Scientia Agricultura Sinica

Risk Assessment and Regulation of Genetically Engineered Crops: History and Reformation

JIA ShiRong

Scientia Agricultura Sinica. 2018, 51(4): 601-612. doi:10.3864/j.issn.0578-1752.2018.04.001

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The crop breeding by genetic engineering (GE) or the widely accepted terms of ‘Precise Breeding’ are the techniques based on the human desire to modify crop traits with precise characteristics of known inserted DNA and protein sequences, anticipated function, and defined insertion sites in the genome of a given species. The acreage of GE crops has reached to 185.1 million hectares world-wide in 2016, which increased almost 100 folds compared with that in 1996, the year of starting commercialization of GE crops. This indicates that the GE is the fastest developing technology in the history of agriculture, which has provided significant economic, social and environmental benefit to the world. In the process of development and commercialization of GE crops during recent three decades, huge amount of scientific data and experiences have also been accumulated regarding their risk assessment and regulation. However, many unscientific burdens still exist that needs to be further reformed. In particular, the rapid development of new products derived from such as genome editing has also raised questions on how to evaluate and regulate them. Theoretically and practically, the GE as a technique, has posed no intrinsic unique or incremental risks comparing with that of the traditional breeding techniques. From scientific point of view, it is common that the GE crops are inadequate- or over-regulated in the recent 30 years. In fact, so far, a risk-based classification and regulation on GE crops has not been fully implemented. Not only the European ‘Process-based’ regulation should be eliminated, but also the US ‘Product-based’ regulation has a space for further improvement. Nowadays, the innovation and reformation in this field based on science has become a common trend internationally. In this paper, some scientific issues associated with environmental and food safety of GE crops are addressed, including what is the proper object for risk assessment (transformed event, variety or species?), the effect of Bt protein on target and non-target organisms, the potential impact of transgene flow, the toxin, allergenicity, compositional change and un-expected effects, and the marker genes and plant pathogen derived DNA sequences in the GE cops. All of these are discussed based on the current scientific knowledge, by which the major focus point in the risk assessment are discussed. Finally, a regulatory framework based on risk classification, which integrally determined by the species that either used as donor or recipient, the genes that confer specific traits and the environment into which the GE crop introduced, is proposed. The possible products belonging to the category of low, mediate or high risk are listed individually. In terms of species, whether the donor or recipient of a transgene has a long history of safe use, does it contain any known toxins, allergens or anti-nutrients should be carefully assayed. Considering the source of transgene, the GE crops can be classified into three categories of intragenesis, cisgenesis and transgenesis. The intragenesis are the gene modifications or transfers within the same species, e.g. the most crops derived from genome editing exhibiting gene silence, knock-out, modification or insertion with no or very low risk. The cisgenesis are the gene transfers between sexual compatible species that may pose low risk. While the transgenesis transfer genes between sexual incompatible species that may present mediate or higher risk. In terms of environmental safety, the risk assessment should be focused on whether there is any sexual compatible wild species or weeds existed in the environment in which the GE crops are going to be released, whether the crossing and backcrossing between crop-to-wild would cause gene introgression that may lead to the selective competitiveness of the hybrids, whether the region in which the GE crops are intend to release is the origin of a given species, etc. All of these issues have to be considered as major focuses in the risk assessment and regulation of GE crops.

Identification and Fine Mapping of an Early Senescent Leaf Mutant es5 in Oryza sativa L.

WANG BeiFang, CHEN YuYu, ZHANG YingXin, LIU QunEn, SUN Bin, XIANG XiaoJiao, CAO YongRun, CHENG ShiHua, CAO LiYong

Scientia Agricultura Sinica. 2018, 51(4): 613-625. doi:10.3864/j.issn.0578-1752.2018.04.002

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【Objective】In this study genetic analysis and gene mapping of es5, a mutant with early senescent leaf in rice, were conducted to explore the molecular mechanism of rice leaf senescence.【Method】 es5 was isolated from the progeny of japonica rice cv. Jiahe212 treated by ethyl methane sulfonate (EMS). F2 population was derived from the cross between es5 and Zhonghui8015. The phenotypes of the F1 and the segregation ratio of the F2 were investigated in Hangzhou and Hainan separately. The recessive individuals in F2 were used to locate the gene by the map cloning method. The SOD activity, MDA content, soluble protein content, chlorophyll content, the net photosynthetic rate, the histochemical analysis and the expression of senescence-related genes were analyzed for es5 and Jiahe212 leaves at heading stage. Several main agronomic traits of es5 and Jiahe212 were analyzed under field conditions at maturation stage in Hangzhou.【Result】There was no difference between Jiahe212 and es5 before 4-leaf-stage. Leaf senescence in es5 occurred at 4-leaf-stage. The etiolated area of the rice leaf increased gradually with the growth and development. At elongation stage, the middle and lower leaves showed serious aging. Compared with the Jiahe212, Chl a, Chl b, carotenoid content and photosynthetic capacity of es5 significantly decreased and the plant height, the number of productive panicles per plant, the number of spikelets per panicle, seed setting rate and 1 000-grain weight also significantly reduced. The results of Evans blue and DAB staining assay indicated that there were more dead cells and H2O2 in leaves of es5. At 7 days to 21 days and heading, the SOD activities increased significantly in es5, MDA content increased significantly in 21days after heading, while the soluble protein content significantly decreased at 7 days and 21 days after heading. Dissolved cytoplasm, abnormal chloroplast structure, dissolved chloroplast membrane, blurred base, loose lamellar lamellae, abnormal thylakoid development and more starches grains and osmiophilic granules were observed in the senescent leaf region of es5 by Transmission Electronic Microscope (TEM). qRT-PCR analysis showed that senescence-associated genes Osh36, Osh69, OsI85, RCCR1 were up regulated significantly in es5. Especially, the expression of Osh69 and OsI85, two marker genes about aging, were up regulated 12.7 and 36.6 times respectively. SGR, a gene about degradation of chlorophyll, was up regulated significantly at the same time. Genetic analysis suggested that the phenotype of es5 was controlled by single recessive nuclear gene, and ES5 was fine mapped in a 52.7 kb interval including 8 open reading frames between BF-10 and RM3664 on chromosome 5 by map-based cloning strategy.【Conclusion】In this study several agricultural traits significantly in es5 for the leaf senescence ES5 was located in a 52.7 kb range between BF-10 and RM3664 by map-based cloning strategy.

