【Objective】 In the present study, the common wheat (Triticum aestivum L.) was exposed to the stress of stripe rust and powdery mildew. Then the cloned TaNAC structural variation transcripts formed by alternative splicing were analyzed. And putative information of TaNAC genes regulated by miRNAs was annotated. It would shed light on the study of TaNAC genes in response to fungal stress of wheat at the post-transcriptional level. 【Method】 After common wheat resistance germplasm N9134 being infected stripe rust and powdery mildew respectively, the leaves were sampled at eight time points. Then a large number of TaNAC transcripts were cloned from the mixed sample pool. Referring to the wheat genome annotation of Chinese Spring (IWGSC RefSeqv1.1), the sequence structure characteristics of TaNAC structural variation transcripts formed by alternative cutting were revealed. Using bioinformatics software and online tools, the coding products derived from these TaNAC transcripts were compared and analyzed, including the functional domain, advanced structure, physical and chemical properties, subcellular localization and other characteristics. And then, one pair of TaNAC structural variation transcripts were selected to further verify the predicted subcellular localizations by onion epidermis transient expression system. Meanwhile, five groups of TaNAC transcripts were conducted transcriptional self-activation experiments in yeast. It was aimed to analyze the effects of structural variation, which caused by alternative splicing on transcriptional regulation activity. Additionally, using the miRBase database, the targeting relationship between TaNAC genes and tae-miRNAs was forecasted and established in wheat. 【Result】 In this study, 35 TaNAC structural variation transcripts were formed by alternative splicing form 13 TaNAC genes, and they all were cloned from common wheat N9134 after infecting by stripe rust and powdery mildew. After analyzing, it was found that there were differences in the nucleic acid sequence structure of different structural variation transcripts from the same TaNAC gene, as well as in the functional domain, advanced structure, physicochemical properties and subcellular localization of their corresponding coding products. And they might be with different transcriptional regulatory activities. Moreover, different TaNAC genes could be with different patterns of alternative splicing, and the coded products of the structural variation transcripts from different TaNAC genes showed diversity in characteristics of structure, physical and chemical properties, transcriptional regulatory activity and so on. By analysis of TaNAC genes and their target tae-miRNAs, which is in the coding region, the result showed that the binding sites of tae-miRNAs were all in the non-alternative splicing region. 【Conclusion】 In conclusion, TaNAC genes might be involved in the response of wheat to fungal stress through the post-transcriptional regulation of alternative splicing. And the tae-miRNAs targeted to TaNAC genes could function post-transcriptional regulation independently of alternative splicing.

【Objective】Transgenic maize NK603 approved for import is an important target of genetically modified organism (GMO) regulation in China. The implementation of GMO regulation requires reference materials (RMs) and standardized detection methods. Establishment of a duplex droplet digital PCR (ddPCR) would provide accurate measurement technology for quantification of NK603 event and development of NK603 RMs. 【Method】 A standard plasmid molecule pUC57-NK603 was constructed by DNA synthetic technique; the primer/probe set of NK603 event was combined with different maize reference genes one by one to select reference gene PCR assay with identical amplification ability to NK603 event-specific PCR assay; main reaction parameters, such as annealing temperature and primer/probe concentration,were optimized in the course of establishing duplex ddPCR; the standard plasmid solution was serially diluted to investigate the limit of detection (LOD), limit of quantification (LOQ) and dynamic range of the duplex ddPCR assay; blinded samples with mass fractions of 100%, 10% and 6% were prepared by mixing NK603 powder with non-GM counterpart, to evaluate the accuracy of quantitative results of the duplex ddPCR. 【Result】 The reference gene zSSIIb was determined to combine with NK603 event to establish the NK603/zSSIIb duplex ddPCR method with the standard plasmid molecule pUC57-NK603 as a quality control after analyzing the fluorescence amplitude of positive droplets, separation between positive and negative droplets, raindrop number, and consistency between measured copy number ratio and expected copy number ratio of NK603 event to reference gene. The primer/probe concentration was optimized to be 400 nmol·L^-1/200 nmol·L^-1 for both NK603 event and zSSIIb gene, and the annealing temperature was determined to be 60°C. The LOD of NK603/zSSIIb duplex ddPCR was estimated to be 2 copies of DNA template, the LOQ was 48 copies of DNA template, both NK603 assay and zSSIIb assay showed good linearity between measured values and theoretical values over the dynamic range from 10 to 60 000 copies of DNA template. The NK603/zSSIIb duplex ddPCR achieved accurate quantitative results of NK603 content in blind maize samples with less than 25% of coefficient of variation; the quantitative results of ddPCR were not significantly different from those of real-time quantitative PCR (qPCR), moreover, the duplex ddPCR showed an advantage over qPCR in term of precision. 【Conclusion】 The selection of reference genes affects the accuracy of quantitative results by ddPCR. Establishment of ddPCR methods should use samples with accurate GMO content as quality controls to evaluate the applicability of reference genes. The NK603/zSSIIb duplex ddPCR method was successfully established using the synthetic standard plasmid molecule pUC57-NK603 as a quality control. NK603 certified reference materials (CRMs) have been successfully developed in China by applying the established NK603/zSSIIb duplex ddPCR assay to characterize the property values.

