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    16 March 2016, Volume 49 Issue 6
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Transcriptome Profiling of Lodicules Before Floret Opening in Oryza sativa L.
    FU Yong-qi, XIANG Miao-lian, JIANG Hai-yan, HE Yong-ming, ZENG Xiao-chun
    Scientia Agricultura Sinica. 2016, 49(6):  1017-1033.  doi:10.3864/j.issn.0578-1752.2016.06.001
    Abstract ( 607 )   HTML ( 5 )   PDF (1114KB) ( 1216 )   Save
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    【Objective】The rice floret opening will be driven by that a pair of lodicules lying between the lemma and the stamen base in the floret will expand rapidly when absorbing abundant water and lever away the rigid lemma. However, little is known about the molecular mechanism of the lodicules regulating the time of floret opening. With the development of sequencing technology, a faster and more effective way is provided to study the mechanism of the responses of lodicules to floret opening time at the whole level of cell.【Method】In this study, conventional indica rice germplasm Zhongzao 25 was used as the material and the transcriptomes of lodicules at 12 h and 1 h before floret opening were sequenced using Illumina sequencing technology. First, clean reads were mapped to the reference sequences of indica 9311 and the unique matched reads were obtained; second, the gene expression level was calculated by using RPKM method; Then, false discovery rate≤ 0.001 and the absolute value of Log2Ratio ≥ 1 were used as the threshold to judge the significance of gene expression differences. Finally, the functions and pathways of differentially expressed genes (DEGs) were annotated by comparing with the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) and Universal Protein Knowledgebase (UniProtKB).【Result】The expressed genes from the two transcriptomes of lodicules at 12 h and 1 h before floret opening were 26 369 and 26 157 respectively, and 3 924 differentially expressed genes (DEGs) were screened between the two transcriptomes. 2 623 DEGs were down-regulated and 1 301 DEGs were up-regulated genes. Moreover, change folds of 105 DEGs were more than 100 folds (|log2Ratio|≥6.7) of lodicules during the two stages before floret opening. Gene ontology enrichment analysis showed that 1 624 DEGs could be mapped as functional molecule, 21.7% DEGs of which had ion-binding activity, 7.5% DEGs of which had oxidoreductase activity and 5.7% DEGs of which had transporter activity. Meanwhile, 1 313 DEGs were involved in biological processes, 15.0% DEGs participated in localization processes, 12.4% DEGs participated in transport process, 4.7% DEGs participated in carbohydrate metabolic process and 4.5% DEGs participated in lipid metabolic process. KEGG pathway enrichment analysis revealed that 2 229 DEGs were involved in 123 different metabolic pathways,which enriched larger numbers of DEGs including biosynthesis of secondary metabolites (339 genes), plant hormone signal transduction (152 genes), nucleotide metabolism (146 genes), and starch and sucrose metabolism (64 genes). The functions of those genes, which were specifically expressed at 1 hour before floret opening and the unique match reads number of which exceeded 30, were mainly concerned in the regulation of physiological processes of cytoskeleton remodeling, membrane stability, energy metabolism, transcription regulation, and signal transduction. Jasmonic acid and analogs have a strong inductive effect on floret opening in rice, 16 DEGs were involved in jasmonic acid biosynthesis pathway and 11 DEGs were involved in jasmonic acid signal transduction pathway, whose expression levels increased significantly as the floret opening time approached.【Conclusion】The transcriptome data of rice lodicules was obtained using RNA sequencing technology. The expression profiling data of DEGs and molecular function of DEGs were also collected. The differential activation or suppression of key DEGs in energy metabolism, carbohydrates metabolism and translocation, cell walls structure modification and hormones metabolism and signaling pathway in lodicules suggested that these DEGs were closely related to water absorption and expansion of lodicules towards the regulating of the floret opening time.
    Molecular Cloning and Functional Analysis of Farnesyl Pyrophosphate Synthase Genes from Taraxacum kok-saghyz Rodin
    CAO Xin-wen, WANG Xiu-zhen, LI Yong-mei, ZHAO Li-jing, MA Hai-xia, YAN Jie
    Scientia Agricultura Sinica. 2016, 49(6):  1034-1046.  doi:10.3864/j.issn.0578-1752.2016.06.002
    Abstract ( 527 )   HTML ( 2 )   PDF (11207KB) ( 1900 )   Save
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    【Objective】 The Farnesyl pyrophosphate synthase (FPS) is a key enzyme in the mevalonate biosynthesis pathway of the crude rubber. The FPS was cloned from Taraxacum kok-saghyz Rodinand transformedinto the wild types, this study will lay a foundation for the research of regulating effects of FPS gene on T.kok-saghyz biosynthesis. 【Method】 Homologous cloning was used to clone the full-length cDNA of this gene, bioinformatic analysis was conducted. Multiple sequence alignment and phylogenetic tree analysis were performed using typical sequences of T.kok-saghyz and other 18 plants FPS proteins. The total RNA was extracted from root, petiole, leaf, peduncle, flower and seed to analysis tissue-specific expression of FPS gene in T.kok-saghyz. The comparation of relative expression levels of the FPS from different growth stages of T.kok-saghyz was processed to research the regulating role of TKFPS in different developing periods of T.kok-saghyz. And this gene was overexpressed in T.kok-saghyz, the expression level of TKFPS and rubber content changes were analyzed after the FPS overexpressed. The rubber was extracted from roots of the wild-type and transgenic strains with Alkali cooking method for determining rubber content. 【Result】 The full-length cDNA of the FPS from T.kok-saghyz was obtained (TkFPS, KJ558350.1). The ORF of 1029 bp encodes 342 amino acid residues with the predicted molecular weight of 39.35 kD, isoeletric point value of 5.41 and average hydrophobicity value of -0.242. Amino acid sequence analysis showed that it contains five conserved domains of FPS gene in higher plants. Phylogenetic tree analysis showed that the T.kok-saghyz had the closest evolutionary relationship with Herba Taraxaci. Subcellular localization analysis of TMHMM and TargetP indicated that it might be targeted to the cytoplasm without transmembrane region. TKFPS gene was expressed in the root, petiole, leaf, peduncle, flower and seed of T.kok-saghyz by the tissue-specificity expression. The expressive content of TKFPS gene in different tissues of T.kok-saghyz was different: the highest was leaf, the next was root, and the lowest level was in flower. Six transgenic strains were obtained after recombinant pCAMBIA2300-35S-TkFPS was transformed to wild T.kok-saghyz by Agrobacterium-mediated transformation. The fluorescent quantitative RT-PCR showed that the relative expression levels of the FPS in transgenic lines were significantly higher, with the increases up to 2.8 times over the wild types. On average, rubber assay results showed that rubber content in roots of six transgenic lines was increased by about 3.92% compared to the wild types. 【Conclusion】 The FPS was successfully cloned from T.kok-saghyz and transformed into the wild types. The obtained transgenic lines had much more rubber than the wilds, this result indicated that TKFPS does play an important role in the biosynthetic pathway of the rubber in T.kok-saghyz.
