Table of Content

01 December 2018, Volume 51 Issue 23

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Isolation and Functional Analysis of Soybean GmLEA in Seed Vigor

ZHOU YaLi,ZHU YaJing,ZHAO FeiYun,WANG Shuang,LIU SuShuang,GUO LingKai,ZHAO HaiHong,MA Hao

Scientia Agricultura Sinica. 2018, 51(23):  4397-4408.  doi:10.3864/j.issn.0578-1752.2018.23.001

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【Objective】 Soybean(Glycine max (L.) Merr.) seed generally form vitality from their physiological maturity period (R6 or R7 period). However, the seed is susceptible to high temperature and humidity (HTH) stress during this period. This will lead to a decline in seed vigor. The results will lay a foundation for further studying the mechanism of seed vigor formation under abiotic stress. 【Method】 Primer Premier 5.0 was used to design primers, and the full length cDNA sequence of GmLEA was isolated by using the cDNA of leaves of cv. Ningzhen No.1 and Xiangdou No.3 as template. The homologous amino acid sequence of GmLEA was searched by BLAST at NCBI, the protein sequences were multiple aligned using MEGA 6.0 and DNAMAN, and the phylogenetic tree was constructed using the N-J algorithm of MEGA 6.0. Yeast two-hybrid experiments were performed to verify the interaction of GmLEA and GmCDPKSK5 in yeast. A subcellular localization and bimolecular fluorescent complementation (BiFC) vector were constructed, The interaction between GmLEA and GmCDPKSK5 in tobacco leaf cells and the subcellular localization of the encoded protein were analyzed by gene-gun-mediated transformation of tobacco leaves. In addition, the tissue-specific expression of GmLEA gene and the expression pattern of GmLEA gene under HTH were analyzed by qRT-PCR, respectively. The pBI121 fusion expression vector was constructed and three homozygous overexpressed Arabidopsis lines were obtained through Agrobacterium- mediated method, and three independent homozygous T₃ transgenic lines were used for analysis. 【Result】 The cDNA sequence of GmLEA gene contains a 1 377 bp open reading frame (ORF), and the subcellular localization result showed that the encoded protein was located on the cell membrane. The results of yeast two-hybrid rotation verification showed that GmLEA could interact with GmCDPKSK5 in yeast. In addition, bimolecular fluorescence complementation (BiFC) experiment showed that GmLEA could interact with GmCDPKSK5 on cell membrane of tobacco leaf cells. The results of tissue-specific analysis showed that GmLEA gene had higher expression levels in developing and mature seeds of both cultivars. The expression level of GmLEA was increased first and then decreased during the development of cv. Xiangdou No. 3 seeds. During the process of seed development of cv. Ningzhen No.1, the level of GmLEA expression was on the rise and reached the highest at 60 days after flowering. After high temperature and high humidity (HTH) stress, the expression of GmLEA was decreased at 96 h in cv. Xiangdou No. 3, and the other time points were increased. However, the expression was decreased at 24 h in cv. Ningzhen No.1. The germination potential, germination rate and seed vigor of GmLEA gene in transgenic Arabidopsis thaliana were significantly (P<0.01) higher than those of the wild type plants under HTH stress. 【Conclusion】 The GmLEA is involved in the formation of seed vigor under HTH stress,and has specific interaction with GmCDPKSK5, It is speculated that they may participate in the formation of seed vigor under HTH stress.

Cloning and Expression Analysis of a Ⅱd Sub-Group WRKY Transcription Factor Gene from Sugarcane

ZHANG Xu,LING Hui,LIU Feng,HUANG Ning,WANG Ling,MAO HuaYing,LI CongNa,TANG HanChen,SU WeiHua,SU YaChun,QUE YouXiong

Scientia Agricultura Sinica. 2018, 51(23):  4409-4423.  doi:10.3864/j.issn.0578-1752.2018.23.002

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【Objective】 WRKY, a group of unique transcription factors in plants, plays an important role in plant physiological regulation and stress response. Through analysis of the role of transcription factor WRKY in sugarcane growth and development and stress resistance, this study will provide excellent gene resources for sugarcane resistance molecular breeding. 【Method】 A unigene sequence of WRKY gene was extracted from the sugarcane transcriptome database, and its full-length cDNA sequence was obtained by RT-PCR amplification. Bioinformatics analysis of this gene sequence and its encoded protein sequence was performed using ORF finder, Smart, ExPaSy, Prabi, NetPhos, Cell-PLOC 2.0 and DNAMAN6.0 softwares, and the phylogenetic tree analysis was constructed using MEGA6.0 software. The fusion expression vector of pCAMBIA1300-WRKY-GFP was constructed and delivered into Nicotiana benthamiana by Agrobacterium mediated method to determine the subcellular localization of WRKY protein in tobacco leaves. Yeast hybridization assay was used to verify whether WRKY possess transcriptional self-activation activity. The tissue specific expression (root, bud, leaf, stem pith and epidermis) of WRKY and its dynamic expression under MeJA (100 μmol·L ^-1), SA (5 mmol·L ^-1), PEG (25%), NaCl (250 mmol·L ^-1), CuCl₂ (500 mmol·L ^-1) and CdCl₂ (500 mmol·L ^-1) stresses in sugarcane variety ROC22 were analyzed by quantitative real-time PCR (qRT-PCR) technique.【Result】 A WRKY transcription factor gene, named ScWRKY6 (GenBank Accession Number: MH393927), was cloned from the sugarcane variety ROC22. This gene sequence was 1 289 bp in full length with a 1 059 bp ORF, encoding 352 amino acids, and contained 45 phosphorylation sites. The theoretical isoelectric point, the instability index and the hydrophilicity of ScWRKY6 protein was 9.73, 50.23 and -0.579, respectively, which is supposed to be an alkaline unstable hydrophilic protein. The ScWRKY6 protein has one WRKY domain and one zinc finger motif (CX5CX23HXH), and its amino acid sequence has the highest homology with Sorghum bicolor WRKY (XP_002464211.1). It is speculated that this gene belongs to the Ⅱd sub-group of WRKY family according to phylogenetic tree analysis. Subcellular localization results showed that the ScWRKY6::GFP fusion protein was located in the nucleus. Yeast transcriptional activation verification experiments indicated that ScWRKY6 protein did not have transcriptional auto-activation activity. qRT-PCR analysis revealed that ScWRKY6 was constitutively expressed in sugarcane, and the expression level in order from high to low were in bud, leaf, root, stem epidermis and stem pith. Its expression in bud, leaf and root were 2.05, 1.55 and 1.37 times higher than that in stem pith, respectively. The expression level of ScWRKY6 was up-regulated under the stresses of NaCl, PEG, MeJA, Cu ²⁺ and Cd ²⁺. Its highest expression was 4.18, 6.88, 3.63, 4.86 times higher than of the control when treated with NaCl for 12 h, PEG for 3 h, CuCl₂ for 24 h and CdCl₂ for 24 h, respectively.【Conclusion】 ScWRKY6 protein was located in the nucleus and did not have transcriptional auto-activation activity. The gene was expressed in different sugarcane tissues and was induced by NaCl, PEG, CuCl₂ and CdCl₂ treatments. It is presumed that the ScWRKY6 may play a role in response to drought stress, salt tolerance and metal ion stress in sugarcane.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Key Growth Stage of Cd Accumulation in Brown Rice Through a Hydroponic Experiment with Cd Stress

