Table of Content

16 December 2020, Volume 53 Issue 24

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Genomic Profiling and Expression Analysis of Phosphatidylinositol- specific PLC Gene Families Among Chinese Spring Wheat

SI XuYang,JIA XiaoWei,ZHANG HongYan,JIA YangYang,TIAN ShiJun,ZHANG Ke,PAN YanYun

Scientia Agricultura Sinica. 2020, 53(24):  4969-4981.  doi:10.3864/j.issn.0578-1752.2020.24.001

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【Objective】To characterize the gene family encoding phosphatidylinositol-specific phospholipase-C (PI-PLC), the gene number, structure, phylogenetic relationship and expression pattern of TaPLC genes were analyzed. In addition, we explored their relative transcription levels in various tissue and the expression pattern under drought and salt stress to further analysis the physiological role of TaPLC genes in response to abiotic stress.【Method】Based on the genome database (Ensembl Plants, http://plants.ensembl.org/index.html), TaPLC genes were identified from Triticum aestivum, and the gene structures were analyzed by some bioinformatics tools, such as Pfam, CDD and SMART. The protein sequence characteristics of the members in TaPLC family were analyzed with the online server ExPASy (http://cn.expasy.org/tools). The subcellular localization of TaPLC genes were predicted using the WoLF PSORT (https://www.genscript.com/wolf-psort.html). Phylogenic tree analysis was performed by software MEGA 7.0. The real-time PCR was used to detect the gene expression levels in different tissues under abiotic stress. 【Result】A total of 11 TaPLC genes members within the heterologous hexaploid wheat genome were identified and analyzed. All TaPLC genes have conserved X-Y catalytic domains and C2 domains, just like other plant PLC genes. Subcellular localization showed that these proteins were located in the chloroplast or mitochondria. Phylogenetic analysis revealed that all of the TaPLC genes were unevenly classified into four numbered, TaPLC1A/TaPLC1D, TaPLC2A/TaPLC2B/TaPLC2D, TaPLC3A/TaPLC3B/TaPLC3D, and TaPLC4A/TaPLC4B/TaPLC4D, which were all orthologous genes of plant PLC genes in plants, and highly conserved during evolution. However, the deletion of TaPLC1 in B subgenome indicates asymmetry evolution in different subgenomes. Further analysis of cis-regulatory elements elucidated that these members of TaPLC family shared similar elements, such as the phytohormone and the stress response cis-elements. Each PLC gene has a unique tissue expression pattern, and they are expressed mainly in roots, leaves, spikes, and gains. In addition, PLC genes mediate the response of wheat to salt and drought stresses, which are induced rapidly by salt or drought stress stimulation. 【Conclusion】The TaPLC family in Triticum aestivum contains 11 members which have conserved X-Y catalytic domains and C2 domains. Although the TaPLC genes were highly conserved, they were asymmetrically evolved in different subgenomes. They had specific expression pattern in different tissues, and were induced by salt or drought stress, which suggested TaPLC genes play a role in response to salt or drought in wheat.

Acquisition and Characteristic Analysis of Transgenic Maize with phyA2, ZmTMT, and Bar

YAO XingLan,YANG WenZhu,LUO YanZhong,CHEN RuMei,WANG Lei,ZHANG Lan

Scientia Agricultura Sinica. 2020, 53(24):  4982-4991.  doi:10.3864/j.issn.0578-1752.2020.24.002

