甘蓝型油菜BnMPK3的序列分析及其对环境信号的响应

为了分析油菜MAP激酶3对各种环境胁迫信号的表达响应,以甘蓝型油菜中双9号为试材,采用同源克隆法克隆了甘蓝型油菜MAP激酶3基因(Bn MPK3)的c DNA序列。序列分析表明,Bn MPK3编码一条370个氨基酸残基的蛋白序列,这个序列含有1个高度保守的TEY磷酸化位点和一个CD锚定区域,与At MPK3的序列相似性达到94%。烟草瞬时表达分析显示,Bn MPK3是1个细胞核定位蛋白;荧光定量PCR分析显示,Bn MPK3在茎以及花和叶组织器官中特异性表达,并且对Na Cl溶液、4℃低温、PEG溶液和K2Cr2O7溶液以及甲基茉莉酸(Me JA)和乙烯前体(ACC)等的处理显著上调表达。结果表明,Bn MPK3是高盐、低温、干旱、重金属以及JA和ET等各种环境胁迫因子的响应基因,建议Bn MPK3可能在调控油菜对各种环境胁迫防御的过程中位于JA和ET相关的信号网络中,数据将为进一步研究Bn MPK3在油菜抗逆中的功能及其机制提供理论线索。     

甘蓝型油菜BnFAD2-C1基因全长序列的克隆、表达及转录调控元件分析

脂肪酸去饱和酶基因(FAD2)是控制植物体中油酸含量的关键基因,在甘蓝型油菜中有4个FAD2基因的拷贝,分别定位在A1、C1、A5、C5染色体上。本文克隆了1个FAD2拷贝基因,依据油菜基因组数据库信息,将其定位到C1染色体上,命名为Bn FAD2-C1,其开放阅读框为1155 bp。采用RACE(rapid-amplification of c DNA ends)技术获得了175 bp的5?UTR序列和212 bp的3?UTR序列。采用荧光定量PCR技术研究发现,Bn FAD2-C1在根、花和角果皮中仅保持本底水平的表达,在种子发育中期呈现高效表达,具有种子特异性诱导表达的特征。根据甘蓝和油菜基因组数据库信息,同源克隆到Bn FAD2-C1基因的启动子(promoter)和内含子(intron)序列,并通过PLACE和Plant CARE网站分析,初步预测到调控该基因转录的潜在顺式作用元件。通过茉莉酸诱导处理,Bn FAD2-C1基因表达量发生变化,推断茉莉酸在Bn FAD2-C1基因的表达过程中可能发挥一定的调控作用。     

棉花GhWRKY106-1的克隆与表达分析

为了研究WRKY转录因子对棉花抗枯萎病的作用机理,发掘抗枯萎病基因资源,本实验以枯萎病菌诱导棉花根部基因表达谱中筛选出的WRKY基因片段为探针,利用电子克隆结合RT-PCR技术,从陆地棉"中棉所12"根部c DNA克隆得到一个WRKY基因,命名为Gh WRKY106-1(登录号:KP202869)。序列分析表明,该基因开放阅读框全长918 bp,编码306个氨基酸,含有1个WRKY结构域和一个典型的C2HC锌指结构,属于典型的第Ⅲ类WRKY家族转录因子。q RT-PCR检测该基因在"中棉所12号"中的表达特性结果分析表明:枯萎病菌诱导后,该基因的表达量表现出先升高后降低的趋势,并且在处理后3 h基因的表达量达到最大。激素处理:茉莉酸(JA)、水杨酸(SA)和乙烯(ET)胁迫后Gh WRKY106-1基因表达量均发生变化,其中茉莉酸在处理后4 h基因的表达量达到最大,而水杨酸和乙烯诱导响应最强烈的时间均在3 h;并且水杨酸诱导Gh WRKY106-1基因的表达较茉莉酸强烈,而乙烯的表达量在处理前期又明显高于水杨酸和茉莉酸,因此推测该基因在棉花对枯萎菌的抗性反应中起着一定作用,可为今后棉花抗枯萎病研究提供依据。     

