Scientia Agricultura Sinica

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Scientia Agricultura Sinica. 2018, 51(11): 0-0.

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Phenotypic Analysis and Gene Mapping of a Floury and Shrunken Endosperm Mutant fse3 in Rice

YU YanFang, LIU Xi, TIAN YunLu, LIU ShiJia, CHEN LiangMing, ZHU JianPing, WANG YunLong, JIANG Ling, ZHANG WenWei, WANG YiHua, WAN JianMin

Scientia Agricultura Sinica. 2018, 51(11): 2023-2037. doi:10.3864/j.issn.0578-1752.2018.11.001

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【Objective】Various types of embryo mutants and endosperm mutants in rice are excellent materials for dissecting embryo development and starch synthesis and regulation pathways. Through large scale screening and functional characterization of floury endosperm mutant with a embryo lethality phenotype, serials of genes involved in embryo development and starch biosynthesis and regulation will be obtained, which will provide the theoretical guidance for the improvement of rice quality.【Method】In this study, we obtained a stably inherited floury and shrunken endosperm mutant fse3 from japonica variety cv Ningjing 3 (NJ3), with a embryo-lethal phenotype. A hybrid F2 population of fse3 and 9311 was developed and the recessive individuals were selected to map the locus. The seeds were soaked in clear water at 30℃ for 24 h and then stained with TTC for 2 h at 35℃ in darkness to detect seed vigor. Observing the seed embryo structure which were swelled at 30℃ for 9 h with a stereo microscope. Separation of mature floury seeds from the heterozygous mutant plants, The physicochemical properties of mature grains which were grinded to brown rice flour after being peeled were analyzed. The structure of mature starch grains was observed by scanning electron microscope. Semi-thin sections were made to observe the starch grain structure of developing endosperm. The expression of starch synthesis related genes during grain filling was determined by qRT-PCR. Western-blot was used to detect the accumulation of proteins related to starch synthesis.【Result】The 1 000-grain weight, grain size, total starch content, and apparent amylose content in mature fse3 seeds were all decreased compared with wildtype, as well as the swelling power of starch. The starch viscosity curve also has significant difference with the wild type. The peak paste viscosity, the hot paste viscosity, the cool paste viscosity and the break down viscosity of the fse3 mutant are significantly higher than those of the wild type. TTC staining showed that the embryo vitality decreased and the mutant embryo can not differentiate. By observing the structure of developing endosperm, we found that a large number of small and irregular single starch granules were produced in the endosperm of fse3 mutant, and the development of compound starch granules was delayed. The cross section observation of mature seeds showed that the starch granules in the fse3 mutant were loosely packed, the size was not uniform, and there was a large gap between the particles. The FSE3 locus was first mapped to a region on the long arm of chromosome 9 with 22 recessive individuals, and then was narrowed down to a 228kb region which includes 28 open reading frames (ORFs) by using 1 400 recessive individuals. In the process of grain filling, several genes related to starch synthesis showed up-regulated and down-regulated in different degrees in the mutant. Immunoblotting showed that the protein accumulation of some amyloid synthase was reduced.【Conclusion】FSE3 is a novel floury endosperm and embryo lethality related gene, it was mapped to a 228 kb region on chromosome 9, which plays an important role in the regulation of embryo and endosperm development of rice seed.

Cloning and Expression Analyses of the mate Gene in Buckwheat

CHANG XueLing, ZHANG ZongWen, LI YanQin, GAO Jia

Scientia Agricultura Sinica. 2018, 51(11): 2038-2048. doi:10.3864/j.issn.0578-1752.2018.11.002

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【Objective】MATE (multidrug and toxic compound extrusion family) is one of the five detoxification output transporter families in organisms. MATEs as one of the largest transporter families in plants，its physiological functions reported to date are xenobiotic efflux, accumulation of secondary metabolites, the translocation and detoxification of metal ions, and plant hormone signaling transduction. The cloning and expression analyses of mate gene in Fagopyrum esculentum provide the basis for the functional study of MATE transporters in buckwheat species. 【Method】The full-length cDNA sequence of mate gene from buckwheat was obtained by 3’ and 5’ RACE cloning. The molecular weight, isoelectric point, secondary structure and transmembrane domain of MATE protein were analyzed using online analysis program. The nonrooted neighbor-joining tree of buckwheat MATE and other species MATE was generated by MEGA program to preliminarily determined the molecular structural model and the function of MATE protein in buckwheat. Quantitative RT-PCR assay were applied to determine the relative expression levels of mate in different tissues spanning developmental stages. In combination with the determination of proanthocyanidins content to investigation the physiological function of MATE transporters in buckwheat species. 【Result】Two full length cDNAs of mate genes from buckwheat were obtained by 3′ and 5′RACE cloning, named Fett12 (GenBank accession No. MG515589) and Femate3 (GenBank accession No. MG515590). Fett12 encodes a protein containing 492 amino acid residues with a molecular weight of 53.81 kD and isoelectric point of 6.75. Femate3 encodes a putative protein of 516 amino acid residues with a molecular weight of 56.12 kD and isoelectric point was 6.52. The phylogenetic tree showed that FeTT12 was belong to the proanthocyanidin MATE transporter and FeMATE3 was belong to the citrate MATE transporter. Multiple alignment and homology analysis revealed that the FeMATE3 had 96.33% identity to FeMATE2, and the FeTT12 was highly homologous to other TT12 proteins. The FeTT12 protein had the highest identity (77.3%) to MnTT12 and had the lowest identity (41.5%) to AtTT12. Furthermore, the gene expression patterns of Fett12 were correlated with the accumulation of proanthocyanidins. Results showed that the expression of Fett12 had obvious spatio-temporal specificity and the transcript of Fett12 was most abundant in buckwheat leaves. As for the accumulation pattern of PAs, the results showed significantly higher accumulation in buckwheat flower. In buckwheat seeds during the maturation, the expression levels of Fett12 increased with the development stages of seeds, and the content of PAs decreased gradually.【Conclusion】 We obtained two mate genes——Fett12 and Femate3. FeTT12 may be involved in the translocation and accumulation of procyanidins in common buckwheat. FeMATE3 may be involved in Al tolerance.

Transcriptome Analysis of Secondary Cell Wall Synthesis Regulation at Different Developmental Stages in Alfalfa (Medicago sativa L.)