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【Objective】Corn seeds are the main feed ingredient for poultry and monogastric animals, but the content of α-tocopherol with high-activity in corn seeds is low, and maize is rich in phosphorus mainly existed in the form of phytate, which cannot be effectively utilized by animals. Therefore, synthesized DL-α-tocopheryl acetate and phosphorus or microbial phytase are supplemented in the feed for optimal animal growth. These methods not only greatly increase the feed cost, but also cause the phosphorus pollution due to the undigested phytate. The aim of this study is to acquire maize seeds rich in α-tocopherol and high in phytase activity, and provide resources for forage maize breeding.【Method】Construct an expression vector with three expression cassette, and introduced into maize through agrobacterium infection method, glufosinate was used for screening; The positive transgenic plants were identified by spraying glufosinate and PCR analysis; RT-PCR analysis of target gene expression at the transcription level; Western blot analysis of target gene expression at the translation level; Phytase activity was measured by spectrophotometric method; vitamin E content was determined by HPLC. At the same time, the differences of other nutrients and agronomic traits between transgenic lines and wild types were compared.【Result】Constructed an expression vector with three expression cassette, phyA2 driven by endosperm-specific promoter 123387, ZmTMT driven by embryo-specific promoter 13387, Bar driven by constitutive promoter CaMV35S, and introduced into maize; Transgenic plants were identified by spraying glufosinate and PCR; Two transgenic homozygous maize lines TPB1 and TPB2 with good target traits were obtained after multiple generations of backcross transfer. RT-PCR and Western blot analysis showed that phyA2, ZmTMT and Bar were all highly expressed in transgenic lines. Phytase activity determination showed phytase activity reached to 10 000-13 000 U·kg ^-1, which is able to meet the needs of animal growth and development, vitamin E content measurement showed more than 90% of γ-tocopherol was transformed to α-tocopherol, and α-tocopherol content increased to 50-70 mg·kg ^-1. In addition, there were no significant differences on agronomic traits between transgenic lines and wild type, and there was no significant difference on nutrient component between TPB1 and wild type, slight difference but without adverse effect between TPB2 and wild type; and the transgenic plants were resistant to BASTA.【Conclusion】These transgenic maize, which are rich in α-tocopherol and phytase, and also with herbicide resistance, can be applied for maize breeding to reduce feed cost, improve the utilization rate of phosphorus and reduce environmental pollution.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Study on the Adaptability of Wheat Reaching the Protein Content Standard of Soft Wheat in Jiangsu Province

XIA ShuFeng,WANG Fan,WANG LongJun,ZHOU Qin,CAI Jian,WANG Xiao,HUANG Mei,DAI TingBo,JIANG Dong

Scientia Agricultura Sinica. 2020, 53(24):  4992-5004.  doi:10.3864/j.issn.0578-1752.2020.24.003

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【Objective】As the material of making biscuits and cakes, the baking properties of soft wheat flour is determined by its content and quality of protein. The grain protein content (GPC, %) is not only determined by genetic factors, but also affected by environment and farming practices. In order to provide suggestions for quality region classification for the soft wheat areas in Jiangsu province, this paper explored the suitable planting areas and influencing factors of soft wheat.【Method】Based on the two-year investigation data related to wheat quality in Jiangsu province, the random forest algorithm was used to screen important indicators, and the meta-analysis of proportions was employed to analyze the impacts of geolocation and meteorological factors on the possibility of wheat GPC reaching the standard of soft wheat under the ordinary farming practices. 【Result】 The average of two-year wheat GPC was 13.92%. In 2018, GPC ranged from 11.06% to 18.09% and the average value was 14.52%, in which GPC of 10% samples was lower than 12.5%. In 2019, the range of GPC was 10.20%-16.50% and the average value was 13.33%, in which GPC of 29.71% samples was lower than 12.5%. With application of random forest algorithm and meta-analysis, it was found that the GPC of wheat growing in the southeastern part along sea and lake of Jiangsu was most likely to meet the standard of soft wheat, and the possibility of which was 92%, followed by the northwestern part along river and the eastern coastal area in Jiangsu. When the plantation was 20-30 km away from the primary river and lake or coastline, the probability of reaching the standard was relatively high, which was 23.95%. In terms of meteorological factors, precipitation had the greatest influence on the formation of soft wheat in Jiangsu province during the early growth stage, especially at emergence stage and joining stage. The impact of accumulated temperature was more important during the later stage of growth stage, especially during grain filling stage and flowering stage. In addition, the sunshine hours at emergence stage and jointing stage and the precipitation at flowering stage were also more important for the formation of soft wheat in Jiangsu province. Among them, the precipitation at emergence stage was positively correlated with the possibility of wheat GPC reaching the standard of soft wheat in Jiangsu province. However, the sunshine hours during emergence stage, the precipitation and the sunshine hours during jointing stage, and the accumulated temperature during filling stage were opposite. 【Conclusion】 The suitable planting areas for soft gluten wheat in Jiangsu province were mainly concentrated in the eastern coastal areas and southeastern coastal and lake areas. With suitable precipitation during the emergence and jointing period and accumulated temperature during flowering and filling stage, the northwest areas along the river would also have high possibility to produce soft wheat. Thus, the geographic location (distribution of river systems, etc.) and climate should be considered when zoning the suitable planting areas for different quality types of wheat.

