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    16 April 2020, Volume 53 Issue 8
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    CRISPR/Cas9 Targeted Editing for the Fragrant Gene Badh2 in Rice
    QI YongBin,ZHANG LiXia,WANG LinYou,SONG Jian,WANG JianJun
    Scientia Agricultura Sinica. 2020, 53(8):  1501-1509.  doi:10.3864/j.issn.0578-1752.2020.08.001
    Abstract ( 882 )   HTML ( 72 )   PDF (2664KB) ( 896 )   Save
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    【Objective】Rice fragrance, a very important trait for quality improvement, is mainly controlled by a recessive gene Badh2. In this study, CRISPR/Cas9-mediated editing were used to generate the gene-edited rice plants with the fragrant Badh2 in the conventional elite rice varieties, and the trait of fragrance was improved.【Method】 The targeted sequences were designed according to the sequence of exon2 and exon7 of the Badh2 by the principle of CRISPR/Cas9 gene editing. Its specificity of the targeted sequence was determined by BLAST analysis, and then constructed into CRISPR/Cas9 expression vector. The callus of Jia58 and Xiushui134 which are widely cultivated in the Zhejiang Province were selected as explants to transform by Agrobacterium-mediated genetic transformation, and the positive transgenic plants were obtained by the screening of hygromycin resistance. Sequencing analysis of transgenic lines was used to detect the presence of the mutation type on the loci of Badh2. Stable marker-free gene-edited lines carrying the mutation on Badh2 were obtained by PCR analysis and identification. The content of 2-AP in the brown rice flour were measured by GC-MS, and the difference between the gene-edited lines and non-transgenic control was determined. 【Result】 Badh2 of the transgenic lines was directionally mutated by the genetic transformation using the expression vector on which the target sequence of the exon2 and exon7 were designed and constructed. A total of 15 T0 gene-edited lines were obtained from Jia58. Eight of them were generated mutation on the exon2 with five different mutations types in which different single base was inserted into different position. Seven of them were generated mutations on the exon7 with five different mutation types in which the base or fragment deletion was produced. A total of 11 T0 gene-edited lines were obtained from Xiushui134, of which five lines were generated mutations on the exon2 with the single base insertion and six lines on the exon7 with the fragment deletion. A total of 16 marker-free gene-edited lines were obtained from 48 T1 Xiushui134, of which five lines were generated mutations on the exon2, and 11 lines on the exon7. The average 2-AP content in brown rice flour of four T2 gene-edited lines were 0.309, 0.347, 0.332 and 0.295 μg·g -1 respectively, which were significantly higher (P<0.01) than that of the non-transgenic control (0.046 μg·g -1). 【Conclusion】 The Badh2 which controlled the rice fragrance trait was directionally edited by using CRISPR/Cas9-mediated technology, and the marker-free gene-edited lines were obtained, of which the fragrance of Badh2 edited lines were significantly improved.

    QTL Mapping and Candidate Genes Screening of Related Traits in Brassica napus L. During the Germination Under Tribenuron-Methyl Stress
    WANG LiuYan,WANG RuiLi,YE Sang,GAO HuanHuan,LEI Wei,CHEN LiuYi,WU JiaYi,MENG LiJiao,YUAN Fang,TANG ZhangLin,LI JiaNa,ZHOU QingYuan,CUI Cui
    Scientia Agricultura Sinica. 2020, 53(8):  1510-1523.  doi:10.3864/j.issn.0578-1752.2020.08.002
    Abstract ( 454 )   HTML ( 25 )   PDF (2660KB) ( 611 )   Save
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    【Objective】The QTLs and tolerance genes related to the germination characters of rape seeds under the stress of tribenuron-methyl were studied, which laid the foundation for screening and cultivating the germplasm of tribenuron-methyl resistant rape and exploring the molecular mechanism of tribenuron-methyl tolerance during the germination of rape seeds.【Method】A high generation RIL population consisted of 175 lines, which were constructed from the synthetic Brassica napus 10D130 and the conventional variety Brassica napus ZS11, was treated with 0.15 mg·kg -1 tribenuron-methyl solution for seed germination test and the control was under the distilled water. The phenotypic data that including relative germination vigor (RGV), relative germination rate (RGR), relative root length (RRL) and relative dry weight (RDW) were analyzed by Excel software. Then, the RIL population was genotyped with 6K SNP chip, and the high-density genetic linkage map was constructed by JoinMap4.0 software. Based on the genetic map, the relative values of four characters were mapped by using Multiple QTL mapping method of MapQTL software. And the genes sequence of Brassica napus were searched according to the confidence interval of each QTL, next, blast with Arabidopsis genome sequence in turn and select the candidate genes that may be related to the tolerance to tribenuron-methyl stress.【Result】The frequency distribution of each traits for RIL population's was continuous with the large variation range, which were consistent with the characteristics of quantitative characters, so it were suitable for the detection of QTL. Correlation analysis showed that there was a significant positive correlation between RGR and RGV, and the correlation coefficient was 0.587. In addition, the constructed genetic map contained 1 897 polymorphic SNP markers covering 3 214.19 cM of the genome of Brassica napus with an average map distance of 1.69 cM. By this map, 22 QTLs related to 4 phenotypic traits were detected and the phenotypic contribution rate was between 6.4% and 12.6%. Among them, there were 6 and 3 QTLs related to RGV and RGR, 8 and 5 QTLs related to RRL and RDW, respectively. Also, the confidence intervals of QTLs for RGV and RGR were found to overlap completely or partially at 64.857 cM, 55.935 cM and 56.645 cM of chromosome A01. Through sequence alignment, 30 candidate genes were screened, including 18 cytochrome P450 family members, 5 glycosyltransferase family genes, 1 GSTF related gene, 1 ABC transporter related gene and 1 ALS gene, all of which were detoxified by accelerating metabolism, and related to the mechanism of herbicide resistance, especially ALS is the target enzyme of sulfonylurea herbicide. Furthermore, others genes were screened, including 1 BHLH gene and 1 JAZ6 gene which could interact to protect against stress; and 1 LSU2 protein gene which was involved in the detoxification of cell oxidants and plant defense response; and 1 MATE family member which was involved in the transport of flavonoids, alkaloids, metal ions, other metabolites and plant stress response caused by toxic substances.【Conclusion】22 QTLs that significantly associated with tribenuron-methyl tolerance related traits and 30 candidate genes for the tolerance to tribenuron-methyl were found. These genes are involved in the stress response caused by toxic substances by accelerating the transport and metabolism of toxic molecules, which may be related to the resistance regulation and response mechanism of plants to tribenuron-methyl.

