Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

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Molecular characterization and roles of AP2 transcription factors on drought tolerance in plants
Jincai LI, Yongsheng ZHANG, Juntao GU, Chengjin GUO, Shumin WEN, Guiru LIU, Kai XIAO
Front Agric Chin    2011, 5 (4): 463-472.
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The APETALA2 (AP2) domain defines a large family of DNA binding proteins. It has been demonstrated that the AP2 proteins have important functions in the transcriptional regulation of a variety of biologic processes related to growth and development in various responses to drought and other abiotic stresses. In this essay, recent researches on the AP2 transcription factors, such as the molecular characterization, expression patterns in responses to drought and other abiotic stresses, the roles of ABA on drought responding which were mediated by AP2 transcription factors, transcription regulation mechanisms, and the roles of overexpression of AP2 transcription factor on plant drought tolerance, etc. have been overviewed. Deepening the understanding of signaling and the corresponding transduction pathways that are initiated via drought stress stimuli will play crucial roles for providing the theoretical basis for variety breeding with promising drought tolerance in the future.

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Comparative mapping of QTLs for H+ secretion of root in maize (Zea mays L.) and cross phosphorus levels on two growth stages
Junyi CHEN, Li XU
Front Agric Chin    2011, 5 (3): 284-290.
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H+ is a root secretion that affects P acquisition and P-use efficiency (PUE) under deficient phosphorus in maize. The secretion of H+, difference value of H+ between deficient and normal phosphorus (DH), and relative H+ (RH) as well as the quantitative trait loci (QTLs) associated with these traits were determined for a F2:3 population derived from the cross of two contrasting maize (BoldItalic L.) genotypes, 082 and Ye107. By using composite interval mapping (CIM), a total of 14, 8, and 9 distinct QTLs were identified for H+, DH, and RH, respectively. Most loci of QTLs for traits H+, DH, and RH had different cross environments. It showed that H+ secretion possessed an environment-sensitive and multi-gene nature. The gene × environment interaction was actually reflected by H+ secretion. One region for QTL of trait H+ was detected at the interval of bnlg2228-bnlg100 (bin 1.08) on chromosome 1. Coincident QTLs in the important genomic region reflected the cross phosphorus levels, different cross growth stages, and two different cross environments. The QTL explained 10% to 14% total phenotypic variance of H+. Therefore, the above segment (bnlg2228-bnlg100) (bin 1.08) identified on chromosome 1 may be used in the future for MAS to improve the phosphorus efficiency in maize.

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Assessment of genetic diversity in glandless cotton germplasm resources by using agronomic traits and molecular markers
LI Zhikun, WANG Xingfen, ZHANG Yan, ZHANG Guiyin, WU Liqiang, CHI Jina, Zhiying MA
Front. Agric. China    2008, 2 (3): 245-252.
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Seventy-one glandless cotton germplasm resources were firstly evaluated genetically by using nine agronomic traits, 33 simple sequence repeat (SSR) primers and ten amplified fragment length polymorphism (AFLP) primer combinations. Principal component analysis (PCA) of the agronomic traits showed that the first six principal components (PCs) explained a total of 86.352% of the phenotypic variation. A total of 329 alleles were amplified for 33 SSR primers, and 232 polymorphic bands in a total of 389 bands were obtained by using ten AFLP primer combinations. The average polymorphic information content (PIC) value was 0.80 and 0.18 for SSR primers and AFLP primer combinations, respectively. The DIST (average taxonomic distance) and DICE (Nei and Li’s pairwise distance) coefficients ranged from 0.373 to 3.164 and 0.786 to 0.948, respectively, for agronomic traits and SSR&AFLP data based on UPGMA analysis. This suggested that there was a higher diversity in the evaluated population for both agronomic traits and molecular markers. The Mantel’s test showed that the correlation between the dendrograms based on agronomic traits and SSR&AFLP data was non-significant.
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Effects of drought on soluble protein content and protective enzyme system in cotton leaves
Dongxiao LI, Cundong LI, Hongchun SUN, Wenxin WANG, Liantao LIU, Yongjiang ZHANG,
Front. Agric. China    2010, 4 (1): 56-62.
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The effects of soil drought on soluble protein content and protective enzyme system of cotton leaves at different positions were studied in the transgenic cotton cultivar, Lumianyan28. The results indicated that the soluble protein content in main stem leaves and in middle-fruit branch leaves under drought treatment were higher than that of CK, the normal soil water management treatment. Lower fruit branch leaves under drought treatment had higher protein and enzyme levels than CK treatments. From June 25 to July 22, the activity of superoxide dismutase (SOD) in main stem leaves under drought treatment was lower than that of CK. Other time periods had varying results. The peroxidase (POD) activity in main stem leaves and in fruit branch leaves was lower at early stage and then higher at late stage, showing a trend of descending first, and then ascending obviously under the drought condition. The catalase (CAT) activity showed an increase-decrease trend, higher in main stem leaves under drought treatment than that of CK. However, it was opposite in the lower fruit branch leaves, and there were no significant differences between the two CAT treatments in the middle branch leaves. It is suggested that the soluble protein and cellular protection enzymes, such as superoxide dismutase, peroxidase activity, and catalase in main stem leaves and fruit branching leaves play important physiological functions in the early growth stage under drought stress.