Inversion of Soil Salinity in Coastal Winter Wheat Growing Area Based on Sentinel Satellite and Unmanned Aerial Vehicle Multi-Spectrum— A Case Study in Kenli District of the Yellow River Delta

XI Xue,ZHAO GengXing,GAO Peng,CUI Kun,LI Tao

Scientia Agricultura Sinica. 2020, 53(24):  5005-5016.  doi:10.3864/j.issn.0578-1752.2020.24.004

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【Objective】 The purpose of this paper was to explore an accurate and efficient remote sensing method for soil salinity extraction of wheat field in the Yellow River Delta, and obtain the degree and distribution of soil salinization of wheat fields.【Method】This study took Kenli District as the research area, and set 77 sample points in winter wheat growing area evenly. At the same time, two representative test areas and 99 grid sample points were set, and the surface soil salinity data in wheat field and the multi-spectral images of UAV in the test area were collected. The sensitive spectral parameters were screened from four spectral bands (red, green, red edge, and near-infrared) and five spectral indexes (SI, NDVI, DVI, RVI, and GRVI). Stepwise regression, partial least squares, BP neural network and support vector machine methods were used to establish models for predicting the soil salinity, and the band ratio mean method was used to obtain the correction coefficient of the corresponding band of sentinel-2A satellite image. And then the selected soil salinity estimation model was converted into an inversion model based on satellite image. After using the data from the wheat field sample points to verify the models, the best soil salinity inversion model in wheat field was selected, and two scales of soil salinity inversion are realized in the test areas and the research area.【Result】The results showed that the four bands of UAV and the spectral indexes NVDI, RVI and SI were significantly correlated with soil salinity. Among the 13 models of the four modeling methods, the four index models established by NDVI, RVI and SI were better than the other models in modeling and verifying R ²; The best inversion model was the spectral index model obtained by partial least square method: Y=-9.4774×NDVI₁+ 0.4794×RVI₁+ 3.0747×SI₁+ 5.0604, and the accuracy R² was 0.513 and RMSE was 1.379. By using this model, the soil salinity distribution map of the test area and the whole wheat area was obtained. Combined with the measured interpolation and the survey, the inversion model and spatial distribution results were proved to be accurate and reliable. 【Conclusion】In this study, the soil salinity inversion model of the coastal wheat area based on the integration of satellite and UAV was constructed, which had positive reference value for the production and management of crops in the coastal saline area.

PLANT PROTECTION

Identification and Expression Pattern Analysis of Septin Gene Family of Setosphaeria turcica

LONG Feng,WANG Qing,ZHU Hang,WANG JianXia,SHEN Shen,LIU Ning,HAO ZhiMin,DONG JinGao

Scientia Agricultura Sinica. 2020, 53(24):  5017-5026.  doi:10.3864/j.issn.0578-1752.2020.24.005

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【Background】Septin, which is widely found in all eukaryotes except plants, is a highly conserved GTP binding protein family and is considered to be the fourth cytoskeletal protein after microtubules, microfilaments and intermediate fibers. Septin proteins of pathogenic fungi are involved in cell polarity determination, morphological shaping and morphological transformation associated with pathogenicity.【Objective】The objective of this study is to identify Septin gene family of Setosphaeria turcica, analyze its expression pattern at different developmental stages, and to lay a foundation for clarifying the relationship between the membrane protein Septin and fungal infection structure development.【Method】The amino acid sequences of six Septin proteins in Bipolaris maydis were used as probe sequences, online Blastp alignment and keyword search were carried out in the S. turcica database to obtain S. turcica candidate Septins. Then bioinformatics analyses including gene structure, physical and chemical properties and transmembrane region structure of the Septins were conducted. The materials of S. turcica that were induced by artificial hydrophobic media at different stages of infective structures development and infected host leaves at different times were collected, and RT-qPCR (real-time fluorescence quantitative PCR) technology was used to analyze the transcription levels of Septins at different stages of infective structures development.【Result】Six candidate Septins were obtained, and among them, four typical Septins contained G1, G3, and G4 motifs, were named by StSep1, StSep2, StSep3 and StSep4, respectively. The expression level of Septins increased slowly under the induction on artificial hydrophobic media. StSep1 was active in the late stage of germ tube formation, and its expression level was 25.69 times of that in the conidia period (P<0.01). The expression level of StSep4 reached a peak in appressorium anaphase, and then the expression level was down-regulated. The change trend of Septin expression in the process of pathogen infection was basically consistent with that under the induction of artificial hydrophobic material. StSep1 expression was significantly up-regulated at 6 h after inoculation (P<0.01). As time went on, StSep4 actively expressed during the period of appressorial formation, and the expression reached a peak at 18 h after inoculation. After that, the expression was down-regulated, but it was still higher than that in the early stage of germination. StSep2 and StSep3 were active at 18 h and 24 h after inoculation, exceeding the initial stage of germination.【Conclusion】There are 4 core Septins in the genome of S. turcica. StSep1 and StSep4 are actively expressed during the formation of germ tube and appressoria, respectively. The results will provide a theoretical basis for further clarifying the function of Septin and the infection regulation mechanism of S. turcica.

