在杂交作物分子育种中利用普通核雄性不育的几个可能途径

由一对隐性基因控制的普通核雄性不育性遗传方式能够满足对植物最佳雄性不育系选育的要求,是水稻等作物杂种优势利用的极好遗传工具。如果能解决其不育系繁殖问题,将优于现有的其他杂种优势利用方式。克隆出普通核雄性不育性的可育基因,通过叶绿体转化,将核雄性不育性可育基因向普通核雄性不育株细胞质转移,创造普通核雄性不育株的保持系;通过种子成熟后表达的启动子;和以位点特异性重组技术为基础的基因开关以及化学诱导启动子的利用,都可能繁殖出100%不育株率的普通核雄性不育系,创造普通核雄性不育性利用的新途径,对植物杂种优势利用产业有十分重要的意义。     

甘蓝型油菜隐性细胞核雄性不育的研究及利用

甘蓝型油菜隐性细胞核雄性不育系育性稳定、无负效应、易转育且恢复源广,在油菜杂种优势利用中被广泛利用.目前我国主要利用的隐性细胞核雄性不育系有2类:双基因隐性细胞核雄性不育以及由2对基因互作控制的核不育类型.随着这2类不育系不育基因相继被克隆,人们对甘蓝型油菜细胞核雄性不育的分子作用机理已经有一定了解.本文综述了在甘蓝型油菜核不育分子机理的研究方面的进展,在此基础上提出了繁殖甘蓝型油菜核不育系全不育系群体可行的途径,展现了油菜细胞核雄性不育杂种优势利用广阔的前景.     

基因工程培育可恢复的植物雄性不育系的研究进展

植物雄性不育是植物杂种优势利用的资源, 具有重要的生产利用价值。植物雄性不育可从自然突变、人工诱变和远缘杂交中发现, 现在可通过细胞工程和基因工程等方法来创造。文章综述了利用基因工程方法制备雄性不育品系及其相应的育性恢复策略, 分为“单组分策略”和“双组分策略”。其中利用“单组分策略”制备的不育植株是条件型雄性不育(可逆转的雄性不育), 它能在特定的条件下实现雄性可育与不育的转换, 实践中可直接作为两用系(不育系和保持系)用于两系法杂交制种; “双组分策略”是利用基因互作和亲本杂交直接培育雄性不育系, 或利用基因互作原理分别研制不育系和恢复系, 用于三系法生产杂交种。文章分析了 “单组分策略”和“双组分策略”的基因工程方法培育雄性不育系及其相应育性恢复策略优缺点, 对以上两种技术路线在实际应用中的现状作了分析和展望。     

应用基因工程技术创造植物雄性不育系

基因工程开辟了创造植物雄性不育系的一个新的途径 ,综述了利用基因工程技术创造植物雄性不育的机制及相关启动子和基因 ;创造雄性核不育和质不育的途径 ;探讨了存在的问题和应用前景。     

植物细胞质雄性不育系育种的反向核置换技术分析

通过不育细胞质为选择背景,在田间事先鉴定出杂种后代的优异完全不育株,用花药培养或诱导孤雌生殖使其纯合,测定其配合力,可以筛选到优良的目标不育系。以下3种方法则可能通过目标不育系而获得其同型保持系：一是通过体细胞变异(花药培养)产生;二是在不育系孕穗期高温或低温处理使其转换成可育,选择仍具有不育保持能力的作为保持系,或作为轮回亲本,将其细胞核换到可育细胞质中;三是用原生质体融合的方式向不育系导入已杀死细胞核的可育细胞质而获得配套保持系。它可以使杂种优势利用变得有预见性,可能提高现有杂种优势水平。在创造雄性不育新质源,排除微效可育基因,进行不育系的定向改造,选育高配合力不育系,以及加速育种进程等方面具有重要价值。     

植物细胞质雄性不育系育种的反向核置换技术分析

通过不育细胞质为选择背景,在田间事先鉴定出杂种后代的优异完全不育株,用花药培养或诱导孤雌生殖使其纯合,测定其配合力,可以筛选到优良的目标不育系.以下3种方法则可能通过目标不育系而获得其同型保持系:一是通过体细胞变异(花药培养)产生;二是在不育系孕穗期高温或低温处理使其转换成可育,选择仍具有不育保持能力的作为保持系,或作为轮回亲本,将其细胞核换到可育细胞质中;三是用原生质体融合的方式向不育系导入已杀死细胞核的可育细胞质而获得配套保持系.它可以使杂种优势利用变得有预见性,可能提高现有杂种优势水平.在创造雄性不育新质源,排除微效可育基因,进行不育系的定向改造,选育高配合力不育系,以及加速育种进程等方面具有重要价值.     