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Identification, characterization and expression analysis of transcription factor () genes in rice ( L.)
CAO Yunfei, WANG Jiaojiao, GUO Li, XIAO Kai
Front. Agric. China    2008, 2 (3): 253-261.
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The acclimation of plants to cold, salt and dehydration is involved in the action of the transcription factor (CBF) cold-response pathway. In this paper, nineteen rice CBF genes, including seven previously released and twelve unpublished novels, were identified and characterized. The multi-members of rice CBFs (OsCBF1 to OsCBF12) were divergent at the nucleotide and amino acid level. Expression analysis shows that five novel rice CBF genes (OsCBF1, OsCBF2, OsCBF3, OsCBF8, and OsCBF9) responded to short-term (1 h or 3 h) stresses of low temperature, salt stress and dehydration. The transcripts of OsCBF2, OsCBF8 and OsCBF9 in the roots were rapidly elevated when the plants were exposed to low temperatures, suggesting that they were possibly involved in low temperature responses in rice plants. Meanwhile, the expression level of OsCBF2 in leaves was enhanced when exposed to salt stress of 1–3 h, implying that OsCBF2 functioned as a transduction component in the salt stress signal cascade. Various expression patterns in OsCBF1, OsCBF2, OsCBF3, OsCBF8, and OsCBF9 under low temperature, salt and drought conditions, together with the different expression patterns between roots and leaves for each of these indicated that every rice CBF gene has unique and non-redundant functions in the response to the abiotic stresses.
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Current progress on genetic interactions of rice with rice blast and sheath blight fungi
Yulin JIA, Guangjie LIU, Stefano COSTANZO, Seonghee LEE, Yuntao DAI
Front Agric Chin    2009, 3 (3): 231-239.
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Analysis of genetic interactions between rice and its pathogenic fungi Magnaporthe oryzae and Rhizoctonia solani should lead to a better understanding of molecular mechanisms of host resistance, and the improvement of strategies to manage rice blast and sheath blight diseases. Currently, dozens of rice resistance (R) genes against specific races of the blast fungus have been described. Among them, ten were molecularly characterized and some were widely used for breeding for genetic resistance. The Pi-ta gene was one of the best characterized rice R genes. Following the elucidation of its molecular structure, interaction, distribution, and evolution, user friendly DNA markers were developed from portions of the cloned genes to facilitate the incorporations of the Pi-ta mediated resistance into improved rice varieties using marker assisted selection (MAS). However, rice blast is still a major threat for stable rice production because of race change mutations occurring in rice fields, which often overcome added resistance based on single R genes, and these virulent races of M. oryzae pose a continued challenge for blast control. For sheath blight, progress has been made on the exploration of novel sources of resistance from wild rice relatives and indica rice cultivars. A major quantitative trait locus (QTL), named qSB9-2, was recently verified in several mapping populations with different phenotyping methods, including greenhouse methods. The ability to identify qSB9-2 using greenhouse methods should accelerate the efforts on the qSB9-2 fine mapping and positional cloning.

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Inheritance and QTL analysis of dough rheological parameters in wheat
Caiying ZHANG, Changhai DONG, Jun MA, Guijun YAN, Chunji LIU, Guangmin LI
Front Agric Chin    2011, 5 (1): 15-21.
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A RIL population from two Australian wheats, Lang and CSCR6, was employed to evaluate the genetic variation and to detect QTL associated with dough rheological characters based on DArT and SSR markers and two environmental experiments. It was showed that the higher variation existed in the RIL for dough rheological characters, and so did much more abundant selection potentials that lacked in Chinese current commercial varieties. Nine additive QTLs for dough rheological characters were identified. Of which those for water absorption (WA) were located on chromosome 2A and 5A, stability time (ST) on 4B and 1B, breaking time (BT) on 1B, degree of softening (DS) on 1B, band width (BW) on 2B (two loci), evaluation value (EV) on 1B. And seven epistatic QTLs were screened out, and non-significant variance was found for the interaction between these epistatic QTLs and the environment. Correlation analysis indicated that there was a significantly positive relation between WA and development time (DT), and EV, whereas negatively related to BW. A significantly positive relation existed between DT, ST, BT and EV each other. They were negatively related to mixing tolerance index (MTI) and degree of softening (DS), both had a markedly positive relation.

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Construction and transformation for the antisense expression vector of the polyphenol oxidase gene in Yali pear
Guiqin LI, Jing QI, Yuxing ZHANG, Zhihua GAO, Dongqian XU, Huixuan LI, Chenmin HUO
Front Agric Chin    2011, 5 (1): 40-44.
