦A不育系)的线粒体基因产物比可育细胞质(农虎26B和丰锦B保持系)缺少一个22KD多肽。它反应了BT型不育细胞质线粒体基因组中有关育性的基因变异。不育系与恢复系杂交后,杂种F_1的育性虽然恢复,但是它的线粒体基因产物中仍然缺少22KD多肽。然而,在杂种F_1的线粒体蛋白质的电泳染色图谱中则显示出一个22KD多肽条带。杂种F_1中的这个22KD多肽是核基因产物。它弥补了线粒体基因组中育性基因的缺陷。     

小麦K型及V型细胞质雄性不育系线粒体DNA的比较分析

采用RFLP（restrictionfragmentlengthpolymorphism）和RAPD（radomlyamplifiedpolymorphicDNA）技术对具有相同核遗传背景的小麦K型不育系K149A、V型不育系V149A及相应保持系149B的线粒体DNA进行了比较分析。结果表明它们之间线粒体DNA的结构显著不同,atpA,atp9,coxII,cob等线粒体功能基因有组织结构上的差异。推测线粒体DNA是小麦K、V型雄性不育系胞质不育困子的载体。但由于不育系与保持系间线粒体DNA差异甚大,难以确定究竞哪些差异确与雄性不育有关。仅仅比较不育系和保持系间线粒体DNA的差异是限制本领域取得突破性进展的症结所在。基于“核质工作”的思路,把“雄性不育-育性恢复”作为一个整体来考虑,并重视比较不育系和杂种F1代之间线粒体DNA的结构和表达的差异,所得结果对寻找和鉴定cms因子,进而阐明cms的形成机理有积极意义。     

BT型细胞质雄性不育水稻及其三系的线粒体DNA研究

用RAPD技术对BT型水稻胞质雄性不育系秀A及其保持系秀B、恢复系湘晴以及杂种F1代的线粒体DNA进行了比较分析。结果表明不育系与其保持系间存在显著差异;不育系与其F1之间mtDNA也存在差异。在引物OPJ－08的扩增产物中,秀A扩增出一条分子量为800bp的多态性片段,在引物OPK－10的扩增产物中,杂种F1扩增出一条分子量为900bp的片段。把这两片段回收、克隆并制备探针,OPJ－08800的Southern杂交结果显示不育系与其F1杂交图谱存在多态性;OPK－10900的Suthern杂交结果显示不育系与其保持系同存在差异。推测这两片段与育性可能有一定的联系。     

玉米S型CMS线粒体DNA R区双向转录模式及其与不育相关性

玉米(Zea mays L.)S型CMS胞质育性基因被认为与其线粒体DNA高频重组R区相关.R区含orf355和orf77两个开放阅读框,其中orf77被认为是重要的胞质候选基因.RT-PCR分析发现R区为双链转录.其中一条为orf77-orf355的模板链,它的转录受植株核育性基因及生长发育时期的影响,在S(rf3rf3)(不育)基因型材料的黄化苗、S(Rf3-)(恢复)基因型材料的黄化苗及雄穗中,RT-PCR检测到这条模板链上R区的转录本完全相同,但S(rf3rf3)(不育)基因型材料雄穗的转录本则与上述三者不同,其转录本在5'端缩短了近238 b的长度.R区的另一模板链与coxⅠ或coxⅡ基因的模板链毗连,转录方向相同,这条链的转录不受核基因型和生长发育时期的影响,不育和恢复基因型材料的黄化苗及雄穗中的转录本完全相同.将orf77进行体外表达,制备抗体后对线粒体蛋白质进行Western杂交分析,未能检测到R区中ORF77蛋白质或多肽.推测orf77无翻译产物而且这可能与R区的双链转录有关.RT-PCR及Western分析结果表明玉米CMS-S可能与R区5'端DNA的转录调控相关.     

小麦T型细胞质雄性不育系与可育系叶片蛋白质双向电泳分析

利用育性恢复基因(Rf3)的近等基因系1031-1、S-165和1031-1与S-165之间的正交与反交,创建了四个实验品系(1031-1、S-165、不育品系、反交品系);采用改进的蛋白质聚丙烯酰胺凝胶双向电泳技术,从发育遗传学的角度,对小麦T型细胞质雄性不育和可育株旗叶表达的相关蛋白产物进行差异分析。通过对旗叶蛋白的双向电泳分析,发现4个品系有相近的蛋白质双向电泳图谱。没有相应穗中差异蛋白质的出现。从蛋白质水平上证实了不育基因与恢复基因表达具有器官特异性特征。     

Maternal inheritance of mouse mtDNA in interspecific hybrids: segregation of the leaked paternal mtDNA followed by the prevention of subsequent paternal leakage.

The transmission profiles of sperm mtDNA introduced into fertilized eggs were examined in detail in F1 hybrids of mouse interspecific crosses by addressing three aspects. The first is whether the leaked paternal mtDNA in fertilized eggs produced by interspecific crosses was distributed stably to all tissues after the eggs'' development to adults. The second is whether the leaked paternal mtDNA was transmitted to the subsequent generations. The third is whether paternal mtDNA continuously leaks in subsequent backcrosses. For identification of the leaked paternal mtDNA, we prepared total DNA samples directly from tissues or embryos and used PCR techniques that can detect a few molecules of paternal mtDNA even in the presence of 10(8)-fold excess of maternal mtDNA. The results showed that the leaked paternal mtDNA was not distributed to all tissues in the F1 hybrids or transmitted to the following generations through the female germ line. Moreover, the paternal mtDNA leakage was limited to the first generation of an interspecific cross and did not occur in progeny from subsequent backcrosses. These observations suggest that species-specific exclusion of sperm mtDNA in mammalian fertilized eggs is extremely stringent, ensuring strictly maternal inheritance of mtDNA.     