WANG QianQian,JIA RunYu,LI HongCheng,ZHOU Hang,YANG WenTao,GU JiaoFeng,PENG PeiQin,LIAO BoHan

Scientia Agricultura Sinica. 2018, 51(23):  4424-4433.  doi:10.3864/j.issn.0578-1752.2018.23.003

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【Objective】 The objectives of this study were to explore the effects of Cd stress on Cd accumulation in brown rice at different growth stages (tillering stage, jointing stage, booting stage, filling stage, dough stage, and maturing stage) of rice plants, to clear and definite the key growth stages of rice plants for Cd accumulating in brown rice, so as to provide directions of rice safety production by taking suitable measures timely for reducing Cd contents in brown rice. 【Method】 Taking Xiangwanxian 13 (a late rice variety) as the studied plant, a hydroponic experiment with rice plants was conducted with seven treatments of exogenous Cd application, including CG (whole growth stages with Cd stress, 102 d), TS (tilling stage with Cd stress, 15 d), JS (jointing stage with Cd stress, 15 d), BS (booting stage with Cd stress, 21 d), FS (filling stage with Cd stress, 18 d), DS (dough stage with Cd stress, 15 d), and MS (maturing stage with Cd stress, 18 d). All seven treatments were repeated 3 times and another treatment of the whole growth stages without Cd stress as control (CK). Exogenous Cd in each treatment was the same as 20 μg·L ^-1. The hydroponic experiment started in July 23, 2017, at the rice experiment base of Central South University of Forestry and Technology in Changsha, Hunan province. Agronomic characters (plant height, tiller number, and biomass of rice tissues) and Cd contents in various rice tissues were determined after harvest in November 19, 2017. Cd amounts and the relative contribution rates of Cd accumulation at different growth stages to Cd accumulation in brown rice at maturing stage were calculated. 【Result】 The results showed that exogenous Cd application at different growth stage of rice plants did not affect plant height, tiller number, and biomass of rice tissues. At the filling stage with Cd stress, Cd contents in brown rice was the highest and reached to 1.05 mg·kg ^-1, which was significantly higher than Cd contents in brown rice with Cd stress at the maturing stage (0.57 mg·kg ^-1), booting stage (0.52 mg·kg ^-1), dough stage (0.38 mg·kg ^-1), jointing stage (0.31 mg·kg ^-1), and tilling stage (0.17 mg·kg ^-1). The range of Cd amounts in brown rice with Cd stress at different growth stages were 0.18-1.56 μg/plant, and the sequence of Cd amounts were: whole growth stages with Cd stress>filling stage with Cd stress>maturing stage with Cd stress>booting stage with Cd stress>jointing stage with Cd stress>tilling stage with Cd stress. The booting stage, filling stage and maturing stage of rice plants were the key growth stages for Cd accumulating in brown rice, and the relative contribution rates of Cd accumulating in brown rice at these three growth stages were 19.7%, 39.3%, and 22.6%, respectively. The contributions of Cd stress to Cd accumulating in brown rice were 2.4%, 4.2%, and 11.9% at the tillering, jointing, and dough stage, respectively, which were relatively lower compared with the other three stages. Cd contents in rice roots, stem, ear, and husk at booting stage and filling stage were all higher except the whole growth stages with Cd stress. There was no significant difference in Cd content in rice leaves (P>0.05) at different growth stages with Cd stress. At the tillering stage and filling stage with Cd stress, Cd amounts in rice roots were 86.09 μg/plant and 79.23 μg/plant, respectively, which was significantly higher than those at the other stages of rice plants (31.55-40.37 μg/plant). Compared to the other stages, Cd amounts in rice plants at the booting stage and filling stage with Cd stress were high, reaching 107.13 μg/plant and 98.35 μg/plant, respectively, which was significantly higher than those at the other four stages of rice plants (42.24-52.47 μg/plant). 【Conclusion】 The booting stage, filling stage, and maturing stage of rice plants were the key growth stages for controlling Cd accumulation in brown rice. With Cd stress at the booting stage and filling stage, the Cd accumulation in the root and brown rice at the maturing stage were higher than those other stages. Therefore, the Cd accumulation in brown rice could be reduced by applying soil amendments at the booting stage and filling stage of rice plants to obstruct root absorbing Cd or reducing Cd transportation from rice root to brown rice.

Effects of Jointing and Booting Low Temperature Treatments on Photosynthetic and Chlorophyll Fluorescence Characteristics in Wheat Leaf

LIU LeiLei,JI HongTing,LIU Bing,MA JiFeng,XIAO LiuJun,TANG Liang,CAO WeiXing,ZHU Yan

Scientia Agricultura Sinica. 2018, 51(23):  4434-4448.  doi:10.3864/j.issn.0578-1752.2018.23.004

