Isolation and Identification of Natural Endophytic Rhizobia from Rice (Oryza sativa L.) Through rDNA PCR-RFLP and Sequence Analysis

Three novel endophytic rhizobial strains (RRE3, RRE5, and RRE6) were isolated from naturally growing surface sterilized rice roots. These isolates had the ability to nodulate common bean (Phaseolus vulgaris). Polymerase chain reaction–restriction fragment length polymorphism and sequencing of 16S rDNA of these isolates revealed that RRE3 and RRE5 are phylogenetically very close to Burkholderia cepacia complex, whereas RRE6 has affinity with Rhizobium leguminosarum bv. phaseoli. Plant infection test using gusA reporter gene tagged construct of these isolates indicated that bacterial cells can go inside and colonize the rice root interiors. A significant increase in biomass and grain yield was also recorded in greenhouse-grown rice plants inoculated with these isolates.     

水稻质体葡萄糖-6-磷酸脱氢酶基因的克隆与表达研究

戊糖磷酸途径是高等植物中重要的代谢途径,主要生理功能是产生NADPH以及供核酸代谢的磷酸戊糖。葡萄糖-6-磷酸脱氢酶（G6PDH）是戊糖磷酸途径的关键酶,广泛存在于高等植物细胞的细胞质和质体中。木研究首次从水稻（Oryza sativa L.）幼苗中分离了核编码的质体G6PDH基因OsG6PDH2,序列分析表明OsG6PDH2编码一个具有588个氨基酸残基的多肽,等电点为8．5,分子量66kDa。OsG6PDH2的N端有1个70个氨基酸的信号肽,推测的裂解位点为Gly55和Val56,表明OsG6PDH2编码产物可能定位于质体。多序列比较的结果表明OsG6PDH2与拟南芥、烟草、马铃薯质体G6PDH的一致性分别达81％、87％、83％。进化关系说明水稻OsG6PDH2与拟南芥（AtG6PDH3）、马铃薯（StG6PDH1）处于高等植物P2型质体G6PDH分支上,暗示了OsG6PDH2可能是一个P2型的质体蛋白。Matinspector程序分析表明,OsG6PDH2在起始密码子上游含有一个bZIP转录因子识别位点、一个ABA应答元件、一个CRT／DRE元件和1个W-box元件。半定量RT-PCR分析表明,OsG6PDH2在水稻根、茎、叶和幼穗组织中都呈低丰度组成型表达,在根部表达较高,在水稻幼苗中的表达显著受暗处理的诱导。将OsG6PDH2的完整开放阅读框构建到大肠杆菌表达载体pET30a（＋）中,pET30a（＋）-OsG6PDH2在大肠杆菌中得到了有效表达。酶活性测定证明,OsG6PDH2的编码产物具有葡萄糖-6-磷酸脱氢酶的功能。     

水稻Ds插入纯合体的筛选和鉴定

采用Basta抗性鉴定、潮霉素抗性鉴定和PCR检测相结合的方法筛选和鉴定了水稻Ds插入纯合体。在T1代236个转化株系中,有16个株系的全部植株表现出对Basta的敏感,其余220个株系的植株表现出对Basta的抗性。经过3代的纯合筛选,共鉴定出Ds插入纯合体203个．这些Ds插入纯合体可用于构建Ac／Ds系统和对Ds插入突变体进行筛选和鉴定,为水稻功能基因组学研究提供了材料。     

Coleoptile Senescence in Rice (Oryza sativa L.)

We investigated the cellular events associated with cell deathin the coleoptile of rice plants (Oryza sativa L.). Seeds germinatedunder submergence produced coleoptiles that were more elongatedthan those grown under aerobic conditions. Transfer of seedlingsto aerobic conditions was associated with coleoptile opening(i.e. splitting) due to death of specific cells in the sideof the organ. Another type of cell death occurred in the formationof lysigenous aerenchyma. Senescence of the coleoptile was alsonoted, during which discolouration of the chlorophyll and tissuebrowning were apparent. DNA fragmentation was observed by deoxynucleotidyltransferase-mediateddUTP nick end labelling (TUNEL) assay, and further confirmedby the appearance of oligonucleosomal DNA ladders in senescentcoleoptile cells. Two nucleases (Nuc-a and Nuc-b) were detectedby in-gel-assay from proteins isolated from coleoptiles. Nuc-a,commonly observed in three cell death phases required eitherCa²⁺or Mg²⁺, whereas Nuc-b which appeared during senescencerequired both Ca²⁺and Mg²⁺. Both nucleases were strongly inhibitedby Zn²⁺. Copyright 2000 Annals of Botany Company Aerenchyma, rice, cell death, coleoptile, fragmentation, nuclease, Oryza sativa, senescence, split, submergence, TUNEL     