【Objective】Bruchids are the main storage pests of cowpea. The discovery of bruchids resistance genes is helpful for breeding resistant varieties and reducing the harm of bruchids to cowpea production.【Method】In this study, a RIL population of 282 lines derived from the cross between Zhongjiang No.1 (a bruchid-susceptible cultivar) and Pant-lobia-1 (a bruchid-resistant cultivar) was used for phenotype identification of bruchids resistance by artificial inoculation of Callosobruchus chinensis and Callosobruchus maculates. The two parents were used to screen polymorphic markers from 3 992 SSR markers of mung bean, adzuki bean and cowpea. Genotypes of 282 lines were analyzed using the polymorphic SSR markers. Based on the phenotype identification and genotype analysis, a genetic linkage map was constructed and the bruchids resistance gene(s) of cowpea was located using Inclusive Composite Interval Mapping (ICIM-ADD). 【Result】The results showed that Zhongjiang No. 1 and F₁ seeds were 100% susceptible to Callosobruchus chinensis and Callosobruchus maculates, and the damage rates of Pant-lobia-1 were 22.5% and 42.5%, respectively. It was speculated that the resistance of Pant-lobia-1 to bruchids was recessive inheritance. 182 polymorphic markers were obtained from 3 992 SSR markers of mung bean, adzuki bean and cowpea. Using those polymorphic SSR markers, a genetic linkage map with 11 linkage groups was constructed. The map covered a total length of 1 065.23 cM with an average interval of 5.85 cM between adjacent markers. Two bruchid-resistant QTLs from linkage groups 1 and 5 were discovered, which were temporarily named as vubr1-1 and vubr5-1. QTL vuvr1-1 was located between markers XD11-44 and HAAS_VR_2274, which genetic distance was 7.6 cM, explaining 7.16% and 6.92% of the phenotypic variation in the two bruchid-resistant tests respectively. QTL vubr5-1 was located between markers XD1-14 and CP185, which genetic distance was 2.9 cM, explaining 6.96% and 6.37% of the phenotypic variation in the two bruchid-resistant tests respectively. 【Conclusion】A genetic linkage map containing 11 linkage groups and 182 polymorphic markers was constructed. Two QTLs linked with bruchids resistance were identified on linkage groups 1 and 5.

【Objective】Water resource scarcity is one of the most prominent constraints for agricultural production in arid irrigation areas, so maize production are mainly mulched with traditional plastic film. However, this process is increasingly challenged by extreme high-temperature and ecological environment pollution. This study investigated the feasibility of maintaining high water use via no tillage with plastic film reusing pattern, and the aim was to prove the theoretical support for the construction of efficient maize production technology with plastic film reduction.【Method】In 2017 and 2018, a field experiment was carried out in Hexi oasis irrigation region from Gansu province to determine the effects of different plastic film mulching and using patterns on soil water utilization of maize field. The treatments included no tillage with plastic film reusing pattern (no tillage with plastic film mulching, NM), no tillage in fall and new plastic film mulching in spring (reduced tillage with plastic film mulching, RM), and conventional tillage with annual new plastic film mulching (conventional tillage with plastic film mulching, the control, CM). 【Result】Compared with CM, the mean soil weight moisture content across the 0-120 cm soil depth was increased by 7.5% and 5.1% with NM and RM before the maize sowing, respectively, which created a good soil moisture environment for sowing of maize. NM had greater mean soil weight moisture content across the 0-120 cm soil depth by 5.0% and 4.7% than thant that under CM from sowing to jointing and silking to early-filling stages, respectively, which made up for the abundant demand of soil moisture for the vigorous growth of maize filling stage. Evapotranspiration under NM was increased until maize big-flare stage, decreased from maize silking to early-filling stage, and increased after maize early-filling stage, which was effectively coordinated with water demand contradiction of maize at each growth stages. Compared with RM and CM, NM increased soil evaporation before silking stage by 11.7% and 26.0%, and increased the ratio of soil evaporation to evapotranspiration (E/ET) by 13.4% and 19.9%, respectively. However, NM reduced soil evaporation after silking stage by 9.2% and 19.4%, and reduced E/ET by 9.7% and 20.7%, respectively. The results indicated that NM was beneficial to enhance the effective utilization of soil water after maize silking stage. Therefore, NM with no tillage and plastic film reduction obtained the same grain yield and water use efficiency for RM and CM.【Conclusion】The results showed that no tillage with plastic film reusing pattern did not lead to a decrease in the grain yield and water use efficiency of maize, compared with conventional tillage with annual new plastic film mulching, but it stabilized grain yield and water use efficiency, thus, this practice was a feasible measure to reduce plastic film input and stabilize productivity of maize production in the arid irrigated area of northwestern China.