Prediction and Analysis of Candidate Secreted Proteins from the Genome of Lasiodiplodia theobromae

XING QiKai,LI LingXian,CAO Yang,ZHANG Wei,PENG JunBo,YAN JiYe,LI XingHong

Scientia Agricultura Sinica. 2020, 53(24):  5027-5038.  doi:10.3864/j.issn.0578-1752.2020.24.006

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【Objective】Lasiodiplodia theobromae is an important phytopathogenic fungus with a worldwide distribution. This species causes severe Botryosphaeria dieback on a wide range of woody plants, which leads to reduced crop quality and tremendous economic losses. The objective of this study is to predict and analyze the candidate secreted proteins in the genome of L. theobromae, clarify their basic characteristics, so as to lay a foundation for the study of the pathogenic mechanism of secreted proteins in this pathogen.【Method】The signal peptide prediction algorithm SignalP v5.0 and subcellular localization prediction algorithm ProtComp v9.0, transmembrane helix prediction algorithm TMHMM v2.0, GPI-anchoring site prediction algorithm big-PI Fungal Predictor, and subcellular protein location distribution algorithm TargetP v2.0 were used to analyze 12 902 protein sequences of L. theobromae published in the previous study. The basic features including the length of the N-terminal signal peptide, the frequency of amino acid usage and cleavage site of the predicted secreted proteins were statistically analyzed. Based on the homology of the protein sequence, biological function annotation of the predicted secreted proteins was clarified by using BLASTP program. The activity of the signal peptide of the selected secreted proteins was detected by yeast secretion and cell translocation assays. Expression patterns of the selected secreted protein genes during L. theobromae infection were analyzed by qRT-PCR technology.【Result】In this study, 522 secreted proteins were verified, accounting for 4.3% of the total proteins present in the genome of L. theobromae. The lengths of amino acids of secreted proteins were ranged from 101 to 400 aa. The distribution length of signal peptides was from 18 to 20 aa and the largest number was 20 aa. The top frequent amino acid was alanine in the signal peptides, and the most frequently incorporated amino acids were non-polar and hydrophobic, accounting for 60.2% of the total amino acids. Further, the amino acids in the position -3 to -1 in the signal peptides were relatively conserved and the signal peptide cleavage site belonged to A-X-A type, which could be recognized and cleaved by Sp I type peptidase. Among them, 336 secreted proteins were identified with a predictive function, which was mostly enzymatic or virulence-associated protein. Besides, there are differences in terms of molecular weight, isoelectric point, the aliphatic index in the candidate secreted proteins. Finally, the predicted signal peptides of the 9 putative L. theobromae secreted proteins were confirmed to have secretory activity by using a yeast invertase secretion assay. qRT-PCR analysis demonstrated that the expression of selected protein genes was differentially regulated during host infection.【Conclusion】A total of 552 candidate secreted proteins of L. theobromae were predicted by a set of computer algorithms. Lengths of the signal peptides vary greatly and the most frequently are mainly non-polar and hydrophobic amino acids. Secreted proteins characterized in this study can be categorized under enzymes related to the degradation of cell wall components, necrosis induction proteins, and chitin-binding proteins which may play an important role in L. theobromae pathogenetic mechanism.

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

Effects of Long-Term Cultivation on Contents of Organic Carbon and Total Nitrogen in Soil Particulate Fraction in Oasis Farmland of Xinjiang

TANG GuangMu,ZHANG YunShu,XU WanLi,MA HaiGang,HU KeLin

Scientia Agricultura Sinica. 2020, 53(24):  5039-5049.  doi:10.3864/j.issn.0578-1752.2020.24.007

