青花菜细胞质雄性不育相关miRNA的鉴定与表达特征分析

植物miRNA是一类内源小分子RNA,在花粉发育和育性调控中具有重要作用。该研究利用高通量测序技术构建了青花菜细胞质雄性不育系及其保持系花蕾的sRNA文库,并通过qRT PCR技术检测育性相关的miRNAs及其预测靶基因的表达特征,为深入探讨miRNA调控青花菜育性机制提供理论依据。结果表明：(1)在青花菜中共鉴定到181个保守miRNA和8个新型miRNA,差异表达miRNA共47个,其中24个已知miRNAs和4个新型miRNAs表达上调,其余为表达下调;发现2个差异miRNA只在不育系中表达,7个差异miRNA为保持系所特有。(2)生物信息学分析表明,共鉴定到2个关联的miRNA mRNA对［miR859 1靶向的花粉外壁蛋白基因(T2/Unigene_BMK.21608)和miR5174e 1调控的氨基酸通透酶基因(T2_Unigene_BMK.31772)］,2个miRNA均负向调控相应的靶基因,均可参与青花菜小孢子发育以及雄性不育的发生过程。(3)qRT PCR分析显示,miR159a 1、miR164a 1、miR319a 1和miRn11在青花菜不育系中的表达量高于其保持系,其中miR164a 1、miR319a 1和miRn11的表达水平随着花蕾的发育不育系逐渐降低,而在保持系的表达呈上升趋势;miR319a 1和miR397a 2在雄蕊中的表达量高,而在其他部位的表达量较低,且miR397a 2在青花菜不育系中的表达量显著低于保持系。研究推测,miRNA可能是通过抑制雄性不育相关的PPR基因表达、调控LAC基因的表达以及调节激素和Ca²⁺介导的信号转导通路等,从而导致青花菜雄性不育的发生。     

青花菜谷胱甘肽S-转移酶的克隆及表达特性分析

本研究采用RT-PCR技术从青花菜自交系‘WN12-95B’中克隆到谷胱甘肽S-转移酶基因。序列分析表明,青花菜GST基因全长690 bp,ORF长度为675 bp,推导编码蛋白含有224个氨基酸,相对分子量为26.15 kD,理论pI值为6.56,属于Tau类家族成员。青花菜GST蛋白的二级结构主要以α-螺旋和延伸链为主。通过构建系统进化树发现,GST基因与油菜、芜菁亲缘关系最近。利用荧光定量PCR检测GST基因在青花菜保持系不同组织中的表达量,结果显示GST基因在根、叶、荚中的表达丰度较高,花蕾中表达丰度较低。青花菜Ogu不育系及其保持系不同发育阶段花蕾时空表达特性分析表明,花蕾发育早期(2 mm)表达量最高,随着发育进程的推移,表达量逐渐下降。同时期不育系的表达量高于保持系。本研究为探讨青花菜GST基因在花粉发育过程中的功能提供一定理论依据。     

辣椒雄性不育系SDH4基因的表达特性分析

为了探究线粒体电子传递复合体Ⅱ的关键酶基因(SDH4)与辣椒细胞质雄性不育的关系,该试验通过GenBank报道的辣椒线粒体基因组序列,特异引物扩增SDH4基因,并通过分析SDH4基因的时空表达及转录本编辑位点,以期找到辣椒细胞质雄性不育系9704A和保持系9704B的差异。结果表明:(1)从辣椒细胞质雄性不育系9704A和保持系9704B中获得的目的基因编码区片段长度一致,全长均为378bp,编码125个氨基酸残基。(2)辣椒保持系不同组织中SDH4基因表达存在差异,种子中表达最高,茎中表达最低。(3)在不同材料花蕾发育的同一时期,SDH4基因表达也不一致,在花粉母细胞减数分裂时期,不育系SDH4基因表达量明显低于保持系;而在造孢细胞增殖期、小孢子单核期和小孢子成熟期的表达量均高于保持系。(4)不育材料中SDH4基因在29位点出现RNA编辑,导致氨基酸由丝氨酸变为亮氨酸,增强了蛋白结构的疏水性能。研究认为,辣椒细胞质雄性不育系9704A和保持系9704B中SDH4基因的表达差异可能引起植物的能量代谢供应出现异常,从而导致雄性不育的产生。     