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To inhibit the browning process in fruits of Yali pear, in this paper, antisense gene techniques were used to reduce the expression of BoldItalic gene. A cDNA fragment of 450 bp, which is located at the 3′ terminal of the polyphenol oxidase (BoldItalic) gene, was amplified from Yali pear using the RT-PCR method, then the antisense expression vector was constructed by inserting the fragment of the Yali pear BoldItalic gene between the CaMV promoter and NOS terminator of the expression vector pBI121 in a reverse orientation. After that, with the agrobacterium-mediated method, the BoldItalic antisense gene was transformed into Yali pear shoots. Northern blot analysis and enzyme activity assay showed that the PPO activities in the transgenic Yali pear shoots were significantly decreased, compared with the non-transformed Yali pear shoots. This lays a good foundation for breeding new varieties of pears with browning resistance in the future.

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Comparative analysis of genomes in Oryza sativa, O. officinalis and O. meyeriana with C0t-1 DNA and genomic DNA of cultivated rice
LAN Weizhen, HE Guangcun, WANG Chenyi, WU Shijun, QIN Rui
Front. Agric. China    2007, 1 (3): 237-242.
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Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) were applied to somatic chromosome preparations of Oryza sativa, O. officinalis/ and O. meyeriana with labeled probes of C0t-1 DNA and genomic DNA from cultivated rice. The coverage percentage (%) and size (Mb) of C0t-1 DNA in O. sativa, O. officinalis and O. meyeriana were 47.1±0.16, 38.61±0.13, 44.38±0.13 and 212.33±1.21, 269.42±0.89, 532.56±1.68, respectively. The coverage percentage and size of probe signals with gen omic DNA from O. sativa in O. officinalis and O. meyeriana were 91.0%, 93.6% and 634 Mb, 1 123 Mb respectively, in which there were 365 and 591 Mb in O. officinalis and O. meyeriana which came from O. sativa genomic DNA not from repetitive sequences of O. sativa, and the uncovered genome size in O. officinalis and O. meyeriana was 64 and 78 Mb, respectively. In addition, karyotype analysis was conducted based on the signal bands of C0t-1 DNA in O. sativa, O. officinalis and O. meyeriana. The results showed that highly and moderately repetitive sequences in Oryza genus were conserved as the functional genes during the evolution process. The repetitive sequence reduplication might be one of the important causes of genome enlargement in O. officinalis and O. meyeriana; the O. officinalis genome enlarged more slowly compared with O. meyeriana. Based on the above results, it is concluded that O. officinalis and O. meyeriana formed by reduplication, rearrangement and gene selective loss during the evolution process.
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Effects of salicylic acid (SA), ultraviolet radiation (UV-B and UV-C) on trans-resveratrol inducement in the skin of harvested grape berries
LI Xiaodong, ZHENG Xianbo, YAN Shutang
Front. Agric. China    2008, 2 (1): 77-81.
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Effects of salicylic acid (SA), ultraviolet radiation (UV-B and UV-C) on the trans-resveratrol (Res) inducement of the skin of harvested grape berries were studied with three grape cultivars Takasuma, Tano Red and Carigane. Split plot design tests were adopted to compare the effects of UV-B and UV-C radiation on Res inducement of different cultivars. Results showed that spraying 100 mg·L-1 SA markedly enhanced Res contents in the skins of harvested berries for the three selected cultivars. However, the effect of SA varied with the cultivars, and Res inducement by SA was more effective to Tano Red than Takasuma and Carigane. UV-B or UV-C irradiation significantly increased Res contents in grape skins and UV-C was more effective than UV-B. The effects of UV types and dosages on Res inducement depended upon cultivars. In the range of 0–3.6 kJ·m-2, the Res contents in the skins of the three grape cultivars were enhanced along with the increase of dosages of UV-B and UV-C.
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GC-MS analysis of the chemical constituents of the essential oil from the leaves of yacon (Smallanthus sonchifolia)
Jingwei LI, Jian LIU, Hai LAN, Mingmin ZHENG, Tingzhao RONG
Front Agric Chin    2009, 3 (1): 40-42.
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The essential oil from the leaves of yacon grown in China was isolated by hydrodistillation and distillation-extraction. Chemical constituents of the essential oil were separated and identified by means of gas chromatography/mass spectrometry (GC/MS) for the first time, and the relative content of each constituent was determined by area normalization. Twenty-one chemical constituents were identified, and their amounts accounted for 96.2% of the total composition. The main components of the essential oil were β-phellandrene (26.3%), β-cubebene (17.7%), β-caryophyllene (14.0%) and β-bourbonene (10.2%). Therefore, in the volatile oil from the leaves of yacon, sesquiterpenes are major compounds, accounting for 52.2%.

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Effects of plastic-film mulching and nitrogen application on forage-oriented maize in the agriculture-animal husbandry ecotone in North China
DU Xiong, BIAN Xiuju, YANG Fucun, ZHANG Lifeng, ZHANG Weihong
Front. Agric. China    2008, 2 (3): 266-273.
