Identification of a novel tillering dwarf mutant and fine mapping of the TDDL(T) gene in rice (Oryza sativa L.)

Rice plant architecture is an important agronomic trait that affects the grain yield. To understand the molecular mechanism that controls plant architecture, a tillering dwarf mutant with darker-green leaves derived from an indica cultivar IR64 treated with EMS is characterized. The mutant, designated as tddl(t), is nonallelic to the known tillering dwarf mutants. It is controlled by one recessive nuclear gene, TDDL(T), and grouped into the dn-type dwarfism according to Takeda’s definition. The dwarfism of ...     

Mapping and characterization of a tiller-spreading mutant<Emphasis Type="Italic">lazy-2</Emphasis> in rice

Tiller angle of rice is an important agronomic trait that contributes to breed new varieties with ideal architecture. In this study, we report mapping and characterization of a rice mutant defective in tiller angle. At the seedling stage, the newly developed tillers of the mutant plants grow with a large angle that leads to a “lazy“ phenotype at the mature stage. Genetic analysis indicates that this tillerspreading phenotype is controlled by one recessive gene that is allelic to a reported mutant la. Therefore, the mutant was named la-2 and la renamed la-1. To map and clone LA, we constructed a large mapping population. Genetic linkage analysis showed that the LA gene is located between 2 SSR markers RM202 and RM229. By using the 6 newly-developed molecular markers, the LA gene was placed within a 0.4 cM interval on chromosome 11, allowing us to clone LA and study the mechanism that controls rice tiller angle at the molecular level.     

Isolation and characterization of rl（t）, a gene that controls leaf rolling in rice

Moderate leaf rolling is one of the most important morphological traits in rice breeding for plant ideotype. Previous studies have shown that the rl（t） gene has a high breeding potential for developing hybrid-rice varieties with an ideal ideotype, because it leads to an appropriate leaf rolling index （LRI） of about 30 % in the heterozygous state, and had a positive effect on grain yield. In this study, we isolated rl（t） and performed a preliminary investigation of its function in regulating leaf rolling in rice. DNA sequencing identified a single base change （G to T） in the finely mapped region （11 kb） containing rl（t）, and this is located in 3′-untranslated region （3′-UTR） of the only predicted gene, Roc5 （Rice outermost cell-specific）. The expression level of Roc5 is significantly higher in the rl（t） mutant than in the wild-type. Using RNAi and overexpression analysis, we found that the expression level of Roc5 correlated with LRI and leaf bulliform area, and wasalso associated with leaf abaxial or adaxial rolling. These results confirmed that Roc5 controls leaf rolling in a dosagedependent manner. Bioinformatics analysis revealed a conserved 17-nt sequence （called the GU-rich element） in the 3′-UTR of HD-GL2 （Homeodomain-Glabra2） family genes including Roc5. Based on the model of this element in regulating mRNA stability in mammals, we speculate that the single nucleotide change in this element accounts for the higher expression level of Roc5 in the rl（t） mutant compared to the wild-type, which ultimately leads to adaxial rolling of the leaf. This discovery further enhances our knowledge of the molecular mechanisms underlying leaf rolling in rice.     

Genetic analysis and fine mapping of an enclosed panicle mutant esp2 in rice （Oryza sativa L.）

The phenomenon of panicle enclosure in rice is mainly caused by the shortening of uppermost internode.Elucidating the molecular mechanism of panicle enclosure will be helpful for solving the problem of panicle enclosure in male sterile lines and creating new germplasms in rice.We acquired a monogenic recessive enclosed panicle mutant,named as esp2 (enclosed shorter panicle 2),from the tissue culture progeny of indica rice cultivar Minghui-86.In the mutant,panicles were entirely enclosed by flag leaf sheaths and the uppermost internode was almost completely degenerated,but the other internodes did not have obvious changes in length.Genetic analysis indicated that the mutant phenotype was controlled by a recessive gene,which could be steadily inherited and was not affected by genetic background.Apparently,ESP2 is a key gene for the development of uppermost internode in rice.Using an F 2 population of a cross between esp2 and a japonica rice cultivar Xiushui-13 as well as SSR and InDel markers,we fine mapped ESP2 to a 14-kb region on the end of the short arm of chromosome 1.According to the rice genome sequence annotation,only one intact gene exists in this region,namely,a putative phosphatidylserine synthase gene.Sequencing analysis on the mutant and the wild type indicated that this gene was inserted by a 5287-bp retrotransposon sequence.Hence,we took this gene as a candidate of ESP2.The results of this study will facilitate the cloning and functional analysis of ESP2 gene.     