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【Objective】 The objective of this paper was to study the effects of low temperature treatment below the daily variation rule of natural temperature on leaf photosynthetic properties of wheat. 【Method】 The environment-controlled phytotron experiments with two different temperature sensitive wheat cultivars were conducted under four low temperature levels and two low temperature durations at jointing and booting stages. During low temperature treatment stage and in 7 days after low temperature treatments, the photosynthetic and chlorophyll fluorescence characteristics in wheat leaves were measured. 【Result】 During low temperature treatment stage, the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), maximum photochemical efficiency (Fv/Fm) , PSⅡ actual photochemical efficiency (ФPSII) and photochemical quenching (qP) decreased with the decreasing temperature. During jointing stage, Pn, Gs, Tr, Fv/Fm, ФPSII and qP decreased firstly and then increased with the increasing of low temperature duration, while the decreased trends were observed in the response of Pn, Gs, Tr, Fv/Fm, ФPSII and qP to low temperature duration when low temperature treatments were conducted at booting stage. After low temperature treatments, except for T4 (T_min/T_max/T_avg, -6℃/4℃/-1℃) at booting stage, Pn, Gs, Tr, Fv/Fm, ФPSII, qP and NPQ in all low temperature treatments could recover to the same status with T1. Moreover, under low temperature treatments, the relative of Fv/Fm, ФPSII and qP were significantly positively correlated to the relative Pn, while the relative NPQ was significantly negatively correlated to the relative Pn. Compared with the relative of Fv/Fm and NPQ, the correlation coefficients between the relative of ФPSII, qP and Pn were more higher. 【Conclusion】 The decreases in photosynthetic rate of leaves under low temperature condition were mainly attributed to the decreased ФPSII and qP, leading to the reduction of dry matter accumulation, finally caused the lower grain yield of wheat.

PLANT PROTECTION

Cloning, Sequence Analysis and Expression of Pheromone Biosynthesis Activating Neuropeptide (PBAN) Gene in Different Development Stages of Pectinophora gossypiella

XU Dong,WANG Ling,CONG ShengBo,WANG JinTao,LI WenJing,WAN Peng

Scientia Agricultura Sinica. 2018, 51(23):  4449-4458.  doi:10.3864/j.issn.0578-1752.2018.23.005

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【Objective】The objective of this study is to clone the pheromone biosynthesis activating neuropeptide (PBAN) gene of pink bollworm (Pectinophora gossypiella), analyze its sequence characteristics, clarify its expression patterns in different developmental stages as well as correlation with mating behavior and cotton volatiles, which will provide a scientific basis for further revealing the biosynthesis and release mechanisms of sex pheromone in P. gossypiella.【Method】The full cDNA sequence of PgosPBAN was cloned by RACE technique. Gene splicing and amino acid sequence were analyzed by the software of DNAMAN 6.0, protein secondary structure prediction of PgosPBAN and bioinformatics information were predicted using Protparam and Chou & Fasman. The expression patterns of PgosPBAN in different developmental stages of P. gossypiella were detected and the effects of mating behavior and cotton volatiles on the expression level of PgosPBAN were analyzed using real-time quantitative PCR (qRT-PCR). 【Result】The full cDNA sequence of PgosPBAN (GenBank accession number: KY987647) is obtained, and the total length of cDNA is 1 461 bp. The open reading frame (ORF) is 618 bp, encoding 205 amino acid residues. The length of 5′-untranslated region (5′ UTR) and 3′-untranslated region (3′ UTR) is 121 and 722 bp, respectively. The amino acid sequence encoded by PgosPBAN contains five peptides, including diapause hormone homolog, α-SGNP, β-SGNP, PBAN and γ-SGNP, and a signal peptide of 23 amino acid residues at the N terminus. The predicted molecular mass and isoelectric point are 2.41 kD and 9.25, respectively. Homology and phylogenetic tree analysis showed that PgosPBAN and PBAN of 15 Lepidoptera insects were located in the same branch, and PgosPBAN had the closest relationship with Chilo suppressalis PBAN (GenBank accession number: ALM30314.1), suggesting that the two genes likely developed from a common ancestral gene. The expression of PgosPBAN was specific in different developmental stages, which was higher in adult stage, second in larval stage, and the lowest in pupal stage. PgosPBAN was expressed in both female and male adults, and the expression of PgosPBAN in male adults was significantly higher than that in female adults from the 1st-day to 5th-day old. The expression level of PgosPBAN in P. gossypiella at 1-3 days after mating was significantly higher than that in virgin moth. After exposed to cotton volatiles for 1-5 days, the expression level of PgosPBAN in male adults had no significant difference compared with the control, but the expression level of PgosPBAN at 1st, 8th day of female adults and 8th day of male adults was significantly lower than that in control. 【Conclusion】The sequence characteristics of nucleotides and amino acids of PgosPBAN were clarified, and the secondary structure characteristics of the protein were analyzed. According to the expression of PgosPBAN in different developmental stages of P. gossypiella and its relationship with mating behavior and the regulation of cotton volatiles, it is speculated that this gene is not only involved in the synthesis and release of female pheromone in P. gossypiella, it may also play an important role in regulating male pheromones and regulating growth and development.

Effect of Adjuvant on Conductivity and Deposition of Electrochargeable Liquid

REN LiRui,CHEN FuLiang,YIN MingMing

Scientia Agricultura Sinica. 2018, 51(23):  4459-4469.  doi:10.3864/j.issn.0578-1752.2018.23.006

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【Objective】The objective of this study is to optimize formula and research the effects of different adjuvants on conductivity and deposition of the electrochargeable liquid and the internal relationship, and to provide scientific guidance for reducing the use of pesticides and achieving sustainable agricultural development.【Method】New environment-friendly solvents HDBE and NCC were used with dimethyl sulfoxide, N-methyl pyrrolidone and isophorone as cosolvents. The contents of cosolvents were set as 0, 3%, 5%, 7%, and 10% respectively, and the traditional solvent S200# was used as control. Qualified formulas were screened according to the technical index of electrochargeable liquid. The effect of the different contents of adjuvants on conductivity of abamectin and dinotefuran electrochargeable liquid was analyzed. At the same time, in order to study the effect of the different contents of adjuvants on the deposition on the front and back of cucumber and tomato leaves, explore the relationship of preparation conductivity and deposition and analyze electrostatic outflank effect. The area of target leaves was measured before pesticide application, and washed repeatedly with acetone after the treatment. The active ingredient on target leaves was determined by high performance liquid chromatography (HPLC) after process of filtration, rotary steaming and dilution to graduation with solvent.【Result】Six environment-friendly electrochargeable liquid formulas that meet the requirements of quality and technical specifications were screened. The conductivity of the formula was not a weighted mean of the individual component, but rather the final result of the interaction of the components. For the electrochargeable liquid prepared by the active components of pesticides with different polarity, the change of the species and contents of the adjuvant had the same effect trend on the conductivity and deposition. It had almost no effect on the overall conductivity of the preparation when the active ingredient was the non-polar pesticide abamectin, whereas it was significantly increased when the polar pesticide dinotefuran replaced abamectin. The conductivity of preparation was nearly 100 times higher than that of abamectin with the same adjuvant. When the solvent was HDBE, the conductivity was small, the conductivity and deposition of the preparation increased rapidly as the content of cosolvents increasing, and the difference was significant. When the solvent was NCC, because of its high conductivity, the conductivity and deposition of the preparation increased steadily as the content of cosolvent increasing, and the difference of conductivity among different formulas was reduced. When the dimethyl sulfoxide was cosolvent, the conductivity and deposition of electrochargeable liquid prepared with HDBE, NCC as solvent were significantly higher than that of the traditional solvent S200#, in which the conductivity was more than 5 times and the deposition amount was more than 1.5 times higher than that of the traditional solvent S200#. The deposition of electrochargeable liquid on the front and back of cucumber leaves was higher than that of tomato. It was found that the ratio of deposition on the front and back of the leaves was between 1.17 and 2.11.【Conclusion】The environment-friendly HDBE and NCC can replace S200# as excellent solvents for electrostatic spray. The species and content of adjuvants had a significant effect on the conductivity and deposition of electrochargeable liquid and the deposition can be significantly improved by increasing the conductivity of the preparation. The prepared electrostatic spray had an excellent electrostatic outflank effect.