    Splicing Property Analyses of the NRSE1 Element from Tobacco PR3b mRNA After Fusion Expression with GUS Gene
    ZHAO Xue,WANG Feng,WANG WenJing,LIU XiaoFeng,BIAN ShiQuan,LIU YanHua,LIU XinMin,DU YongMei,ZHANG ZhongFeng,ZHANG HongBo
    Scientia Agricultura Sinica. 2020, 53(8):  1524-1531.  doi:10.3864/j.issn.0578-1752.2020.08.003
    Abstract ( 334 )   HTML ( 23 )   PDF (1403KB) ( 821 )   Save
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    【Objective】Previously, a post-transcriptional splicing of tobacco PR3b gene was observed in the low-nicotine mutants (nic1, nic2) of Nicotiana tabacum L. cv. Burley 21, yet the mechanism underlying this phenomenon is still unclear. In this study, we developed transgenic plants expressing the fusion of the alternative splicing element NRSE1 (nicotine-synthesis related splicing element 1) from PR3b and the GUS gene to investigate the splicing properties of the NRSE1 element after excising from PR3b mRNA, in order to reveal its regulatory mechanism. 【Method】The NRSE1 element was amplified from PR3b cDNA by PCR amplification, and the vector for expressing the fusion of NRSE1 element and the GUS gene was constructed by molecular methods. And, the vector was used to develop transgenic plants expressing the fusion of NRSE1 element and GUS gene with wild type tobacco and the low-nicotine mutants nic1 and nic2 via agrobacterium (LBA4404) mediated transformation method. The transgenic plants were identified by RT-PCR and GUS staining, and the splicing of the fusion of NRSE1 element and GUS gene in the transgenic plants of wild type tobacco and low-nicotine mutants were then analyzed by RT-PCR. Seedlings of the transgenic plants were treated with ethylene (ET) and jasmonic acid (JA), respectively. And, the effects of ET and JA treatment on the GUS activity and the splicing of the fusion of NRSE1 element and GUS gene in the transgenic plants were analyzed by GUS staining and RT-PCR, respectively. The effects of ET and JA treatment on the expression level of the fusion of NRSE1 element and GUS gene were analyzed as well. 【Result】A set of transgenic wild type tobacco and low-nicotine mutants expressing the fusion of NRSE1 element and GUS gene were identified by RT-PCR and GUS staining. Further RT-PCR and sequencing analyses showed that the NRSE1 element could be alternatively spliced at higher levels in the low-nicotine mutants after fusion with the GUS gene, in a pattern consistent with its alternative splicing in the PR3b mRNA as previously report. ET and JA treatments could alter the GUS activity in the transgenic plants, but did not affect the inducible splicing of the NRSE1 element or the expression level of the fusion of NRSE1 element and GUS gene in the transgenic plants. 【Conclusion】 A highly splicing of the NRSE1 element was observed in the low-nicotine mutants after fusion expression with GUS gene. The alternatively splicing of NRSE1 element is independent of the rest regions of PR3b mRNA. ET and JA treatments had an effect on the GUS activities of the transgenic plants expressing the fusion of NRSE1 element and GUS gene, which may result from a translational regulation.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Response of Non-Photochemical Quenching in Bundle Sheath Chloroplasts of Two Maize Hybrids to Drought Stress
    LIU WenJuan,CHANG LiJuan,YUE LiJie,SONG Jun,ZHANG FuLi,WANG Dong,WU JiaWei,GUO LingAn,LEI ShaoRong
    Scientia Agricultura Sinica. 2020, 53(8):  1532-1544.  doi:10.3864/j.issn.0578-1752.2020.08.004
    Abstract ( 580 )   HTML ( 23 )   PDF (6341KB) ( 868 )   Save
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    【Objective】 Non-photochemical chlorophyll fluorescence quenching (NPQ) of photosystem II (PSII) is the most rapid photoprotective mechanism of higher plants that responds to a changing environment. Maize has two distinctly different classes of chloroplasts in mesophyll and bundle sheath cells. In the present study, the drought tolerance of two maize hybrids was compared to explore the significance of non-photochemical quenching in bundle sheath chloroplasts to maize tolerance. 【Method】 The experiment was conducted with two maize hybrids, Chengdan 30 and Zhongyu 3, and consisted of three soil moisture regimes, including 70%-80% of field water capacity (FWC) (sufficient irrigation), 50%-60% FWC (moderate drought stress), and 35%-45% FWC (severe drought stress). Physiological and biochemical parameters of maize leaves, including relative water content, chlorophyll contents, reactive oxygen species (ROS) accumulation, lipid peroxidation, and gas exchange, were measured. Fv/Fm and NPQ of PSII in mesophyll and bundle sheath chloroplasts of two maize hybrids were investigated through a chlorophyll fluorescence kinetic microscope. The steady-state levels of PSII subunit S (PsbS) in mesophyll and bundle sheath cells were analyzed using method of western blotting. PSII complexes levels were detected by blue native PAGE. 【Result】 The stomatal conductance and transpiration rate of maize leaves decreased under drought stress. There were no remarkably difference in decline degree of stomatal index between Chengdan 30 and Zhongyu 3. However, under severe drought conditions, Chengdan 30 showed better leaf water status, lower ROS damages, and higher photosynthetic efficiency compared with Zhongyu 3. NPQ levels and PsbS contents in bundle sheath chloroplasts increased more markedly than that in mesophyll chloroplasts when maize plant suffered with drought treatment, which was especially outstanding in Chengdan 30. The PSII complexes contents of Zhongyu 3 reduced obviously under drought stress, while the steady-state levels of light-harvesting complex II (LHCII) trimer of Chengdan 30 enhanced after severe drought. 【Conclusion】 The responses of photosynthetic mechanism to stomatal limitation were no significant difference in two maize hybrids under drought stress. However, compared with Zhongyu 3, Chengdan 30 had a higher non-photochemical quenching capacity in bundle sheath chloroplasts, which might play a positive influence on its superior drought tolerance of non-stomatal limitation.

    Weed Identification in Maize Field Based on Multi-Spectral Remote Sensing of Unmanned Aerial Vehicle
    ZHAO Jing,LI ZhiMing,LU LiQun,JIA Peng,YANG HuanBo,LAN YuBin
    Scientia Agricultura Sinica. 2020, 53(8):  1545-1555.  doi:10.3864/j.issn.0578-1752.2020.08.005
    Abstract ( 688 )   HTML ( 73 )   PDF (4433KB) ( 831 )   Save
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    【Objective】In order to reduce the application rate of herbicides and to make the maize planting management more effective, the accurate identification of weeds in maize fields was investigated based on multi-spectral remote sensing of unmanned aerial vehicles (UAV). 【Method】In this paper, a Red Edge-M multi-spectral camera was mounted in a six-rotor UAV to acquire five single-band images of blue, green, red, red edge, and near-infrared, and the application was taken in Zibo, Shandong province, China to acquire multi-spectral images of a maize field in July 14, 2018. In order to separate the vegetation and non-vegetation pixels in the image, 7 vegetation indices were calculated, the OTSU method was used to obtain the non-vegetation area, and the multi-spectral image was masked. Then multi-spectral image was transformed by principal component analysis, retaining the first three principal component bands with the most information. The experimental region was divided into 3 training areas and 1 verification area. 675 maize and 525 weed samples were selected in the training areas to train the supervised classification model, and 240 maize and 160 weed samples were selected in the verification area to evaluate model classification accuracy. The 7 vegetation indices, 24 texture features of the 3 principal component bands and 10 reflectivity of multi-spectral image bands which were filtered, and a total of 41 features were taken as features of maize and weed. Support vector machines-feature recursive elimination (SVM-RFE) algorithm and Relief algorithm were applied to selecting 14 features from 41 features to constitutes a feature subset separately, and supervised classification for weed detection was performed using support vector machine (SVM), K-nearest neighbor (KNN), Cart decision tree (Cart), random forest (RF) and artificial neural network (ANN) .【Result】SVM and RF performed a better classification with all features and SVM-RFE & Relief feature subsets. The overall accuracy of SVM was 89.13%-91.94%, Kappa>0.79, and overall accuracy of random forest was 89.27%-90.95%, Kappa>0.79.【Conclusion】 SVM-RFE feature selection algorithm was better than the Relief feature algorithm for reducing the original features. SVM model had the highest classification accuracy for identification of weed and maize at regional canopy scales.