Genetic analysis and gene cloning of a triangular hull 1 (tri1) mutant in rice (Oryza sativa L.)

Grain shape and size are two key factors that determine rice yield and quality. In the present study, a rice triangular hull mutant (tri1) was obtained from the progeny of japonica rice variety Taipei 309 treated with 60Co γ-rays. Compared to the wild type, the tri1 mutant presents a triangular hull, and exhibits an increase in grain thickness and protein content, but with a slight decrease in plant height and grain weight. Genetic analysis indicated that the mutant phenotype was controlled by a recessive nuclear gene which is stably inherited. Using a map-based cloning strategy, we fine-mapped tri1 to a 47-kb region between the molecular markers CHR0122 and CHR0127 on the long arm of chromosome 1, and showed that it co-segregates with the molecular marker CHR0119. According to the rice genome sequence annotation there are six predicated genes within the mapped region. Sequencing analysis of the mutant and the wild type indicated that there was a deletion of an A nucleotide in exon 3 of the OsMADS32 gene, which could result in a downstream frameshift mutation and premature termination of the predicted polypeptide. Both semi-quantitative and real-time RT-PCR analyses showed that this gene expressed highly in young inflorescences, while expressed at very low levels in other tissues. These results implied that the OsMADS32 gene could be a candidate of TRI1. Taken together, the results of this study lay the foundation for further investigation into the molecular mechanisms regulating rice caryopsis development.     

Clustered spikelets 4, encoding a putative cytochrome P450 protein CYP724B1, is essential for rice panicle development

Rice （Oryza sativa L.） inflorescence （panicle） architecture is an important agronomic trait, serving as one of the determinants of rice yield. A number of genes related to panicle development have been cloned and functionally characterized so far. However, more information is needed for fully understanding of the mechanism underlying the panicle development. In the present study, we identified a clustered spikelets 4 （cl4） mutant in the 93-11 genetic background. Compared to its wild-type 93-11, cl4 mutant has a typical clustered spikelets phenotype with all primary branches clustered on the base of the main rachis and 2-3 abnormal spikelets clustered on the primary branches. Moreover, cl4 mutant also shows shorter plant height than that of the wild type. Map-based cloning strategy is per- formed to clone the CL4 gene. As a result, CL4 is demonstrated to encode a putative cytochrome P450 protein CYP724B1, which is involved in brassinosteroid biosynthesis. To confirm our mapping result, the CL4 RNAi transgenic plants are generated. And the transgenic plants also show similar phenotype as the cl4 mutant. These results provide strong evidence that CL4 plays an important role in rice panicle development as well as plant height regulation.     

Morphological and physiological analysis of narrow and striped leaf 1 （nsl1） mutant of rice （Oryza sativa L.） and the gene mapping

