水稻逆境响应基因OsMsr15的表达分析与克隆

应用Affymetrix水稻表达芯片,分析了苗期和孕穗期的培矮64S水稻基因在高温、干旱和低温逆境处理条件下的表达模式,发现多个与逆境反应相关基因.其中,OsMsr15(Oryza sativa L.multiple stresses responsivegene 15)受高温、干旱和低温胁迫诱导,表达水平均显著上调.实时定量PCR分析结果与芯片数据基本吻合.序列分析表明,该基因无内含子,ORF(open reading frame)全长为717 bp,编码一个由238个氨基酸残基组成、具有典型C2H2结构域的锌指蛋白,推测相对分子质量约为2.46 kD,pI值约为8.90,蛋白分子的氨基端和羧基端分别存在一个推测的核定位信号B-box和EAR(ERF-associated amphiphilic repression)基序.对OsMsr15可能的启动子区域进行分析,发现多种与逆境诱导相关的调控元件.该基因编码的蛋白在不同物种中存在高度相似性,显示OsMsr15基因可能作为一个保守的耐逆基因,参与植物的多种耐逆反应过程.     

Effects of Cadmium on Enzymatic and Non-Enzymatic Antioxidative Defences of Rice (Oryza Sativa L.)

The effects of 60-d cadmium (Cd) exposure on enzymatic and non-enzymatic antioxidative system of Oryza sativa L. seedlings at tillering stage were studied using soil culture experiment. Research findings showed that chlorophyll content of Oryza sativa L. declined with the increase in soil metal concentration. Cd pollution induced the antioxidant stress by inducing O₂ ^?1 and H₂O₂, which increased in plants; at the same time, MDA as the final product of peroxidation of membrane lipids, accumulated in plant. The antioxidant enzyme system was initiated under the Cd exposure, i.e. almost all the activities of superoxide dismutase (SOD), peroxidase, catalase, glutathione peroxidase, and ascorbate peroxidase were elevated both in leaves and roots. The non-protein thiols including phytochelatins and glutathione to scavenge toxic free radicals caused by Cd stress was also studied. The contents of phytochelatins and glutathione were about 3.12–6.65-fold and 3.27–10.73-fold in leaves, against control; and the corresponding values were about 3.53–9.37-fold and 1.41–5.11-fold in roots, accordingly.     

Physiological Responses of Rice Plants Supplied with Silicon to Monographella albescens Infection

This study investigated the effect of silicon (Si) on the resistance of rice plants of the cv. ‘Primavera’ cultivar that were grown in a nutrient solution with 0 (?Si) or 2 mm (+Si) Si to leaf scald, which is caused by Monographella albescens. The leaf Si concentration increased in the +Si plants (4.8 decag/kg) compared to the ?Si plants (0.9 decag/kg), contributing to a reduced expansion of the leaf scald lesions. The extent of the cellular damage that was caused by the oxidative burst in response to the infection by M. albescens was reduced in the +Si plants, as evidenced by the reduced concentration of malondialdehyde. Higher concentrations of total soluble phenolics and lignin‐thioglycolic acid derivatives and greater activities of peroxidases (POX), polyphenoloxidases (PPO), phenylalanine ammonia‐lyases (PAL) and lipoxygenases (LOX) in the leaves of the +Si plants also contributed to the increased rice resistance to leaf scald. In contrast, the activities of chitinases and β‐1,3‐glucanases were higher in the leaves of the ?Si plants probably due to the unlimited M. albescens growth in the leaf tissues, as indicated by the larger lesions. The results of this study highlight the potential of Si in decreasing the expansion of the leaf scald lesions concomitantly with the potentiation of phenolic and lignin production, and the greater activities of POX, PPO, PAL and LOX rather than simply acting only as a physical barrier to avoid M. albescens penetration.     