芥菜型油菜BjA09.TT8和BjB08.TT8基因调节类黄酮的合成

bHLH类转录因子TT8具有调控植物类黄酮合成的功能。本研究采用同源克隆法,在芥菜型油菜紫叶芥中获得了2个TT8拷贝,分别命名为BjA09.TT8和BjB08.TT8,它们分别编码521和517个氨基酸。定量表达分析表明,这2个拷贝在叶中的表达量均显著高于茎和根中,且都响应茉莉酸(JA)信号,在50μmol L–1的茉莉酸甲酯处理0.5 h后表达量均达到峰值;利用毛状根体系过表达发现,BjA09.TT8和BjB08.TT8分别可使紫叶芥和绿叶芥菜型油菜四川黄籽的毛状根中总黄酮含量升高,同时对类黄酮合成基因Bj.CHS的表达具有促进作用,说明二者在功能上具有冗余性。分别在野生型拟南芥和拟南芥tt8突变体中过表达BjA09.TT8和BjB08.TT8发现,过表达拟南芥植株叶片颜色变紫,植株总黄酮和原花色素含量显著升高。本研究表明,BjA09.TT8和BjB08.TT8基因能够促进芥菜型油菜类黄酮的合成,为进一步解析芸薹属植物原花色素合成的调控机制提供了参考。     

水稻OsMED7基因的克隆及表达分析

转录中介体复合物(Mediator com-plex)是一个由多亚基组成的RNA聚合酶II重要辅助因子,参与真核生物基因的转录调控。通过RT-PCR方法从水稻中克隆了拟南芥MED7的同源基因,命名为OsMED7,该基因的开放阅读框全长为531bp,编码一个含176个氨基酸的蛋白,含有一个MED7保守结构域。用水杨酸处理水稻幼苗后OsMED7基因的表达受到抑制,而茉莉酸的处理对OsMED7基因的表达没有影响,水稻接种白叶枯病菌24h后OsMED7基因的表达开始下降。结果表明,OsMED7亚基通过SA介导的信号转导途径参与水稻对白叶枯病菌的应答。     

樱桃番茄Cf9基因调控元件预测及同源基因系统发育分析

为探明Cf9基因的表达调控元件和同源基因进化关系,从樱桃番茄品种京番红星中克隆了Cf9上游序列,利用PlantCARE启动子预测工具对转录起始位点上游1500 bp序列进行顺式调控元件分析。结果表明,Cf9启动子不仅含有TATA-box和CAAT-box等核心元件,而且含有光响应元件、茉莉酸甲酯响应元件、水杨酸响应元件等。Cf9基因可能受光、水杨酸和茉莉酸甲酯等诱导表达。Cf9同源基因系统进化分析结果表明,当同源指数为0.65时,番茄、马铃薯、辣椒、烟草中含有Cf9同源基因,同源基因既包括旁系同源基因,也包括直系同源基因,分析结果也进一步说明番茄与马铃薯的亲缘关系最近,其次为辣椒、烟草。     

棉花GhTGA1与GhTGA9.2基因克隆及表达特征分析

为了揭示棉花GhTGA1与GhTGA9. 2基因的生物学功能,利用RT-PCR技术,从抗枯萎病棉花品种中棉所12号中克隆了TGA转录因子基因GhTGA1与GhTGA9. 2。生物信息学分析表明,2个基因开放阅读框(ORF)长分别为1 281,1461 bp,分别编码405,486个氨基酸。序列分析表明,2个基因均含有b ZIP和DOG1结构域,属于b ZIP亚家族TGA转录因子基因。利用实时荧光定量(qRT-PCR)技术进行表达特征分析表明,GhTGA1基因能被枯萎病菌、乙烯(ET)、茉莉酸(JA)诱导上调表达,水杨酸(SA)诱导后,在各处理时间点,该基因表达量均低于Mock,以上结果表明,GhTGA1基因可能通过参与乙烯(ET)和茉莉酸(JA)信号通路调控棉花对枯萎病的抗性;枯萎病处理后,GhTGA9. 2基因在各时间点表达量均低于Mock,呈下调表达,但能被激素(ET、JA、SA)诱导上调表达。结果表明,GhTGA9. 2基因可能通过激素(ET、JA、SA)途径参与胁迫应答,通过对GhTGA1与GhTGA9. 2基因表达特征分析为后续研究提供基础。     

番茄系统创伤信号因子研究进展

当植物受到机械损伤或害虫伤害时,植物体会在受伤部位产生创伤信号分子启动防御基因的系统表达.许多研究表明系统素(或其前体原系统素)在受伤部位激活茉莉酸的合成,使之达到系统反应的水平,应对外来伤害;茉莉酸或其衍生物是重要的系统伤信号分子,它诱导伤防御基因的系统表达.自从系统素和茉莉酸被认为是伤诱导防御基因表达的信号分子后,番茄成为研究植物系统伤信号很好的模式植物.本文介绍了番茄的茉莉酸合成突变体、茉莉酸感知突变体和系统素功能突变体,及它们之间相互作用,并推测了茉莉酸和系统素在系统创伤信号调节中的作用.     