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

Quantifying Carbon Sink by Biochar Compound Fertilizer Project for Domestic Voluntary Carbon Trading in Agriculture

SUN JianFei,ZHENG JuFeng,CHENG Kun,YE Yi,ZHUANG Yuan,PAN GenXing

Scientia Agricultura Sinica. 2018, 51(23):  4470-4484.  doi:10.3864/j.issn.0578-1752.2018.23.007

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【Objective】Industrial system of straw pyrolysis - biochar compound fertilizer (BCF) - ecological agriculture is emerging in China. As a suggested measure which could promote soil organic carbon pool and mitigate greenhouse gas emissions through replacing chemical fertilizer, BCF application has the potential to participate in China’s ongoing carbon trading of voluntary emission reduction (VER). Development of a measurable, reportable and verifiable net greenhouse gas (GHG) reduction quantification methodology is the basis for the implementation of VER carbon trading. The objective of this study was to discuss and develop a methodology for quantifying carbon sequestration and GHG emission mitigation in BCF project, which might provide scientific basis and methodology support for BCF project to attend the VER carbon trading. 【Method】Based on the theoretical framework of the methodology for VER projects, a discussion of how to develop a methodology for BCF project was performed from the aspects of project eligibility, baseline, boundary, carbon pool, key GHG sources, leakage and net carbon sink quantification by incorporating the recorded VER methodologies, the existing frameworks of carbon sequestration and GHG reduction quantification in cropland, and the BCF scientific research basis. In addition, a case study was conducted to quantify the net carbon sink in BCF project under different cropping systems by using the data from literature collection and field survey, which would assess the feasibility of the discussed methodology by this study. 【Result】Through analyzing and discussing, this study indicated that the baseline scenario of BCF project should be local conventional fertilization management in the methodology, and the boundary could be determined according to the difference of farmland operation mode, such as the boundaries of smallholder operated farmland and factory-farmland system intensive operated by enterprises. The key GHG sources and carbon pool considered in the methodology were suggested as farmland N₂O and CH₄ emissions, and soil organic carbon pools, respectively. The extra GHG emissions induced by the transportation of BCF or the changes in original straw utilization method could be considered as leakage. According to the case study, the net carbon sink of 1 439.77 kg CO₂-eq·hm ^-2 and 281.58 kg CO₂-eq·hm ^-2 for the growing seasons of winter wheat and rice production could be obtained, respectively, when the boundary was set as smallholder operated farmland. However, in the boundary of factory-farmland system intensive operated by enterprises, the carbon sink obtained in the farmland might offset by the GHG emissions in the process of BCF production, and the net carbon sink of 1 479.01 kg CO₂-eq·hm ^-2 and 340.43 kg CO₂-eq·hm ^-2 for the growing seasons of winter wheat and rice production could be obtained, respectively, once the BCF production process was optimized. Given these, the optimizing of BCF production process by recycling the by-products would make the carbon trading of VER by BCF project feasible. 【Conclusion】A theoretical framework and a set of methods were proposed to quantify the net carbon sink for BCF project. The case study indicated that the developed methodology could be well applied into the quantification of net carbon sink for BCF project, and it was found that dry cropping system had the higher net carbon sink than paddy rice cropping system under BCF project, while optimizing the production process of BCF was an important pathway to obtain the considerable amount of carbon sink under factory-farmland system operated by enterprises. This study indicated that a national or industry standard of BCF should be established as soon as possible to provide a theoretical basis for project eligibility identification; meanwhile the attentions should be paid to the development of regional specific N₂O and CH₄ emission reduction factors and soil carbon sequestration factors for different types of BCFs applied.

Effects of the Different Crop Straw Biochars on Soil Bacterial Community of Yellow Soil in Guizhou

HOU JianWei,XING CunFang,LU ZhiHong,CHEN Fen,YU Gao

Scientia Agricultura Sinica. 2018, 51(23):  4485-4495.  doi:10.3864/j.issn.0578-1752.2018.23.008

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【Objective】The objective of the experiment was to determine the effects of different straw biochars on bacterial community structures and composition in yellow soil, and to find main environmental factors as the changes in order to provide information for soil amelioration and proper management of straw residue.【Method】Through a laboratory incubation experiment and used a zonal yellow soil of Guizhou province, the influences of corn, rice and rape straw biochar that were pyrolyzed at 500℃ on bacterial communities were investigated by a high-throughput sequencing (Illumina Hiseq). Correlation and factor analysis of the bacterial community structure with environmental factors were followed. The experiment consisted of four treatments: control soil (CK), soil amended with 500℃ corn (BC1), rice (BC2) and rape (BC3) straw biochar. 【Result】The results showed that the gene copy numbers of bacterial 16S rRNA were closely related with soil total nitrogen (TN), pH and total carbon (TC) (r=0.78**, 0.62* and 0.66*, respectively). Biochar addition to soil increased the richness and diversity of dominant bacteria at phylum and class level, which were a strong positive with pH and C/N. The analysis of bacterial community at phylum level showed that Actinobacteria, Cyanobacteria and Chloroflexi were dominant bacteria, occupying 68.5% of all phyla. Factor analysis showed that soil total nitrogen (TN), C/N ratio, pH, available phosphorus (AP) and cation exchange capacity (CEC) were main environmental factors on the soil bacterial community structure, total explaining 80.8% of the community changes. The order of contribution rate was soil C/N>pH>AP>TN>CEC.【Conclusion】This study provided clear evidence that community composition and chemical properties of bacterial were changed due to biochar addition to yellow soil. And soil TN, C/N, pH, AP and CEC had a greater contribution than environmental factors on the change of the bacterial community structure, in addition, TN and pH were more efficient on improving soil richness and diversity of bacterial community.

