Jun 2008, Volume 2 Issue 2
    

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  • BAI Yunfeng, YANG Hongchun, QU Lin, ZHENG Jun, ZHANG Jinpeng, WANG Maoyan, XIE Wan, ZHOU Xiaomei, WANG Guoying
    RNA silencing is a post-transcriptional gene-silencing phenomenon induced by double-stranded RNA (dsRNA). In an attempt to generate dsRNA-mediated transgenic maize plants resistant to sugarcane mosaic virus (SCMV), we cloned SCMV NIb gene-specific sequences and inserted it into the binary vector p3301 in the sense and antisense orientations (named SCMVirNIb), which could produce RNAs capable of duplex formation in plant cells. Maize immature embryos were co-cultured with Agrobacterium carrying two vectors, one marker-free vector harboring the SCMVirNIb and one vector harboring bar gene as the selective marker. Resistant calli were recovered by selection on medium containing Biolaphos. Among the regenerated plantlets from resistant calli, 14 plants have been certified to contain SCMVirNIb by PCR amplification and DNA dot blot. T1 plants derived from the 14 plants were challenged in a greenhouse with SCMV inoculums and the percentages of resistant plants in 11 T1 lines were higher than 60%. One plant in the T1 line was found to carry SCMVirNIb without bar gene by PCR assay.T2 plants derived from T1 SCMV resistant transgenic plants were challenged with SCMV inoculums in field. The percentages of resistant plants from 3 lines, including the line derived from the marker-free transgenic plant, were higher than 85%. The non-transgenic control plants were all susceptible. Further molecular analysis confirmed that the resistant plants from the marker-free transgenic line contained SCMVirNIb but not the bar gene.
  • YIN Jing, WANG Guangjin, MA Fengming, ZHANG Hongji, XIAO Jialei, SUN Yan, DIAO Yanling, HUANG Jinghua, GUO Qiang
    Wheat (Triticum aestivum L.) stem rust caused by Puccinia graminis f. sp. tritici is one of the main diseases of wheat worldwide. Wheat mutant line D51, which forms a highly susceptive cultivar ‘L6239’ to the three races notated and cultured with immature embryos, shows resistance to prevailing races 21C3CPH, 21C3CKH, and 21C3CTR of P. graminis f. sp. tritici in China. In this study, the number and the expression stages of the resistance genes in mutant D51 were studied using inoculation identification and microsatellite (SSR) marker analysis. Two F1 populations from the crosses of D51 × L6239 (60 individuals) and D51 × Chinese Spring (60 individuals), their F2 populations (185 and 175 individuals respectively) at the seedling stage, and one F2 population derived from the cross of D51 × L6239 (194 individuals) at the adult stage were inoculated with pathogen race 21C3CPH to test for resistance. All F1 individuals of the two crosses were immune to stem rust at both seedling and adult stages. The response pattern of the three F2 populations showed that the R:S segregation ratio was 3:1, suggesting that the stem rust resistance of D51 is controlled by a single dominant gene, and is expressed during the entire growth period. The identification of the stem rust resistance by the F3 progeny test confirmed the credibility of the F2 population test. Segregating populations and small population analyses were used to identify chromosomal regions and molecular markers linked to the gene by the SSR marker method. A total of 675 SSR markers and 185 individuals of the D51 × L6239 F2 population were used to search genetically linked markers to the target gene. Using Mapmaker 3.0 and Map-draw with Kosambi’s function and other options set at default values, molecular mapping revealed that the gene was located on chromosome 5DS, linked with and flanked by two SSR markers, Xgwm190 and Xwmc150, at 18.58 and 21.33 cM, respectively. It has been reported that only one stem rust resistant gene, Sr30, is located on the wheat chromosome 5DL, and that it has no resistance to 34C2MKK and 34C2MFK, while the parent L6239 of mutant D51 has no resistance to 21C3CPH, 21C3CTK and 21C3CTR, but has resistance to 34C2MKK and 34C2MFK. The results above indicate that the gene identified in the study might be a novel resistance gene to stem rust, tentatively designated as SrD51.