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To counter the actual problems of forage shortage and low quality existing in the agriculture–animal husbandry ecotone in North China, a research was conducted to study the effects of plastic-film mulching and nitrogen application on the production of forage-oriented maize with the aim of producing water-saving forage with high-yield and good quality. Field experiments combined with laboratory experimental estimation and analysis was adopted. Plastic-film mulching increased the dry biomass of forage-oriented maize by 23.8% with effectively improving the maize’s nitrogen absorption so that the apparent utilization ratio and output-input ratio of nitrogen were enhanced. The content of crude protein in maize plant was increased and thus, forage nutritive quality was improved. Plastic-film mulching remodeled the maize field water consumption scheduling pattern and increased the water use efficiency by over 10%. Nitrogen application to forage-oriented maize co-improved the biomass and the nutritive quality with the nutritive matter (percentage and yield) several times of the biomass. Nitrogen application increased maize biomass production by 36.1%–39.5% and it increased the contents of crude protein and crude fat in maize plant by 109% and 145%, respectively. The yields of the two nutritive matters increased by 160% and 210%. Nitrogen application at the rate of about 200 kg·hm-2 to the uncovered field and the rate less than 300 kg·hm-2 to the field with film mulching were considered as the most proper rates to guarantee high yield and good quality of forage-oriented maize and were the rates to keep the available nitrogen balanced in the soil. Plastic-film mulching and nitrogen fertilizer application to forage-oriented maize was an effective way of producing forage with high yield and good quality, relieving the shortage of animal forage and accelerating ecological recovery and economic development in this ecotone in North China.
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Study on the culture of cut plants in wheat haploid embryo induction by a wheat × maize cross
GU Jian, LIU Kun, LI Shaoxiang, TIAN Yuxian, YANG Hexian, YANG Mujun
Front. Agric. China    2008, 2 (4): 391-395.
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The wheat × maize system is one of the most effective ways to produce haploids in wheat. Whether and how it could be successfully applied in practical breeding mostly depends upon the efficiency of haploid embryo production. To perfect the protocols of haploid embryo induction, the efficiency of haploid embryo production between in vitro culture of cut plant and intact plant growth for hybrid spikes with two F1 wheat hybrids and two maize varieties was compared. Effects of different cutting plant times and formulas of nutrient solutions for cut plant culture on haploid embryo formation were also studied. Results indicated that the embryo rate of in vitro culture was 3.29 times that of intact plant growth, with the figures of 31.6% vs 9.6%, respectively. The optimal time for cut plant culture was 24 h after pollination. Formulas of nutrient solutions significantly affected the efficiency of haploid embryo induction. With an embryo rate of 0–35.5%, adding calcium phosphate in the culture solution at 3 g·L-1 could raise the caryopsis and embryo rates. According to this study, the best medium for cut plant culture was: 100 mgL-1 2,4-D + 40 gL-1 sucrose + 10 mgL-1 silver nitrate + 8 mLL-1 sulfurous acid + 3 gL-1 calcium phosphate, with which a caryopsis rate of 95% and an embryo rate of about 30% could be obtained.
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Comparative QTL mapping of resistance to sugarcane mosaic virus in maize based on bioinformatics
LÜ Xiangling, LI Xinhai, XIE Chuanxiao, HAO Zhuanfang, JI Hailian, SHI Liyu, ZHANG Shihuang
Front. Agric. China    2008, 2 (4): 365-371.
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The development of genomics and bioinformatics offers new tools for comparative gene mapping. In this paper, an integrated QTL map for sugarcane mosaic virus (SCMV) resistance in maize was constructed by compiling a total of 81 QTL loci available, using the Genetic Map IBM2 2005 Neighbors as reference. These 81 QTL loci were scattered on 7 chromosomes of maize, and most of them were clustered on chromosomes 3 and 6. By using the method of meta-analysis, we identified one “consensus QTL” on chromosome 3 covering a genetic distance of 6.44 cM, and two on chromosome 6 covering genetic distances of 16 cM and 27.48 cM, respectively. Four positional candidate resistant genes were identified within the “consensus QTL” on chromosome 3 via the strategy of comparative genomics. These results suggest that application of a combination of meta-analysis within a species with sequence homology comparison in a related model plant is an efficient approach to identify the major QTL and its candidate gene(s) for the target traits. The results of this study provide useful information for identifying and cloning the major gene(s) conferring resistance to SCMV in maize.
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Isolation and identification of the antagonistic strain DM-54 of Bacillus amyloliquefacien against Verticillium dahliae, and optimization of antifungal protein producing conditions
Ying ZHU, Shuna LI, Hongshui YUAN, Xiaojun GUO, Baocheng ZHU
Front Agric Chin    2009, 3 (1): 16-23.
