Table of Content

16 January 2019, Volume 52 Issue 2

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Phenotypic Analysis of Epoxygenase-Transgenic Soybeans

HAO QingTing,ZHANG Fei,JI XiaJie,XUE JinAi,LI RunZhi

Scientia Agricultura Sinica. 2019, 52(2):  191-200.  doi:10.3864/j.issn.0578-1752.2019.02.001

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【Objective】 Epoxy fatty acids (EFAs) derived from plants are renewable materials for production of high-valued chemical products. Such unusual fatty acids (UFAs) are only highly synthesized and accumulated in some wild plant seeds, with difficult in utilization on a large scale. This study was conducted to assembly epoxy fatty acid biosynthesis pathway in developing seeds of soybean (Glycine max (L.) Merr) for commercial production of these unusual fatty acids. 【Method】 In this paper, SlEPX, an epoxygenase gene from Stokesia laevis (a high accumulator of EFAs) was cloned into pCAMBIA1301 expression vector driven by a seed-specific promoter. Soybean (cv. Jack) was genetically transformed using the particle bombardment method based on somatic embryogenesis system. The high-generation lines of SlEPX-transgenic soybean with stable phenotypes were obtained by continuous selection and identification. The integration of heterologous SlEPX gene and its expression profiles were examined by PCR and Real-time quantitative PCR, respectively. Seed phenotypic examinations were statistically analyzed including seed morphology, size, 100-seed weight and germination rate. Seed oil and protein contents and other physiological properties were measured by gas chromatography and Kjeldahl method. 【Result】 The results showed that SlEPX gene was stably integrated into soybean genome, with its accurate and effective expression in developing seeds of high-generation soybeans. EFAs were newly synthesized but low content (2.9%) in SlEPX-transgenic soybean seeds, and linoleic acid (18﹕2Δ9,12）was accordingly reduced by 8%. Compared to the control, the transgenic soybean seeds were a little longer and wrinkled seed coat. The percentage of small seeds (diameter <4mm) was increased significantly in the transgenic soybean. Seed germination rate had no difference between transgenic and control whereas the transgenic plant exhibited slow growth. The oil and protein content as well as 100-seed weight of transgenic soybean seeds were reduced by 5%, 6% and 8.28%, respectively. Further biochemical analysis demonstrated that newly-synthesized vernolic acid (one kind of EFAs) in the transgenic seeds mostly bound to phosphatidylcholine (PC) (12.6% of total fatty acid) while only a small amount existed in triacylglycerol (TAG) (2.3%). These data indicated that heterologous SlEPX enzyme did correctly catalyze oleic acid (18﹕2Δ9,12) to vernolic acid (Va)(12-epoxy-18﹕1Δ9) in the transgenic soybean seeds. However, most of Va molecules were accumulated in PC (the major cell membrane lipid) but not in storage TAG. A large amount of Va bound to PC could damage cell membrane homeostasis, causing unfavorable phenotypes in transgenic soybeans. 【Conclusion】 The present study revealed that the overexpression of a heterologous epoxygenase alone in soybean developing seeds can catalyze biosynthesis of EFAs at small amount, but results in some undesirable agronomy traits. It is needed to further co-express the acyltransferase that can specifically transfer the Va-acyl group from PC into TAG molecules in SlEPX-transgenic soybean for enriching epoxy fatty acids in TAG and simultaneously, to eliminate negative effect caused by EFA accumulation in cell membrane.

Cloning Expression Analysis and Transformation of MsGAI Gene from Medicago sativa L

ZHANG Han,WANG XueMin,LIU XiQiang,MA Lin,WEN HongYu,WANG Zan

Scientia Agricultura Sinica. 2019, 52(2):  201-214.  doi:10.3864/j.issn.0578-1752.2019.02.002

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【Objective】 DELLA, one of GRAS family proteins, is an important transcription factor, which negatively regulates the gibberellin (GA) signal pathway. In this study, the Medicago sativa L. DELLA gene MsGAI was cloned, and its secondary structure was also predicted through bioinformatics. To illustrate the molecular function of MsGAI in M. sativa L. gibberellin (GA) signal transduction pathway, we clarify the spatio-temporal expression pattern of MsGAI and its response to different treatments; and the transgenic alfalfa over-expressing MsGAI were obtained by Agrobacterium-mediated transformation to explore gene function as well.【Method】 MsGAI was cloned by homologous cloning and sequence characteristics of MsGAI were analyzed by online bioinformatics tools. The phylogenetic tree of MsGAI and its homologous genes from other species was constructed by MEGA 7.0. The real-time quantitative PCR (qRT-PCR) was used to detect the spatio-temporal expression pattern of GAI and its response to abiotic including PEG, NaCl, GA, ABA and dark. The plant overexpression vector 35S:MsGAI-gus was transferred into Agrobacterium GV3101 strain, and transgenic alfalfa were obtained by Agrobacterium-mediated callus transformation. The transgenic positive seedlings were verified by PCR and GUS histochemical staining. Three transgenic line (L5, L8, L11) were selected to analysis MsGAI expression under PEG, and NaCl treatment.【Result】 Sequence analysis showed that 605 amino acids constitute MsGAI and it contains the conserved DELLA and VHYNP sequences in N-terminal regulatory region, and SAW in C-terminal. Multiple sequence alignment and phylogenetic tree analysis showed that the sequence similarity with other DELLA protein was as high as 80%, and it has the closest relationship with M. truncatula GAI, followed by the dicotyledonous such as Cicer arietinum and Trifolium pratense, and is far away from monocotyledon such as Hordeum vulgare. Real-Time PCR analysis showed that MsGAI had the highest expression in roots and after PEG, NaCl, GA and ABA treatment, the response was obvious while dark treatment significantly inhibited the expression of MsGAI. The GUS tissue staining showed that the positive plants were blue and the control group was white and the expression of MsGAI was upregulated in positive plants under PEG and NaCl treatments.【Conclusion】 The cloning and overexpression vector of DELLA protein in alfalfa was successfully constructed and the MsGAI could respond to stress treatments.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Characteristics of Good Taste and High Yield Type of Single Cropping Late Japonica Rice in Southern China

HU Lei,ZHU Ying,XU Dong,CHEN ZhiFeng,HU BingQiang,HAN Chao,QIU Shi,WU Pei,ZHANG HongCheng,WEI HaiYan

Scientia Agricultura Sinica. 2019, 52(2):  215-227.  doi:10.3864/j.issn.0578-1752.2019.02.003

