雄性不育枸杞花药比较转录组分析

为筛选宁夏枸杞(Lycium barbarum L.)雄性不育花药发育相关的差异表达基因,本研究开展了雄性不育品种‘宁杞5号’和可育品种‘宁杞1号’成熟花粉时期花药转录组分析,共获得差异表达基因1 759个,在‘宁杞5号’中有753个基因上调表达,1 006个基因下调表达。KEGG富集表明,这些差异表达基因主要富集到氨基酸的生物合成、植物激素信号传导、苯丙素生物合成、淀粉和蔗糖代谢、脂肪酸代谢等相关通路。对有注释信息的差异表达基因进行NR和Uniprot数据库检索,筛选到28个花药发育相关基因,其中有20个在‘宁杞5号’中下调表达,8个上调表达。选取其中的5个差异表达基因进行RT-qPCR验证,结果与转录组数据趋势一致。甘油醛-3-磷酸脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase, GAP)、束状阿拉伯半乳糖蛋白3(Fasciclin-like arabinogalactan protein 3,FLAs)和花药特异表达启动子LAT52系统发育树分析表明,3个基因分别与同科的物种在同一个进化分支上且同源性较高。这些差异表达基因可能在宁夏枸杞‘...     

atp1基因在小麦BNS雄性不育系和自身转换系中的差异表达

atp1基因在植物中是线粒体基因组编码,其产物ATP1是线粒体ATP合酶F1的α亚基,在小麦BNS的雄性不育系和它的转换系中差异表达。为了检测该基因的表达丰度,探讨与BNS不育性的联系,以BNS不育系和它的转换系的幼穗、花药和穗轴等组织为材料,利用实时荧光定量PCR方法,测定和比较该基因在不同组织中的表达水平,结果表明,该基因在各组织中的总表达量比内参甘油醛-3-磷酸脱氢酶基因表达量高3~4个数量级;在幼穗及各穗轴营养组织中表达量一致;花药与营养组织比较,在不育系四分体和单核期花药中表达均上调;在不育系中,该基因表达量在单核期花药中显著下调。这些结果说明,线粒体atp1基因在小麦中是一个高水平表达基因,在BNS营养组织中组成型表达,在花药中特异性上调表达,在不育系中表达受到抑制,表现显著下调,表明atp1基因的下调表达与BNS不育性有关。     

小麦遗传型与生理型雄性不育花药蛋白质双向电泳分析

为了研究小麦雄性不育蛋白表达机制, 以具有异质同核的3个亲本: 即遗传型不育系ms(S)-西农1376、对应的保持系(A)-西农1376和生理型(化学杀雄剂SQ-1诱导)雄性不育系ms(A)-西农1376为材料, 利用IEF/SDS-PAGE凝胶电泳技术, 对发育到单核后期的花药全蛋白特异性进行了比较分析, 得到320~350个清晰的蛋白点。结果表明: 遗传型和SQ-1诱导的生理型不育系蛋白质图谱与正常保持系在蛋白质(多肽)表达量上存在一定的差异, 同时发现有几种蛋白质的表达有明显的特异性, 两个不育材料2D胶上共有几个明显的特异蛋白点, 而在保持系中未发现; 两个不育材料又分别具有自己的特异蛋白表达, 不育机理不同; 比较分析可知, 不育系中某些蛋白的表达受到了抑制, 并开启了与花药败育有关的特定蛋白的表达, 可能使物质能量代谢受阻,导致雄性不育的发生。     

杀雄剂SQ-1诱导小麦生理型雄性不育小花完整叶绿体差异蛋白质的鉴定

为了在蛋白质水平揭示小麦雄性不育的分子遗传机制,以小麦经杀雄剂SQ-1诱导的生理型雄性不育系,以及对应正常可育系所构建的等生理差异系为试材,应用差速离心和蔗糖密度梯度离心,首先分离出供试材料小花的完整叶绿体,制备蛋白样品后采用固相IEF/SDS-PAGE双向凝胶电泳技术对完整叶绿体蛋白质进行了分离、银染,得到了重复性较好的双向电泳图谱.PDQuest2DE软件分析识别出约150个较为清晰的蛋白质点,采用MALDI-TOF鉴定出的6个差异表达蛋白质分别是:PAP-fibrillin、ATRABB1A、底物同源结构域蛋白/RhoGAP结构域蛋白、铜锌超氧化物歧化酶(Cu/Zn-SOD)、R2R3-MYB转录因子及1个假定蛋白质.生物信息学功能分析暗示,这些蛋白直接参与了花药内激素调节、蛋白质转运、蛋白质互作、活性氧积累及花药的发育,表明SQ-1诱导的小麦生理型雄性不育其败育机理可能就与这些生理代谢过程的变异直接相关.     