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【Objective】Particulate organic carbon and total nitrogen are the important components of soil organic carbon (SOC) and total nitrogen (TN), which has an effect on SOC and TN turnover and sequestration. Therefore, studies exploring the changes in soil particulate organic carbon and total nitrogen under different tillage years could aid the understanding of the mechanism of regional soil carbon and nitrogen fixation, so as to provide a scientific basis for improving land productivity. 【Method】In this study, soil samples from the farmlands with different tillage years (0, 5, 10, 15, and 20 years) in the three typical oases (Lanzhouwan, 31 corps, and Puhui farms) of Tianshan were collected, and the SOC and TN contents of different particle components were determined. And then, the effects of long-term tillage on SOC and TN contents of different particle components were analyzed. 【Result】Results indicated that long-term tillage practices increased SOC and TN accumulation. SOC and TN contents increased rapidly during the first five years of tillage and then tended to be stable after five years of tillage. SOC contents increased to 76.4% (Lanzhouwan), 286.2% (31 corps), and 145.6% (Puhui farms) of the SOC contents in uncultivated land. Similarly, TN contents increased to 14.7%, 58.9%, and 75.0%, respectively. The effects of long-term tillage practices on OC contents in different particles components were different. The organic carbon (OC) contents in sand showed a downward trend after reaching a maximum value (10-15 years tillage), and then showed a increasing trend, but it remained higher than that of an uncultivated land. After 20 years of tillage, OC contents in sand were increased by 0.63 g·kg^-1 (Lanzhouwan), 0.89 g·kg^-1 (31 corps), and 1.56 g·kg^-1 (Puhui farms). While, the contents of OC in silt and clay showed a continuous increasing trend with tillage time, After 20 years of tillage, OC contents in silt and clay were increased by 0.42-2.39 g·kg^-1 (Lanzhouwan), 2.64-3.39 g·kg^-1 (31 corps), and 1.36-2.72 g·kg^-1 (Puhui farms). However, the effect of long-term tillage practices on TN in different particles components was complex. The OC contents in sand showed a continuous increasing trend, After 20 years of tillage, TN contents in sand were increased by 0.24 g·kg^-1 (Lanzhouwan), 0.40 g·kg^-1 (31 corps), 0.29 g·kg^-1 (Puhui farms).. The content of TN in silt decreased (0-10 years of tillage) initially and then increased (10-20 years). TN in clay increased by 67.6% in Lanzhouwan, 306.3% in 31 corps, and 91.3% in Puhui farms during 0-10 years of tillage and decreased after 10 years of tillage. With regard to particle components, OC and TN in silt had the largest proportions, accounted for 43.3%-56.1% and 30.2%-72.2% of SOC and TN, respectively. Tillage affected the distribution proportions of OC and TN in different components. The distribution proportion of OC in sand increased during the first 10 years and decreased in the subsequent 10 years. The distribution proportion of TN in sand was increased by 14.8% in Lanzhouwan, 19.8% in 31 corps, and 29.0% in Puhui farms after 20 years of tillage. Soil C/N was increased by 40.3%-142.9% during 0-5 years of tillage, and no significant change was observed after 5 years of tillage. Tillage changed C/N ratio in different particles components, and the largest C/N ratio was obtained in sand during 0-10 years of tillage. During the subsequent 10 years, the largest C/N ratio was obtained in silt. 【Conclusion】The SOC and TN contents and their stocks increased over tillage years in the oasis farmland of Xinjiang. The contents and its distribution proportions of OC and TN in different particles components were also changed. We found that the contents of OC and TN in silt fraction were the largest components of the fixed SOC and TN in the study area.

Greenhouse Gas Emission During the Initial Years After Rice Paddy Conversion to Vegetable Cultivation

WU Lei,HE ZhiLong,TANG ShuiRong,WU Xian,ZHANG WenJu,HU RongGui

Scientia Agricultura Sinica. 2020, 53(24):  5050-5062.  doi:10.3864/j.issn.0578-1752.2020.24.008