T-DNA Integration Category and Mechanism in Rice Genome

T-DNA integration is a key step in the process of plant transformation, which is proven to be important for analyzing T-DNA integration mechanism. The structures of T-DNA right borders inserted into the rice (Oryza sativa L.) genome and their flanking sequences were analyzed. It was found that the integrated ends of the T-DNA right border occurred mainly on five nucleotides "TGACA" in inverse repeat (IR)sequence of 25 bp, especially on the third base "A". However, the integrated ends would sometimes lie inward of the IR sequence, which caused the IR sequence to be lost completely. Sometimes the right integrated ends appeared on the vector sequences rightward of the T-DNA right border, which made the TDNA, carrying vector sequences, integrated into the rice genome. These results seemingly suggest that the IR sequence of the right border plays an important role in the process of T-DNA integration into the rice genome, but is not an essential element. The appearance of vector sequences neighboring the T-DNA right border suggested that before being transferred into the plant cell from Agrobacterium, the entire T-DNA possibly began from the left border in synthesis and then read through at the right border. Several nucleotides in the T-DNA right border homologous with plant DNA and filler DNAs were frequently discovered in the integrated position ofT-DNA. Some small regions in the right border could match with the plant sequence, or form better matches, accompanied by the occurrence of filler DNA, through mutual twisting, and then the TDNA was integrated into plant chromosome through a partially homologous recombination mechanism. The appearance of filler DNA would facilitate T-DNA integration. The fragments flanking the T-DNA right border in transformed rice plants could derive from different parts of the inner T-DNA region; that is, disruption and recombination could occur at arbitrary positions in the entire T-DNA, in which the homologous area was comparatively easier to be disrupted. The structure of flanking sequences of T-DNA integrated in the rice chromosome presented various complexities. These complexities were probably a result of different patterns of recombination in the integrating process. Some types of possible integrating mechanism are detailed.     

一个水稻多逆境响应基因OsMsr3克隆与表达分析

为深入探讨水稻对逆境的反应机理并寻找新的植物耐逆基因,采用Affymetrix水稻表达芯片(含51279个转录本)分析了培矮64S全基因组在不同逆境(高温、干旱、低温)胁迫下、不同生育时期叶片和穗中的表达谱,从中筛选出一个受多种逆境诱导表达的基因OsMsr3(Oryza sativa L.multiple stresses responsive gene3,GenBank登陆号为 FJ383169).定量实时PCR分析结果进一步证实了此基因在逆境条件下的诱导表达模式.用 RT-PCR方法扩增获得了包含其完整开放阅读框的cDNA克隆,序列分析表明,其ORF大小为480 bp,编码一个具有160个氨基酸残基的低分子量热激蛋白,推测分子量约为18.0 kD;pI约为6.8.对其编码的蛋白质进行分析,发现其羧基端存在一个HSP20的蛋白保守结构域,与其他植物中的低分子量热激蛋白的相似性介于33.7%～97.5%.对其可能的启动子序列分析,发现6类与逆境反应有关的顺式作用元件.推测该基因在逆境反应中起着重要的作用,进一步的研究正在进行中.     

Phenotype Analysis and Fine Mapping of the Male Sterile Mutant <i>ms10</i> in Rice (<i>Oryza sativa</i> L.)

There is a positive correlation between fertility and yield, and the decrease of fertility is bound to a greatly reduced crop yield. Male sterile mutants can be used in hybrid rice. Therefore, rice male sterility has an important value in research and application, and the study of related mutants is also very vital. The mutant ms10 (male sterile 10) reported in this study was induced by ethyl methane sulfonate (EMS) in the indica maintainer line Xinong 1B. There was no significant difference between the ms10 and wild type in the vegetative growth stage. However, in the reproductive growth stage, ms10 showed that the plant became shorter, the anther became smaller and the color became lighter, and finally showed the phenotype of male sterility in comparison to the wild type. I₂-KI staining showed that the pollen was malformed and only a little was active. Scanning electron microscopy observation showed that the exine waxy layer of the ms10 anther decreased, suggesting that the protective effect on pollen was decreased. This may be one of the reasons leading to the phenotype of male sterility. Finally, the pollen showed shrinkage and collapsed, and the structure of germinating pore cover disappeared. This may be the result of sterility. Genetic analysis showed that the male sterility phenotype of the mutant was controlled by a single recessive nuclear gene. MS10 was mapped between the molecular markers IND37 and IND51 on chromosome 4, with a physical distance of 178.6 kb. These results lay the foundation for further studies on MS10.     