杀雄剂SQ-1诱导小麦生理型雄性不育小花完整叶绿体差异蛋白质组学的鉴定

为了在蛋白质水平揭示小麦雄性不育的分子遗传机制,以小麦经杀雄剂SQ-1诱导的生理型雄性不育系,以及对应正常可育系所构建的等生理差异系为试材,应用差速离心和蔗糖密度梯度离心,首先分离出供试材料小花的完整叶绿体,制备蛋白样品后采用固相IEF/SDS-PAGE双向凝胶电泳技术对完整叶绿体蛋白质进行了分离、银染,得到了重复性较好的双向电泳图谱．PDQuest 2DE软件分析识别出约150个较为清晰的蛋白质点,采用MALDI-TOF鉴定出的6个差异表达蛋白质分别是：PAP-fibrillin、ATRABB1A、底物同源结构域蛋白/Rho GAP结构域蛋白、铜锌超氧化物歧化酶（Cu/Zn-SOD）、R2R3-MYB转录因子及1个假定蛋白质．生物信息学功能分析暗示,这些蛋白直接参与了花药内激素调节、蛋白质转运、蛋白质互作、活性氧积累及花药的发育,表明SQ-1诱导的小麦生理型雄性不育其败育机理可能就与这些生理代谢过程的变异直接相关．     

猪链球菌2型强毒株05ZYH33转录调控因子Rgg基因敲除突变体与野生株的基因表达差异分析

目的:为了解猪链球菌2型强毒株05Z33转录调控因子Rgg的调控作用,用基因芯片方法分析野生株与rgg基因敲除突变体之间的差异表达基因。方法:用猪链球菌2型全基因组序列点样制备芯片,将芯片运用于rgg敲除株与野生株的基因表达差异研究,采用定量real-time PCR(qRT-PCR)验证表达谱结果。结果:在突变体中共发现45个基因表达量变化在2倍以上,其中19个基因表达上调,26个基因表达下调。这些基因在细菌毒力、免疫抗原、DNA合成和修复、基础代谢和ABC转运系统等方面起着重要作用。结论:转录调控因子Rgg是一个全局调控因子,但rgg敲除后并不影响猪链球菌的毒力。     

蓝标型核不育小麦花药转录物组学及花色素苷合成相关基因的表达分析

为了揭示蓝标型小麦核雄性不育的分子机制,更好地利用隐性核不育小麦杂种优势,本研究以蓝标型白粒小麦WS（不育）和浅蓝粒小麦WF（育性正常）植株花药为试验材料,利用转录物组学技术对两者差异表达基因进行了分析,并对其中涉及花色素苷合成相关基因进行了验证。结果表明： WF与WS相比,共检测到2 352个差异表达基因,这些基因经GO功能注释分为3大类43个小类,主要涉及生物合成、苯丙烷代谢、L-苯丙氨酸分解代谢、膜组成部分、质膜、细胞质、ATP结合和蛋白质丝氨酸/苏氨酸激酶活性等。 KEGG通路分析结果显示,苯丙烷类生物合成通路富集基因最多,有159个,其次是苯丙氨酸代谢通路,包含136个显著差异表达基因,其他还涉及多种氨基酸代谢、嘌呤代谢、嘧啶代谢及糖代谢通路;与花青素代谢直接相关的通路中,多个控制关键酶结构基因存在差异表达,且大多数在WF中上调表达,只有黄烷酮3-羟化酶基因（flavanone 3-hydroxylase,F3H）和无色花青素双加氧酶基因（anthocyanin dioxygenase,ANS）下调表达;实时荧光定量分析显示,10个与花青素代谢相关基因实际表达情况和转录物组测序数据中基因表达情况具有相同的上下调趋势;差异基因序列同源性分析显示,筛选出的2个转录因子（DN48762c2g1、DN25944c0g1）与玉米、水稻及拟南芥花色素苷合成调控转录因子聚为同一簇,可能是蓝标型小麦浅蓝粒植株蓝色糊粉层性状的候选基因。并且荧光定量分析表明,DN48762c2g1和DN25944c0g1在WF中的表达量要明显高于WS。综上认为,花青素的生物合成途径相关基因不仅与籽粒蓝色性状有关,而且可能参与了蓝标型核不育系的花药败育。     