水稻体细胞无性系变异

水稻体细胞无性系变异研究取得了很大进展 ,获得了大量抗病、抗逆、优质、矮杆等突变体。对这些突变体遗传分析表明 ,大多数突变性状由 1对或 2对基因控制。水稻体细胞无性系变异的发生与基因型、性状、继代时间、培养方式等有关 ,并具有内在的机制 ,点突变和反转录转座子插入可能是引起水稻无性系变异的两个重要原因。     

QTL Analysis of Aluminum Resistance in Rice (Oryza sativa L.)

Aluminum (Al) toxicity is considered as one of the primary causes of low-rice productivity in acid soils. In the present study, quantitative trait loci (QTLs) controlling Al resistance based on relative root elongation (RRE) were dissected using a complete linkage map and a recombinant inbred lines (RILs) derived from a cross of Al-tolerant japonica cultivar Asominori (Oryza sativa L.) and Al-sensitive indica cultivar IR24 (O. sativa L.). A total of three QTLs (qRRE-1, qRRE-9, and qRRE-11) were detected on chromosomes 1, 9, and 11 with LOD score ranging from 2.64 to 3.60 and the phenotypic variance explained from 13.5 to 17.7%. The Asominori alleles were all associated with Al resistance at all the three QTLs. The existence of these QTLs was confirmed using Asominori chromosome segment substitution lines (CSSLs) in IR24 genetic background (IAS). By QTL comparative analysis, the two QTLs (qRRE-1and qRRE-9) on chromosomes 1 and 9 appeared to be consistent among different rice populations while qRRE-11 was newly detected and syntenic with a major Al resistance gene on chromosome 10 of maize. This region may provide an important case for isolating genes responsible for different mechanisms of Al resistance among different cereals. These results also provide the possibilities of enhancing Al resistance in rice breeding program by marker-assisted selection (MAS) and pyramiding QTLs.     

水稻分蘖角度的ＱＴＬs分析

分蘖角是水稻株型构成的重要性状之一,在育种上人有极其重要的意义,利用分蘖角度差异显著的１对籼粳亲本,将其杂交Ｆ１代花培加倍,构建了１个ＤＨ群体,考察了１１５个ＤＨ株系的分蘖角度,并使用该群体构建的分子图谱进行数量性状座痊（ＱＴＬs)分析。分别在第９和１２个染色体上检测３个ＱＴＬs(qTA-9a、qTA－９b和qTA－１２）,贡献率分别为２２．７％、１１．９％和２０．９％,其加性效应均为负,表明由分蘖角度较大的窄占青８号的基因控制,并讨论了这种由主产和微效基因控制的分蘖性状在育种学上的应用。     

水稻纹枯病抗性QTL分析

对灿稻窄叶青8号（ZYQ8）和粳稻京系17（JX17）以及由它们构建的加倍单倍体（DH）群体,分别在杭州和海南岛,采用注射器接种法进行纹枯病抗性鉴定,并使用该群体的分子链锁图谱进行数量性状座位（QTL）分析。共检测到4个抗纹枯病的QTL（qSBR-2、qSBR-3、qSBR-7和qSBR-11）,分别位于第2、第3、第7和第11染色体。其中qSBR-2、qSBR-3、qSBR-7的抗性基因由抗病亲本ZYQ8贡献,而qSBR-11的抗性基因来自感病亲本JX17。qSBR-2、qSBR-3、qSBR-7在杭州和海南岛都能检测到,而qSBR-11只在杭州检测到。在杭州的实验中,纹枯病病级与秆长和抽穗期呈显著负相关;在控制秆长和抽穗期的QTL中,控制秆长的qCL-3与qSBR-3位于同一染色体区域,其余QTL与抗纹枯病的QTL之间无连锁关系。     

Isolation and Characterization of Matrix Associated Region DNA Fragments in Rice (Oryza sativa L.)