【Objective】In order to explore the growth dynamic prediction model and its characteristic parameters of summer maize plant height and leaf area index (LAI) based on effective accumulated temperature with different nitrogen, phosphorus and potassium treatments, in order to provide a theoretical basis for using effective accumulated temperature to quantitatively simulate the growth and development of summer maize.【Method】Based on the two-year field experiment in Langfang, Hebei province (2019-2020), this study uses Zhengdan 958 as the experimental material and is divided into three single-factor fertilizer efficiency experiments of nitrogen, phosphorus, and potassium. Each factor is set at 4 levels, respectively no fertilizer, low fertilizer, moderate fertilizer and high fertilizer treatments. The Logistic mathematical model was used to fit the dynamic equation of summer maize plant height and leaf area index based on effective accumulated temperature under different nitrogen, phosphorus and potassium nutrition levels, and the growth rate curve and its characteristic parameters were used to quantitatively analyze the growth and development characteristics of summer maize.【Result】(1) Under the conditions of this experiment, compared with other treatments, the maximum summer corn plant height was the largest in the treatments with proper amount of fertilizer (N2, P2 and K2). Excessive application of potassium fertilizer has a significant inhibitory effect on the maximum plant height of summer corn. Appropriate fertilization treatment of summer maize plant height requires accumulated temperature of 952.43-958.83℃·d. proper fertilization can effectively increase summer maize leaf area index, and excessive or insufficient nutrients will affect the formation of leaf area. Appropriate fertilization treatment of summer maize leaf area index required accumulated temperature to enter the plateau period is 849.18-952.43 ℃·d.(2) The fit R² of the summer maize plant height and leaf area index equations established with effective accumulated temperature as the independent variable under each fertilization treatment condition were 0.9949-0.9970 and 0.9840-0.9939, respectively, and the equations reached extremely significant levels and had biological significance. The correlation coefficient (r) between the simulated value and the measured value based on the plant height fitting equation of the effective accumulated temperature is between 0.9961-0.9983; the r of the simulated value and the measured value of the leaf area index fitting equation based on the effective accumulated temperature is 0.9815-0.9981.(3) Under various fertilization conditions, the growth rate of summer maize plant height and leaf area index showed a “single-peak curve”. Under the conditions of appropriate fertilization, the growth rate curve showed the characteristics of rapid rise and fall. The growth rate curve of the treatments without nitrogen fertilizer, phosphate fertilizer and potassium fertilizer showed the characteristics of slow rise and slow decline. (4) Under the condition of appropriate fertilization, the accumulated temperature of summer maize plant height entering the rapid growth period, the accumulated temperature entering the slow growth period and the accumulated temperature reaching the maximum growth rate are 394.17℃·d, 776.63℃·d and 585.40℃·d, respectively, which were significantly different from N0, P0 and K0 treatment. The maximum growth rate and average growth rate of plant height in the rapid growth period are 0.4907 cm·(℃·d)^-1 and 0.4302 cm·(℃·d)^-1, respectively, which are not significantly different from N0, P0 and K0 treatment. (5) Under the condition of appropriate fertilization, the accumulated temperature of summer maize leaf area index entering the rapid increase period, the accumulated temperature entering the slow increase period and reaching the maximum growth rate are 609.69℃·d, 855.08℃·d and 732.38℃·d, respectively. The maximum growth rate of leaf area index and the average growth rate of rapid increase period are 0.0135℃·d and 0.0118℃·d, respectively.【Conclusion】Insufficient nutrient supply can increase the effective accumulated temperature required for summer corn plant height and leaf area index to enter the plateau period. The Logistic model based on the effective accumulated temperature can well simulate and predict the dynamic changes of summer maize plant height and leaf area index under different N, P, and K treatments. The degree of fit and stability of the fitting equation under the condition of proper fertilization is better than that of the fitting equation with excessive or insufficient nutrients. Compared with the treatment with no fertilization, the plant height and LAI of summer corn reach the required accumulated temperature during the critical period (accumulated temperature required to enter the rapid increase period, accumulated temperature required to enter the slow increase period, and accumulated temperature required for the maximum growth rate). The growth rate (the maximum growth rate, the average growth rate during the rapid growth period) has decreased significantly. This study provides a theoretical basis for the effective accumulated temperature to quantitatively simulate the growth and development of summer maize.

As the importance of making an accurate rate to the sustainable development of agricultural insurance programs, lots of literature on agricultural risk assessment and agricultural insurance pricing had been conducted since the 1980s. Yet, uncertainty still existed regarding the risk assessment results and/or the agricultural insurance premium. With the purpose of improving the credibility of Chinese agricultural insurance pricing, we firstly conduct a literature review on the recent development in the field of agricultural risk assessment and insurance pricing, and then put forward the uncertainty sources for agricultural insurance pricing, followed by a solution. It is found that the data scarcity, the fuzziness in dealing with technical issues, and the unmatched spatial scale of risk assessment and pricing are the three reasons for the uncertainty of agricultural risk assessment and insurance pricing, and improving the agricultural insurance pricing credibility has been emerging as a hot topic in recent literature. Reducing the uncertainty of agricultural insurance pricing can be achieved in the big data era with the help of data mixing technology and data-intensive research. While making a sound agricultural insurance rate cannot overcome the essential adverse selection problem which could hamper the agricultural insurance sustainable development, however, it can be partly addressed by providing more flexible agricultural insurance products with alternative coverage levels.

【Objective】Apple Alternaria blotch occurs in all major apple producing areas in China, causing serious economic loss. Apple Alternaria blotch is a worldwide airborne disease caused by Alternaria alternata f. sp. mali. The objective of this study is to investigate the diversity of dsRNA of A. alternata f. sp. mali in China, and to provide new biological control resources for the disease and new understanding of virus diversity and evolutionary.【Method】Tissue samples with typical symptom were collected from eight provinces in China. Pure culture was obtained by tissue isolation and single spore separation. The diversity of dsRNA carried by A. alternata f. sp. mali in China was determined by dsRNA extraction and gel electrophoresis. The biological characteristics of dsRNA-carrying pathogens were determined by evaluation of culture characteristics, mycelial growth rate, and pathogenicity on fruits and leaves. By high throughput sequencing and molecular cloning techniques, the whole genome sequence, genome structure and phylogenetic for the mycovirus carried by QY-2 strain were analyzed.【Result】The pure culture of 102 strains of A. alternata f. sp. mali was obtained and five strains with obvious dsRNA bands were found. The dsRNA virus of A. alternata f. sp. mali in China could be divided into four types, there were about 8, 2.5 and 1.5 kb dsRNA in type I (strain YT-3-7), about 8 kb dsRNA in type II (strain SJZ-4), about 3 and 0.8 kb dsRNA in type III (strain QY-2), and about 2.5 and 1.5 kb dsRNA in type IV (strains CL-2-6 and SQ-1-1). The strains carrying dsRNA had diverse culture characteristics, and there was no significant relationship between the colony characteristics, growth rate and whether dsRNA carrying or not and its type. The strains carrying dsRNA were mostly of low pathogenicity type. It was found that QY-2 strain showed weak pathogenicity on leaves and fruits, so QY-2 was selected for virus identification. The genome of the dsRNA virus carried by the strain QY-2 consists of five dsRNA fragments, dsRNA1-dsRNA5, with the sequence sizes of 3 665, 3 054, 2 824, 2 819 and 831 nt, which were submitted to GenBank. The accession numbers are MK672910, MK672913, MK672912, MK672911, and MK836314. The molecular weights of coding proteins are 124, 83, 84, 83 and 13 kD, respectively. Phylogenetic analysis showed that it was closely related to AaCV1, so it was identified as Alternaria alternata chrysovirus belonging to Chrysoviridae and Betachrysovirus. The tentative designation is Alternaria alternata chrysovirus 2 (AaCV2). 【Conclusion】There are diverse dsRNAs in A. alternata f. sp. mali in China, and there is no significant correlation between dsRNA diversity and host pathogen culture traits. Most of the strains carrying dsRNA showed low pathogenicity. QY-2 strain with the least pathogenicity carries AaCV2. The low pathogenicity characteristic of this strain may have potential application value, which provides a new resource for biological control of apple Alternaria blotch.