苗期水稻响应褐飞虱取食的基因差异表达分析

【目的】褐飞虱Nilaparvata lugens (St?l)是水稻的重要害虫之一,主要刺吸水稻韧皮部汁液为害,对水稻生产造成严重的产量和经济损失。为研究水稻响应褐飞虱取食的分子机制,对褐飞虱取食6 h后的苗期水稻进行转录组测序及基因差异表达分析。【方法】采用illumina二代测序技术获得褐飞虱取食前后水稻组织的转录组数据,利用RSEM软件进行基因表达定量和DEseq2进行差异表达分析;从差异表达基因中随机选取20个基因采用荧光定量PCR技术进行验证;采用GeneMerge软件对差异表达基因进行KEGG和GO富集分析。【结果】褐飞虱取食后,水稻转录组中的1 104个基因出现了差异表达,其中435个基因表达上调,669个基因表达下调。荧光定量PCR结果显示,20个差异表达基因中18个基因的表达变化趋势和测序结果一致,证明了转录组分析结果可靠。GO和KEGG富集分析表明,表达上调基因主要与水稻氧化应激、海藻糖合成及次生化合物代谢有关,显著富集在14个KEGG通路和30个GO功能分类中;而表达下调基因主要参与水稻纤维素、蛋白质及脂肪酸合成过程,显著富集在29个KEGG通路和26个GO功能分类。在差异表达基因中,分别有61个转录因子和13个水杨酸和茉莉酸信号通路相关基因。【结论】褐飞虱取食激发了水稻的应激反应和保护机制,同时还降低了营养合成的过程,是飞虱为害造成水稻减产的原因之一。本研究初步揭示了苗期水稻响应褐飞虱取食的差异表达基因,为研究水稻-褐飞虱互作机制以及褐飞虱抗性水稻品种培育提供了参考和依据。     

施芝麻饼肥对烟草叶片基因差异表达的影响

施用芝麻饼可提高烟叶油份和质量,但对其机理了解很少.为了解施用芝麻饼肥条件下烟草基因表达的影响,采用拟南芥基因芯片检测了施用芝麻饼肥后的烟草叶片的基因谱.在22810个基因微矩阵点中,有效差异表达的基因有54个,上调32个,下调22个.其中包括一些与烟叶质量形成关系密切的基因,如与促进脂类分解的类黄酮合成有关的4-二氢黄酮醇还原酶基因表达上调,与糖类物质合成有关的糖基转移酶基因表达下调,与根毛形成代谢有关的CslD3基因表达上调,推动各种离子和小分子代谢产物进行跨膜运输的ATPase subunit 6基因表达上调,促进氨基酸合成的γ - 谷氨酰转肽酶基因上调,还检测到与光合系统有关的基因如psaK、PsbI以及多个参与复制、转录和翻译过程基因的差异表达,如SWIM锌指家族蛋白、H4组蛋白、mTERF-related等 .此外检测到19个未知基因,它们的差异表达可能和施用饼肥有关.通过分析这些特异表达基因,了解芝麻饼肥促进烟草生长及品质形成的有关机理,揭示出一些潜在的生物学规律,为研究烟草栽培生理提供有价值的信息.     

NOR1基因转染对肝癌细胞HepG2基因表达谱的影响

NOR1基因是一在正常组织中广泛表达且在肿瘤组织中表达下调的新基因.为进一步研究NOR1基因的功能和寻找其下游基因,利用脂质体技术将NOR1基因转染进HepG2细胞,采用cDNA微阵列技术分析其基因表达谱的改变.试验表明NOR1基因的转染能使Grb2,HBP17,TNFRSF11B等59个基因上调,同时也下调Bik,MAp2K6,ZFP95等103个基因.随后用实时荧光定量PCR对cDNA 微阵列结果中上述3个上调表达基因进行验证,结果表明,基因表达差异具有统计学意义(P<0.05),荧光定量PCR结果与微阵列结果相符.这些结果提示,NOR1基因对肝癌HepG2细胞的生物学行为的影响可能与它对细胞信号转导,细胞周期调控,转录、翻译调控相关基因的表达影响有关.     