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The strains capable of resistance against Verticillium dahliae Kleb were isolated and screened from the soils of cotton fields from several different provinces in China. A strain, coded DM-54, with a rather high antagonistic activity was obtained. Its morphological characteristics, physiological and biochemical properties and a 16 S rDNA sequence of this strain were further studied. The DM-54 strain was finally identified as a kind of Bacillusamyloliquefacien. Through a single factor experiment and an orthogonal experiment, the optimal shaking flask fermentation condition of strain DM-54 was found to be: media composed of 5% dextrin, 3% soy peptone, 0.02% MgSO4, 0.01% CaCl2, initial pH 7.0 and 10% inoculum volume, media volume 30/250 (mL/mL), fermentation temperature at 32oC, rotating speed 200 r?min-1, fermentation time of 48 h. Its antagonistic activity was distinguished to be elevated, at about 39.9%. Our research offers an effective means for the massive production of antagonistic proteins.

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Inheritance and molecular markers for the seed coat color in Brassica juncea
Mingli YAN, Zhongsong LIU, Chunyun GUAN, Sheyuan CHEN, Mouzhi YUAN, Xianjun LIU
Front Agric Chin    2009, 3 (1): 1-6.
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To elucidate the inheritance of seed coat color in Brassica juncea, Sichuan Yellow inbred (PY) was crossed with the Ziyejie inbred, and their F1, F2 and BC1 and BC2 progenies, derived from backcrossing to PY, were phenotyped for seed coat color. Results showed that the yellow seed coat was controlled by two independent recessive loci. Seven brown-seeded near-isogenic lines were developed by successive backcrosses to PY and by selfing. One of the BC6F2 populations segregated for a single locus controlling seed coat color was used for mapping. Using the 88 primer pairs from sequence-related amplified polymorphism and the 500 random primers, two markers were found to be linked to the gene for brown seed coat, which were designated as SCM57 and SCM1078. The crossover between these markers and the brown seed coat loci was 2.35% and 7.06%, respectively. A sequence characterized amplified region (SCAR) marker according to Negi et al. (2000), designated as SZ1-331, was found to be linked to the gene for brown seed coat, with a cross-over estimate of 2.35%. The markers were located on the same side of the brown seed coat loci and 2.41, 7.51 and 2.41 cM away from the brown seed coat locus. The seven brown-seeded near-isogenic lines were classified into two groups by three DNA markers. They were located at the same linkage group of the marker RA2-A11 previously published by Padmaja et al. (2005).

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Analysis of the apple fruit acid/low-acid trait by SSR markers
YAO Yuxin, ZHAI Heng, ZHAO Lingling, YI Kai, LIU Zhi, SONG Ye
Front. Agric. China    2008, 2 (4): 463-466.
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It is necessary to find out the genetic characteristics of malic acid in the course of apple genomic research and breeding. In this study, the SSR marker linked to the acid/low-acid trait in apple fruit was identified from 140 SSR primer pairs, using 91 F1 population hybrids from the intra-specific cross between apple cultivar ‘Dongguang’ and ‘Fuji’ as the experimental materials. Of 140 SSR primer pairs, only primer SDY085 produced a polymorphic band linked to acid trait, and the linkage distance was 8.89 cM. Also, the titrated acid and malic acid in different developmental stages were determined. The SSR marker analysis, coupled with the change of the total acid and malic acid contents, revealed that the acid/low-acid trait was governed by a major gene and acid trait was completely dominant.
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Gene cloning: exploring cotton functional genomics and genetic improvement
LIU Diqiu, ZHANG Xianlong
Front. Agric. China    2008, 2 (1): 1-9.
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Cotton is the most important natural fiber plant in the world. The genetic improvement of the quality of the cotton fiber and agricultural productivity is imperative under the situation of increasing consumption and rapid development of textile technology. Recently, the study of cotton molecular biology has progressed greatly. A lot of specifically or preferentially expressed cotton fiber genes were cloned and analyzed. On the other hand, identification of stress response genes expressed in cotton was performed by other research groups. The major stress factors were studied including the wilt pathogens Verticillium dahliae, Fusarium oxysporum f. sp. vasinfectum, bacterial blight, root-knot nematode, drought, and salt stress. What is more, a few genes related to the biosynthesis of gossypol, other sesquiterpene phytoalexins and the major seed oil fatty acids were isolated from cotton. In the present review, we focused on the major advances in cotton gene cloning and expression profiling in the recent years.
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Correlation between specific fine root length and mycorrhizal colonization of maize in different soil types
Wenke LIU
Front Agric Chin    2009, 3 (1): 13-15.
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A pot experiment was conducted in a glasshouse to investigate the correlation between specific fine root length (SFRL) and root colonization (RC) of maize inoculated with six arbuscular mycorrhizal fungi (AMF) in three soil types. The results showed that six AMF associated with maize presented different abilities in RC and effects on SFRL. In addition, there was a significant correlation between SFRL and RC of arbuscular mycorrhizal maize in Beijing soil (Cinnamon soil), but no significant correlation in Hubei soil (Brunisolic soil) and Guangdong soil (Red soil). It is concluded that mycorrhizal colonization decreased the SFRL of maize, and the correlation between SFRL and RC of mycorrhizal maize depended on soil type.

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Preliminary proteomics analysis of the total proteins of flower bud induction of apple trees
CAO Shangyin, GUO Junying, CHEN Yuling, XUE Huabai, ZHANG Qiuming, ZHU Zhiyong
Front. Agric. China    2008, 2 (4): 467-473.