缺磷条件下不同水稻品种磷素吸收特性的研究

缺磷条件下,供试粳稻品种植株的单株鲜重、干重、全磷含量和单株磷累积量等具有较大差异,从中筛选出磷高效品种TP309和优质8号,其中TP309较磷低效品种早88-1的全磷含量和单株磷累积量分别增加37.50%~40.00%和82.76%~102.00%。单株根数、平均根长和根系体积与单株磷素吸收量的相关程度较小,光合速率(Pn)、叶片可溶蛋白含量和叶片可溶性糖含量均表现为随着吸磷量的增加而不断增大。     

水稻不同品种的穗发芽及其对外源激素的反应

选择8份早籼稻品种在开花后不同天数取穗子进行保湿培养,观察其抗穗发芽能力的差异。结果显示不同品种之间的抗穗发芽能力存在较大差异;处理以开花后22d左右的穗子保湿培养6d考查穗发芽,就能较好地比较出品种之间的差异。以3种不同浓度的赤霉素(GA3)和脱落酸(ABA)进行溶液培养,30mg／kg的赤霉素有明显促进穗发芽的效果,脱落酸抑制穗发芽的效果在不同品种之间表现出比较大的差异。     

栽培稻旱胁迫叶片相关性状的遗传解析

利用籼稻窄叶青8号(ZYQ8)和粳稻京系17(JX17)衍生的加倍单倍体(DH)群体127个株系,2002年在杭州采用田间断水法栽培,在水分胁迫下,对叶片的卷叶、相对含水量和电导率3个性状进行了评价和QTL分析。结果表明,3个性状在DH群体中均存在双向超亲分离,接近正态分布,受数量性状基因的控制;检测到影响这些性状的6个QTL,其中卷叶3个(qLR—1,qLR—5和qLR—11)、相对含水量2个(qRWC—1和qRWC—6和电导率1个(qERC—6）。旱胁迫时,目测卷叶方便易行,适于对大批品种或资源筛选,对抗旱栽培稻品种的筛选和利用具有一定的指导意义。     

Active Oxygen Damage Effect of Chlorophyll Degradation in Rice Seedlings Under Osmotic Stress

The changes of chlorophyll (Chl) content and contents of protochlorophyllide (Pchl), superoxide radical (O2-) and hydrogen peroxide (H2O2), malondialdehyde (MDA), ascorbic acid (ASA), glutathione (GSH), carotenoid (CAR) and the binding capacity of chlorophyll-protein (Chl-Pro) in rice (Oryza sativa L. ) seedlings exposed to osmotic stress induced by PEG 6000 (–0. 5 MPa, –0.8 MPa) were investigated to explore the relationship between Chl degradation and active oxygen effect. Under osmotic stress, Chl degradation was accompanied by the increase of contents of O2-, H2O2 and MDA and the decrease of contents of antioxidants AsA, GSH and CAR. The binding of Chl-Pro was loosened with the change of time and intensity of osmotic stress. Pretreatment with scavengers for active oxygen, such as AsA, α-tocopherol and mannitol retarded lipid peroxidation and reduced the oxidative injury of Chl, but Fe2+, H2O2 and Fenton reaction promoted the formation of MDA. The Fenton reaction accelerated the degradation of Chl. The results indicate that Chl degradation in rice seedlings induced by osmotic stress may be mainly due to the formation of more active hydroxyl radicals ('OH) through Fenton reaction and Haber-Weiss reaction.     

