水稻H3.2型组蛋白基因RH3.2A的克隆与盐胁迫下的表达分析

组蛋白H3与其他类型的组蛋白分子H2A,H2B,H4共同构成了真核生物核小体的八聚体核心。研究发现组蛋白H3的多种翻译修饰,如甲基化、乙酰化、磷酸化等在调控基因转录过程种发挥了重要的作用。本研究从盐胁迫处理的水稻幼苗组织中分离了一个新的水稻组蛋白H3基因RH3．2A,编码具有136个氨基酸残基的多肽,与多种植物的组蛋白H3蛋白具有高度的氨基酸一致性。多序列比较发现,除了基因结构差异之外,还有3个位置的氨基酸残基（32、88、91）在H3．1与H3．2型组蛋白H3中存在差异。研究了RH3．2A基因在高盐和ABA胁迫下的表达,结果发现在水稻根部RH3．2A基因受高盐的强烈诱导,而在叶片RH3．2A基因的表达则不受高盐诱导,此外RH3．2A基因也受外源ABA的诱导,结合启动子分析的结果,我们认为RH3．2A基因可能参与了依赖于ABA的高盐胁迫应答反应。文章讨论了植物组蛋白H3基因在高盐胁迫应答反应中可能的作用。     

Isolation and Expression Analysis of Plastidic Glucose-6-phosphate Dehydrogenase Gene from Rice (Oryza sativa L.)

Glucose-6-phosphate dehydrogenase is a rate-limiting enzyme of pentose phosphate pathway, existing in cytosolic and plastidic compartments of higher plants. A novel gene encoding plastidic glucose-6-phosphate dehydrogenase was isolated from rice (Oryza sativa L.) and designated OsG6PDH2 in this article. Through semiquantitative RT-PCR approach it was found that OsG6PDH2 mRNA was weakly expressed in rice leaves, stems, immature spikes or flowered spikes, and a little higher in roots. However, the expression of OsG6PDH2 in rice seedlings was significantly induced by dark treatment. The complete opening reading frame (ORF) of OsG6PDH2 was inserted into pET30a (+), and expressed in Escherichia coli strain BL21 (DE3). The enzyme activity assay of transformed bacterial cells indicated that OsG6PDH2 encoding product had a typical function of glucose-6-phosphate dehydrogenase.     

Silicon decreases chloride transport in rice (Oryza sativa L.) in saline conditions

Silicon can alleviate salt damage to plants, although the mechanism(s) still remains to be elucidated. In this paper, we report the effect of silicon on chloride transport in rice (Oryza sativa L.) seedlings in saline conditions. In the absence of salinity, silicon enhanced the growth of shoots, but not roots in three cultivars (cv. GR4, IR36, and CSR10). Salinity reduced the growth of both shoots and roots in all three genotypes. In saline conditions, addition of silicon to the culture solution again improved the growth of shoots, but not of roots. Under these saline conditions, the concentrations of chloride in the shoot were markedly decreased by adding silicon and the ratio of K⁺/Cl⁻ was significantly increased, while the concentration of chloride in the roots was unchanged. The decrease in chloride concentration in the shoot was correlated with the decrease in transpirational bypass flow in rice, as shown by the transport of the apoplastic tracer trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS). Addition of silicon increased the net photosynthetic rate, stomata conductance, and transpiration of salt-stressed plants in cv. IR36, indicating that the reduction of chloride (and sodium) uptake by silicon was not through a reduction in transpiration rate. Silicon addition also increased the instantaneous water use efficiency of salt-stressed plants, while it did not change the relative growth rate of shoots. The results suggest that silicon addition decreased transpirational bypass flow in the roots, and therefore decreased the transport of chloride to the shoot.     

Mapping QTL for Biomass Yield and Its Components in Rice (Oryza sativa L.)

Addicive effects, additive by additive epistatic effects, and their environmental interactions of QTLs are important genetic components of quantitative traits. Genetic architecture underlying rice biomass yield and its two component traits (straw yield and grain yield) were analyzed for a population of 125 DH lines from an inter-subspecific cross of IR64/Azucena. The mixed-model based composite interval mapping approach (MCIM) was used to detect QTLs, There were 12 QTLs detected with additive main effects, 27 QTLs involved in digenic interaction with aa and/or aae effects, and 18 QTLs affected by environments with ae and/or aae effects. It was revealed that epistatic effects and QE interaction effects existed on biomass yield and its component traits in rice. In addition, the genetic basis of relationships among these traits were investigated. Four QTLs and one pair of epistatic QTLs were detected to be responsible for the positive correlation between biomass yield and straw yield. Three QTLs might be responsible for the negative correlation between straw yield and grain yield. This result could partially explain the genetic basis of correlation among the three traits, and provide useful information for genetic improvement of these traits by marker-assisted selection.     

