Inheritance of two endosperm protein loci in rice (Oryza sativa L.)

Summary Previous studies indicated two types of phenotypic protein markers as two minor bands of SDS-PAGE for rice storage protein. A variant derived from a Pakistani variety, Dular, was found to show a mobility variant with Band 11, a relatively faster-moving band as compared to Band 10, while most of the other cultivated rices exhibited Band 10 at a molecular weight of around 100–110 K. Band 11 was also observed in several wild rice species. How this variant occurred is not known. Another marker is characterized by the presence of either Band 56 (slower-migrating band) or Band 57 (faster-migrating band) in most cultivars at a molecular weight of about 28–27 K. Most indica varieties developed in Taiwan have Band 57 and japonica varieties have Band 56. Genetic analysis of F₁, F₂ and F₃ seeds from interstrain crosses indicated that Band 10 versus Band 11 and Band 56 versus Band 57 are due to codominant alleles at two loci. Tests of independent inheritance between these two loci (Band 10/11 versus Band 56/57) indicated that there is no linkage between them. Both of these two protein loci encode for endosperm proteins and mostly belong to the minor polypeptide subunits of the glutelin fraction of rice seed proteins. Studies on reciprocal crosses indicate dosage effects as exhibited in band patterns. Variations in band intensity were frequently observed when the maternal genotype was different.     

水稻新种质资源的耐盐性鉴定评价

用0.5%的NaCl溶液作灌溉水,对2000-2002年江苏省水稻区域试验参试品系和近年引进的部分水稻种质资源进行苗期耐盐性鉴定.结果表明:株高矮化是苗期盐胁迫的一种形态特征;耐盐性鉴定易受环境影响;就整体而言,籼稻种质资源的苗期耐盐性好于粳稻;综合2年结果,籼156和64608两种质资源的苗期耐盐性较强.     

Ammonium nutrition increases photosynthesis rate under water stress at early development stage of rice (Oryza sativa L.)

An hydroponic experiment with a simulated water stress induced by PEG (6000) was conducted in a greenhouse to study the effects of nitrate (NO₃ ⁻), ammonium (NH₄ ⁺) and the mixture of NO₃ ⁻ and NH₄ ⁺, on water stress tolerance of rice seedlings. Rice (Shanyou 63) was grown under non- or simulated water stress condition (10% (w/v) PEG, MW6000) with the 3 different N forms during 4 weeks. Under non-stressed condition no difference was observed among the N treatments. Under simulated water stress, seedlings grown on N-NO₃ ⁻ were stunted. Addition of PEG did not affect rice seedling growth in the treatment of only NH₄ ⁺ supply but slightly inhibited the rice seedling growth in the treatment of mixed supply of NO₃ ⁻ and NH₄ ⁺. Simulated water stress, when only N-NH₄ ⁺ was present, did not affect leaf area and photosynthesis rate, however, both parameters decreased significantly in the NO₃ ⁻ containing solutions. Under water stress, Rubisco content in newly expanded leaves significantly increased in the sole NH₄ ⁺ supplied plants as compared to that in plants of the other two N treatments. Under water stress, the ratio of carboxylation efficiency to Rubisco content was, respectively, decreased by 13 and 23% in NH₄ ⁺ and NO₃ ⁻ treatments, respectively. It is concluded that, water stress influenced the Rubisco activity than stomatal limitation, and this effects could be regulated by N forms. Responsible Editor: Herbert Johannes Kronzucker. Shiwei Guo and Gui Chen contributed equally to this paper.     

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.     

Cloning and characterization of a novel splicing isoform of the iron-superoxide dismutase gene in rice (Oryza sativa L.)