    PLANT PROTECTION
    Effect of Transcription Factor CsWRKY61 on Citrus Bacterial Canker Resistance
    LONG Qin,DU MeiXia,LONG JunHong,HE YongRui,ZOU XiuPing,CHEN ShanChun
    Scientia Agricultura Sinica. 2020, 53(8):  1556-1571.  doi:10.3864/j.issn.0578-1752.2020.08.006
    Abstract ( 461 )   HTML ( 30 )   PDF (4975KB) ( 436 )   Save
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    【Background】Citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc) is one of the most serious citrus diseases in the world, which is a quarantine disease. Due to the relatively backward research of citrus molecular pathology, the available resistance gene resources are relatively scarce. WRKY transcription factor is involved in plant responses to biotic and abiotic stress. The previous study has found that citrus WRKY transcription factor may play an important role in regulating host disease resistance response.【Objective】The objective of this study is to evaluate the canker resistance of transgenic citrus (Citrus sinensis) with over-expression of CsWRKY50, CsWRKY61 and CsWRKY72, clarify the biological function and disease resistance breeding value of these genes in citrus in response to Xcc. RNA-seq was further used to analyze the signaling pathway regulated by CsWRKY61.【Method】Agrobacterium-mediated method was used to obtain transgenic citrus plants with over-expression of CsWRKY50, CsWRKY61 and CsWRKY72. Real-time quantitative PCR (qRT-PCR) was used to analyze the expression level and copy number of the target genes. In vitro pinprick inoculation was used to evaluate the resistance of transgenic plants to canker disease. The molecular mechanism of CsWRKY61 improving citrus bacterial canker resistance was investigated by transcriptome sequencing analysis of over-expression CsWRKY61 and wild-type (WT) plants.【Result】The plant expression vectors of CAMV 35S promoter controlling the expression of CsWRKY50, CsWRKY61 and CsWRKY72 were constructed, and 6, 8 and 6 transgenic lines were obtained by GUS staining and PCR identification, respectively. The expression of the target gene in transgenic plants increased in different degrees. The copy number of exogenous genes in most transgenic plants was 1. Only the transgenic plants with over-expression of CsWRKY61 had significantly enhanced canker disease resistance, and the lesion area was significantly smaller than that of WT plants, while over-expression of CsWRKY50 and CsWRKY72 transgenic plants had no significant difference in disease resistance compared with WT. Transcriptomics analysis showed that biotic stress related pathways (including pathogen recognition, respiratory burst, transcription factors, defense genes, hormones, cell wall and secondary metabolism, etc.) and signal transduction-related pathways (mainly kinase receptors) were significantly activated in over-expression of CsWRKY61 transgenic plants. 【Conclusion】Over-expression of CsWRKY61 can activate pathways related to biotic stress and signal transduction, enhance citrus bacterial canker resistance. It is suggested that CsWRKY61 has potential application value in citrus disease resistance breeding.

    Investigation on Occurrence of Citrus Huanglongbing and Virus Diseases, and Prophage Genetic Diversity of Huanglongbing Pathogen in Meizhou, Guangdong
    CUI YiPing,PENG AiTian,SONG XiaoBing,CHENG BaoPing,LING JinFeng,CHEN Xia
    Scientia Agricultura Sinica. 2020, 53(8):  1572-1582.  doi:10.3864/j.issn.0578-1752.2020.08.007
    Abstract ( 403 )   HTML ( 10 )   PDF (1077KB) ( 657 )   Save
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    【Objective】The objective of this study is to investigate the impacts of citrus Huanglongbing (HLB) and virus diseases on citrus production in Meizhou City of Guangdong Province, and to reveal the local ‘Candidatus Liberibacter asiaticus’ (CLas, the pathogen of HLB in China) strains genetic diversity based on the prophage region.【Method】The primers were designed with 16S rDNA as the template, and the incidence of HLB in different sampling sites in Meizhou City in the 3 years from 2017 to 2019 was detected by qPCR. After RNA extraction, RT-PCR followed by ordinary PCR were applied to detect Citrus tristeza virus (CTV), Citrus exocortis viroid (CEVd), Citrus chlorotic dwarf-associated virus (CCDaV), Citrus tatter leaf virus (CTLV) and Citrus yellow vein clearing virus (CYVCV) using their own reported primers, separately. Two pairs of primers (Lap-TJ-F/Lap-TJ-R1 and Lap-TJ-F/Lap-TJ-R2) were designed based on the supervariant gene regions corresponding to two prophage types (SC1 and SC2), and the genetic diversity of CLas prophage regions was analyzed using ordinary PCR method.【Result】The results of CLas detection showed that only the sample from one orchard (Honey pummelo base in Shunxing Company in Dapu County) in September, 2018 was free, other inspected areas in Meizhou all had CLas. Overall, the CLas detection rate of navel orange in Meizhou was 55.3%, the honey pummelo was 61.5%, and the Shatian pummelo was 61.7%. Among them, the CLas detection rate of honey pummelo and Shatian pummelo collected from Datuo Town, Pingyuan County in 2017 was 100%, the detection rate of honey pummelo and Shatian pummelo in Baima Town, Xingning City was 100% and 80%, respectively. The detection rate of samples in other sampling areas was 16.7%-83.3%. Although the detection rate in some areas of Meizhou was high, the content of pathogenic bacteria was not high, most of them were at the early and middle stages of HLB, which belonged to a preventable and controllable stage. In the same region, the total detection rate of citrus viruses in Meizhou City was not high. CTV, CTLV and CYVCV were the main viruses in Meizhou, CEVd and CCDaV were not detected. The detection rate of CTV, CTLV and CYVCV on navel orange was 78.9%, 7.9% and 21.1%, on honey pummelo was 15.4%, 25.0%, and 9.6%, on Shatian pummelo was 6.4%, 2.1%, and 4.3%, respectively. Through prophage genetic diversity analysis of CLas strains in Meizhou City, four CLas prophage amplicon types (SC1-1, SC1-2, SC2-1 and SC2-2) were observed in the CLas from all the tested samples, SC2-1 represented the predominant CLas type in navel orange and Shatian pummelo in Meizhou City, and SC1-1 was predominant in honey pummelo.【Conclusion】The citrus HLB in Meizhou citrus producing area is currently at a controllable stage. The prophage of its pathogenic CLas strain is unique in different varieties. Due to the existence of citrus virus diseases during spot inspection, seedling supervision needs to be strengthened during the planting process and effective measures should be taken to prevent and control HLB and Diaphorina citri.