湖北光感核不育水稻的发现、鉴定及其利用途径

1973年在湖北发现了一株不育水稻,它在长日照条件下不育,在短日照下则可育。经鉴定,它的不育性是受一对对光照长度敏感的隐性雄性不育主基因控制,是在植物界首次被发现,是生理遗传的典型例子;用于理论研究有很大的科学价值,作为杂种优势利用,可以在短日照下自交繁殖,在长日照下杂交制种,一般粳稻品种都可以作为父本使用,杂种一代在任何生长季节下都能发挥其增产作用。这株水稻被命名为“湖北光感核不育水稻”,这个对光照长度敏感的隐性雄性不育基因被命名为msh。     

合成甘蓝型油菜中双隐性雄性核不育材料的发现及遗传规律分析

从甘蓝型油菜与白菜型油菜的种间杂交获得的甘蓝型油菜(Brassica napus)中发现了雄性不育单株,兄妹交株系和不育株与甘蓝型油菜常规杂交F1和F2株系的育性分离分析表明,该不育材料属于双隐性雄性核不育类型.利用育性分离株系的可育株自交和可育株与不育株间兄妹交等方法筛选出7个纯合可育株系,等位测验表明这7个纯合可育株系(B1～B7)中存在两种基因型:Ms1Ms1ms2ms2和ms1ms1Ms2MS2.该材料对油菜核不育基因定位和杂种优势利用研究有重要意义.     

辣椒细胞核雄性不育材料GMS702AB的鉴定

本研究发现1个具有雄性不育与单性结实特征的细胞核雄性不育系辣椒材料,并对该材料的农艺性状、单性结实坐果率、不同发育时期的不育系单性结实与可育系单性结实内源激素进行测定;利用田间鉴定和显微镜镜检,分析了F2群体的遗传分离情况,并利用辣椒细胞核雄性不育基因ms-3、msw、ms、msms、msc-1开发的分子标记,分析了群体的育性分离比。结果表明：不育系单性结实果实纵横径较大,可育系与不育系单性结实坐果率明显不同,可育系坐果率为22%,不育系坐果率为43%;不同时期不育系单性结实果实的赤霉素（GA4）含量显著高于可育系单性结实果实;田间鉴定和分子标记检测表明,F2群体中可育系有97株,不育系有30株,分离比为3.23∶1,符合孟德尔遗传规律,确定其育性由隐性单基因控制,将该辣椒细胞核雄性不育系命名为GMS702AB。本研究提供新的辣椒不育系,有助于辣椒育种和种子生产。     

谷子显性雄性核不育基因的发现与利用

植物界的雄性不育现象绝大部分都是由隐 性雄性不育基因控制的。1978年我们在澳大利 亚谷和吐鲁番谷的杂交后代中发现一份雄性不 育材料。从1978-1984年7个世代的遗传表 现为：和400个谷子普通品种杂交,其不育株 自由授粉、其不育株和分离出来的可育株杂交、 和原父回交,F,就出现育性分离,不育与可育的 比率都为1：1;其可育株后代育性不分离;其不 育株自交子一代育性分离,不育与可育的比率 为3:1。如此证明,这份雄性不育材料的不育 性是受显性雄性不育基因控制的。     

A biotechnology‐based male‐sterility system for hybrid seed production in tomato

Incorporating male sterility into hybrid seed production reduces its cost and ensures high varietal purity. Despite these advantages, male‐sterile lines have not been widely used to produce tomato (Solanum lycopersicum) hybrid seeds. We describe the development of a biotechnology‐based breeding platform that utilized genic male sterility to produce hybrid seeds. In this platform, we generated a novel male‐sterile tomato line by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9)‐mediated mutagenesis of a stamen‐specific gene SlSTR1 and devised a transgenic maintainer by transforming male‐sterile plants with a fertility‐restoration gene linked to a seedling‐colour gene. Offspring of crosses between a hemizygous maintainer and the homozygous male‐sterile plant segregated into 50% non‐transgenic male‐sterile plants and 50% male‐fertile maintainer plants, which could be easily distinguished by seedling colour. This system has great practical potential for hybrid seed breeding and production as it overcomes the problems intrinsic to other male‐sterility systems and can be easily adapted for a range of tomato cultivars and diverse vegetable crops.     