李传友研究组等揭示MYC2调控茉莉酸信号终止的机制

<正>作为一种重要的植物激素,茉莉酸调控植物的防御反应和适应性生长。当植物遭遇病虫侵害或其他逆境胁迫时,活性茉莉酸被受体COI1(CORONATINE-INS ENS ITIVE 1)识别而释放核心转录因子MYC2的活性,MYC2与转录中介体亚基MED25形成功能复合物而在全基因组范围内激活茉莉酸响应基因的表达,产生防御反应。但茉莉酸信号的过度激活会大量消耗自身能     

巴西橡胶树产胶基因HbREF、HbHMGR1与MYCs转录因子互作

天然橡胶主要来自巴西橡胶树,巴西橡胶树是海南省主要的经济作物之一。胶乳在乳管中合成储存,乳管数量影响胶乳产量。乳管分化一直被证明与茉莉酸信号途径相关,本实验室已在前期研究中鉴定了茉莉酸信号途径中的关键转录因子MYCs与G-box顺式作用元件的互作,为了验证HbMYCs是否与含G-box(CACGTG)顺式作用元件的产胶相关基因互作,本研究克隆了两个产胶相关基因HbREF与HbHMGR1的全长启动子序列,并设计了相应的单荧光素酶报告基因实验,经生物信息学分析发现,2个基因的启动子上均含G-box (CACGTG)顺式作用元件,单荧光素酶报告基因实验的结果表明,HbREF和HbHMGR1分别与Hb MYC2、Hb MYC3、Hb MYC4互作,且受HbMYCs转录因子的正向调控,说明茉莉酸信号通路中的转录因子可以调控橡胶产排胶基因的表达,对阐明橡胶树产排胶机理具有重要意义。     

You reap what you sow – or do you? – volunteers in organic row-sown and broadcast-sown oilseed rape fields

The frequency and origin of oilseed rape volunteers in organic row and broadcast-sown oilseed rape fields was evaluated using DNA markers (ISSR) for analysis of potential volunteers in nine organic oilseed rape fields with different history. Using the software AFLPOP, the potential volunteers were assigned to variety by comparing their ISSR fingerprints with those of present and previous varieties. Results showed that two of the five row-sown fields, had high numbers of volunteer in the row (5 and 9 volunteers m^?2), and as inter-row volunteers were also present (0.9 and 3.6 volunteers m^?2), the recommended inter-row hoeing should not be neglected. Potential volunteers were also identified in the broadcast-sown fields, but their identity could not be proven. In a scenario where GM oilseed rape is cultivated previous to the organic oilseed rape, the present results indicate that the frequency of GM volunteers would exceed the threshold of 0.9%, and thus GM labelling would be demanded. Some of the volunteers belonged to varieties cultivated 8–11 years previously, indicating a long persistence of volunteer populations. What you reap might be added in the past – with consequences for organic farmers’ production and earning.     

分离和鉴定油菜D型MAP激酶基因BnMPK9

为了应对不良的外界环境,植物形成了一整套复杂的信号网络体系以适应变化的外界环境。其中MAPK级联是其中一个重要而保守的信号传导系统。采用RT-PCR策略,从干旱处理的油菜cDNA中克隆出全长的细胞分裂原激活的蛋白激酶基因。该基因命名为BnMPK9(GenBank登录号为AY737714),包含一个蛋白激酶区和一个保守的CD区。该基因与拟南芥AtMPK9高度同源,其激酶的磷酸化位点为TDY,同为D型MAPK亚家族基因。Southern杂交结果表明该基因在油菜基因组中拷贝数不少于2个。Northern杂交结果显示该基因能在不同的植物组织中表达。其表达受到甘露醇、紫外线和双氧水诱导上调表达,但低温和水杨酸处理后下调表达。实时RT-PCR分析表明甘露醇和双氧水能长时间诱导该基因高表达。在根中,甘露醇也能促进其上调表达。BnMPK9连接到pYES2.1酵母表达载体中转化酵母,发现增强了酵母对600 mmol L^–1甘露醇和0.2 mmol L^-1 tBuOOH的抗性。以上结果说明BnMPK9是MAPK基因家族中的一员,涉及真核生物细胞渗透和活性氧胁迫,并增强其抗性。     