    The Analysis of the Parental Combining Ability and Heterosis on Two-Line Hybrid Flax
    WANG Li-min, ZHANG Jian-ping, DANG Zhao, PEI Xin-wu, DANG Zhan-hai
    Scientia Agricultura Sinica. 2016, 49(6):  1047-1059.  doi:10.3864/j.issn.0578-1752.2016.06.003
    Abstract ( 481 )   HTML ( 3 )   PDF (376KB) ( 799 )   Save
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    【Objective】 The combining ability of parents and heterosis of agronomic and quality traits in two-line hybrid flax were analyzed to provide theoretical basis for a two-line flax crossbreeding program.【Method】 A incomplete diallel cross using eighteen flax genotypes including seven thermo-sensitive male sterile lines and eleven restorer lines with 77 (7×11) hybrid combinations was carried out to estimate effects of the general combining ability (GCA), special combining ability (SCA) and heterosis for fourteen main agronomic and quality traits in two-line hybrid flax. Data analysis was processed with DPS (Data Processing System). 【Result】Analysis of variances for the studied traits indicated highly significant differences among the genotypes. The general combining ability (GCA) effects was significant rather than specific combining ability (SCA) for most traits, including plant height, technical length, branches per plant, stems per plant, seeds per capsule, 1000-seed weight, oil content, oleic acid, linoleic acid, stearic acid and palmitic acid. The heredity of these traits was mainly controlled by additive genes. Both GCA and SCA effects were significant for some traits, including capsules per plant, yield per plant, and linolenic acid. The heredity of these three traits was controlled by additive and non-additive genes, of which the additive genes played a dominant role. The narrow-sense heritability of agronomic and quality traits was ranged as follows: technical length>1000-seed weight>plant height>seeds per capsule>branches per plant >yield per plant>stems per plant>capsules per plant, and oil content>linolenic acid>stearic acid>palmitic acid>linoleic acid> oleic acid. The general combining ability (GCA) and special combining ability (SCA) have greater differences among different traits with the same parent/cross or among different parents/crosses with the same trait. Meanwhile, there is no direct relationship between GCA and SCA. Among eighteen parents, the male sterile line 113S, 16-1-1-2, 24-1-1-1, T-11, 1S and restorer line Longya10, Tianye9, Lunxuan3, Macbeth, AC Watson were good parents with better GCA effects on some agronomic and quality traits. The heterosis over male parent and over control cultivar were widely observed in seventy-seven hybrid combinations. The correlation analysis revealed that heterosis of main agronomic and quality traits in two-line hybrid flax was significantly related to general combining ability (GCA) and specific combining ability (SCA). Both or one of parents have high GCA or have high SCA in strong heterosis combinations. Meanwhile, the parental GCA was significantly greater than SCA, and the GCA in restorer lines was significantly greater than in male sterile lines.【Conclusion】Heterosis in two-lines hybrid flax was closely related to the general combining ability (GCA) and special combining ability (SCA). Selection for restorer lines with higher general combining ability (GCA) was the key of parent selection in two-line flax cross breeding program.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY
    Effects of Exogenous 6-BA and Nitrogen Fertilizers with Varied Rates on Function and Fluorescence Characteristics of Wheat Leaves Post Anthesis
    LUO Yong-li, YANG Dong-qing, YIN Yan-ping, CUI Zheng-yong, LI Yan-xia, CHEN Jin, ZHENG Meng-jing, WANG Yu-zhu, PANG Dang-wei, LI Yong, WANG Zhen-lin
    Scientia Agricultura Sinica. 2016, 49(6):  1060-1083.  doi:10.3864/j.issn.0578-1752.2016.06.004
    Abstract ( 653 )   HTML ( 11 )   PDF (539KB) ( 931 )   Save
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    【Objective】The purpose of this experiment was to investigate the effects of exogenous cytokinin (6-BA) and nitrogen application with varied rates on photosynthetic characteristics of wheat post anthesis and provide a theoretical basis for enhancing photosynthetic productivity of wheat. 【Method】The experiment was conducted using a stay-green cultivar (Wennong 6) and a non-stay-green cultivar (Jimai 20) with three nitrogen rates, namely, N0(0), N1(240 kg·hm-2) and N2(360 kg·hm-2). Exogenous 6-benzyladenine (6-BA) (25 mg·L-1) and lovastatin (300 mg·L-1) were sprayed on the leaves at a rate of 100 mL·m-2 for 3 days after anthesis (DAA). Then chlorophyll content, MDA content and antioxidant enzyme activity were determined using flag leaf samples. The content of four kinds of endogenous hormones was determined using high performance liquid chromatography method (HPLC). The chlorophyll fluorescence kinetics parameters of flag leaves induced by different treatments were determined using pulse modulation type fluorometer. 【Result】Spraying exogenous 6-BA significantly enhanced the max photochemical efficiency (Fv/Fm), actual photochemical efficiency (ΦPSII), photosynthetic electron transport rate (ETR) and photochemical quenching coefficient (qP) of flag leaves of two wheat cultivars at different growth stages post anthesis. The chlorophyll fluorescent parameters were significantly decreased by spraying lovastatin. It was found that under N0 treatment, spraying exogenous 6-BA increased ΦPSII of flag leaves of Jimai 20 by 12.08%, 6-BA combined with N1 treatment and N2 treatment increased ΦPSII by 14.21% and 9.43%, respectively. ΦPSII of flag leaves of Wennong 6 was increased by 12.44%, 14.84% and 11.58%, respectively, in above three treatments. In addition, in N0 treatment, spraying exogenous 6-BA enhanced photosynthetic electron transport rate (ETR) of flag leaves in Jimai 20 by 16.57%, 6-BA combined with N1 treatment and N2 treatment enhanced ETR by 25.81% and 18.83%, respectively. ETR of flag leaves of Wennong 6 was increased by 13.88%, 23.58% and 22.80%, respectively in above three treatments. Other indicators of flag leaves in both wheat cultivars showed the following rule that in 6-BA combined with N1 treatment, fluorescence parameters of flag leaves were increased more than 6-BA combined with N0 treatment or 6-BA combined with N2 treatment. Meanwhile, the cultivar, nitrogen fertilizer, single hormone effect, hormone combined with nitrogen fertilizer affected ΦPSII, ETR and qP significantly. In addition, Fv/Fm was affected significantly by cultivar, hormone but not affected by combined application of nitrogen fertilizer and hormone significantly. Besides, in N0 treatment, foliar application of the inhibitor of cytokinin lovastatin reduced ETR of flag leaves of Jimai 20 by 22.71%, lovastatin combined with N1 treatment and N2 treatment decreased ETR by 12.06% and 11.92%, respectively. Other fluorescence parameters including Fv/Fm, ΦPSII and qP of the two cultivars showed a downward trend. Increasing nitrogen application rate could slow down the decrease of fluorescence parameters of both wheat cultivars caused by the reduction of the synthesis of cytokinin. Exogenous 6-BA had significant effects on the content of endogenous hormones in two cultivars of wheat. Spraying exogenous 6-BA significantly increased the zeatin riboside (ZR) content and the auxin (IAA) content of flag leaves post anthesis in both cultivars. The gibberellins (GA3) content was significantly increased at 21-28 DAA. On the contrary, abscisic acid (ABA) was significantly decreased after spraying exogenous 6-BA. After spraying exogenous lovastatin, changes of endogenous hormones contents were on the contrary with the above results. With the increase of nitrogen application rate, the ZR and IAA content was enhanced at 21-28 DAA. The overall trend of the ABA content was decreased with the increase of nitrogen rate. In N1 treatment, the GA3 content was higher than that under the other two nitrogen rates at 7-14 DAA. Meanwhile, the ZR content was significantly affected by the cultivar, nitrogen fertilizer, single hormone effect, combined application of nitrogen fertilizer and hormone. In addition, in 6-BA combined with N1 treatment, chlorophyll content and antioxidant enzyme system activity of flag leaves were increased more than 6-BA combined with N0 treatment or 6-BA combined with N2 treatment. Combined application of nitrogen fertilizer and hormone had significant effects on chlorophyll content and antioxidant enzyme system activity. Lovastatin combined with N0 treatment reduced chlorophyll content and antioxidant enzymes activity more than that lovastatin combined with N1 and N2 treatments. This shows that increasing nitrogen fertilizer application could mitigate the senescence of the photosynthetic structure. Spraying 6-BA significantly affected the grain weight and yield (P<0.01), and had no significant effect on the spike number and grain number, and combined application of nitrogen fertilizer and exogenous 6-BA significantly affected the number of spike, grain number per spike, 1000-grain weight and yield. 【Conclusion】Obvious combined effects between nitrogen fertilizer and cytokinin exists, under 6-BA combined with appropriate nitrogen rate, the photosynthetic performance of wheat was improved higher than 6-BA combined with no N application or 6-BA combined with N2. The improvement of photosynthetic performance significantly improved grain yield.