Superoxide dismutases (SODs) are ubiquitous metalloenzymes in aerobic organisms that play a crucial role in protecting organisms against ROS. Here, we report the molecular cloning and functional characterization of a novel alternatively spliced variant of the iron-superoxide dismutase gene, OsFe-SODb, from a rice panicle cDNA library. The alternative splicing event occurred in the fourth exon of the OsFe-SOD gene, and led to the translation of two isoforms of different sizes. The 5′ flanking region of the OsFe-SOD was cloned and many cis-acting regulatory elements were found that are involved in light responsiveness, including a G-box and an I-box. RT-PCR analysis showed that the two alternative forms of OsFe-SOD were expressed in both the vegetative and reproductive tissues of Cpslo17. Moreover, accumulation of both isoforms was upregulated by light induction. In addition, the alternative splicing of OsFe-SOD mRNA was sensitive to low temperature. High yield production of the two recombinant OsFe-SOD isoforms was achieved in Escherichia coli. SOD assays showed that C-terminal truncation in OsFe-SODb did not result in a loss of SOD enzyme activity.     

水稻株高构成因素的QTL剖析

利用水稻籼粳杂交 (圭 6 30× 0 2 42 8) F1 的花药离体培养建立的一个含 81个 DH家系的作图群体 ,对水稻株高构成因素 (穗长、第 1节间长、……、第 5节间长 )进行基因定位。DH群体中株高构成因素均呈正态分布。相邻的构成因素间呈极显著的正相关 ,而相距较远的构成因素间的相关较弱或不显著。采用 QTL(Quantitative trait lo-cus)分析 ,定位了影响株高构成因素的 6个 QTL:qtl7同时影响穗长和第 1、2、3节间长 ,qtl1 和 qtl2 同时影响第 4和第 5节间长 ,qtl1 0 a和 qtl1 0 b仅影响第 1节间长 ,qtl3 仅影响第 3节间长。采用 QTL 互作分析 ,检测到 19对显著的互作 ,每个构成因素受 2个或 2个以上的 QTL 互作对的影响。并且还发现 ,同一个 QTL 互作对可能影响不同的性状 ,以及一个 QTL 可以分别与不同的 QTL 产生互作而影响同一个性状或影响不同的性状 ,但总的看来 ,加性效应是主要的。这些结果揭示了株高构成因素间相关的遗传基础 ,在水稻育种中运用这些 QTL 将有助于对株高 ,以及对穗长和上部节间长度进行精细的遗传调控。     

The expression of the rice (Oryza sativa L.) homologue of Snm1 is induced by DNA damages

We isolated and characterized the rice homologue of the DNA repair gene Snm1 (OsSnm1). The length of the cDNA was 1862bp; the open reading frame encoded a predicted product of 485 amino acid residues with a molecular mass of 53.2kDa. The OsSnm1 protein contained the conserved beta-lactamase domain in its internal region. OsSnm1 was expressed in all rice organs. The expression was induced by MMS, H(2)O(2), and mitomycin C, but not by UV. Transient expression of an OsSnm1/GFP fusion protein in onion epidermal cells revealed the localization of OsSnm1 to the nucleus. These results suggest that OsSnm1 is involved not only in the repair of DNA interstrand crosslinks, but also in various other DNA repair pathways.     

Sink anatomy in relation to solute movement in rice (Oryza sativa L.): A summary of findings

The results of studies on assimilate and water transport in the developing caryopsis of rice are summarised. Evidence is presented for a symplastic movement of solutes as far as the aleurone layer. However, transport into the apoplast at the nucellus/aleurone interface appears to be a necessary step due to the absence of plasmodesmata at this site. It is suggested that water leaves the caryopsis during grain filling by the isolated cell walls of the pigment strand, the suberised walls of these cells functioning to isolate the apoplast from the symplast and thereby allowing opposing fluxes of water and assimilates to occur in the dorsal region of the grain.     