Genetic Diversity and Population Structure of Important Chinese Maize Breeding Germplasm Revealed by SNP-Chips

ZHAO JiuRan, LI ChunHui, SONG Wei, WANG YuanDong, ZHANG RuYang, WANG JiDong, WANG FengGe, TIAN HongLi, WANG Rui

Scientia Agricultura Sinica. 2018, 51(4): 626-634. doi:10.3864/j.issn.0578-1752.2018.04.003

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【Objective】 Understanding the genetic diversity and population structure of representative maize accessions are of importance in breeding practice for the guidance and reference. 【Method】A total of 344 maize inbred lines were selected, including American heterotic group, local germplasm, New germplasm used in maize breeding in China in recent years which were broadly representative. These lines were genotyped by 3 072 SNP markers which were developed by Maize Research Center, BAAFS to reveal the genetic diversity and population structure. 【Result】For 3 072 high-quality SNPs, the gene diversity averaged 0.442, ranging from 0.028 to 0.646, and the PIC averaged 0.344, ranging from 0.028 to 0.570. The result of population structure based on a model-based method indicated that these 344 lines could be divided into eight groups, including Lüda red cob, Huangzaosi improved lines, Iodent, Lancaster, P group, Improved Reid group, Reid and X group. The seven groups above were well-known, and the X group was selected from the populations constructed from X1132X. Among the eight groups, the Fst ranged from 0.319 to 0.512, and the genetic distance ranged from 0.229 to 0.514. AMOVA results indicated that 38.6% of the total genetic variation occurred among groups, 58.1% within groups and 3.3% within lines. PCA results showed that X group had higher genetic differentiation with Huangzaosi improved lines and Lancaster, but lower with Iodent. The genetic diversity of subpopulations indicated that with the increase of breeding years, the average of genetic diversity in each subpopulation was decreased, and among them, X group had the highest genetic diversity. Further analysis showed that the genetic diversity of core accessions in American heterotic group and local germplasm were higher decreased compared with that in P group and Improved Reid group. However, the genetic diversity of core accessions in X group was no decreased, which indicated that the core accessions of X group still maintained higher genetic diversity and had potential application in breeding.【Discussion】X group was different from the other seven known groups, which can be defined as an independent group. Furthermore, X group had further genetic relationship with Huangzaosi improved lines which indicated the strong heterosis pattern of "X group × Huangzaosi improved lines" had application potential.

Dynamic and Associative Transcriptomic Analysis of Glucosinolate Content in Seeds and Silique Walls of Brassica napus

TIAN ZhiTao, ZHAO YongGuo, LENKA Havlickova, HE Zhesi, ANDREA L Harper, IAN Bancroft, ZOU XiLing, ZHANG XueKun, LU GuangYuan

Scientia Agricultura Sinica. 2018, 51(4): 635-651. doi:10.3864/j.issn.0578-1752.2018.04.004

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【Objective】To assess the dynamic and underlying genetic control of glucosinolate (GS) content in seeds and silique walls at different developing stages. We conducted Associative Transcriptomics (AT) in a panel of European winter oilseed rape. 【Method】The glucosinolate content in developing and mature seeds and silique wall samples from the rapeseed panel (n=113) was determined by HPLC. Using mRNA-seq data derived from the young leaves of B. napus, functional genotypes were inferred for the panel, which included both sequence variation (i.e. Single Nucleotide Polymorphism, SNP) and transcript abundance (Gene Expression Markers, GEM). A total number of 355 536 SNPs and transcript abundance were scored across the rapeseed panel using a reference sequence of 116 098 ordered coding DNA sequence (CDS) gene models for resolving the genetic factors controlling GS variation. All significantly associated markers were further traced back to a CDS gene model, of which the function can be inferred by sequence blast and prediction using public Brassica and Arabidopsis databases. 【Result】The GS content in seeds and silique walls 15 days after pollination (DAP) was very low, with an average of 7.58 μmol?g^-1 (1.69-20.45 μmol?g^-1) and 4.81 μmol?g^-1 (1.47-25.23 μmol?g^-1), respectively. The GS content for mature seeds, seeds at 25 DAP and silique walls at 25 DAP varied from 8.87 to 111.83 μmol?g^-1, 2.17 to 147.21 μmol?g^-1 and 0.73 to 130.77 μmol?g^-1, respectively. The observed extensive phenotypic variations for GS content at these two stages made them suitable for AT analysis. In total, 256,397 informative SNPs with minor allele frequency (MAF) ＞ 0.01 and 53,889 GEMs with mean expression level＞0.4 were plotted after AT analysis. With these SNPs markers, a total number of 167, 158 and 3 SNPs were shown to be significantly associated (-log₁₀P＞6.71) with GS content of mature seeds, seeds at 25 DAP, and silique walls at 25 DAP, respectively. Among these, 5 association peaks distributing across chromosome A2, A9, C2, C7 and C9 were found for mature seeds. Meanwhile, 127, 16 and 24 GEMs were significantly (-log₁₀P＞6.03) associated with GS content of mature seeds, seeds at 25 DAP, and silique walls at 25 DAP, respectively, which also unambiguously defined 5 association peaks in A2, A8, A9, C2, and C9. The association peaks in A9, C2 and C9 were common for both mature seeds and silique walls at 25 DAP. Based on the Arabidopsis homologous gene annotations, 25 genes derived from the significant SNPs or GEMs were inferred and predicted to be involved in GS metabolic pathways. The other genes derived from AT analysis, though not directly involved in GS metabolism, were classified as transcription factors, factors responding to stimulus or involved in cellular process, catalytic activity, and thus were also predicted to play an important role in GS accumulation in the different tissues of Brassica napus. 【Conclusion】The GS content in silique walls was found to be high and positively correlated with that of seeds, indicating that silique wall was an important organ for GS synthesis or translocation. A total number of 328 SNPs and 144 GEMs were found to be significantly associated with GS content for different tissues. From these, 25 genes were predicted to be directly involved in GS accumulation or translocation, and 73 genes with uncharacterized function were also inferred.

Study on Changes of Sesame Protein Content and Its Components of Yuzhi 11 Sesame Seed During Growth Period

HAN YaFei, WANG XueDe, ZHENG YongZhan, MEI HongXian, WEI AnChi, LIU YanYang

Scientia Agricultura Sinica. 2018, 51(4): 652-661. doi:10.3864/j.issn.0578-1752.2018.04.005

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【Objective】The purpose of this study was to explore the objective of sesame in growth period and the effect of surroundings on yield and constituent of sesame protein to provide reference for sesame breeding and protein application.【Method】Sesame seeds（Yuzhi 11） were planted in Nanyang, Zhumadian and Zhoukou of Henan province in the same growing season. About 5 000-flowers bloom at same time were marked in one day, and samples were acquired after ten days later and every five days then until sesame seeds mature. And then, content, constituent, relative molecular mass, and amino acid of sesame protein were measured.【Result】The content of sesame protein increased gradually with the growth period, and the protein content in sesame seeds reached the maximum at thirty-fifth days after pollination, and then the protein content was stable; The protein content of Zhoukou test site was lower than that in Nanyang and Zhumadian. The change of globulin was consistent with that of the total protein content of sesame, and the content of albumin showed the trend of increasing at first and then decreasing. The content of albumin in mature sesame seeds did not show regional difference; When the sesame seeds were pollinated for 15 days, bands appeared in gel chart at 20, 30 and 55 kD, and they grew deeper with the extension of the growth period. With the growth and development of sesame, the percentage of essential amino acids in total amino acids decreased gradually, and the proportion of essential amino acids in Nanyang and Zhumadian test site lower than that in Zhoukou significantly; Valine, isoleucine, leucine and phenylalanine + tyrosine ratio coefficients were greater than 1.【Conclusion】Surrounding has a significant effect on sesame protein content, globulin content and albumin content of sesame in growth period. Protein molecules synthesis in different time from kinds, and the main relative molecule weight is in 20-30 kD. With the growth and development of sesame, the percentage of essential amino acids in total amino acids decreased gradually, which indicates that sesame protein could be supplement on valine, isoleucine, leucine and phenylalanine and tryosine.