    Effects of Rainfall Harvesting Planting on Temporal and Spatial Changing of Soil Water and Temperature, and Yield of Spring Maize (Zea mays L.) in Semi-Arid Areas
    LI Yu-ling, ZHANG Peng, ZHANG Yan, JIA Qian-min, LIU Dong-hua, DONG Zhao-yun, JIA Zhi-kuan, HAN Qing-fang, REN Xiao-long
    Scientia Agricultura Sinica. 2016, 49(6):  1084-1096.  doi:10.3864/j.issn.0578-1752.2016.06.005
    Abstract ( 461 )   HTML ( 3 )   PDF (1455KB) ( 551 )   Save
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    【Objective】To provide a theoretical evidence for high yield and water use efficiency (WUE) of maize, the experiment was conducted to test soil moisture transportation and distribution, soil temperature, yield and WUE of dryland spring maize for determining the effects of rainfall harvesting under different planting methods.【Method】Four different plastic film mulching planting treatments were used in arid areas of Pengyang country in Ningxia in 2013-2014: double ridges and furrows both mulched with plastic film and planting in furrows (D), flat planting of narrow film mulching (F), plastic film mulching on ridge and planting in film-side (R1) and plastic film mulching on ridge and planting in narrow furrow (R2). A conventional flat field without mulching was used as the control (CK). Soil water and temperature, WUE, and yield at different growth stages of spring maize were analyzed in this study. The experiment was designed by random blocks and repeated three times. The plastic film width of R1 and F treatments were both 60 cm, furrow width of R1 was 10 cm and ridge width was 50 cm, the big-ridge width of D was 70 cm and ridges height was 15 cm, the other small one width and height were 50 cm and 10 cm. Planting density of different treatments were 75 000 plants/hm2. The amount of based fertilizer applied before sowing was N 102 kg·hm-2and P2O5 90 kg·hm-2 , and topdressing was N 153 kg·hm-2. 【Result】 The results showed that the status of soil water and temperature had improved significantly in all the four plastic film mulching planting treatments compared with CK. In maize seedling period (0-30 d), the soil water storage at depths of 0-200 cm soil layer with D, F, R1 and R2 increased by 10%, 8.9%, 10.9% and 14.4%, respectively, compared with CK. At maize mid-late stage (90-120 d), affected by rainfall precipitation and different planting treatments, the maize water consumption was different. In addition, soil water storage in 0-200 cm soil profile showed differences: the soil water storage in the plastic film mulching planting treatments were significantly lower compared with CK in 2013 (precipitation was 309.4 mm in July and August), there was no significant difference between treatments, however, the soil water storage in R1 and R2 treatments were significantly higher than other plastic film mulching planting treatments. However, in the 2 years experiment, the soil water storage in 0-40 cm soil profile of R1 was significantly higher than other treatments, with an average of increasing of 5%; The soil temperature at the depth of 0-25cm soil layer increased by 3.5, 2.3, 0.9 and 1.1℃ in D, F, R1 and R2 treatments compared with CK. Total dry matter accumulation of maize during the whole growth period showed a "S" curve type, during the first two months, the dry matter accumulation was small, each treatment accounted for only 4.3%-15.4% of the whole growth period, with the order from high to low: D>R2>F>R1>CK. About 74.5% of the dry matter accumulation was formed in the next two months (flare opening to filling period ), the dry matter accumulations were 30.93 and 3.241 kg·hm-2·d-1 in the treatments of D, R2. WUE and maize yield of D, F, R2 increased by 13.4%, 21.2%, 13.3% and 18.0%, 11.2%, 20.3% compared with CK in 2013 (when annual rainfall was 594.1 mm). However, the WUE and maize yield in D, R1, R2 treatments increased by 31.1%, 33.8%, 35.1% and 42.5%, 39.9%, 40.8% in 2014 (when annual rainfall was 341.9 mm). There were no significant differences among D, R1, and R2 treatments. The yield and WUE of plastic film mulching planting treatments increased significantly in a less precipitation year, and among all of which, the effect in the treatment of R2 was obvious. 【Conclusion】 The plastic film mulched ridge and sowing furrow planting changed the shallow soil water condition significantly, improved soil temperature, increased dry matter accumulation in semi-arid areas of southern Ningxia. Furrow and ridge mulching planting treatments gave obvious advantages in the less precipitation years. the WUE and maize yield of D and R1 were the best. This study enriched the plastic film mulching plating treatments in the semi-arid areas of southern Ningxia, and provided a basis for the sustainable developing treatments which could improve the yield and stabilize the yield in the semi-arid areas.