RFLP analysis of rice (Oryza sativa L.) introgression lines

Summary Fifty-two introgression lines (BC₂F₈) from crosses between two Oryza sativa parents and five accessions of O. officinalis were analyzed for the introgression of O. officinalis chromosome segments. DNA from the parents and introgression lines was analyzed with 177 RFLP markers located at approximately 10-cM intervals over the rice chromosomes. Most probe/enzyme combinations detected RFLPs between the parents. Of the 174 informative markers, 28 identified putative O. officinalis introgressed chromosome segments in 1 or more of the introgression lines. Introgressed segments were found on 11 of the 12 rice chromosomes. In most cases of introgression, O. sativa RFLP alleles were replaced by O. officinalis alleles. Introgressed segments were very small in size and similar in plants derived from early and later generations. Some nonconventional recombination mechanism may be involved in the transfer of such small chromosomal segments from O. officinalis chromosomes to those of O. sativa. Some of the introgressed segments show association with genes for brown planthopper (BPH) resistance in some introgressed lines, but not in others. Thus, none of the RFLP markers could be unambiguously associated with BPH resistance.     

Molecular mapping of the ge s gene controlling the super-giant embryo character in rice (Oryza sativa L.)

The giant-embryo character is useful for quality improvement in rice. Three alleles controlling embryo size have been reported at the ge locus. Based on trisomic analysis, this locus is known to reside on chromosome 7. The objective of the present study was to identify linkage between molecular markers and the ge ^s gene using an existing molecular map of rice and an F₂ population derived from Hwacheongbyeo-ge ^s (super-giant embryo)/Milyang 23. The bulked-segregant method was used to screen 38 RFLPs and two microsatellite markers from rice chromosome 7. RZ395 and CDO497 flanked the ge ^s gene, at 2.4 cM and 3.4 cM, respectively. The two microsatellite markers, RM18 and RM10, were linked with ge ^s at 7.7 cM and 9.6 cM, respectively. The availability of molecular markers will facilitate selection of this grain character in a breeding program and provide the foundation for map-based gene isolation.     

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.     

Molecular cloning and nucleotide sequence cDNA encoding nucleoside diphosphate kinase of rice (Oryza sativa L.)

We isolated a rice cDNA encoding nucleoside diphosphate kinase (NDK, EC 2.7.4.6). The deduced amino acid sequence of the rice NDK shows highest homology to spinach NDK-I. The rice NDK gene exhibits a strong codon bias (73.8% GC) in the third position of the codon. DNA blot analysis indicated that at least single NDK gene is present in rice genome.     

Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification

Summary Rice cells were precultured for 10 d in medium containing 60 g/L sucrose and subsequently for 1 d in medium supplemented with 0. 4 M sorbitol. After loading with 25%PVS2 at 22°C for 10 min and dehydration in 100%PVS2 at 0°C for 7. 5 min,they were plunged into liquid nitrogen directly. Survival was 45. 0 ±5.1% (based on the reduction of triphenyl tetrazolium chloride)following warming and unloading. For regrowth, cells were plated on semi-solid medium replenished with 40%(w/v) starch for 2d prior to reculture. Cell suspensions were reestablished and plants were regenerated from recovered cells. Twenty eight plants set seeds in the greenhouse.Abbreviations PVS plant vitrification solution - P preculture - LN liquid nitrogen - TTC triphenyl tetrazolium chloride - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - EG ethylene glycol - BSA bovine serum albumin     

Identification and genetic analysis of semidwarfism-related proteins in rice (Oryza sativa L.)

Summary By transferring a semidwarf gene (sd-1) from Taichung Native 1 into a tall Japanese cultivar, Norin 29, through seven backcrosses, a semidwarf near-isogenic line SC-TN1 was obtained. The proteins of the embryo in Norin 29 and SC-TN1 were separated by two-dimensional electrophoresis. Most of the proteins showed the same electrophoretic pattern. However, it was found that there was a difference in the appearance of two basic glycoproteins designated as SRP-1 and SRP-2. These proteins exhibited the same molecular mass, but different isoelectric points. Hybridization results indicated that a single locus controls SRP-1 and SRP-2 with codominant alleles. The gene symbol Srp was given to this locus, with alleles Srp-1 and Srp-2 responsible for SRP-1 and SRP-2, respectively. Srp-2 was found in all of the semidwarf cultivars and lines having sd-1, except a tall cultivar Tsaiyuan-chung. This finding suggests that Srp-2 may be closely linked with sd-1. The amounts of these proteins markedly increased after water absorption of the seed, suggesting that these proteins may be related to the early development of the plant.     