The shape and color of rice leaves are impor- tant agronomic traits that directly influence the proportion of sunlight energy utilization and ultimately affect the yield and quality. A new mutant exhibiting stable inheritance was identified as derived from ethyl methane sulfonate （EMS）-treated restorer Jinhui 10, tentatively named as narrow and striped leaf 1 （nsll）. The nsll displayed pale white leaves at the seeding stage and then white striped leaves in parallel to the main vein at the jointing stage. Meanwhile, its leaf blades are significantly narrower than the control group of Jinhui 10. The chloroplast structures of cells in the white striped area of the nsll mutant break down, and the photosynthetic pigments are significantly lower than that of the wild type. Moreover, fluorescence parameters, such as Fo, Fv/Fm, ФpsⅡ, qP, and ETR, in the nsll mutant are significantly lower than those of the wild type, and the photosynthetic efficiency is also significantly decreased. These changes in leaf color and shape, together with physiological changes in the nsll, result in smaller plant height and a decrease in the most important agro- nomic traits, such as the number of grains per panicle, grain weight, etc. Genetic analysis shows that the narrow and striped traits of the nsll mutant are controlled by a single recessive nuclear gene, which is located between InDel 16 and InDel 12 in chromosome 3. The physical distance is 204 kb. So far, no similar genes of such leaf color and shape in this area have been reported, This study has laid asolid foundation for the gene cloning and function analysis of NSL 1.     

Molecular cloning and characterization of a cotton phosphoenolpyruvate carboxylase gene

Phosphoenolpyruvate carboxylase （PEPC） plays diverse physiological functions during plant development. In this study, a new phos- phoenolpyruvate carboxylase gene GhPEPC2 is isolated from cotton （Gossypium hirsutum cv, zhongrnian 35） by RACE-PCR, The cloned cDNA of GhPEPC2 is 3364 bp in length, and has an open reading frame of 2913 bp, encoding for 971 putative amino acids with a calculated molecular mass of 110,6 kD and pI of 5,56. The deduced amino acid sequence of GhPEPC2 shares high similarity with other reported plant PEPCs, Southern blot analysis indicates that the cotton PEPC exists as a small gene family and the GhPEPC2 might have two copies in the cotton genome, The semi-quantitative RT-PCR reveals that GhPEPC2 constitutively expresses in all the tissues of cot- ton and accumulated highly in roots, flowers and embryos but relatively low in stems and fibers, In addition, the recombinant GhPEPC2 has been purified by expressing it in Escherichia coli and the catalytic properties of it were also investigated. The results showed that GhPEPC2 is a typical C3 PEPC with a higher Km （83,6 p.M） and lower Vmax （8,0 p.mol min^-1 mg^-1） compared with the C3 PEPCs previously reported.     

Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP （green fluorescent protein） fusion expression results indicate that Mtp 1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Amtpl mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.     

Genetic analysis and gene mapping of a narrow leaf mutant in rice ( Oryza sativa L.)

A narrow leaf mutant was obtained after T-DNA transformation conducted on a rice variety Zhonghua 11. Several abnormal morphological characteristics, including semi-dwarf, delayed flowering time, narrow and inward rolling leaves, and lower seed-setting, were observed. The rate of net photosynthesis (under saturate light) of flag leaves in the mutant was significantly lower than that of the wild type. Moreover, the leaf transpiration rate and stomatal conductance in the mutant flag leaf were lower than those of the wild type at the grain filling stage. It was found that the mutant phenotype was not caused by the T-DNA insertion. Genetic analysis showed that the mutant was controlled by a single recessive gene, designated as nal3(t). A genetic linkage map was constructed using a large F₂ mapping population derived from a cross between nal3(t) and an indica variety Longtefu B with 6 polymorphic markers on chromosome 12 identified from 366 SSR markers by the BAS method. Gene nal3(t) was mapped between the markers RM7018 and RM3331. Fine mapping of nal3(t) locus was conducted with 22 newly developed STS markers based on the sequence diversity around the region harboring nal3(t) between Nipponbare and 93–11, and nal3(t) was finally mapped to a 136-kb region between the STS markers NS10 and RH12-8. Supported by National High Technology Research and Development Program of China (863 Program) (Grant No. 2006AA10A102), National Natural Science Foundation of China (Grant No. 30600349) and Natural Science Foundation of Zhejiang Province (Grant No. Y306149)     