水稻中水杨酸和茉莉酸特异诱导蛋白激酶基因OsSJMK1的分离和鉴定

裂原激活蛋白激酶（MAPK）级联系统负责把接受自胞外或胞内的信号进一步传递和放大而最终作用于特异的转录因子,从而启动或调控基因的表达。MAPK信号级联系统在细胞分裂、分化、生物胁迫和非生物胁迫等多种信号传递途径中起着十分重要的作用。该文以一个茉莉酸（JA）诱导的表达序列为基础,在水稻中分离到了一个裂原激活蛋白激酶基因OsSJMK1的全长cDNA。序列比较分析表明该基因编码498个氨基酸,蛋白质等电点为8．43,包括完整的MAPK家族的蛋白激酶结构域。OsSJMK1与所有物种的MAPK一样包括蛋白激酶的全部11个次级结构域,在Ⅶ和Ⅷ次级结构域间一个双磷酸化位点;该位点苏氨酸（T）和酪氨酸（Y）残基之间为天冬氨酸（D）,而不是其他MAPK中常见的谷氨酸（E）、脯氨酸（P）或甘氨酸（G）。除了典型的MAPK激酶功能域外,在羧基端还有一段长约150个氨基酸残基的可能参与蛋白互作的结构域。以上这些结构特征表明OsSJMK1属于植物中第v类MAPK家族成员。蛋白激酶结构域序列比较表明OsSJMK1与报道的稻瘟病菌和机械伤害诱导的BWMK1的序列相似性高达81％,而且基因内含子和外显子的组成也非常相似,属于同一亚类,但在蛋白质序列的C端差异却很大。与BWMK1不同,OsSJMK1的表达不受伤害诱导,而受稻瘟病轻微诱导,但在JA和SA（水杨酸）处理早期表达量却迅速升高。在JA处理后1h,OsSJMK1转录水平升高到最大,而12h后回落到处理前的本底水平;在SA处理后30min转录水平就开始上升,2h达到最高值,而随后开始下降到处理前的本底水平。SA类似物BTH也能诱导OsSJMK1的表达。其他一些激素处理（如ABA）和非生物胁迫（如干旱、盐胁迫）都不对基因的表达产生任何影响,而且在植物大部分组织中的表达量都非常低。这些结果说明OsSJMK1可能特异性的参与JA和SA介导的防卫反应。     

Effects of Abscisic Acid and Water Stress on the Senescence of Detached Rice Leaves

The effects of abscisic acid (ABA) and water stress on senescence and enzyme activities of oxygen scavenging enzymes of detached rice leaves were compared. Exogenously applied ABA exhibited water stress-like effects by promoting senescence, by decreasing the activities of catalase, peroxidase, ascorbate peroxidase and superoxide dismutase. It seems that the effects of water stress on senescence and enzyme activities are possibly mediated through increased content of endogenous ABA. This revised version was published online in July 2006 with corrections to the Cover Date.     

Basal and Foliar Treatment using an Organic Fertilizer Amendment Lowers Cadmium Availability in Soil and Cadmium Uptake by Rice on Field Micro-Plot Experiment Planted in Contaminated Acidic Paddy Soil

In the present study, field micro-plot experiments were conducted to investigate the basal and foliar application of a tested organic fertilizer amendment (OFA) for decreasing the risk of Cd accumulating in rice. The results showed that applications of OFA significantly increased rice yields in Cd-polluted soil and reduced the level of Cd in rice plants, especially in rice grain. In addition, three application methods of OFA were investigated (single basal application (B1, B2, and B3), combined basal application (+LM, +D, and +Z), and foliar application (F1, F2)). Treat B, F, +LM, +D were all higher than control on rice yield with 25.03, 28.05, 30.61, 22.50 g pot^?1 on average, respectively. Among which, rice cadmium depress to 0.33 mg kg^?1 in foliar application is considered to be a more efficient and economical method of heavy metal remediation. The mechanism of foliar application to alleviate the accumulation of Cd in brown rice may be related to the probable Cd sequestration in the leaves and straws. And the doses of the foliar application were 2.25–3.75 kg hm^?2, approximately 1.0–2.5% of the basal application amount yet with more effect (0.10 mg kg^?1 more than single basal; 0.23 mg kg^?1 more than combined basal) on Cd reduction.     

施硅提高水稻叶片对紫外线胁迫的抗性

采用加硅与缺硅营养液培养的方法,首次研究证明硅能提高水稻(Oryza sativa L.)叶片抗紫外线胁迫的能力.结果表明,在紫外胁迫条件下,缺硅水稻叶片表面出现明显的棕色伤害斑点,而加硅叶片未出现伤害症状.硅在水稻表皮细胞壁及细胞内部的积累明显促进了紫外吸收物质在表皮细胞中的聚集,使表皮中可溶性酚类物质含量提高17%,不溶性紫外吸收物质的含量增加65%左右.荧光显微镜观察表明,在表皮细胞外壁或胞内沉积的水合二氧化硅固体中包含着大量不溶性的酚类化合物,它们与可溶性酚类物质一起在叶片的上、下表皮细胞中形成了吸收紫外线的屏障.     