【Objective】 Metarhizium anisopliae, an entomopathogenic fungus, is found also an endophyte. MAD2 is known to be one of the two adhesin proteins of M. anisopliae, which plays a vital role in adhesion and colonization in plants, but its functional mechanism is poorly understood. The objective of this study is to explore the functional effect of MAD2 protein on the characteristics of growth, virulence, adhesion and inducing plant responses in M. anisopliae by construction of the mad2 mutant strain (Δmad2) of M. anisopliae strain Ma9. 【Method】 The genomic DNA sequences of mad2 anteroposterior were obtained from NCBI, and specific primers were designed to amplify mad2 homologous arm genes S1 and S2 by PCR based on genomic DNA template of M. anisopliae strain Ma9. Meanwhile, Hyg-F/R primer pair was designed to amplify hygromycin gene with promoter sequence based on pKH-KO vector DNA template. Then, homologous knockout boxes S1H and S2H of mad2 were constructed by overlap PCR. Finally, mad2-knockout strains with stable inheritance were obtained by PEG-mediated protoplast transformation of the homologous knockout boxes. By comparing the biological characteristics of the knockout strains (Δmad2) to the wild-type strains (WT), the effects of MAD2 protein on the characteristics of M. anisopliae growth, virulence, adhesion and inducing the peanut response of symbiosis genes were analyzed. 【Result】 Homologous recombination transformants with mad2 knockout were obtained by protoplast transformation. The spore germination rate of Δmad2 was significantly decreased and spore semi-germination time was significantly prolonged 5.47 h compare to WT, as well as the mycelium length of Δmad2 was significantly shorter than WT in 12 h and 14 h incubation, which occupied 77.8% and 76.3% of WT, respectively. The sporulation in 12-day incubation was reduced by 33.3% compared to WT. The ability of Δmad2 strain adhesion in onion was significantly decreased but showed no difference in adhere to the underwings of locust. In addition, mad2-knockout did not affect the virulence of M. anisopliae to silkworm. In the peanut inoculated mad2-knockout strain for 12 h, the transcription level of symbiosis receptor gene SYMRK, calcium signal decoding related genes (CAM, CCaMK, DELLA), lipid and nitrogen transfer related genes (LTP1, NRT24, ABCC2) was significantly down-regulated compared to the treatment of WT. While compared with blank control, Δmad2 still had certainly up-regulated SYMRK transcription level, significantly inhibited the transcription levels of CAM, CCAMK and DELLA, but had no effect on the transcription level of ABCC2, LTP1 and NRT24. 【Conclusion】 M. anisopliae adhesin protein MAD2 affects spore germination, initial growth of mycelium, sporulation quantity and plants adhesion, while has no effect on insect adhesion and virulence of strain, and MAD2 triggers the transcription of peanut symbiotic genes at the initial stage of interaction between the strain and peanut.

【Objective】The objective of this study is to clone the full-length cDNA of the G protein-coupled receptor kinase 2 (AlGRK2) in Apolygus lucorum, clarify its expression profiles and the effect of exogenous ecdysterone hormone 20E on the expression of AlGRK2, analyze the role of AlGRK2 in the growth and development of A. lucorum, and to provide a preliminary research basis for further study of its function in the ecdysone signaling transduction pathway. 【Method】The AlGRK2 was cloned and obtained by RACE method. Using the qRT-PCR method, the relative expression levels of AlGRK2 in different days (1-16 day-old) and tissues (head, thorax, wing, leg, midgut, ovary and fat body) in female adults in A. lucorum were determined. Finally, the response of the AlGRK2 mRNA expression in A. lucorum after exogenous 20E induction and RNAi treatment and their effects on the main parameters (development progress, nymphs weight and adult emergence rate) in the growth and development of the A. lucorum were analyzed.【Result】The full length of AlGRK2 cDNA is 2 715 bp, and ORF is 2 106 bp, which encodes 701 amino acids. ExPASy predicts that the protein molecular weight is 80.2 kD and the theoretical isoelectric point is 6.56. Protein structure analysis shows that AlGRK2 contains 4 domains: G protein signalling domain (RGS, 54-175 aa), serine/threonine protein kinases domain (S-TKc, 191-454 aa), extension to Ser/Thr-type protein kinases (S-TK-X, 455-534 aa) and pleckstrin homology domain (PH, 558-655 aa), and the PH domain is a typical protein feature in the GRK2 subtype. Phylogenetic analysis showed that GRK2 in A. lucorum had the closest genetic relationship with Halyomorpha halys GRK2. qRT-PCR results showed that AlGRK2 was expressed in 1-day-old to 16-day-old nymphs of A. lucorum, and the mRNA expression showed a fluctuating downward pattern. The expression level of AlGRK2 was higher in the initial instar, but decreased significantly in the last instar of A. lucorum. The AlGRK2 was highly expressed in the ovary and fat body of female adults and less expressed in the thorax and leg. After exogenous 20E treatment, the expression of AlGRK2 was significantly down-regulated in nymphs at the 1-day-old and 3-day-old. The relative expression of AlGRK2 in all tissues of females was up-regulated after treating with 20E which compared with ethanol control, especially in ovary and fat body. In contrast, the expression of AlGRK2 treated with U73122 (PLC inhibitor) was down-regulated. Compared with ethanol control, the developmental duration of nymph, the body weight of the last instar nymph and the emergence rate of adult in A. lucorum were significantly decreased after 20E treatment. On the contrary, the development duration of nymph stage in U73122 treatment group was significantly prolonged. In addition, compared with the dsGFP group, AlGRK2 expression level of A. lucorum was significantly decreased after injection of dsAlGRK2, and nymphs mortality and developmental stages were significantly increased, while adult emergence rate and weight of the 5th instar nymph were significantly decreased. 【Conclusion】The expression profile of AlGRK2 in A. lucorum showed the specificity of the developmental stage and the tissue. Exogenous 20E inhibitor and RNAi treatment can inhibit the expression of AlGRK2, and have adverse effects on the growth and development of A. lucorum, such as delaying the development progress of A. lucorum, reducing the weight of 5th instar nymph and adult emergence rate.

