The serine carboxypeptidase like gene family of rice (Oryza sativa L. ssp. japonica)

Serine carboxypeptidases (SCPs) comprise a large family of protein hydrolyzing enzymes and have roles ranging from protein turnover and C-terminal processing to wound responses and xenobiotic metabolism. The proteins can be classified into three groups, namely carboxypeptidase I, II and III, based on their coding protein sequences and the fact that each family is characterized by a central catalytic domain of unique topology designated as the “α/β hydrolase fold”. The available SCP protein sequences have been utilized as datasets to build a HMM (hidden Markov model) profile, which is used to search the rice (Oryza sativa L. ssp. japonica) proteome. A total of 71 SCP and serine carboxypeptidase-like (SCPL) protein-coding genes exist in rice. The intron-exon structure, chromosome localization, expression and characteristics of encoded protein sequences of the 71 putative genes are reviewed.     

Daylength responses of various morphogenetic events after panicle initiation in late japonica rice (Oryza sativa L. ssp. japonica)

Plant morphogenesis is an intimate cooperation between environmental signals and a genetically-encoded developmental programme. Although all stages subsequent to floral induction may be influenced by daylength, the information available has come from diverse species during different morphogenetic events. This makes it difficult to investigate how photoperiodic signalling is integrated into various steps of the developmental programme. Here, we report that, in late japonica rice lines, morphogenetic events, including 12th leaf, axis of the main panicle, awn of the glum, anthers, elongation rate of internodes, and pollen fertility of the photoperiod-sensitive genic male-sterile (PGMS) rice, are significantly affected by daylength after panicle initiation. These data indicate that daylength affects many morphologic events, and that changes in morphology are mainly determined by the characteristics of the events themselves. These findings lay a foundation for future investigations into how potentially common photoperiodic signalling system(s) are integrated with diversified developmental events. In addition, we discussed the essential nature of PGMS rice.     

Heavy genetic load associated with the subspecific differentiation of japonica rice (Oryza sativa ssp. japonica L.)

Genetic load in the genome of the model species, rice, was genetically dissected by mapping quantitative trait loci (QTLs) affecting the radiosensitivity of 226 recombinant inbred lines (RILs) to gamma-ray- and spaceflight-induced radiation. The parents and RILs varied considerably in their radiosensitivity to gamma-ray irradiation. A total of 28 QTLs affecting the two index traits, seedling height (SH) and seed fertility (SF), of radiosensitivity were identified. The japonica parent, Lemont, was much more sensitive to gamma-ray irradiation than the indica parent, Teqing, and its alleles at almost all QTLs were associated with increased radiosensitivity, suggesting a much higher genetic load in the japonica genome of rice. Six QTLs (QSh2a, QSh2b, QSh5a, QSh7, QSf3b, and QSf10b) were located in the genomic regions particularly sensitive to radiation and thus might represent possible 'mutation hot spots' in the japonica genome. Detailed characterization of these genomic regions may shed light on the evolution and subspecific differentiation of rice.     

The Accumulation of Glycine Betaine Is Dependent on Choline Monooxygenase (OsCMO), Not on Phosphoethanolamine N-Methyltransferase (OsPEAMT1), in Rice (Oryza sativa L. ssp. japonica)

Glycine betaine (GB) is an important osmoprotectant, which improves plant tolerance to various abiotic stresses. In higher plants, GB is synthesized through two-step oxidations of choline, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Choline, the precursor of GB, is synthesized by phosphoethanolamine N-methyltransferase (PEAMT). Rice is known as a typical non-GB-accumulated species. However, the underlying mechanism related to GB accumulation remains elusive. Here, we determined whether the endogenous accumulation of choline is sufficient to GB biosynthesis in rice and whether the rice CMO protein has the function of oxidizing choline to generate betaine aldehyde. The results showed that overexpression of the rice PEAMT1 gene (OsPEAMT1) resulted in increased levels of choline, while GB content remained unchanged in the transgenic rice plants overexpressing OsPEAMT1. However, the intracellular GB level and the tolerance to salt stress of the transgenic lines overexpressing OsCMO were significantly enhanced. Immunoblotting analysis demonstrated that abundant functional OsCMO proteins with correct size were detected in OsCMO-overexpressing transgenic rice plants, but rarely accumulated in the wild type. Collectively, these results implicated that the endogenous accumulation level of choline is not the major factor leading to non-GB accumulation in rice. Instead, the defective expression of OsCMO resulted in non-GB accumulation.     