Modeling the Effects of Post-Anthesis Waterlogging Stress Under Different Fertilizer Levels on Rapeseed Yield and Its Formation

SONG ChuWei, CAO HongXin, ZHANG WenYu, ZHANG WeiXin, CHEN WeiTao, FENG ChunHuan, GE SiJun

Scientia Agricultura Sinica. 2018, 51(4): 662-674. doi:10.3864/j.issn.0578-1752.2018.04.006

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【Objective】Water-logging is one of the main meteorological disasters in rapeseed production areas in the Yangze River valley. This study aimed at quantifying the effects of waterlogging at anthesis stage under different fertilizer levels on rapeseed grain yield and yield components, which could provide scientific basis for waterlogging stress assessment, forecasting, and early warning in rapeseed production. 【Method】 The experiments in two-growing season were conducted during 2014-2016 with different waterlogging durations at anthesis stage under different nitrogen levels at Jiangsu Academy of Agricultural Sciences. Using two rapeseed cultivars (Ningyou 18, and Ningza 19) as experimental materials, the treatments were waterlogging at anthesis stage of 0, 3, 6, and 9 days in pot, and 0, 3, and 6 days in pool under different fertilization conditions, including N1 (N 0.018 kg·m^-2, P₂O₅ 0.012 kg·m^-2, K₂O 0.018 kg·m^-2, and borax 0.0015 kg·m^-2), and N0 (N 0 kg·m^-2). 【Result】The results of this study showed that: (1) The yield per plant and pods per plant for Ningyou 18 and Ningza 19 under pot experiment without fertilizer significantly decreased after 3 days waterlogging at anthesis, but the 1000-grain weight, and the grain number per pod significantly decreased after 6 days or 9 days waterlogging at anthesis; the responses of the yield per plant and pods per plant for Ningyou 18 and Ningza 19 under pot experiment with fertilizer to waterlogging at anthesis existed obvious differences, i.e., the yield per plant for Ningyou 18 significantly decreased after 3 days waterlogging at anthesis, while Ningza 19’s significantly decreased after 6 days waterlogging at anthesis. The yield per plant for Ningyou 18 under pool experiment without fertilizer significantly decreased after 6 days waterlogging at anthesis, but which of Ningza 19 decreased after 3 days waterlogging at anthesis; the yield per plant for Ningyou 18, and Ningza 19 under pool experiment with fertilizer significantly decreased after 6 days waterlogging at anthesis. The dry matter weight, grain yield, and harvest index (HI) of the aboveground of rapeseed were decreased with the increase of waterlogging duration. There were no significant differences in dry matter weight of aboveground, and yield components under 3 days of waterlogging treatments, but the yield decreased significantly. Under the condition of 6 days of waterlogging treatment, the aboveground dry weight, yield, and yield components all decreased significantly. (2) In accordance with mixed linear model analysis, with each 1 d increasing of waterlogging days at flowering time, the HI decreased at 0.008, the number of pods per plant decreased at 14.71, the grain number per pod decreased at 0.29, the 1000-grain weight decreased at 0.04 g, and the yield per plant reduced at 1.52 g/plant. The interaction between waterlogging and fertilization could significantly increase the yield of 3.17 g/plant, and the number of pods per plant of 39.16, which could alleviate the effects of waterlogging on yield. When the waterlogging was up to 6 days, the interaction between waterlogging and fertilization could reduce the yield of 2.29 g/plant, but it did not reach the significant with P<0.05, so which had no significant effect on the yield components. When the waterlogging was up to 9 days, the interaction between waterlogging and fertilization could significantly reduce the yield of 4.78 g/plant, i.e., it could increase the yield loss.【Conclusion】We concluded that the waterlogging stress at anthesis stage reduced the rapeseed grain yield and yield components, and the reduction was strengthened with the waterlogging duration expand. The pod number per plant was more sensitive to waterlogging stress than the grain number per pod and 1000-grain weight, the responses of Ningyou 18 for waterlogging were more sensitive than Ningza 19, and the responses of pot cultivation for waterlogging were more sensitive than pool cultivation. According to the model analysis in this study, the negative effects of waterlogging at anthesis within short duration on the yield, HI, and number of pods per plant can be alleviated by fertilizing, the alleviation role can be weakened with waterlogging duration expand, and the effects of 9 days of waterlogging at anthesis on the yield, HI, and number of pods per plant for no fertilizer and fertilizer treatments can be arrived to uniformity; With each 1 d increasing of waterlogging at flowering period, the HI decreased, the number of pods per plant, the grain number per pod, the 1000-grain weight, and the yield per plant all reduced obviously, the harm of 3 days of waterlogging on rapeseed under fertilizer condition was small, while under 6 days, and 9 days of waterlogging conditions, the weaken effects of fertilizer on waterlogging at anthesis can be reduced. Under normal water management, the application of fertilizer could significantly increase the yield and yield components, however, with the prolongation of waterlogging duration, the effects of fertilizer for alleviating the impact of waterlogging on the yield was declining.

An Efficient Hierarchical Indicator Based on the Correction Area of Remote Sensing Identification Error for Planting Acreage Sampling——A Case Study of Winter Wheat

YANG JunWen, ZHANG JinShui, PAN YaoZhong, SUN PeiJun, ZHU Shuang

Scientia Agricultura Sinica. 2018, 51(4): 675-687. doi:10.3864/j.issn.0578-1752.2018.04.007

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【Objective】Remote sensing has a strong superiority in crop planting area acquisition. It is critical to design an efficient hierarchical indicator in a stratified sampling investigation. Traditional method neglects the classifying error of the classification results, which reduces the sampling efficiency. Therefore, a new hierarchical indicator (S_correct) that can correct the error area is presented in this paper, which is in favor of raising the efficiency of the sampling survey. 【Method】The yield of winter wheat in Tongzhou and Daxing district of Beijing was as a study object. The data source was GF-1 imagery (resolution: 16 m, phase: 2015-04-04) obtained in winter wheat growing season. This paper designed the S_correct which was based on an algorithm involved the error of omission and the error of commission at pixel scale. This indicator summed all pixels’ correct scale in a sampling unit to correct the area scale indicator (S). A certain plan was devised for Multiple sampling by using S_correct and the area scale indicator (S). This plan constructed regular square grids (size: 90-300 m) as the sampling frames, set the number of layers, determined the hierarchical boundary method and the allocation method for sample capacity, etc. In order to testify the validity of S_correct, this paper analyzed the correlation between this indicator and true value, estimated the total winter wheat area by multiple sampling, after that, compared S_correct with S in sampling variance, average relative error ( ), CV value, etc. The typical corrected regions for pixel with error were selected to compare and analyze for verifying the effectiveness at pixel scale. 【Result】 (1) By comparing and analyzing the typical corrected regions, the error distribution could accord with the actual circumstance for both the error of omission and the error of commission. This indicator could correct error pixel. With the sizes of the sampling unit, S_correct was better than S in the correlation, and the value was greater than 0.7, which can ensure a high and stable correlation with the truth value. The validity of S_correct had been verified by experiments. (2) S_correct was better than S in the stability, accuracy and CV value. Its total variance was 1.70×10¹³-2.41×10¹³, and the total variance obtained by using S was 2.05×10¹³-3.11×10¹³. Its was 4.21%-5.00%. The obtained by using S was 4.87%-5.98%. Therefore, nearly 1% could be enhanced stability in accuracy. Besides, S_correct could reduce CV value nearly 0.8%. 【Conclusion】The effectiveness and advantages of S_correct had been proved by the experimental results. The indicator can improve the accuracy of sampling and the stability of inference in a certain extent.