Effect of polyamines on ribonuclease activity of rice (Oryza sativa L.)

The RNA content and RNase activity were determined during maturation and germination of rice seeds. The RNA content reached a maximum after the 16th day from anthesis but RNase activity steadily increased up to the last stage of maturation. During germination RNA content was greatest after 24 hr and associated with a very low level of RNase activity. Maximum RNase activity was observed at 72 hr from germination and it afterwards gradually declined. During germination, exogenous application of polyamines decreased the level of RNase activity. RNase was partially purified (448-fold) from 72 hr germinated embryonic axis. Effects of polyamines and other divalent cations were observed on the purified enzyme.     

Identification of salt-stress responsive genes in rice (Oryza sativa L.) by cDNA array

To identify salt stress-responsive genes, we constructed a cDNA library with the salttolerant rice cultivar, Lansheng. About 15000 plasmids were extracted and dotted on filters with Biomeck 2000 HDRT system or by hand. Thirty genes were identified to display altered expression levels responding to 150 mmol/L NaCl. Among them eighteen genes were up-regulated and the remainders downregulated. Twenty-seven genes have their homologous genes in GenBank Databases. The expression of twelve genes was studied by Northern analysis. Based on the functions, these genes can be classified into five categories, including photosynthesis-related gene, transportrelated gene, metabolismrelated gene, stress-or resistancerelated gene and the others with various functions. The results showed that salt stress influenced many aspects of rice growth. Some of these genes may play important roles in plant salt tolerance.     

Indole-3-acetyl-myo-inositol in kernels of Oryza sativa

IAA-myo-inositol was isolated from kernels of Oryza sativa and characterized by its chromatographic properties and its mass spectral fragmentation pattern. This is the first demonstration of the occurrence of a myo-inositol ester of IAA in a plant other than Zea mays.     

Identification of Related QTLs at Late Developmental Stage in Rice (Oryza sativa L.) Under Two Nitrogen Levels

QTL underlying related traits at the late developmental stage under two different nitrogen levels were investigated in rice using a population of chromosome segment substitution lines (CSSL) derived from a cross between Teqing and Lemont. A total of 31 QTLs referring 5 traits, that is, plant height (PH), panicle number per plant (PN), chlorophyll content (CC), shoot dry weight (SDW) and grain yield per plant (YD), were detected. Under normal N level, 3 QTLs were detected for each trait, while under low N level, 5,4, 5 and 2 QTLs were detected for PH, PN, CC and SDW respectively. Most of the QTLs were located on chromosome 2, 3, 7, 11 and 12. QTLs controlling different traits or the same trait under different N levels were mapped on the same or adjacent intervals, forming several clusters in rice chromosomes. More than two traits were controlled by QTLs on one of four intervals (RM30-RM439, RM18-RM478, RM309-RM270, and RM235-RM17), suggesting that there were some pleiotropic effects. It was supposed that some QTLs only detected at low N level might be associated with the ability to tolerate the low N stress in rice.     

水稻金属耐受蛋白基因OsMTP2生物信息学及表达分析

为了解水稻(Oryza sativa)响应重金属污染及对重金属积累的机制,利用生物信息学手段对水稻金属耐受蛋白(metal tolerance protein, MTP) OsMTP2的结构特征进行预测,并分析OsMTP2基因的表达特征。结果表明,该蛋白由415个氨基酸组成,具有很强的疏水性。mRNA原位杂交结果表明,OsMTP2在叶片输导组织中有较强的表达,但在根、花药中没有检测到明显的杂交信号。这说明水稻金属耐受蛋白基因OsMTP2主要在输导组织中表达,并在金属离子运输中发挥重要作用。     

角类肥蛛Larinioides cornuta组蛋白H3基因保守序列克隆与分析

本文利用常规的PCR技术,克隆、测序园蛛科Araneidae角类肥蛛Larinioides cornuta组蛋白基因H3.将其H3基因与皿蛛科Linyphiidae 11个种的组蛋白H3基因的序列,在1级结构、2级结构、蛋白质水平进行了比较,分析发现:组蛋白基因H3的序列A、T、C、G碱基的百分含量比较平均,分别为24.9%、23.2%、25.7%、26.3%;简约信息位点数74,多数分布在第3位点;平均替换率为0.15.平均转换/颠换比(R)为1.8,大于0.5;G-C, C-T间的替换是该区域进化的另一个显著特征;DNA 1级结构高度保守的区段都不分布在2级结构"茎"区,不存在补偿性突变,2级结构的"环"(loop)区差异也很大;在氨基酸水平上所有成员非常保守.     