LIU XiQiang, ZHANG Han, GONG Pan, GONG WenLong, WANG Zan

Scientia Agricultura Sinica. 2018, 51(11): 2049-2059. doi:10.3864/j.issn.0578-1752.2018.11.003

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【Objective】The aim of the study was to investigate the gene network regulation dynamics and expression pattern of secondary cell wall synthesis and to identify the key candidate genes and transcription factors in regulatory network for the understanding of molecular mechanism of the secondary cell wall thickening in alfalfa.【Method】The stem samples collected at vegetative stage (S1), flower bud stage (S2), early flower stage (S3) and late flower stage (S4) were used to determine the main substance contents in the secondary cell wall of the ‘Zhongmu No.1’ cultivar of alfalfa using a near-infrared-reflectance spectroscopy. The high-throughput transcriptome sequencing of these tissues were performed using an Illumina HiSeq^TM 2500 platform. The genome of Medicago truncatula was used as a reference to align the sequences and construct the Unigenes. The gene expression level was calculated in the term of Fragments Per Kilobase of transcript per Million fragments (FPKM). The thresholds to judge the significance of gene expression differences between three adjacent growth periods (S2 vs S1, S3 vs S2 and S4 vs S3) were set as fold change ≥2 or≤0.5 for up or down regulation with a false discovery rate (FDR) ≤ 0.001. The Gene Ontology database and KEGG Pathway database were used to annotate the functions and metabolic pathways of differentially expressed genes. 【Result】A total of 41 734 Unigenes were identified at different developmental stages of alfalfa in this study, of which 27 differentially expressed genes (DEGs) were annotated with functions closely related to cellulose and lignin biosynthesis. The expression levels of these DEGs gradually increased along with the accumulation of cellulose and lignin over the developmental stages. The early flower stage was found to be a transitional period at which the contents of cellulose and lignin in the secondary cell wall and expression of synthetic genes were all increased significantly. For instance, the expression of cellulose synthase genes of MTR_2g016630 (Ces) and MTR_7g103590 (Ces A1) were significantly increased at the early flower stage, the expression of lignin biosynthesis genes of MTR_1g064090 (PAL1), MTR_1g111240 (C4H) and MTR_2g104960 (CCR) were significantly up-regulated more than 10 times at the early or late flower stage compared to the vegetative stage. A total of 27 transcription factors were differentially expressed at different developmental stages of alfalfa which were related to plant growth and development, of which 18 belonged to NAC and MYB transcription factor family while the rest were of WRKY, BHLH, ERF and C3H transcription factor family.【Conclusion】We acquired the gene expression profiling data of the stem at four developmental stages of the ‘Zhongmu No. 1’ cultivar of alfalfa. A total of 54 DEGs were identified, of which 24 were up-regulated while 30 were downregulated. These genes were postulated to be involved in the synthesis and regulation of the secondary cell wall in alfalfa.

Activities of Key Enzymes in Root NADP-Dehydrogenase System and Their Relationships with Root Vigor and Grain Yield Formation in Wheat

ZHOU Yan, YANG XiWen, ZHOU SuMei, WANG YanJing, YANG Rui, XU FengDan, MEI JingJing, SHEN GuanYu, LI QiuJie, HE DeXian

Scientia Agricultura Sinica. 2018, 51(11): 2060-2071. doi:10.3864/j.issn.0578-1752.2018.11.004

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【Objective】Field experiments were conducted to explore activities of key enzymes in wheat (Triticum aestivum L.) root NADP-dehydrogenase system and their relationships with root vigor and grain yield under the condition of reducing nitrogen fertilization in the typical field on the Huang-Huai Plain, in China.【Method】A split block design was employed with 2 semi-winterness wheat cultivars, Aikang 58 and Zhoumai 27, and 6 nitrogen fertilizer application rates: 0 (N0), 135 (N1), 157.5 (N2), 180 (N3), 202.5 (N4), and 225 kg N·hm^-2 (N5). Activities of NADP-ICDH, NADP-ME and (G6PDH+6PGDH) and root vigor by improved TTC method were measured prior to wintering, and at re-growing, jointing, heading, grain filling and late dough stage. And then, the relationships between activities of these key enzymes with root vigor at different growth stages and grain yield were analyzed.【Result】The results showed that dynamics of root vigor had a “high-low-high-low” trend during the whole wheat growing period. Activities of NADP-ICDH, NADP-ME and (G6PDH+6PGDH) were all increased at first and then decreased. However, difference in activities of these key enzymes was not significant at 5% probability level after grain-filling stage. Before jointing stage, activities of key enzymes in the NADP-dehydrogenase system were greater in N0 than those in nitrogen application treatments N1-N5. However, from jointing to heading stage, activities of key enzymes and root vigor under the treatments from N1 to N5 were significantly higher than that under N0. Further analysis pointed out that both activity of NADP-dehydrogenases and root vigor were decreased with the decrease of nitrogen fertilization rate. Compared with N5, decrease both in activities of NADP-ICDH, NADP-ME and (G6PDH+6PGDH), and in root vigor under N4 were the least. Nitrogen application rate significantly affected wheat grain yield and its components. Compared with N0, wheat spikes per unit area and grains per spike were significantly increased under nitrogen treatments N1-N5. Average grain yield in 2 experimental years indicated that yield under N5 was the highest, 9 238.02 kg·hm^-2. Compared with N5, the wheat grain yield under N4 was only decreased by 0.3% while those under treatments of N3-N0 were decreased significantly. Statistical analyses showed that there were significant or extremely significant positive correlations between activities of NADP-ICDH, NADP-ME, and (G6PDH+6PGDH) at different growth stages. And there were significant or extremely significant positive correlations between activities of key enzymes in root NADP-dehydrogenase system with root vigor in middle and late wheat growing period. Grain yield had significant or extremely significant positive correlations with root vigor, activities of NADP-ICDH, NADP-ME, and (G6PDH+6PGDH). Path analysis further showed that activities of NADP-ICDH, NADP-ME and (G6PDH+6PGDH) at both jointing and heading stages had greater positive effects on grain yield in two years. Direct effects of root vigor at heading stage on yield were smaller, however, indirect positive effects of root vigor by means of increase in activities of NADP-ME and (G6PDH+6PGDH) on grain yield were significant or extremely significant.【Conclusion】Under the experimental conditions in this study, reducing nitrogen application rate from N5 to N4 could be optimum at present in wheat field in the Huang-Huai Plain, considering dynamics of activities of key enzymes in root NADP-dehydrogenase system, root vigor and grain yield. NADP-ICDH, NADP-ME and (G6PDH+6PGDH) were closely related to the "root vigor" determined by the improved TTC method. Particularly, NADP-ME and (G6PDH+6PGDH) were the most important enzymes. It was concluded that wheat root vigor could be improved by high expression of NADP-ME and (G6PDH+6PGDH) in roots, which could be achieved by means of molecular breeding and effective cultivation operations.