水稻锌指蛋白基因OsBBX6的克隆、表达及生物信息学分析

锌指蛋白作为植物体内一类重要的转录因子,对植物生长发育、基因调控以及响应外界环境变化方面发挥重要作用。Os BBX6基因属于水稻锌指蛋白B-Box基因家族成员,启动子元件分析发现其含有高温应答元件(HSE)、干旱应答元件(MBS)及非生物胁迫响应元件(TC-rich repeats)等逆境相关元件。组织特异性定量表达分析表明,Os BBX6在叶片中表达最高,根其次,茎和幼穗中表达最低。胁迫处理后的荧光定量PCR发现其受低温诱导上调,受高温、干旱、盐胁迫等抑制表达,表明其正向响应低温胁迫,负向响应高温、干旱、盐胁迫等。另外,本研究还克隆了OsBBX6基因,并对其进行了系统进化、蛋白跨膜、蛋白亚细胞定位及OsBBX6基因共表达等分析,为进一步研究其生物学功能奠定基础。     

Genetic Identification of Quantitative Trait Loci for Contents of Mineral Nutrients in Rice Grain

In present study, Fe, Zn, Mn, Cu, Ca, Mg, P and K contents of 85 introgression lines （ILs） derived from a cross between an elite indica cultivar Teqing and the wild rice （Oryza rufipogon） were measured by inductively coupled argon plasma （ICAP） spectrometry. Substantial variation was observed for all traits and most of the mineral elements were significantly positive correlated or independent except for Fe with Cu. A total of 31 putative quantitative trait loci （QTLs） were detected for these eight mineral elements by single point analysis. Wild rice （O. rufipogon） contributed favorable alleles for most of the QTLs （26 QTLs）, and chromosomes 1,9 and 12 exhibited 14 QTLs （45%） for these traits. One major effect of QTL for zinc content accounted for the largest proportion of phenotypic variation （11%-19%） was detected near the simple sequence repeats marker RM152 on chromosome 8. The co-locations of QTLs for some mineral elements observed in this mapping population suggested the relationship was at a molecular level among these traits and could be helpful for simultaneous improvement of these traits in rice grain by marker assisted selection.     

水稻耐淹涝性状的遗传分析和SSR标记的研究

淹涝胁迫对水稻生产造成了严重影响, 发掘可应用于耐淹涝辅助选择的分子标记(MAS), 将有助于水稻耐淹涝性状的遗传改良。应用耐淹涝材料FR13A和淹涝敏感材料IR39595-503-2-1-2为亲本做正反交获得F₁和F₂代群体。对正反交的F₁群体的耐淹涝性状进行遗传分析, 发现正反交的F₁代群体在耐淹涝性状上没有显著差异, 说明耐淹涝性状是核基因控制。从两次淹涝处理中F₂代群体的分离情况来看, 来源于FR13A的耐淹特性表现出数量-质量性状遗传的特点。当淹涝胁迫压力比较轻时表现为数量性状遗传, 具有微效多基因的作用。当淹涝胁迫压力增大时, 表现为主效基因控制的质量性状。在SSR分析中, 187对SSR引物中有73对引物在两亲本间有明显的差异, 差异率为39%。用这73对差异引物, 对F2群体进行多态筛选, 结果筛选到一个与耐淹涝性状连锁的标记RM219, 验证了耐淹涝性状确实由主效基因Sub1控制, 因此, RM219在水稻耐淹涝育种中具有利用价值。     

Rice Mitogen-activated Protein Kinase Gene Family and Its Role in Biotic and Abiotic Stress Response

The mitogen-activated protein kinase （MAPK） cascade is an important signaling module that transduces extracellular stimuli into intracellular responses in eukaryotic organisms. An increasing body of evidence has shown that the MAPK-mediated cellular signaling is crucial to plant growth and development, as well as biotic and abiotic stress responses. To date, a total of 17 MAPK genes have been Identified from the rice genome. Expression profiling, biochemical characterization and/or functional analysis were carried out with many members of the rice MAPK gene family, especially those associated with biotic and abiotic stress responses. In this review, the phylogenetic relationship and classification of rice MAPK genes are discussed to facilitate a simple nomenclature and standard annotation of the rice MAPK gene family. Functional data relating to biotic and abiotic stress responses are reviewed for each MAPK group and show that despite overlapping in functionality, there is a certain level of functional specificity among different rice MAP kinases. The future challenges are to functionally characterize each MAPK, to identify their downstream substrates and upstream kinases, and to genetically manipulate the MAPK signaling pathway in rice crops for the Improvement of agronomically important traits.