  • TIAN Feifei, ZHU Jiehua, WANG Shutong, ZHANG Weihong, SUN Mingqing, JIANG Jizhi
    Pieris rapae extract was sprayed on the surface of cucumber leaf to determine the induction of resistance to anthracnose. The enzyme activities of peroxidase (POD) and polyphenoloxidase (PPO) were detected on cucumber leaves after P. rapae extract induction and pathogen challenge. The results showed that the disease index of cucumber anthracnose was significantly decreased after the cucumber was induced with the P. rapae extract at a concentration of 5.0 mg·mL-1. The POD and PPO activities in foliar-applied P. rapae extract without pathogen inoculation (PETO) or with pathogen inoculation (PETI) were relatively higher than those with no-P. rapae extract treatment and without pathogen inoculation (CONO) or with pathogen inoculation (CONI), respectively. The results suggested that the increased levels of POD and PPO activities in PETO and PETI play an important role in the induction of resistance to cucumber anthracnose.
  • ZHANG Haina, GUO Chengjin, LI Cundong, XIAO Kai
    Superoxide dismutases (SODs) play an important role in catalyzing the conversion of O2- to H2O2, which can reduce the amount of harmful reactive oxygen specie (ROS) generated by the adverse environments, and alleviate the damage to plants. As one class of SODs, CuZnSODs have vital functions in preventing the ROS-generated cell damage and the death in aerobically growing organisms. In this study, two novel CuZnSOD genes in wheat, referred to TaSOD1.1 and TaSOD1.2 were identified, cloned and characterized. TaSOD1.1 and TaSOD1.2 were 780 bp and 1121 bp, respectively, predicting all to encode 201 amino acids. A 45-aa length of transit peptide at the N-terminal and a 79-aa conserved CuZn-SOD domain were respectively located in TaSOD1.1 and TaSOD1.2. Phylogenetic analysis indicated that the query SODs, most of CuZnSODs, could be classified into four subgroups. Compared with the control (CK), the abundance of TaSOD1.1 transcripts did not change under drought, salt, low and high temperature conditions, but the TaSOD1.2 transcripts were strongly induced by the above abiotic stresses, which was in accordance with the elevated SOD activities in leaves in the above stress treatments to some extent, suggesting its involvement in the plant’s acclimation and tolerance to the above abiotic stresses by possibly reducing the amount of the harmful ROS from enhancement of the SOD activity.
  • ZHAO Sha, YAO Jialing
    A systematic cytological comparison of the anther development of photoperiod sensitive genic male sterile (PSGMS) rice with its normal fertility counterpart was conducted. The results showed that pollen abortion in PSGMS rice occurred first no later than the pollen mother cell (PMC) stage and continued during the entire process of pollen development till pollen degradation. This abortive process was closely associated with the abnormal behavior of tapetum. Although tapetum degeneration in the PSGMS rice initiated as early as at the PMC stage, it proceeded slowly and did not complete until the breakdown of the pollen, in sharp contrast to the rapid disintegration of the tapetal layer during the late microspore to the bicellular pollen stage in the fertile rice. Such cytological observation was supported by the results of the TUNEL (TdT2 mediated dU TP Nick End Labeling) assay that detects DNA fragmentation resulting from programmed cell death (PCD), indicating that the tapetum degeneration occurs in the process of PCD.
  • YE Xingguo, QIN Hua
    Obtaining marker-free plants with high efficiency will benefit the environmental release of transgenic crops. To achieve this point, a binary vector with three T-DNAs was constructed using several mediate plasmids, in which one copy of BAR gene expression cassette and two copies of VIP1 gene expression cassette were included. EHA101 Agrobacterium strain harboring the final construct was applied to transform soybean cotyledon nodes. Through 2–3 months regeneration and selection with 3–5 mg·L-1 glufosinate, transgenic soybean plants were obtained at 0.83%–3.16%, and the co-transformation efficiency of both genes in the same individual reached up to 86.4%, based on the southern blot test. Using PCR analysis, southern blot and northern blot tests, as well as leaf painting of herbicide in T1 progenies, 41 plants were eliminated of BAR gene with the frequency of 7.6%. Among the T1 populations tested, the loss of the alien genes happened in 22.7% lines, the silence of the BAR gene took place in 27.3% lines, and VIP1 gene silence existed in 37.1% marker-free plants. The results also suggested that the plasmid with three T-DNAs might be an ideal vector to generate marker-free genetically modified organisms.