    PLANT PROTECTION
    Screening and Identification of the Interacting Protein of Cytokinin Response Regulator VvRR2 in Grapevine
    YU Yi-he, LI Xiu-zhen, GUO Da-long, YANG Ying-jun, LI Xue-qiang, ZHANG Guo-hai
    Scientia Agricultura Sinica. 2016, 49(6):  1097-1105.  doi:10.3864/j.issn.0578-1752.2016.06.006
    Abstract ( 450 )   HTML ( 4 )   PDF (1644KB) ( 765 )   Save
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    【Objective】The objective of this study is to clone cytokinin response regulator VvRR2 in grapevine, obtain the interaction protein of VvRR2, and elucidate the mechanism of VvRR2 in the disease resistance response in Vitis vinifera. 【Method】 Powdery mildew was inoculated on grapevine, total RNA extraction and reverse transcription, the transcripts of VvRR2 response to powdery mildew was detected by real-time quantitative PCR at transcription level. The transient expression vector pBI221-VvRR2- GFP was constructed, Arabidopsis protoplasts was transformed and the subcellular localization was analyzed. The yeast expression vector pGBKT7-VvRR2 was constructed and transformed into yeast strain AH109, and the transcriptional activity of VvRR2 was detected. The yeast cDNA library was constructed, VvRR2 was used as a bait to screen the interaction protein by Mating method. The candidate sequence was analyzed by Blast. The candidate protein VvTGA sequence was cloned into pGADT7 vector to form recombinant vector pGADT7-VvTGA, with the recombinant bait plasmid pGBKT7-VvRR2 were transformed into yeast, VvRR2 and VvTGA were verified with yeast two-hybrid methods. VvTGA full-length sequence was cloned into pSPYNE (R) 173 vector to form the recombinant vector pSPYNE-VvTGA, VvRR2 full-length sequence was cloned into pSPYCE (M) vector to form the recombinant vector pSPYCE-VvRR2. Then, the two recombinant vectors were co-transformed into Arabidopsis protoplasts, VvRR2 interaction with VvTGA were verified with bimolecular fluorescence complementary technology.【Result】After inoculation of the powdery mildew, the expression pattern of VvRR2 was regulated by the powdery mildew. The VvRR2 was located in the nucleus of Arabidopsis protoplasts, and the transcriptional activation experiment results showed VvRR2 had transcriptional activation activity in yeast. On the culture medium containing 60 mmol?L-1 3-AT, the self-activating activity of VvRR2 bait vector could be inhibited, and the bait vector of VvRR2 had no toxicity to the host yeast. VvRR2 was used as the bait to screen the yeast cDNA library, and 287 clones were obtained. The Blast analysis showed that these genes were involved in protein synthesis and degradation, signal transduction, light reaction and biological clock rhythm, growth development and the stress tolerance. The yeast two-hybrid assays showed that the yeast containing empty vector (pGADT7 or pGBKT7) in four deficient medium (containing 3-AT) couldn’t grow. However, the yeast containing two kinds of recombinant plasmid in four deficient medium (containing 3-AT) could grow, also showed color in four deficient medium (containing X-α-Gal). Bimolecular fluorescence complementation assays results showed co-transformation pSPYCE-VvRR2 and pSPYNE (R)173, pSPYNE-VvTGA and pSPYCE (M) into protoplasm, the transformed protoplasm had not yellow fluorescence. However, the transformed protoplasm containing pSPYCE-VvRR2 and pSPYNE-VvTGA recombinant vectors showed yellow fluorescent. The expression of VvTGA was similar to VvRR2, presented by pathogen induced expression patterns. 【Conclusion】 Grapevine cytokinin response regulator VvRR2 is a transcription factor, the expression is induced by powdery mildew, VvRR2 interacts with VvTGA, and the expression of VvTGA induced by powdery mildew.
    Cloning, Sequence Analysis and Expression Profile of an Odorant Binding Protein Gene in Western Flower Thrips (Frankliniella occidentalis)
    ZHANG Zhi-ke, WU Sheng-yong, LEI Zhong-ren
    Scientia Agricultura Sinica. 2016, 49(6):  1106-1116.  doi:10.3864/j.issn.0578-1752.2016.06.007
    Abstract ( 340 )   HTML ( 2 )   PDF (2858KB) ( 638 )   Save
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    【Objective】The objective of this study is to clarify the sequence properties and distribution of odorant binding protein (OBP) in the western flower thrips (Frankliniella occidentalis), which would be favorable for throwing light on the mechanism of insect olfaction perception and lay a basis for controlling the target pests by interfering with insect chemoreception.【Method】The OBP gene from F. occidentalis (FoccOBP1) was cloned using RT-PCR and RACE-PCR strategies. Nucleotide sequence was analyzed using DNAMAN software. Homology was analyzed using BLAST. A phylogenetic tree was constructed using neighbor-joining of MEGA 6.0. The secondary structure and three-dimensional model of FoccOBP1 were predicted using Chou & Fasman and SWISS MODEL, respectively. Expression profiles of FoccOBP1 at different developmental stages and in different tissues of the adults were assayed using real-time quantitative PCR. 【Result】 The OBP gene in F. occidentalis was cloned and named as FoccOBP1. The number in GenBank is KM527948. The full length of FoccOBP1 cDNA is 660 bp, contains a 450 bp open reading frame (ORF) encoding a putative protein of 149 amino acids with a molecular mass of 16.39 kD and an isoelectric point of 7.46. The non-coding district of 3′ and 5′ end is 172 bp and 38 bp, respectively, there is a polyA structure of eukaryotes. The deduced amino acid sequence possesses a putative signal peptide of 22 amino acid residues at the N terminus and contains the typical six-cysteine signature of insect OBPs. The arrangement of C1-X26-C2-X3-C3-X40-C4-X9-C5-X8-C6 conform to six conservative cysteine site of the typical OBPs structural model. There are three lipotropism areas in amino acid sequence. A lipotropy pocket significantly was formed by 74-83 amino acid residues, which could be a fat-soluble binding site of odor molecules. Three OBPs of Hemiptera insects, 10 OBPs of Homoptera insects and FoccOBP1 amino acid sequence were clustered into one group by distance tree, and among them FoccOBP1 protein had high homolog with Apolygus lucorum AlucOBP8 (GenBank number is AFJ54049.1), Adelphocoris lineolatus AlinOBP5 (GenBank number is ACZ58031.1) and Lygus lineolaris LlinOBP2 (GenBank number is AHF71029.1) of Hemiptera insects, and Aphis gossypii AgosOBP7(GenBank number is AGE97637.1), Aphis glycines AglyOBP7 (GenBank number is AHJ80893.1), Rhopalosiphum padi RpadOBP7 (GenBank number is AHL30243.1) and so on of Homoptera insects, suggesting that these genes likely developed from a common ancestral gene. Secondary structure of FoccOBP1 predicted showed that FoccOBP1 had more α-helices, followed by beta-fold and less angle. The results of FoccOBP1 expression at different development stages revealed that this gene was highly expressed in nymph and 1-day-old adult. Expression levels reduced gradually with ages of adult and had differences with adult sex. The results of FoccOBP1 expression in different tissues revealed this gene was specifically expressed in the antennae. The reconstruction of expression plasmid pET-30a (+)/FoccOBP1 was constructed successfully. 【Conclusion】The sequence characteristics of nucleotides and amino acids of FoccOBP1 were clarified. The secondary structure and three-dimensional model of FoccOBP1 were analyzed. Temporal and spatial expression pattern of FoccOBP1 was clarified, indicating that FoccOBP1 may play an important role in olfactory reception, locating food sources, synthesis of pheromone and mating of F. occidentalis. The reconstruction of expression plasmid pET-30a (+)/ FoccOBP1 was constructed successfully, which is a basis of expression, purity and function of FoccOBP1 protein.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Analysis of Cultivated Land and Grain Production Potential in China
    CHEN Yin-jun, YI Xiao-yan, FANG Lin-na, YANG Rui-zhen
    Scientia Agricultura Sinica. 2016, 49(6):  1117-1131.  doi:10.3864/j.issn.0578-1752.2016.06.008
    Abstract ( 559 )   HTML ( 12 )   PDF (536KB) ( 826 )   Save
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    【Objective】The purpose of this study is to reveal grain production potential and Cultivated land resources and provide a reference for national food security decisions.【Method】The time series forecasting method was used to forecast the arable land area, cropping index, food and non-food crops area ratio, crop planting structure in 2020 by using the cultivated land area and grain production series data in 1980/1996-2013 as sampled and based on the results of the second survey of land resources. Then the potential of the nation's grain yield increase was analyzed from the multi-angle of view the high-yield demonstration area, the unit-yield level in variety regional test. 【Result】By 2020, the national cultivated land area will be 1.32×108 hm2, grain crops and non-crops area will account for 66:34, grain crops acreage will be 1.12×108 hm2. The potential of national grain production will be 68.9% from high-yield demonstration area and 35.5% from regional trial variety. By 2020, the potential of national grain production will reach 6.34×108-6.53×108 t ,with 5.3%-8.5% increase compared to 2013.【Conclusion】In the future, the cultivated land and grain crops area will be decreased, but the national total grain production capacity will be continued show a upward trend with the pulling of crop yield increasing.
