不结球白菜同源四倍体Pol CMS及其保持系花药发育的解剖学研究

以秋水仙素人工诱变获得的不结球白菜同源四倍体Pol CMS及其保持系为材料,采用石蜡切片法研究其花药发育过程.结果显示:四倍体Pol CMS与保持系花药发育差异明显,不育系花药发育受阻于孢原细胞分化期,不形成药室,属无花粉囊型.极少数温敏型花药能产生1~2个花粉囊,但四分体时期绒毡层提早退化,导致成熟小孢子数目减少.四倍体保持系花药发育过程与二倍体基本一致,同一时期的可育小孢子四倍体比二倍体大,二倍体花粉粒都为3个萌发孔,而四倍体多为4个萌发孔.四倍体保持系同一花粉囊中花粉粒发育有不同步现象,花粉粒败育频率比二倍体高.研究表明,不结球白菜同源四倍体Pol CMS比其二倍体Pol CMS不育更彻底,更具应用性.     

不同倍性不结球白菜Pol CMS及保持系生理生化特性比较

对二、四倍体不结球白菜Pol CMS及其保持系花蕾和薹叶进行生理生化特性比较分析,结果表明:不育系花蕾中可溶性蛋白、可溶性糖、脯氨酸含量均显著低于保持系,且各指标在不育系中四倍体低于二倍体,而在保持系中则四倍体高于二倍体;不育系MDA含量,POD、SOD、CAT活性均高于保持系,不育系中四倍体均高于二倍体.而保持系中四倍体均低于二倍体;薹叶中MDA含量,POD、SOD、CAT活性均为不育系高于保持系,不育系中四倍体高于二倍体;POD、EST同工酶显示不育系与保持系间均具特异酶带,但不同倍性间并无差异.     

大白菜细胞质雄性不育的分子生物学研究

细胞质雄性不育(cytoplasmic male sterility,CMS)是作物杂种优势利用的一 条重要途径。对其机制的研究不仅具有重要的理论意义,而且具有重要的应用价值。对大白菜而言,利用CMS最关键的环节是要育成不育系和其相应的保持系。利用不育系和其它材料杂交产生F1代种子;用不育系和保持系杂交来繁殖不育系;保持系自交繁殖种子。但采用常规的方法来选 育不育系和其相应的保持系周期长、见效慢,远不能满足生产发展的需要。现代生物技术为创造新的不育系和保持系提供了便利。本论文对大白菜细胞质雄性不育的分子机理和 RAPD分析的有关问题进行了研究。     

高粱同源四倍体及四倍体杂交种

将二倍体高粱诱变成同源四倍体高粱,一般都表现籽粒增大,蛋白质含量显著提高,但结实率下降。将二倍体高粱三系都诱变成四倍体并配成四倍体杂交种以后,不但籽粒大,蛋白质含量高,而且结实率显著提高,其它农艺性状也有所改善,因此利用三系来改良多倍体在育种上有重要意义。高粱同源四倍体和二倍体相比,减数分裂不够正常,结实率不同的四倍体品系减数分裂过程的异常程度也有所不同。     

大白菜细胞质雄性不育系和其保持系的RAPD分析

利用RAPD技术对大白菜细胞质雄性不育系CMS341-7和其保持系3411-7的基因组DNA进行了比较分析,共使用了269个随机引物,其中有163个引物在两系之间都得到了扩增产物,79个引物扩增结果在两系之间表现出了遗传多态性。找到了不育系和保持系的特异扩增条带CMSOPL01670和MOPB04600。并对这些特异片段的来源及其在细胞质雄性不育中的作用进行了讨论。     

大白菜orf224基因植物表达载体的构建及遗传转化

为了创制新的大白菜雄性不育材料,双酶切重组质粒pMD18-T-CMS7311-orf224和表达载体pWR306后,将CMS7311-orf224基因与线性表达载体pWR306进行定向连接,构建了细胞质雄性不育线粒体基因CMS7311-orf224的植物表达载体pWR-CMS7311-orf224,通过酶切和PCR验证pWR-CMS7311-orf224载体构建正确.采用快速冻融法,将表达载体导入农杆菌EHA105,转化大白菜材料06J28,对转化植株的GUS、PCR和RT-PCR检测表明,获得了2个大白菜转基因植株.     