    Prokaryotic Expression of dsRNA of Zucchini yellow mosaic virus and Its Control Efficacy on ZYMV
    XIE KunLun,LIU LiMing,LIU Mei,PENG Bin,WU HuiJie,GU QinSheng
    Scientia Agricultura Sinica. 2020, 53(8):  1583-1593.  doi:10.3864/j.issn.0578-1752.2020.08.008
    Abstract ( 461 )   HTML ( 18 )   PDF (3016KB) ( 446 )   Save
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    【Objective】Zucchini yellow mosaic virus (ZYMV) is the most prevalent virus that harms watermelon. The objective of this study is to realize the prevention and treatment of ZYMV by external application of viral gene dsRNA on watermelon. 【Method】Based on the existing ZYMV virus sequence in the laboratory, the NCBI database was used to find the similar sequences for different gene fragments. Then the software DNAMAN8 was used to perform multiple alignment analysis to find the conserved regions. Based on the analysis results, 5 gene fragments of ZYMV 3′UTR, 6K2, HC-Pro, P3 and NIb with a length range of 200-350 bp were selected, at the same time, a GUS gene fragment with a length of 190 bp was selected as a control. After the 6 fragments were amplified by PCR, they were inserted into the vector L4440 containing double T7 promoters by homologous recombination method. The RNAIII enzyme-deficient strain E. coli-HT115 was used to construct a prokaryotic expression system for dsRNA production, dsRNA was released by ultrasonic disruption and dissolved in TE buffer (10 mmol·L -1 Tris-HCl and 1 mmol·L -1 EDTA). The concentration of IPTG, induction time and ultrasonic breaking time in the production process were compared and analyzed. The dsRNA was applied on watermelon plants by spraying method, two experiments for spraying dsRNA first, then inoculating virus (prevention), and inoculating virus first, then spraying dsRNA (treatment) were designed, and the efficacy of dsRNA prevention and treatment against ZYMV was evaluated by statistical comparison and analysis of the incidence at 21 days after virus inoculation. 【Result】The production system for ZYMV 3′UTR, 6K2, HC-Pro, P3, NIb 5 gene fragments and GUS gene fragment was established, which could express and release dsRNA efficiently and stably. The cells could efficiently produce and release dsRNA after IPTG induced concentration of 8 mmol·L -1, induction time of 7 h, and disruption for 15 min under the condition of Ningbo Xinzhi brand ultrasonic cell disrupter 3ø and 60% output power. In the prevention experiment, it was found that the disease incidence of both GUS gene fragment dsRNA and TE buffer spraying treatments was 100%. The best control efficacy on ZYMV was HC-Pro gene fragment, after 21 days of virus inoculation, the efficacy reached 95%, the control efficacy of NIb gene fragment (80%) was relatively poor. On this basis, the efficacy of prevention and treatment of HC-Pro fragment on ZYMV was analyzed in depth. When the ZYMV was inoculated on the 3rd, 5th and 7th day after spraying the dsRNA of HC-Pro fragment, the incidence of disease was 16%, 63% and 63%, respectively. The dsRNA of the HC-Pro fragment was sprayed on the 1st, 3rd, 5th, and 7th day after inoculation of ZYMV, the results show that there is no obvious therapeutic efficacy, and only the onset of disease was delayed.【Conclusion】A prokaryotic expression system was established for dsRNA targeting different gene fragments of ZYMV, and it was found that dsRNA based on HC-Pro gene fragment has the best control efficacy on ZYMV, which can reduce the incidence of disease, delay the onset time of disease, and have the potential for application.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Change Characteristics of Organic Carbon and Total Nitrogen in Water-Stable Aggregate After Conversion from Upland to Paddy Field in Black Soil
    MA Yuan,CHI MeiJing,ZHANG YuLing,FAN QingFeng,YU Na,ZOU HongTao
    Scientia Agricultura Sinica. 2020, 53(8):  1594-1605.  doi:10.3864/j.issn.0578-1752.2020.08.009
    Abstract ( 409 )   HTML ( 22 )   PDF (495KB) ( 604 )   Save
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    【Objective】 The objectives of this study were to analyze the composition and stability of soil aggregate, the changes of organic carbon (OC), total nitrogen (TN) content, natural abundance of 13C and 15N in different-sized aggregates, to explore the sequestration and stability of soil aggregate organic C, TN, and to reveal the evolution of soil aggregate organic C, TN changes after the conversion from upland to paddy field in black soil region of Northeast China.【Method】 Soil samples were collected from upland (soybean planted for over 60 years) in typical black soil and paddy soil with different years (3, 5, 10, 17, 20 and 25 years, soybean was planted in all the fields before conversion to paddy field). The dynamic characteristics of OC and TN in soil aggregates were studied by using wet-sieving method and stable isotope analysis technology.【Result】In the 0-60 cm soil layers, compared with the control treatment, the composition of 2-0.25 mm aggregates in the soil of different years after rice planting was decreased, which of 0.25-0.053 mm and <0.053 mm aggregates was increased. There was no obvious change in the composition of >2 mm aggregates, but the different years of dry land change to paddy fields were dominated by 2-0.053 mm aggregates; the mean weight diameter (MWD) of aggregates was significantly positive correlated with the proportion of >2 mm aggregates (P<0.01), and significantly negative correlated with the proportion of 0.25-0.053 mm and <0.053 mm aggregates (P<0.01 or P<0.05). The change of aggregate composition was significantly affected by different rice planting years and soil depth, whereas the MWD was significantly affected by soil depth. Compared with the control soil, in the 0-40 cm soil layer, the OC and TN contents in the size of 2-0.25 mm and 0.25-0.053 mm aggregates were declined in the 3 years, however there showed increased trend with the extension of rice cultivation in 3-25 years. Generally, OC and TN were mainly accumulated in the 2-0.25 mm and 0.25-0.053 mm aggregates. There existed significant positive correlation between OC and TN contents and aggregate composition in > 2 mm aggregates (P<0.01 or P<0.05) in the 0-60 cm soil layers, as well as 2-0.25 mm aggregates in the 0-20cm soil layer (P<0.01 or P<0.05). The OC and TN contents variation in <2 mm aggregates were significantly affected by rice cultivation time, while soil depth significantly affected >0.25 mm aggregates OC and TN contents. Compared with the control soil, the δ 13C in each size of aggregates significantly increased in 3 rice planting years and decreased in 5 rice planting years, respectively, while there was no significant change in the 5-25 rice planting years, and the δ 15N in all size of aggregates decreased slightly during the 25 years of rice replanting. In general, the δ 13C and δ 15N of soil in aggregates were significantly affected by rice cultivation time and soil depth, which increased with the decreasing of aggregate size. The δ 13C increased with soil depth in the same year, while δ 15N had no significant change.【Conclusion】After the conversion from dry land to paddy field for 25 years, non-water-stable macro-aggregates in the soil were damaged and formed into small sized aggregates. The 2-0.053 mm water-stable aggregates were the main carrier of OC and TN sequestration, while OC in small size aggregates more stable, and its stability was increased by the rice cultivation time and soil depth increased.