    Evolution Characteristics of Soil Available Phosphorus and Its Response to Soil Phosphorus Balance in Paddy Soil Derived from Red Earth Under Long-Term Fertilization
    HUANG Jing, ZHANG Yang-zhu, XU Ming-gang, GAO Ju-sheng
    Scientia Agricultura Sinica. 2016, 49(6):  1132-1141.  doi:10.3864/j.issn.0578-1752.2016.06.009
    Abstract ( 430 )   HTML ( 12 )   PDF (460KB) ( 640 )   Save
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    【Objective】 In order to reveal the impact of various fertilization treatments on the characteristics of paddy soil phosphorus (P) evolution, and its response to soil P balance. We analyzed the annual variation of soil available P content, total P content, soil P balance, and phosphorus activation coefficient (PAC). 【Method】 This study was based on a long-term different fertilization experiment of paddy soil in subtropical China that was established in 1982. The different fertilization treatments included non-fertilization (CK), cattle manure (M), chemical nitrogen (N), P and potassium (K) fertilizer (NPK), NPK with M (NPKM), N and P fertilizer with M (NPM), N and K fertilizer with M (NKM), and P and K fertilizer with M (PKM). The annual variation characteristics of soil available P content, total P content, soil P balance, and PAC from 1982 to 2012 were analyzed. 【Result】 Soil available P content improved efficiently through fertilizer application. The change rate of soil available P content of M, NKM, NPK, NPM, NPKM, and PKM was 0.18, 0.20, 0.83, 1.35, 1.46, and 1.62 mg·kg-1·a-1, respectively. The soil total P content was on the decline under non-fertilization. The change rate of the soil total P content of M, NPK, PKM, NPM, and NPKM was 4.3, 15.4, 16.0, 18.3, and 22.9 mg·kg-1·a-1, respectively. All the fertilizer treatments had a P surplus no matter whether applying chemical fertilizer or cattle manure. The P apparent balance was significantly correlated with the Olsen-P increment (P<0.05). With an average surplus of 100 kg P ·hm-2, the soil Olsen-P increased by 0.4, 0.7, 1.9, 2.1, 2.2, and 3.2 mg·kg-1, in the M, NKM, NPM, NPKM, PKM, and NPK treatments, respectively. The PAC of NPK was significantly higher than M and NKM (P<0.05), while there was no significant difference in the soil P surplus amount among these treatments. 【Conclusion】Applying chemical P fertilizer plus cattle manure can significantly improve the soil available P, total P content, and PAC, compared with the treatments that applied chemical fertilizer or cattle manure alone.
    Effect of Biochar Amendment on Soil Microbial Biomass Carbon and Nitrogen and Enzyme Activity in Tier Soils
    SHANG Jie, GENG Zeng-chao, WANG Yue-ling, CHEN Xin-xiang, ZHAO Jun
    Scientia Agricultura Sinica. 2016, 49(6):  1142-1151.  doi:10.3864/j.issn.0578-1752.2016.06.010
    Abstract ( 572 )   HTML ( 4 )   PDF (450KB) ( 740 )   Save
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    【Objective】 The effect of biochar on soil microbial biomass carbon and nitrogen and enzyme activity of Tier soils in the Guanzhong region of China was studied. It may provide a theoretical guidance for biochar to improve soil quality as a soil amendment. 【Method】Biochar used in the field trail was produced by the pyrolysis of fruit tree stems and branches under the condition of 450°C and limited oxygen. Then biochar was mixed with the surface horizon of the soil (0-20 cm) with different dosages (0, 20, 40, 60, and 80 t·hm-2 which was expressed as B0, B20, B40, B60, and B80, respectively.). After two years of summer maize and winter wheat rotation, the soil biological activity and physicochemical properties of different soil layer was measured. The effect of biochar on soil enzyme activity and microbial biomass carbon and nitrogen in Tier soils was studied by the principal component analysis. 【Result】(1) In the 0-20cm soil layer, the soil microbial biomass carbon (SMBC) and soil microbial biomass nitrogen (SMBN)of the treatments with the 40 or 60 t·hm-2biochar were higher than those of the other treatments, while in the 20-30cm soil layer, SMBC and SMBN reached the maximum with the treatment of 80 t·hm-2 biochar, and this principle was true when the biochar amendment and the control was compared. (2) In the different soil layers, the variation regularity of soil enzyme activities were the same, the overall trend was an increase and then decrease with the increase of the amount of biochar. The soil enzyme index (SEI) indicated that the application of biochar significantly increased the SEI. In the 0-10 cm soil layer, compared with the control, the SEI of the treatments of biochar amendment was significantly increased by 1.6-2.7 times. In the 10-20 cm and 20-30 cm soil layers, this index was significantly increased by 26.6%-39.5% and 18.7%-21.7%, respectively. However, SEI was significantly decreased with the increase of biochar to 80 t·hm-2. (3) By principal component analysis, the eight indexes in this study can be divided into two categories, the soil active factor and the soil strength factor. The comprehensive scores of different soil layers were 0-10 cm >10-20 cm >20-30 cm. In 0-10 cm and 10-20 cm soil layer, the comprehensive scores of different treatments were B60>B40>B20>B80>B0, and in 20-30 cm soil layer, the composite score was B60>B80>B40>B20>B0, respectively.【Conclusion】The application of biochar may lead to the increase of soil microbial biomass, soil enzyme activities, and the soil biological environment. Overall, the treatment with 60 t·hm-2 biochar led to the best comprehensive performance in this study.
