不同氮素供应下水稻酚类物质代谢关键酶基因差异表达

运用实时荧光定量PCR技术探讨了不同供氮条件下强化感与弱化感水稻苯丙烷代谢途径中9个关键酶基因的表达差异。结果表明,与正常氮素供应相比,低氮胁迫引起强化感水稻‘P1312777’中与酚类代谢途径相关的9个关键酶基因表达量均上调,表达量增幅在1.9～5.4倍之间,且以PAL基因上调倍数最大。而弱化感水稻‘Lemont’则相反,只有2个基因(苯丙氨酸裂解酶基因和肉桂酰CoA基因)表达上调,但上调倍数分别是强化感水稻对应的基因的22%和74%,其余的7个基因表达均下调,降幅在29%～72%之间,表明低氮胁迫诱发的水稻化感抑草能力增强与其体内酚类物质合成代谢增强有关。     

不同胁迫条件下化感与非化感水稻PAL多基因家族的差异表达

水稻苯丙氨酸解氨酶(PAL)调控酚酸类化感物质的合成代谢。编码PAL的基因是一个基因家族,包含至少11个基因成员,并受不同环境条件的调控。为了明确PAL基因家族中调控水稻化感作用的特定基因成员,本研究运用实时荧光定量PCR技术分析了低氮及稗草胁迫条件下强化感水稻PI312777与非化感水稻Lemont中根系的11个PAL成员基因的表达差异。结果表明,低氮和稗草胁迫条件下,PI312777和Lemont中的 PAL4和PAL10均不表达,其余9个PAL基因成员发生了不同程度的表达变化。其中,PAL11均上调表达,其分别在低氮处理和稗草胁迫的PI312777中上调3.29倍和1.07倍,而在相同处理下的Lemont中上调3.92倍和1.08倍;PAL3和PAL9则仅在低氮和稗草胁迫条件下的PI312777中上调表达,低氮胁迫分别为1.83倍和2.66倍,稗草胁迫为1.46倍和2.65倍;而这两个基因在相同处理下的Lemont中表达下调,低氮胁迫下调1.05和1.24倍,稗草胁迫下调1.14和1.16倍,推测PAL3和PAL9可能与胁迫初期调控水稻化感作用有关。     

不同化感潜力水稻(Oryza sativa L.)苗期的氮素养分效率及相关基因的表达

为了阐明养分水平引起水稻(Oryza sativa L.)化感抑草潜力变化的生理生态机制,研究了不同N素营养处理下,不同化感潜力水稻苗期对N素营养逆境的响应特性及N素养分效率的差异,并运用实时荧光定量RT-PCR技术(FQ-PCR)检测与N素代谢和次生代谢关键酶的基因表达.结果表明:弱化感水稻品种Lemont对N素营养胁迫较敏感,强化感水稻品种PI312777对资源波动的适应性较强,N素养分效率较高.FQ-PCR分析结果显示,在低N条件下Lemont中的亚硝酸还原酶基因(nir),谷氨酰胺合成酶基因(gs)相对表达量均有不同幅度的下调, PI312777分别下调了1.2倍和1.4倍,而Lemont分别下调了3.0倍和1.8倍, Lemont下调的幅度分别是PI312777的2.5倍和1.3倍,但对于苯丙氨酸解氨酶基因(pal)与3-羟基-3甲基戊二酰辅酶A还原酶基因(hmgr)而言, PI312777叶组织中的pal和hmgr均上调表达,与对照相比上调了6.0倍和1.6倍,而Lemont中对应的基因均下调表达,分别下调了1.3倍和6.8倍,佐证了上述差异的分子生态学特性.     

低磷营养胁迫下水稻化感抑草潜力的变化特性及其作用机理

在低磷(0.5 mg·L^-1) 营养胁迫下,运用生理生化方法分析了化感水稻PI312777（PI）与非化感水稻Lemont (Le) 对稗草抑制作用潜力的变化特性及其内在机理.结果表明,在低磷营养胁迫下,化感水稻品种PI对受体稗草根干重的抑制能力明显提高,在处理后的5 、10和15 d,其对稗草地下部干重的抑制率分别增加了5.64%、3.89%和12.13%,增加幅度比非化感水稻品种Le显著.生理生化分析结果表明,与正常营养条件相比,用低磷营养下生长的化感水稻PI的根系分泌物处理稗草5、10和15 d,受体稗草叶片中POD活性的促进率分别提高了20.19%、15.47%和6.68%,吲哚乙酸氧化酶活性的促进率分别提高了18.08%、17.71%和12.50%,硝酸还原酶活性的抑制率分别增加了13.89%、18.60%和2.10%. 在低磷营养胁迫下,化感水稻通过抑制受体植物的硝酸还原酶活性,影响其对氮营养的吸收,同时显著提高了吲哚乙酸氧化酶活性,减缓了受体稗草的生长速度,提高了其抑草作用潜力.     