青花菜两类雄性不育系花器官形态结构的比较

青花菜是一种重要的十字花科蔬菜作物,具有良好的抗癌功效。随着青花菜栽培面积的不断扩大和抗癌药物的开发,对青花菜种子的需求量也不断增加。利用雄性不育系生产青花菜F1杂交种子是其杂种优势利用的重要途径,但常用的细胞质雄性不育系在生产中存在前期死蕾较重、花蜜量少和种子产量不高等问题。本课题组用优良的青花菜自交系为父本,以甘蓝显性细胞核雄性不育材料79-399-3和细胞质不育材料OguraCMSR3629为不育源,通过回交转育的方法获得了多个青花菜显性细胞核雄性不育系和细胞质不育系,为了进一步有效利用其青花菜雄性不育系,寻求更好的不育源。本研究以青花菜高代自交系(保持系)8554、8590、93219及由其经多代回交转育而成的两类雄性不育系为试材,对其死蕾数、花器官形态和蜜蜂访花情况进行了比较研究。结果表明,在花蕾长、花蕾直径、单枝死蕾数、花冠直径、雄蕊长、花瓣长、花药长、蜜蜂访花次数、来访蜜蜂数及访花时间等方面,经相同自交系转育而成的显性细胞核雄性不育系(DGMS)与细胞质雄性不育系(CMS)间的差异有统计学意义,总体表现为DGMS优于CMS。     

辣椒细胞质雄性不育系和保持系过氧化物酶同工酶的比较分析

运用辣椒细胞质雄性不育系21A和保持系21B为试材,比较分析了过氧化物酶（POD）同工酶在幼叶和不同发育时期花蕾中的表达特征。结果表明,辣椒花蕾中的POD同工酶谱带均比叶片中的更丰富,表现出明显的组织特异性。不育系21A和保持系21B间的酶谱在幼叶和小花蕾（纵径0．8～1．5mm）中基本无差异,但从中花蕾（纵径2～3mm）开始,保持系21B的一些酶带（POD4b和POD4c）染色开始减弱。在大花蕾（纵径4．5～5mm）和特大花蕾（纵径6．8～7mm）时期,不育系比保持系分别多2条酶带（POD2c和POD4b）和1条酶带（POD2c）,表明辣椒细胞质雄性不育系21A与保持系21B花蕾中的POD同工酶表达与小孢子的发育过程相关,其表达差异发生在细胞学上观察到的败育之前。     

辣椒雄性不育系葡萄糖-6-磷酸脱氢酶基因的克隆及表达分析

为了深入研究辣椒雄性不育与能量代谢之间的关系,该研究以辣椒近缘物种番茄的葡萄糖-6-磷酸脱氢酶基因(G6PDH)同源序列为基础,采用电子克隆的方法克隆出辣椒CaG6PDH基因。利用荧光定量PCR技术,对辣椒雄性不育系9704A与其保持系9704B花蕾发育的不同阶段,以及保持系9704B不同组织(茎、叶、花、果皮、胎座、种子)中CaG6PDH基因进行表达分析。结果表明:两系中获得的CaG6PDH基因的编码序列一致,全长1 533bp,编码510个氨基酸残基;辣椒CaG6PDH基因在保持系不同组织中表达量存在差异,胎座中表达量最高,茎中表达量最低;辣椒CaG6PDH基因的表达量在花蕾发育的不同阶段雄性不育系均高于保持系,此种差异在小孢子发育的单核期与成熟期尤为明显,这种差异可能使雄性不育系能量代谢供应出现异常,从而影响小孢子的正常发育而导致雄性败育。     