Ecological Emergy Analysis of Different Paddy Ecosystems in Hunan Province

ZHOU Jiang,XIANG PingAn

Scientia Agricultura Sinica. 2018, 51(23):  4496-4514.  doi:10.3864/j.issn.0578-1752.2018.23.009

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【Objective】Hunan paddy ecosystem is mainly one- and double-cropping rice patterns ecosystem. By evaluating the effects of the adjustment of cropping patterns on the planting efficiency of rice grain in different seasons, this paper was expected to provide decision basis for the sustainable management of paddy.【Method】In this paper, raw data on the input and output of environmental resources and economic resources in the ecosystem of early rice, semilate rice and late rice in Hunan from 2002 to 2016 were obtained through relevant statistical yearbook. emergy analysis theory and method were used to analyze the dynamic development status and comprehensive development level of the different seasons paddy ecosystem. Then the input and output efficiency of paddy were evaluated by emergy analysis index and compared with the traditional economic profit rate index.【Result】From 2002 to 2016, the input emergy of natural resources in Hunan paddy ecosystem was relatively stable, and the input emergy was mostly dependent on purchasing emergy and tended to increase. The emergy input structure was adjusted to be mechinery>labor+animal>fertilizer>pesticide or seed>fuel>organic fertilizer. Agricultural mechanization gradually replaced the artificial, animal - based production mode. Purchasing emergy input per 1000 hm ²planting area: the density of unrenewable industrial emergy showed a significant growth trend, and the density of machinery operation emergy was the highest contribution rate in the unrenewable industrial emergy inputs. The density of fertilizer emergy inputs was the semilate rice ecosystem>early and late rice, and it stayed high for years. The density of pesticide emergy inputs was semilate and late rice ecosystem>early rice, and it tended to increase. Density of renewable organic emergy inputs had been tended to reduce. Although the labor emergy decreased significantly, its contribution rate was still the highest in the renewable organic emergy inputs. The density of rice seeds emergy inputs for early rice ecosystem>late rice>semilate rice, and early rice and late rice tend to increase. The density of animal emergy had been converted into semilate rice ecosystem>early and late rice, and the trend was decreasing. Organic fertilizer inputs tended to reduce unceasingly. The purchase emergy inputs had been converted into the semilate rice>early>late rice ecosystem from 2012. The emergy outputs of the unit planting area, ecological and economic average profit margin of rice were the semilate rice ecosystem>late rice>early rice. The planting area of early rice was always lower than that of late rice. The variation trend of the emergy index of Hunan paddy ecosystem was: the emergy input ratio (EIR) in the late rice ecosystem>early and semilate rice; the emergy yield ratio (EYR) in the semilate rice>late rice>early rice. The ecosystem puts less pressure on the environment, but the environmental load ratio (ELR) increases rapidly and it was the late rice ecosystem>early and semilate rice. The sustainable development index (ESI) had dropped significantly to<2, and converted into the semilate rice ecosystem>early and late rice after 2008. 【Conclusion】The rice production mode in Hunan paddy ecosystem had been modernized day by day. The ecosystem was dynamic but its potential was declining. The rice production mode belonged to extensive operation, which large inputs of industrial emergy cause short-term ecosystem pressure in environment, ecological and economic profit margins declining. It was not conducive to long-term sustainable development ecosystem. Although the semilate rice ecosystem’s ELR, ESI, average rate of profit is still better than the early and late rice ecosystem, but it’s EYR, ecological and economic profit margins were in the larger decline by using more labor, animal power, chemical fertilizers and pesticides and its mechanical emergy efficiency was low, leading to the competitive advantage to reduce. The seed emergy input of early rice ecosystem was high and the emergy yield density and profit rate were low. The comprehensive benefit of purchasing emergy of late rice ecosystem was higher. The contradictions in regional unbalanced development of modern paddy planting technology were still outstanding. Regardless of the grain in different seasons, the cost-benefit method which based on market value underestimates the real value of the paddy ecosystem. The different incentive policies should be formulated by government according to early, semilate and late rice, so as to safeguard farmers’ interests and realize sustainable paddy production.

HORTICULTURE

Molecular Mechanism of Apple MdWRKY18 and MdWRKY40 Participating in Salt Stress

XU HaiFeng,YANG GuanXian,ZHANG Jing,ZOU Qi,WANG YiCheng,QU ChangZhi,JIANG ShengHui,WANG Nan,CHEN XueSen

Scientia Agricultura Sinica. 2018, 51(23):  4514-4521.  doi:10.3864/j.issn.0578-1752.2018.23.010

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【Objective】 In order to improve the molecular mechanism of salt stress, we studied several aspects of MdWRKY18 and MdWRKY40 in apple WRKY transcription factors, including the protein structure, the expression level and the function in salt stress. 【Method】 We cloned the MdWRKY18 and MdWRKY40 genes in ‘Hongcui No.2’ apple and analysed their protein structure. The expression levels of MdWRKY18 and MdWRKY40 were studied by the qRT-PCR under the salt stress, and their promoter activities were analyzed using the GUS staining. We analyzed the interaction relationship between MdWRKY18 and MdWRKY40 proteins by yeast two-hybrid and verified their function by transgenosis. 【Result】 Analysis of protein structure showed that both MdWRKY18 and MdWRKY40 proteins contained a WRKY, Cx5C and HxH structural domains. The expression levels and promoter activities of MdWRKY18 and MdWRKY40 were induced by the 150 mmol·L ^-1 NaCl. The yeast two-hybrid experiments showed that MdWRKY18 and MdWRKY40 could respectively interact with itself to form homodimers, and MdWRKY18 could also interact with MdWRKY40 to form heterodimers. When MdWRKY18 and MdWRKY40 was overexpressed respectively in orin callus, they could increase the callus weight under salt stress and promote the expression of MdSOS1 and MdNHX1. When MdWRKY18 and MdWRKY40 were co-overexpressed in orin callus, it could also promote the expression of MdSOS1 and MdNHX1, however, the weight of callus was heavier than the weight of callus overexpressing MdWRKY18 or MdWRKY40. 【Conclusion】 MdWRKY18 and MdWRKY40 were induced by the salt stress, and they could form homodimers or heterodimers, overexpressing MdWRKY18 or MdWRKY40 in orin callus could increase its salt tolerance.