Construction of a Near Infrared Model for Detecting Stem Fiber Component Content and Lignin Monomer G/S in Rapeseed

CHEN XuePing, LIU ShiYao, YIN NengWen, JING LingYun, WEI LiJuan, LIN Na, XIAO Yang, XU XinFu, LI JiaNa, LIU LieZhao

Scientia Agricultura Sinica. 2018, 51(4): 688-696. doi:10.3864/j.issn.0578-1752.2018.04.008

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【Objective】The aim of this study is to explore the feasibility of rapidly detecting the content of fiber composition and monomer G/S with near-infrared spectroscopy (NIRS) in rapeseed(Brassica napus L.). 【Method】 After the NIRS was collected, 103 samples for fiber component content and 75 for monomer G/S in rapeseed were selected by removing outlier and redundant samples according to measure distance. Fiber component content was measured with Van Soest method and lignin monomer G/S was measured with GC-MS. Subsequently, data analysis showed that the little difference for the chemical test, and the range of variation in the calibration samples was wide enough for building the NIRS models. By using the optimum spectrum pretreatment and regression method along with internal cross validation, the calibration models were established, and the model accuracy was tested. 【Result】The best regression method for prediction of fiber component and lignin monomer G/S in rapeseed was modified partial least-squares (MPLS) by using near infrared spectroscopy. The cross-validation correlation coefficient (1-VR) of NIRS model for neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) and monomer G/S model was 0.864, 0.861, 0.872 and 0.920, respectively, and the determination coefficient (RSQ) was 0.892, 0.891, 0.907 and 0.953, respectively; The results of testing the validation set showed that external validation correlation coefficient (RSQ) was 0.837、0.818、0.870 and 0.935, respectively, and square errors of prediction (SEP) was 0.680, 0.636, 0.348 and 0.054, respectively. 【Conclusion】These results indicated that the model established in our research had relatively high accuracy in prediction tests for stem fiber component content and lignin monomer G/S in rapeseed, which could provide support for rapeseed disease and lodging resistance in research and breeding project.

Advances in Trehalose Metabolism and Its Regulation of Insect Chitin Synthesis

TANG Bin, ZHANG Lu, XIONG XuPing, WANG HuiJuan, WANG ShiGui

Scientia Agricultura Sinica. 2018, 51(4): 697-707. doi:10.3864/j.issn.0578-1752.2018.04.009

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Trehalose as a non-reducing disaccharide is widely found in bacteria, algae, fungi, plants, and invertebrates. Moreover, trehalose is regarded as ‘blood sugar’, since it is the primary carbohydrate substances in insects, which plays a key role in normal physiological processes, such as growth, development, moulting and so on. The trehalose in insects is synthesized by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP). Trehalase (TRE) can hydrolyze trehalose into glucose rapidly and provide energy when energy is needed. Chitin is the main component of insect epidermis, midgut peritrophic membrane and tracheal system. During developmental stages, insects need to shed old epidermis for forming new epidermis. This process has always been an important target for pest control. TRE is the first enzyme in the chitin biosynthesis pathway, which has significant functions of influencing the chitin metabolism by regulating chitin synthesis pathway. So how does trehalose metabolism regulate chitin synthesis pathways to control insect molt and chitin metabolism? With further research on the function of trehalose metabolism related genes over the world, these results showed that the providing of trehalose in insects plays a pivotal role in chitin synthesis. In addition, trehalase can be divided into soluble and membrane-bound types. The soluble TRE and TPS have multiple homologous genes in different insect species, indicating that the trehalose metabolic evolution of insects is diversified. Second, trehalose metabolism can regulate the chitin synthesis directly. Whether the low expression of TPS or TRE can break the supply balance of trehalose, resulting in chitin synthesis pathway blocked, especially the expression of chitin synthase gene decreased which lead to low chitin content, and further cause molting difficulties, developmental deformities, even death. Furthermore, trehalase inhibitors can inhibite the activities between soluble and membrane-bound TRE, resulting in a significant decrease in chitin synthesis pathway-related genes and chitinase gene expression, low chitin content and high proportion of death. These results indicate that once the supply balance of insect trehalose metabolism is broken, it will directly affect the chitin synthesis of insects which related to the process of molting and development. In this report, the research progress on trehalose metabolism and the regulation of chitin biosynthesis are introduced and summarized, in the desire to provide a theoretical basis for the development of green pesticides represented by trehalase inhibitor and trehalose-6-phosphate synthase pathway inhibitor aimed at pest control.

Allelopathy and fertilizer efficiency of compost made from Ageratina adenophora on wheat

FAN Qian, HUANG JianGuo

Scientia Agricultura Sinica. 2018, 51(4): 708-717. doi:10.3864/j.issn.0578-1752.2018.04.010

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【Objective】 After coming into our country from Myanmar in 1940s, Ageratina adenophora is now widespread in agricultural lands, forests and pastures in Southwest China, which has produced huge economic loss and ecological disaster. The objective of this study is to realize the allelopathic effects of this invasive plant on wheat and the efficiency of organic fertilizer made from A. adenophora, and to provide scientific information for the effective control and resource utilization.【Method】A microbial inoculum was made with Pseudomonas putita and Clostridium thermocellum to compost A. adenophora in situ. Then pure culture and pot experiments were carried out to compare the allelopathic effects of extracts from fresh and decomposed A. adenophora on wheat seed germination, young seedling growth and nutrient uptake, and field trial to realize soil microbes, wheat grain yield and quality as affected by organic fertilizer made from A. adenophora.【Result】In the culture experiment, the higher concentration of the extracts from fresh A. adenophora (EFA) significantly inhibited the seed germination and decreased the growth rate of the seedlings. In contrast, the seed germination rate, germination index, vigor index, and seedling height increased by 4.73%, 13.06%, 6.58%, and 29.87%, respectively, which were treated by decomposed extracts of A. adenophora (EDA). Meanwhile, EDA treatment increased the chlorophyll concentration, nitrate reductase activity, and root vitality, which was beneficial to photosynthesis, nitrogen assimilation, and nutrient uptake. In the process of wheat germination, EFA significantly slowed the hydrolysis of macromolecular inclusions such as protein, starch and inositol phosphates in wheat seed endosperm. Conversely, EDA increased free amino acids, soluble sugars, and inorganic phosphorus in the seeds, which could be used as the building materials for new plants and supply seedlings with nutrients resulting in fast plant growth. In pot experiment, using EFA to water wheat seedlings, nitrogen absorption by plants decreased by 74.78%, phosphorus decreased by 58.55%, and potassium decreased by 70.77%, and the seedling biomass decreased by 47.77%-58.70%, respectively. However, EDA promoted plant nutrient uptake and the seedling biomass increased by over than 27.53% compared with control. In the field trial, compared with the control (no fertilizer), there was no significant effect of chemical fertilizer application on microbial carbon and nitrogen content, dehydrogenase activity, and Shannon-Wiener diversity index in wheat rhizosphere soil. However, chemical fertilizer in combination with organic fertilizer made from A. adenophora, greatly increased the three microbial parameters above compared with only chemical fertilizer application. Application of this organic fertilizer increased by 8.87% of wheat grain yield and by 10.02% of seed protein in comparison with only chemical fertilizer application.【Conclusion】 Fresh A. adenophora contained allelopathic toxins, inhibited the hydrolysis of macromolecular inclusions in seed endosperm, and decreased seed germination rate, nutrient absorption by seedlings, including nitrogen, phosphorus and potassium, and inhibited seedling growth. Composted A. adenophora with microbial inoculum contained no toxicant, stimulated seed germination, promoted nutrient uptake, accelerated seedling growth, and enhanced the numbers, activities, and biodiversity of microbes in the rhizosphere of wheat, resulting in wheat yield increment and seed quality improvement. In artificial control and removal of A. adenophora, therefore, to produce organic fertilizer by composting A. adenophora with microbial inoculum in situ could realize harmless treatment and resource utilization.