  • CHEN Fabo, YANG Kecheng, RONG Tingzhao, PAN Guangtang
    In this study, analyses of phenotypic characters, SSR molecular markers and pedigrees were done to study the genetic diversity in 186 maize hybrids that were tested in regional trials in Sichuan and Southwest China. The results showed that there were differences in the variation coefficients of different characteristics, but all of the variation coefficients changed within a narrow range. Sixty pairs of simple sequence repeat (SSR) primer distributed on the ten chromosomes of maize produced stable amplified bands and 608 alleles were detected among the hybrids. The average number of alleles per locus was 10.1 ranging from 3 to 23. The values of polymorphism information content (PIC) for each SSR locus varied from 0.5179 to 0.9256 with an average of 0.7826. The genetic similarities of SSR marker pattern among the 186 hybrids ranged from 0.6067 to 0.9162, with an average of 0.7722. There were 16499 pairs of genetic similarity, in which 96.9% were 0.70000 to 0.9256. The cluster analysis showed that the hybrids could be classified into ten clusters, with 88.2% of the hybrids included in Cluster 4, Cluster 8 and Cluster 10. The analysis of pedigree sources of 51 hybrids showed that 36 hybrids had close genetic relationships with the hybrids developed by the Pioneer Company in the late 1980s and early 1990s in the United States, such as Y78599, Y7865 and Y78698, accounting for 70.58%. Meanwhile, 13 hybrids had close genetic relationships with Y78599, accounting for 8.66%. The genetic similarities of SSR marker pattern among the 51 hybrids ranged from 0.66192 to 0.8799, with an average of 0.7686. There were 1196 pairs of genetic similarity ranged between 0.7000 to 0.8796, accounting for 93.80% of all the genetic similarity pairs. The cluster analysis showed that 88.2% of the 51 hybrids were in Cluster 4, Cluster 8 and Cluster 10, which indicated that similarity was high and genetic diversity narrow among the 186 hybrids. This showed that it is necessary to broaden the genetic basis of breeding germplasm in maize.
  • LIU Kai, TANG Cheng, WANG Zhiqin, YANG Jianchang, YE Yuxiu
    The objectives of this study were to investigate ethylene and 1-aminocylopropane-1-carboxylic acid (ACC) in rice grains and root bleeding sap during the grain filling period and their relationship to the grain filling rate. Two high lodging-resistant rice (Oryza sativa L.) cultivars were grown in pots or tanks. Three treatments, including well watered (WW), moderate soil-drying (MD) and severe soil-drying (SD), were conducted from 9 days of post-anthesis until maturity. The effects of chemical regulators on the concentrations of ethylene and ACC in the grains were also studied. The results show that MD significantly increased the grain-filling rate and grain weight, whereas SD significantly reduced the grain-filling rate and grain weight. Concentrations of ethylene and ACC in the grains were very high at the early grain filling stage and then sharply decreased during the linear period of grain growth. MD reduced the ACC concentrations and ethylene evolution rate, whereas SD remarkably increased the ACC concentrations and ethylene evolution rate. Both the ethylene evolution rate in rice grains and the ACC concentrations in the root-bleeding sap were significantly and positively correlated with the ACC concentrations in rice grains. The ethylene evolution rate was significantly and negatively correlated with the grain-filling rate. The application of amino-ethoxyvinylglycine (AVG), an inhibitor of ethylene synthesis, at 9–13 days of post-anthesis significantly reduced the ACC concentrations and ethylene evolution rate of grains, but significantly enhanced the activities of sucrose synthase, ADP glucose pyrophosphorylase and soluble starch synthase. The results were reversed when ethephon, an ethylene-releasing agent, was applied. The results suggest that moderate soil drying during the grain-filling period in rice could inhibit the production of ethylene and ACC and therefore accelerate grain filling and increase grain weight.