【Objective】 Excessive application of phosphorus has not only caused the depletion of phosphate rock resources, but also increased the risk of environmental pollution. Livestock manure has always been used as a good alternative resource for chemical phosphorus. However, due to the complex composition and various influencing factors in the transformation process of phosphorus in animal manure, its effectiveness compared with that in chemical fertilizers has long been controversial. Clarifying the phosphorus replacement value of livestock manure, and the accurate proportion of organic fertilizer phosphorus replacing chemical fertilizer phosphorus, could provide the supporting data for rational application of organic fertilizer phosphorus.【Method】In this study, composted pig manure, chicken manure and cattle manure as well as chemical fertilizer were selected as the research materials. The application rate of P₂O₅ was set at 6 levels, which were 0, 20, 40, 60, 80 and 100 mg·kg^-1 dry soil, respectively. A soil column experiment was conducted to investigate the effects of phosphorus in manure and chemical fertilizer on wheat yield, phosphorus uptake and the content of soil available phosphorus. Furthermore, the relative substitution equivalent of phosphorus in three kinds of manure was statistically analyzed.【Result】 (1) The content of organic and inorganic phosphorus in the three kinds of manure varied much. The proportion of organic phosphorus accounted for 25.9%, 17.6% and 56.5% of total phosphorus in pig, chicken and cattle manures, respectively. The liable phosphorus (H₂O-P and NaHCO₃-P) was the main phosphorus fraction for the cattle manure, while there was more phosphorus fraction attributed to highly stable phosphorus (HCl-P) in chicken manure and pig manure. (2)There was no significant difference in grain yield between different phosphorus supplies with the same application rate. The phosphorus uptake of wheat with chemical fertilizer was slightly higher than that treated with three kinds of manure. (3) In this study, when the seasonal recovery rate of phosphorus was used as a reference index, the chemical fertilizer equivalent value in chicken, pig and cattle manures to the commercial mineral phosphorus (super phosphate) were 80.3%, 84.3% and 90.4%, respectively. When the relative substitution equivalents of three kinds of manure were calculated by using the regression function between the chemical phosphorus and grain yield, biomass, phosphorus uptake of grain or total phosphorus uptake, the relative substitution equivalents of pig manure were 90.0%, 93.6%, 80.6% and 80.2%, respectively; The relative substitution equivalents of chicken manure were 78.4%, 87.9%, 73.4%, 67.6%, and that of cattle manure were 89.6%, 99.9%, 90.0%, 87.3%. (4) Both the manure and chemical fertilizer could increase the content of available phosphorus in soil, but the manure showed a slight effect than the chemical fertilizer. 【Conclusion】 Based on the integrated methods for calculating substitution equivalent of the present experimental condition, the phosphorus in pig manure could replace 85.7% of equivalent chemical fertilizer P, while chicken manure and cow manure could replace 77.6% and 91.4%, respectively.

【Objective】 To date, the responses of the soil ecosystem stability to soil disturbance after long-term no-tillage practice are still unknown. In this study, soil nematodes were served as a model to study the response of soil nematode community structure to before and after disturbance under a long-term no-tillage practice, so as to provide a scientific support for the rational selection of farming measures in the black soil area of Northeast China.【Method】Two different phases of long-term no-tillage practices, including no-tillage (NT) and disturbed no-tillage (DNT), were set up on a black soil experimental station of Northeast Institute of Geography and Agricultural Ecology, Chinese Academy of Sciences, Dehui City, Jilin Province. Before planting the crops, the soil samples collected from the 0-15 cm soil layer were used to isolate and identify the nematodes. Using the conventional tillage (CT) as the control, the effect value (lnR) was calculated to measure the effect size of NT and DNT on the number of genus and the abundance and ecological index of nematodes under different rotation systems (corn-soybean rotation, CS; corn continuous cropping, CC). Principal component analysis (PCA) and redundancy analysis (RDA) were used to determine the composition of soil nematode communities and their driving factors among different tillage practices.【Result】Compared with CT, NT had a positive effect on the abundance of omnivores-predators across crop rotation systems, while DNT significantly (P<0.05) improved the genus number and abundance of bacterivores, the abundance of fungivores and the total nematodes under CS, and exerted negative effects on the genus number and abundance of all nematode trophic groups under CC. The composition of nematode community of NT and DNT was completely different from that of CT, and the main driving factors were total nitrogen content and bulk density, which could explain 10.5% and 11.4% of the variation in nematode community, respectively. In addition, compared with CT, NT significantly (P<0.05) increased the value of structure footprint (Fs) across all crop rotation systems, while DNT significantly (P<0.05) decreased almost all ecological indexes (except the maturity ratio of free-living nematodes to plant-parasitic nematodes, MI/PPI). Furthermore, the decrease degree of ecological index varied with crop rotation systems. CS instead of CC exerted a significant positive effect on MI/PPI. 【Conclusion】Long-term no-tillage practice could form a relatively stable soil nematode community structure in all crop rotation systems. This stability was easily gone away with the soil disturbance, including mouldboard ploughing and the removal of residues. However, crop rotation could mitigate the instability caused by soil disturbance.