Effects of Urea Ammonium Nitrate Solution on Grain Yield and Nitrogen Uptake of Spring Maize in Black Soil Region

WANG Yin, XU Zhuo, LI BoNing, GAO Qiang, FENG GuoZhong, LI CuiLan,YAN Li, WANG ShaoJie

Scientia Agricultura Sinica. 2018, 51(4): 718-727. doi:10.3864/j.issn.0578-1752.2018.04.011

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【Objective】Urea ammonium nitrate solution (UAN) is a liquid nitrogen (N) fertilizer with nitrate-N, ammonium-N and amide-N and owns advantages of these N sources. This study aimed to investigate maize yield responses and N use efficiency of UAN in black soil region, and thereby provided scientific references for application and promotion of UAN in local maize production.【Method】Two field experiments were conducted in black soil region of Jilin province in 2015 and 2016, including seven fertilization treatments, i.e. No N fertilizer application (N0); 200 kg N·hm^-2of urea as base fertilizer (U200); 200 kg N·hm^-2of UAN as base fertilizer (UAN200); 80 kg N·hm^-2of urea as base fertilizer with 120 kg N·hm^-2 of urea as topdressing (U80-120); 80 kg N·hm^-2of UAN as base fertilizer with 120 kg N·hm^-2 of urea as topdressing (UAN80-120); 64 kg N·hm^-2of urea as base fertilizer with 96 kg N·hm^-2 of urea as topdressing (U64-96); 64 kg N·hm^-2of UAN as base fertilizer with 96 kg N·hm^-2 of urea as topdressing (UAN64-96). All the topdressing were applied at jointing-bellbottom stage, and the fertilization depth was 12 cm below soil surface. Grain yield, yield characteristics, N uptake in plant, and soil mineral N content were measured at harvesting, as well as further N balance and the percentage of apparent recovery, residual and loss were also calculated.【Result】The results showed that N fertilization increased significantly grain yield and N uptake of maize in 2015 and 2016. In these two years, both the highest yields (10.3 and 11.9 t·hm^-2) and N uptake (187.4 and 288.2 kg·hm^-2) were observed under UAN200 treatment, while the lowest values were obtained under U64-96 treatment, which were 9.14, 10.2 t·hm^-2 and 159.1, 243.8 kg·hm^-2, respectively. With same application rates and methods, grain yields under UAN treatments were generally higher than those under urea treatments. Yield advantages was 6.1% for UAN200, 2.0% for UAN80-120 and 5.3% for UAN64-96 compared with the responding urea treatments in 2015, and these were 0.1%, 7.8% and 7.4% in 2016, respectively. The yield advantage in UAN treatments was mainly attributed to increased kernels number per ear and reduced bare tip length at maturity. With same application rates and methods, UAN treatments showed higher N uptake in plant but lower mineral N residual and surplus in soil after harvesting, thus obtained higher N use efficiency. UAN64-96 treatment reduced N input of 40 kg·hm^-2compared with UAN200 treatment, but it still obtained grain yields of 9.6 and 11.0 t·hm^-2 in the two years, respectively, and which was similar with UAN200 treatment in 2015. Moreover, both the highest N recovery rates and lowest N loss rates were observed under UAN64-96 treatment, the former was 42.6% in 2015 and 52.0% in 2016, and the latter were 2.4% and 4.4%, respectively.【Conclusion】Compared with urea treatments, UAN treatments showed equal or higher maize grain yields and plants N uptake in black soil region when applied with same rates and methods, meanwhile soil N residual and surplus were relatively lower, and thereby resulting in higher N use efficiency and better environmental effect. Based on the integrated consideration of N application rate, grain yield, N use and loss, 160 kg N·hm^-2 of UAN was recommended to be applied for maize production in black soil region with two splits, i.e. 40% before planting and 60% at jointing-bellbottom stage.

Heteroskedasticity Diagnosis and Feasible Generalized Least Squares Regression Modeling of Ternary Fertilizer Efficiency Model

KONG QingBo, ZHANG MingQing, LI Juan, XU WenJiang, ZHANG ZanDe, YAO JianZu

Scientia Agricultura Sinica. 2018, 51(4): 728-737. doi:10.3864/j.issn.0578-1752.2018.04.012

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【Objective】 This paper presented the new strategy to develop fertilizer efficiency models, so as to improve the scientificity of fertilizer effect models and the success rate of the field experiment. 【Method】 According to the results from the 171 “3414” NPK field experiments with early rice, using the modern regression analysis theory, the reason of the ternary model heteroscedasticity and the statistical test methods were studied in the paper. Feasible generalized least squares (FGLS) was used to eliminate the heteroscedasticity effect. 【Result】 According to the professional characteristics of fertilizer efficiency models, the field experiment design and its management practices, soil fertility and its spatial heterogeneity were the key reasons for the models’ heteroscedasticity. Based on the White test, Park test and KB test, the heteroscedasticity of the 25.15% ternary fertilizer efficiency model showed the statistical significantly level. In view of the serious heteroscedastic of the field experimental data, FGLS regression model could convert part of field experimental results into significant level, whereas the Ordinary Least Square (OLS) could not; or the FGLS could convert the no-typical model into typical model; the probability of typical models increased from 27.68% to 34.99%. For the typical ternary model, the recommended fertilization rate of FGLS regression model roughly was equate to the rate of OLS model. Compared to the OLS modeling, the FGLS regression modeling could decrease the Mean Square Error (MSE) greatly, at the same time, which also could decease the variance from the parameters. Thus the FGLS regression model could improve the fitting precision and the predication ability. 【Conclusion】 FGLS is an effect way to eliminate or ease the heteroscedasticity from the ternary quadratic polynomial model, and can obviously improve the success rate of the field experimental data modeling.