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【Objective】 The objective of this study was to explore the differences in the yield and quality of the single cropping late japonica rice between different levels of taste value and the types of yield in southern China, and to elucidate the characteristics of good taste and high yield varieties in southern China. 【Method】 48 single cropping late japonica rice varieties were used as materials in the article, and they were divided into four types of good taste and high yield, good taste and medium yield, medium taste and high yield, medium taste and medium yield according to the yield and taste value. In order to reveal the characteristics of single cropping late japonica rice varieties with good taste and high yield in southern China, three types of good taste and high yield, good taste and medium yield, medium taste and high yield were selected for comparative study of yield and quality according to the actual production demands. 【Result】 The comprehensive of the type of good taste and high yield was 26.69% higher than the type of medium taste and high yield. Appearance, viscosity and balance degree indexes in comprehensive were 38.81%, 36.30% and 37.40% higher than the type of medium taste, respectively. The results showed that, the brown rice rate, milled rice rate, head milled rice rate, chalkiness rate and chalkiness degree of the type of good taste and high yield were 0.34%, 6.92%, 7.13%, 40.39% and 47.56% higher than that of the type of good taste and medium yield, respectively. The length of the gel consistency of the type of good taste and high yield was 19.92% longer than the type of medium taste and high yield, while the amylose and protein content of that were 37.67% and 33.08% lower, respectively. Compared with the type of good taste and medium yield, the proportions of the number of grains per panicle, seed setting rate, 1000-grain weight, sunshine duration during the grain filling period, earing rate, grain density, and the bioaccumulation from heading to maturity accounted for the total biomass of the type of good taste and high yield were 4.47%, 3.29%, 5.72%, 16.25%, 11.09%, 15.89% and 21.49% higher, respectively, while the decreasing rate of leaf area was 13.51% lower. In the period from heading to maturity, good taste and high yield type’s crop growth rate, net assimilation rate and photosynthetic potential were 24.46%, 14.62% and 19.01% higher than that of good taste and medium yield type, respectively. 【Conclusion】 Good taste and high yield type of single cropping late japonica rice had the following characteristics: The milling quality had reached the first level of Chinese standard; The transparency level was 3; The chalkiness rate and the chalkiness degree were between 50% to 85%, and 20% to 35%, respectively; The content of amylose and protein were around 10.0% and 8.0%, respectively; The length of gel consistency was over 90 mm; The setback was below -500cP and the consistence was between 550cP and 650cP. The seed setting rate of the good taste and high yield type was about 90.0%, the 1000-grain weight had more than 25.0 g. It was also important to maintain the suitable dry matter accumulation in the early stage of heading period, and to ensure the leaf area index, dry matter accumulation and the proportion of the total growth period with high levels after the heading period.

Difference of Traits Relating to Lodging Resistance in Hulless Barley Genotypes

BAI YiXiong,YAO XiaoHua,YAO YouHua,WU KunLun

Scientia Agricultura Sinica. 2019, 52(2):  228-238.  doi:10.3864/j.issn.0578-1752.2019.02.004

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【Objective】 Lodging has become one of the main factors affecting the production and yield of hulless barley. Screening the traits closely related to the lodging resistance and constructing the lodging resistance evaluation system is an important theoretical basis for the breeding of hulless barley varieties. 【Method】 23 agronomic traits in roots, stems, and ears related to the lodging of 35 hulless barley germplasms were analyzed by statistical method. The variance analysis was carried out to identify the differences among different traits, and correlation analysis was used to screen out the traits which closely related to the lodging resistance. The indexes were finally constructed by principal component analysis and linear stepwise regression analysis to construct a hulless barley resistance evaluation system. 【Result】 The results showed that different genotypes of barley cultivars had larger differences among the same traits, and the differences of genotypes among the phenotypic traits were extremely significant, among this, the genetic variation of lodging rate was the most abundant. The agronomic traits of the same genotypes were greatly affected between different ecoregion, and the genetic variation of the genotypes in Haibei alpine farming-pastoral ecotone was abundant. There were significant interaction effects between genotypes of various traits and environmental factors (P<0.05). Correlation analysis showed that the stem strength was most closely related to the lodging resistance of the hulless barley, which was used lodging resistance index to construct the lodging resistance evaluation system. When the plant had more tiller numbers, longer third and fourth stem segments, which caused the lodging, and made the number of grains reduced, and the ear weight became lighter. When the root dry weight was heavier, the stem was heavier, the wall thickness was thicker, and the stem strength was larger, which made the plants have the stronger ability to retain and lodging resistance. 【Conclusion】 Based on tiller numbers, ear weight, stem length, stem weight and stem strength, a comprehensive evaluation system for the resistance indexes of barley was constructed. The results showed that the system was reliable and could be used for the evaluation of lodging resistance of hulless barley germplasms.

PLANT PROTECTION

Identification, Distribution and Occurrence of Viruses in the Main Vegetables of China

LIU Yong,LI Fan,LI YueYue,ZHANG SongBai,GAO XiWu,XIE Yan,YAN Fei,ZHANG AnSheng,DAI LiangYing,CHENG ZhaoBang,DING Ming,NIU YanBing,WANG ShengJi,CHE HaiYan,JIANG Tong,SHI XiaoBin,HE ZiFu,WU YunFeng,ZHANG DeYong,QING Ling,YAN WanRong,YANG XueHui,TANG YaFei,ZHENG HongYing,TANG QianJun,ZHANG SongBai,ZHANG DongFang,CAI Li,TAO XiaoRong

Scientia Agricultura Sinica. 2019, 52(2):  239-261.  doi:10.3864/j.issn.0578-1752.2019.02.005