梨花和油菜花挥发性气味物质的鉴定及差异分析

为了解花朵中挥发性气味物质对吸引传粉昆虫采集所起到的作用,采用固相微萃取(SPME)和气相色谱-质谱联用(GC-MS)技术,检测梨花和油菜花全花、花药、花蜜中挥发性气味物质成分,并进行主成分及与昆虫采集相关性分析。鉴定梨花中挥发性气味物质成分有9种,氧烷类为主要挥发性气味物质,占44.44%,通过主成分分析,气味贡献物主要来自于二甲基硫醚、十四甲基环七硅氧烷、苯丙腈、苯甲醛等化合物。油菜花中挥发性气味物质成分有23种,酯类和氧烷类为主要气味挥发性物质,分别占8.7%和16.09%,气味贡献物主要来自于反-2-己烯醛、异硫氰酸-2-苯乙酯、4-异硫氰酸根合-1-丁烯、二甲基三硫醚、二甲基二硫醚等化合物;花药的挥发性气味物质种类比花蜜中更丰富。传粉昆虫调查发现,试验地共有传粉昆虫4目10科11种,梨和油菜传粉昆虫数量上有显著差异(P<0.05),但各成分与采集昆虫数量均没有显著相关性(P>0.05)。研究发现梨花和油菜花挥发性气味物质成分有显著差异,将为研究气味物质主要贡献物在吸引传粉昆虫中发挥的作用提供参考。     

Leaf glucosinolate profiles and their relationship to pest and disease resistance in oilseed rape

Summary Glucosinolates are sulphur-containing glycosides which occur within vegetative and reproductive tissue of oilseed rape. Following tissue damage, glucosinolates undergo hydrolysis catalysed by the enzyme myrosinase to produce a complex array of products which include volatile isothiocyanates and several compounds with goitrogenic activity. Many of these products have been implicated in the interaction betweenBrassica and their pests and pathogens and some may have a role in defence mechanisms. Low glucosinolate (00) oilseed rape cultivars have been shown to possess similar concentrations of leaf glucosinolates as high glucosinolate (0) oilseed rape cultivars. Likewise, despite considerable speculation to the contrary, 00 cultivars have been shown not to be more susceptible to pests and pathogens than 0 cultivars. The potential to enhance pest and disease resistance of oilseed rape by manipulating the leaf glucosinolate profile without reducing seed quality is discussed.     

湖南主要大田作物系统投入产出的能值分析

比较不同作物系统的投入产出,是优化作物生产结构和设计农田可持续经营政策的重要前提。采用能值分析法,对湖南油菜、棉花、烤烟和水稻系统的投入产出情况进行分析,并比较各系统的能值评价指标。研究发现,整体上这4种作物系统的投入对自然资源依赖程度较低,主要依靠市场投入,具体表现为机械与燃料能值投入增加,农用化学品投入占比基本不变,人力投入与有机肥投入减少。尽管4种作物系统的环境负载率均小于3,但都呈上升趋势。这4种作物系统的能值可持续发展指数顺序为油菜>棉花>水稻>烤烟。本研究表明,传统利润率严重低估油菜、棉花和水稻系统对环境的贡献。鉴于油菜系统对环境压力较小,在注意控制其环境负载率的前提下,可适当扩大其种植规模。     

江苏省冬油菜生育期内低温事件的年际变化及对产量的影响

为探讨气候变化对我国油菜生产带来的可能影响,选取江苏省地区52个气象站1961—2012年日气温资料及冬油菜产量逐年数据,采用M-K趋势检验探求冬春季节8种低温指数的变化趋势,并依托一阶差分法探讨低温指数与产量的数量关系。结果表明：（1）1961-2012年间省内大部分地区冬春季节低温事件的总天数、连续低温事件的场次、强度及持续性呈现出显著的减弱趋势;（2）不同类型低温指数中,FI1（越冬期日最低气温<-5℃的总天数）与CI4（蕾薹期连续日最低气温<0℃天气的最长持续时间）对冬油菜产量的负效应最为显著;（3）各站点产量对FI1及CI4敏感性分析表明江苏省北部地区冬油菜种植对低温灾害事件更为敏感;（4）1961-2012年间江苏大部分地区FI1及CI4的减少趋势给研究区冬油菜产量带来了一定幅度的增产。     