Introgressive hybridization of human and rodent schistosome parasites in western Kenya

Hybridization and introgression can have important consequences for the evolution, ecology and epidemiology of pathogenic organisms. We examined the dynamics of hybridization between a trematode parasite of humans, Schistosoma mansoni, and its sister species, S. rodhaini, a rodent parasite, in a natural hybrid zone in western Kenya. Using microsatellite markers, rDNA and mtDNA, we showed that hybrids between the two species occur in nature, are fertile and produce viable offspring through backcrosses with S. mansoni. Averaged across collection sites, individuals of hybrid ancestry comprised 7.2% of all schistosomes collected, which is a large proportion given that one of the parental species, S. rodhaini, comprised only 9.1% of the specimens. No F1 individuals were collected and all hybrids represented backcrosses with S. mansoni that were of the first or successive generations. The direction of introgression appears highly asymmetric, causing unidirectional gene flow from the rodent parasite, S. rodhaini, to the human parasite, S. mansoni. Hybrid occurrence was seasonal and most hybrids were collected during the month of September over a 2-year period, a time when S. rodhaini was also abundant. We also examined the sex ratios and phenotypic differences between the hybrids and parental species, including the number of infective stages produced in the snail host and the time of day the infective stages emerge. No statistical differences were found in any of these characteristics, and most of the hybrids showed an emergence pattern similar to that of S. mansoni. One individual, however, showed a bimodal emergence pattern that was characteristic of both parental species. In conclusion, these species maintain their identity despite hybridization, although introgression may cause important alterations of the biology and epidemiology of schistosomiasis in this region.     

Genome-wide analysis of maize cytoplasmic male sterility-S based on QTL mapping

Three types of sterile cytoplasm in cytoplasmic-male-sterility (CMS) maize, T, C and S, can be identified according to their fertility-restoration and mitochondrial DNA RFLP patterns. CMS-S, which is the least stable among the three types of CMS, is controlled by sterile cytoplasm interactions with certain nuclear-encoded factors. We constructed a high-resolution map of loci associated with male-restoration of CMS-S in BC1 populations of maize. The map covers 1730.29 cM, including 32 RFLP, 51 SSR 62 RAPD and 21 AFLP markers. Genome-wide QTL analysis detected 6 QTLs with significant effects on male fertility as assessed by their starch-filled pollen ratios. Four QTLs out of six were located between the SSR markers MSbnlg1633-Mrasg20, MSbnlg1662-Msume1126, MSume1230-MSumc1525, and RAPD marker MraopQ07-2-MraopK06-2 on chromosome 2. Two other minor loci were mapped between MraopK16-1-Mraopi4-1, on chromosome 9, and between Msuncbnlg1139-MraopR10-2, on chromosome 6. The Rf3 nuclear restoring gene was precisely located on the chromosome 2, 2.29 cM to the left of umc1525 and 8.9 cM to the right of umc1230. The results provide important markers for marker-assisted selection of stable CMS-S maize.     

Linkage analysis of the nuclear homologues of mitochondrial plasmid-like DNAs in rice.

A genetical study on the nucleotide sequences of the nuclear DNAs which share homology with rice mitochondrial plasmid-like DNAs, B1, B2, B3 and B4 was carried out. Restriction fragments of the nuclear DNAs hybridized with these plasmid-like DNAs showed polymorphisms in their length between Indica and Japonica rice cultivars. The hybridized signals found specifically in Indica or Japonica cultivars segregated in the F2 population derived from a cross between these two subspecies. The observed ratio of the nuclear homologues in the F2 population demonstrated that they were transmitted according to the Mendelian inheritance. The co-segregation of homologues was examined and the linkage was detected between the B1-nuclear homologue of Japonica and the B4-nuclear homologue of Indica, and also between the nuclear homologues of B2 and B3 of Indica. The linkage between the B1-nuclear homologue of Japonica and the B4-nuclear homologue of Indica was conserved in the different rice cultivars.     

Reorganization of mitochondrial genomes of cytoplasmic revertants in cms-S inbred line WF9 in maize

Summary Cytoplasmic reversion to fertility in cms-S maize has been previously correlated with changes in mitochondrial genome organization, specifically with loss of the autonomously replicating linear plasmid-like DNAs, S1 and S2, and with accompanying alterations in the high molecular weight mtDNA (main genome) that specifically involved S1 and S2 sequences. These studies, however, dealt with cytoplasmic revertants occurring in the cms-VG M825 inbred line and in the cms-VG M825/Oh07 F₁ hybrid. This paper deals principally with patterns of mitochondrial DNA reorganization accompanying cytoplasmic reversion to fertility in the WF9 inbred line nuclear background. Here the free S1 and S2 plasmid-like DNAs are retained in the revertants. Mitochondrial DNA analysis by Southern hybridization using cloned fragments of S1 and S2 shows altered organization around S-homologous regions in the main mitochondrial genome of revertants as compared with that of the male-sterile parental controls, but the pattern of main genome changes involving these regions differs from that of the cytoplasmic revertants that occurred in M825 and M825/Oh07 backgrounds. Similar experiments using a clone of the cytochrome oxidase I (COX I) gene of maize as a probe indicate that reorganization in this region is also involved in the changes in mtDNA that accompany cytoplasmic reversion to male fertility in cms-S WF9. The heterogeneity in patterns of reorganization of the main mtDNA genome that accompany cytoplasmic reversion in the same and different nuclear backgrounds are discussed in relation to cytoplasmic male sterility (CMS).