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【Objective】 The objective of this study is to determine the pathogens, dominant viruses, distributions of Solanaceae, Cucurbitaceae, Leguminosae, and Cruciferae vegetables viral diseases, and to analyze the expansion trend of some important viruses in China.【Method】 Viral disease survey and virus detection using a combination of serological and molecular biology methods on Solanaceae, Cucurbitaceae, Leguminosae, and Cruciferae vegetables were carried out in 31 provinces (municipalities, autonomous regions) of mainland China from 2013 to 2017.【Result】 A total of 41 653 suspected virus disease samples of Solanaceae, Cucurbitaceae, Leguminosae and Cruciferae vegetables were collected from the 31 provinces (municipalities, autonomous regions) across the country. A total of 63 viruses were detected, of which 40 viruses were detected in solanaceous vegetable crops, as well as 26 viruses in cucurbitaceous vegetable crops, 19 viruses in leguminous vegetable crops, and 14 viruses in cruciferous vegetable crops. Among them, 33 and 25 viruses were detected in pepper and tomato of solanaceous samples, respectively; 22 and 19 viruses were detected in pumpkin and cucumber of cucurbitaceous samples, respectively; 14, 12, 12 and 7 viruses were detected in cowpea, kidney beans, radish and Chinese cabbage, respectively. Mixed infections were common in the vegetable crops with two to six viruses respectively, most of the co-infected crops were infected with two viruses and some of the pepper, tomato and eggplant plants were infected with six viruses. Tomato mottle mosaic virus (ToMMV), Zucchini tigre mosaic virus (ZTMV), Pepper vein yellows virus (PeVYV), Pepper cryptic virus 1 (PCV-1) and Pepper cryptic virus 2 (PCV-2) were firstly recorded in China. ToMMV, Melon aphid-borne yellows virus (MABYV), Cucurbit aphid-borne yellows virus (CABYV) were firstly detected on pepper plants, ZTMV and Tobacco mild green mosaic virus (TMGMV) were firstly recorded on cucumber and pumpkin plants, respectively. While Tomato spotted wilt tospovirus (TSWV) was firstly detected on celery, Datura stramonium, cowpea, pea, Codonopsis pilosula, dahlia, Tropaeolum majus, Solanum indicum, and so on. Chilli veinal mottle virus (ChiVMV) was firstly found infecting Solanum yingjiangense. Meanwhile, pepper and tomato plants were found as the new natural hosts of Tobacco bushy top virus (TBTV). 【Conclusion】 Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Turnip mosaic virus (TuMV) and Broad bean wilt virus 2 (BBWV2) are the dominant viruses currently in China due to their widely distribution and serious damage on vegetable crops nationwide. Among them, CMV occurs in all of the 31 provinces (municipalities, autonomous regions), and is also the dominant virus in the all regions. CMV and TMV are the dominant viruses on solanaceous vegetable crops, while CMV, Zucchini yellow mosaic virus (ZYMV), Cucumber green mottle mosaic virus (CGMMV) and TMV are the dominant viruses on cucurbitaceous vegetable crops. CMV and BBWV2 are the dominant viruses of leguminous vegetable crops, and TuMV, CMV, TMV are the dominant viruses of cruciferous vegetable crops. Tomato chlorosis virus (ToCV), TSWV, CGMMV and ToMMV, which occurring seriously in some provinces and spreading rapidly, may have the risk of national epidemic.

Analysis of Main Pathogens and Dominant Species of Maize Stalk Rot in the Main Summer Maize Producing reas of Huang-Huai-Hai

LIU ShuSen,MA HongXia,GUO Ning,SHI Jie,ZHANG HaiJian,SUN Hua,JIN Ge

Scientia Agricultura Sinica. 2019, 52(2):  262-272.  doi:10.3864/j.issn.0578-1752.2019.02.006

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【Objective】 The objective of this study is to determine the main pathogens and dominant species of maize stalk rot in the main summer maize producing areas of Huang-Huai-Hai, and to provide a basis for understanding the pathogenic mechanism, resistant breeding, and ultimately maize stalk rot management.【Method】 In 2014-2017, 850 samples of maize stalk rot were collected from three provinces (Hebei, Henan and Shandong) in the main summer maize producing areas of Huang-Huai-Hai. The isolated fungi or oomycetes were morphologically characterized and confirmed by using universal and specific primers, those primers were also used for detecting pathogens in diseased tissues. The combined results of isolate identification and tissue molecular detection were analyzed to determine the main pathogens and the dominant species. The detection rates of main pathogens in different provinces and different years of the same province were analyzed to reveal the population dynamics. The detection rates of pathogens in individual samples were also analyzed to determine the coexistence patterns of multiple pathogens.【Result】 Fungi or oomycetes were detected in 667 samples, accounting for 78.47% of all samples. The detection rates were different among years, less than 50% in 2014 and more than 90% in 2015-2017. Detected fungi or oomycetes were classified to 46 species of 20 genera. Fusarium spp. had the highest detection rate of 89.96%, including F. graminearum species complex, F. proliferatum, F. verticillioides, F. chlamydosporum, F. subglutinans, F. oxysporum, F. culmorum, F. fujikuroi, F. incarnatum, F. equiseti and F. solani. In addition, Pythium spp. had the second highest detection rate of 34.18%, including P. aristosporum, P. graminicola, P. acanthicum, P. amasculinum and P. oligandrum. The detection rates of four main pathogens, F. verticillioides, F. graminearum species complex, P. aristosporum and F. proliferatum, were 62.07%, 46.93%, 29.09% and 28.04%, respectively. It indicated that the dominant species was F. verticillioides. There were some differences in the detection rates of the four main pathogens among provinces. The detection rate of F. verticillioides in Hebei Province was 73.98%, which was significantly higher than that in Henan and Shandong Provinces, and the detection rates of F. graminearum species complex in the three provinces were similar. The detection rates of F. proliferatum and P. aristosporum in Shandong Province were 35.78% and 34.31%, respectively, which were higher than those in other two provinces. The detection rates of the four main pathogens from the same province were dynamic in different years, and any one of them can be the dominant species. Furthermore, several pathogens could be detected in a single sample. While 38.38% samples were colonized by only one pathogen, 29.24% and 19.04% samples were colonized by two and three pathogens. The patterns of two or multiple pathogens in single samples were mainly coexistence of Fusarium spp. and Pythium spp., or coexistence of Fusarium spp. and Fusarium spp..【Conclusion】 The main pathogens of maize stalk rot in the main summer maize producing areas of Huang-Huai-Hai are F. verticillioides, F. graminearum species complex, P. aristosporum and F. proliferatum, and the dominant species is F. verticillioides. The detection rate of F. verticillioides in Hebei Province and that of F. proliferatum and P. aristosporum in Shandong Province are the highest, and the detection rate of F. graminearum species complex in the three provinces is similar. There is a coexistence pattern of two or multiple pathogens in a single sample.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Spatial Differentiation and Impact Factors of Grain Yield Per Hectare in Weibei Plateau Based on GWR Model: A Case Study of Binxian County, Shannxi