Effects of Non-Uniform Salt Stress on Growth Characteristics of Alfalfa

XIONG Xue, LUO JianChuan, WEI YuQi, ZHOU JiQiong, ZHANG YingJun

Scientia Agricultura Sinica. 2018, 51(11): 2072-2083. doi:10.3864/j.issn.0578-1752.2018.11.005

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【Objective】Soil salinity is often non-uniform in salt fields. Previous studies found that non-uniform salt stress could alleviate salt damage to plants compared to uniform salt treatment. The study on the response of alfalfa under non-uniform saltinity was an important supplement to plant physiology under uniform saltinity, so as to provide references for cultivation and management in salt fields.【Method】The root system of alfalfa was divided into two parts, including salt free treatment (0/0, NaCl concentrations were zero in both root systems）, non-uniform salt stress with a NaCl concentration of zero in one side of the roots (0/S: 0/100, 0/150, 0/200, 0/250), non-uniform salt stress with a NaCl concentration of 50 in one side of the roots (50/S: 50/100, 50/150, 50/200, 50/250) and uniform salt stess (S/S: 50/50, 100/100, 150/150, 200/200, 250/250 ) treatments ( “/” on both sides represent different salt stress concentrations, indicated by NaCl concentration mmol·L^-1). After treatments for 15 days, the plant growth rate, biomass, water uptake, sodium and potassium ion concentration and malondialdehyde content were measured, and the effects of non-uniform salt stress on the growth characteristics of alfalfa were studied.【Result】Salt stress inhibited the growth of alfalfa, and the plant growth rate, biomass, water uptake decreased, chlorophyll contents declined, proline contents increased, membrane lipid peroxidation aggravated, Na⁺concentrations increased, K⁺ concentrations decreased, showed a lower K⁺/Na⁺ratio. Compared with the uniform high salt S/S treatments, the plant growth rate and aboveground biomass of 0/S, 50/S treatments increased by 57.05%-369.34% and 15.47%-42.57%, respectively, and the Na⁺concentrations decreased by 15.85%-55.93%, alleviating the toxic effect of Na⁺. And 70%-92% water uptake from non-uniform salt stress came from the no-saline or low-saline part, increased water uptake of the whole plant.【Conclusion】The non-uniform salt stress treatments with a higher K⁺/Na⁺ increased the chlorophyll contents of alfalfa and decreased the degree of membrane lipid peroxidation compared with the uniform salt stress treatments. The no-saline or low-saline part showed compensated water uptake and the compensatory growth of the roots, which promote the growth of plants, increase the shoot and root biomass. The more difference between the two sides of the roots, the effects on relieve salt damage were more obvious.

Agricultural Pest Identification Based on Multi-Feature Fusion and Sparse Representation

ZHANG YongLing, JIANG MengZhou, YU PeiShi, YAO Qing, YANG BaoJun, TANG Jian

Scientia Agricultura Sinica. 2018, 51(11): 2084-2093. doi:10.3864/j.issn.0578-1752.2018.11.006

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【Objective】In agricultural pest forecasting, it is often necessary to identify several important pests from a large number of insects. At present, most of the researches on agricultural pest identification are based on limited pest species and limited sample sizes. In order to identify nine species of rice forecasting pests from a large number of agricultural insect images, a method based on multi-feature and sparse representation for pest image identification was proposed in this paper. 【Method】 Firstly, in order to obtain an optimal identification model of agricultural pests, all pest images were rotated to make insect head upright, cropped with 1:2 aspect ratio to make insect center and take up a large portion of the image, and scaled to a uniform size with 48×96 pixels. The HSV color features, LBP features, Gabor features and HOG (histogram of oriented gradient) features of each image were extracted. Then, the overcomplete dictionaries based on single features or multi-feature were constructed and each column vector represented a training sample. The same training sample species were in the same subspace. a testing sample was sparsely represented by an overcomplete dictionary and a sparse solution was obtained by solving the optimization of the l₁ norm to make different training sample species with zero or near-zero coefficient. Finally, the threshold value of sparse concentration index was used to determine the validity of the testing sample. If the sparse concentration index of a testing sample was greater than the threshold value, the pest was identified as the species with the minimizing reconstruction error. Otherwise, the testing sample was judged to be a non-forecasting pest. The support vector machine (SVM) classifiers were trained on the same features and training samples to compare with the sparse representation identification model of agricultural pests. 【Result】 In the sparse representation identification models trained on single feature, the model based on HOG features could get the higher identification rate of 87.0% and lower false detection rate of 7.5% in the nine rice forecasting pests. The sparse representation identification model based on color and HOG features obtained the highest identification rate of 90.1% and lowest false detection rate of 5.2%. The identification rate decreased and the false detection rate rose in the sparse representation models based on color, HOG and Gabor features. The identification rates of the SVM classifiers were lower and the false detection rates were higher than the sparse representation models trained on the same features. 【Conclusion】 The sparse representation pest identification model based on color and HOG features obtained the higher identification rate and lower false detection rate of agricultural forecasting pests. The threshold value of sparse concentration index could effectively exclude the non-forecasting insects. The sparse representation pest identification model could automatically identify the forecasting pests from a large number of insects.

Mining and Characterization of MicroRNAs Associated with Pathogenicity by Different Sweet Potato Viruses

LI HuaWei, LIU ZhongHua, ZHANG Hong, XU YongQing, LI GuoLiang, LIN ZhaoMiao, QIU YongXiang, LUO WenBin, JI RongChang, TANG Hao, QIU SiXin

Scientia Agricultura Sinica. 2018, 51(11): 2094-2105. doi:10.3864/j.issn.0578-1752.2018.11.007