Cloning, Characterization and Expression Analysis of K ⁺/H ⁺ Antiporter Genes in Grape

WANG ZhuangWei,WANG QingLian,XIA Jin,WANG XiCheng,SONG ZhiZhong,WU WeiMin

Scientia Agricultura Sinica. 2018, 51(23):  4522-4534.  doi:10.3864/j.issn.0578-1752.2018.23.011

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【Objective】 Isolation and characterization of KEA family genes from grape. Analysis of the tissue-specific expression patterns of KEA family genes and response to K ⁺ depletion, ABA, NaCl and sorbitol treatments. Screen the potential major KEA genes in grape. 【Method】 By carrying out homology-based cloning, putative KEA family genes were isolated and characterized from grape. A phylogenetic tree was constructed by multiple alignment of KEA family proteins from 9 known plants (grape, Arabidopsis, rice, maize, sorghum, slender false brome, polar, pear, and apple) using the neighbor-joining method via MEGA7.0 software. Details of grape KEA family genes and encoded proteins were analyzed with the help of bioinformatical analysis softwares. By screening the EST database, electrical expression profiles of grape KEA genes were determined. Quantitative real-time PCR (qRT-PCR) was carried out to analyze the expression patterns of KEA family genes and response to K ⁺ depletion, ABA, NaCl, and sorbitol treatments, and obtained the major genes. 【Result】 Four KEA family genes were isolated from grape, entitled by VvKEA1—VvKEA4, which were all containing the K/H exchanger and TrkA-N functional domains that belonging to the classic plant KEA family antiporters. The amino acid sequences of KEA proteins from 9 plants shared an overall identity of 33.10%. These KEA members were classified into 2 major groups (Groups Ⅰ and Ⅱ), and VvKEA1and VvKEA2 belong to Group Ⅰ that containing 7 Motifs, while VvKEA3 and VvKEA4 belong to GroupⅡ that just containing 4 Motifs. Phylogenetic tree analysis showed that VvKEA1, VvKEA 2 and VvKEA 4 of grape were closely clustered with AtKEA2, AtKEA 3 and AtKEA 5 of Arabidopsis, respectively, and VvKEA3 was clustered with PbrKEA5 of pear and MdoKEA7 of apple. KEA members of 4 grass family plants (rice, maize, sorghum and slender false brome) were prone to clustered together, while three woody plants (polar, apple and pear) KEA members were prone to clustered together. Mainly localized in plasma membrane, all predicted VvKEA proteins possessed similar tertiary structures, contained 12 or 13 transmembrane domains (TMs), and the theoretical isoelectric point (pI) were all less than 7.0. In particular, only VvKEA3 possessed the signal peptide. Fifteen cis-acting regulatory elements, including the stress response, nutrition and development, hormone response and circadian rhythm regulations, et al., were identified in the promoter region of VvKEA genes. Expression profile analysis showed that VvKEA family genes were expressed in different tissues or organs in grape, and the highest percentage was predicted in fruit, followed by leaf, seed, root and pistil. qRT-PCR analysis showed that VvKEA3 was the most abundant expressed gene during different parts of 8-year-old ‘Rosario Bianco’ on the whole, especially in fruitlet, and the other 3 genes were less expressed with similar amount. In grape seedlings, VvKEA1—VvKEA4 genes were more sensitive to ABA treatment, whose expression were all induced in both tested shoots and roots, but had no response to NaCl treatment. The expression of VvKEA3 in both shoots and roots and VvKEA1 in shoots were up-regulated by K ⁺ depletion treatment, and the expression of VvKEA3 in both shoots and roots and VvKEA4 in roots were increased by sorbitol treatment. 【Conclusion】 Four predicted KEA family genes were cloned and characterized from grape, which were majorly expressed in fruit, leaf and seed. Notably, VvKEA3 was the most abundant gene in 8-year old grape tree, especially in fruitlet, whose expression was prone to be regulated by K ⁺ depletion, ABA, and sorbitol osmotic stress. VvKEA3 may be a crucial K ⁺/H ⁺ antiporter during grape fruit development.

FOOD SCIENCE AND ENGINEERING

Using Neural Network Coupled Genetic Algorithm to Optimize the SPME Conditions of Volatile Compounds in Korla Pear

ZHANG Fang,WEI ZhiSheng,WANG Peng,LI KaiXuan,ZHAN Ping,TIAN HongLei

Scientia Agricultura Sinica. 2018, 51(23):  4535-4547.  doi:10.3864/j.issn.0578-1752.2018.23.012

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【Objective】 In order to lay the foundation for fitting and regulation of natural flavors of Korla fragrant pear-related products, the extraction and analytical method for Korla pear aroma compounds was established by the optimization of solid phase microextraction (SPME) conditions (extraction fiber type, sample amount, extraction temperature, extraction time, and salt addition amount) with the back propagation neural network coupled genetic algorithm (BPNN-GA). 【Method】The type of extraction fiber and the best extraction parameters of sample amount, extraction temperature, extraction time, and salt addition amount were determined by single factor test. On the basis of the single factor test, the central composite design was applied to further investigate the effects of sample amount, extraction temperature, extraction time, and salt addition amount on the SPME of Korla pear juice by using the content of the key aroma substances in Korla pear juice (1-Hexanal, butanoic acid ethyl ester, 1-nonanal, hexanoic acid ethyl ester, acetic acid ethyl ester, (E)-2-hexenal, 1-hexanol, (Z)-2-hexenal, and propanoic acid ethyl ester) as evaluation indicators. The model of BPNN was used to debug fitness of the function based on the results of central composite design, and then the sample amount, extraction temperature, extraction time, salt addition amount and the content of the key aroma substances in Korla pear juice were used as initial population, input values, and output value, respectively. The generalization ability of BPNN was validated using the data set outside the central composite design. The global optimal extraction conditions within the experimental level were predicted using the genetic algorithm (algorithm parameter setting: maximum evolution algebra 100, population size 20, mutation probability 0.2, and crossover probability 0.4).【Result】The result of statistical analysis showed that the extraction fibers had a significant (P<0.05) effect on the extraction efficiency of SPME, and the 65 μm PDMS/DVB fiber was the best extraction fiber of SPME for volatile compounds of Korla pear juice. The sample amount, extraction temperature, extraction time, and salt addition had significant (P<0.05) effect on the SPME efficiency. The topology of the BPNN was ‘4-15-1’. The Root Mean Square Error (MSE) of the verification data and the training data were both greater than 0.017, and the correlation coefficients of training, test and verification data were 0.990, 0.951 and 0.973, respectively, indicating that the BPNN prediction model had good accuracy and could be used for the prediction of the result of SPME of the aroma substances in Korla pear juice. The fitting degree of the prediction value of BPNN and the verification experiment result validating the generalization ability of the BPNN was 0.992, which indicated the established BPNN model had good generalization ability and could accurately predict the output value of datasets outside the training datasets. Using genetic algorithm after evaluation of data for 100 generations determined the best individual (3.41 μg?g ^-1) and optimum condition: sample amount 5.33 g, extraction temperature 44.7℃, extraction time 25.22 min and salt addition 0.63 g. The verification test were conducted under these conditions as closely as possible (sample amount 5.33 g, extraction temperature 45℃, extraction time 25 min and salt addition 0.63 g), and the content of 9 key volatile substances in Korla pear juice was (3.37±0.23) μg?g ^-1, which compared with the predicted value, and the error was -1.19%.【Conclusion】 The above results indicated that BPNN-GA was a new method with better accuracy to optimize the solid-phase microextraction parameters, and the method of BPNN-GA provided a new way to solve the nonlinear problem. And the optimum extraction conditions for SPME of Koral pear juice were as follows: sample amount 5.33 g, extraction temperature 45℃, extraction time 25 min and salt addition 0.63 g.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