Genetic relationships among resynthesized,semi-resynthesized and natural <Emphasis Type="Italic">Brassica napus</Emphasis> L. genotypes

The allopolyploidization event that created cultivated oilseed rape Brassica napus L, followed by intense breeding, reduced its genetic diversity. Resynthesized (RS) B. napus L. obtained by interspecific hybridization between genotypes of B. rapa L. and B. oleracea L. can be a valuable source for broadening genetic diversity in cultivated oilseed rape. In this study, we determined the extent of DNA polymorphism among natural accessions of oilseed rape, resynthesized B. napus, their parental species and double-low quality semi-RS lines carrying the Rfo gene. Using 10 selected primer combinations, 522 polymorphic AFLP markers were scored in the complete set of 100 Brassica sp. To detect relationships between these genotypes, a cluster analysis was performed using the Jaccard’s distance. Resynthesized allopolyploids clustered directly between their diploid parents. Cultivated accessions of oilseed rape created a compact group away from resynthesized allopolyploids as well as semi-RS lines. The natural oilseed rape group, which consists of 49 cultivars and breeding lines of oilseed rape, is characterized by lower genetic diversity than the group of 33 accessions of resynthesized oilseed rape, and the analysis showed that the double-low quality semi-RS lines represent a specific genetic variation of B. napus. The de novo resynthesized B. napus lines and the semi-RS lines of double-low quality generated from them, provide a significant opportunity for enrichment the gene pool of oilseed rape.     

Occasional loss of expression of phosphinothricin tolerance in sexual offspring of transgenic oilseed rape (Brassica napus L.)

The commercial and economic value of genetically modified crops is determined by a predictable, consistent and stable transmission and expression of the transgenes in successive generations. No gene inactivation is expected after selfings or crosses with non-transformed plants of homozygous transgenic oilseed rape plants if the expression of the transgene in homozygous or hemizygous nature in such plants is stable. The segregation ratios of phosphinothricin (PPT) tolerance in successive generations of selfings and mutual crosses of a few independent transgenic PPT-tolerant oilseed rape plants indicated a dominant, monogenic inheritance. In within-variety and between-variety crosses no transgene inactivation was observed. However, after selfings and backcrosses with non-transgenic oilseed rape infrequent loss of the expression of the PPT tolerance transgene was observed independent from its homozygous or hemizygous nature. Molecular analysis of PPT-susceptible plants showed that the loss of expression was due to gene inactivation and not to the absence of the transgene. Methylation and co-suppression are mechanisms that might cause reduced or even loss of expression of the transgene in later generations. The implications of this observation for seed multiplication of varieties and breeding activities with transgenic oilseed rape are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Plasticity of seed weight compensates reductions in seed number of oilseed rape in response to shading at flowering

Understanding the response of the number of seeds and seed weight to the availability of assimilates is crucial for designing breeding strategies aimed to increase seed and oil yield in oilseed rape. This study aims to answer the questions: i) do seed number and seed weight in oilseed rape differ in their plasticity in response to the availability of assimilates at flowering? and ii) how sensitive are oil and protein concentrations to the availability of assimilates during flowering?A spring oilseed rape hybrid was sown in two field experiments and the treatments were combinations of i) two plant densities and ii) shading or no shading between the beginning and end of flowering. Seed yield was not affected by plant density or by shading. Lower plant density was compensated by an increase in seed number per plant, without effects on single-seed weight. However, the negative effect of shading during flowering on seed number per area was fully compensated by an increase in single-seed weight by 47–61%. The plasticity of single-seed weight observed in the present study of oilseed rape has never been reported for annual seed crops. Shading at flowering increased both the seed filling rate and the duration of the seed filling period at all positions in the canopy. We also observed that the reduction of the source-sink ratio at flowering increasing seed weight does not necessarily modify oil or protein concentrations. Thus, single-seed weight could be targeted to increase seed yield in oilseed rape without compromising oil content.