【Objective】The aim of this study was to identify ACA genes family from Brassica rapa, and to analyze the commonness and characteristics of those genes, which provided data support for further revealing the evolutionary relationship of ACA family. At the same time, the expression of BraACAs after self-pollination under abiotic stress was investigated, which laid the foundation for further exploring how BraACAs as regulate calcium to perform some biological functions. 【Method】Based on Arabidopsis genome database, the ACA gene family of Brassica rapa was identified by homologous alignment. The molecular weight, theoretical isoelectric point and other physicochemical properties were predicted by online software Expasy. The MEGA 5.0 software was used to construct phylogenetic tree, and the online software GSDS 2.0 was employed to draw gene structure map. According to the family gene location information, chromosome mapping was carried out in TBtools. Then, McscanX software was used to carry out the collinearity analysis of ACA family genes in Arabidopsis and Brassica rapa, and the online software PlantCARE was used to predict and analyze the promoter elements of BraACAs. The protein conserved domains were analyzed by online tools Pfam and MEME. The expression of BraACAs gene in different tissues, abiotic stress and self-pollination were detected by qRT-PCR. 【Result】A total of 18 ACA genes were systematically identified from Brassica rapa, which were distributed on 10 chromosomes. According to the phylogenetic tree, four groups were classified, including 3, 4, 4 and 7 members. According to promoter element analysis, there were many light, abiotic stress and hormone response elements in the promoter of ACA family genes in Brassica rapa, which indicated that ACA family genes had potential biological function of resisting stress. According to the analysis of protein domains, most of the ACA gene family proteins in Brassica rapa had four functional domains unique to the ACA family, 13 of which had N-terminal autoinhibitory domains. The results of qRT-PCR showed that BraACAs were mainly expressed in flowers and pods, and the expression of Bra002762 and Bra035649 genes were up-regulated under chill stress, suggesting that Bra002762 and Bra035649 had potential biological functions in response to chill stress; the expression of Bra031701 gene was up-regulated under salt stress, suggesting that Bra031701 had potential biological functions in response to salt stress. There were significant differences in the expression of Bra003276 and Bra024117 genes between self-pollination and cross-pollination, suggesting that Bra003276 and Bra024117 responded to the self-incompatibility of Brassica rapa. The subcellular localizations of these five genes were all located in the plasma membrane to verify their function of balancing ion concentration on the membrane. 【Conclusion】BraACAs protein structures contained four highly conserved domains. The expression of BraACAs was specific in tissues, and five ACA family gene members encoded proteins located on the cell membrane, of which Bra0002762, Bra035649, Bra031701 were associated with chilling and salt stress response, while Bra003276 and Bra024117 were associated with self-incompatibility reaction.

【Objective】Browning is the main element affecting the commercial value of Luffa cylindrica. The principal consideration of browning is linked to the metabolism of phenolic acids. Studying the changes of phenolic acid during the browning process of Luffa is helpful to understand the physiological mechanism of browning, so as to provide the basis for the storage and processing, breed selection and utilization of germplasm resources of Luffa. 【Method】Browning-resistant Luffa cultivar 2D-2 and browning-prone cultivar 35D-7 were used as experiment materials. Their browning degree, total phenol content and polyphenol oxidase activity after fresh cutting for different time were measured. Ultra high-performance liquid chromatography-mass spectrometry (UPLC-MS) was utilized to analyze the metabolomic of Luffa pulp before and after browning in boiling water bath. 【Result】 After fresh-cut browning treatment, the content of polyphenols and the polyphenol oxidase activity in the flesh of both two Luffa cultivars were increased. Luffa 35D-7 had severer browning degree. The total phenol content after 24 h storage was much more than 3 times of that of 2D-2, and the polyphenol oxidase activity achieved the top at 12 h, reaching 174.23 U·g^-1·min^-1. Based on metabolomics, the substrate of enzymatic browning reaction and the difference of metabolites in browning-prone Luffa 35D-7 before and after browning were analyzed. A total of 420 metabolites were detected, 229 of which were differential metabolites. After browning, the contents of 140 kinds of differential metabolites were substantially decreased, while 89 kinds were increased. KEGG pathway enrichment analysis demonstrated that metabolites related to phenylpropanoid biosynthesis, proline metabolism and flavonoid biosynthesis displayed significant changes. The results of qualitative and quantitative analysis of metabolomics suggested the content of p-coumaric acid was the highest in the phenolic acids identified in the flesh of the tested Luffa, followed by coniferin and gentisic acid. After browning treatment, 35 different metabolites were classified as phenolic acid metabolites. Among them, the content of coniferaldehyde, 3-aminosalicylic acid and phenethyl caffeate decreased most obviously, while the content of syringin, 2,5-dihydroxy benzoic acid O-hexside and isochlorogenic acid A increased significantly, and the log2 fold change was more than 10. 【Conclusion】The main phenolic substances in the flesh of 35D-7 were p-coumaric, and the main phenolic acid metabolites involved in the browning process of 35D-7 were coniferaldehyde, syringin and isochlorogenic acid A etc. The results could help further reveal the physiological mechanism of Luffa browning and provide basis for the improvement of Luffa variety.