玉米隐性核不育突变体ms14的遗传分析与基因定位

玉米雄性不育材料是一种宝贵的种质资源,不育基因的遗传分析与定位研究对玉米分子育种和杂种优势利用具有重要价值。通过对从美国引进的玉米雄性不育突变体材料ms14进行雄花育性鉴定和花药I₂-KI染色,表明该突变体是无花粉型雄性不育;通过不育突变体ms14与正常自交系郑58、昌7-2杂交获得F₁,然后自交构建两个F₂遗传分离群体（ms14×郑58和ms14×昌7-2）,并进行雄花育性调查、数据统计和遗传分析,发现可育株数与不育株数的分离比是3∶1,表明该突变体由隐性单基因控制;通过SSR等分子标记与不育位点的连锁分析,将ms14基因定位在玉米第1染色体的SSR标记umc2025和umc1676之间,遗传距离分别是2.2cM和0.3cM。对玉米不育基因ms14的遗传分析和初步定位,为该基因的精细定位和克隆、不育机理的解析及其产业化应用奠定了基础。     

Distinguishing among male sterile and fertile lines of tomatoes (Lycopersicon esculentum Mill.) by means of electrophoretic protein patterns

Vertical block SDS-PAAG electrophoresis of individual tomato seeds was used to examine the potential of seed proteins as gene markers for distinguishing male sterile from fertile lines and cultivars. Seeds of three genic male sterile lines and four fertile cultivars and lines were investigated. The line 6944 is carrier of the gene ms 10 ³⁵for pollen male sterility and the lines P-1a and PPS-3 carry the gene ps-2 for positional sterility. A modified extract solution which includes 1M tris — buffer pH 7.0, 7 % SDS, dimethylformamide and distilled water was applied. Qualitative variation in the A protein locus which could be used as a marker for distinguishing male sterile from fertile tomato lines was established.     

以基因枪介导获转ps1—barnase基因的工程雄性不育水稻植株

以ps1-barnase(brn)为目的基因,pHcintG(PG)为选择/标记基因进行共转化,以PDS-1000-氦气基因枪介导,将brn及PG基因转化到水稻台北309及秋光的核DNA中,得到了转ps1-barnase基因的工程雄性不育植株。以悬浮细胞作为基因枪轰击的靶材料,转化植株再生频率较初级愈伤组织的为高。转brn基因植株的其他主要性状与供体亲本无显著差异,但却表现不育。其不育的程度在不同的植株之间表现不同。在转brn基因植株中观察到全不育(占全部brn阳性植株的40.6％)、高不育(占15.6％)及半不育的个体(占43.7％)。全不育的转基因植株自交完全不能结实(结实率为零),除个别植株外,花粉完全不被I-KI染色;而人工授以正常的花粉则可以获得杂交种子。而brn基因的阴性植株及未进行转化的对照植株则完全可育,表明转基因植株之雄性不育乃brn基因所致。结果表明,brn基因在水稻中是完全可以正常表达的,其表达的时期推测在花粉母细胞减数分裂前至花粉形成之间的整个时期。     

Generation of a transgenic mouse in which Cre recombinase is expressed under control of the type II collagen promoter and doxycycline administration.

The ability to generate tissue-specific ablation of gene expression has been extremely useful in connective tissue biology, as it can potentially overcome the early embryonic lethal phenotype often associated with universal gene knockout. The value of tissue-specific knockouts can be enhanced by also allowing gene ablation to occur at specific times during development, growth or aging. In the present work a transgenic mouse has been generated in which expression of Cre recombinase is under control of both the type II collagen promoter to allow cartilage-specific expression and a doxycycline response element to permit temporal control of expression. This mouse has been crossed with the Rosa26R reporter mouse, which possesses a floxed repressor element associated with a lacZ transgene, in order to validate the functional efficacy of the conditionally expressed Cre. The results demonstrate that excision of the floxed element can be achieved specifically in cartilage at different times during embryonic and juvenile development. The conditional Cre transgenic mouse should be a valuable tool to all interested in skeletal development.     