RNAi-directed downregulation of betaine aldehyde dehydrogenase 1 (OsBADH1) results in decreased stress tolerance and increased oxidative markers without affecting glycine betaine biosynthesis in rice (Oryza sativa)

Chemically inducible gene switches can provide precise control over gene expression, enabling more specific analyses of gene function and expanding the plant biotechnology toolkit beyond traditional constitutive expression systems. The alc gene expression system is one of the most promising chemically inducible gene switches in plants because of its potential in both fundamental research and commercial biotechnology applications. However, there are no published reports demonstrating that this versatile gene switch is functional in transgenic monocotyledonous plants, which include some of the most important agricultural crops. We found that the original alc gene switch was ineffective in the monocotyledonous plant sugar cane, and describe a modified alc system that is functional in this globally significant crop. A promoter consisting of tandem copies of the ethanol receptor inverted repeat binding site, in combination with a minimal promoter sequence, was sufficient to give enhanced sensitivity and significantly higher levels of ethanol inducible gene expression. A longer CaMV 35S minimal promoter than was used in the original alc gene switch also substantially improved ethanol inducibility. Treating the roots with ethanol effectively induced the modified alc system in sugar cane leaves and stem, while an aerial spray was relatively ineffective. The extension of this chemically inducible gene expression system to sugar cane opens the door to new opportunities for basic research and crop biotechnology.     

粳稻SRAP分子标记遗传群的构建与分析

用超级稻品种‘沈农606’和普通粳稻‘丽江新团黑谷’为亲本杂交获得的102份F_2代单株,通过SRAP分子标记遗传分析,构建了包含14个连锁群,由129个多态性位点组成的水稻连锁图谱,此图谱覆盖基因组长度1671.5 cM,平均图距13.0 cM。连锁群上有17.2%的多态性位点表现偏分离,偏分离标记在连锁群上存在热点区域。     

籼稻品种93-11同义密码子的使用偏性

利用籼稻品种93－11的全基因组序列及相应的EST数据,对影响同义密码子用法的若干因子进行了详细分析。指出93－11基因的表达水平（mRNA丰度）与3个同义密码子偏性指标CAI、CPP和ENC相关极显著（r=0.227^**,0.145^**和-0.147^**),表明高表达的基因其同义密码子非随机使用的程度越大;基因长度与CAI和CPP极显著负相关（r=-0.413^**和-0.480^**),与ENC极显著正相关（r=0.210^**),暗示较短的基因具有更高的转录活性;编码区G＋C含量对其同义密码子偏性的贡献率远高于mRNA丰度和基因长度,G＋C含量与CAI、CPP和ENC相关系数分别高达0.877^**,0.832^**和-0.740^**;起始编码区内A、T、C、G4种碱基呈明显的3周期振荡,尤以ATG下游第一个密码子所在的3个位点（＋4、＋5和＋6）偏置最强烈,由此认为在这3个特殊位点有较高的自然选择压存在;93－11中25个最优密码子的首次确定将对水稻转基因具有指导意义。     

光敏核不育水稻'农垦58S'精细胞的分离

文章对常用的渗透压冲击法作了改进,并从水稻成熟花粉粒中分离出大量精细胞。显微镜下的2个精细胞具有二型性,用显微操作仪从中分别挑选出2个细胞群体,并以荧光素二醋酸酯(FDA)染色的结果表明,分离的精细胞是活的。     

Proteomics characterization of different bran proteins between aromatic and nonaromatic rice (Oryza sativa L. ssp. indica)