QIU MengLong,CAO XiaoShu,ZHOU Jian,FENG XiaoLong,GAO XingChuan

Scientia Agricultura Sinica. 2019, 52(2):  273-284.  doi:10.3864/j.issn.0578-1752.2019.02.007

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【Objective】 This research was conducted to reveal the spatial differentiation characteristics and influencing factors of grain yield per hectare on the county scale in the Loess Plateau of Weibei, and to provide scientific references for similar researches on small scale and improvement of regional grain output. 【Method】 The spatial distribution characteristics of grain yield per hectare and spatial heterogeneity of its influencing factors were analyzed by using spatial autocorrelation, least square method and geographically weighted regression model in Binxian county of Shannxi province -a main grain producing county in Weibei Plateau. 【Result】 The Moran's I index of grain yield per hectare in Binxian County was 0.328, and the Z value of significance test was 5.51, and the characteristics of local spatial agglomeration were north high and south low. Slope, plough layer thickness, soil organic matter, road density and cost of fertilization had a positive effect on the grain yield in Binxian County. Soil type, erosion degree and groundwater depth had a negative influence on the grain yield in Binxian County. The relative range of regression coefficients for explanatory variables was between 0.55-14.11. In space, plough layer thickness, soil type, erosion degree, soil organic matter and road density had a stronger influence on the grain yield of the hilly and gully areas in the South and southeast in Binxian County than that in the northern Loess Plateau; while slope, groundwater depth and cost of fertilization showed opposite spatial non-stationary characteristics. The significance of regression coefficient of OLS model was negatively correlated with the relative range of regression coefficient of GWR model. The R ² of the GWR model was 0.04 higher than that of the OLS model, and the AIC value was reduced by 11.04. 【Conclusion】 There was a significant positive spatial correlation in grain yields per hectare of Binxian County. Soil organic matter, cost of fertilization and groundwater depth were the most important factors influencing grain yield per hectare in the county of Weibei Plateau. The influence degree of influencing factor on grain yield per hectare was of great difference in different spatial location, and the spatial non-stationarity of the influencing factors was the main reason for the lower significance level of regression coefficient of OLS model. The GWR model had better explanatory power and accuracy in modeling spatial non-stationary data than OLS model. And the spatial visualization of model estimation parameters could be realized by GWR model.

Response of Maize Roots to Different Additive Amounts of Weathered Coal Humic Acids

ZHOU LiPing,YUAN Liang,ZHAO BingQiang,LI YanTing,LIN ZhiAn

Scientia Agricultura Sinica. 2019, 52(2):  285-292.  doi:10.3864/j.issn.0578-1752.2019.02.008

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【Objective】 Weathered coal humic acid possesses abundant reserves in China, which has multiple active functional groups. The response of roots to humic acid, which acts as a humic acid acting on the first contact organ of plant, is the initial motive force to promote plant growth. Hence researching the effect of humic acid on the growth and development of maize roots is of significance for crops’ production increase and quality improvement, and can provide a theoretical basis for the effective utilization of weathered coal resource of China. 【Method】 Zhengdan958 maize cultivar was used as the tested cultivar, and Hogland nutrient solution hydroponics was adopted in this experiment. When maize seedlings grew to two leaves and one terminal bud, they were moved to the hydroponic basin for one day for readaption time. Then, the effect of applying different concentrations of humic acid (0, 5, 10, 15, and 20 mg C·L ^-1) on the growth of maize biomass, root-shoot ratio, root systematic architecture and root nutrient were studied. 【Result】 (1) Humic acids could significantly increase the root dry weight, shoot dry weight, root-shoot ratio, root activity, and root TTC reducing amount of maize roots. Compared with the control treatment (0 humic acid), the average value of the above indicators with humic acid treatment increased by 42.31%, 19.33%, 18.18%, 46.54%, and 81.01%, respectively. (2) Humic acid could improve the root morphology of maize roots by increasing root length, root number, root volume, root surface area, and average diameter of the maize roots. Compared with the control, the average value of the total root length, total root number, root volume, root surface area and average diameter of maize with humic acid treatment increased by 13.51%, 16.74%, 69.62%, 14.68%, and 49.28%, respectively. (3) The addition of humic acid increased the number of axial roots, length of axial roots and number of lateral roots in maize roots. The average value above of maize roots with the addition of humic acids increased by 16.28%, 21.65% and 16.80%, respectively, compared with the control treatments. (4) The promoting effect of humic acid on the growth of maize roots showed the tread of first increase and then decrease with the increasing additive amount of humic acid. When the medium concentration was 10 mg C/L, the promotion effect on maize roots was the most significant. Compared with the control, the root dry weight and root activity of maize roots increased by 69.36% and 69.07%, respectively. (5) The addition of humic acids (10 mg C·L ^-1) could increase the content of carbohydrates, lipids, proteins, peptides, amino acids, and nucleic acids in maize roots. 【Conclusion】 The addition of humic acid could significantly increase the biomass, root activity and root-shoot ratio of maize. In addition, it could also improve the root architecture of the maize and the content of main chemical components in maize roots. The medium concentration (10 mg C·L ^-1) of humic acid had the most significant effect on the promotion of maize roots.

Environmental Risks for Application of Phosphogysum in Agricultural Soils in China

WANG XiaoBin,YAN Xiang,LI XiuYing,JI HongJie

Scientia Agricultura Sinica. 2019, 52(2):  293-311.  doi:10.3864/j.issn.0578-1752.2019.02.009