狗尾草属野生近缘种的染色体鉴定

对来自多个国家和地区的10个种(青狗尾草S.viridis,法氏狗尾草S.faberii,轮生狗尾草S.verticillata、S.verticillifor-mis,金色狗尾草S.glauca、S.pumila、S.grisebachii、S.leucopila、S.parviflora、S.queenslandica等)50份狗尾草材料进行了染色体计数及倍性鉴定。发现狗尾草属中的青狗尾草均为二倍体,金色狗尾草S.glauca有四倍体和八倍体,轮生狗尾草S.verticillata有二倍体和四倍体,法氏狗尾草S.faberii为四倍体,S.pumila有二倍体和四倍体,S.grisebachii为二倍体,S.leucopila为二倍体,S.queenslandica为四倍体。本研究中对S.grisebachii、S.leucopila、S.queenslandica3个种是首次染色体倍性观察。发掘近缘种的有益基因是作物育种的重要途径之一,本研究搜集的谷子近缘野生种对谷子远缘杂交育种和谷子起源进化分析有重要意义。     

甘蓝型油菜新型不育系花器特征及mtDNA的RAPD分析

对新选育的甘蓝型油菜PL CMS不育系和Pol CMS及Ogu CMS不育系材料花器形态进行比较,并对3种不育系10份材料mtDNA进行RAPD分析.结果表明,新选育的PL CMS不育系与Pol CMS、Ogu CMS在花瓣长和宽、花冠直径、雄蕊大小等形态特征方面有极显著差异;mtDNA分析结果表明,新发现的细胞质雄性不育材料L04-02A、L04-05A、L04-01A为同一类型的细胞质雄性不育系,它们与Pol CMS及Ogu CMS不育材料有很大差异,由此推知PL CMS不育系是一种新型甘蓝型油菜不育系.     

青花菜雄性不育系选育研究初报

以具有细胞质雄性不育基因的青花菜不育材料CMS94008为不育源,通过杂交和连续回交的方法选育出不育性稳定、经济性状良好的一批青花菜胞质雄性不育系,并以CMS92100、CMS93-2、CMS9905、CMS95234等不育材料及其转育父本为代表,对不育系的主要特征及农艺性状进行系统的观察鉴定。     

大白菜细胞质雄性不育系RAPD特异扩增片段的克隆及其序列分析

利用RAPD技术从大白菜细胞质雄性不育系CMS3411-7的DNA中得到1个特异扩增片段CMSO-PL01670,进一步以该片段为探针进行Dot和Southern杂交证实了该片段为不育系所特有。回收该特异扩增片段并将其克隆到pGEM-T Easy载体上进行序列测定。测序结果表明该片段全长673bp,其碱基组成为A T=67.16％。通过与GenBank EMBL DDBJ PDB中的455 972个序列进行比较,同源性均小于30％,表明该片段为一新发现序列。并且该序列的 4 - 216的区域内含有1个可编码70个氨基酸的开放阅读框架。上述结果为今后从分子水平进一步研究大白菜细胞质雄性不育打下了坚实的基础。     

Development and utilization of an InDel marker linked to the fertility restorer genes of CMS-D8 and CMS-D2 in cotton

Molecular Biology Reports - The cytoplasmic male sterility (CMS) system is a useful tool for commercial hybrid cotton seed production. Two main CMS systems, CMS-D8 and CMS-D2, have been recognized...     

Transfer of wild abortive cytoplasmic male sterility through protoplast fusion in rice

Wild abortive cytoplasmic male sterility has been extensively used in hybrid seed production in the tropics. Using protoplast fusion between cytoplasmic male sterile and fertile maintainer lines; we report here, transfer of wild abortive cytoplasmic male sterility to the nuclear background of RCPL1-2C, an advance breeding line which also served as maintainer of this cytoplasm. In total, 27 putative cybrids between V20A and RCPL1-2C and 23 lines between V20A and V20B were recovered and all of them were sterile. DNA blots prepared from the mitochondrial DNA of the cybrid lines from both the sets were probed with orf155 that is known to exhibit polymorphism between the mitochondrial DNA of the male-sterile and fertile maintainer lines. Hybridization of orf155 to 1.3 kb HindIII-digested mitochondrial DNA fragment of the cybrids showed transfer of mitochondrial DNA from wild abortive cytoplasmic male-sterile line to the maintainers, viz. RCPL 1-2C and V20B. Expression of male sterility was confirmed by the presence of sterile pollen grains and the lack of seed setting due to selfing in all the cybrid lines. These cybrids, on crossing with respective fertile maintainers set seeds that in turn, produced sterile BC1 plants. DNA blots from HindIII-digested mitochondrial DNA of these BC1 plants when probed with orf155 again exhibited localization of orf155 in wild abortive cytoplasm-specific 1.3 kb HindIII-digested mitochondrial DNA fragments. This demonstrated that the cytoplasmic male sterility transferred through protoplast fusion retained intact female fertility and was inherited and expressed in BC1 plants. Fusion-derived CMS lines, on pollination with pollen grains from restorer, showed restoration of fertility in all the lines. The results demonstrate that protoplasts fusion can be used for transferring maternally inherited traits like cytoplasmic male sterility to the desired nuclear background which can, in turn, be used in hybrid seed production programme of rice in the tropical world.     