玉米黄早4雄性不育系、保持系过氧化物酶同工酶的比较研究

在玉米黄早4雄性不育系、保持系的10个组织(叶、根、茎、芽鞘、胚轴、苞叶、穗轴、花丝、雌蕊、花药)中共检测出18种正、负极向过氧化物酶,其中有7个组织不育系与保持系间过氧化物酶没有差异,只有在3个组织中(叶、茎、花药)不育系与保持系间的过氧化物酶存在差异,说明玉米黄早4雄性不育系及保持系的过氧化物酶可能由细胞核基因编码。不育系与保持系个别组织内过氧化物酶存在差异,可能是由于核内编码过氧化物酶的基因表达异常所引起,而这种表达异常,可能是与不育系中不育细胞质基因调控核基因的表达有关。     

辣椒胞质雄性不育系线粒体基因CaATP9的克隆与表达

通过克隆辣椒胞质雄性不育系线粒体基因CaATP9及其表达情况,为研究辣椒胞质雄性不育机理提供依据。以辣椒胞质雄性不育系9704A和同型保持系9704B为试验材料,研究CaATP9的一级结构在2个材料之间的差异和RNA编辑现象,并利用实时荧光定量PCR研究了该基因的时空表达。结果表明,CaATP9的一级结构在2个材料之间没有差异;CaATP9在转录过程中存在5个C→T的编辑位点,导致4个亲水性氨基酸(DNA模板)变换成4个疏水氨基酸,增强了蛋白质的稳定性;CaATP9在辣椒的不同组织中均有表达,但表达量存在一定差异,繁殖器官中的表达量要普遍高于营养器官;在花蕾发育过程中,胞质雄性不育系中CaATP9的表达量与同型保持系存在差异,推测这种表达差异导致胞质雄性不育系花蕾能量供应紊乱,影响小孢子的正常发育而表现不育。     

辣椒胞质雄性不育系和保持系内源激素含量的比较

以2个辣椒品系(199807、199803)的胞质雄性不育系和相应保持系为实验材料,采用酶联免疫吸附法(ELISA)测定IAA、(Z ZR)、GA3和ABA等内源激素含量,用气相色谱分析仪测定乙烯(ETH)释放量,对辣椒胞质雄性不育系和相应保持系内源激素含量变化规律进行研究.实验结果表明:在四分小孢子之前,花药中的IAA含量不育系显著高于保持系,在四分小孢子时期花药和花蕾中的IAA含量出现转折,到花粉粒成熟期的花蕾和花药以及开花期叶片中的IAA含量均是不育系显著低于保持系;小孢子各发育时期花药以及花期叶片中GA3含量均是不育系高于保持系,但花粉粒成熟期化蕾中GA3含量为不育系低于保持系;小孢子不同发育时期的花药以及花期叶片中ABA含量始终足不育系显著高于保持系,而花粉粒成熟期花蕾中ABA含量不育系与保持系没有显著差异;花粉粒成熟期的花蕾和花期叶片中ETH释放量表现为不育系显著高于保持系.同时,花粉粒成熟期的花蕾、花药和叶片中IAA/ABA、(Z ZR)/ABA、GA3/ABA、IAA/GA3、(Z ZR)/GA3等5个激素的比值均有不育系低于保持系的趋势.本实验结果说明辣椒的育性表现与花器和叶片等组织中内源激素的含量变化有关,花药和花期叶片中IAA亏缺、GA3和ABA增加以及化蕾和叶片中ETH过度产生,都有可能导致辣椒雄性不育.     

不结球白菜Pol胞质雄性不育系和保持系蕾期基因的差异表达

以不结球白菜Pol胞质雄性不育系及其保持系为材料,利用cDNA-AFLP技术分析它们蕾期基因的差异表达,以获得与胞质不育相关的差异表达基因.结果共得到7条在GenBank中有同源性的差异表达片段,5条存在于不育系中,2条存在于保持系中.序列分析发现,在不育系花蕾克隆到的5条差异片段,分别与芥菜胞质雄性不育系线粒体orf108和atpA、拟南芥焦磷酸酶基因、拟南芥的锚定蛋白家族基因、甘蓝型油菜中的水胁迫蛋白、大白菜叶绿体基因片段有较高同源性;在保持系花蕾克隆到的2条差异片段分别与拟南芥未知蛋白基因有较高同源性.采用实时定量PCR验证其中5条差异片段在不育系和保持系花蕾中的表达水平,结果表明bcA19T15、bcA7T9在不育系中特异表达,bcA6T9、bcA19T8、bcA12T19在不育中表达比保持系中略强.     