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Apple is one of the most important fruit trees in the world. Nevertheless, mainly due to its long juvenile period, its breeding work constantly falls far behind other crops. So the aim of this study is to reveal the mechanism of apple flower bud differentiation, shorten the juvenile period and accelerate its breeding process. Proteomics technology (including two-dimensional gel electrophoresis (2-DE), biomass spectrometry and bioinformatics) was applied to work on the specific protein of flower bud and leaf bud after the brachyblasts of ‘Fuji’ stopped growth for 3–9 weeks. The results showed that the morphodifferentiation of flower bud did not begin until the seventh week after the brachyblast stopped growth. Furthermore, compared with the leaf bud, flower bud had significant changes in the expression of 283 protein spots in quality and quantity on 2-DE maps. Among the 283 protein spots, four protein spots (16.4, 30.2, 40.3 and 65.1 kD) were characteristic of the flower bud in the archae-stage (initial inflorescence appeared) at the beginning of flower-bud differentiation, three (39.3, 60.2 and 66.3 kD) in the post-stage (Lateral-flower appears) and one (77.1 kD) in the sepal stage on 2-DE maps. Analysis by peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry also identified and forecasted functionally by blasting different databases. In the four specific proteins, it was found that spots No. 256 (16.4 kD) and 298 (30.2 kD) were unknown proteins, spot Nos. 327 (40.3 kD) was identified as the synthesis enzyme protein and spot No. 367 (40.3 kD) was identified as a RNA-binding protein involved in transcription. When flower bud started to differentiate morphologically, we detected four specific proteins which were 16.4, 30.2, 40.3 and 65.1 kD. Three specific proteins 39.3, 60.2 and 66.3 kD were observed at side flower-appearing stage. When calyx began to emerge, there was one specific protein: 77.1 kD. The proteins 16.4 kD and 30.2 kD were two unknown specific proteins. The 40.3 kD protein was related to methionine synthetase while 65.1 kD protein, a RNA-binding protein, was related to transcription.
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Effects of different fertilization on microbial biomass carbon from the red soil in tea garden
XU Huaqin, XIAO Runlin, SONG Tongqing
Front. Agric. China    2008, 2 (4): 418-422.
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The present study examined the influence of the different fertilization on the dynamic of soil microbial biomass carbon (SMBC) of red soil in tea gardens. The results showed that straw mulching, intercropping, chemical fertilizer could all improve the amount of the soil microbial biomass C. The annual variation of microbial biomass C showed the tendency of “low–high–low–high”, and the influences were variable with the time. For the annual average of soil microbial biomass C, Treatment 1(T1) (straw mulching + 100% organic manure), Treatment 2 (T2) (straw mulching + 75% organic manure + 25% fertilizer), Treatment 3 (T3) (straw mulching + 50% organic manure + 50% fertilizer), Treatment 4 (T4) (straw mulching + 25% organic manure + 75% fertilizer), Treatment 5 (T5) (100% fertilizer),Treatment 6 (T6) (intercropping white clover) were 17.05%, 32.38%, 32.05%, 24.30%, 26.23%, 24.63% higher, respectively, than CK, and the differences among all the treatments were significant (P < 0.05). The correlation of the SMBC with the active organic matter, the total nitrogen, the microbial biomass N, the microbial biomass P were remarkable, but no significant correlation was found with available nitrogen, total phosphorus, total potassium and moisture. Compared with other treatments, those mixed with organic matter and chemical fertilizer were more advantageous to enhance the soil fertility.
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Protein extraction from rice (Oryza sativa L.) root for two-dimensional electrophresis
Xiaoliang XIANG, Shuju NING, Xia JIANG, Xiaogui GONG, Renlei ZHU, Lanfang ZHU, Daozhi WEI
Front Agric Chin    2010, 4 (4): 416-421.
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An efficient protein extraction method for two-dimensional gel electrophoresis (2-DE) from plant samples is usually challenging due to the low protein content and high level of interfering compounds. Proteomic analyses of rice (Oryza sativa L.) roots are limited by the lack of an efficient protein extraction method. To establish an effective protocol of protein extraction suitable for 2-DE analysis in rice roots, we evaluated three protein extraction methods (trichloroacetic acid [TCA]/acetone, Mg/NP-40/TCA, and tris-base/acetone). Our results showed that the Mg/NP-40/TCA extraction method had the highest protein yield and is the best resolution of protein separation among the three methods. The TCA/acetone method exhibited clear ?protein? profiles? and ?detected? more ?protein ?spots with the highest intensity in the region of high Mr (above 45 kDa) than the other methods. However, this method was unable to detect proteins with low-Mr (less than 24.0 kDa). The Tris-base/acetone method showed the poorest resolution of protein separation. Our results suggest that the Mg/NP-40/TCA method was the most effective among the three methods and may provide enhanced proteomic information for rice and other crop roots.

