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28 December 2007, Volume 14 Issue 4 Previous Issue    Next Issue
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Research Paper

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Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture ZHU Li, FU Ya-ping, LIU Wen-zhen, HU Guo-cheng, SI Hua-min, TANG Ke-xuan, SUN Zong-xiu 2007, 14(4): 239-246 .  Abstract ( )   PDF (382KB) ( )   The ‘double T-DNA’ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene (hpt) in one T-DNA region and three target genes (hLF, SB401, RZ10) in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

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Construction and Testing of a Primary Microsatellite Database of Major Rice Varieties in China YING Jie-zheng, SHI Yong-feng, E Zhi-guo, ZENG Rui-zhen, CHEN Jie, ZHU Zhi-wei, ZHUANG Jie-yun 2007, 14(4): 247-255 .  Abstract ( )   PDF (380KB) ( )   Sixty-three major inbred varieties and parental lines of major F1 hybrids used in the commercial rice production in China were assayed with rice microsatellites screened in a previous study and additional microsatellites on four chromosomes. A set of 24 markers was selected and proposed for its application in the variety identification of rice, which are distributed on all the 12 rice chromosomes with 2 markers on each chromosome. The 63 major varieties and parental lines, as well as 41 major F1 hybrids, were genotyped with the markers. Alleles detected in each line at each marker locus were verified. By matching marker genotypes of corresponding F1, maternal and paternal lines of hybrid rice, high reliability of the maternal lines was verified, data on the paternal lines were modified, and a false hybrid was removed. A database containing genotype data of 103 major rice varieties and parental lines at the 24 marker loci was constructed and analyzed.

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RiceDB: A Web-Based Integrated Database for Annotating Rice Microarray HE Fei, SHI Qing-yun, CHEN Ming, WU Ping 2007, 14(4): 256-264 .  Abstract ( )   PDF (105KB) ( )   RiceDB, a web-based integrated database to annotate rice microarray in various biological contexts was developed. It is composed of eight modules. RiceMap module archives the process of Affymetrix probe sets mapping to different databases about rice, and aims to the genes represented by a microarray set by retrieving annotation information via the identifier or accession number of every database; RiceGO module indicates the association between a microarray set and gene ontology (GO) categories; RiceKO module is used to annotate a microarray set based on the KEGG biochemical pathways; RiceDO module indicates the information of domain associated with a microarray set; RiceUP module is used to obtain promoter sequences for all genes represented by a microarray set; RiceMR module lists potential microRNA which regulated the genes represented by a microarray set; RiceCD and RiceGF are used to annotate the genes represented by a microarray set in the context of chromosome distribution and rice paralogous family distribution. The results of automatic annotation are mostly consistent with manual annotation. Biological interpretation of the microarray data is quickened by the help of RiceDB.

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Natural Homologous Triploidization and DNA Methylation in SARII-628, a Twin-seedling Line of Rice (Oryza sativa) PENG Hai, ZHANG Hong-yu, LI Yun, XU Pei-zhou, WANG Xu-dong, WU Xian-jun 2007, 14(4): 265-271 .  Abstract ( )   PDF (87KB) ( )   A total of five pairs of diploid-triploid twin-seedlings (a diploid seedling and a triploid seedling emerged from a grain) were selected out from 4500 pairs of seedlings from SARII-628, a twin-seedling rice line. SSR analysis indicated that no difference between the diploid seedling and corresponding triploid seedling in a twin-seedling was found at the 310 loci, indicating that there was no obvious change in DNA primary structure. A modified AFLP technique ‘MSAP (methylation-sensitive AFLP)’ was used to analyze methylation mutation. Although no methylation mutation was noted among the five diploids, 29 methylation mutation loci were found from the corresponding triploids. This suggested that methylation mutation happened rapidly on M0 generation after natural homologous triploidization. The mutations were classified into 10 types, including 3 increased types, 3 decreased types and 4 undecided types of methylation-degrees. The bands of 22 loci were sequenced and then those sequences were searched through website. The result showed that the methylation mutation involved into the whole rice genome and the 12 pairs of chromosomes. The mutation trend was site-related and there were different mutation loci for different triploids, which foretold that SARII-628 would have different evolution fates after natural homologous triploidization.