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Phosphogypsum (PG) is an industrial by-product gypsum obtained from wet process production of phosphoric acid, and it is one of the largest solid waste emissions in the chemical industry. With the development of phosphate and compound fertilizer industry at the late 1950s, the PG waste residue discharged from the production process had continuously increased in China. To solve the environmental problems caused by massive PG stockpiling, at present, PG was mainly used as for building materials, such as cement retarder, and gypsum-building materials, as well as for filling mine pits and road construction. Some researches have also tried to use PG instead of natural gypsum for saline-alkali land amendment in agriculture since 1990s. However, several harmful elements in phosphate rock were preserved or enriched in PG in the process of wet process phosphoric acid production, resulting in high concentration of hazardous pollutants, including heavy metals, fluoride (F), and radioactive element radium ( ²²⁶Ra) in PG, and to some extent, even far beyond the limits of national standards for soil environmental quality and for groundwater quality. For example, the concentrations of heavy metals Cr, As, Cd and Hg in PG exceed the limits (about 20%-67%) of Soil Environmental Quality Risk Control Standard for Soil Contamination of Agricultural Land (GB 15618-2018); and the leachable concentrations of Hg, Cd, Pb, Ni, Cr and Be in PG exceeded the limits (IV-V) of Standards for Groundwater Quality (GB/T 14848-2017). The concentrations of total F and water-soluble F in PG exceeded the critical values of total and soluble F (about 89% and 100%, respectively) in soils of endemic fluorosis-affected areas in China, and the leachable concentration of F in PG exceeded the limit (V) of Standards for Groundwater Quality, and some exceeded the limit of Identification Standards for Hazardous Wastes Identification for Extraction Toxicity (GB 5085.3-2007). According to the concentration of pollutants in PG, as compared with the limits of Soil Environmental Quality Risk Control Standard (GB 15618-2018), the long-term impacts of PG application (if PG input was at rate of 22.5 t·hm ^-2·a ^-1, and with no leaching) on the accumulations of pollutants in the soil could be estimated. For instance, the accumulation of Cd, Hg, F and ²²⁶Ra from un-contaminated to contaminated soils would need 1, 5, 4 and 25 years, respectively. Some field experiments have shown that PG could lead to the risk of excessive pollutants (such as F, As, Cd, Hg, Pb and Zn) enriched in some agricultural products. In 2017, the Identification Standards for Solid Wastes General Rules (GB 34330-2017) was issued in China, which stipulated clearly that those by-products produced in the production process, including PG produced in the inorganic chemical production process, were solid wastes; and those directly used for soil amendment, land reconstruction, land restoration and other land use methods by solid waste disposal were still managed as solid wastes. In order to ensure soil health, food safety, and environmental quality, it was suggested that those industrial waste like PG without any harmless treatment of pollutants, and with harmful elements far beyond the limit standard should not be allowed to directly use as for soil remediation or conditioning in the farmlands by solid waste disposal methods, to prevent hazardous pollutants from entering food chain and harming to human health.

HORTICULTURE

Cloning of Adβgal-1 and Adβgal-2 Genes and Their Roles During Fruit Softening of Kiwifruit

FENG Xin,LAI RuiLian,GAO MinXia,CHEN WenGuang,WU RuJian,CHEN YiTing

Scientia Agricultura Sinica. 2019, 52(2):  312-326.  doi:10.3864/j.issn.0578-1752.2019.02.010

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【Objective】 Beta-galactosidase is one type of glycosidases involving in cell wall degradation, which plays an important role in plant growth and fruit ripening and softening. In this paper, the identification and expression analysis of two β-galactosidase genes in kiwifruit, as well as their functions during fruit softening, were carried out to provide new insights for the mechanism of kiwifruit softening.【Method】 Actinidia chinensis var. deliciosa ‘Miliang-1’ was used as material to amplify the β-galactosidase genes by RT-PCR, then their characteristics, functional domains, phylogenetic relationships, gene structures and miRNAs were analyzed by using online databases. qRCR was applied to analyze their expression patterns in different tissues, different stages of softening, different storage temperatures, and ABA treatment. Enzymes activities were also analyzed to clarify the function of these two β-galactosidase genes during fruit softening.【Results】Two β-galactosidase genes (Adβgal-1 and Adβgal-2) were obtained from kiwifruit. Their accession numbers were MH319788 and MH319789, respectively. Adβgal-1 had an open reading frame (ORF) of 2 280 bp and encoded 759 amino acids, while the ORF of Adβgal-2 was 2 025 bp encoding 674 amino acids. Protein domain analysis showed that both Adβgal-1 and Adβgal-2 harbored a functional domain (Glyco_hydro_35) of plant glycoside hydrolase family 35. Gene organization analysis revealed that Adβgal-1 had 18 exons and 17 introns, while Adβgal-2 harbored 17 exons and 16 introns. Moreover, Adβgal-1 and Adβgal-2 were clustered in different evolutionary branching in the phylogenetic tree, which was due to the great difference in their sequences. This implied that Adβgal-1 and Adβgal-2 might originate from different ancestral genes. qPCR analysis showed that Adβgal-1 and Adβgal-2 were expressed in all tested tissues (root, stem, leaf, flower, young fruit and ripe fruit) although their expression levels were different. The expression levels of both Adβgal-1 and Adβgal-2 were increased at the start stage of fruit softening, and the amount of gene expression continued to increase as the firmness of fruit decreased. When stored at 25℃, both the Adβgal-1 and Adβgal-2 were upregulated at 3 d, and the amount of gene expression continued to increase as time over. When stored at 4℃, the expression of Adβgal-1 was inhabited, while that of Adβgal-2 was like wavy. ABA induced the expression of Adβgal-1 and Adβgal-2.【Conclusion】 Both Adβgal-1 and Adβgal-2 participated in the process of fruit softening of kiwifruit. The effect of storage temperatures and ABA on fruit softening might be achieved by changing the expression of β-galactosidase genes.

Segmentation of Navel Orange Surface Defects Based on Mask and Brightness Correction Algorithm

ZHANG Ming,LI Peng,DENG Lie,HE ShaoLan,YI ShiLai,ZHENG YongQiang,XIE RangJin,MA YanYan,LÜ Qiang

Scientia Agricultura Sinica. 2019, 52(2):  327-338.  doi:10.3864/j.issn.0578-1752.2019.02.011

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【Objective】 The purpose of this study was to effectively solve the problem that some defects of fruit images with defective peels were mistakenly divided into backgrounds when removing background, and it was difficult to effectively segment and extract fruit surface defects.【Method】 Taking Newhall navel orange as the research object, this paper proposed to remove the background based on HSI color space model method to construct the mask template with I component image, and to select a single threshold (T=75) by bimodal method according to its gray histogram information and filled the holes to obtain a mask template. At last, the mask template and I component image were obtained by dot multiplication to obtain I component image from which the background was removed. A multi-scale Gaussian function image brightness correction algorithm was proposed to correct the brightness of I component image after removing the background. By constructing a multi-scale Gaussian function filter, I component image with the background removed and the constructed multi-scale Gaussian function filter were convoluted to obtain the surface illumination component image of I component image after the background was removed. Finally, the I component image after removing the background and the obtained illumination component image were subjected to dot division operation to obtain a luminance correction image of the I component image after the background was removed. At last, the surface defects of navel orange were extracted by a single global threshold method.【Result】 The background was removed based on the HSI color space model method, and the surface information of the navel orange could be preserved while the background was effectively removed, which was beneficial to subsequent operations. The image brightness correction algorithm based on multi-scale Gaussian function was used to extract the defects of the six common navel orange defects, and then the single-threshold method was used to extract the defects. Therefore the surface defects of navel oranges with different gray levels were successfully segmented at one time, and the segmentation rate was up to 100%, the lowest was 88.5%, and the total was 92.7%. Through experimental analysis, it was found that the cause of partial mis-segmentation or leakage segmentation was mainly due to the fact that some defects were lighter in color, and the difference in gray level from normal region was smaller, resulting in leakage segmentation. There were still some defects due to the small defect area, which was mistaken for noise removal during image morphology processing. At the same time, the false positive rate of normal fruit was also found to be 10.8%. It was found that the fold of a part of the normal fruit epidermal tissue area was located in the edge area of the image, which was mistaken for the defect of the edge area, resulting in misjudgment.【Conclusion】 The experimental results showed that image removal based on HSI color space model and image brightness unevenness correction algorithm based on multi-scale Gaussian function had achieved good results for background image segmentation of Newhall navel orange image and I component image surface brightness correction after background removal. It provided technical support for the precise grading of navel oranges and also provided a new idea for the rapid detection of other fruit surface defects.