Characteristics of Nitrogen Loss in Sloping Cropland of Purple Soil During Maize Growth Stage Under Rainstorm

FENG XiaoJie, ZHENG ZiCheng, LI TingXuan, FAN Li

Scientia Agricultura Sinica. 2018, 51(4): 738-749. doi:10.3864/j.issn.0578-1752.2018.04.013

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【Objective】The characteristics of soil erosion and nitrogen loss in sloping cropland of purple soil during maize (Zea mays L.) growth stage under the rainstorm were investigated to provide a theoretical basis for the predicting and effective preventing of nitrogen loss.【Method】 Simulated rainfall combined with runoff plot experiments were employed during maize seedling stage (1th May), elongation stage (26th May), tasseling stage (27th June) and maturity stage (4th August). According to the feature of the stormy summer months in the purple soil region of Sichuan province, the characteristics of nitrogen loss in surface runoff, interflow, and sediment were studied during maize growth stage under rainfall intensity of 1.5 mm·min^-1 and the slope gradient of 15°. 【Result】(1) The rate of surface runoff and sediment yield increased with the increasing rainfall duration during maize growth stage, which were the highest within seedling stage, and the lowest within tasseling stage, but the rate of interflow within tasseling stage and maturity stage were the highest and lowest, respectively. (2) The rate of nitrogen loss in surface runoff increased with the increasing rainfall duration and became stable after the rainfall duration of 36 min. The average total nitrogen and dissolved total nitrogen were up to 5.24 mg·m^-2·min^-1 and 4.74 mg·m^-2·min^-1 in seedling stage, respectively. The rate of nitrate nitrogen reached stable after the rainfall duration of 30 min, and the rate of ammonium nitrogen showed volatility with rainfall duration. The rate of nitrate nitrogen and ammonium nitrogen were up to 3.90 mg·m^-2·min^-1 and 0.14 mg·m^-2·min^-1 in elongation stage, respectively. The regression relationship between total nitrogen, dissolved total nitrogen, nitrate nitrogen and surface runoff during maize growth stage were extremely significant. (3) The rate of nitrogen loss in the interflow increased slowly with increasing the rainfall duration. The rate of dissolved total nitrogen and nitrate nitrogen showed the same trend with nitrogen loss within seedling stage, elongation stage and maturity stage, while the rate of ammonium nitrogen showed volatility with time. The rate of total nitrogen, dissolved total nitrogen, nitrate nitrogen and ammonium nitrogen were up to 25.04, 20.34, 16.20 and 0.22 mg·m^-2·min^-1 within elongation stage, respectively. The regression relationship between total nitrogen, dissolved total nitrogen, nitrate nitrogen and interflow during maize growth stage were significant, and the slope of liner was the biggest within elongation stage. (4) The rate of nitrogen loss in sediment yield increased with the increasing rainfall duration, but the increase was the highest in seedling stage, which was up to 0.92 mg·m^-2·min^-1. The regression relationship between nitrogen loss and sediment yield during maize growth stage were extremely significant. (5) The forms of nitrogen loss in the surface runoff were reached the highest in seedling stage and elongation stage. Nitrogen loss in the interflow reached maximum in elongation stage and tasseling stage. Nitrogen loss in sediment yield reached the highest in seedling stage. Interflow was the main source for nitrogen loss and accounted 64.07%−83.39% of the total nitrogen loss. Dissolved total nitrogen was the main form of nitrogen loss in surface runoff and interflow, and nitrate nitrogen was the main form.【Conclusion】 Under the rainfall intensity of 1.5 mm·min^-1 nitrogen loss in surface runoff and interflow were the highest within seedling stage and elongation stage, respectively. Dissolved total nitrogen and nitrate nitrogen were the highest in elongation stage, which could easily lead to the eutrophication in waters. Thus, it is necessary to control surface runoff in seedling stage and interflow in elongation stage to reduce nitrogen loss in the purple soil region.

Effects of Five Dwarfing Interstocks on Shoot Growth, Fruiting and Accumulation of Mineral Elements in Leaves of Yishui Red Fuji Apple

HE Ping, LI LinGuang, WANG HaiBo, CHANG YuanSheng

Scientia Agricultura Sinica. 2018, 51(4): 750-757. doi:10.3864/j.issn.0578-1752.2018.04.014

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【Objective】Five dwarfing interstocks were evaluated on tree growth, yield, fruit quality and accumulation of mineral elements in leaves of Yishui red Fuji apple（Malus domestica Mill）for three years in order to select the suitable dwarfing interstock with independent intellectual property rights. 【Method】The Yishui red Fuji apple tree seedlings with different dwarf interstock (M26, SH6, Qing 2, Liao 2 and M9T337) planted in spring of 2009 were used as experimental trees, and the dynamic changes of tree growth, yields and fruit qualities and accumulation of mineral elements in leaves were investigated from 2014 to 2016. 【Result】The results showed that the interstock Liao 2 had better growing compatibility with the rootstock and the scion. The shoot growth of Yishui red Fuji trees grafted on SH6 was significantly higher than that of on other four interstocks，although the shoot growth tendency of Yishui red Fuji trees grafted on different interstocks was similar. The ratio of short shoots of SH6 dwarfing interstock apple tree was the highest (65.2%), and the ratio of long branches was the lowest (11.1%). The average yield per plant of M26 dwarfing interstock apple trees was significantly higher than the other four interstock, but the proportion of big fruits (the fruit weight more than 200 g) from high to low in order was Qing 2＞Liao 2＞SH6＞M9T337＞M26. There was no significant difference among five dwarfing interstock apple trees in average weight of fruit weight, fruit shape, fruit hardness, the content of soluble solid and solid-acid ratio. During the growing stages, the contents of N，P，Ca，and Mg in the leaves of the scion trees grafted on Qing 2, Liao 2 and SH6 were relatively higher, and high Zn content was found on Liao 2 interstock tree. However，the contents of Ca in leaves of the Yishui red Fuji trees grafted on M9T337 were slightly higher in later growth stage. 【Conclusion】According to the 3 years survey data, Yishui Red Fuji apple trees with Qing 2, Liao 2 and SH6 dwarf interstocks showed better growing compatibility, small tree, moderate growth, early bearing, high and stable yield, and good fruit quality characters.

Molecular Cloning and Expression Analysis of Genes MaGDHs Encoding Glutamate Dehydrogenase in Mulberry

ZHI Shuang, REN YanHong, TANG Xing, XU FengXiang, WANG ChuanHong, ZHAO AiChun, WANG XiLing

Scientia Agricultura Sinica. 2018, 51(4): 758-769. doi:10.3864/j.issn.0578-1752.2018.04.015