    HORTICULTURE
    Effects of Different Ratios of Nitrate and Ammonium on the Dynamic Kinetic and Growth for Eriobotrya japonica Lindl. Seedlings
    MA Jian, FAN Wei-guo
    Scientia Agricultura Sinica. 2016, 49(6):  1152-1162.  doi:10.3864/j.issn.0578-1752.2016.06.011
    Abstract ( 468 )   HTML ( 2 )   PDF (797KB) ( 532 )   Save
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    【Objective】 The effect of nitrate ( NO3--N ) and ammonium ( NH4+-N) on the growth and nitrogen absorption kinetics of loquat seedlings was studied to determine the absorption and utilization of nitrogen forms and to provide a scientific basis for the management of loquat. 【Method】 Taking loquat seedlings as materials and using the ion depletion method, the loquat seedlings roots of different nitrate and ammonium absorption kinetics parameters were tested. The yellow soil with pH at 7.35 was used as the cultivation medium, and five different ratios of nitrate and ammonium were designed. The effect of different ratios of nitrate and ammonium on the growth and the root morphology were studied. 【Result】 At different NH4+ and NO3- ion concentrations and ratios of ammonium nitrate in the nutrient solution, the regular pattern of the absorption of ammonium, nitrate and total nitrogen in the root system of loquat seedlings was followed with the Michaelis-Menten dynamic equation. No matter how the NH4+ and NO3- ion concentrations changed, the inherent potential absorption, affinity and roots of flow rate of loquat seedlings roots for NH4+ absorption were greater than NO3-. Under the pure nitrate supply conditions, loquat absorption of NO3- did not get the promotion. During the treatment supplied different proportions of ammonium nitrate, with the increasing of the proportion of ammonium, total nitrogen for loquat seedlings roots of maximum uptake rate (Imax) and the root of the velocity (α) increased significantly, while the Michaelis constant value (Km) significantly reduced. The Imax and α of these value declined and the Km values increased with the increasing in the proportion of nitrate. The ratio of increasing in the proportion of ammonium can promote roots of loquat seedlings for nitrogen absorption, but increasing in the proportion of nitrate absorption of loquat roots has an adverse effect. Ammonium is the preferred absorption nitrogen forms of loquat. Under soil culture conditions, applying different proportions of ammonium nitrate, plant height, basal diameter, dry weight biomass, shoot ratio, root morphology index and total leaf area of loquat seedlings were different significantly. The proportion of ammonium fertilizer significantly increased plant height, basal diameter, dry weight biomass, root to shoot ratio, total leaf area and total root length, total surface area, total volume, average diameter, total count the number and root apical fractal dimension. In 100% of ammonium treatment, the above indicators were the largest, but in 100% of nitrate treatment, the above indicators were the smallest. 【Conclusion】 In a single source of nitrogen and different ratios of ammonium nitrate mixture for nitrogen conditions, the maximum absorption rates of loquat seedlings roots on NH4+ were larger than NO3-, and the inflow rate of roots for NH4+ were faster than NO3-, and the affinity of roots for NH4+ were stronger than NO3-, and showed significant absorption preferences for ammonium. Loquat likes ammonium nutrition absorption characteristics, which is the preferred form of loquat absorption. So ammonium can obviously promote the growth development of loquat seedlings and strengthen the loquat seedlings for nitrogen uptake, but nitrate can inhibit the growth of loquat seedlings. Under the conditions of mixed supply of ammonium and nitrate, adding ammonium could promote the growth development of loquat seedlings.
    STORAGE·FRESH-KEEPING·PROCESSING
    Effects of Different Drying Methods on Quality Changes during Processing and Storage of Tremella fuciformis
    Scientia Agricultura Sinica. 2016, 49(6):  1163-1172.  doi:10.3864/j.issn.0578-1752.2016.06.012
    Abstract ( 397 )   HTML ( 4 )   PDF (443KB) ( 649 )   Save
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    【Objective】 The objective of this study is to provide theoretical basis for the production of high-quality Tremella fuciformis by studying the effect of different drying methods on quality changes during processing and storage of it. 【Method】Shrinkage, rehydration rate, sensory evaluation of color, contents of protein, reducing sugar, total sugar and polysaccharide, scavenging activity to superoxide anion and hydroxyl radicals of polysaccharides, water content, total bacteria and fungus count, contents bongkrekic acid, hydrogen peroxide and SO2 were taken as evaluation index. Vacuum drying, vacuum freeze drying and hot air drying were adopted to dry Tremella fuciformis, and then the effects of three drying methods on texture quality and related physicochemical indexes during processing and storage of Tremella fuciformis were studied. 【Result】 For effects of different drying methods on physical properties, Tremella fuciformis processed byhot air had the highest shrinkage rate, browning degree, and the lowest increasing amplitude. Tremella fuciformis dried by vacuum freeze presented the lowest browning degree and shrinkage rate and the highest rehydration rate. As for nutritional ingredient, Tremella fuciformis dried by vacuum freeze drying had the highest contents of soluble protein, total sugar and polysaccharide. Tremella fuciformis dried by vacuum contained the highest reducing sugar content and the lowest contents of soluble protein and polysaccharide. Reducing sugar obtained from hot air dried Tremella fuciformis was lowest. The strongest scavenging activity to superoxide anion and hydroxyl radicals was found for polysaccharides obtained from vacuum freeze dried Tremella fuciformis, while the weakest scavenging activity was observed in Tremella fuciformis dried byvacuum. In aspect of the physical and chemical changes of Tremella fuciformis processed by three different drying methods, in all samples, the content of water and reducing sugar presented an increase trend, while the contents of protein, total sugar and polysaccharide showed a decrease trend with the storage time. After 90 days storage, the largest increase amplitude of contents of water and reducing sugar and the lowest decrease amplitude were observed on Tremella fuciformis dried by hot air. Tremella fuciformis dried by vacuum freeze presented the largest decrease amplitude of contents of protein and polysaccharide and the lowest increase amplitude of reducing sugar content. The lowest amplitude of contents of water and protein were observed in Tremella fuciformis processed by vacuum. As for metabolite of microorganism, the sum of bacteria and fungus, the contents of bongkrekic acid, hydrogen peroxide and SO2 in Tremella fuciformis processed by three different drying methods all presented an increase trend with storage time. After 90 days storage, the largest increase amplitude of count of bacteria and fungus and contents of bongkrekic acid were observed in Tremella fuciformis dried by vacuum freeze. Tremella fuciformis dried by hot air presented the largest increase amplitude of hydrogen peroxide contents. The lowest increase amplitude of count of bacteria and fungus and hydrogen peroxide contents were observed in Tremella fuciformis processed by vacuum. There was no significant difference among SO2 contents of samples processed by three drying methods.【Conclusion】The results indicated that the best quality and nutrition was found in Tremella fuciformis processed by vacuum freeze drying, followed by vacuum drying and hot air drying. Tremella fuciformis dried by vacuum freeze was susceptible to microorganisms and had the largest decrease amplitude of nutrition substances.