Inducing male sterility of tomato using two component system

Production of hybrid seeds and pursuing heterosis breeding of many crops have been accomplished using male sterile lines. However, not all crops have valuable male sterile lines due to instability of male sterility and absence of a restorer system. In this study, male sterile lines have been induced using a two-component system. The extracellular ribonuclease Barnase was cleaves into two inactive yet complementary fragments, designated as ??Bn-5?? and ??Bn-3??. Both components were controlled by a TA29 promoter. They were transferred into the tomato inbred line ??Yellow tomato?? by Agrobacterium method. Southern blotting identified that 11 transgenic Bn-5 plants (T₀) and 10 transgenic Bn-3 plants (T₀) were obtained. The vegetative phenotypes of all T₀ plants were similar to wild-type, and they were capable of producing viable pollen grains and normal fruit with seeds, indicating that Barnase had lost its function after it being split two partial fragments. After self-pollination, homozygous progenies (T₁) of transgenic Bn-5 and Bn-3 plants were chosen to cross each other, Barnase could be reconstituted and co-expressed in the same cell, which caused the hybrid plants to produce collapsed pollen grains with no viability and thus100?% male sterile plants were obtained. Stamens of male sterile plants were shorter than those of the wild type plants. PCR detection demonstrated that all male sterile plants contained Barnase, but male fertile plants did not. The male sterile plants were crossed with the male fertile inbred lines, and the result showed that hybrid (F₁) plants were capable of producing normal fruit with seeds, and their pollen grain fertility was restored. The co-segregation ratio of Bn-5 and Bn-3 fragments showed 1:1 among hybrid plants. In conclusion, the results verified that the male sterility could be generated by two component system and be used in hybrid seed production. The F₁ between the male sterile plant and the inbred line showed heterotic comparing to both parents. This system needs not breed restoration line.     

Cre/ lox site-specific recombination controls the excision of a transgene from the rice genome

The Cre/lox site-specific recombination controls the excision of a target DNA segment by recombination between two lox sites flanking it, mediated by the Cre recombinase. We have studied the functional expression of the Cre/lox system to excise a transgene from the rice genome. We developed transgenic plants carrying the target gene, hygromycin phosphotransferase (hpt) flanked by two lox sites and transgenic plants harboring the Cre gene. Each lox plant was crossed with each Cre plant reciprocally. In the Cre/lox hybrid plants, the Cre recombinase mediates recombination between two lox sites, resulting in excision of the hpt gene. The recombination event could be detected because it places the CaMV 35S promoter of the hpt gene adjacent to a promoterless gusA gene; as a result the gusA gene is activated and its expression could be visualized. In 73 Cre/lox hybrid plants from various crosses of T0 transgenic plants, 19 expressed GUS, and in 132 Cre/lox hybrid plants from crosses of T2 transgenic plants, 77 showed GUS expression. Molecular data proved the excision event occurred in all the GUS⁺ plants. Recombination occurred with high efficiency at the early germinal stage, or randomly during somatic development stages. Received. 2 April 2001 / Accepted: 29 June 2001     

TA29-barnase基因转化菜心

利用根癌农杆菌导入法, 以菜心带柄子叶为外植体, 对TA29-barnase基因转化菜心进行研究。获得转化植株,进行PCR、Southern blotting杂交和半定量RT-PCR检测, 表明目的基因已经整合到转化植株中, 并且目的基因在转基因植株花蕾中得到表达, 但是表达水平在不同转基因植株间存在差别; 转基因植株开花后, 均表现雄性不育, 不能产生花粉或产生没有活力的少量花粉, 自交不能结实; 用未转化植株正常花粉对雄性不育植株进行授粉, 能够正常结实; 保持系(未转化植株)与不育株杂交后代中雄性不育株与可育株的比例为1:1, 在杂交后代植株子叶期, 喷洒10 mg/L的PPT可以完全杀死可育株; 利用其他菜心品种为父本与不育株进行杂交, 获得的F1植株在生长势和产量方面表现优势, 表明开展菜心优势育种具有一定的潜力。