    Soil Nutrient Status of Oilseed Rape Cultivated Soil in Typical Winter Oilseed Rape Production Regions in China
    REN Tao,GUO LiXuan,ZHANG LiMei,YANG XuKun,LIAO ShiPeng,ZHANG YangYang,LI XiaoKun,CONG RiHuan,LU JianWei
    Scientia Agricultura Sinica. 2020, 53(8):  1606-1616.  doi:10.3864/j.issn.0578-1752.2020.08.010
    Abstract ( 520 )   HTML ( 27 )   PDF (894KB) ( 550 )   Save
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    【Objective】 The objective of the study was to investigate the current soil nutrient status of oilseed rape-cultivated soil in typical winter oilseed rape production region in China, especially for soil micronutrients status. It would provide critical reference for optimizing fertilizer strategy of winter oilseed rape. 【Method】 430 soil samples distributed in the typical winter oilseed rape production regions in 14 provinces around the Yangtze River Basin were sampled from April to May, 2018. Soil chemical properties, including soil organic matter, total nitrogen (N), soil available phosphorus (P) and potassium (K), pH, soil available calcium (Ca), magnesium (Mg), sulfur (S), iron (Fe), manganese (Mn), copper (Cu), zinc (Zn) and boron (B), were determined. Referring to the second national soil survey and the classification index of soil available P, K and B in rapeseed-cultivated soil, soil nutrient status of rapeseed-cultivated soil was clarified, and the soil nutrient characteristics under different regions (upper, middle and lower Yangtze River Basin), planting systems (paddy-oilseed rape and upland-oilseed rape rotation) and seed yield levels (<2 000 kg·hm -2, 2 000-3 000 kg·hm -2 and >3 000 kg·hm -2) were also analyzed. 【Result】 Results showed that the average soil organic matter, total N, soil available P and K, pH, soil available Ca, Mg, S, Fe, Mn, Cu, Zn and B content in the typical winter oilseed rape production region around the Yangtze River Basin were 25.9 g·kg -1, 1.47 g·kg -1, 27.5 mg·kg -1, 131.1 mg·kg -1, 6.04, 2 436.1 mg·kg -1, 225.7 mg·kg -1, 22.6 mg·kg -1, 212.3 mg·kg -1, 89.7 mg·kg -1, 3.84 mg·kg -1, 4.03 mg·kg -1and 0.45 mg·kg -1, respectively. More than two-thirds of soil organic matter and total N content belonged to the medium class or above. For soil available P content, the proportion of the rich, medium and deficient class accounted for one-third, respectively. While 63.8% of soil available K content was deficient. For soil available Fe, Mn and Cu, all soils belonged to the medium class or above. Only about 8.4% and 12.2% of soils were soil deficient Ca and Zn soil, respectively. The proportions of deficient Mg, S and B soils accounted for 24.2%, 36.0% and 83.5%, respectively. Soil nutrient contents in the upper, middle and lower Yangtze River Basin were different, however, the distributions of soil nutrient status in different regions were similar. There were significant differences on soil nutrient content between paddy and upland soils. The rapeseed-planting soils in paddy-oilseed rape rotation showed significant higher soil organic matter, total N, soil available S, Fe and Zn content. Soil nutrient characteristics under different seed yield levels were slight different. Soils with high rapeseed yield (>3 000 kg·hm -2) revealed higher soil available K, Ca, Mg and B content compared with the soils with low rapeseed yield (<2 000 kg·hm -2). 【Conclusion】 Soil nutrients content in the typical winter oilseed rape planting area around the Yangtze River Basin was increasing, nevertheless, the percentages of deficient K and B soils were still huge, and soil available Mg and S were gradually becoming the potential limiting factors of winter oilseed rape. Therefore, in the current production of winter oilseed rape in the Yangtze River Basin, we should pay more attention to the rational application of chemical fertilizers, applying N fertilizer continuously and reasonably, increasing K and B fertilize application, reducing P fertilization rate in western Yunnan, northern Guangxi and southern Hunan depending on soil available P content, and focusing on the application of S and Mg fertilizer in northern Guangxi, southern Hunan, and northern Jiangxi.

    SPECIAL FOCUS: MEAT QUALITY
    Effect of Nitric Oxide Synthetase Inhibitor on Yak Meat Quality During Post-Mortem Aging
    LI XueRu,SHI XiXiong,WANG JianZhong,ZHANG PanGao,TIAN Zhu,HAN Ling
    Scientia Agricultura Sinica. 2020, 53(8):  1617-1626.  doi:10.3864/j.issn.0578-1752.2020.08.011
    Abstract ( 477 )   HTML ( 14 )   PDF (452KB) ( 582 )   Save
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    【Objective】The effect of nitric oxide on the yak meat quality of post-mortem was studied to provide a theoretical basis for improving the yak meat quality. 【Method】 The Longissimus Doris (LD) muscles of 3-5-year-old castrated Gannan yak were used as the material. After removing the fascia and fat, the LD was cut into uniform-sized slices (8 cm × 8 cm), and uniformly pierced with a 20 G needle. Subsequently, a part of each sample were immersed at deionized water (as the control group), and the remainders of the muscle samples were immersed at 1, 10, and 100 mmol L -1nitric oxide synthase (NOS) inhibitor (N-nitro-L-arginine methyl ester hydrochloride) then aged at 4℃ for 0, 1, 3, 5, and 7 d, which served as treatment group. Then, the NOS activity, the content of nitric oxide, sulfhydryl, and carbonyl, the myofibrillar fragmentation index (MFI), pH and color were analyzed. 【Result】 The NOS activity and nitric oxide content of the meat sample in NOS inhibitor group were significantly decreased; at 7 d, the NOS activity of each NOS inhibitor treatment group was 30.9%, 43.6% and 74.7% lower than that of the control group, respectively. Nitric oxide levels were 4.7%, 12.5%, and 21.5% lower than the control group, respectively. The pH in the treatment group significantly decreased; at 7d, the treatment groups were 0.8%, 5.7%, and 15.2% lower than the control group, respectively. The 10 and 100 mmol L -1 NOS inhibitor treatment groups were significantly different compared with the control group. The treatment significantly reduced the carbonyl content of the yak meat; at 7 d, treatment groups were 4.1%, 19.0%, and 22.2% lower than the control group, respectively. The MFI of the treatment group increased significantly; at 7 d, treatment groups were 31.1%, 23.3%, and 9.6% lower than the control group, respectively. The total sulfhydryl content was significantly increased in the treatment groups; at 7d, the control group was 3.2%, 3.7%, and 2.7% lower than the NOS inhibitor treatment groups. In addition, the treatment significantly reduced the a* value and significantly increased the L* value of samples; at 7d, the a* values of each treatment groups were 7.1%, 40.2%, and 30.7% lower than the control group, respectively. The L* value of treatment groups were 1.1%, 2.0%, and 1.1% higher than the control group, respectively. 【Conclusion】 Nitric oxide promoted protein oxidation of yak meat post-mortem, inhibited tenderness of yak meat, decreased L * value and increased a * value, which adversely affected the quality of yak meat during post-mortem aging; NOS inhibitor inhibited NOS activity in muscle.