The Effects of Artemisia annua Extracts on the Rumen Fermentation in Dairy Cows

WANG LiFang, ,AO ChangJin

Scientia Agricultura Sinica. 2018, 51(23):  4548-4555.  doi:10.3864/j.issn.0578-1752.2018.23.013

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【Objective】The effects of daisy plant (Artemisia annua) extracts (AAE) on the fermentation indexes such as acetic acid, propionic acid and butyric acid in the rumen in dairy cows were studied in this paper, and furthermore the effects on B. fibrisolvens and C. proteoclasticum for regulating CLA synthesis in the rumen were researched. The purpose of this study was to explore the effects of AAE on rumen fermentation in dairy cows, and to investigate the part mechanism of effecting CLA synthesis from the rumen.【Method】Fifteen dairy cows (600±29) kg, parity 2-3, in lactating (158±3) d and milk yield (22.8±1.8)kg·d ^-1 were used in completely random design in 40 d experimental periods. The experimental dairy cows were integrated management, free ingestion, free drinking water, feeding 2 times per day, milking 2 times (4:30 a.m., 16:30 p.m.). The experiments groups comprised the control and two treatments (treatment one and treatment two, the doses of AAE for supplementation was 96 g·d ^-1 and 160 g·d ^-1 for every cow, respectively). The experimental period comprised a 9-d adaptation and a 31-d the formal period. 【Result】 The results showed as follows: ruminal pH values tended to increase in two treatments for AAE supplementation compared with the control, which increased by 2.63% and 8.61%, respectively (P>0.05). The concentrations for acetic acid, propionic acid and butyric acid decreased significantly in two treatments except for isovaleric acid and valeric acid in the low dose of AAE compared with the control (P<0.05). AAE supplementation increased the relative percentage of B. fibrisolvens in rumen mixture and rumen liquid (P<0.05), and decreased the relative percentage of B. fibrisolvens in rumen solid (P>0.05). AAE supplementation decreased the relative percentage of C. proteoclasticum in rumen liquid and rumen solid (P<0.05), and increased the relative percentage of C. proteoclasticum in rumen mixture (P>0.05). 【Conclusion】 It was concluded that AAE supplementation had adverse effect on rumen fermentation characteristics, and had different effects on B. fibrisolvens and C. proteoclasticum on different contents of the rumen, but the general trend was increasing the relative percentage of B. fibrisolvens, and decreased the relative percentage of C. proteoclasticum, which ws beneficial to CLA synthesis.

Effects of Temperature and Relative Humidity on the Growth Performance and Blood Index of Goats

LI JinPeng,WANG GuoJun,ZHAO Tian,ZHOU GuangChen,YANG YuXin

Scientia Agricultura Sinica. 2018, 51(23):  4556-4574.  doi:10.3864/j.issn.0578-1752.2018.23.014

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【Objective】The objective of this study was to provide a theoretical reference for breeding disease diagnosis and mitigation measures for goats under high temperature and high humidity condition. This study was carried out to investigate the effects of temperature and relative humidity (RH) on growth performance, routine urine, blood routine and serum biophysiological- biochemical indexes in goats. 【Method】According to 4×4 factorial design of four temperatures (26℃, 30℃, 34℃ and 38℃ ) and four RH levels (35%, 50%, 65% and 80%), eight goats of one and a half years old from first filial generation of healthy Boer goat × Sannen dairy goat with similar body weight were allotted, and randomly divided into 2 groups (each group contained four replicates with one goat per replicate). The adjustment period lasted for 30 days in the natural environment. The experiment period of every group was 5 days and was treated 24 hours a day. And the experiment was carried out by turns in artificial environment chambers according to the processing method. Every goat’s weight was recorded before and after trial begin, and feed intake and water intake were also recorded every day. On days 3 and 5, blood was drawn from the jugular vein to determine the blood routine, and the serum samples were used to determine the serum indexes. Data were analyzed using IBM SPSS Statistics 21.0 software.【Result】(1) The interaction of temperature and RH significantly affected average daily feed intake (ADFI) and average daily water intake (ADWI) (P<0.05), but no significantly affected on average daily gain (ADG), weight ratio and blood routine indexes (P>0.05). ADG of goats at every treatment group of 38℃ was significantly lower than that at every treatment group of 26℃ (P<0.05). ADFI of goats at 38℃ was significantly lower than that at 26℃ and 30℃ (P<0.05). The white blood cell counts (WBC) of goats at 65% and 80% RH were significantly lower than those at 35% RH (P<0.05). (2) On day 3, the interaction of temperature and RH significantly affected content of potassium (K), activity of alanine aminotransferase (ALT), and aspertate aminotransferase (AST) in serum of goats (P<0.05). The content of K in serum of goats at 38℃ and 80% RH was significantly lower than that at other groups (P<0.05). The glucose (GLU), globulin (GLO), urea nitrogen (BUN), calcium (Ca) and alkaline phosphatase (ALP) in serum of goats at 38℃ was significantly lower than that at 26℃ and 30℃ (P<0.05). The GLU and BUN in serum of goats at 80% RH was significantly lower than that at 35% RH (P<0.05). On the day 5, the interaction of temperature and RH significantly affected content of total protein (TP), albumin (ALB), GLO, BUN, Ca, K, phosphorus (P), sodium (Na) and activity of lactate dehydrogenase (LDH) in serum of goats (P<0.05). The GLU, chlorine (Cl), ALT, AST, ALP and creatine kinase (CK) in serum of goats at 34 and 38℃ was significantly lower than that at 26℃(P<0.05). The GLU and Cl in serum of goats at 60% and 80% RH were significantly lower than those at 35% RH (P<0.05). (3) On the day 3 and 5, the concentrations of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalsae (CAT) in serum of goats at 38℃, 80% and 38℃, 65% were significantly lower than those at every group at 26℃ (P<0.05). The concentration of malonaldehyde (MDA) in serum of goats at 34 and 38℃ was significantly higher than that at 26℃ (P<0.05).(4)As the temperature rose, the ADFI, ADG, TP, GLO, BUN, GLU, ALT, T-AOC, SOD and CAT in serum were deceased, but average daily water intake and MDA in serum were risen. As the RH rose, the ADFI, GLU, Cl, T-AOC and GSH-Px in serum were deceased, but TP, LDH and MDA in serum were risen. (5) The TP, ALB, GLO, BUN, GLU, TC, TG, Ca, P, ALT, AST, ALP, LDH and CK in serum on the day 5 was lower than that on the day 3, but the MDA in serum was higher on the day 3 and T-AOC, SOD and GSH-Px in serum on day 5 in 34 and 38℃ was higher on day 3.【Conclusion】Temperature, RH and their interaction affected the average daily intake, average daily water intake, serum biochemical indicators and antioxidant indicators of goats. The high temperature and high humidity environment had adverse effects on the growth performance and antioxidation function of goats, and the effect was the most serious for the goats at 38℃ with 80% RH. Moreover, with the extended duration of higher temperature and humidity, the goats showed more extensive stress effect.