Proteomic approach is applied for the analysis of seed brans of 14 rice varieties (Oryza sativa L. ssp. indica) which can classify to five aromatic rice and nine nonaromatic rice. The two-dimensional electrophoresis (2-DE) protein patterns for 14 rice varieties were similar within pH ranges of 3-10 and 4-7. To characterize aromatic group-specific proteins, we compared 2-D gels of aromatic rice to nonaromatic rice using PDQUEST image analysis. Four out of six differential spots were identified as hypothetical proteins, but one (SSP 7003) was identified by matrix assisted laser desoption/ionization-quardrupole-time of fight (MALDI-Q-TOF) as prolamin with three matching peptides based on NCBI database. Prolamin is a class of storage proteins with three different polypeptides of 10, 13, and 16 kDa. Spot SSP7003 was identified as a 13 kDa polypeptide of prolamin by combination of mass spectroscopy and N-terminal sequence analyses. In contrast, one sulfur-rich 16 kDa polypeptide of prolamin was found in extremely high intensity in brans of deep-water rice compared to nondeep-water rice. Our results suggest that proteomics is a powerful step to open the way for the identification of rice varieties.     

Gene flow from transgenic rice to red rice (Oryza sativa L.) in the field

In this study, we simulate a transgenic rice crop highly infested with red rice to examine transgene transfer from a transgenic line (A2504) resistant to glufosinate ammonium to cohabitant red rice. The red rice was sown along with the transgenic line at the highest density found in naturally infested crops in the region. Agricultural practices similar to those used to control red rice infestation in northern Italy rice fields were used to reproduce the local rice production system. During the first 2 years, the field was treated with herbicide at the appropriate time; in the first year the dosage of herbicide was three times the recommended amount. In this first year, detectable red rice plants that escaped herbicide treatment were manually removed. Nevertheless, two herbicide‐resistant hybrid plants (named 101 and 104) were identified in the experimental field during the second year of cultivation. Phenotypic and molecular characterisation suggests the hybrid nature of these two plants, deriving from crossing events involving A2504, respectively, with red rice (plant 101) and the buffer cultivar Gladio (plant 104). The progeny of two subsequent generations of the two plants were examined and the presence of the transgene detected, indicating stable transfer of the transgene across generations. In conclusion, despite control methods, red rice progeny tolerant to the herbicide can be expected following use of transgenic rice and, consequently, difficulties in controlling this weed with chemicals will emerge in a relatively short time.     

Genetics of amylose content in rice (Oryza sativa L)

Inheritance of amylose content was studied in crosses involving very low-, intermediate-, and high-amylose parents. The single-grain analysis of parents, F₁, F₂, B₁F₁, and B₂F₁ seed from a single-season harvest, showed that the parental mean difference of 14–17 % in IR37307-8/BPI 121-407 or IR37307-8/IR24632-34 and about 20% in the cross IR37307-8/IR8 were controlled by a single gene with major effect, along with some minor genes and/or modifiers. The appearance of segregants inbetween the two parents was attributed to gene dosage effects in the endosperm. The results indicate that selection for amylose level can effectively be done in early segregating generations. Selection for intermediary segregants would be ineffective because the dosage effects would dissipate in further generations.     

粳稻米粒延伸性的QTL剖析

考察粳稻品种‘沈农265’和‘丽江新团黑谷’为亲本的F23,群体及双亲的米粒延伸率相关性状,并用该群体的分子连锁图谱进行QTL分析,共检测到8个与稻米延伸性有关的QTL,包括1个米粒型QTL、1个饭粒型QTL、2个延伸率QTL、3个膨胀率QTL和1个延伸指数QTL,分别位于第1、2、6和11染色体,单个QTL对性状表型贡献率在15．4％-37．5％之间。与其他研究结果比较表明,主效QTL在不同群体和不同环境中的重演性较好,这些QTL受水稻籼粳分化这一演化过程的影响较小。     

Developmental Changes in the Free Amino Acid Pool of Rice(Oryza sativa L. subsp,japonica) Bmbryos