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.     

A comparative study of the atp9 gene between a cytoplasmic male sterile line and its maintainer line and further development of a molecular marker specific for male sterile cytoplasm in kenaf (Hibiscus cannabinus L.)

mtDNA was isolated from cytoplasmic male sterility (CMS) line P3A and its maintainer P3B of kenaf (Hibiscus cannabinus L.). The atp9 gene and its two flanking sequences were obtained using homology cloning and high-efficiency thermal asymmetric interlaced PCR methods. The coding sequences showed only two base pairs difference between the CMS and its maintainer, and shared a homology of over 87 % with atp9 genes from other species in GenBank. However, when comparing the flanking sequences, a 47-bp deletion was characterized at the 3′ flanking sequence of atp9 in the CMS line. Quantitative PCR analysis indicated that the expression level of atp9 in the CMS line was 0.937-fold that of its maintainer. Furthermore, the respiratory rate of anthers in the CMS line was markedly lower than that of its maintainer. The results indicated that the 47-bp deletion at the 3′ flanking sequence of atp9 and/or down-regulated expression of the atp9 gene in the CMS line might be closely related to CMS in kenaf. To confirm whether the 47-bp deletion was specific to cytoplasm of male sterile lines, another 21 varieties were used for further analysis. The results showed that the 47-bp deletion was specific to male sterile cytoplasm (MSC) of kenaf. Based on these, a specific molecular marker was developed to distinguish the MSC from male fertile cytoplasm of kenaf.     

Small mitochondrial DNA molecules of wild abortive cytoplasm in rice are not necessarily associated with CMS

Summary Mitochondrial DNA was isolated from leaf tissue of both the cytoplasmic male sterile line of Indica rice variety V41, which carries wild abortive (WA) cytoplasm, and from the corresponding maintainer line. In addition to the main mitochondrial DNA, four small plasmid-like DNA molecules were detected in both the male sterile and fertile lines. Restriction analysis of total mitochondrial DNA from the male sterile and fertile lines showed DNA fragments unique to each. Our findings suggest that the four small mitochondrial DNA (mtDNA) molecules are conserved when WA cytoplasm is transferred into different nuclear backgrounds. However, there is no simple correlation between the presence/ absence of small mitochondrial DNA molecules and the expression of WA cytoplasmic male sterility (CMS).     

罗汉果花芽分化过程中形态及其激素水平变化特征

采用石蜡切片和酶联免疫法(ELISA)对罗汉果雄性、雌性、两性花芽分化过程的形态和激素水平变化进行观测,为罗汉果开花调控和品种选育提供科学依据。结果表明:(1)罗汉果雄性、雌性、两性花的花芽分化过程均可分为花芽未分化期、花芽分化初期、花序分化期、萼片原基分化期、花瓣原基分化期、雄蕊原基分化期和雌蕊原基分化期7个阶段。雄蕊原基分化期前,3种花芽分化过程无明显差异,各时期形态特征均依次为:茎端呈圆锥状(花芽未分化期)→茎端经半球形变成扁平状(花芽分化初期)→距茎端5~7节位处分化出穗状花序(花序分化期)→小花原基周围形成5个萼片原基(萼片原基分化期)→萼片原基内侧形成5个花瓣原基(花瓣原基分化期)。雄蕊和雌蕊原基分化期,3种花芽分化过程存在明显差异,雄蕊原基内侧出现雌蕊原基后,雄花芽雄蕊原基继续发育成雄蕊,雌蕊原基停滞生长,退为一个小突起;雌花芽雌蕊原基继续发育成雌蕊,雄蕊原基生长缓慢,退化为小花丝;两性花芽雌蕊和雄蕊原基均继续发育,形成外观正常的雌蕊和雄蕊。(2)内源激素脱落酸(ABA)、赤霉素(GAs)和玉米素核苷(ZR)含量在3种花芽分化过程中变化规律相似,即ABA含量在花芽生理分化期降低,花芽形态分化期升高,而GAs和ZR含量则基本保持不变;吲哚乙酸(IAA)含量在3种花芽分化过程中变化存在明显差异,雌花芽IAA含量在花芽生理分化期升高,花芽形态分化期逐渐降低,而雄性和两性花芽的IAA含量则基本保持不变。ABA/GAs、ABA/IAA、ZR/IAA和ZR/GAs激素含量比值在3种花芽分化过程中变化规律相似,ABA/GAs在花芽生理分化期降低,花芽形态分化期升高,而BA/IAA、ZR/IAA和ZR/GAs则基本保持不变。研究认为,罗汉果花芽分化过程经历一个"两性期",高ABA含量和ABA/GAs比值有利于罗汉果花芽分化,IAA可能对罗汉果花性分化具有重要作用。     