【Objective】Seedlessness is an important agronomic trait in table grape breeding studies. The accuracy of molecular markers linked to seedless genes will substantially influence the early selection of seedless grapes. In this study, five previously reported seedless molecular markers against 183 genotypes were assessed to determine their efficacy for the selection of seedless plants and to provide alternative strategies for the breeding of seedless grapevines based on marker-assisted selection. 【Method】 Leaf samples for DNA extraction were obtained from 96 grape cultivars (63 seedless and 33 seeded cultivars) and 87 F₁ hybrids (61 seedless and 26 seeded hybrids) from a cross between ‘Red Globe’ and ‘Dawn Seedless’. The previously reported primer pairs for five markers (SCF27-2000, GSLP1-569, VMC7f2, p3_VvAGL11, and 5U_VviAGL11) were used for PCR amplification. The presence of markers in cultivars and hybrid genotypes was detected by running the PCR products on 1.5% agarose gels, 8% polyacrylamide gels, or by fluorescence capillary electrophoresis. 【Result】 The primer for the SCAR marker GSLP1-569 amplified a 569-bp segment in the 96 grape cultivars, which showed 40.6% accuracy and 63.6% efficiency in the detection of seedless cultivars. The primers for the SCAR marker SCF27-2000 amplified a 2000-bp segment in the 96 grape cultivars and 87 F₁ hybrids, with an accuracy of 71.9% and an efficiency of 76.54% for detection of the seedless trait in the 96 cultivars, and an accuracy of 70.5% and efficiency of 78.5% in the 87 F₁ hybrids. Among the 96 assessed cultivars, the SSR marker VMC7f2 identified eight alleles, a 189-bp allele of which was found to be most significantly associated with seedlessness based on a Chi-square (χ²) independence test, and detected seedless genotypes with an accuracy of 85.4% and an efficiency of 85.5%. Among the 96 cultivars, the SSR marker p3_VvAGL11 identified seven alleles, among which, a 187-bp allele was found to be most significantly associated with seedlessness and detected seedless genotypes with an accuracy of 89.6% and efficiency of 90.7%. Among the F₁ hybrids, the accuracy and efficiency of this marker were 87.65% and 91.1%, respectively, whereas it showed false-positive and false-negative rates of 6.17% and 6.17%, respectively. Among the 96 grape cultivars, the SSR marker 5U_VviAGL11 identified 17 alleles, of which, a 306-bp allele was found to be most significantly associated with the detection of seedlessness, identifying seedless genotypes with an accuracy of 88.5% and an efficiency of 90.6%. Among the F₁ hybrids, the accuracy and efficiency of 5U_VviAGL11 were 88.89% and 92.7%, respectively, with false-positive and false-negative rates of 4.94% and 6.17%, respectively. 【Conclusion】 The SSR markers 5U_VviAGL11 and p3_VvAGL11 showed higher accuracy and efficiency with respect to the detection of grape germplasm and genetic population. These markers should be given priority for future molecular marker-assisted selection based on seedless genes in the grapevine.

【Objective】 This study was designed to investigate the effects of ultrasonic treatment on the structure and physicochemical properties of pea protein isolate (PPI), and to explore the regulation mechanism of ultrasonic treatment on the emulsifying characteristics of PPI, so as to provide the theoretical basis for the application of PPI as natural emulsions or related products in the food field. 【Method】 The ultrasonic-pea protein isolate (U-PPI) was prepared by ultrasonic wave with frequency of 20 kHz and power of 600 W under different times (0, 20, 30, 40 and 60 min), and then U-PPI emulsions were prepared by high pressure homogenization. The effects of ultrasonic treatment on the physicochemical properties of PPI were investigated by the tests of free amino group, total sulfhydryl group, particle size, solubility and SDS-PAGE. The changes in secondary and tertiary structure of U-PPI were analyzed by circular dichroism and intrinsic tryptophan fluorescence, respectively. The emulsifying ability and emulsion stability of U-PPI were characterized by emulsion activity index, particle size, protein distribution at the interface of emulsion, Zeta potential and apparent viscosity. The microstructure of the emulsions was observed by using laser confocal fluorescence microscopy. 【Result】 Ultrasonic treatment significantly modified the structure of PPI, i.e., a short time (30 to 40 min) ultrasonic treatment significantly reduced the α-helix content and improved the β-sheet content of PPI, which made the structure of the PPI more flexible and more hydrophobic groups exposed to the interface. Simultaneously, the depolymerization effect of ultrasonic also caused the decrease of the average particle size and the significant increase of the solubility of PPI. Therefore, the modification of PPI by a short time ultrasonic treatment was conducive to the formation of a dense and stable protein film at the oil/water interface, which effectively improved the emulsifying activity of PPI and the stability of the emulsions, and the microstructure of the emulsions also showed that the particle sizes of emulsions stabilized with U-PPI were smaller, and the droplet distribution was more uniform. However, a long time (60 min) ultrasonic treatment caused the hydrophobic repolymerization and insolubility of PPI, which was not conducive to the adsorption and rearrangement of PPI in the oil/water interface and thus reduced the emulsifying activity and emulsion stability. 【Conclusion】 The cavitation effect and mechanical effect produced by a short time (30, 40 min) ultrasonic treatment had significant depolymerization effect on PPI, which promoted the protein structure more flexible and was beneficial to the adsorption and rearrangement of PPI at the oil/water interface, and thus significantly improved the emulsifying properties of PPI.