    Effects of Multiplex Antistaling Agent Coating on Postharvest Physiology, Storage Quality and Storage Diseases of Pomegranate Fruit
    ZHANG Run-guang, TIAN Cheng-rui, ZHANG You-lin
    Scientia Agricultura Sinica. 2016, 49(6):  1173-1186.  doi:10.3864/j.issn.0578-1752.2016.06.013
    Abstract ( 440 )   HTML ( 2 )   PDF (2414KB) ( 554 )   Save
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    【Objective】 Several unexpected phenomenon can be easily occurred on postharvest pomegranate fruit such on peel browning, aril deterioration, softening, decay and so on during storage. The storage period of pomegranate fruit is short, its quality becomes poor and the commodity value drops after storage. The best formula of multiplex antistaling agent for pomegranate fruit was optimized. In order to provide the theoretical basis and technical parameters for pomegranate storage, the effects of antistaling agent on postharvest physiology, storage quality and storage diseases of pomegranate fruit were studied in this study. 【Method】‘Jingpitian’ pomegranate fruit was used as raw material. The composition proportion of multiplex antistaling agent was optimized by orthogonal experiment, and the best formula was determined by physiological changes and sensory evaluation. The pomegranate fruit coated with multiplex antistaling agent was stored under the conditions of temperature (1.0±0.5)℃, relative humidity (RH) 90%-95%. The fruit without antistaling agent treatment was used as the control. The changes of fruit respiration rate, relative conductance rate, firmness, peel polyphenoloxidase (PPO) activity, peroxidase (POD) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity and arils soluble solids content, total sugar content, titratable acidity content of each treatment were measured during storage. Moreover, the browning index, decay index, weight loss rate and commodity fruit rate were calculated in different periods of storage. The sensory characteristics such as taste, color and smell of the fruit were overall evaluated. The storage disease pathogenic fungi were identified by Koch’s validation, ITS sequence analysis and morphological characteristics. The inhibition rate, semi lethal concentration (EC50) and minimal inhibitory concentration (MIC) of multiplex antistaling agent on pathogenic fungi of pomegranate fruit were also determined during storage. 【Result】 The multiplex antistaling agent was prepared by 7 types of components including chitosan oligosaccharide, natamycin, glucono-δ-lactone, citric acid, ascorbic acid, sodium hexametaphosphate and sodium caseinate. The results generally showed that the coating treatment of multiplex antistaling agent could reduce the fruit respiration rate, slow down the rise of the peel browning index and relative conductance rate, inhibit the activities of PPO and POD, keep the activities of CAT and SOD at a high level, maintain the soluble solids content, titratable acidity content, total sugar content of arils and fruit firmness well, decrease the fruit decay index and weight loss rate, improve the commodity fruit rate, and keep the good sensory quality of pomegranate fruit. Botryotinia fuckeliana and Penicillium spinulosum were the pathogenic fungi that led to main diseases during storage. Multiplex antistaling agent could inhibit the growth of pathogenic fungi obviously. The storage life could reach up to 160 days with the browning index being 0.21, decay index being 0.16 and commodity fruit rate being 90.2%. After long-term storage, the pomegranate fruit had a colorful appearance and glassy arils and its sensory quality was excellent and the fresh-keeping effect was perfect.【Conclusion】 The optimum formula of multiplex antistaling agent included chitosan oligosaccharide 0.2 g, natamycin 0.02 g, glucono-δ-lactone 0.08 g, citric acid 3 g, ascorbic acid 2 g, sodium hexametaphosphate 0.1 g, and sodium caseinate 0.6 g. All the components were mixed and dissolved in distilled water to prepare the 160 mg·L-1 antistaling agent. After pre-cooling at (5.0±0.5)℃ for 3 days, the pomegranate fruit was immersed in the above mentioned antistaling agent for 10-20 s, then was taken out and dried naturally, then was packed with plastic bag individually, and then was stored at (1.0±0.5)℃ and RH 90%-95%.
    ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT
    Isolation and Characterization of Ammonia Nitrogen-Degrading Microbe from Chicken Manure
    LIU Zhi-yun, LIU Guo-hua, CAI Hui-yi, ZHANG Shu, CHANG Wen-huan, XIE Qing, SI Yan-pei
    Scientia Agricultura Sinica. 2016, 49(6):  1187-1195.  doi:10.3864/j.issn.0578-1752.2016.06.014
    Abstract ( 458 )   HTML ( 6 )   PDF (529KB) ( 797 )   Save
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    【Objective】Ammonia is one of the most harmful gases in animal production. In order to inhibit the production of ammonia, we planned to screen microbes with the ability of ammonia removal and investigate its functional features.【Method】Ammonium sulfate was used as the sole nitrogen source to isolate the ammonia nitrogen-degrading microbe from chicken manure. Enrichment cultivation was conducted ten times. The final enrichment culture was streaked on the isolation agar after a ten-time step dilution. The colonies with different morphological characters were isolated and purified. The strain with the highest efficiency on ammonia nitrogen-degrading is our choice. The morphology, molecular biology and physiological and biochemical identification method was used to identified the target strain. Single factor experiments were conducted to identify its optimum degradation conditions, such as temperature (20, 25, 30, 35, 40℃), pH (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0), carbon source (starch, mannitol, Na-Citrate, glucose, sodium acetate, sodium bicarbonate), C/N (5, 10, 20, 40) and initial ammonium concentrations (100, 300, 600,  1 200 mg·L-1). The bacterial suspension was inoculated into chicken manure with the inoculation of 10%. The ammonia emissions and nitrogen forms from chicken manure were studied.【Results】Fifteen strains with the ability of ammonium-degrading were isolated from the chicken manure. One strain named LSA, showed high efficient degradation ability. The strain was identified as Candida krusei and 99% sequence similarity with 18S rDNA of Candida krusei isolate EM12(JF274497.1)was revealed compared with homologous genes in GenBank. The GenBank accession number of the strain LSA was KT025851. Further degradation studies showed that the optimal conditions for the ammonium degrading of the strain LSA were pH 3.0–7.0, 20–40°C, C/N 20 with glucose as carbon source. The removal ef?ciency increased with increasing initial NH4+-N concentrations. When the initial NH4+-N concentration was 327.20 mg·L-1, the NH4+-N removal rate was 71.88% and the OD600 was 2.45 within 60h. When the initial NH4+-N concentration was 1 105.26 mg·L-1, the NH4+-N removal rate was 57.44% and the OD600 was 2.96 within 96h. The ammonium concentration and ammonia emissions from chicken manure with LSA were lower than that in the control group. The reduction rates in the treatment groups were 22.30% and 15.92%. The total nitrogen in chicken manure inoculated with strain LSA is higher than that in the control group, while the ammonia nitrogen/ total nitrogen is lower. 【Conclusion】Strain LSA has good performance in ammonium degrading and displays good potential for future applications of ammonia emission control from chicken manure.