Differential proteomic analysis of polyubiquitin-related proteins in chemical hybridization agent-induced wheat (Triticum aestivum L.) male sterility

Protein polyubiquitination is a significant regulator of diverse physiological functions, including sexual reproduction, in plants. Chemical hybridizing agents (CHA) SQ-1 has been shown to induce male sterility in wheat (Triticum aestivum L.) through inhibition of pollen development. This mechanism by which CHA induces male sterility in wheat is unclear. In this study, differential proteomic analysis of polyubiquitinated proteins associated with wheat male sterility was investigated. Wheat plants of the same genetic background were treated with or without CHA. Ubiquitinated proteins were then extracted and enriched for proteomic analysis. Differentially expressed polyubiquitinated proteins in trinuclear stage anther were identified by nanospray liquid chromatography/tandem mass spectrometry. A total of 127 and 131 differentially expressed polyubiquitinated proteins, including heat shock protein 70, ATPase subunit, glycosyltransferase, ubiquitin-related enzyme, and 20S proteasome subunit, were successfully identified by searching against wheat protein database and NCBInr database, respectively. Most of these proteins are related to photosynthesis, carbohydrate and energy metabolism, and multiple metabolic processes. These findings show that alteration of polyubiquitinated proteins is associated with male sterility in wheat.     

用DDRT-PCR技术对太谷核不育小麦基因差别表达的研究

采用DDRT—PCR技术对太谷核不育小麦的一对近等基因系进行了差别表达分析,以找出与太谷核不育基因Tα1表达有关的基因并研究其引起雄性不育的机制。共用30对随机引物进行差异显示,从展示的近1000条cDNA片段中找出30条特异表达的cDNA片段,其中包括可育特异和不育特异,这些片段可能与Tα1基因的表达有关。     

Effectiveness of SC2053 as a chemical hybridizing agent for winter wheat

The use of chemical hybridizing agents (CHA) allows production of hybrid wheat seeds. We evaluated the effectiveness of a new CHA (SC2053) to induce male sterility on winter wheat in controlled growth conditions. CHA effectiveness was measured with the application of 4 doses (0–400–700–1000 g.ha^–1) at 7 stages. These stages were defined by the length of the main stem spike (1–4–7–11–15–20–40 mm). At heading, individual ears were isolated with a greaseproof paper bag. The seeds formed were counted on treated and control ears. The spikes' sterility was calculated three weeks after flowering. The sterility of the main stem's spike reached 95% to 100% for application of 700 g.ha^–1 and 1000 g.ha^–1 for main stem spike length of 7 mm to 20 mm. The effects of ear tillering (5 tillers per plant) on CHA effectiveness were also investigated. We observed a significant delay of ear development between the main stem and tillers so that complete sterilities were not reached for each dose. Since tillering in field conditions rarely exceeds 3 ears per plant, CHA effectiveness was studied on plants bearing 3 ears. The mean sterility of the first 3 ears was close to 100% for applications with 700 g.ha^–1 and 1000 g.ha^–1 at stages from 11 mm to 20 mm of main stem spike length.     

化学杂交剂诱导的小麦生理型雄性不育花药的活性氧代谢

以化学杂交剂SQ-1诱导的生理型小麦雄性不育及其对照植株花药为材料,研究了不同花粉发育时期花药中超氧阴离子(O-·2)生成速率、过氧化氢(H2O2)和丙二醛(MDA)含量以及主要抗氧化酶活性的变化,以探明小麦花药活性氧代谢和生理型雄性不育的关系.结果表明,在幼穗时期,O-·2生成速率、H2O2和MDA含量、超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)活性均高于相应对照,而过氧化物酶(POD)和过氧化氢酶(CAT)活性则低于或显著低于对照;在单核早期以及花粉败育主要发生期(单核后期和二核初期),O-·2生成速率、H2O2和MDA含量极显著高于对照,而SOD、POD、CAT和APX酶活性却极显著低于对照;在败育后的花药中,O-·2生成速率和H2O2含量与对照之间差异幅度缩小,但MDA含量依然加大,同期的几种抗氧化酶活性依然极显著低于对照.在败育的关键期,品种'西农1376'处理株花药的活性氧升高幅度比'西农2611'处理株较大,抗氧化酶活性降低幅度也较大,且'西农1376'处理株的相对雄性不育率也较高.可见,化学杂交剂SQ-1能诱导小麦花药中O-·2和H2O2大量积累以及SOD、POD、CAT和APX活性的极显著降低,引起花粉关键败育期花药活性氧代谢严重失衡和严重膜脂过氧化,导致大量花粉母细胞发育受到严重抑制,最终造成小麦生理型雄性不育.     