  • SHI Yu, YU Zhenwen
    Application of nitrogen (N) fertilizer is one of the most important measures that increases grain yield and improves grain quality in winter wheat (Triticum aestivum L.) production. Presently, there is a large number of investigations (experiments) in the field on different nitrogen fertilizer application regimes. However, there still exists a serious problem of low nitrogen use efficiency, especially in winter wheat high yield conditions: unsuitable nitrogen fertilizer, which often leads to lower yield and large accumulation of nitrate in the soil, bringing a potential risk to the environment. In order to explore the optimal regime of nitrogen fertilizer application suitable for environment and economy, a field experiment on the different rate and ratio of base and topdressing of nitrogen fertilizer at the different growth periods of winter wheat was conducted. The field experiment was undertaken from the fall of 2003 to the summer of 2004 in the village of Zhongcun in Longkou city, in the Shandong Province of China. The field experiment with three repeats for each treatment was designed in a split-plot. The major plot was applied with urea at a nitrogen fertilizer rate of three levels, namely, 0 kg·hm-2 (CK), 168 kg·hm-2 (A), and 240 kg·hm-2 (B). In the sub-plot, the ratios of base and topdressing nitrogen fertilizer at the different development periods of wheat were 1/2:1/2 (A1 and B1), 1/3:2/3 (A2 and B2) and 0:1 (A3 and B3). Treatment B1 was under a regime used now in the local region. It was found that the amount of N accumulation in plants had no significant difference between treatments applied with nitrogen fertilizer. The grain yield and grain protein content were all elevated remarkably by applying nitrogen fertilizer compared with those of treatment CK. There was no significant difference in the grain yield and grain protein content between A2 and B2 and B3. However, when compared with those of B2 and B3, in A2 there was an increase in nitrogen use efficiency and residual soil NO3--N and N losses were reduced. Under the condition of the same rate of nitrogen fertilizer, increasing topdressing nitrogen rate clearly elevated the grain yield, grain protein content and nitrogen use efficiency. The results indicated that the residual soil NO3--N in A1 and B1 accumulated higher than that of CK in 80–160 cm soil layers at the jointing stage, but that of A2 had no significant difference compared with that of CK in 0–200 cm soil layers. At the maturity stage, more residual soil NO3--N was detected in B2, B3 and A3 than that in CK in 120–180 cm soil layers, which could not be absorbed by the roots of wheat, but led to be eluviated easily. The amount of soil NO3--N accumulation in treatment A2 had no significant difference compared with that of treatment CK in the 100–200 cm soil layer. In conclusion, A2, whose nitrogen fertilizer rate was 168 kg·hm-2 and the ratio of base and topdressing was 1/3:2/3, had a higher grain yield and grain protein content, and heightened N use efficiency and minimized the risk of NO3--N leaching. This should be one of the most appropriate nitrogen fertilizer application regimes in wheat production in local regions in China.
  • YUAN Kejun, LIU Qingzhong, LI Bo, ZHANG Lisi
    Primer pairs were designed to amplify the genomic DNA sequence of the alpha-farnesene synthase (AFS) gene by PCR. The PCR products were sequenced, spliced and compared to cDNA sequences in the GenBank (accession No. AY182241). The genomic sequence and intron-exon organization of the AFS gene were thus obtained. The AFS genomic sequence has been registered in the GenBank (accession No. DQ901739). It has 6 introns and 7 exons, encoding a protein of 576 amino acids. The sizes of the 6 introns were 108 bp, 113 bp, >1000 bp, 125 bp, 220 bp and 88 bp, and their phases were 0, 1, 2, 2, 0, 0, respectively. The sizes of the deduced amino acids of the 7 exons were 57, 89, 127, 73, 48, 83 and 99, respectively. The AFS protein contained three motifs: the RR(X8)W motif encoded by a sequence in exon 1, and the RxR motif and DDxxD motif encoded by two sequences in exon 4. After comparing the AFS genomic sequence (accession No. DQ901739) to the cDNA sequence (accession No. AY523409) in the GenBank, it was found that there were 6 single-nucleotide polymorphisms between the two sequences, four of which caused mutations at the amino acid level. Interestingly, one amino acid mutation (291R → G) was found in the RxR motif, and further investigation is needed to determine whether the alpha-farnesene synthesis ability and superficial scald susceptibility of apples are influenced by this amino acid mutation and other mutations.