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【Objective】Various viruses had been identified from sweet potato. Infections with different sweet potato viruses may result in different pathological symptoms of plants. The objective of this study is to investigate the microRNAs (miRNAs) in response to different pathogens from sweet potato viruses in sweet potato.【Method】Illumina RNA sequencing was performed to mine and identify the differentially expressed miRNAs among samples with different infection pathological symptoms (yellowing and deformity, herpes, chlorotic and dwarf, and curl; sample was named Fj01, Fj02, Fj03 and 1H, respectively) of ‘Longshu 9’ from Quanzhou, Fujian, China. Total and differentially expressed miRNAs among samples were identified. Real-time PCR analysis of several miRNAs and viruses was performed to validate the quality of sequencing data. Target predication and functional enrichment for these miRNAs were analyzed. Association between miRNAs and virus infections would be discussed.【Result】 After the alignment to the virus database of sweet potato, 3 samples (without 1H) were simultaneously infected with common sweet potato viruses, including Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus 2 (SPV2), Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG), and Sweet potato virus C (SPVC), although they showed different pathological symptoms. In addition, uncommon viruses were identified in different samples. Using PCR analysis of SPFMV and SPVC viruses, the consistence with sequencing data were validated. Using sequencing analysis, 679 known miRNAs and 1 004 novel miRNAs were found in these 4 samples. With comparative analysis, it was identified that 288 known miRNAs and 433 novel miRNAs were differentially expressed among these 4 samples. These miRNAs showed different expression profiles among samples, such as the members of miR-156, miR-157, and miR-166 families. qRT-PCR analysis showed these expression profiles of several miRNAs (including miR-156, novel-miR-40 and miR-319m) were in consistence with their profiles by sequencing. In comparison with that in virus-free seedling, the expression of 3 miRNAs (miR-160a, miR-2096 and miR-5387b) was differentially upregulated in these 4 sequencing samples. These results suggested that these miRNAs might be related to virus infections. Further target predication found that most of these miRNA targets were transcription factors, including genes encoding ZFP, WD, Myb, and SPL domain-containing proteins. Enrichment analysis for those targets showed that they were associated with plant growth and development, morphogenesis, and stress resistance via regulating genes, signaling pathways, and antigen recognition. These represented the multipotent functions of these miRNAs’ targets.【Conclusion】Different sweet potato viruses induced differences in miRNA profiling, and these miRNAs were mainly associated with plant growth, resistance and plant defense.

Differential Expressed Genes and their Pathways of the Resistance to Flubendiamide in Plutella xylostella

WANG ChengHua, SUN ShiQing, XU JuLong, ZHAO XiaoLong, XUE ChaoBin

Scientia Agricultura Sinica. 2018, 51(11): 2106-2115. doi:10.3864/j.issn.0578-1752.2018.11.008

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【Objective】With the widespread use of diamide insecticides, such as flubendiamide, the resistance of Plutella xylostella to these insecticides became more and more prominent. The objective of this study is to research the resistant mechanism to flubendiamide in P. xylostella from the transcriptome perspective, identify the key genes and pathways that lead to resistance to flubendiamide in P. xylostella, and to lay a foundation for revealing the mechanism of resistance to flubendiamide in P. xylostella.【Method】 The resistance to flubendiamide in P. xylostella was determined by the RNA sequencing technique (RNA-Seq) using the flubendiamide-resistant strains (R_h28), the field resistant population (R_z36) and the susceptible strain (S). The differential expressed genes (DEGs) and significantly up-regulated resistant genes were obtained in the strains/populations. The enrichment analysis of Gene Ontology (GO) and the enrichment analysis of Kyoto Encyclopedia of Gene and Genome (KEGG) were used to identify DEGs. The major biological functions, the major biochemical metabolic and signaling pathways of the DEGs were elaborated. 【Result】The different numbers of DEGs were obtained by comparing RNA-Seq data of the different resistant strains/populations with susceptible strains. GO analysis of the biological process (BP) and pathway for differential gene enrichment showed that they were mainly focused on response to stimulus, catalytic activity and metabolic process items. KEGG analysis found that the differential genes were mainly concentrated in the metabolic pathway, with the highest number of genes (17.84%). the genes with significantly up-regulated expression were further obtained, and GO analysis was carried out on 218 of them, which still mainly focused on the metabolic process, response to stimulus and biological regulation items. KEGG analysis of significantly up-regulated expression genes found that the DEGs were still mainly in the metabolic pathway. The genes of glutathione-S-transferase, cytochrome P450 detoxification enzymes, and involved in a variety of insect physiological responses to heat shock protein (Hsp) family genes were significantly up-regulated compared with the susceptible strain. The results also showed that most of the genes with significant up-regulation were concentrated in protein processing in endoplasmic reticulum, tyrosine metabolism, caffeine metabolism and the Wnt signaling pathway by heat map and clustering analysis.【Conclusion】the DEGs and the significantly up-regulated expression genes were obtained in resistant strain of P. xylostella, which of them were mainly involved in the metabolic process, the response to stress and the response to stimulus. The synergistic regulation of these genes is an important mechanism of resistance to flubendiamide in P. xylostella.

The Situation and Prospect of Research on Efficient Fertilization

BAI YouLu

Scientia Agricultura Sinica. 2018, 51(11): 2116-2125. doi:10.3864/j.issn.0578-1752.2018.11.009

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For long time, the purpose of fertilization was to increase crop yield and ensure the demand for food of human, the invention of chemical fertilizer greatly alleviated the contradiction between population growing and food demanding. However, the relationship of fertilizer application and the safety of agricultural products and ecological environment have aroused wide concern. How to find a balance between improving fertilizer benefit and reducing fertilizer application to ensure environmental safety has become an important problem for fertilizer application. For them, the main way is efficient fertilization. This paper expounds the connotation of efficient fertilization, and considers that efficient fertilization needs to be understood from four aspects: efficiency for grain products, efficiency for production, efficiency for resource and efficiency for environment. Efficient fertilization can be defined as, in every link of crop nutrient supply, the use of modern technical means to maximize the efficiency of fertilizer utilization so as to ensure the improvement of crop yield and quality, reduce the waste of resources and protect the ecological environment. This paper introduces in detail several kinds of efficient fertilization techniques which have been widely used in worldwide, such as soil testing and fertilizer recommendation, precision fertilization, fertigation technology, simple and labor saving fertilization. In the field of soil testing and fertilizer recommendation, this paper introduces the current application of this technique in the United States and Europe, and also introduces the situation of soil testing fertilizer recommendation in China since the 1970s. In the field of precision fertilization, this paper introduces its theory, core technology, development process and the technical ways to realize it, and analyzes the economic benefits of precision agriculture and its application in worldwide. In the field of fertigation, the advantages, pattern, and research process of fertigation are introduced, and emphatically introduced the surface fertigation technology, drip fertigation technology and micro-spraying fertigation technology. The paper put forward that in the foreseeable future, efficient fertilization will be developing towards to information, automation, intelligence and simplification, in particular, it is necessary to carry out in-depth research on efficiency nutrition diagnosis technology, digital nutrient management system and nutrient efficient gene screening, and we also should pay attention to research the integrated management of nutrition chain and the crop nutrition regulation technology.