水稻Argonaute 2蛋白的原核表达与多克隆抗体制备

为了制备水稻Argonaute 2(AGO2)的多克隆抗体,该研究采用RT-PCR扩增OsAGO2蛋白165~401aa片段和440~570aa片段的编码序列,并构建了2个原核表达载体。诱导表达重组蛋白后注射家兔,制备了相应的多克隆抗体,最后利用Western blot初步分析水稻AGO2蛋白的表达模式。结果表明:成功构建2个表达载体,通过诱导获得了分子量约为30kD和23kD的重组蛋白。其中,以440~570aa片段为抗原所制备的多克隆抗体免疫印记效果较好。Western blot表明在水稻花药、愈伤组织及小穗中检测到OsAGO2表达。该研究为进一步深入探讨水稻OsAGO2基因的特性与功能奠定了基础。     

Transport of DMAA and MMAA into rice (Oryza sativa L.) roots

Arsenate (As(V)) transport into plant cells has been well studied. A study on rice (Oryza sativa L.) showed that arsenite is transported across the plasma membrane via glycerol transporting channels. Previous studies reported that the dimethylarsinic acid (DMAA) and monomethylarsonic acid (MMAA) uptake in duckweed (Spirodela polyrhiza L.) differed from that of As(V), and was unaffected by phosphate (H₂PO₄). This article reports the transport mechanisms of DMAA and MMAA in rice roots. Linear regression analysis showed that the DMAA and MMAA uptake in rice roots increased significantly (p ≤ 0.0002 and ≤0.0001 for DMAA and MMAA, respectively) with the increase of exposure time. Concentration-dependent influx of DMAA and MMAA showed that the uptake data were well described by Michaelis-Menten kinetics. The MMAA influx was higher than that of DMAA. The DMAA and MMAA uptake in rice roots were decreased significantly (p ≤ 0.0001 and ≤0.0077 for DMAA and MMAA, respectively) with the increase of glycerol concentration indicating that DMAA and MMAA were transported into rice roots using the same mechanisms of glycerol. Glycerol is transported into plant cells by aquaporins, and DMAA and MMAA are transported in a dose-dependent manner of glycerol which reveals that DMAA and MMAA are transported into rice roots through glycerol transporting channels. The DMAA and MMAA concentration in the solution did not affect the inhibition of their uptake rate by glycerol.     

Mapping and Marker-assisted Selection of a Brown Planthopper Resistance Gene bph2 in Rice (Oryza sativa L.)

Nilaparvata lugens Stål (brown planthopper, BPH), is one of the major insect pests of rice (Oryza sativa L.) in the temperate rice-growing region. In this study, ASD7 harboring a BPH resistance gene bph2 was crossed to a susceptible cultivar C418, a japonica restorer line. BPH resistance was evaluated using 134 F_2:3 lines derived from the cross between “ASD7” and “C418”. SSR assay and linkage analysis were carried out to detect bph2. As a result, the resistant gene bph2 in ASD7 was successfully mapped between RM7102 and RM463 on the long arm of chromosome 12, with distances of 7.6 cM and 7.2 cM, respectively. Meanwhile, both phenotypic selection and marker-assisted selection (MAS) were conducted in the BC₁F₁ and BC₂F₁ populations. Selection efficiencies of RM7102 and RM463 were determined to be 89.9% and 91.2%, respectively. It would be very beneficial for BPH resistance improvement by using MAS of this gene.     

不同渗氧能力水稻品种对砷的耐性和积累

水稻是目前世界上(尤其是东南亚)最主要的粮食作物之一,也是砷(As)通过食物链进入人体的主要途径。日益加剧的土壤砷污染,严重影响了稻米的产量和品质,进而威胁着人体健康。通过温室实验,研究CNT 87059-3、玉香油占和巴西陆稻3种不同渗氧能力的水稻品种在不同砷浓度处理下的生长情况和砷积累特征,结果表明:(1)渗氧能力强的玉香油占砷耐性指数最高,砷处理浓度为2 mg/L时耐性指数高达0.71,而CNT 87059-3的耐性指数为0.55,巴西陆稻仅有0.17;(2)随着砷处理浓度的升高,3种水稻品种的生物量呈现下降趋势,但渗氧能力强的玉香油占较其它两品种生物量的下降幅度小;(3)在不同砷浓度处理下水稻地下部分的砷含量有显著性差异(P0.001),且同种砷浓度处理下不同水稻品种的地下部分砷含量也存在显著性差异(P0.01),渗氧能力较强的水稻品种与渗氧能力较弱的品种相比能显著降低砷在根部(地下部分)的积累。水稻渗氧能力与其砷耐性和砷积累有显著相关性,渗氧能力越强,水稻的砷耐性越强,砷的积累量越少。因此,通过筛选渗氧能力强的水稻品种,有望降低污染农田水稻的砷含量和健康风险。     