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Coordinate induction of antioxidant defense and glyoxalase system by exogenous proline and glycinebetaine is correlated with salt tolerance in mung bean
Mohammad Anwar HOSSAIN, Mirza HASANUZZAMAN, Masayuki FUJITA
Front Agric Chin    2011, 5 (1): 1-14.
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The purpose of this study was to assess the synergistic effects of exogenously applied proline and glycinebetaine (betaine) in antioxidant defense and methylglyoxal (MG) detoxification system in mung bean seedlings subjected to salt stress (200 mmol·L-1 NaCl, 48 h). Seven-day-old mung bean seedlings were exposed to salt stress after pre-treatment with proline or betaine. Salt stress caused a sharp increase in reduced glutathione (GSH) and oxidized glutathione (GSSG) content in leaves, while the GSH/GSSG ratio and ascorbate (AsA) content decreased significantly. The glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glyoxalase II (Gly II) activities were increased in response to salt stress, while the monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT) and glyoxalase I (Gly I) activities sharply decreased with an associated increase in hydrogen peroxide (H2O2) and lipid peroxidation level (MDA). Proline or betaine pre-treatment had little influence on non-enzymatic and enzymatic components as compared to those of the untreated control. However, proline or betaine pre-treated salt-stressed seedlings showed an increase in AsA, GSH content, GSH/GSSG ratio and maintained higher activities of APX, DHAR, GR, GST, GPX, CAT, Gly I and Gly II involved in ROS and MG detoxification system as compared to those of the untreated control and mostly also salt-stressed plants with a simultaneous decrease in GSSG content, H2O2 and MDA level. These results together with our previous results suggest that coordinate induction of antioxidant defense and glyoxalase system by proline and betaine rendered the plants tolerant to salinity-induced oxidative stress in a synergistic fashion.

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Primary studies on tissue culture from mature embryos in diploid and tetraploid wheat
BI Ruiming, WANG Honggang
Front. Agric. China    2008, 2 (3): 262-265.
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Using mature embryos (MEs) as the explants, the callus induction, embryogenic callus differentiation, plantlet regeneration and culture efficiency in diploid and tetraploid wheat of four genotypes were studied. The tested four genotypes wheat included cultivable emmer wheat (Triticum dicoccum Schuble), durum wheat (Triticum durum Desf.) and the common wheat progenitors Triticum dicoccoides and Triticum aegilopides. Results indicated that there were significant differences in the efficiency of callus induction, callus differentiation and plant regeneration among the tested genotypes. The efficiency of differentiation and regeneration shows strong genotype dependence. The rates of callus induction, embryogenic callus differentiation, plantlet regeneration and culture efficiency respectively were 95.00%, 90.00%, 32.40%, and 27.70% in cultivable emmer wheat, which were significantly higher than other tested genotypes. Therefore, this study has provided a basis for genetic transformation, gene cloning and molecular plant breeding in wheat and other related species.
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Improvement of megaprimer method for site-directed mutagenesis and its application to phytase
Haiqiang LU, Hongwei YU, Runfang GUO, Yingmin JIA
Front Agric Chin    2009, 3 (1): 43-46.
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Site-directed mutagenesis is used extensively for probing gene function. In this paper we describe an improved megaprimer method to the site-directed mutagenesis of phytase from Aspergillus niger, which allowed the mutations to be performed more efficiently in less time than other traditional methods. Three rounds of PCR and two pairs of primers were required in this method, and additionally, the restriction enzyme Dpn I was used for the elimination of template instead of the gel purification in this process. The entire procedure was performed in one tube. Moreover, this method was easier for obtaining large mutant genes than other methods. We successfully carried out the site-directed mutagenesis of phytase by adopting this method.

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Cloning of endo-β-glucanase I gene and expression in Pichia pastoris
Yu BAI, Runfang GUO, Hongwei YU, Long JIAO, Shuli DING, Yingmin JIA
Front Agric Chin    2011, 5 (2): 196-200.
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Total RNA of Thermoascus aurantiacus was isolated from its mycelium and acted as template for RT-PCR. The full-length cDNA encoding an endo-β-glucanase I was cloned via RACE-PCR method and the cDNA contained an ORF of 1005 bp encoding 305 amino acids. A recombinant plasmid, pPIC9k-egI, was constructed by inserting the ORF sequence of endo-β-glucanase I gene (egI) into the yeast expression vector pPIC9k and transformed to Pichia pastoris GS115. The results showed that the recombinant endo-β-glucanase I was excreted into the fermentation medium. The highest activity of endo-β-glucanase I and the protein content were up to 45.42 U/mL and 788.26 μg/mL at incubation time of 144 h. The optimal temperature and pH for the recombinant endo-β-glucanase I were found to be 70oC and 3.5, respectively.

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Some factors affecting the concentration of the aroma compound 2-acetyl-1-pyrroline in two fragrant rice cultivars grown in South China
Piebiep GOUFO, Meiyang DUAN, Xiangru TANG, Sugunya WONGPORNCHAI
Front. Agric. China    2010, 4 (1): 1-9.