Effects of Applying Biochar-Based Fertilizer and Biochar on Organic Carbon Fractions and Contents of Brown Soil

GAO MengYu, JIANG Tong, HAN XiaoRi, YANG JinFeng

Scientia Agricultura Sinica. 2018, 51(11): 2126-2135. doi:10.3864/j.issn.0578-1752.2018.11.010

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【Objective】 As an essential indicator of soil quality, soil organic carbon and its fractions play an important role in land productive capacity and crop yield. Based on a 4-year long-term experiment, effects of Biochar-based fertilizer and Biochar treatments on the contents of total organic carbon and its fractions in Brown soil were analyzed to provide scientific basis for controls of soil fertility and organic carbon management. 【Method】 Since 2011, a field experiment was conducted with five different fertilizing treatments: no fertilizer (CK), low amount biochar (C15), high amount biochar (C50), chemical fertilizer (NPK), and biochar-based fertilizer (BBF). Among them, C15 and BBF had the same level of carbon content, and NPK and BBF had the same level of nutrient. At the 4th year (autumn 2014), soil samples at 0-20 cm depth of different fertilization treatments were collected after harvest. Contents of soil total organic carbon, its fractions and peanuts yield were determined by chemical analysis. 【Result】 The results showed that compared with initial soil, soil total organic carbon content (TOC) was significantly increased by 10% and 8% under the biochar-based fertilizer and biochar treatment, respectively. It was increased from 2% to 15% (in biochar-based fertilizer treatment) under the same level carbon or NPK nutrition input. Soil free particulate organic carbon (FPOC) and soil occluded particulate organic carbon (OPOC) contents were significantly increased by applying biochar-based fertilizer or biochar. Compared with the same level carbon input treatments, FPOC and OPOC increased was 43% and 17% by applying biochar-based fertilizer, respectively; compared with the same level NPK nutrition input treatments, FPOC and OPOC increased was 40% and 43%, respectively. Soil mineral organic carbon (MOC) content was increased slightly in all fertilizer treatments, but the difference was not significant. Soil dissolved organic carbon (DOC) content was significantly increased under biochar-based fertilizer and biochar treatments, but there was no significant difference between them. The peanut yield of continuous application of fertilization was199.4-232.9 kg/667m², which was higher than no fertilizer treatment. The peanut yield was the highest when BBF was applied in soil and obviously higher than C15 and NPK treatment 17% and 10%, respectively. 【Conclusion】 Soil total organic carbon, FPOC, OPOC contents were significant increased by applying biochar-based fertilizer or biochar for more years, and the increasing amount under biochar-based fertilizer treatment was more than that under same amount carbon or NPK nutrition input. Soil DOC content was also increased, but there was no significant difference between same amount carbon and NPK nutrition input. Soil MOC content was not affected by applying biochar or fertilizer. The effect of improving peanuts yield was the best when BBF continuously was applied in soil, and it was better than the same level of nutrient treatment and the same level of carbon treatment.

Crop Yield and Soil Fertility Response to Commercial Organic Fertilizer Substituting Chemical Fertilizer

WEN YanChen, ZHANG YueDong, YUAN Liang, LI Wei1, LI YanQing, LIN ZhiAn, ZHAO BingQiang

Scientia Agricultura Sinica. 2018, 51(11): 2136-2142. doi:10.3864/j.issn.0578-1752.2018.11.011

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【Objective】The three-year continuous field experiment under winter wheat-summer maize rotation system was carried out in North China Plain, to study the effects of commercial organic fertilizer substituting chemical fertilizer on crop yields and soil fertility. Thus, the aim of present study was achieved to provide academic and practical evidence for fertilizer application.【Method】The treatments included the following three treatments: control (T1, no fertilizer added), conventional dosage of inorganic fertilizer (T2), and commercial organic fertilizer partial substituting chemical fertilizer (T3) (The substituting ratio of N, P₂O₅ and K₂O were 11.3%, 13.7%, 58.8%, respectively). In addition, the application amount of total nitrogen, phosphorus, potassium between T2 and T3 kept the same. During the mature period, crop yield, yield composition and soil fertility under different treatment were investigated. 【Result】 The results were shown as: (1) The three-year average yield of winter wheat and summer maize under T3 treatment were 7 700 kg·hm^-2and 9 175 kg·hm^-2, respectively (higher than no fertilizer treatment by 259%, 161%, respectively). (2) Compared with chemical fertilizer treatment, commercial organic fertilizer partially substituting chemical fertilizer application had no significant difference in spike numbers, grains per spike and 1 000-grain weight of winter wheat or summer maize. (3) After three-year cultivation, compared with chemical fertilizer application alone, soil organic carbon and total nitrogen content of commercial organic fertilizer partially substituting chemical fertilizer treatment increased by 19.5% and 12.3%, respectively.【Conclusion】Compared with no fertilizer treatment, fertilizer application could significantly increase crop production. Moreover, commercial organic fertilizer partially substituting chemical fertilizer maintained crop yield, and also improved soil fertility, which was beneficial to the sustainable utilization of soil resource. Therefore, commercial organic fertilizer partially substituting chemical fertilizer is a scientific strategy for fertilizer application in North China Plain.

Effects of a Mineral Conditioner on the Forms of Cd in Paddy Soil and Cd Uptake by Rice

LI Chao, AI ShaoYing, TANG MingDeng, LI LinFeng, WANG YanHong, LI YiChun

Scientia Agricultura Sinica. 2018, 51(11): 2143-2154. doi:10.3864/j.issn.0578-1752.2018.11.012

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【Objective】 The purpose of this study was to explore the effects of application of a mineral conditioner on the forms of Cd in paddy soil and Cd uptake by rice and to provide directions of field application of this conditioner. 【Method】 An alkaline mineral source composite material was selected as the tested conditioner and a pot experiment with dynamic sampling pattern was carried out to research Cd forms in paddy soil and to analyze the translocation of Cd in rice plant at different growth stages.【Result】The mineral conditioner effectively increased soil pH, reduced soil DTPA-Cd content, and continuous shifted Cd from exchangeable Cd fractions to hard usable and residual fractions. The conditioner significantly reduced Cd content in rice root, stem and leaf, glume and hulled rice, and dosages of conditioner showed negative correlation with Cd content in different parts of rice. The Cd content of different rice parts were significantly positively correlated with soil DTPA-Cd and soil Ex-Cd, but negatively correlated with soil Ox-Cd at returning green and booting stage. The growth period of rice significantly affected Cd content of root and stem leaf. In different growth periods of CK treatment, the sequence of Cd content in rice root was mature stage ≈ green stage＞jointing stage＞tillering stage＞botting stage, but the sequence of Cd content in stem leaf of CK treatment showed green stage＞mature stage＞tillering stage＞jointing stage＞botting stage; compared with CK treatment, there had largest decline of Cd content in root, stem and leaf, rice hull and hulled rice at mature stage, with 92.0%, 94.1%, 86.3%, 80.6%, respectively; when the dosage of conditioner reached to 5 g·kg^-1, the Cd content of pulled rice decreased to the national standard of food safety (0.2 mg·kg^-1). The sequence of Cd content in different rice organs showed root>>stem and leaf>>hulled rice＞rice hull. For the bio-transportation factor of Cd transforms from root to stem and leaf at different periods, by conditioner, the resistance control effect on Cd at early growth stage was superior than that in late growth stage.【Conclusion】Tested conditioner could shift Cd from high active fractions to low active fractions, and effectively restrict Cd uptake in each rice parts.