Analysis of Differentially Expressed Circular RNAs and Their Regulation Networks During the Developmental Process of Apis mellifera ligustica Worker’s Midgut

GUO Rui,CHEN HuaZhi,XIONG CuiLing,ZHENG YanZhen,FU ZhongMin,XU GuoJun,DU Yu,WANG HaiPeng,GENG SiHai,ZHOU DingDing,LIU SiYa,CHEN DaFu

Scientia Agricultura Sinica. 2018, 51(23):  4575-4590.  doi:10.3864/j.issn.0578-1752.2018.23.015

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【Objective】Circular RNA (circRNA) plays a primary role in alternative splicing, transcription regulation and expression regulation of parental gene. The objective of this study is to investigate the profile expression of circRNAs and differentially expressed circRNAs (DEcircRNAs) during the developmental process of the midguts of Apis mellifera ligustica workers, and to explore the role of DEcircRNAs in the development of midgut at the transcriptional level. 【Method】 On basis of the whole transcriptome data from 7- and 10-day-old worker’s midguts of A. m. ligustica (Am7 and Am10), find_circ software was used to predict circRNAs based on the filtered sequencing data. The circRNA expression level was normalized by RPM algorithm. Differential expression analysis for circRNAs was conducted via DEGseq software following standards fold change≥2.0, P<0.05 and false discovery rate (FDR) <0.05. Source genes of DEcircRNAs were annotated to GO and KEGG databases to gain function and pathway annotations by using BLAST. DEcircRNAs-miRNAs and DEcircRNAs-miRNAs-mRNAs networks were predicted with TargetFinder software and visualized using Cytoscape v.3.2.1 software. RT-qPCR was conducted to verify the reliability of sequencing data.【Result】 On average, 19 616 356 anchors reads were obtained from each A. m. ligustica worker’s midgut sample. Pearson correlations between different biological repeats within Am7 and Am10 groups were ≥0.950. In total, 256 DEcircRNAs including 105 up-regulated circRNAs and 151 down-regulated circRNAs were predicted. Novel_circ_009675 and novel_circ_013879 were highly expressed in Am7 and Am10, respectively. Source genes of DEcircRNAs could be annotated to 32 GO terms including binding, single-organism process and cellular process, among them 35, 35 and 7 source genes were involved in catalytic activity, metabolic process and stress response. Additionally, these source genes could be annotated to 35 KEGG pathways, in which 5, 5 and 4 source genes were associated with Hippo signaling pathway, endocytosis and phagosome, respectively; further investigation showed that 1, 2 and 2 source genes could be annotated to material metabolisms such as phosphoinositol metabolism, starch and sucrose metabolism and galactose metabolism; 5, 4, 3, 1 and 1 source genes could be annotated to immune signaling pathways including endocytosis, phagosome, lysosome, ubiquitin-mediated proteolysis and MAPK signaling pathway, respectively. These results suggested that the corresponding DEcircRNA was involved in the development, metabolism and immune defense of the midgut of A. m. ligustica. DEcircRNA-miRNA regulation network analysis showed that 141 DEcircRNAs could link to 107 miRNAs, most of these DEcircRNAs could only bind to 1-2 miRNAs, but novel_circ_011577 and novel_circ_010719 could respectively bind to 32 and 28 miRNAs. In addition, the number of DEcircRNAs combined with mir-136-y, ame-miR-6001-3p and mir-136-y was the highest (15, 14 and 14, respectively), which indicated that the corresponding DEcircRNA could play roles during the developmental process of A. m. ligustica worker’s midgut as competing endogenous RNAs. Furthermore, DEcircRNAs-ame-miR-6001-3p-mRNA network was constructed and analyzed, and the result indicated that 14 DEcircRNAs could jointly link to ame-miR-6001-3p, implying they were likely to indirectly regulate division and differentiation of stem cells in A. m. ligustica worker’s midgut via regulation of ame-miR-6001-3p. Six DEcircRNAs were randomly selected for RT-qPCR assay, the result showed the alteration trend of expression levels of 5 DEcircRNAs was consistent with that of the sequencing data, which proved the reliability of trancriptome data.【Conclusion】Through the deep investigation of DEcircRNAs during the developmental process of A. m. ligustica worker’s midgut, the expression profile and differential expression information of circRNAs in the development of midgut of worker bee were provided, and the role of DEcircRNAs in the development of midgut was revealed. It provides a basis for the screening and functional study of key circRNAs associated with the development of the midgut.