Genome-Wide Identification, Classification, and Expression Analysis of Autophagy-Associated Gene Homologues in Rice (Oryza sativa L.)

Autophagy is an intracellular degradation process for recycling macromolecules and organelles. It plays important roles in plant development and in response to nutritional demand, stress, and senescence. Organisms from yeast to plants contain many autophagy-associated genes (ATG). In this study, we found that a total of 33 ATG homologues exist in the rice [Oryza sativa L. (Os)] genome, which were classified into 13 ATG subfamilies. Six of them are alternatively spliced genes. Evolutional analysis showed that expansion of 10 OsATG homologues occurred via segmental duplication events and that the occurrence of these OsATG homologues within each subfamily was asynchronous. The Ka/Ks ratios suggested purifying selection for four duplicated OsATG homologues and positive selection for two. Calculating the dates of the duplication events indicated that all duplication events might have occurred after the origin of the grasses, from 21.43 to 66.77 million years ago. Semi-quantitative RT–PCR analysis and mining the digital expression database of rice showed that all 33 OsATG homologues could be detected in at least one cell type of the various tissues under normal or stress growth conditions, but their expression was tightly regulated. The 10 duplicated genes showed expression divergence. The expression of most OsATG homologues was regulated by at least one treatment, including hormones, abiotic and biotic stresses, and nutrient limitation. The identification of OsATG homologues showing constitutive expression or responses to environmental stimuli provides new insights for in-depth characterization of selected genes of importance in rice.     

高盐低温胁迫下水稻叶细胞ROS清除系统的相关基因表达

采用Affymetrix水稻表达谱芯片,分析高盐和低温胁迫下水稻叶细胞内ROS清除系统的相关基因表达状况,探讨在响应非生物胁迫过程中水稻植株体内抗氧化防卫体系的积极作用。结果表明:(1)水稻叶细胞内ROS清除系统涉及187个基因和/或EST,由抗氧化的非酶类物质如抗坏血酸、谷胱甘肽、生育酚等和抗氧化酶如超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)等组成。(2)在低温逆境下,籼稻(i-93-11)叶细胞表达上调基因(2倍以上,下同)有5个,下调(0.5倍以下,下同)基因有2个;粳稻(j-NJ-1)叶细胞表达上调基因5个,下调基因6个。(3)在高盐胁迫下,籼稻(i-93-11)叶细胞表达上调基因有31个、下调基因13个;粳稻(j-NJ-1)叶细胞表达上调基因27个,下调基因25个。(4)高盐和低温胁迫下水稻叶细胞ROS清除系统相关基因的表达在籼粳稻间存在较大差异,并根据水稻基因表达谱芯片数据构建了水稻响应高盐和低温胁迫ROS清除网络图。     

Molecular mapping of two semidwarf genes in an indica rice variety Aitaiyin3 (Oryza sativa L.)

Genetic analysis established that Aitaiyin3, a dwarf rice variety derived from a semidwarf cultivar Taiyin1, carries two recessive semidwarf genes. By using simple sequence repeat (SSR) markers, we mapped the two semidwarf genes, sd-1 and sd-t2 on chromosomes 1 and 4, respectively. Sd-t2 was thus named because the semidrawf gene sd-t has already been identified from Aitaiyin 2 whose origin could be traced back to Taiyin1. The result of the molecular mapping of sd-1 gene revealed it is linked to four SSR markers found on chromosome 1. These markers are: RM297, RM302, RM212, and OSR3 spaced at 4.7 cM, 0 cM, 0.8cM and 0 cM, respectively. Sd-t2 was found to be located on chromosome 4 using five SSR markers: two markers, SSR332 and RM1305 located proximal to sd-t2 are spaced 11.6 cM, 3.8 cM, respectively, while the three distally located primers, RM5633, RM307, and RM401 are separated by distances of 0.4 cM, 0.0 cM, and 0.4 cM, respectively. __________ Translated from Acta Genetica Sinica, 2005, 32 (2) [译自: 遗传学报, 2005,32(2)]     

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.