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【Objective】In this paper, Genes of GDH were cloned from mulberry and characteristics of their structures, tissue expressions，regulation factors were studied and GDH proteins were obtained in order to reveal the role of glutamate dehydrogenase (GDH) in nitrogen metabolism. The results could provide data for studying other perennial woody plants on GDH.【Method】Primers were designed according to the homologous sequences of GDH downloaded from the Morus genome database. Using Guisangyou 62 (M. atropurpurea Roxb.) cDNA as template, MaGDHs were cloned by RT-PCR. The similarity of GDH sequences was analyzed by BLASTN and converse structure domains were analyzed by CDD on line of NCBI. The composition of the amino acid, theoretical molecular mass and pI were deduced by Expasy software online. A phylogenetic tree was constructed by MEGA 5.0 software performed with the neighbor-joining (NJ) method. Expressions of MaGDHs under the condition of different concentrations of sucrose, NH4+ and 6-BA solutions were measured by qRT-PCR. The data were analyzed by StepOne Software V2.1 using method of 2^-△△Ct. MaGDHs recombinant plasmids were constructed with vectors of pET-28a(+), pET-32a(+), pcold-TF and transformed to E. coli to produce MaGDHs recombinant protein.【Result】Both two obtained genes MaGDHs were 1236 bp in length with an open reading frame encoding a polypeptide of 411 amino acids, named MaGDH1 and MaGDH2. The predicted molecular mass of MaGDH1 was 44.1 kD and a predicted isoelectric point was 5.84. The predicted molecular mass of MaGDH2 was 44.2 kD and a predicted isoelectric point was 6.68. Both two sequences have 9 exons and 8 introns including a mitochondrial transfer peptide and two features domains: a Glu/aKG binding domain and an NAD(P) binding domain. The MaGDHs protein showed identity of 99% with Morus notabilis, about 90% with dicotyledons plants and 85% with monocotyledons plants. The proteins of MaGDHs were successfully expressed in E.coli BL21 (DE3) in the form of inclusion bodies. After purification and renaturation, active enzymes were obtained and the activity of recombinant protein pET-28a(+)-MaGDH1 was the highest with 10.07 nmol?min^-1?mL^-1. MaGDH1 and MaGDH2 showed organ specificity but distinct transcript intensity. The expression level of flower was the highest and the expression level of young leaf was taken the second place. They hardly expressed in fruit. Transcript abundance of MaGDHs were up-regulated by sucrose in this study, MaGDHs were also expressed without NH₄⁺ in media and excess NH4+ promoted the transcript of MaGDHs. 6-benayl aminopurine first inhibited and then promoted the expressions of MaGDHs and MaGDH1 was promoted after 24 h and MaGDH2 was promoted after 48 h.【Conclusion】Two genes of GDH were obtained from mulberry named MaGDH1 and MaGDH2 respectively encoding β and α subunit. The proteins of MaGDHs were successfully expressed in E. coli BL21 (DE3) in the form of inclusion bodies. The activities of recombinant proteins were different with different vectors. The expression of MaGDHs showed organ specificity and expressed higher in vigorous organs than others. They were up-regulated by excess NH₄⁺. MaGDH1 were promoted by 6-BA earlier than MaGDH2.

Generation and Propagation of Cluster of Differentiation 163 Biallelic Gene Editing Pigs

WEI YingHui, LIU ZhiGuo, XU Kui, Evanna HUYHN, Paul DYCE, LI JiLiang, ZHOU WeiLiang, DONG ShuRen, FENG BaoLiang, MU YuLian, JuLang LI, LI Kui

Scientia Agricultura Sinica. 2018, 51(4): 770-777. doi:10.3864/j.issn.0578-1752.2018.04.016

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【Objective】 Porcine reproductive and respiratory syndrome (PRRS), commonly known as “blue ear disease”, is a highly fatal infectious disease with porcine reproductive and respiratory syndrome virus (PRRSV) being the causative pathogen. PRRSV causes major economic losses in the pork industry world-wide. The genetic variability of PRRSV is high and an ideal vaccine to prevent the occurrence of this disease is not available. Cluster of differentiation 163 (CD163) is the important receptor for the entry of PRRSV into the porcine alveolar macrophage (PAM) cells. The aim of this study was to generate CD163 gene edited Large White pigs by using the CRISPR / Cas9 and somatic cell nuclear transfer (SCNT) techniques.【Method】 CRISPR/Cas9 vector was constructed for editing the exon 7 of the porcine CD163 gene; The constructed vectors were transfected into pig fetal fibroblasts to obtain gene edited positive cell colonies; CD163 gene edited fibroblasts and in vitro matured porcine oocytes were employed as nuclear donors and nuclear receptors respectively to obtain reconstructed embryos; For obtaining CD163 gene edited pigs the reconstructed embryos ere transferred into recipient sows and performing the subsequent propagation experiment. 【Result】 The designed gRNAcould effectively recognize the intended site. Genotyping analysis of cloned cell showed that 21 colonies had mutations in the CD163 gene, of which 14 colonies had either a monoallelic mutation or a biallelic heterozygous mutation, and 7 colonies had a biallelic homozygous mutation. Through SCNT, we successfully obtained CD163 biallelic edited Large White pigs. Successful breeding allowed us to obtain F₁ generation CD163 gene edited piglets, and they are all in good health. It is anticipated that more F₁ piglets will be produced soon. 【Conclusion】The CD163 biallelic edited Large White pigs that do not harbor a drug resistant gene in their genome were produced and they can thus safely and quickly serve as a gene donor for breeding of PRRSV resistant pigs.

Effects of Zearalenone on Expression of Heat Shock Protein 70 and Morphology of Uterus Tissues of Post-Weaning Piglets

ZHOU Min, ZHOU XueMei, YANG LiJie, HUANG LiBo, FENG Lei, SHAO MingHui, YANG Chen, YANG WeiRen, YANG ZaiBin, JIANG ShuZhen

Scientia Agricultura Sinica. 2018, 51(4): 778-788. doi:10.3864/j.issn.0578-1752.2018.04.017

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【Objective】 The aim of this study was to investigate the effects of zearalenone (ZEA) on uterus morphology, distribution and mRNA relative expression of heat shock protein 70 (HSP 70) in post-weaning piglets. 【Method】 A total of 40 healthy female piglets (Duroc×Landrace×Large White) aged approximately 25-28 d were selected for study. The piglets were transferred to single cages after 10 day adaptation on the obstertric table, then they were allocated into four treatments. Each treatment consisted of 10 replicates of 1 pig per replicate according to the age (35-38) and the average body weight (14.01 ± 0.86 kg). After 10 d of adaptation piglets were fed a normal basal diet supplemented with purified ZEA of 0 (Control), 0.5 (0.52±0.07), 1.0 (1.04±0.03) and 1.5 (1.51±0.13) mg·kg^-1 for 35 d. Animals were housed individually with free access to feed and water. At the end of the feeding trial, piglets were deprived with feed for 12h and sacrificed. Uterus were then isolated under sterile conditions. Two samples of uterus tissues from each pig were quickly collected, one of which was stored at -80℃ to detect relative mRNA expression of HSP70, and the other was fixed promptly in Bouin’s for histological examination and immunohistochemical analysis. 【Result】 Results showed that the myometrium and endometrium became thicker and the number and density of uterine glands increased with the higher level of ZEA. The thickness of myometrium and endometrium increased linearly (P＜0.001) and quadratically (P＜0.001) with higher level of ZEA. The thickness of myometrium and endometrium from ZEA1.5, 1.0 and 0.5 treatments were significantly higher than that of ZEA1.0, 0.5 and control treatments, respectively (P＜0.05). The results of immunohistochemistry indicated that HSP70 immunoreactive substance was mainly distributed in the cytoplasm of uterine smooth muscle cells, uterine glandular epithelial cells, luminal epithelial cells and vascular endothelial cells, but nuclear staining was rarely observed. The light yellow immunereactive substance was shown in the tissues of negative control samples, the color of the positive reaction of HSP70 was enhanced to yellow or brown of block shape with higher level of ZEA. The immunoreactive integrated optic density (IOD) of HSP70 and the total measured part of the uterus in weaning gilts increased linearly (P＜0.001) and quadratically (P＜0.05) in myometrium, luminal epithelium, gland epithelium with rising level of ZEA. In general, the IOD of ZEA1.5 treatment was significantly higher than that of ZEA0.5, while the IOD of ZEA0.5 and ZEA1.0 treatment was significantly higher than that of control. Relative mRNA expression of HSP70 in the uterus of weaning gilts increased linearly (P＜0.05) and quadratically (P＜0.05) with the rising level of ZEA. Although the difference between ZEA1.5 and ZEA1.0 was not significant (P＞0.05), the ZEA1.5 treatment was significantly higher than that of ZEA0.5 treatment (P＜0.05), while ZEA0.5 treatment was significantly higher than that of control (P＜0.05). 【Conclusion】 In conclusion, ZEA at 0.5 mg·kg^-1was sufficient to make the myometrium and endometrium to become thicker, while at the time the concentration could also increase the number of uterine glands and changed the morphology of the uterus; under the condition of this experiment, the positive reaction of HSP70 was enhanced with the higher concentration of ZEA, which means the higher level expression of HSP70 may prevent damage of uterine cells by toxin stress.