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【Objective】In recent years, with the rapid development of social economy, the improvement of people’s living standards and shifting diets and the increasing demands of vegetables result in a considerable share of rice paddy fields conversion to vegetable production in China, thus influencing soil carbon and nitrogen cycling and associated greenhouse gas (GHG) emissions. Therefore, it is necessary to investigate the impacts of land-use conversion from rice into vegetable cultivation on methane (CH₄) and nitrous oxide (N₂O) emissions and their key regulating factors, particularly during initial period upon conversion. 【Method】In this study, six rice paddies subjected to long-term double-rice planting were chosen, and the half of them were converted into vegetable cultivation (Veg) and the remaining still for rice production (Rice), with three replicates of each treatment. The Veg and Rice were managed according to local practices. The fluxes of CH₄ and N₂O from the rice paddy and converted vegetable fields were measured with static chambers from December 2012 to December 2015, so as to investigate the characteristics and inter-annual variation of CH₄ and N₂O emissions and to identify the key factors regulating the two GHGs during the initial period upon conversion. 【Result】Rice paddy acted as an important source of CH₄, and CH₄ emission was significantly lower in the first year (183.91 kg CH₄-C·hm^-2?a^-1) relative to the later two years (241.56-371.50 kg CH₄-C·hm^-2?a^-1), mainly attributed to enhanced precipitation increasing soil water content during the latter two years. Conversion from rice to vegetable cultivation substantially reduced CH₄ emission from Veg by 83%-100% as compared to Rice over the study period. Annual CH₄ emissions from Veg were significantly higher in the first year (31.22 kg CH₄-C·hm^-2) relative to any later years (0.45-0.89 kg CH₄-C·hm^-2), suggesting that this land-use conversion had strong legacy effect on CH₄ emission. Paddy soil acted as a minor source of N₂O (1.35-3.49 kg N₂O-N·hm^-2?a^-1). Rice conversion to vegetable cultivation led to substantial N₂O emission, particularly in the first year during which the cumulative emissions were significantly larger (95.12 kg N₂O-N·hm^-2) than that in the second (38.28 kg N·hm^-2) and third year (40.07 kg N₂O-N·hm^-2). N₂O fluxes from Veg were significantly and positively related to soil heterotrophic respiration rates (R_h), and the dependence of N₂O fluxes on R_h was greater in the first year relative to the subsequent two years. These results suggested that soil organic matter mineralization contributed to N₂O emissions during the first year upon land-use conversion from rice to vegetable production. Land-use conversion from rice to vegetable cultivation significantly increased the global warming potential (GWP) of Veg by 390% and 98% in the first and second year, respectively, relative to Rice, primarily due to the increased GWP of N₂O emission far outweighing the decreased GWP of CH₄ emission. In contrast, the GWP of rice (14.84±1.39 Mg CO₂-eq·hm^-2) was similar to that of Veg (16.72±3.25 Mg CO₂-eq·hm^-2) in the third year after conversion, due to the decreased GWP of CH₄ emission fully offsetting the increased GWP of N₂O emission. These results suggested that land-use conversion from rice to vegetable cultivation had significant impacts on the GWP only at the initial stage upon conversion. 【Conclusion】Land-use conversion from rice to vegetable cultivation significantly decreased CH₄ while increasing N₂O emissions over the whole study period, and increased the GWP only in the first and second year upon conversion. Soil organic matter mineralization significantly contributed to increased N₂O emission from the converted vegetable field. This study suggested that soil GHG emissions in the first years upon conversion were the most important, therefore, which should be considered when evaluating the environmental consequences of land-use conversion. This study also helped us develop effective options to alleviate the effects of land-use conversion on GHG emissions, and for sustainable agricultural production and GHG mitigation.

Research on Spatial and Temporal Difference of Agricultural Carbon Emission Efficiency and Its Influencing Factors in Hubei Province

TIAN Yun,WANG MengChen

Scientia Agricultura Sinica. 2020, 53(24):  5063-5072.  doi:10.3864/j.issn.0578-1752.2020.24.009

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【Objective】As a traditional agricultural province, Hubei Province has a heavy dependence on agricultural inputs such as chemical fertilizers and pesticides, which has objectively led to its relatively high carbon emissions in the agricultural production. Clarifying its agricultural carbon emission efficiency and influencing factors could provide necessary references and policy implications for the practical promotion of agricultural low-carbon production in Hubei Province. 【Method】The DEA-Malmquist decomposition method was employed to effectively measure the agricultural carbon emission efficiency of Hubei Province, and its temporal and spatial characteristics were analyzed. On this basis, Tobit model was adopted to explore the key factors affecting the change of its carbon emission efficiency. 【Result】Since 2011, the agricultural carbon emission efficiency of Hubei has been overall increasing but with certain interannual fluctuations, and the average annual growth rate was 2.9%. From the perspective of driving sources, its enhancement mainly depended on the progress of frontier technology rather than technical efficiency improvement. Further decomposition of technical efficiency showed that the pure technical efficiency had an obvious trend of deterioration, while the scale efficiency has been slightly improved. There were apparent differences in agricultural carbon emission efficiency among cities and prefectures in Hubei Province, among which Wuhan had the highest of 1.584 while Jingmen has the lowest of 0.803. Among to numerical differences, 15 regions could be divided into three different groups: high-speed growth, low-speed growth and decline. Frontier technological progress played a more obvious role in promoting the agricultural carbon emission efficiency for all regions, while the improvement of technical efficiency played a relatively small role. By decomposing the technical efficiency, it could be found that the influencing direction of pure technical efficiency and scale efficiency varied by regions, but the latter had a slightly greater effect than the former. Rural economic development level, urbanization level and rural electricity consumption had a significant and positive impact on agricultural carbon emission efficiency of Hubei Province, that is, under the premise that other conditions remained unchanged, the higher farmers’ net income per capita, the higher the urbanization level, or the greater the rural electricity consumption, the higher the agricultural carbon emission efficiency. However, the situation of agricultural industrial structure was exactly the opposite. Specifically, the higher proportion of the output value of planting industry was not conducive to the improvement of agricultural carbon emission efficiency. 【Conclusion】The agricultural carbon emission efficiency in Hubei Province was generally on the rise but with interannual fluctuations, and there were great differences among cities and prefectures. Whether it was Hubei Province or cities and prefectures, the enhancement of agricultural carbon emission efficiency depended more on frontier technological progress than technical efficiency improvement, which also required us not only to pay attention to the research and development of new technologies but to strengthen the rational use of various technologies in the process of promoting agricultural low-carbon production in Hubei Province. Considering the realistic situation that rural economic development, urbanization level, rural electricity consumption and agricultural industrial structure all had a significant impact on agricultural carbon emission efficiency, in practice, the enhancement of agricultural carbon emission efficiency could be effectively ensured by means of prospering rural economic development, improving urbanization level, ensuring rural electricity demand, optimizing agricultural industrial structure, improving the legal system construction and institutional support, etc.

