大豆雄性不育系与其保持系不同器官蛋白质比较

采用双向凝胶电泳技术对大豆质核互作雄性不育系NJCMS2A及其保持系NJCMS2B的种子、叶片和花药等不同器官蛋白质进行比较分析.结果显示,不育系NJCMS2A与其保持系NJCMS2B的花药2-DE图谱间存在较多差异表达蛋白点,种子2-DE图谱间仅有少量差异表达蛋白点,而叶片2-DE图谱间基本没有差异表达蛋白点.结果表明,不育基因表达具有时空性和器官特异性,与育性有关的蛋白主要在花药中表达.     

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.     

生物膜形成中间状态下副溶血弧菌的基因转录谱分析

【目的】探究生物膜形成中间状态下副溶血弧菌的差异基因表达情况,为今后研究生物膜形成调控机制提供基因信息。【方法】以非生物膜形成条件下为参照,采用Illumina HiSeq测序平台进行转录组测序（RNA sequencing,RNA-seq）研究,分析生物膜形成中间状态下副溶血弧菌的基因表达情况,并采用实时定量PCR （quantitative real-time PCR,qPCR）进行验证。【结果】本研究共获得979个差异显著性表达基因（differentially expressed gene,DEG）,其中下调基因379个,上调基因600个。基因本体（gene ontology,GO）分类分析结果显示,共有363个DEGs注释到分子功能、细胞组分和生物学过程3个一级分类和30个二级分类;京都基因和基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）代谢途径分析结果显示,共有706个DEGs归到37个代谢通路中（Q value<0.05）,差异表达基因主要集中在细胞代谢和转运通路上;蛋白相邻类的聚簇（clusters of orthologous groups,COG）分类结果显示,有888个DEGs可归为20个类别,涉及DEGs最多的为氨基酸转运与代谢、一般功能预测基因、能量产生与转换以及未知功能基因。qPCR验证挑选的DEGs变化趋势均与RNA-seq的结果一致。此外,从转录组数据中共筛选出10个c-di-GMP代谢相关基因、1个侧生鞭毛蛋白基因（lafA）、13个极生鞭毛合成相关基因、6个荚膜多糖合成相关基因、6个胞外多糖合成相关基因、5个IV型菌毛合成相关基因、膜融合蛋白（membrane fusion protein,Mfp）基因（cpsQ-mfpABC）、14个III型分泌系统1（T3SS1）相关基因、14个Vp-PAI基因（1个tdh2和13个T3SS2基因）、3个VI型分泌系统1（T6SS1）相关基因、6个T6SS2基因、45个推定调控子基因和15个推定的外膜蛋白基因。【结论】生物膜形成引起副溶血弧菌基因表达谱发生明显变化,差异表达基因中包含生物膜形成相关基因、关键毒力基因和许多推定调控子基因等,这为后续研究生物膜形成调控机制提供重要信息。     

Map-based cloning of a fertility restorer gene, Rf-1, in rice (Oryza sativa L.)

A rice nuclear gene, Rf-1, restores the pollen fertility disturbed by the BT-type male sterile cytoplasm, and is widely used for commercial seed production of japonica hybrid varieties. Genomic fragments carrying Rf-1 were identified by conducting chromosome walking and a series of complementation tests. Isolation and analysis of cDNA clones corresponding to the fragments demonstrated that Rf-1 encodes a mitochondrially targeted protein containing 16 repeats of the 35-aa pentatricopeptide repeat (PPR) motif. Sequence analysis revealed that the recessive allele, rf-1, lacks one nucleotide in the putative coding region, presumably resulting in encoding a truncated protein because of a frame shift. Rice Rf-1 is the first restorer gene isolated from cereal crops that has the property of reducing the expression of the cytoplasmic male sterility (CMS)-associated mitochondrial gene like many other restorer genes. The present findings may facilitate not only elucidating the mechanisms of male sterility by the BT cytoplasm and its restoration by Rf-1 but also isolating other restorer genes from cereal crops, especially rice.     

Transcriptomic analysis of differentially expressed genes in flower-buds of genetic male sterile and wild type cucumber by RNA sequencing

Cucumber (Cucumis sativus L.) pollen development involves a diverse range of gene interactions between sporophytic and gametophytic tissues. Previous studies in our laboratory showed that male sterility was controlled by a single recessive nuclear gene, and occurred in pollen mother cell meiophase. To fully explore the global gene expression and identify genes related to male sterility, a RNA-seq analysis was adopted in this study. Young male flower-buds (1–2 mm in length) from genetic male sterility (GMS) mutant and homozygous fertile cucumber (WT) were collected for two sequencing libraries. Total 545 differentially expressed genes (DEGs), including 142 up-regulated DEGs and 403 down-regulated DEGs, were detected in two libraries (Fold Change ≥ 2, FDR < 0.01). These genes were involved in a variety of metabolic pathways, like ethylene-activated signaling pathway, sporopollenin biosynthetic pathway, cell cycle and DNA damage repair pathway. qRT-PCR analysis was performed and showed that the correlation between RNA-Seq and qRT-PCR was 0.876. These findings contribute to a better understanding of the mechanism that leads to GMS in cucumber.     

Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function.     

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.     

青花菜细胞质雄性不育相关LncRNAs的鉴定与表达分析

细胞质雄性不育系的应用可有效提高杂交种质量。该研究利用illumina测序技术鉴定青花菜细胞质雄性不育相关LncRNAs,对其靶基因和表达特征进行分析,并随机选取16个LncRNAs用qRT-PCR技术检测其表达特征,为进一步阐明LncRNA参与青花菜雄性不育发生机制提供新的路径。结果表明：(1)鉴定获得青花菜雄性不育相关LncRNAs共4 326个,其中37个LncRNA在不育系及其保持系中差异表达。(2)差异LncRNAs可预测得到370个cis靶基因,这些靶基因部分为雄性不育相关的转录因子和生物蛋白。(3)XLOC＿006651、XLOC＿016660、XLOC＿003494和XLOC＿013121在不育系和保持系花蕾发育早期表达量较高,之后随着花蕾的发育,表达量逐渐下降;XLOC＿021769和XLOC＿038964呈先降后升的表达模式,且XLOC＿038964和XLOC＿012613在花蕾不同发育阶段不育系的表达量均高于保持系。(4)16个LncRNA均可在花梗、花萼、花瓣、雄蕊及雌蕊中表达,且XLOC＿038964、XLOC＿011575、XLOC＿013157、XLOC＿...