FOOD SCIENCE AND ENGINEERING

Preparation and Stability of Sorghum ACE Inhibitory Peptides by Extrusion-Enzyme Synergistic Method

ZHOU JianMin,YIN FangPing,YU Chen,TANG XiaoZhi

Scientia Agricultura Sinica. 2019, 52(2):  339-349.  doi:10.3864/j.issn.0578-1752.2019.02.012

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【Objective】 Sorghum ACE inhibitory peptides were prepared by extrusion-enzyme synergistic method in order to provide a technical guidance to enhance the utilization efficiency of sorghum protein.【Method】 Sorghum flour was introduced into an extruder, and then treated by α-amylase and alkaline protease to obtain the ACE inhibitory peptides. Effects of water content in sorghum flour, extrusion temperature and enzyme activity on the degree of hydrolysis and the activity and stability of ACE inhibitory peptides were investigated.【Result】 With the increasing moisture content and extrusion temperature, the specific mechanical energy (SME) decreased. During extrusion, the interaction between starch and protein in sorghum became loose, which broke the starch-protein complex, and the spherical protein in sorghum was broken up, and thus the sensitivity of sorghum protease was enhanced and more ACE peptides were obtained after alkaline protease treatment. The moisture content, extrusion temperature and the activity of α-amylase presented a significant effect on the degree of hydrolysis and the inhibition rate of ACE inhibitory peptides. With the increasing moisture content, the assemble degree of protein decreased, which increased the degree of hydrolysis and the inhibition rate of ACE inhibitory peptides. When the moisture content reached to 19%, the damage degree of the starch around protein decreased, leading to the gentle increase trend of the hydrolysis degree and the inhibition rate of ACE inhibitory peptides. When the extrusion temperature increased from 120℃ to 180℃, the damage degree of starch-protein complex in sorghum increased. Meanwhile, the denaturation degree of protein also increased. The degree of hydrolysis increased from 7.42% to 11.06%, and the inhibition rate of sorghum protein ACE inhibitory peptides increased from 46.57% to 53.41%. The sorghum flour was treated by a-amylase to remove the starch around the protein after extrusion, and then it was found that when the activity of α-amylase increased, the damage degree of protein-starch complex in sorghum increased, which provided more raw materials for the preparation of sorghum protein ACE inhibitory peptides, leading to the higher the degree of hydrolysis and the activity of ACE inhibitory peptides. When the activity of α-amylase increased to 2.0 U·g ^-1, the binding of α-amylase to starch reached saturation, and the degree of hydrolysis and the inhibition rate of ACE inhibitory peptides tended to be stable. The activity of ACE inhibitory peptide fluctuated within 68.1%-71.31% after being treated by different storage temperatures and pH, suggesting a good inhibitory activity. After in vitro simulated gastrointestinal digestive enzymes digestion, the inhibitory activity of ACE inhibitory peptides was higher than 73%, which still maintained high value. The stability test indicated that sorghum ACE inhibitory peptides had good resistance to thermal, acid and alkaline treatment, and intestinal enzymes digestion.【Conclusion】 The degree of hydrolysis and the inhibition rate of ACE inhibitory peptides all increased significantly by extrusion-enzyme synergistic method, while sorghum ACE inhibitory peptides had good stability, thus this work provided a new approach for the utilization of sorghum and the preparation of sorghum protein and ACE inhibitory peptides with potential use as functional food ingredients.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

Research Progress of Omics Technologies in Cow Mastitis

LI GuangDong,LÜ DongYing,TIAN XiuZhi,JI PengYun,GUO JiangPeng,LU YongQiang,LIU GuoShi

Scientia Agricultura Sinica. 2019, 52(2):  350-358.  doi:10.3864/j.issn.0578-1752.2019.02.013

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Dairy mastitis, a common and complex disease with a high incidence, takes its toll on the development of world dairy industry, brings economic losses of billions of dollars per year. Clinical and subclinical mastitis, caused by pathogens such as Staphylococcus aureus, Escherichia coli and Streptococcus agalactiae, posed a huge security risk to milk industry. In recent years, with the continuous breakthrough of sequencing technology and decline of sequencing cost, the research of life science has entered into the Omics era. The traditional methods such as histopathological screening, somatic cell counting, milk PH value detection, detection of milk conductivity, enzyme activity test, infrared thermal imaging can be employed for clinical diagnosis of dairy cow mastitis, but these methods are not powerful enough to elucidate the pathogenesis in a cellular or molecular view. Omics technologies are mainly composed of genomics, proteomics and metabolomics. Genomics can not only reveal the phenotypic variation and genetic basis of the complex trait of dairy mastitis at the transcriptional level, but also reveal the molecular patterns of the mastitis from the aspects of transcriptional regulation (miRNAs, LncRNAs, etc.) and epigenetic modification (DNA methylation, histone modification, etc.). Genomic analysis of mastitis can also dig out the related changes of DNA, RNA and the rules of multi-molecule interaction, which accounts for a better understanding of the immune mechanism of the host against the pathogen, so as to screen and identify the signal pathways and key candidate genes of mastitis resistance, thus improving the accuracy of genome prediction or selection. Proteomics can not only compare milk protein type and abundance but also analyze protein interaction and post-translational modification in breast tissues under different states and environments. The differentially expressed proteins are annotated by COG (Cluster of Orthologous Groups of protein) function followed by database comparison, GO and Pathway enrichment analysis, which help bring to light the complex regulatory mechanism of mastitis occurrence and defense process at protein level. Proteomic analysis can also be used to find molecular marker of mastitis diagnosis, which will provide a potential precise target for the development of therapeutic drugs. Metabolomics, an important part of the system biology, can detect metabolites of low molecular weight (such as amino acids, lipids, carbohydrates, etc.) of the specific tissues or organs in specific environment or specific physiological states. Efficient qualitative and quantitative analysis will elucidate the relevant metabolic pathways. As the ultimate downstream of gene expression, metabolomics technology enables small changes in gene expression and protein synthesis to be amplified at metabolite levels to fully reflect cellular functions, whose application in dairy mastitis will be able to identify related biomarkers and reveal the physiological and pathological changes of dairy breasts. In conclusion, applying Omics or multi-Omics association analysis techniques to mastitis can further reveal the pathogenic defense mechanism, which will provide valuable reference for disease prediction, diagnosis and treatment. This paper reviews the latest research progress about application of Omics in the field of cow mastitis, aiming to provide solid theoretical bases and practical references for cow health and safety of dairy industry in China.