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The fragrance potential of two fragrant rice (Oryza sativa L.) cultivars grown in South China was investigated in this study using headspace SPME and static headspace in conjunction with GC-MS. About a five-fold difference of 2-acetyl-1-pyrroline (2-AP) levels were observed among the two fragrant rice cultivars, with Guixiangzhan having the highest content (3.86&#8201;&#181;g&#183;g&#8722;1) comparable to that obtained with Thai KDML 105 rice. Other compounds instead of 2-AP were assumed to contribute to the characteristic aroma of Peizaruanxiang. The two cultivars were subjected to two preharvest treatments (planting density and harvest date) and different storage conditions (3 to 6 months at &#8722;4, 8, 20, and 30&#176;C). Results were discussed in terms of grain yield, milling quality, grain appearance, and amylose and protein contents of rice samples associated with differing treatments. Highest 2-AP concentrations were obtained for Guixiangzhan and Peizaruanxiang with lower planting densities, the earliest harvesting time of 10 days after heading, the shortest storage time of 3 months, and the lowest storage temperature of &#8722;4&#176;C. These findings indicate that manipulating pre- and postharvest treatments can greatly improve the specific attributes of the domestically produced cultivars. With that in mind, China could effectively increase its share of the domestic market of fragrant rice and even tap into the international market.
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The allelopathy of (L.) Kuntze, an invasive weed species
XU Jiao, XU Wenchao, YANG Yajun, TAO Bu, ZHANG Jinlin
Front. Agric. China    2008, 2 (4): 446-450.
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To identify the allelopathic effect of Flaveria bidentis (L.) Kuntze (F. bidenti) on other plants, the effects of different extracts from F. bidentis on the growth of several plants were studied by bioassay. Results showed that the water extracts inhibited the growth of corn (Zea mays L.), wheat (Triticum aestivum L.), cotton (Gassypium Hirsutum L.), soybean (Glycine hispida L.), peanut (Arachi shypogaea L.), rice (Oryza sativa L.), crabgrass (Digitaria sanguinalis L.) and rigweed (Amaranthus retroflexus L.), with the most reactive indexes found in root and stem of cotton at -0.85 and -0.88, respectively, at a concentration of 0.2 g·mL-1. However, the water extracts accelerated the growth of rice. In addition, the reactive indexes of the extracts of petroleum ether chloroform, ethyl acetate, acet and alcohol were higher than that of the water extracts, and that of the acet extracts was the highest. The melting point of the refined acetone extract ranged from 192.5°C to 193.5°C, and its maximum absorbing wavelength was 220 nm. This extract was found to be herbicide-active and played an inhibitory role in the growth of crabgrass and rigweed at concentrations of 1000, 500, 100 and 50 mg·L-1.
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Construction of a molecular map for melon ( L.) based on SRAP
WANG Jianshe, YAO Jianchun, LI Wei
Front. Agric. China    2008, 2 (4): 451-455.
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A molecular map of melon (Cucumis melo L.) was constructed with SRAP (Sequence-Related Amplified Polymorphism) markers using a population consisting of 114 F2 individuals derived from the cross of 4G21 (C. melo var. chinensis) and 3A832 (C. melo var. saccherinus). Twenty-nine primer pairs were used and 187 polymorphic loci were produced. The map consists of 12 linkage groups that include 152 genetic markers and cover 2077.1 cM with an average genetic distance of 13.67 cM. Every linkage group has 6–32 genetic markers with average genetic distance of 9.72–19.19 cM. The length of linkage group is 85.3–496.1 cM.
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Effects of phosphate fertilizer and manure on Chinese cabbage yield and soil phosphorus accumulation
LIAO Wenhua, LIU Jianling, Wang Xinjun, JIA Ke, MENG Na
Front. Agric. China    2008, 2 (3): 301-306.
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The yield response of Chinese cabbage to phosphate fertilizer and manure was studied. The effect of over-application of phosphate fertilizer and manure on plant total phosphorus content and phosphorus accumulation in soil was also investigated. The experiment was arranged in a plastic barrel in the field for two years. Application of phosphate fertilizer at the rates of 150–600 mg·kg-1 gave a yield increase of 14.9%–21.5% of Chinese cabbage. Application of manure at the rates of 33.3–133.2 g·kg-1 gave a yield increase of 18.2%–25.9% of the crop. There was no significant difference of yield response at the rates of 150, 300 and 600 mg·kg-1 phosphate fertilizer, and no significant yield response to the application of phosphate fertilizer after applying manure. The total P content in Chinese cabbage was increased gradually with the rate increase of phosphate fertilizer and manure. Phosphorus was absorbed luxuriously by the plant with over-application phosphate fertilizer and manure. The content of total-P, Olsen-P, water-soluble P, biological available P in the soil was increased with the rate of phosphate fertilizer and manure. Organic phosphorus in the soil was increased by the application of manure. Olsen-P had high correlations with water-soluble-P and biological available-P, but there was a poor relationship between Olsen-P and organic-P.
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