【Objective】 This experiment was conducted to determine the effects of dietary supplemental pattern of trace elements on growth performance, carcass traits and meat quality of broiler chicks, so as to provide the experimental basis for the reasonable addition of trace elements to broiler diets.【Method】 A single-factor completely randomized design was adopted in this experiment. A total of 240 one-day-old Arbor Acres (AA) broiler chicks were randomly allotted by body weight to 1 of 5 treatments with 6 replicate cages of 8 birds per cage. The trace elements were added to the corn-soybean basal diet as follows: the inorganic trace elements according to NRC (1994) recommendation for broiler chicks (T1: the added levels of copper (Cu), Iron (Fe), manganese (Mn), zine (Zn) and selenium (Se) were 8, 80, 60, 40 and 0.15 mg·kg^-1 during 1-42 days, respectively), the inorganic trace elements according to the recommendation for broiler chicks in Chinese Feeding Standard of Chicken (NY/T 33-2004) (T2: the added levels of Cu, Fe, Mn, Zn and Se were 8, 100, 120, 100 and 0.3 mg·kg^-1 for 1-21 days old, respectively; and 8, 80, 120, 80 and 0.3 mg·kg^-1 for 22-42 days old, respectively), the inorganic trace elements according to the previous results of trace elements requirements from our lab (T3: the added levels of Cu, Fe, Mn, Zn and Se were 4, 40, 110, 60 and 0.35 mg·kg^-1 during 1-21 days, respectively; and 0, 30, 80, 40 and 0.35 mg·kg^-1 during 22-42 days, respectively), the decrement levels of organic trace elements according to the previous results of from our lab (T4: the added levels of Cu, Fe, Mn, Zn and Se were 2, 30, 80, 40 and 0.25 mg·kg^-1 during 1-21 days, respectively; and 0, 30, 80, 40 and 0.25 mg·kg^-1 during 22-42 days, respectively), and the organic trace elements according to the recommendation for broiler chicks in NY/T 33-2004 (T5: the added levels of Cu, Fe, Mn, Zn and Se during 1-21 and 22-42 days were the same as those in T2), respectively. The inorganic trace element sources (feed grade) were Cu sulfate pentahydrate, Fe sulfate monohydrate, Mn sulfate monohydrate, Zn sulphate monohydrate and sodium selenite, and the organic trace element sources (feed grade) were Cu mothionine, Fe glycine, Mn methionine, Zn glycinate and Se yeast, respectively. The experiment lasted for 42 days.【Result】The results showed that those different supplemental patterns of trace elements had no significant effects (P>0.05) on the average daily feed intake and average daily gain. Broilers from T2 had higher (P<0.05) feed to gain ratio during 22-42 days than those from T1, T4 and T5, and no difference was detected between T2 and T3 (P>0.05). Broilers from T2 had higher (P<0.05) feed to gain ratio during 1-42 days than those from other groups, and there were no differences (P>0.05) among other groups. The different supplemental patterns of trace elements had no significant effects on (P>0.05) the carcass traits, L* and a* values, pH values and drip losses of breast and thigh muscles. The breast muscle b* value of broilers from T5 was higher (P<0.05) than that of broilers from T1 and T3, and no difference was observed (P>0.05) between T5 and T4. The shear force of thigh muscle from T4 was lower (P<0.05) than that from T1 or T5, and the muscle tenderness was relatively well. 【Conclusion】Under this experimental conditions, the group with decrement supplement of organic trace elements based on our previous results (T4: the added levels of Cu, Fe, Mn, Zn and Se were 2, 30, 80, 40 and 0.25 mg·kg^-1 during 1-21 days, and 0, 30, 80, 40 and 0.25 mg·kg^-1 during 22-42 days, respectively) was better than other groups in improving the growth performance and meat quality of broiler chicks.

【Objective】 This experiment was conducted to study the effects of glucose oxidase (GOD) on growth performance, immune functions and intestinal health of ducks challenged by Escherichia coli (E. coli) O₈₈ and its mechanism to find a substitute for antibiotics to prevent duck colibacillosis_. 【Method】 A total of 144 one-day-old healthy male lean Peking ducklings were selected and randomly divided into three groups with six replicates and eight ducks per replicate. Ducks were fed the three diets supplemented with nothing (Control group), 30 mg·kg^-1virginiamycin (Antibiotic group) and 200 U·kg^-1 GOD in basal diet, respectively. On day 7, all ducks were orally taken 0.2 mL E. coli O₈₈ (3×10⁹ CFU/mL) twice, 8 hours apart. The experiment lasted for 28 days. 【Result】The results showed as follows: 1) Compared with the control group, adding antibiotic or GOD in the diet significantly increased the average daily gain and average daily feed intake of 1 to 14-day-old attacking ducks (P<0.01). 2) The number of serum white blood cell of 28-day-old attacking ducks was significantly reduced and the percentage of serum lymphocyte was significantly increased by adding antibiotic and GOD in diet (P<0.01), and the number of serum red blood cell of 28-day- old attacking ducks was significantly reduced by adding GOD in diet (P<0.05). 3) Serum MDA at day 14 (P<0.05) and CAT contents at day 28 (P<0.01) were significantly reduced by adding antibiotic and GOD, serum T-AOC at day 28 was significantly increased (P<0.05), and CAT at day 14 showed a tendency to decrease by adding GOD (P=0.087). 4) Supplementation of antibiotic and GOD significantly decreased endotoxin of 14 and 28-day-old attacking ducks (P<0.01). 5) Adding antibiotic or GOD significantly decreased the concentration of IL-1β and IL-6 of ducks at day 14 and 28 and TNF-α at d 28 (P<0.05), no significant differences between the GOD and the antibiotic groups（P>0.05）. 6) The concentration of serum diamine oxidase and D-lactic of 14 and 28-day-old attacking ducks were significantly reduced by adding antibiotic and GOD (P<0.05), no significant differences between the GOD and the antibiotic groups (P>0.05). 7) The addition of GOD increased the number of unique OTUs in ileum, reduced the contents of E. coli, and increased the relative abundance of probiotics such as Lactobacillus and Bifidobacterium. 【Conclusion】 In conclusion, addition of GOD in the diet can balance the structure of intestinal flora, reduce the production of bacterial endotoxin and decrease oxidative stress, and then maintain the integrity of intestinal mucosa and prevent activation of inflammatory signal pathway by endotoxin and occurrence of inflammatory reaction, so as to improve intestinal health and growth of ducks infected with E. coli. The GOD can be used as antibiotics substitute to prevent or alleviate colibacillosis of ducks.