Role of Cysteine in Enhancing Androgenesis and Regeneration of Indica Rice (Oryza sativa L.)

The effects of amino acid cysteine to culture systems of microspore-derived callus induction as well as plantlet regeneration were studied. Isolated pollen along with anther walls of basmati cultivars, Pusa basmati 1, Basmati 370 and Basmati 386 were cultured in a medium based on N₆ salts supplemented with or without cysteine following pollen embedment in agarose. The induction and regeneration medium with cysteine gave twice as effective androgenesis and plantlet regeneration in recalcitrant basmati rice cultivars as compared with medium lacking cysteine. Unlike the highly responsive model systems, most of the indica cultivars responded rather poorly in anther culture. So the study may accelerate the introgression of desirable genes into basmati rice using anther culture as a breeding tool. Response of microspores in androgenesis, plant regeneration and albinism was genotype specific. Regeneration of Indica rice varieties remains a limiting factor for researchers undertaking transformation experiments.     

Simple dehydration treatment promotes plantlet regeneration of rice (Oryza sativa L.) callus

Summary To increase plantlet regeneration frequency, rice callus was dehydrated in a Petri dish with a single layer of filter paper prior to transfer to the regeneration medium. With a 24 h dehydration treatment, the regeneration frequency was increased to 47 %, while the regeneration frequency of the untreated control was less than 5 %. This relatively simple method provides an alternative method for improving the regeneration frequency of rice callus.     

Molecular cloning, expression analysis and chromosomal mapping of salt-responsive cDNAs in rice ( Oryza sativa L.)

By using differential display PCR (DD-PCR) technique, two salt-inducible and one salt-repressed cDNA fragments were isolated from rice. The three cDNA fragments were characterized respectively as partial sequence of rice S-adenosylmethionine decarboxylase (SAMDC) gene, a new member of translation elongation factor 1A gene (namedREF1 A), and a novel gene whose function is unknown (namedSRG1). The full-length cDNA of SAMDC gene (namedSAMDC1) was further isolated by RT-PCR approach and the deduced polypeptide was found to be homologous to SAMDC proteins of other plants, yeast and buman. Northern hybridization revealed that expression of SAMDCl and REFlA was induced, while SRGl was dramatically repressed, by salinity stress. Southern blot analysis demonstrated that SAMDCl and SRGl were present as a single copy gene in rice genome, whereas riceREF1 A gene was organized as a gene family. TheREF1 A,SAMDC1, andSRG1 genes were located on chromosome 3,4, and 6 respectively by RFLP mapping approach using ZYQ8/JX17 DH population and RFLP linkage maps. Project supported by the National “863” High-Technology Program.     

Purification and characterization of carbonic anhydrase of rice (Oryza sativa L.) expressed in Escherichia coli

Rice carbonic anhydrase (CA) was successfully expressed as a glutathione-S-transferase (GST) fusion protein in an Escherichia coli expression system. The optimal induction concentration of IPTG and growth temperature was found to be 1.0mM and 28 degrees C. To obtain milligram amounts of homogeneous active recombinant proteins, 150mM NaCl and Mg-ATP solution were used during the purification procedures. After improving the conditions of expression and the purification procedures, final yield of recombinant proteins was 1.3mg/g wet cell weight after enzymatic cleavage of the GST tag, and the molecular weight was about 29kDa. The purified protein had CO(2) hydration activity, and had no detectable esterase activity in vitro. Addition of zinc improved the CO(2) hydration activity of the rice CA produced by E. coli. The effects of acetazolamide (AZ) and the anions N3-, NO3-, I(-), Br(-), and Cl(-) on CO(2) hydration activity of CA were studied. AZ and N3- were found to be strong inhibitors of rice CA. The inhibitory activity of AZ and ions was in the order AZ>N3->NO3->I(-)>Br(-)>Cl(-).