QTL Fine Mapping and Candidate Gene Prediction for Growth Traits in G.41×Malus sieversii

DONG Jun, WANG Jing, LIANG Wei, MA BaiQuan, DONG LiJuan, MA FengWang, FU XuanChang, LI CuiYing

Scientia Agricultura Sinica. 2018, 51(11): 2155-2163. doi:10.3864/j.issn.0578-1752.2018.11.013

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【Objective】Dwarf rootstock is an important germplasm to be utilized in the production of high and stable yields of apples by inducing dwarf scions. Mapping the genes that regulate the growth of apple trees has important theoretical and practical significance for improving apple's yield and quality. 【Method】 In this study, 188 F₁ progenies derived from the cross G.41×Malus sieversii were used to perform QTL mapping, and ‘Fuji Champion’ were grafted on every F₁plant. Subsequently, based on SSR markers, a genetic map of apple was constructed using Join Map 4.0 software. Using Map QTL 5.0 mapping software, the growth traits, including scion height and cross-sectional area of the scion were used as phenotypic data in QTL preliminary analysis. Combined with information from the apple genome sequence and newly developed SSR markers using Primer 5.0 software, the QTL has been defined and the candidate genes were predicted. 【Result】 In this study, a total of 108 out of 361 SSRs showed a variable degree of polymorphism, and the polymorphism rate was 29.9%. A genetic map was constructed using 95 of 108 polymorphism SSRs and four QTLs for the growth trait were detected in linkage group No.5. Two markers (L05024 and Hi09b04) were closely linked with the height and the cross-sectional area of scion. Combined with the genome sequence, 24 new SSR markers were developed, which contained 10 polymorphism markers. Subsequently, the linkage group No.5 was analyzed again using these 10 markers. The reconstructed high density linkage group, consisted of 21 SSR markers, spanning 86.0 cM with an average of 7.52 cM per marker. The QTLs responsible for the height and the cross-sectional area of the scion were defined and two relevant SSR markers (L05024 and Hi09b04) were identified accounting for 19.2% and 51.7% of the observed phenotypic variation. According to the ‘Gold Delicious’ genome sequence, this locus has been defined in a region of 543 kb that contains 16 candidate genes and a gene (MDP0000323212) was considered as the candidate gene for plant growth in this QTL interval. 【Conclusion】 In this study, genes responsible for dwarfing were located in a 4.048-4.591 Mb interval on apple chromosome No.5 and two high linked SSR markers (L05024 and Hi09b04) were identified. A gene (MDP0000323212) was identified as a potential candidate gene for growth traits.

Effects of Exogenous Citric Acids on Nutrient Activation of Calcareous Yellow Soil and Promotion effects of Nutrient Absorption and Growth of Rosa roxburghii Seedlings

GONG FangFang, FAN WeiGuo

Scientia Agricultura Sinica. 2018, 51(11): 2164-2177. doi:10.3864/j.issn.0578-1752.2018.11.014

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【Objective】In order to provide a scientific basis for nutrient regulation of Rosa roxburghii culture in calcareous yellow soil of Guizhou Karst region, the effects of exogenous citric acid on the nutrient activation of calcareous yellow soil were analysed and the effects of exogenous citric acid on nutrient absorption and growth of Rosa roxburghii on calcareous yellow soil were explored.【Method】 Taking calcareous yellow soil and 'Guonong 5' Rosa roxburghii seedlings as materials，three treatments of citric acid 40mg, 80mg and 120mg per 1000g of dry soil were set up by pot experiment method. The available nutrients, microbial numbers, enzyme activities and low molecular organic acids of treated calcareous yellow soil, and on the parameters for nutrient uptake and growth of Rosa roxburghii seedling were measured. 【Result】 The application of exogenous citric acid to calcareous yellow soil showed that the pH of soil was obviously decreased, and the contents of available N, P, K, Ca, Fe, Zn and B were significantly increased. Among the three treatments, the contents of available P, K, Ca and B in the soil were the highest in 80 mg·kg^-1 citric acid treatment, the effective content of N, Fe and Zn in soil were the highest in 120 mg·kg^-1 citric acid treatment, but the contents of Mn and Cu in soil were significantly decreased, and the content of Mg was almost no change. The total number of bacteria and the numbers of phosphate solubilizing bacteria and potassium solubilizing bacteria in soils increased with the increasing of applied citric acid amount, and the numbers of fungi and actinomycetes decreased. The numbers of phosphate-solubilizing bacteria and potassium-dissolving bacteria were the largest under the treatment of 80 mg·kg^-1 citric acid. The activities of soil enzymes were significantly enhanced after application of exogenous citric acid in limestone yellow soil, and the activities of alkaline soil phosphatase, protease and sucrase were highest in the treatment of 80 mg·kg^-1 citric acid, and the activities of urease, nitrate reductase and the activity of iron reductase increased with the increasing of citric acid application, and the amylase activity decreased with the increasing of citric acid application. The contents of citric acid, tartaric acid, malic acid and acetic acid in the soil increased obviously after exogenous citric acid was applied to calcareous yellow soil, and the contents of tartaric acid, malic acid and total content of organic acid were the highest in treatment of 80 mg·kg^-1citric acid, the contents of oxalic acid in all treatments were significantly lower than the control. After exogenous citric acid application on the calcareous yellow soil, the uptaking ratio of N, P, K, Ca, Mg, Fe, Zn and B for Rosa roxburghi seedlings were significantly increased, and the highest value was measured under 80 mg·kg^-1 citric acid concentration. The absorption of Cu for Rosa roxburghi seedlings showed no significant change with the increasing of exogenous citric acid. Exogenous citric acid application significantly reduced the content of citric acid and oxalic acid in roots of Rosa roxburghi seedlings. The applications increased the root activity and the contents of malic acid, tartaric acid, acetic acid and succinic acid, and also significantly increased the activities of nitrate reductase, alkaline phosphatase, secreted alkaline phosphatase, iron reductase and glutamine synthetase (P＜0.05). Among three treatments, the activities of root vitality, alkaline phosphatase, glutamine synthetase and exudative alkaline phosphatase were the strongest under the treatment of 80 mg·kg^-1 citric acid. The activities of nitrate reductase and iron reductase were increased with the increasing of citric acid application. The growth, root development and root morphology of Rosa roxburghi seedlings were significantly improved with all three exogenous citric acid treatments, and the largest biomass and plant height, root and shoot ratio, total root surface area, total volume and total root tip number were foundin 80 mg·kg^-1 citric acid-treated seedlings, and the above indexes were significantly larger than those of the control (P＜0.05).【Conclusion】 The application of exogenous citric acid to calcareous yellow soil with pH higher than 8 can improve the soil ecological environment and activate soil nutrients. It can enhance the activity of various soil enzymes and increase the contents of tartaric acid, citric acid, malic acid and acetic acid and total bacteria, phosphorus-solubilizing bacteria and potassium-solubilizing bacteria, and promote the growth of Rosa roxburghi seedling.