HORTICULTURE

Relationship Between LAC Gene Expression and Core Browning of Yali Pear

WANG ZiYu,ZHANG YinYin,LI YueYuan,LI Ling,YOU LingLing,LI XiaoYan,JIN Zhao,YAN ShiJie

Scientia Agricultura Sinica. 2020, 53(24):  5073-5080.  doi:10.3864/j.issn.0578-1752.2020.24.010

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【Objective】 The aim of this study was to explore the relationship between LAC gene expression patterns and core browning of Yali pear under different maturity and cooling storage methods, so as to provide a theoretical basis for further analysis of the mechanism of Yali core browning. 【Method】 Using Yali pear as the material, the Yali pears of different maturity levels (early harvesting, middle harvesting and late harvesting) were treated with rapid cooling and slow cooling to observe the core of the pear during storage. For browning situation, the activity of LAC and the relative expression of LAC gene were determined, and the role of LAC gene in the browning process of Yali pear core was studied. 【Result】 When the Yali pear samples were refrigerated for 60 days, the late-harvested pears browned, and the browning index of late-harvested pears gradually decreased was 0.32, which was 2.56 times the rapid temperature drop during the same period; after 90 days of storage, the middle-harvested browning index decreased slowly, and the index was 0.24, and the browning index of the medium mining was only 0.01. During the storage period, the LAC activity of each treatment group increased gradually and then decreased. The late harvested fruits showed a peak activity and browning occurred after 60 days of storage. The peak of LAC activity of early harvesting and middle harvesting Yali pears appeared in 90 days, and the degree of browning was lower than that of late harvesting Yali. During the storage period, the activity of slow cooling LAC was higher than that of rapid cooling. The LAC14 and LAC7 gene expressions of Yali pears increased first and then decreased. LAC6 realized a change trend of first decline, then increased and then decreased; the expression of LAC14 and LAC7 genes reached the highest when the middle and late harvested pears were stored for 60 days. 【Conclusion】 Compared with slow cooling, rapid cooling treatment reduced the browning of Yali fruit core. During the whole storage period, the activity of LAC showed a trend of rising first, then falling, and then rising again, and the order of activity at its peak was: late harvesting>medium harvesting>early harvesting, which was consistent with the browning trend of the core. LAC was in the Yali fruit Up-regulated expression during heart browning. The rapid cooling was slower than the slow cooling, and the expression of LAC7, LAC14 and LAC6 genes was lower. A timely harvesting with the rapid cooling could inhibit the up-regulated expression of LAC and reduce the browning of Yali pear core.

Effect of UV-C on the Browning of Fresh-Cut Huangguan Pear

CHEN Chen,JIANG AiLi,LIU ChengHui,ZHAO QiQi,ZHANG YanHui,HU WenZhong

Scientia Agricultura Sinica. 2020, 53(24):  5081-5090.  doi:10.3864/j.issn.0578-1752.2020.24.011