利用RNA指纹技术分析大白菜胞质雄性不育相关基因的差异表达

采用改进的RNA指纹技术(RAP-PCR)对两套大白菜胞质雄性不育材料及其杂种F1花蕾的总RNA进行了分析, 通过对186条随机引物的筛选, 获得4个重复性较好的差异片段S47-412, S93-622, S176-343, S199-904, 并对其克隆、测序。根据各差异条带的测序结果合成长引物进行验证, S47, S93所对应的差异消失, 而S176, S199长引物验证的结果与RAP-PCR相似, 序列分析表明: S176-343, S199-904两差异片段均和油菜Polima不育胞质线粒体基因组orf224/atp6位点有着极高的同源性, 两差异片段序列部分重叠, 这说明两差异片段可能和大白菜胞质雄性不育有很高的相关性。     

白菜OguCMS相关MYB家族新基因BcMYBogu的克隆与特征分析

为研究CMS核质互作的分子机理, 将甘蓝型油菜(Brassica napus L.)和白菜(B. campestris L. ssp. chinensis Makino)杂交并连续回交6代获得白菜OguCMS, 在与保持系花药细胞学比较的基础上, 运用cDNA-AFLP筛选得到白菜OguCMS早、中期花蕾提早表达的MYB-like差异片断, 利用RACE克隆得到该片断的cDNA全长, 命名为BcMYBogu(GenBank 登录号: EF127861), 对其氨基酸序列和表达特征进行研究。结果表明, 白菜OguCMS绒毡层在四分体后增生, 高度液泡化, 导致小孢子花粉外壁异常, 细胞质同外壁分离并降解; 花药变白; BcMYBogu具有典型的MYB DNA结合域—W残基和SH[AL]QKY[RF]基序; 系统进化分析显示BcMYBogu与AtMYB26, AtMYB32和AtMYB4等聚类在同一分枝; RT-PCR分析表明BcMYBogu在莲座叶、花茎和花蕾中均有表达, 但在OguCMS花蕾中表达量显著上升。由此推测BcMYBogu是一个新的与白菜OguCMS相关的MYB家族新成员。     

A new cytoplasmic male sterility system for hybrid seed production in Indian oilseed mustard Brassica juncea

We report a novel cytoplasmic male sterility (CMS) system in Brassica juncea (oilseed mustard) which could be used for production of hybrid seed in the crop. A male sterile plant identified in a microspore derived doubled haploid population of re-synthesized B. napus line ISN 706 was found to be a CMS as the trait was inherited from the female parent. This CMS, designated ‘126-1’, was subsequently transferred to ten different B. juncea varieties and lines through inter-specific crosses followed by recurrent backcrossing. The F₁s of inter-specific crosses were invariably partially fertile, but irrespective of the variety/line used, the recipient lines became progressively male sterile over five to seven generations and could be maintained by crossing the male sterile lines with their normal counterparts. The male sterile lines were found to be stable for the trait under both long and short day conditions. CMS lines when crossed with lines other than the respective maintainer line were restored for fertility, implying that any variety could act as a restorer for ‘126-1’ cytoplasm in B. juncea. These unique features in maintenance and restoration of CMS lines coupled with near normal floral morphology of the CMS lines have allowed the use of ‘126-1’ cytoplasm for hybrid seed production. The uniqueness of ‘126-1’ has been further established by Southern hybridization with mitochondrial DNA probes and by a histological study of the development of male sterile anthers.     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

Plant regeneration from protoplasts of cytoplasmic male sterile lines of rice (Oryza sativa L.)

This study compared plant regeneration from protoplasts isolated from suspension cultures of threeJaponica rice (Oryza sativa L.) lines with different male sterile cytoplasms. More than 180 green plants were regenerated from protoplasts from 5–8 month old suspensions of IR58024A, a line with the WA type of cytoplasmic male sterility (CMS). About 40% of the calli recovered from protoplasts produced green plants. ShuangbaiA (BT type of CMS) and Tai2A (Dian I type of CMS), both from Zhejiang province of China, responded less well in culture. ShuangbaiA produced green plants from 6.6% of calli, although initial protoplast yield per gram fresh weight was higher than for IR58024A. Tai2A showed lower protoplast yield, and only 1.1% of the calli produced green plants. Flow cytometric analyses of nuclear DNA content indicated that many of the regenerated plants were tetraploid. The percentage of tetraploids varied in the different lines. The male sterile characteristics of the original lines were maintained in the regenerated plants. Pollen abortion occured earliest in IR58024A and latest in Tai2A. IR58024A is a promising rice genotype for use as a recipient in direct gene transfer experiments.Abbreviations BAP 6-benzylaminopurine - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxyacetic acid - IRRI International Rice Research Institute - LS Linsmaier and Skoog's (1965) medium - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid - WA wild abortive