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Effects of 1,2,4-Trichlorobenzene and Mercury Ion Stress on Ca2+ Fluxion and Protein Phosphorylation in Rice GE Cai-lin, WANG Ze-gang, WAN Ding-zhen, DING Yan, WANG Yu-long, SHANG Qi, LUO Shi-shi, MA Fei 2007, 14(4): 272-282 .  Abstract ( )   PDF (176KB) ( )   The effects of 5 mg/L 1,2,4-trichlorobenzene (TCB) and 0.1 mmol/L mercury ion (Hg2+) stresses on Ca2+ fluxion and protein phosphorylation in rice seedlings were investigated by isotope exchange kinetics and in vitro phosphorylation assay. The Ca2+ absorption in rice leaves and Ca2+ transportation from roots to leaves were promoted significantly in response to Hg2+ and TCB treatments for 4-48 h. The Ca2+ absorption peaks presented in the leaves when the rice seedlings were exposed to Hg2+ for 8-12 h or to TCB for 12-24 h. Several Ca2+ absorption peaks presented in the roots during rice seedlings being exposed to Hg2+ and TCB, and the first Ca2+ absorption peak was at 8 h after being exposed to Hg2+ and TCB. The result of isotope exchange kinetic analysis confirmed that short-term (8 h) Hg2+ and TCB stresses caused Ca2+ channels or pumps located on plasmalemma to open transiently. The phosphorylation assay showed that short-term TCB stress enhanced protein phosphorylation in rice roots (TCB treatment for 4-8 h) and leaves (TCB treatment for 4-24 h), and short-term (4-8 h) Hg2+ stress also enhanced protein phosphorylation in rice leaves. The enhancement of protein phosphorylation in both roots and leaves corresponded with the first Ca2+ absorption peak, which confirmed that the enhancement of protein phosphorylation caused by TCB or Hg2+ stress might be partly triggered by the increases of cytosolic calcium. TCB treatment over 12 h inhibited protein phosphorylation in rice roots, which might be partly due to that TCB stress suppressed the protein kinase activity. Whereas, Hg2+ treatment inhibited protein phosphorylation in rice roots, and Hg2+ treatment over 12 h inhibited protein phosphorylation in rice leaves. This might be attributed to that not only the protein kinase activity, but also the expressions of phosphorylation proteins were restrained by Hg2+ stress.

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Characterization of Leaf Photosynthetic Properties for No-Tillage Rice CHEN Song, XIA Guo-mian , ZHAO Wei-ming, WU Fei-bo, ZHANG Guo-ping 2007, 14(4): 283-288 .  Abstract ( )   PDF (240KB) ( )   A study was conducted to determine the influence of no-tillage cultivation on leaf photosynthesis of rice plants under field conditions. Experiments with the treatments, no-tillage and conventional tillage were carried out at three locations (Jiaxing, Hangzhou, and Xiaoshan, Zhejiang Province, China) for two years (2005 and 2006). Grain yield was constant in Jiaxing, but slightly higher in Hangzhou and Xiaoshan under no-tillage cultivation than that under conventional cultivation. In comparison with the conventional cultivation, no-tillage cultivation showed less biomass accumulation before heading and higher capacity of matter production during grain filling. A significantly higher leaf net photosynthetic rate was observed for the plants under no-tillage than for those under conventional tillage. The fluorescence parameter (Fv/Fm) in leaf did not show any difference between the two cultivations. The effect of cultivation management on transpiration rate (Tr) and SPAD value of rice leaf was dependent on the location and year.

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Effects of Different Nitrogen Fertilizer Levels on Fe, Mn, Cu and Zn Concentrations in Shoot and Grain Quality in Rice (Oryza sativa) HAO Hu-lin, WEI You-zhang, YANG Xiao-e, FENG Ying, WU Chun-yong 2007, 14(4): 289-294 .  Abstract ( )   PDF (100KB) ( )   In a pot experiment, effects of N fertilizer application on the concentrations of Fe, Mn, Cu and Zn in shoot of rice and the quality of brown rice were studied. In the treatments with N fertilizer application, the concentrations of Fe, Mn, Cu and Zn in most parts of rice shoot increased compared with control (no N fertilizer application). This indicated that the transportation ability of microelements from root to shoot in rice was improved with N fertilizer application. Effect of N fertilizer on IR68144 was similar to that of on IR64, but the concentrations of the microelements in plant differed, suggesting that the characteristic expression of the two rice genotypes was not controlled by the amount of N fertilizer supplied. The concentrations of those microelements in brown rice increased at first and then decreased with increasing N fertilizer application, reaching the highest at 160 kg/ha, at which the Fe, Mn, Cu and Zn concentrations in brown rice increased by 28.96%, 41.34%, 58.31% and 16.0% for IR64, and by 22.16%, 13.75%, 8.75% and 20.21% for IR68144 compared with control, respectively. Moreover, N fertilizer promoted the accumulation of protein, decreased the accumulation of amylose in grain, and enhanced gel consistency of brown rice. These results indicate that appropriate N fertilizer management could increase micronutrient contents in grain and improve nutrition quality of rice.