  • ZHANG Maojun, LI Baojiang, WANG Qiang, DING Lihua, YAN Xingkai, XING Guojie
    To understand the mating compatibility of Pyrus ussuriensis Maxim., we studied the fertility of pollen and conducted a hand-pollination trial in the field on some pear varieties and wild types. The results showed that about 53% of varieties among 32 tested genotypes were male sterile. Not only did the pollen vitalities in normal varieties show distinct differences, but pollen vitalities from flower forcing in a glasshouse were found to be lower than those from natural flowering in the field, which had no apparent effect on fruit setting of tested varieties. Most of the tested genotypes such as Nanguoli, Pingxiangli, and Hanxiangli showed self-incompatibility (SI). Honghuagaili could bear fruit after hand pollination, but there were abnormal seeds in its fruits. So we suggested it was a recessive SI that happened during embryo development. Longxiangli has the capacity of self-compatibility (SC) to some extent, its fruit setting rate of inflorescence could reach 23.3%. Manual self-pollination during bud flowering could improve the fruit setting rate of part tested genotypes with SI, but had no effect on the fruit setting rate 3 days after flowering. Mating between female parents with the variety selected from F1 generation showed that the majority of their combinations were compatible. There was one-way SC when Nanguoli was crossed with Hanhongli, while no fruits could be found after Hanhongli was crossed with Nanguoli. It may be related to the S-genotype or haplotype of Nanguoli. In addition, mating between the varieties derived from bud mutation with the female parent appeared incompatible. We concluded that P. ussuriensis Maxim. is similar to other grown pear systems with the characteristics of SI, the fruit setting rate of self pollination in some varieties and wild types can be improved by artificial self-pollination during bud flowering, and fruit cannot be developed through pollination between the varieties from bud mutation and the female parent.
  • Yuecun MA, QIN Hongling, YIN Chunmei, GAO Wangsheng, SUI Peng, CHEN Yuanquan, LI Xiangdong, ZHANG Hongsheng
    The dynamics of soil water content under different tillage systems was studied throughout the growing period of oat (Avena sativa L.). The treatments included tillage system (zero tillage, minimum tillage, and conventional tillage), residue cover (with and without cover), and crop rotation (continuous cropping and crop rotation). The results indicated that soil water content and crop water use efficiency were improved under zero tillage with cover. When crop stubble was removed, soil water content under zero tillage was reduced, especially in the surface soil layer. Compared to conventional tillage, minimum tillage increased soil water content and its storage, either with cover or without cover. For all the three tillage treatments, soil water content with cover was significantly higher than that of without cover. Furthermore, soil water content and crop water use efficiency under crop rotation was consistently higher than continuous cropping. Therefore, it is concluded that minimum tillage with cover is the optimum management system in this area. At present, however, a combination of crop rotation and minimum tillage is a viable option, since there are not enough crop residues available for cover of land.
  • YANG Yajun, TAO Bu, ZHANG Weihong, ZHANG Jinlin
    In order to screen and isolate microorganisms capable of degrading nicosulfuron, five strains of microorganisms coded as YF1, YB1, YB2, YB3, and YB4 that can take nicosulfuron as the only source of carbon, nitrogen, and energy were obtained by enrichment culture. Of the five strains, YF1 was a fungus and the others were bacteria. All of the microorganisms were inoculated into the culture media with different concentrations of nicosulfuron and shaking culture was performed for 5 days at 30°C and 150 r·min-1. High Performance Liquid Chromatography (HPLC) was used to detect the concentration of nicosulfuron and calculate the degradation efficiency. The results showed that the degradation rates of the five strains of microorganisms were higher in low concentrations than in high concentrations of nicosulfuron. YF1 had the highest degradation rate of 80.31%, followed by YB1 and YB2 with degradation rates of 78.18% and 73.72%, respectively. However, YB3 and YB4 had lower degradation rates of 36.82% and 25.75%, respectively. Upon primary identification of the three strains of microorganisms with higher degradation rates, it was discovered that YF1 was Aspergillus niger, while YB1 and YB2 were Bacillus sp.