水稻化感作用及其生理生化特性的研究

选用具有强化感作用的6个水稻品种为供体,大田稗草为受体,研究了水稻化感作用及生理生化特性,结果表明,提高水稻叶片浸提液浓度,可以相应增强对稗草生长的抑制效果,这种抑制效果与杂草的种植密度呈负相关;化感水稻叶片浸提液能显著抑制物质稗草体内超氧化物歧化酶（SOD）和过氧化氢酶（CAT）的活性,从而影响其生长;苯丙氨酸氨解酶（PAL）的活性大小与酚类物质的含量吴正相关;多种酚类物质的化感作用之间可能是增效的,也可能是拮抗的。     

外源水杨酸调控水稻化感抑草作用及其分子生理特性

为了探讨外源水杨酸（SA）调控水稻化感抑草效应的可行性,研究了不同浓度的外源SA对强化感水稻PI312777抑草效应的影响及其生理生化特性,并运用实时定量RT-PCR(FQ-PCR)技术检测SA介导的关键酶苯丙氨酸解氨酶基因（ZB8）的相对表达量.结果表明：外源SA能够诱导水稻化感抑草效应增强,而且这种诱导效应与SA的浓度和处理时间相关.叶面喷施SA后,PI312777对稗草的抑制率显著提高,其根系活力、超氧化物歧化酶（SOD）和过氧化物酶（POD）活性增强,过氧化氢酶（CAT）活性降低;而伴生杂草稗草的相应生理指标的变化趋势则相反.PI312777植株中的苯丙氨酸解氨酶（PAL）活性增强,总酚含量升高.0.2 mmol·L^-1的SA诱导水稻化感抑草效应最显著,该浓度下目的基因ZB8的相对表达丰度随处理时间先上调后下调,在24 h达到表达高峰.     

应用差异蛋白质组学方法分析作物化感作用的分子机理

试验旨在分析运用分子标记技术(QTL)和差异蛋白组学技术研究作物化感作用分子机理的差异性。首先运用差异蛋白组学技术探讨在生物胁迫(稗草)下水稻化感作用潜力变化的内在分子机理。分别用稗草和水稻的根系分泌物培养切自一株5叶龄化感水稻P I312777植株并经恢复的2个分蘖。7d后,提取处理和对照相同叶位叶片的全蛋白质并进行双向电泳,每张电泳胶片上获得800多个电泳胶点,其中差异表达的蛋白质点有4个。采用M ALD I-TOF-M S对各差异蛋白质点进行肽质量指纹图谱分析,经过SW ISS-PROT数据库查询,结果表明化感水稻P I312777在稗草胁迫下的特异蛋白分别与苯丙氨酸氨解酶(PAL)、硫还原型蛋白(T rx-m)、3-羟基-3-甲基戊二酰辅酶A还原酶(HM GR)和过氧化物酶(POD)相匹配。根据编码以上4个差异蛋白质的DNA序列,发现编码以上4个差异蛋白的基因分别位于水稻染色体4、7、8和12上的特定克隆位点,这就是与化感作用相关基因。前人也运用QTL方法开展作物化感作用的分子机理研究,但由于所采用的供体材料、受体植物及对表型性状的评价方法等的不同,定位结果存在较大的差异。综合比较两种方法后认为,运用差异蛋白组学技术分析水稻化感作用的分子机理,比QTL技术更加直接和深入。因为比较胁迫处理和对照植物组织的2-DE图谱将能鉴定出由表达候选基因编码的胁迫蛋白质,氨基酸残基序列的测定将揭示那些功能与胁迫性状密切相关的蛋白质,这种编码的基因就是兼具功能与表达的候选基因。     

青香蕉苹果MdAFS基因序列与表达模式分析

以青香蕉苹果叶片为材料,研究青香蕉苹果‘White Winter Pearmain’α-法尼烯合酶(α-farnesene synthase,Md AFS)全长基因结构特征及编码蛋白质性质,分析Md AFS基因的诱导表达模式。采用生物信息学软件对Md AFS及其编码蛋白质进行详细分析,应用MEGA 6.0对不同物种来源的AFS构建系统进化树,利用q RT-PCR技术研究Md AFS诱导表达模式。α-法尼烯合酶基因(Gen Bank No:AY563622)全长1 731 bp,由576氨基酸组成,是α螺旋为主要构成元件的混合型蛋白。不同物种的α-法尼烯合酶氨基酸序列相似度低,但α-法尼烯合酶N端RRx8W和C端的H-α1loop具有高度保守性。非生物胁迫和激素信号能诱导Md AFS基因上调表达,且MV、ABA和Me JA诱导该基因上调表达更加明显。α-法尼烯合酶氨基酸序列相似度低,但蛋白三维结构相似;α-法尼烯合酶在蛋白结构和功能上具有单萜合酶特征,初步揭示了非生物胁迫和激素信号能诱导青香蕉苹果α-法尼烯代谢途径的关键酶Md AFS基因上调表达。     