To investigate the interactions between chromosomal DNA andnuclear matrices in higher plants, matrix associated regions(MARs) of rice (Oryza sativa L.) DNAs were cloned. First, weprepared nuclear matrices from isolated nuclei by digestingthem with EcoRl and then extracting with 2 M NaCl. About 6%of the total DNA remained in the nuclear matrices after thisdigestion and extraction. The residual DNA fragments in thenuclear matrices were cloned. Some of the cloned DNA fragmentsshowed binding to certain nuclear proteins. One of the MAR fragmentscontained sequences related to known consensus motifs and ahairpin loop structure. A method is presented for isolationof matrix associated region (MAR) DNAs from plant cells. (Received January 13, 1997; Accepted July 10, 1997)     

Comparison of Rice (Oryza sativa L.) Seed Proteins from Several Varieties

Enzymatically modified proteins (EMP) with different methionine levels were produced from soy protein isolate using an improved plastein reaction. The products having methionine at approximate levels of 4%, 7%, and 14%, designated as EMP₄, EMP₇, and EMP₁₄, respectively, were investigated to characterize their chemical properties particularly in terms of the state and location of the methionine residues. Leucine aminopeptidase treatment of the EMP products did not find any significant amount of methionine residues at the N-terminals, but carboxypeptidase A treatment liberated methionine efficiently in accordance with the methionine levels in the EMP products. Treatment with LiBH₄ reduced the methionine content of EMP₁₄ by approximately 64%. A significant amount of homoserine was produced when EMP₁₄ was treated with BrCN. All these data indicate that the covalently attached methionine molecules are localized at or near the C-terminals of the EMP molecules, probably as oligomers.     

Mechanism of Anther Dehiscence in Rice (Oryza sativa L.)

This paper presents a new explanation of the mechanism of antherdehiscence in rice during the period from floret opening topollen dispersal. The theca dehisced on the stomium in the apicalpart and the anther wall in the basal part of the large locule.Comparison of the anther dehiscence process under various airhumidity conditions showed that the process, until the splittingat the apical and basal parts, was a moisture-requiring processwhereas the widening of the splits in both parts was a desiccatoryprocess. Observation of the anther transverse section, revealedthe marked development of the U-shaped thick cell wall in theendothecium adjacent to these two splits. From these observations,the anther dehiscence mechanism may be explained as follows.At the time of anthesis, pollen grains swell rapidly in responseto the floret opening and cause the theca to bulge, rupturingthe septum. The pollen pressure combined with the inward bendingof the locule walls adjacent to the stomium causes splittingof the stomium in the apical part of the theca. At the sametime, the septum rupture extends to the bottom of the largelocule supported by the pollen pressure. After these processes,the locule walls adjacent to both splits straighten probablydue to their water loss. This straightening widens the splitsand the swollen pollen grains overflow from the widened splits.Copyright1999 Annals of Botany Company Anther dehiscence, Oryza sativa L., pollen grain swelling, rice, septum, stomium, theca.     

水稻米粒延伸性的遗传剖析

以籼稻ZYQ8与粳稻JX17为亲本的DH群体作为研究材料,考察DH群体及双亲的米粒延伸率相关性状,并使用该群体的分子连锁图谱进行QTL分析.共检测到14个与稻米延伸性有关的QTL,包括2个粒长QTL、7个饭粒长QTL和5个米粒延伸率QTL,分别位于第1、2、3、5、6、7、10、11和12染色体.所有QTL的LOD值介于2.26～9.25,分别解释性状变异的5.31%～17.21%.在第3染色体上的G249～G164、第6染色体上的G30～RZ516和第10染色体上的G1082～GA223区间同时检测到控制饭粒长和米粒延伸率的QTL.米粒延伸性受多基因控制,Wx基因与位于第6染色体上的qCRE-6的G30～RZ516区间相近,对米饭的延伸性具重要影响.     

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.     