Aroma Enhancement of Ecolly Dry White Wine by Co-inoculation of Selected Pichia fermentans and Saccharomyces cerevisiae

WANG QianQian, QIN Jie, MA DeCao, TAO YongSheng

Scientia Agricultura Sinica. 2018, 51(11): 2178-2192. doi:10.3864/j.issn.0578-1752.2018.11.015

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【Objective】It is aimed to evaluate the aroma enhancement of Ecolly dry white wine produced by simultaneous inoculation of one selected Pichia fermentans and Saccharomyces cerevisiae yeast, and to optimize fermentation process of Ecolly dry white wine.【Method】The selected strain P. fermentans (H5Y-28) was co-inoculated in synthetic grape must with a strain S. cerevisiae yeast (F5) at the ratios of 10:1, 4:1, 1:1, 1:4, 1:10, respectively; and single inoculation of S. cerevisiae was set as control. Yeasts biomass and viable cells count of all the samples were monitored during fermentation and the results were used to build yeasts growth kinetics. For fermentation test of Ecolly dry white wine, same ratios of P. fermentans (H5Y-28) and S. cerevisiae yeast (F5) were inoculated on Ecolly must, single inoculation of S. cerevisiae and single inoculation of S. cerevisiae with extracellular enzyme extract from 72h P. fermentans (H5Y-28) culture were used as controls. Volatile aroma compounds analysis and sensory analysis of Ecolly wine samples were conducted by SPME-GC–MS and a well-trained tasting panel in the next April.【Result】Yeasts growth kinetics showed that the amount of viable cells and the survival duration of the selected strain obviously increased with the higher inoculation ratio of P. fermentans in model fermentation, but 10:1 and 4:1 inoculation lowered the total amount of S. cerevisiae yeast. Sensory analysis of Ecolly dry white wine samples revealed that mixed fermentation could improve intensities of fruity and flower characteristics, particularly tropical fruity note. However, higher inoculation ratios of P. fermentans (10:1) caused intense scent of green trait while wine samples of 1:1 and 4:1 only induced weak scent of green trait. Volatile aroma compounds analysis showed that the total concentration of varietal aroma compounds increased significantly as the inoculation ratio of P. fermentans increased, especially terpenols and C13-norisoprenoids. Moreover, the addition of extracellular enzyme extract improved the total level of varietal aroma compounds, especially C13-norisoprenoids (90% higher than the single use of S. cerevisiae). In terms of fermentative aroma compounds, mixed fermentation remarkably elevated the concentration of acetate, C6-C12 fatty acids and their corresponding ethyl esters, isoamyl alcohol and phenyl ethanol. Compared with the wine sample of single use of S. cerevisiae, inoculation ratio of 1:1 improved the contents of varietal aroma compounds and fermentative aroma compounds by 26% and 39%, respectively, and the total content of medium-chain fatty acids ethyl esters in wine sample of 1:1 was notably 40% higher than the control (single use of S. cerevisiae). 【Conclusion】 Mixed fermentation at the ratio 1:1 of P. fermentans and S. cerevisiae has no negative effect on the growth of S. cerevisiae yeast,andhas a great potential to improve the quality of the wine aroma of Ecolly dry white wine.

Effects of the Milk Replacer on the Development of Intestine, the Flora Diversity and the Relative Expression of Glucose Transporter Gene of Early Weaned Yimeng Black Goat Lambs

LI YongZhu,JIN TaiHua, HAN ZhaoQing, XIA ChunFeng, CHAO HongYu, ZHANG NaiFeng, WANG ShiQin, DIAO QiYu

Scientia Agricultura Sinica. 2018, 51(11): 2193-2205. doi:10.3864/j.issn.0578-1752.2018.11.016

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【Objective】 Thisstudy was conducted to evaluate the effects of the milk replacer on the intestinal development, the flora diversity and the relative expression of glucose transporter gene of Yimeng black goat lambs in order to provide theoretical basis for the cultivation of early stage Yimeng black goat lambs. 【Method】 A total of 36 twin born Yimeng black goat lambs were randomly divided into 2 groups, each with 6 replicates and each replicate 3 lambs. The lambs in the control group (group I) were fed with breast milk for 75 days, while the lambs in the experimental group (group II) were weaned on the 10th day and fed with milk replacer thereafter. The weight of duodenum, jejunum, ileum as well as live body of the lambs in the two groups were recorded on the 10, 15, 25, 45 and 75th day, and meanwhile, the small intestinal contents and mucous tissue samples were collected. The colonization of small intestinal flora and the expression of glucose transporter (SGLT-1, GLUT-2) gene mRNA were analyzed by the PCR-DGGE of 16S rDNA and real-time fluorescence quantitative PCR, respectively. 【Result】 The results showed that the average daily gain and the weight of jejunum and ileum of the lambs in group II on the 45th day were significantly higher than those in group I (P＜0.05), while on the 75th day, the average daily gain and ileum weight of the lambs in group II were highly significantly higher than those in group I (P＜0.01). The flora distribution difference between group I and group II was significant in the duodenum on the 45 and 75th day, in the jejunum on the 15th day and in the ileum on the 10th day. The flora diversity in the ileum was higher than that in the jejunum and duodenum, and the microbiota diversity in the jejunum was higher than that in the duodenum. The bacterium lacticum dissolving starch and prevotella were dominant bacteria in the whole intestinal tract of Group I and II. The number of probiotics in group I decreased with the growth of age, while an opposite dynamic was found in the group II. The expression of SGLT-1 and GLUT-2 genes in the group II was the lowest at the 10 d, but their expression increased on the 25th day, meanwhile the two genes took on different expression profiles in different intestinal segments. The fluctuation of expression level of SGLT-1 was the highest and lowest in the jejunum and duodenum, respectively, while that of GLUT-2 was the biggest and lowest in the duodenum and ileum, respectively.【Conclusion】 In conclusion, feeding milk replacer could promote early colonization of probiotics in the jejunum and ileum of early weaned Yimeng black goat lambs on the 25th day and the glucose absorption and transport could be changed by regulating glucose transporter gene expression, which further affected later development of intestinal tissue and the growth of the lambs.