甾醇生物合成中的关键酶-环氧角鲨烯环化酶的分子生物学研究

植物体中环氧角鲨烯环化酶催化2,3-环氧角鲨烯形成一系列三萜烯,为甾醇和三萜化合物的生物合成提供前体。这一催化反应被认为是甾醇和三萜化合物生物合成分支形成的关键位点．综述了甾醇和三萜化合物生物合成中的关键酶——环氧角鲨烯环化酶（OSCs）家族的生物学功能,基因克隆与属性,酶的细胞定位与酶活的表达调控等分子生物学研究进展．     

不同生态条件下华抗草78水稻对杂草的干扰控制作用

对不同水稻叶龄、水层深度、水稻密度和保水时间条件下水稻对杂草控制效果进行研究.结果表明,具有化感特性的水稻品系华抗草78对稗草、鳢肠、异型莎草等杂草的抑制率显著优于无化感特性品种Lemont;华抗草78对0～1.5叶期稗草和0～0.3叶期异型莎草的控制效果优于1.5～2.4叶期稗草和0.8～2.0叶期异型莎草;移栽时叶龄与栽插密度互作有利于提高华抗草78对杂草的防效,并明显高于移栽时叶龄分别与水层深度和保水时间互作效应.适当提高水稻移栽时的叶龄、增加移栽密度可显著提高华抗草78对供试杂草的抑制率.     

Analysis of gene expressions associated with increased allelopathy in rice (Oryza sativa L.) induced by exogenous salicylic acid

The defense characteristics of allelopathic rice accession PI312777 and its counterpart Lemont induced by exogenous salicylic acid (SA) to suppress troublesome weed barnyardgrass (BYG) were investigated using the methods of suppression subtractive hybridization (SSH) and real-time fluorescence quantitative PCR (qRT-PCR). The results showed that exogenous SA could induce the allelopathic effect of rice on BYG and this inducible defense was SA dose-respondent and treatment time-dependent. PI312777 exhibited higher inhibitory effect than Lemont on BYG after treated with different concentrations of SA. The activities of cell protective enzymes including SOD, POD and CAT in the BYG plants co-cultured with PI312777 treated by SA were highly depressed compared with the control (co-cultured with rice without SA-treatment). Similar but lower depression on these enzymes except for CAT was also observed in the BYG plants when co-cultured with Lemont treated by SA. It is therefore suggested that allelopathic rice should be more sensitive than non-allelopathic rice to exogenous SA. Seventeen genes induced by SA were obtained by SSH analysis from PI312777. These genes encode receptor-kinase proteins, ubiquitin carrier proteins, proteins related to phenylpropanoid metabolism, antioxidant related proteins and some growth-mediating proteins. The differential expressions of these genes were validated in part by qRT-PCR in the two rice accessions. Our work elucidated that allelopathic rice possesses an active chemical defense and auto-detoxifying enzyme system such as the up-regulated enzymes involved in de novo biosynthesis of phenolic allelochemicals and the glutathione-S-transferase (GST) associated with xenobiotic detoxification.     

Separation of allelopathy from resource competition using rice/barnyardgrass mixed-cultures

Plant-plant interference is the combined effect of allelopathy, resource competition, and many other factors. Separating allelopathy from resource competition is almost impossible in natural systems but it is important to evaluate the relative contribution of each of the two mechanisms on plant interference. Research on allelopathy in natural and cultivated plant communities has been hindered in the absence of a reliable method that can separate allelopathic effect from resource competition. In this paper, the interactions between allelopathic rice accession PI312777, non-allelopathic rice accession Lemont and barnyardgrass were explored respectively by using a target (rice)-neighbor (barnyardgrass) mixed-culture in hydroponic system. The relative competitive intensity (RCI), the relative neighbor effect (RNE) and the competitive ratio (CR) were used to quantify the intensity of competition between each of the two different potentially allelopathic rice accessions and barnyardgrass. Use of hydroponic culture system enabled us to exclude any uncontrolled factors that might operate in the soil and we were able to separate allelopathy from resource competition between each rice accession and barnyardgrass. The RCI and RNE values showed that the plant-plant interaction was positive (facilitation) for PI312777 but that was negative (competition) for Lemont and barnyardgrass in rice/barnyardgrass mixed-cultures. The CR values showed that one PI312777 plant was more competitive than 2 barnyardgrass plants. The allelopathic effects of PI312777 were much more intense than the resource competition in rice/barnyardgrass mixed cultures. The reverse was true for Lemont. These results demonstrate that the allelopathic effect of PI312777 was predominant in rice/barnyardgrass mixed-cultures. The most significant result of our study is the discovery of an experimental design, target-neighbor mixed-culture in combination with competition indices, can successfully separate allelopathic effects from competition.     