    Selection of Water-Soluble Compounds by Characteristic Flavor in Chahua Chicken Muscles Based on Metabolomics
    ZHAO WenHua,WANG GuiYing,XUN Wen,YU YuanRui,GE ChangRong,LIAO GuoZhou
    Scientia Agricultura Sinica. 2020, 53(8):  1627-1642.  doi:10.3864/j.issn.0578-1752.2020.08.012
    Abstract ( 448 )   HTML ( 19 )   PDF (2232KB) ( 521 )   Save
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    【Objective】 The biomarkers in the chest and thigh muscles of Chahua Chicken before and after heat treatment were screened by using metabolomics combined with multivariate statistical methods, the aim of this study was to screen and characterize water-soluble compounds in different parts of Chahua Chicken, so as to provide scientific and theoretical basis for establishing evaluation standards of high-quality local chicken.【Method】Using liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry (LC-Q-Exactive-MS) and 300-day-old Yunnan native Chahua Chicken, the water-soluble compounds in the chest and thigh muscles before and after heat treatment were tested. Also, the spectrum was identified based on the network database Metlin, the relative content of each substance were calculated by the concentration of DL-o-Chlorophenylalanine, and the multivariate statistical analysis was used to detect substances. The relationship model between metabolite expression and sample groups was established by using orthogonal partial least squares discrimination analysis (OPLS-DA) to predict the sample category. The representative biomarkers was screened by S-plot to determine whether the model had over-fitting by observing the slope of the line of the 200 test plots of the permutations, and to discriminate the influence intensity and interpretation ability of each metabolite expression pattern on the classification and discrimination of each group of samples by the variable important of the projection (VIP). Therefore, the study achieved the purpose of screening markers: combining the calculation for Taste Activity Value (TAV) by relative content results with sensory evaluation results evaluate the characteristic flavor of Chahua Chicken muscles.【Result】The results showed that 106 species and 43 metabolites were detected in the chest and thigh muscles of Chahua Chicken before and after heat treatment in the positive ion mode and the negative ion mode, including amino acids, vitamins, nucleotides, organic acids, and fatty acids. Among them, creatine, carnosine, amino acid, and nucleotide were water-soluble compounds which had differences between before and after heat treatment of Chahua Chicken muscles, which could be used as a biomarker for the characteristics of the tribute. The TAV value results showed that the largest TAV value in the chest muscles was gamma-aminobutyric acid (TAV=23.00), and the followed by Histidine (TAV=1.14). The highest TAV value in the thigh muscles was gamma-aminobutyric acid (TAV=37.50), and the followed by Betaine (TAV=2.00). Through sensory evaluation, it was found that the overall taste of the chest muscles and thigh muscles was mainly sweet and umami.【Conclusion】After metabolomics analysis, the characteristic taste substances were obtained, and through the taste theory and sensory evaluation values, the study determined that the taste of the breast and leg muscles of Chahua chicken was mainly sweet and fresh meat, and the leg muscles were superior to the chest muscles.

    Improving Quality and Delaying Oxidation in Goat Meat Refrigeration by Polyphenols from Thinned Young Kiwifruit
    GU MingHui,LIU YongFeng,SHEN Qian,QIAO ChunYan
    Scientia Agricultura Sinica. 2020, 53(8):  1643-1654.  doi:10.3864/j.issn.0578-1752.2020.08.013
    Abstract ( 410 )   HTML ( 18 )   PDF (741KB) ( 501 )   Save
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    【Objective】The objective of this study was to research the function of thinned young kiwifruit discarded in “Blossom and Fruit Thinning” in goat meat cold storage and to provide useful information for lengthening the shelf life of refrigerated goat meat, maintaining goat meat quality and delaying the oxidation of goat meat. 【Method】The goat meat was sprayed with 1 mg·mL -1 polyphenols from thinned young kiwifruit, epicatechin and potassium sorbate solution, control group was treated with distilled water, then preserved at 4℃ refrigerator in ziplock bags. The microstructure, texture characteristics, pH, fatty acid content, thiobarbituric acid reactive substance assay (TBARS) and volatile base nitrogen (TVB-N) of goat meat were measured during 8 d refrigeration. 【Result】As time went on, the goat meat microstructure of the four groups became tender and muscle fibers were gradually loosened and relaxed. During the cold storage period, the hardness of goat meat in four groups showed a decreasing trend. In the first 3 days, the hardness values were ranked as follows: potassium sorbate group> epicatechin group> blank group> thinned kiwifruit polyphenol group. After 5 days, the hardness values of the groups became similar. At the end of cold storage, the hardness of the thinned kiwifruit polyphenol group, epicatechin group, potassium sorbate group and blank group decreased by 50.52%, 41.73%, 39.63% and 51.56%, respectively. The springing of goat meat in four groups was first increased and then remained stable. In the first 3 days, the springing value of the polyphenols group was lower than that of the other three groups. At the end of cold storage, the springing values of the thinned kiwifruit polyphenol group, epicatechin group, potassium sorbate group and blank group were reduced by 45.23%, 33.34%, 44.64% and 41.31%, respectively; the chewiness of goat meat in four groups showed a downward trend as a whole, and the chewiness of the thinned kiwifruit polyphenol group, epicatechin group, potassium sorbate group and blank group decreased by 72.06%, 61.46%, 62.00% and 67.37%, respectively. The cohesiveness and resilience of the thinned kiwifruit polyphenol group were relatively stable, and the cohesiveness of the potassium sorbate group was in the same trend. With the prolongation of time, the content of polyunsaturated fatty acids (PUFA) in the thinned kiwifruit polyphenol group was remained stable, and the PUFA content of epicatechin and potassium sorbate groups was peaked at 6 d, while the content of blank group showed a downward trend. At the end, the PUFA content of thinned kiwifruit polyphenol group was 1.11, 1.40 and 1.86 times of the epicatechin, potassium sorbate and blank groups, respectively. The content of monounsaturated fatty acid (MUFA) in the thinned kiwifruit polyphenol group was increased first and then decreased. The highest value of MUFA content in the thinned kiwifruit polyphenol group was reached in 4 d, while other groups showed a downward trend. At the end of cold storage, the MUFA content of thinned kiwifruit polyphenol group was 1.10, 1.17 and 1.21 times of the epicatechin, potassium sorbate and blank groups, respectively. The content of the saturated fatty acids (SFA) and total fatty acids (TFA) in the thinned kiwifruit polyphenol, epicatechin and blank groups was decreased, while the content of potassium sorbate group decreased first and then stabilized. The TFA content of thinned kiwifruit polyphenol group was higher than that of the other three groups in 2-4 days. The TFA content of the thinned kiwifruit polyphenol and epicatechin groups was higher than that of the other two groups in 4-8 days. The TBARS values of goat meat in four groups decreased firstly after storage, and after 3 days, their values were ranked as follows: thinned kiwifruit polyphenol group < epicatechin group < blank group < potassium sorbate group. At the end of cold storage, the TBARS values of thinned kiwifruit polyphenol group were 90.84%, 56.83% and 59.45% of the epicatechin, potassium sorbate and blank groups, respectively. With the prolongation of time, the TVB-N value of goat meat gradually increased while the pH increased first and then stabilized. Compared with the epicatechin, potassium sorbate and blank groups, the chilled time of goat meat in the thinned kiwifruit polyphenol group was extended by 0.48, 1.13 and 1.61 d, respectively. 【Conclusion】Thinned young kiwifruit polyphenols could prolong the shelf life of meat and improve meat quality by delaying the rise of goat meat pH, inhibiting lipid oxidation, slowing down PUFA oxidation, and helping maintain cohesiveness and resilience stability.