Expression and Evaluation of Protective Efficacy of No-toxic Clostridium perfringens ε Toxin Derivative

DU JiGe, XUE Qi, ZHU Zhen, LI QiHong, YIN ChunSheng, YAO WenSheng, KANG Kai, CHEN XiaoYun

Scientia Agricultura Sinica. 2018, 51(11): 2206-2215. doi:10.3864/j.issn.0578-1752.2018.11.017

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【Objective】This experiment was conducted to obtain no-toxic Clostridium perfringens ε toxin (ETX) derivative and subsequently evaluate the virulence and immunogenicity of it. 【Method】The ETX gene of Clostridium perfringens type D strain was optimizedaccording to Escherichia coli (E. coli) expression system codon preferences. At the same time, H106 and F199 were substituted with proline and glutamic acid, respectively, following with synthesis of GETX_m. And this synthetic fragment was then cloned into prokaryotic expression vector pET30a-(＋) . Subsequently, pET30a- GETX_m2 was transformed into BL21 (DE3) competent cells and induced by IPTG at 15℃ and 37℃ for 16 h and 4 h, respectively. The supernatant and the precipitate of the cells broken by ultrasonic were collected and subjected to SDS-PAGE and Western blot to detect the expression and solubility of recombinant protein, rETX_m2. rETX_m2 expressed in a soluble form was then purified by Ni-IDA chromatograph. The reactivity of rETX_m2 with antiserum of Clostridium perfringens type D was detected by Western blot. Meanwhile, rETX_m2 was diluted with cell maintenance medium up to 100 and 10 μg·ml^-1 and then incubated with Canine Kidney (MDCK) cells to detect the cytotoxicity of it. Moreover, rETX_m2 and rETX_m2activated by trypsin were tested for the virulence to mice by tail vein injection at doses of 0.0625, 0.625 and 6.25 mg·kg^-1, respectively. According to the method prescribed in Chinese Veterinary Pharmacopoeia (2015), four rabbits were immunized subcutaneously with 100 μg of rETX_m2 emulsified with oil adjuvant of ISA 201 twice (at an interval of 2 weeks). Meanwhile, rabbits of adjuvant control group were immunized with mixture of Montanide ISA 201 adjuvant and PBS. Serum samples were collected 14 d after the first immunization and 21 d after last immunization to detect the neutralizing titer against the Clostridium perfringens type D toxin. At the same time, rabbits were challenged with 1 rabbit MLD Clostridium perfringens type D toxin through the ear marginal veins 21 days after the second immunization to detect the protective efficacy of rETX_m2. 【Result】 rETX_m2 was expressed in both soluble and insoluble form (inclusion bodies) after induced at 15℃ and 37℃. Considering the expression level, solubility and time of induction, rETX_m2expressed as soluble form induced at 37℃ was purified. The results of gray scale scanning showed that the rate of rETX_m2 expressed as soluble form was up to 30%. And it could react with the antiserum of Clostridium perfringens type D with specific band. Cytotoxicity assays showed that there was no cytopathic effect (CPE) after incubation in cell culture medium with rETX_m2 at a concentration of 100 μg·ml^-1 for 24 h, whereas Clostridium perfringens Type D toxin with 2 000-fold dilution induced apparent CPE, characterized by predominant lysis. At the same time, rETX_m2 with the injection volume of 6.25 mg·kg^-1 still was not fatal to mice. After the first immunization, sera from rabbits immunized with rETX_m2could neutralize 450-750 mice MLD Clostridium perfringens type D toxin per ml, and 2 500-4 000 mice MLD after twice immunization. Moreover, rabbits in rETX_m2 immunized group fully survived at the dose of 1 rabbit MLD of Clostridium perfringens type D toxin challenge, whereas all of the rabbits died (4/4) in the control groups. 【Conclusion】The results suggest that rETX_m2 without virulence retains the good immunogenic antigen, which is an ideal candidate antigen for genetic engineering subunit vaccine of Clostridium perfringens.

Protein Components of Degumming Bombyx mori Silk

ZHANG Yan, DONG ZhaoMing, XI XingHang, ZHANG XiaoLu, YE Lin, GUO KaiYu, XIA QingYou, ZHAO Ping

Scientia Agricultura Sinica. 2018, 51(11): 2216-2224. doi:10.3864/j.issn.0578-1752.2018.11.018

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【Objective】The objective of this study is to explore the changing pattern of protein component in the degummed silk fiber of silkworm (Bombyx mori), which will lay a foundation for understanding the distribution feature of silk proteins in the silk fiber, and will also provide references for improving silk processing technology.【Method】Sericins were removed from the silk fiber using various methods (including papain, sodium carbonate, urea, neural soap, and alkaline water). SDS-PAGE was used to detect the degumming degree and degumming effect on the fibroins by using different methods and different silk products. Silk proteins were extracted using LiSCN and then digested into peptides by using FASP method. Liquid chromatograph-tandem mass spectrometer was explored to identify the protein component in the degummed cocoon silk by using different methods and different silk products. The identified silk proteins were annotated by using bioinformatics. The abundances of silk proteins in different samples were compared by label-free quantitative method. 【Result】 Degumming of cocoon silk was performed by using five methods. Protein electrophoresis result showed that papain could degrade sericins to remove them from silk, but could not degrade the fibroins. Degummed by sodium carbonate caused slight degradation of sericins and serious degradation of fibroins, and degummed by neural soap and urea could strongly degrade both the sericins and fibroins, while degummed by alkaline water was not efficient. Proteomics research found that degummed silks by using papain and neural soap contained little sericins, but had fibroins, seroin 1 and glycine-rich protein. Degummed silk by using urea or sodium carbonate had fewest proteins when compared with the degummed silk by other methods, the major component of which were fibroins and the glycine-rich protein. Proteins in six silk products were detected by protein electrophoresis. The result showed that fibroins were relatively integrate in the raw silk and raw silk fabric, and partly degraded in the silk fabric, silk satin, and silk habotai, while serious degraded in the old silk clothes. The proteome analysis showed that raw silk and raw silk fabric mainly contained three fibroins, sericins 1, seroin 1, osiris 9a, glycine-rich protein and serine protease inhibitor SPI51, and silk fabric, silk satin, and silk habotai mainly had three fibroins and seroin 1, while old silk clothes mainly had three fibroins and the glycine-rich protein. 【Conclusion】 Degumming using papain could keep the maximum integrity of silk fiber, and degumming by sodium carbonate and urea are the best methods to removing sericins. Raw silk and raw silk fabric contain many sericins, and have integrated silk fiber. Silk fabric, silk satin, and silk habotai contain little sericins, and have partly-degraded silk fiber. After complete degumming, the silk still contain fibroin heavy chain, fibroin light chain, fibroin p25, seroin 1 and glycine-rich protein.

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