水稻柱头外露率的QTL分析

利用高柱头外露率的籼稻窄叶青8号(ZYQ8)和极低外露率的粳稻京系17(JX17)以及由它们构建的加倍单倍体(DH)群体,在海南对各DH株系的柱头外露率进行调查,并使用该群体的分子连锁图谱进行数量性状座位(QTL)分析。共检测到2个控制水稻柱头外露率的QTL(qPES-2,qPES-3),分别位于第2、第3染色体;并发现控制柱头单边外露率的QTL与柱头外露率完全一致,而控制柱头双边外露率的QTL在第2染色体上检测到;其增效基因均来源于ZYQ8。同时定位的控制穗粒数的QTL位于第6染色体和第8染色体上,与柱头外露率之间没有连锁关系。     

Ethylene-Induced Polyamine Accumulation in Rice (Oryza sativa L.) Coleoptiles

The light-harvesting complex (LHC) of photosystem II is composed of several different pigment-binding apoproteins. We have identified a cDNA clone LHCIIa-1 encoding the 31-kilodalton LHC IIa (CP29, Chl a/b-P1) apoprotein of barley (Hordeum vulgare). Direct protein microsequencing of an internal peptide fragment from the LHC IIa apoprotein has been used to identify unequivocally the cDNA clone as that coding for the LHC IIa apoprotein. Microsequencing of the 28-kilodalton LHC IIc protein (CP26) showed only minor sequence similarity to the LHC IIa protein, indicating that they are two different gene products. LHCIIa-1 codes for a protein of 286 amino acid residues (molecular weight, 31,308), which displays strong similarities to other pigment-binding LHC proteins, and yet contains an additional 42 amino acid residue segment. Two regions of strong intramolecular sequence similarity are also observed.     

水稻分蘖角度的QTL定位和主效基因的遗传分析

利用水稻籼粳亚种间组合Asominori×IR24重组自交系(RIL)群体71个株系和相应的全基因组染色体片段置换系(Chromosomesegmentsubstitutionline,CSSL)群体65个株系,在2种环境下对分蘖角度性状进行了数量性状基因座(QTL)定位和上位性效应的遗传分析。在两种群体中都出现了分蘖角度的超亲分离。在RIL群体中发现了5个主效QTLs和3对上位性双位点互作标记基因座,控制水稻分蘖角度。其中在第9染色体上位于XNpb108~C506RFLP分子标记区间的qTA-9基因座在2种环境中同时出现,其贡献率平均为28·6%,增加分蘖角度的等位基因来自籼稻品种IR24。利用CSSL群体图示基因型分析,证实在第9染色体上含有RFLP标记C609和C506约15cM的染色体区段,存在增加分蘖角度的基因,来源于染色体片段供体亲本IR24,在Asominori的遗传背景中能增加分蘖角度约15°,该基因的位置与RIL群体在第9染色体上定位的QTL相同,证实了qTA-9的存在。F1表型测定及F2代遗传分析表明,来自IR24的等位基因是一个不完全显性基因。除一对上位性位点存在显著的环境互作效应外,未发现其他位点存在与环境的互作效应。不同基因的加性效应和双位点的上位性效应的共同作用可能是造成水稻分蘖角度超亲分离的主要原因。     

Chronopathological Aspects of Disease Incidence in Rice (Oryza sativa L.)

Rice seedlings maintained under uncontrolled glasshouse conditions were inoculated with conidial suspensions of a fungal pathogen, Helminthosporium oryzae, at various times during the 24 h. Significant increase in the percent germination and germ tube length of conidia were observed in the rice samples inoculated at 02:00 and 06:00h. The 24 h temporal variation in leaf temperature was positively correlated with variation in stomatal movements. The results indicate a 24 h rhythm in the behavior of the fungal pathogen on the host in relation to the conditions of the growing environment. In all the inoculated seedlings, the appearance of a large number of brown leaf spots was confined to the light span. Among the plants inoculated, earlier initiation of brown leaf spot appearance, maximum number of leaf spots, and highest disease severity were observed when plants were inoculated at 02:00h. There was a positive correlation between disease severity of the host and in vivo values of percent germination of conidia and germ tube length of the pathogen in plants inoculated between 02:00 and 06:00h. The findings of this study implicate that light intensity and temperature could play a predominant role in controlling disease susceptibility rhythms in plants.