Screening and Analysis of Anti-BmNPV Cytokines in Silkworm (Bombyx mori)

WANG Fei, LI XianYang, HUA XiaoTing, XIA QingYou

Scientia Agricultura Sinica. 2018, 51(4): 789-799. doi:10.3864/j.issn.0578-1752.2018.04.018

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【Objective】The objective of this study is to investigate the role of BmRelish, a NF-kB-like transcriptional factor in immune response against infection of Bombyx mori nucleopolyhedrovirus (BmNPV), and to screen and analyze the potential antiviral cytokines to better understand the antiviral immunity in silkworm (Bombyx mori).【Method】The generation of BmRelish active form (BmRelish_act) from full-length BmRelish (BmRelish-FL) in BmE cells after BmNPV infection was examined by Western blot, the relative level of viral DNA in BmRelish_act-expressing cells was evaluated by quantitative PCR and the amount of EGFP-positive cells was compared with control cells which did not express BmRelish_act after infection with EGFP-labeled BmNPV (BmNPV-EGFP) to investigate whether BmRelish participates in antiviral immunity. Naive cells were then incubated with supernatant medium of BmRelish_act-expressing cells in Transwell co-culture system followed by BmNPV infection, and the relative level of viral DNA in those cells was evaluated to determine whether antiviral cytokines were produced and secreted from BmRelish_act-expressing cells. Next, the supernatant medium of BmRelish_act-expressing cells was fractionated by ultrafiltration, and the filtrate and retenate fractions were incubated with naive cells respectively, the relative level of viral DNA in those cells was then evaluated to estimate the molecular mass of antiviral cytokines. This assay was also performed with heat-treated filtrate and retenate to determine the biological properties of antiviral cytokines. Finally, the supernatant medium of BmRelish_act-expressing cells was analyzed by LC-MS/MS.【Result】After BmNPV infection, BmRelish-FL was partially processed into its active form (BmRelish_act), and the relative level of viral DNA as well as the amount of BmNPV-EGFP positive cells in BmRelish_act-expressing cells was significantly lower than control cells. Incubation with supernatant medium of BmRelish_act-expressing cells also led to a remarkable decrease in the relative level of viral DNA in naive cells after BmNPV infection. Incubation with filtrate fractions from the supernatant medium of BmRelish_act-expressing cells fractionated by centrifugal filter with cut-off size of 100 and 3 kD maintained the antiviral activity, while retenate fractions did not. The antiviral activity can be removed by heating. A total of 67 peptides consisting of 9-45 amino acids and derived from 32 proteins were identified by LC-MS/MS from the filtrate fraction of supernatant medium. Those proteins all have molecular mass＞3 kD and 9 of them contain signal peptide.【Conclusion】BmRelish is activated in response to BmNPV infection and participates in antiviral immunity by promoting the production of antiviral cytokines, which are secreted into the supernatant medium and enhance the anti-BmNPV immunity of naive cells. Those anti-BmNPV cytokines are small peptides with molecular mass＜3 kD cleaved from larger proteins.

Analyses of Physicochemical Properties of NFC Apple Juices from Sixteen Cultivars

GOU XiaoJu, TIAN You, GUO YuRong, YANG Xi, HOU YanJie

Scientia Agricultura Sinica. 2018, 51(4): 800-810. doi:10.3864/j.issn.0578-1752.2018.04.019

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【Objective】The eight physicochemical properties of the not from concentrate (NFC) apple juices from sixteen cultivars were investigated in this study. Correlation analysis of all measured variables was applied for simplifying the evaluating indicators of NFC apple juices. Moreover, multivariate data analyses were applied to categorize apple juices, in hope of finding out the alternative cultivars in NFC juice production and intelligent formulation, and thus providing theoretical basis for juice industry.【Method】NFC juices from sixteen cultivars were prepared through squeezing, inactivating enzymes, pasteurization and hot filling. Eight physicochemical properties of sixteen juices, including the total soluble solids (TSS) content, pH value, titratable acidity (TA), TSS/TA, apparent viscosity, turbidity, total phenol content (TPC) and color quality (L^*, a, b, C, ^*h°) were determined, and their differences and correlations of the properties were evaluated using variance analysis and correlation analysis methods, respectively. Additionally, principal component analysis (PCA), liner discriminant analysis (LDA) and cluster analysis (CA) were applied to analyze the data matrix of physicochemical properties to classify sixteen juices.【Result】 The results showed that physicochemical properties of sixteen juices were significantly different. The cultivar ‘Xinshijie’ had the highest TSS content (15.7 °Brix) and turbidity (3 720 NTU), while ‘Granny Smith’ possessed the lowest pH (2.74), ‘Jonathan’ possessed the highest TA value (9.6 g·L^-1, malic acid equivalent), and ‘Qinyan’ had the highest TSS/TA (39.24). Besides, three cultivars ‘Delicious’, ‘Empire’ and ‘Cox’s orange pippin’ showed the highest apparent viscosity (3.75 mPa·s), and ‘Fuji’ possessed the highest TPC (775.94 mg·L^-1, gallic acid equivalent). pH value and TSS/TA were extremely significantly positive correlation (R²=0.844), and turbidity and L^* value also showed extremely positive correlation (R²=0.967, P<0.01). However, pH and TA (R²=-0.896), as well as TSS/TA and TA (R²=-0.918) showed extremely negative correlation (P<0.01). The results from PCA, LDA and CA provided the totally same classification, and the sixteen cultivars were classified into three categories.【Conclusion】The original eight physicochemical properties could be simplified to five indicators, they are TSS, TA, color quality (L^*, a^*, b^*, C^*, h°), apparent viscosity and TPC. These five indicators can be used to reflect overall the quality of NFC apple juices. Based on the classification of juice quality, the cultivar ‘Yuhuazaofu’ and ‘Jonagold’ formed the first group, ‘Xinshijie’ and ‘Qinguan’ constituted the second group, and ‘Starkrimson’, ‘Fuji’, ‘Cox’s orange pippin’, ‘Ziyou’, ‘Qinyan’, ‘Granny Smith’, ‘Qiuxiang’, ‘Pink lady’, ‘Delicious’, ‘Empire’, ‘Liangxiang’, ‘Jonathan’ formed the third group. The cultivars in the same group can be the alternatives in NFC juice production and intelligent formulation.

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