籼型多分蘖矮秆突变体的遗传分析及其相关生理特性

籼型多分蘖矮秆突变体“佳禾丛矮”(Jiahecongai,JHCA)是本研究室在进行成熟花粉辐照诱变育种过程中发现的.遗传分析表明,JHCA同时携带互不等位的半矮秆基因sd-1和另一个由核基因隐性突变造成的多分蘖矮秆基因,暂命名为xmd(t).从JHCA与野生型高秆品种“广场13”(GC13)的杂交后代中分离出只带xmd(t)且遗传稳定的单基因型突变品系“新佳丛”( Xinjiacong,XJC).为揭示xmd(t)突变所造成的多分蘖与茎秆矮化协同出现的机理,对XJC的相关生理特性进行了分析.全生育期去除分蘖芽的试验表明,突变体植株的矮化是由于或部分由于过多分蘖的发生所造成的,该突变体的实质是多分蘖突变体.显微分析和田间分蘖动态观察表明,突变体多分蘖特性的形成是由于分蘖芽发生更早、分糵级数多、分蘖节位更高,且分蘖持续时间更长所造成的.本研究也表明,多分蘖矮秆突变体是研究水稻分蘖分子机理的理想材料.     

Modified fiber qualities of the transgenic cotton expressing a silkworm fibroin gene

A silkworm gene for fibroin was introduced into the upland cotton WC line by Agrobacterium-mediated transformation. PCR detection for fibroin, nptII and gus genes, Kanamycin (Km)-resistance analysis and GUS-histochemical assay were conducted on 30 regenerated plants from 9 callus lines, and 17 positive plants were obtained by these 5 screening methods. By Km-resistance analysis and PCR for fibroin, 6 homozygous lines in T₃ were obtained. Southern blot and Northern bolt demonstrated that the fibroin gene was inserted into the genome of these 6 lines, stably inherited and expressed. Compared to the control, the surface structure of mature fiber in the 6 lines was significantly distorted and an increased number of convolution was observed by scanning electron microscopy (SEM). Fiber quality traits analysis indicated that fiber elongation of the 6 homozygous lines was all increased and fiber strength of 3 lines was enhanced. These results indicated that fibroin expression influenced cotton fiber structure and quality, suggesting that fibroin has great potential for improving cotton fiber quality by genetic engineering. Supported by National High Technology Research and Development Program of China (Grant No. 2006AA100105), Science & Technology Pillar Program of Jiangsu Province (Grant No. BE2008310) and Programme of Introducing Talents of Discipline to Universities (Grant No. B08025) Contributed equally to this work     

Magnetoelectric and converse magnetoelectric res ponses in TbxDy1-xFe2-y alloy ＆ Pb（Mg1/3Nb2/3）（1-x）TixO3 crystal laminated composites

Measured results of magnetoelectric （ME） and converse magnetoelectric （CME） effects of TbxDy1-xFe2-y/ Pb（Mg1/3Nb2/3）（1-x）TixO3/TbxDy1-xFe2-y （TD/PMNT/TD） and PMNT/TD/PMNT laminated composites are presented. ME effect was determined by measuring laminate voltage output under a Helmholtz-generated AC field biased by a DC field （0-1 kOe） （1Oe = 79.58 A/m）. The CME effect was measured by recording the voltage induced in a solenoid encompassing the ME sample while exposed to a DC bias field and PMNT layer driven by a 10 V AC source. The ME and CME responses in the two laminated structure are linear. The highest values of ME coefficients in TD/PMNT/TD and PMNT/TD/PMNT composites are 384 mV/Oe and 158 mV/Oe, respectively, while the highest values of CME coefficients in the two composites are 118 mG/V and 162 mG/V （1 G=10^-4 T）, respectively.     