    Influence of MyoD1 Promoter Activity by MyoD Family of Guangling Cattle
    ZHANG Wen, XU Hou-qiang, CHEN Wei, CHEN Xiang, ZHAO Jia-fu, HUAN Cong-cong, XIA Dan, ZHOU Di
    Scientia Agricultura Sinica. 2016, 49(6):  1196-1206.  doi:10.3864/j.issn.0578-1752.2016.06.015
    Abstract ( 387 )   HTML ( 1 )   PDF (1599KB) ( 573 )   Save
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    【Objective】The myogenic determination gene (MyoD) family is an important one in the molecular regulation of the muscle generation process. It includes MyoD1, Myf5, MyoG and Myf6 genes. They only express in mature skeletal muscle cells and its early cells, but will be suppressed in other than muscle cells. In this family, MyoD1 is responsible for the activation of early embryo muscle progenitor cells and participates in the adjustment of growth, development and repair of skeletal muscle after the embryonic stage, which in order to maintain the relatively stable of individual skeletal muscle. MyoD1 is an important factor for starting and maintaining skeletal muscle cell differentiation and growth, such that it has become a research hotspot. At present, the research on MyoD1 in polymorphism and correlation analysis is more, the expression and regulation of it are mainly on the formation mechanism of muscle cells in mice, chicken and pig. In cattle, the researches on MyoD1 are mainly in the expression levels of transcription and translation, but the mechanism of MyoD1 on transcriptional regulation is not clear. This study was to research the effect of MyoD family on the promoter of MyoD1 in Guangling Cattle, in order to lay the foundation of probing into the expression regulation mechanism of cattle MyoD1. 【Method】 Specified primers were designed to amplify CDS regions of MyoD family and MyoD1 promoter fragments P1 and P2 of Guangling Cattle. At the same time, the CDS regions and promoter fragments were connected to pcDNA3.1(+)and pGL3-Basic framework by double enzyme digestion, respectively, to construct eukaryotic expression vectors of pcDNA3.1(+)-Myf5, pcDNA3.1(+)-Myf6, pcDNA3.1(+)-MyoD, pcDNA3.1(+)-MyoG and report carrier pGL3-P1, pGL3-P2 which contain the firefly luciferase gene as the reporter gene. After the recombinant plasmids were identified by double enzyme digestion and sequencing, the expression vector and report carrier were transfected into myoblast cell line C2C12 of mice by cotransfection. After 30 hours, we cracked the cells and tested the double luciferase activity of cell lysis buffer. According to the relative luciferase activity to analyze the effect of the MyoD1 promoter activity by the MyoD family. 【Result】The sequences of the cloned CDS regions of MyoD family and MyoD1 promoter of Guangling Cattle were validated by DNA sequencing. Restriction analysis and sequencing confirmed that the vectors pcDNA3.1(+)-Myf5, pcDNA3.1(+)-Myf6, pcDNA3.1(+)-MyoD, pcDNA3.1(+)-MyoG, pGL3-P1 and pGL3-P2 were successfully constructed. Compared with the control group with the same dose of pcDNA3.1 (+), the relative luciferase activities of pGL3-P1 were obviously enhanced after transfected pcDNA3.1(+)-Myf5, pcDNA3.1(+)-Myf6, pcDNA3.1(+)–MyoD. Among them,when transfection concentration was 200 ng, the potentiation was maximal. There were significant difference (P<0.05). Luciferase activity of pGL3-P1 was enhanced after transfected pcDNA3.1(+) –MyoG. but not have significant difference (P>0.05). The luciferase activity of pGL3-P2 had no obvious change after transfected pcDNA3.1(+)-Myf5, pcDNA3.1(+)-Myf6, pcDNA3.1(+)–MyoD and pcDNA3.1(+)-MyoG (P>0.05). 【Conclusion】 In myoblast cell line C2C12 of mice, exogenous overexpression transcription factors MyoD, Myf5 or Myf6 could significantly improve the transcription activities of Guanling cattle MyoD1 promoter P1 (P<0.05). But exogenous overexpression transcription MyoD family could not significantly improve the transcription activities of Guanling cattle MyoD1 core promoter P2. This observation helps explain that the action sites of transcription factors MyoD, Myf5 and Myf6 of Guanling cattle with MyoD1 promoter are not on its core promoter region P2.
    Characterization and Expression Analysis of Serine Protease BmSP141 from the Silkworm (Bombyx mori ) in Response to Starvation
    LIU Hua-wei, LI You-shan, TANG Xin, ZHANG Xiao-lu, ZHAO Ping
    Scientia Agricultura Sinica. 2016, 49(6):  1207-1218.  doi:10.3864/j.issn.0578-1752.2016.06.016
    Abstract ( 364 )   HTML ( 3 )   PDF (4765KB) ( 416 )   Save
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    【Objective】The objective of this study is toanalyze the sequence of BmSP141, clear its spatial and temporal expression patterns, and explore its potential function by starvation and re-feeding in silkworm (Bombyx mori). 【Method】The nucleotide sequence of serine protease gene BmSP141 coding region was obtained by TA cloning. Biology online software was used to analyze the amino acid sequence, molecular weight, functional domain and other information of the coding region of this gene. Sequence alignment and phylogenetic tree analysis of BmSP141 with serine proteases from other species were done by ClustalX1.8 and MEGA5.02 software. Prokaryotic expression vector p28-BmSP141 was constructed and introduced into Rosetta (DE3) to express the target protein, which was purified by nickel affinity chromatography. The expression profile of BmSP141 at mRNA and protein levels in different tissues and development stages were analyzed by semi-quantitative reverse transcription PCR (RT-PCR), quantitative real-time PCR (qPCR) and Western blot. Immunofluorescence localization was used to detect the location of BmSP141 protein in the 4th instar larvae. The impacts of starvation and re-feeding on the expression of BmSP141 mRNA and protein were detected by qPCR and Western blot. 【Result】The BmSP141 encoded a 292 amino acid protein with a 17-residues signal peptide. The predicted molecular mass and isoelectric point of BmSP141 mature protein are 25.9 kD and 7.8, respectively. Multi-sequence alignment showed that BmSP141 share a highly sequence identity (62% and 63%) with the serine proteases of Heliothis virescens and Mamestra configurata,respectively. These homologs have conserved catalytic triad and similar serine protease catalytic motifs, but the substrate specificity pocket sites of BmSP141 are relatively different compared with the conversed pocket sites of chymotrypsin. Furthermore, the recombinant protein BmSP141 was expressed as inclusion body at 37, and purified by nickel affinity chromatography. Then, the purified protein was used to generate the BmSP141-specific polyclonal antibodies. RT-PCR analysis of BmSP141 revealed that BmSP141 mRNA was mainly expressed in the larval midgut and highly expressed at the larval feeding stage. Whereas its expression level was lower in the pupae and moth than in the larvae. Western blot analysis showed that BmSP141 protein was only expressed in the midgut and the expression level was increased first and then decreased from 5th instar to wandering stage. Immunohistochemical analysis further confirmed that BmSP141 protein was predominately present in the cytoplasm of midgut epithelium cell. The expression of BmSP141 mRNA and protein was down-regulated after starvation but up-regulated by re-feeding. As a result, the expression of BmSP141 was induced by the food in the midgut.【Conclusion】BmSP141 was highly expressed in the midgut of larval feeding stage and lower expressed in the pupae and moth stages than in the larvae stage. The expression of BmSP141 mRNA and protein was down-regulated after starvation but up-regulated by re-feeding, which suggested that this gene might be involved in food protein digestion in the midgut during larval development.