The Safety Evaluation of Cefalonium Intramammary Infusion (Dry Cow)

HUA WeiYi,LIU YiMing,XU Fei,LU YongQiang,KONG Mei,WANG HaiTing,HUANG HuiLi,WANG HongLei,WU LianYong,LI XiuBo

Scientia Agricultura Sinica. 2019, 52(2):  359-366.  doi:10.3864/j.issn.0578-1752.2019.02.014

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【Objective】 The purpose of this paper was to investigate the safety of cefalonium intramammary infusion(dry cow) for target animals. 【Method】 Six primiparous and six multiparous healthy dairy cows were selected, which have not been treated with any antibiotics by systemic or intramammary administration 30 days before. Their daily milk yield was 15-35 kg. At day 1 and day 0 prior to administration of the tested drug, the daily milk production and body temperature of the tested animals were recorded, and their milk samples were collected for somatic cell count analysis. For milk sampling,the udder was rinsed with clean water and the nipples and the close skin were disinfected with 75% ethanol. Milk samples were collected in sterilized test tubes (milking by hand and discarding the first three times of milking). The collected sample were stored at low temperature (4℃) and sent to laboratory for testing within 6 h. For drug infusion, each quarter of udder was cleaned with a disinfected towel and the teats were soaked for 30 seconds with disinfectant. The drug was slowly inject into the quarter so that it could be evenly distributed. Each quarter was injected with a single dose of cefalonium (Dry Cow) (250 mg). Samples of each quarter were collected at 1, 3, 5, 7, and 10 days after the drug administration. Individual milk production, quantitative somatic cell count (SCC) and body temperature were recorded. Day 0 and day 10 milk samples were cultivated with selective medium to isolate bacteria. The isolated strains were identified according to their colony morphology, staining and biochemical characteristics. The main pathogens analyzed were Staphylococcus aureus, Streptococcus (Streptococcus agalactiae, Streptococcus uberis), Escherichia coli. 【Result】 During the whole test period, there was no clinical symptoms such as swelling, erythema, pain, or heat. On day 1, 0 prior to and 1, 3, 5, 7 and 10 day after administration of the drug, somatic cell count was 250-400 thousand per milliliter. The mean somatic cells count for the seven time points were 332.6, 327.4, 327.0, 316.3, 312.4, 306.2, 300.4 thousand per milliliter respectively, but there is no significant difference (P>0.05) among them by analysis of repeated measures anova. Daily milk yield was 23-33 kg. The average daily milk yield for the seven time points was 27.30, 27.35, 27.25, 27.40, 27.64, 27.83, 28.00 kg, respectively. It had no significant difference (P>0.05) by repeated measures anova. The rectal temperature of all tested cows before and after administration was within the normal range. The mean values of rectal temperature were 38.79, 38.82, 38.83, 38.77, 38.71, 38.71 and 38.69 ℃ respectively, and there was no significant difference (P>0.05). Therefore, according to the recommended dose of a single administration, it had no significant effect on somatic cell count, daily milk yield and rectal temperature of the tested animals. The results for bacteria isolation showed that there were 2, 3 and 4 strains of Streptococcus, Staphylococcus and Escherichia coli, respectively on day 0 of the administration of the drug, and there were 1, 1 and 0 strains of Streptococcus, Staphylococcus and Escherichia coli on day 10 after administration, respectively. The number of pathogens was significantly decreased through the treatment of the drug. 【Conclusion】 The recommended dose of cofalonium had no adverse effect on rectal temperature, milk yield and somatic cell counts for dairy cows after mammary administration. It is safe for the drug to be used in dairy cows by intramammary infusion (Dry Cow).

Residue Depletion Study and Withdrawal Period for Cefalonium Intramammary Infusion (Dry cow) in Bovine Milk

YAN ChaoQun,LI ShuaiPeng,ZHANG Shen,XIE Shun,WEI KaiYun,HUANG XianHui

Scientia Agricultura Sinica. 2019, 52(2):  367-375.  doi:10.3864/j.issn.0578-1752.2019.02.015