Cytological and Comparative Proteomic Analyses on Male Sterility in Brassica napus L. Induced by the Chemical Hybridization Agent Monosulphuron Ester Sodium

Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Monosulphuron ester sodium (MES), a new acetolactate synthase-inhibitor herbicide belonging to the sulphonylurea family, has been developed as an effective CHA to induce male sterility in rapeseed (Brassica napus L.). To understand MES-induced male sterility in rapeseed better, comparative cytological and proteomic analyses were conducted in this study. Cytological analysis indicated that defective tapetal cells and abnormal microspores were gradually generated in the developing anthers of MES-treated plants at various development stages, resulting in unviable microspores and male sterility. A total of 141 differentially expressed proteins between the MES-treated and control plants were revealed, and 131 of them were further identified by MALDI-TOF/TOF MS. Most of these proteins decreased in abundance in tissues of MES-treated rapeseed plants, and only a few increased. Notably, some proteins were absent or induced in developing anthers after MES treatment. These proteins were involved in several processes that may be crucial for tapetum and microspore development. Down-regulation of these proteins may disrupt the coordination of developmental and metabolic processes, resulting in defective tapetum and abnormal microspores that lead to male sterility in MES-treated plants. Accordingly, a simple model of CHA-MES-induced male sterility in rapeseed was established. This study is the first cytological and dynamic proteomic investigation on CHA-MES-induced male sterility in rapeseed, and the results provide new insights into the molecular events of male sterility.     

小麦雄性不育遗传及基因定位研究进展

雄性不育的研究对于杂种优势的利用具有重要意义。本文综述了小麦雄性不育遗传及基因定位研究进展,介绍了小麦雄性不育的基因工程,对小麦雄性不育的应用进行了讨论。 Abstract:The study of plant male sterility plays an important role on utilization of heterosis.This paper reviews the current status of the studies of the heredity and mapping of the male sterile genes in wheat and the gene engineering of wheat male sterility.The application of male sterility in wheat breeding is discussed.     

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。     

Abnormal Development of Tapetum and Microspores Induced by Chemical Hybridization Agent SQ-1 in Wheat

Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.     

哒嗪类化合物9403诱导小麦雄性不育的初步研究

9403是国内新合成的一种哒嗪类化合物,经初步研究证明,9403具有诱导小麦雄性不育的作用。试验以GENESIS化学杂交剂为对照,对9403诱导小麦雄性不育的作用进行了研究。结果表明;9403对试验的两个品种西农6419与BAU95在其花药发育的药隔期,用剂量0.75kg/hm^2进行一次叶面喷施,诱导雄性不育率可达到96%以上,人工饱和授粉证明9403对雌蕊的育性没有影响,平均人工饱和授粉结实率为88.6%,与GENESIS相比,9403的杀雄效果较不彻底且具有一定的药害。     

两个小麦14-3-3基因的特征、亚细胞定位及其对非生物胁迫的响应(英文)

为了探讨14-3-3基因在小麦逆境胁迫应答中的调控作用,利用RACE技术克隆了两个包含完整编码框的14-3-3基因(命名为Ta14R1和Ta14R2),其中Ta14R1 cDNA长999 bp,编码262个氨基酸,而Ta14R2 cDNA长897 bp,编码261个氨基酸。Ta14R1/Ta14R2-GFP融合载体瞬时表达结果显示,Ta14R1和Ta14R2蛋白均定位于细胞质和细胞膜,但不在叶绿体中。荧光定量PCR分析表明,Ta14R1和Ta14R2均在萌发1 d的胚芽鞘中表达量最高;在高温、低温、模拟干旱和ABA处理下,两个基因在小麦的根和叶中都受胁迫诱导而且显著上调表达,推测这两个14-3-3基因通过依赖ABA的非生物胁迫响应途径发挥作用,可能参与了小麦中高温、低温和干旱胁迫的耐受调节过程。