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Changes in Activities of Three Enzymes Degrading Galactomannan During and Following Rice Seed Germination REN Yan-fang, HE Jun-yu, WANG Xiao-feng 2007, 14(4): 295-301 .  Abstract ( )   PDF (120KB) ( )   To investigate the relationships among β-mannanase, β-mannosidase and α-galactosidase required for degrading galactomannan in cell wall during and following rice seed germination, the activities of the three enzymes and the effects of ABA and GA3 on them were surveyed. The activities of β-mannosidase and α-galactosidase presented in dry and pre-germinated rice seeds, and increased slowly during and following germination. However, the activity of β-mannanase was detected only after germination. GA3 could promote the activities of β-mannanase and α-galactosidase. ABA had little effect on the activities of β-mannosidase and α-galactosidase, but it could seriously inhibit the activity of β-mannanase.

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Host Active Defense Responses Occur within 24 Hours after Pathogen Inoculation in the Rice Blast System WANG Zhong-hua, JIA Yu-lin, LIN Hui, Adair INTERN, Barbara VALENT, J. Neil RUTGER 2007, 14(4): 302-310 .  Abstract ( )   PDF (372KB) ( )   Phenotypical, cytological and molecular responses of rice to the fungus Magnaporthe grisea were studied using rice cultivars and lesion mimic plants. The cultivar Katy was susceptible to several virulent M. grisea isolates, and a Sekiguchi like-lesion mimic mutant of Katy (LmmKaty) showed enhanced resistance to these isolates. Lesion mimic phenotype of LmmKaty was rapidly induced by virulent M. grisea isolates or by avirulent ones only at high levels of inoculum. Autofluorescence (a sign of an active defense response) was visible under ultraviolet light 24 h after localized inoculation in the incompatible interaction, whereas, not evident in the compatible interaction. Autofluorescence was also observed in LmmKaty 20 h after pathogen inoculation, indicating that rapid cell death is a mechanism of LmmKaty to restrict pathogen invasion. Rapid accumulations of defense related (DR) gene transcripts, phenylalanine ammonia lyase and ß-glucanase, were observed beginning at 6 h and were obvious at 16 h and 24 h after inoculation in an incompatible interaction. Rapid transcript accumulations of PR-1 and chitinase had occurred by 24 h after inoculation in an incompatible interaction. Accumulations of these transcripts were delayed in the compatible interaction. These results indicate that host active defense responses occur 24 h after pathogen inoculation and that LmmKaty exhibits enhanced resistance to M. grisea. It is suggested that the autofluorescence and expression of the DR genes after heavy inoculation are important cytological and molecular markers respectively for early determination of the host response to M. grisea in the rice blast system.

Short Communication

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Difference of Phytic Acid Content and its Relation to Four Protein Composition Contents in Grains of Twenty-nine japonica Rice Varieties from Jiangsu and Zhejiang Provinces, China WU Wei, CHENG Fang-min, LIU Zheng-hui, WEI Ke-su 2007, 14(4): 311-314 .  Abstract ( )   PDF (132KB) ( )   Twenty-nine japonica rice varieties collected from Jiangsu and Zhejiang Provinces, China were planted in Hangzhou, China, to investigate the phytic acid content in brown rice and its frequency distribution as well as the correlation among the contents of phytic acid, total protein and four protein compositions in brown rice. The phytic acid content in brown rice ranged from 0.699% to 1.034%, with a mean of 0.868% for the 29 tested rice varieties. Xiushui series rice varieties generally exhibited lower phytic acid level than Wuyujing and Huai series rice varieties. A rough normal distribution, with a mean of 8.722%, was observed for the total protein contents in the tested varieties. Of the four protein compositions, the glutelin, globulin and albumin contents had larger coefficient of variation than the prolamin content, although the difference in prolamin content was genotype-dependent. No significant correlation was found between the phytic acid and four protein composition contents, whereas the total protein content was significantly and positively related to the glutelin content in brown rice.

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Discussion on Photoreceptor for Negative Phototropism in Rice Roots WANG Yue-xia, WANG Zhong, SUO Biao, GU Yun-jie, WANG Hui-hui, CHEN Yong-hui, DAI Yun-xia 2007, 14(4): 315-318 .  Abstract ( )   PDF (239KB) ( )   To properly explore the photoreceptor for the negative phototropism in rice (Oryza sativa L.) root, lights with different wavelengths were applied to investigate the effect of light quality on phototropic bending. The phototropic bending could be induced prominently by blue/ultraviolet light, whereas not by red or far-red light. The absorption spectrum of the extracted solution from rice root cap had two peaks at 350 nm and 450 nm, respectively, and the molecular weight of the 120 kD protein in the root cap under unilateral light was larger than that under the dark. It suggested that the blue light receptor might be the photoreceptor for the negative phototropism in rice root.