    Prediction of Center Temperature of Beijing Roast Duck Based on Quality Index
    LIU YanXia,WANG ZhenYu,ZHENG XiaoChun,ZHU YaoDi,CHEN Li,ZHANG DeQuan
    Scientia Agricultura Sinica. 2020, 53(8):  1655-1663.  doi:10.3864/j.issn.0578-1752.2020.08.014
    Abstract ( 378 )   HTML ( 17 )   PDF (499KB) ( 611 )   Save
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    【Objective】 The aim of this study was to solve the problem in detecting center temperature of Beijing roast duck during traditional Gua-lu roasting accurately and timely. 【Method】 The prediction models of center temperature were established by using multiple linear regression, partial least-squares regression, support vector regression and artificial neural network according to the quality indicators. 【Result】 The results showed that the models were effective to identify center temperature of Beijing roast duck by L*, a*, b*, deoxymyoglobin, oxymyoglobin, metmyoglobin, moisture and fat content, as well as protein secondary structure of duck breast. The R 2C of multiple linear regression and partial least-squares regression were 0.9543 and 0.9384, and SEC of 5.8205℃ and 6.7634℃, respectively. The prediction effect of multiple linear regression was better than partial least-squares regression, while the prediction model of support vector regression was superior to artificial neural network. R 2C and R 2CV of support vector regression were 0.9837 and 0.9496, SEC and SECV were 3.5215℃ and 6.1236℃, respectively, so the support vector regression was the best prediction model of center temperature. The R 2V of the verified models of support vector regression was 0.9748, and the SEV was 5.5204℃. The model obtained by support vector regression together with the modeling results could accurately predict the center temperature of Beijing roast duck. 【Conclusion】 The color, myoglobin, water content, fat content and protein secondary structure of the breast of Beijing roast duck could effectively identify the central temperature. The SVR model was the most accurate prediction model for the center temperature.

    ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT
    Isolation, Culture, Identification and Biological Characteristics of Pig Skeletal Muscle Satellite Cells
    QIN BenYuan,YANG Yang,ZHANG YanWei,LIU Min,ZHANG WanFeng,WANG HaiZhen,WU YiQi,ZHANG XueLian,CAI ChunBo,GAO PengFei,GUO XiaoHong,LI BuGao,CAO GuoQing
    Scientia Agricultura Sinica. 2020, 53(8):  1664-1676.  doi:10.3864/j.issn.0578-1752.2020.08.015
    Abstract ( 1053 )   HTML ( 49 )   PDF (3925KB) ( 970 )   Save
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    【Objective】 The aim of this study was to establish a method for isolation, purification and identification of porcine skeletal muscle satellite cells in vitro, and to explore its biological characteristics, in order to provide a reliable cell model for further research of muscle growth and development in pigs. 【Method】 In this study, the longissimus dorsi muscle of 1 day old pig was selected and cut into meat emulsion in aseptic state. After that, it was digested with 0.2% type I collagenase for 90 min, and then digested with 0.25% trypsin for 30 min at 37 ℃. After termination of digestion, filtration and resuspension, the isolated cells were cultured in a 37 ℃ and 5% CO2 cell incubator. The skeletal muscle satellite cells were purified by repeated differential adherence technique. The first purification selection was performed after cell culturing for 2 h, and non-adherent cells were transferred to a new culture dish. After the supernatant was further cultured for 18 h, the satellite cells were purified again. The cells were subcultured or frozen when the cell density reached 70% - 80%. Cell immunofluorescence technique was used to detect the protein expression of marker genes of Pax7 and MyoD of P2 satellite cell, and the growth curve was determined. The satellite cells were differentiated into myocytes, adipocytes and osteoblasts by adding different inducing differentiation fluids. The protein expression of myoblast differentiation marker gene MHC was detected by using cell immunofluorescence to identify myotube formation in satellite cells. Oil red O staining and triglyceride content were quantified to identify the adipogenic differentiation effect in satellite cells. Alizarin red staining was used to identify the osteogenic differentiation ability in satellite cells, which were detected the expression of key genes during myogenesis, adipogenesis and osteogenesis by qRT-PCR. 【Result】 The results showed that the satellite cells with higher purity were isolated and purified by two-step enzymatic digestion and repeated differential adherence technique. The cells that were originally isolated with highly refractive, and were fusiform or spindle-shaped after adherence, after which the cells extended and began to proliferate. The results of cell immunofluorescence identification of satellite cells specific marker proteins Pax7 and MyoD were positive, indicating that the isolated cells were skeletal muscle satellite cells. Skeletal muscle satellite cell proliferation underwent the incubation period, and the growth period and finally reached the plateau phase. The satellite cells were self-fusion when the cells grew to 90% density. After myogenic induction and differentiation of the satellite cells, a large number of myotubes were formed by the adjacent satellite cells. Multinucleated myotubes were regularly arranged and the myoblast marker protein MHC staining was positive. The qRT-PCR results showed that the marker genes of MyoD and MyoG both increased first and then decreased during the process of myoblast differentiation. After adipogenic induction, the cell morphology changed into triangle, and lipid droplets appeared and aggregated into large lipid droplets with continuous induction. Oil red O staining observed a large number of red grape-like lipid droplets. Oil red O staining quantitative results showed that the triglyceride content steadily increasing during the adipogensis. There were extremely significant differences among each time point (P<0.01). The qRT-PCR results showed that the expression level of PPARγ gene was high in the middle and the late stage of induction. The FABP4 gene reached the highest at the 6th day of induction and was significantly higher than that at other time points (P<0.01). A similar dynamic was observed with the relative expression level of CEBP/β and HSL in the differentiating cells. Their expression tended to increase first and then decrease. After inducing osteogenic differentiation, it was found that the cell morphology became irregular. Cells formed bone nodules after inducing, compared with the period without induction, the alizarin red staining showed that the number and density of round opaque calcified nodules were significantly increased. The results showed that the cells appeared osteogenic differentiation. The expression levels of osteogenic marker genes BGLAP and RUNX2 also showed a steady upward trend during the inducing procession, which was significantly different from that without inducing cells (P<0.01). 【Conclusion】 This study established a method for isolation and purification of pig skeletal muscle satellite cells based on combined enzyme digestion and differential adherent technique. The obtained cells had strong proliferation ability and multi-directional differentiation potential. The results provided a technical platform for pig skeletal muscle satellite cells as seed cells for future tissue engineering research.