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【Objective】 Cefalonium is a first generation semi-synthetic cephalosporin with broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The cefalonium is used intramammarily during the dry period of cattle at a recommended dose of 250 mg per quarter to treat existing sub-clinical infections and to prevent new infections. The bactericidal activity of cefalonium is a result of its inhibitory action on bacterial cell wall synthesis due to binding to one or more penicillin binding proteins located under the cell wall susceptible bacteria. The resulting high internal osmotic pressure leads to rupture of the cytoplasmic membrane. The objective of this study was to establish a withdrawal period for cefalonium in milk, following intramammary infusion treatment.【Method】 Twenty healthy dairy cows in the dry period were injected with a cefalonium injection into each breast area. The average dry period was 50 days, and they were mixed with the samples of 4 milk areas collected per cow at 6, 12, 24, 36, 48, 60, 72, 96 and 120 h after the start of milking. Residues of cefalonium were detectable in milk for up to 6, 12, 24, 36, 48, 60, 72, 96 and 120 h post-treatment, respectively. Phenomenex Luna C18 (150 mm×2 mm, 5 μm) was used. Acetonitrile-0.1% formic acid aqueous solution was used as the mobile phase. The gradient elution procedure was used. The flow rate was 0.25 mL·min ^-1, the column temperature was 35°C, and the injection volume was 5.0 μL. Phenomenex Luna C18 (150 mm×2.1 mm,3.5 μm) was used. Acetonitrile-0.1% formic acid aqueous solution was used as the mobile phase. The gradient elution procedure was used. The flow rate was 0.25 mL/min, the column temperature was 35°C, and the injection volume was 5.0 μL. MS conditions of detection method: ESI ion source, positive ion scan, multiple reaction monitoring, ion spray voltage: 4kV, the pressure of GS1: 55 psi, the pressure of CUR: 15 psi, the pressure of GS2: 35 psi, TEM: 600℃, the quantitative ion is: cefalonium m/z→459.4/337.3. The sample was thawed naturally, 5g of milk was accurately pipetted into a 50 mL centrifuge tube, 20 mL of acetonitrile was added, vortexed and shaken for 2min, and centrifuged at 10 000 r/min for 10 min. The supernatant was extracted once with 15 mL of 75% acetonitrile aqueous solution, and the extracts were combined twice, and 10 mL of acetonitrile saturated n-hexane was added, shaken for 1 min, the n-hexane was discarded, and the extract was transferred to a 100 mL heart bottle. Acetonitrile was removed by rotary evaporation at 40 ° C using a rotary concentrator. To the above sample solution, 20 mL of a sodium dihydrogen phosphate buffer solution was added, followed by adjustment to pH = 8.5 with a sodium hydroxide solution. The sample was passed through the HLB solid phase extraction column, then the heart bottle was washed with 5 mL of sodium dihydrogen phosphate buffer solution and passed through the column, and the column was washed with 2 mL of water. It was eluted with 2 mL of acetonitrile, dried at 40 °C with a nitrogen concentrator, dissolved in 2 mL of water, shaken, passed through a 0.22μm microporous membrane, LC-MS/MS detection analysis.【Result】 Cefalonium showed a good linearity in the concentration range of 2-200 μg·kg ^-1, with a correlation coefficient of 0.991-0.997, a detection limit of 0.5 μg·kg ^-1, and a limit of quantification of 2 μg·kg ^-1. The relative recovery rate of this method at the four levels of addition of 2, 20, 40 and 200 μg·kg ^-1 was 82.21%-89.08%. The coefficient of intra-assay coefficient of variation was 1.85%-10.41%, and the inter-assay coefficient of variation was 3.41%-8.97%. The experimental method has high sensitivity and simple operation, and can be used for the residue depletion study of cefalonium in milk. Milk samples from these cows after calving were analyzed for antibiotic residues by a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) procedure. Primiparous dairy cows that used cefalonium intramammary infusion (dry cow) were below the limit of quantitation at 96 hours after calving. Dairy cows were below the limit of quantitation at 36 hours after calving.【Conclusion】 After the cefalonium intramammary infusion (dry cow) was administered at the recommended dose of 4 milk areas (250 mg/milk area), the withdrawal period in milk was 24 h.

Screening and Identification of Candidate Proteins Interacting with BmHSP60 in the Silkworm (Bombyx mori)

DONG ZhanQi,JIANG YaMing,PAN MinHui

Scientia Agricultura Sinica. 2019, 52(2):  376-384.  doi:10.3864/j.issn.0578-1752.2019.02.016

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【Objective】 HSP60 is an important member of heat shock proteins, which plays an important role in the innate immunity of insects, and is also an essential factor for insect growth and development. Previous studies have proved that BmHSP60 is involved in the invasion process of Bombyx mori nucleopolyhedrovirus (BmNPV). The objective of this study is to identify the interaction proteins of BmHSP60, and to provide a basis for further exploring the mechanism in the innate immunity of B. mori.【Method】 BmHSP60 overexpression vector was constructed and fused with the Flag tag for co-immunoprecipitation and silver-linked-mass spectrometry (LC-MS/MS). After transfected into the B. mori cells for 48 h, the cell lysates was collected for co-immunoprecipitation. The antibody was incubated with the magnetic beads to capture the interaction proteins. After silver staining, the distinct bands could be detected. The differential strips were stored at -80℃, and then the differentially expressed peptides were analyzed by mass spectrometry. The size of the bands was aligned with the NCBI database to screen for candidate interaction proteins according to the mass spectrometry results. Finally, the cloning vector of candidate interaction proteins was constructed and fused with HA tag, and transferred into B. mori cells with BmHSP60, respectively. Co-immunoprecipitation and fluorescence co-localization confirmed the interaction between BmHSP60 and candidate proteins. The co-localization of BmHSP60 candidate interaction proteins with mitochondria was analyzed by Mito-Tracker-Green.【Result】 Mass spectrometry showed that there were 5 distinct bands in the vicinity of 110, 90, 75, 60 and 35 kD. A total of 32 differentially expressed proteins were identified by comparing the results of silver-linked mass spectrometry with those of silkworm genome database and NCBI database. The 5 bands were mixed into a protein pool for mass spectrometry, and 5 interaction proteins were screened consistent with the differential bands in silver staining according to the number of peptides and molecular weight of proteins. Candidate proteins were ADP/ATP translocase (ANT), Actin, Alpha-tubulin, Elongation factor 1-alpha (EF-1α) and HSP90, respectively. The BmHSP60 candidate interaction protein clone expression vector was constructed and fused with HA tag, and co-transfected with BmHSP60 into B. mori cells. Immunocoprecipitation (IP) was incubated with α-Flag antibody, immunoblotting (IB) was incubated with α-Flag antibody, and BmHSP60 expression was detected by IP. IB incubated with α-HA antibody showed that only BmANT and BmHSP90 could detect the corresponding bands, while Alpha-tubulin and EF1α did not detect the corresponding bands with BmHSP60, indicating that only BmANT and BmHSP90 had direct interaction with BmHSP60, and Alpha-tubulin and EF1α had no direct interaction with BmHSP60. Further analysis of fluorescence co-localization showed that BmANT and BmHSP90 could co-locate with BmHSP60 in cytoplasm. Mitochondrial marker Mito-Tracker-Green co-localization results showed that BmHSP60, BmANT and BmHSP90 were co-localized with mitochondria.【Conclusion】 Heat shock protein BmHSP60 of B. mori was identified to interact with BmANT and BmHSP90, and co-localized in mitochondria. It can be used to study the mechanism of BmHSP60 in antiviral immunity of B. mori.