Genomic analysis of allelopathic response to low nitrogen and barnyardgrass competition in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To explore the molecular mechanism of allelopathic rice in response to low nitrogen (N) supply or accompanying weed stress, allelopathic rice PI 312777 and its counterpart Lemont were grown under low N supply or co-cultured with barnyardgrass [Echinochloa crus-galli (L.) Beauv.] in hydroponics. The suppression subtractive hybridization (SSH) technique was employed to isolate the up-regulated genes in the treated rice accession. The results indicated that the expression of the genes associated with N utilization was significantly up-regulated in allelopathic rice PI 312777, and the higher efficiency of N uptake and its utilization were also detected in PI 312777 than that in Lemont when the two rice accessions were exposed to low N supply. This result suggested that the allelopathic rice had higher ability to adapt to low N stress than its non-allelopathic counterpart. However, a different response was observed when the allelopathic rice was exposed to accompanying weed (barnyardgrass) co-cultured in full Hoagland solution (normal N supply). It showed that the expression of the genes associated with allelochemical synthesis and its detoxification were all up-regulated in the allelopathic rice when co-cultured with the target weed under normal N supply. The results suggested that the allelopathic rice should be a better competitor in the rice-weed co-culture system, which could be attributed to increasing de novo biosynthesis and detoxification of allelochemicals in rice, consequently resulting in enhanced allelopathic effect on the target and preventing the autotoxicity in this process. These findings suggested that the accompanying weed, barnyardgrass is not only the stressful factor, but also one of the triggers in activating allelopathy in rice. This implies that the allelopathic rice is sensible of the existing target in chemical communication.     

不同水层和密度条件下化感作用水稻对无芒稗的干扰控制作用

不同水层下水稻对无芒稗的干扰控制作用研究表明,水稻化感作用品种吓一跳、谷梅2号和中156对无芒稗的株高抑制作用比无化感作用品种秀水63和春江11呈显著性差异．随盆栽水稻密度增加,水稻对无芒稗的干扰控制作用加大．在每盆4株栽植密度下,水稻化感作用品种鸡早籼、谷梅2号显著抑制无芒稗植株株高;在每盆32株密度时,水稻化感作用品种吓一跳、PI312777、TN1等对受控无芒稗的干扰控制与无化感作用品种春江11相比,呈显著性水平．     

不同供氮条件下水稻的化感抑草作用与资源竞争分析

为探讨化感水稻在不同氮素水平下的生物干扰现象,提出了一种新的测试方法———化感竞争分离法(allelopathyandcompetitionseparation basedbioassay,ACS),成功地区分了在稻/稗共生系统中水稻化感作用与资源竞争及其在不同N条件下的反应特性.结果表明,水稻PI312777具有较强的生物干扰能力.在不同N水平下,其资源竞争能力较强且表现稳定,但化感作用潜力则随供N水平的下降而明显增强.水稻Lemont不具化感作用潜力,在供N水平正常或充足条件下,其资源竞争能力减弱,但在N胁迫下却明显增强.这是在环境资源贫乏时,稻/稗共生系统中生态位竞争加剧的结果.     

水稻和稗草共生土壤微生物生物量碳及酶活性的变化

以稻田稗草、化感水稻PI312777和普通水稻辽粳9为试材,研究了田间稗草和水稻1∶1共生条件下,土壤微生物生物量碳及脱氢酶、脲酶和转化酶活性的变化.结果表明：在稗草 的干扰下,化感水稻PI312777根区土壤微生物生物量碳含量比单作减少了 50.52％（P<0.01）,而行间土壤微生物生物量碳含量增加;普通水稻辽粳9根区土壤 微生物生物量碳含量比单作减少了38.99％（P<0.01）,但其行间土壤微生物生物量碳含量无明显变化.两个水稻品种根区土壤脱氢酶活性均被显著抑制（P<0.05）,下降率都在20％以上;PI312777根区土壤脲酶和转化酶活性均被显著促进（P<0.01）;而辽粳9根区土壤转化酶活性也被显著抑制（P<0.01）,但脲酶活性无明显变化.化感水稻根区土壤微生物生物量碳含量的显著减少及脲酶、转化酶活性的增加是其化感特性的表现,表明土壤微生物和酶均参与了水稻和稗草的种间作用,化感水稻具有抗稗草干扰的明显优势.