    Comparative Analysis of miRNA Expression Profiles in the Hearts of Tibetan Cattle and Xuanhan Cattle
    CHEN LuLu,WANG Hui,WANG JiKun,WANG JiaBo,CHAI ZhiXin,CHEN ZhiHua,ZHONG JinCheng
    Scientia Agricultura Sinica. 2020, 53(8):  1677-1687.  doi:10.3864/j.issn.0578-1752.2020.08.016
    Abstract ( 336 )   HTML ( 13 )   PDF (1271KB) ( 384 )   Save
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    【Objective】As a kind of non-coding RNA, miRNA is widely involved in various life activities of the organism. This study was aimed to explore the differential expression profiles of miRNA in the heart tissues between Tibetan cattle and Xuanhan cattle, so as to provide the basic data for further study on molecular mechanism of hypoxia adaptation in Tibetan cattle. 【Method】Each three healthy Tibetan and Xuanhan cattle were randomly selected for heart tissue sampling. RNA was extracted from tissues using the Trizol method. An 18 to 30nt fragment was selected by agarose gel electrophoresis, and 3' connector and 5' liner was ligated and then the fragment was enlarged. After gel electrophoresis, three Tibetan cattle and Xuanhan cattle libraries were established, respectively. High-throughput sequencing was performed by using the Illumina HiSeq4000 sequencing platform. The sequence was then filtered and the differentially expressed miRNA of Tibetan cattle and Xuanhan cattle were screened by comparing GenBank and Rfam databases. Functional annotation and signal pathway enrichment analysis of differentially expressed miRNA in Tibetan cattle and Xuanhan cattle. Finally, in order to verify the accuracy of the sequencing data, 8 miRNAs were randomly selected and the expression level of miRNA was detected by RT-qPCR. 【Result】The results showed that Tibetan cattle and Xuanhan cattle had high-quality reads of 17 463 446 and 13 662 812, respectively, while the clean reads were 16 552 296 and 12 055 304, respectively. The highest enrichment of high-quality nucleic acid sequences in Tibetan cattle and Xuanhan cattle were 21 nt, which were 37.5% and 32.1%, respectively. A total of 219 differential expressed miRNAs (48 up-regulated and 171 down-regulated) were obtained. There were 22 terms in the GO function annotation that significantly enriched in the molecular function of differentially expressed miRNAs target genes, such as GO: 0005488 (binding), GO: 0005515 (protein binding) and GO: 0043167 (ion binding). GO: 0005623 (cell), GO: 0044464 (cell component) and GO: 0005622 (cell) were among the 20 terms, which were significantly enriched in the cellular components. While there were 13 terms, which were significantly enriched in biological processes, such as GO: 0035556 (intracellular signal transduction), GO: 0032774 (RNA biosynthesis process) and GO: 0006351 (transcription, DNA templated). Analysis of KEGG signaling pathways revealed that miRNA target genes were significantly enriched to 232 signaling pathways, including the insulin signaling pathway (139 target genes), the mTOR signaling pathway (38 target genes) and the HIF-1 signaling pathway (92 target genes). Among them, 12 miRNA target genes worked together on these three signaling pathways. These results suggested that the differentially expressed miRNAs might participate in the regulation of hypoxia adaptation in Tibetan cattle through these three signaling pathways. Eight miRNAs were randomly selected for RT-qPCR, and the expression profiles were consistent with the sequencing data, indicating that the high-throughput sequencing data was reliable. 【Conclusion】Taken together, the expression profiles of miRNAs in the heart tissues of Tibetan and Xuanhan cattle were obtained in the present study, which laid a foundation for further research on the hypoxia adaptation mechanism of Tibetan cattle.

    Effects of Chronic Ammonia Stress on Serum Metabolites of Broilers Based on GC-MS
    SUN YongBo,WANG Ya,SA RENNA,ZHANG HongFu
    Scientia Agricultura Sinica. 2020, 53(8):  1688-1698.  doi:10.3864/j.issn.0578-1752.2020.08.017
    Abstract ( 317 )   HTML ( 26 )   PDF (2178KB) ( 543 )   Save
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    【Objective】With the development of intensive and large-scale broiler breeding, the impact of environmental conditions on the growth of livestock and poultry has become increasingly prominent. Under high-density feeding conditions, a large amount of toxic and harmful gases is generated constantly, among which ammonia is the most harmful gas which reduces the performance of livestock and poultry and threatens animal health. The purpose of this experiment was to study the effects of chronic ammonia stress on serum metabolites in broiler chickens, and to explore the changes of metabolism caused by ammonia stress in terms of small molecules and metabolic pathways, so as to provide data support for reasonable regulation of ammonia in the poultry house. 【Method】Ninety-six 21-day-old healthy AA broilers were randomly divided into control group and experimental group by a single factor completely random design, with 4 replicates in each group and 12 chickens per replicate. The control group ammonia concentration was 0 mg·kg -1, the test group ammonia concentration was 45 mg·kg -1. The experiment was carried out in an artificial simulated respiratory chamber, which was fully automated to control the temperature and humidity and the other environmental conditions. Broilers were raised in the nets and the feed and water were provided ad libitum during the entire experimental period. The trial lasted 21 days with 24 h lighting program. They were routinely managed according to the AA broiler feeding manual and immunized according to the routine procedures. Two broiler chickens pre replicate from each treatment were chosen randomly and blood samples were collected from the wing vein at 42 day-old. After pretreatment of serum samples, the metabolites were detected by GC-MS technology, and identified by mass spectral database. Principal component analysis (PCA), partial least-squares discriminant analysis (PLSDA), orthogonal partial least squares discriminant analysis (OPLSDA) and t-test were used to find the different metabolites, and then the bioinformatics methods was used to analyze the differential metabolic pathways. 【Result】Results showed that: (1) A total of 204 metabolites were detected in serum of broiler chickens by GC-MS combining with rapid identification of the detected metabolites performed with a mass spectrometry database. Metabolomics data were imported into SIMCA-P software for multivariate statistical analysis, and then t-test. Twenty-three different metabolites were identified, of which 19 were up-regulated and 4 were down-regulated. (2) Differential metabolites mainly involved energy metabolism (lactic acid, α-ketoglutaric acid), amino acid metabolism (L-Allothreonine acid, L-Homoserine, Nicotinylglycine), fatty acid metabolism (stearic acid, linolenic acid, linoleic acid, cholesterol) and nucleotide metabolism (hypoxanthine, uracil, thymine) and so on. (3) Metabolic pathway enrichment analysis showed that ammonia stress mainly affected the fat metabolism pathway in serum of broiler chickens, including linoleic acid metabolism, arachidonic acid metabolism and α-linolenic acid metabolism. 【Conclusion】 It can be seen that GC-MS can detect serum metabolites comprehensively, and screen differential metabolites accurately. Chronic ammonia stress significantly affected serum metabolites content of broilers, mainly affected fat metabolism pathway.