Evolution of the serum resistance-associated SRA gene in African trypanosomes

Serum resistance-associated (SRA) protein, a protein unique for Trypanosoma brucei rhodesiense, is responsible for resistance of this parasite to the lysis by normal human serum (NHS) and is a vital molecular marker to distinguish this species from other African trypanosomes. We cloned and sequenced the SRA basic copy (SRAbc) gene from T. b. rhodesiense and related species and found that this gene is confined to the subgenus Trypanozoon. The average 82% identity among the sequenced SRAbc genes indicates that they may have a common origin and are highly conserved. Since SRAbc coexists in the T. b. rhodesiense genome with SRA, we propose that SRAbc might be the ‘donor VSG’, which after duplication became inserted into the expression site by recombination. Under natural selection, SRAbc could reform into SRA following mosaic formation. Supported by National Natural Science Foundation of China (Grant Nos. 30570245, 30670275), Changjiang Scholars and Innovative Research Team in University (Grant No. DPCKSCU/IRT0447), International Foundation for Science of Sweden (Grant No. B/4318-1), Grant Agency of the Czech Republic (Grant No. Z60220518) and Education Foundation of the Czech Republic (Grant No. 2B06129)     

Microcalorimetric investigation of effect of berberine alkaloids from Coptis chinensis Franch on intestinal diagnostic flora growth

The inhibitory effect of three berberine alkaloids (BAs) from Coptis chinensis Franch, a traditional Chinese medicinal (TCM) herb, on Bifidobacterium adolescentis growth was investigated by microcalorimetry. The power-time curves of B. adolescentis with and without BAs were acquired, meanwhile the extent and duration of inhibitory effect on the metabolism were evaluated by the growth rate constant (k), half inhibitory ratio (IC₅₀), maximum heat-output power (P _max), peak time of maximum heat-output power (t _p) and total heat production (Q _t). k, P _max and Q _t decreased, and t _p was prolonged with the increase of BAs concentration. The IC₅₀ of BAs is 806 μg/mL for berberine, 341 μg/mL for coptisine and 236 μg/mL for palmatine. The sequence of antimicrobial activity of BAs is berberine < coptisine < palmatine. Combined with previous studies, it could be shown that the sequences of antimicrobial activity of BAs on both Bacillus shigae and Escherichia coli are berberine > coptisine > palmatine. The structure-function relationship of BAs indicates that the functional group methylenedioxy or methoxyl at C2 and C3 might be the major group inducing the activities of BAs on E. coli and B. adolescentis. Meanwhile, the substituent groups at C2, C3, C9 and C10 almost have equal effect on B. shigae. Supported by the National Natural Science Foundation of China (Grant Nos. 30625042 and 30873385) and Key Projects of National Science & Technology Pillar Program of China in the 11th Five-Year Period (Grant No. 2007BAI40B05)     

Functional characterization and mapping of two MADS box genes from peach （Prunus persica）

Previously an AGAMOUS gene homologue PpMADS4 and a FRUITFULL gene homologue PpMADS6 were isolated from peach （Prunus persica）, and both genes were shown to express in the developing floral and fruits. To gain insight into their function, the two genes were constitutively expressed in Arabidopsis thaliana and their effects on plant growth and floral organ development were studied in this work. The transgenic plants all displayed early flowering and conversion of inflorescence to floral meristem. However, the two genes had different effects on the floral organ structures in A. thaliana. The transgenic plants overexpressing PpMADS4 displayed homeotic conversion of floral organs, and particularly the perianth abscission was inhibited. The plants overexpressing PpMADS6 showed early flowering, produced higher number of carpels, petals, and stamens than nontransgenic plants, and pod shatter was prevented; significantly, the transgenic plants yielded more than one siliques from a single flower. A SSR molecular marker was developed for PpMADS4, and it was then assigned into the G5 linkage group of Prunus sp. Both PpMADS4 and PpMADS6 genes were located at the same region in the G5 linkage group. Our results showed the potential application of these two MADS box genes for crop and fruit tree improvement.     

Development and characterization of interspecific hybrids between <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Subscript>O. latifolia</Subscript> by <Subscript>in situ</Subscript> hybridization

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization （GISH） and fluorescent in situ hybridization （FISH） analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase Ⅰ of the hybrid pollen mother cell, poor chromosome pairing was identified and most of the chromosomes were univalent, which resulted in complete male sterility in the hybrid.