The level of/various amino acids in rice embryos rose sharply during embryo differentiation (7–9 days after anthesis). Then it increased steadily or tended to become stable at mid-maturation stage (13–18 days after anthesis), thereafter continuing to increase, except that the contents of aspartate and glutamate decreased significantly. The total free amino acid pool expanded rapidly during the differential stage. There after the pool capacity showed only a slightly increase until the end of embryogenesis. Both on embryo cell and dry weight bases, the capacity reached the maximum at the 9th day after anthesis, then decreased at the 13th day, and later remained stable. We deemed that the establishment of the free amino acid pool is one of the events which occur in the process of rice embryo differentiation. By the fulfillment of the differentiation (the 13th dray after anthesis), the pool capacity within the embryo cells remained stable on the whole. The free amino acid pool was dominated by serine, alanine, aspartate and glutamate during the differentiation stage. In the maturation stage, serine, alanine, arginine and lysine were the main components. These predominant; amino acids may play an important role in regulating the availability of the whole amino acid pool.     

Evidence for genomic changes in transgenic rice (Oryza sativa L.) recovered from protoplasts

The occurrence of genomic modifications in transgenic rice plants recovered from protoplasts and their transmission to the self-pollination progeny has been verfied with the random amplified polymorphic DNA (RAPD) approach. The plant was the Indica-type rice (Oryza sativa L.) cultivar Chinsurah Boro II. The analysed material was: (1) microspore-derived embryogenic rice cells grown in suspension culture, (2) transgenic plants recovered from protoplasts produced from the cultured cells and (3) the self-pollination progeny (two successive generations) of the transgenic plants. DNA purified from samples of these materials was PCR-amplified with different random oligonucleotide primers and the amplification products were analysed by agarose gel electrophoresis. Band polymorphism was scored and used in band-sharing analyses to produce a similarity matrix. Relationships among the analysed genomes were expressed in a dendrogram.The extensive DNA changes evidenced in cultured cells demonstrate the occurrence of somaclonal variation in the material used to produce protoplasts for gene transfer. Quantitatively reduced DNA changes were also found in the resulting transgenic plants and i their self-pollination progenies.While confirming the stability of the foreign gene in transgenic plants, this work gives molecular evidence for the occurrence of stable genomic changes in transgenic plants and points toin vitro cell culture as the causative agent. RAPDs are shown to be a convenient tool to detect and estimate the phenomenon at the molecular level. The methodology is also proposed as a fast tool to select those transgenic individuals that retain the most balanced genomic structure and to control the result of back-crosses planned to restore the original genome.     

Novel pleiotropic loci controlling panicle architecture across environments in japonica rice （Oryza sativa L.）

To identify quantitative trait loci (QTLs) controlling panicle architecture in japonica rice, a genetic map was constructed based on simple sequence repeat (SSR) markers and 254 recombinant inbred lines (RILs) derived from a cross between cultivars Xiushui 79 and C Bao. Seven panicle traits were investigated under three environments. Single marker analysis indicated that a total of 27 SSR markers were highly associated with panicle traits in all the three environments. Percentage of phenotypic variation explained by single locus varied from 2% to 35%. Based on the mixed linear model, a total of 40 additive QTLs for seven panicle traits were detected by composite interval mapping, explaining 1.2%-35% of phenotypic variation. Among the 9 QTLs with more than 10% of explained phenotypic variation, two QTLs were for the number of primary branches per panicle (NPB), two for panicle length (PL), two for spikelet density (SD), one for the number of secondary branches per panicle (NSB), one for secondary branch distribution density (SBD), and one for the number of spikelets per panicle (NS), respectively. qPLSD-9-1 and qPLSD-9-2 were novel pleiotropic loci, showing effects on PL and SD simultaneously. qPLSD-9-1 explained 34.7% of the phenotypic variation for PL and 25.4% of the phenotypic variation for SD, respec- tively. qPLSD-9-2 explained 34.9% and 24.4% of the phenotypic variation for PL and SD, respectively. The C Bao alleles at the both QTLs showed positive effects on PL, and the Xiushui 79 alleles at the both QTLs showed positive effects on SD. Genetic variation of panicle traits are mainly attributed to additive effects. QTL × environment interactions were not significant for additive QTLs and additive × additive QTL pairs.