Development of a SCAR marker for early identification of S-cytoplasm based on mitochondrial SRAP analysis in pepper (Capsicum annuum L.)

Molecular markers, coxII SCAR, atp6-2 SCAR and accD-U, have been used for marker-assisted selection of cytoplasmic male sterility (CMS) in pepper. However, the presence of these markers at the sub-stoichiometric level in maintainer lines affects the reliable selection of male sterile (S-) cytoplasm. This study aimed to develop a new CMS-specific molecular marker, SCAR₁₃₀, for reliable identification of S-cytoplasm in pepper, while the new and three previous molecular markers were used to determine the cytoplasm types of pepper lines. Based on mitochondrial genome sequence related amplified polymorphism (SRAP) analysis of the CMS lines and the maintainer lines, SCAR₁₃₀ was developed from a 10-bp deletion at the SRAP primer binding site in the CMS line (130 bp) compared with that in the maintainer line (140 bp). S-cytoplasm could be unambiguously selected from the pepper lines by the different length of the marker bands. Application of the four molecular markers to various pepper lines revealed that SCAR₁₃₀ is more reliable than the other three previous markers, orf507, ψatp6-2 and accD-U. Homology alignment with BLAST showed that the marker was located between trnE and trnS in the Nicotiana tabacum mitochondrial genome. Furthermore, expression of the marker-linked gene was significantly higher at the pollen abortive stage in the CMS line (HW203A) than in the maintainer line, which indicated that the marker was closely related to male sterility. Hence, factors other than orf507 and ψatp6-2 may exist for the regulation of male sterility in pepper.     

Alterations of RNA Editing for the Mitochondrial ATP9 Gene in a New orf220-type Cytoplasmic Male-sterile Line of Stem Mustard （Brassica juncea var. tumida）

RNA editing for the mitochondrlal ATP9 gene of encoding regions has been observed in both cytoplasmic malesterile and maintainer lines of stem mustard, where its editing capacity varied spatially and temporally in the cytoplasmic male sterility （CMS） line. There were four RNA editing sites for the mitochondrial ATP9 gene according to Its normal editing sites in mustard, of which three sites occurred as C-to-U changes and one as a U-to-C change. As a result, the hydrophobicity of deduced ATP9 protein was reduced due to the conversions at its 17th, 45th and 64th positions. Meanwhile, the conservation of deduced ATP9 protein was enhanced by changes at the 56th position. Loss of a specific editing site for ATP9 was observed in juvenile roots, senile roots, senile leaves and floret buds of the CMS line. Comparatively, complete RNA editing for ATP9 gene was retained in juvenile roots, juvenile leaves and floret buds of its maintainer line; however, the loss of a specific editing site for ATP9 gene occurred at senile roots and senile leaves in its maintainer line. These observations allow us to produce a hypothesis that the dysfunction of a specific mitochondrial gene arising from RNA editing could probably be a factor triggering CMS and organ senescence through unknown cross-talk pathways during development.     