  • HOU Rong, LAI Songjia, ZHU Qing, ZHANG Ming, XIAN Hong
    Is it possible that Giant Panda (Ailuropoda melanoleuca) post-thawed sperm fertilization is improved with pentoxifylline (PF), platelet activated factor (PAF) and prostaglandin F 2-alpha (PGF2?)? In our study Giant Panda post-thawed sperm was incubated in Ham’s F-10 medium with different concentration of PF, PAF and PGF2? under 37°C. The effects of PF, PAF and PGF2? on Giant Panda post-thawed sperm fertility were evaluated through sperm motility, survival time, sperm membrane integrity, acrosome state and heterospecific egg penetration. The results were that PF, PAF and PGF2? all can affect Giant Panda post-thawed sperm in vitro fertilizing capability. In the experiment: 1 mg·mL-1 PF was most suitable for improving Giant Panda post-thawed sperm in vitro fertilizing capability. In the 1 mg·mL-1 PF group, sperm survival time was (15.33 ± 4.73) h, the heterospecific egg penetration was 51.44% after incubating for 4 hours, the heterospecific egg penetration was 7.49% after incubating for 6 hours. The results of the 1 mg·mL-1 PF group were significantly higher than those of the control group (P < 0.01). The results were also higher than those of the other treatment groups; Treated Giant Panda post-thawed sperm with 50 ng·mL-1 PAF had a better effect than 100 ng·mL-1 PAF, but the sperm fertilizing capability was damaged when incubation time exceeded 2 hours; 50 ng·mL-1 PGF2? had no significant effect on Giant Panda post-thawed sperm, but when the PGF2? treated concentration was increased, sperm in vitro fertilizing capability decreased because of the damaged motility and declined acrosomal reaction rate. The conclusions suggest that it is possible to improve post-thawed Giant Panda sperm fertility with 1 mg·mL-1 PF.
  • ZHANG Hao, WU Changxin, LING Yao, CHAMBA Yangzom, JI Suling
    Tibetan chicken is a precious resource in Qinghai-Tibet Plateau, China. In order to study its growth rhythm and heterosis of its hybrid, three groups comprising Tibetan chicken (T), Dwarf Recessive White (D) and Tibetan × Dwarf Recessive White (TD) were reared under the same management conditions at low and high altitudes. Body weight and shank length were measured, and growth curves were fitted using Richards model. The results showed that the model fitted well with the chickens’ growth courses in weight and shank with R2 at more than 0.99. The high-altitude might retard the growth of chickens, with decreases in inflection point values, final values and maximal growth rates, and prolongation of inflection point time. The final weight, inflection point weight, and maximal growth rates of Tibetan chicken were 1008.3 g, 477.9 g and 11.69 g respectively at low-altitude, and 525.3 g, 229.5 g, and 5.12 g at high-altitude. This showed a decline of 47.9%, 52.0%, and 56.8% at the high-altitude; the degree of influence of altitude was less than that of Dwarf Recessive White. The TD had high heterosis in weight gain, and at high-altitude the heterosis of final weight, inflection point weight, and maximal growth rates were 59.5%, 56.8% and 52.3% respectively. Therefore, the Tibetan × Dwarf Recessive White cross was good for improving the Tibetan chicken.
  • GU Youfang, WANG Bingyun, ZHANG Hongying, MAO Xinzhi, SHEN Yonglin
    To study the changes of IL-2 and TNF-? in serum and the IL-2 secreted by peripheral blood lymphocytes of goats infected with Fasciola hepatica, thirty six clinically healthy white goats were randomly divided into groups coded as I, II, III, every 12 goats. Goats in group I and II were inoculated with a single oral dose of 200 and 500 encysted Fasciola hepatica metacercaria, respectively, while those in group III were untreated controls. Blood samples were collected from the jugular vein once a week for 11 weeks. Changes in the serum levels of IL-2 and TNF-? and the ability of peripheral blood lymphocytes to secrete IL-2 were detected. Serum IL-2 levels decreased in group I, but increased in group II. The levels of TNF-? and secreted IL-2 were elevated by varying degrees in both groups I and II. Lymphocytes displayed reactivity during the first week after infection with a significantly increased response to both specific and non-specific challenges. TNF-? may be involved in the hepatic injury process caused by Fasciola hepatica. The infective dose of Fasciola hepatica greatly influenced IL-2 levels in the host playing a vital role in the initiation of host defense.