细胞质雄性不育水稻幼穗和花粉发育期的蛋白酶活性变化

水稻细胞质雄性不育系珍汕97A,在花粉母细胞形成期以后可溶性蛋白含量迅速降低。单核期仅为花粉母细胞形成期的56％,二核期和三核期分别为花粉母细胞形成期的36．4％和30．3％。保持系珍汕97B虽在花粉母细胞形成期后可溶性蛋白含量降低,但在二核期则有所增高,表现为鞍形变化过程。在花粉母细胞形成期后,珍汕97A的酪蛋白水解酶活性增高,单核期为前一时期的1倍,而珍汕97B的酶活性增幅较小。第一次枝梗分化期珍汕97A的内肽酶活性较珍汕97B高,而雌雄蕊形成期两者的酶活皆降低。整个发育期中珍汕97A的内肽酶活性较珍汕97B高15％至55．5％。珍汕97A在幼穗第一次枝梗分化期时氨肽酶活性较珍汕97B高。第二次枝梗分化期后,不育系和保持系的氨肽酶活性均下降。但花粉母细胞形成期后,珍汕97A的氨肽酶活性迅速增高,三核期达最高水平,这可能意味着此时大量蛋白质被水解。蛋白酶类抑制剂的实验表明,在雌雄蕊形成期后不育系和保持系含有半胱氨酸型氨肽酶和约10％含金属型氨肽酶;而在三核期,保持系的氨肽酶则主要为半胱氨酸型。不育系三核期含有的BAPA内肽酶包括半胱氨酸型、丝氨酸型,以及部分含金属酶类;而保持系三核期的内肽酶仅为丝氨酸型。不育系肽酶类型的变化,可能反映酶基因编码的去抑制。     

Organelle Simple Sequence Repeat Markers Help to Distinguish Carpelloid Stamen and Normal Cytoplasmic Male Sterile Sources in Broccoli

We previously discovered carpelloid stamens when breeding cytoplasmic male sterile lines in broccoli (Brassica oleracea var. italica). In this study, hybrids and multiple backcrosses were produced from different cytoplasmic male sterile carpelloid stamen sources and maintainer lines. Carpelloid stamens caused dysplasia of the flower structure and led to hooked or coiled siliques with poor seed setting, which were inherited in a maternal fashion. Using four distinct carpelloid stamens and twelve distinct normal stamens from cytoplasmic male sterile sources and one maintainer, we used 21 mitochondrial simple sequence repeat (mtSSR) primers and 32 chloroplast SSR primers to identify a mitochondrial marker, mtSSR2, that can differentiate between the cytoplasm of carpelloid and normal stamens. Thereafter, mtSSR2 was used to identify another 34 broccoli accessions, with an accuracy rate of 100%. Analysis of the polymorphic sequences revealed that the mtSSR2 open reading frame of carpelloid stamen sterile sources had a deletion of 51 bases (encoding 18 amino acids) compared with normal stamen materials. The open reading frame is located in the coding region of orf125 and orf108 of the mitochondrial genomes in Brassica crops and had the highest similarity with Raphanus sativus and Brassica carinata. The current study has not only identified a useful molecular marker to detect the cytoplasm of carpelloid stamens during broccoli breeding, but it also provides evidence that the mitochondrial genome is maternally inherited and provides a basis for studying the effect of the cytoplasm on flower organ development in plants.     

一个辣椒功能性雄性不育系的花器形态及遗传研究

本报道了一个新型辣椒功能性雄性不育系的花器形态和遗传规律。研究表明,新发现的辣椒雄性不育花冠退化,大部分雄蕊萎缩,没有花丝、具发育不完整的花药;虽然花内１～２枚发育正常的雄蕊有少量可育花粉,但由于花萼畸长紧闭,不能开花。雌蕊发育正常,人工辅助自花或异花杂交可正常结果并产生种子。遗传杂交分析表明,该功能性雄性不育遗传上受一对隐性核基因控制,用基因的符号ｆｍｓ表示。