Abstract ( 343 )   HTML ( 18 )   PDF (1151KB) ( 332 )   Save

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【Objective】In order to provide an experimental basis for the preservation of fresh-cut pears and enlarging the application of UV-C, the effects of pre-processing and post-processing UV-C treatment on the browning inhibition of fresh-cut pears and its possible physiology mechanism were studied.【Method】Fresh-cut pears were separately treated by pre-processing and post-processing UV-C (254 nm) for 5 min, and then the physiological parameters were evaluated, including browning degree (BI), the activities of polyphenol oxidase (PPO), phenylalnine ammonialyase (PAL), total phenolic content (TPC), H₂O₂ and MDA content, the activities of antioxidant enzymes (superoxide dismutase, SOD, catalase CAT, ascorbate peroxidase APX, glutathione reductase GR) and non-enzymatic antioxidant capacities (DPPH, ABTS radical scavenging activities and reducing power).【Result】The results showed that both pre-processing and post-processing UV-C treatment could effectively delay the browning of fresh-cut pears. Post-processing UV-C treatment exhibited better browning controlling effect than that of pre-processing UV-C treatment. Both two kinds of UV-C treatments could improve the PPO activity, reduce the PAL activity and TPC, as well as increase the enzymatic and non-enzymatic antioxidant activities of fresh-cut pears and decrease the accumulation of H₂O₂ and MDA. Statistical analysis indicated that BI were significantly negatively correlated with CAT, APX and GR activities, and non-enzymatic capacities (P<0.01). While there was a positive correlation between BI and H₂O₂, MDA content (P<0.01).【Conclusion】These findings suggested that UV-C treatment delayed browning of fresh-cut pears during storage mainly through improving the antioxidant defense system. Post-processing UV-C treated fresh-cut pears possessed higher antioxidant activity than that of pre-processing treated samples, therefore, which exhibited better browning controlling effect.

Cloning and Function Characterization of Chalcone Synthase Gene AgCHS1 in Ampelopsis grossedentata

XU Ming,LIN ShiQiang,NI DongXin,YI HenJie,LIU JiangHong,YANG ZhiJian,ZHENG JinGui

Scientia Agricultura Sinica. 2020, 53(24):  5091-5103.  doi:10.3864/j.issn.0578-1752.2020.24.012

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【Objective】Chalcone synthase(CHS) is the first key enzyme in the biosynthesis of plant flavonoids. In this study, the AgCHS1 was cloned from Ampelopsis grossedentata, the gene sequence and tissue specific expression were analyzed, and the gene function was characterized via in vitro enzymatic activity assay and transformation of N. tabacum, aiming to provide a theoretical basis for further elucidating the flavonoid accumulation of the A. grossedentata. 【Method】The primers were designed according to the transcriptome of A. grossedentata and the leaf cDNA and genomic DNA were used as templates to amplify the AgCHS1 with PCR. The sequence features were analyzed with bioinformatic methods. The MEGA6 and DNAMAN were used for multiple sequences alignment and phylogenetic tree construction. The AgCHS1 recombinant protein was obtained via prokaryotic expression, and the enzymatic activities of the protein on substrates were assayed by identifying the catalytic products with HPLC-MS. The expression of AgCHS1 in different organs was quantified by qRT-PCR, and the content of total flavonoids was determined by Al(NO₃)₃ colorimetric method. The overexpression vector was constructed and transformed to N. tabacum by using the leaf disk method. The transgenic plants were screened and the contents of anthocyanin and flavonol in the petal of T₂ strain were measured.【Result】The ORF of AgCHS1 had a length of 1 182 bp and encoded 393 amino acids. The genomic DNA spanned 1 315 bp, containing 2 exons and 1 intron. The bioinformatic analysis showed that AgCHS1 was a stable hydrophilic protein. The results of sequence alignments indicated that the AgCHS1 possessed the signature sequence and enzymatic site residues of the chalcone synthase gene superfamily, including the binding site of malonyl-CoA and triple active center, which share a high similarity with the CHS proteins of other species. The phylogenetic analysis showed that AgCHS1 had a close relationship with and was in the same clade with the CHSs of Vitis vinifera and Vitis amurensis. The results of fluorescent qPCR showed that AgCHS1 expressed highest in the mature leaf and the flower, and lowest in the old leaf. There was a positive correlation between the expression level of AgCHS1 gene and the content of total flavonoids in different organs of A.grossedentata. The in vitro enzymatic analysis showed that the recombinant AgCHS1 was able to catalyze the substrates p-coumaroyl-CoA and CoA malonyl-CoA to produce naringenin, demonstrating the activity of chalcone synthase. 5 transgenic N. tabacum lines were obtained, 2 of which showed a significant deeper leaf color. The anthocyanin contented of the transgenic lines OE3 and OE4 increased by 56.6% and 25.3%, respectively, compared to the control. The flavonol content of line OE3 showed no significant difference, which of the line OE4 were increased by 39.1%.【Conclusion】The AgCHS1 was the key enzyme in the flavonoid biosynthesis of A. grossedentata, and the overexpression of AgCHS1 increased the contents of anthocyanin and flavonol in transgenic plants.

FOOD SCIENCE AND ENGINEERING

Antibacterial Mechanism of Cold Plasma Against Listeria monocytogenes

DOU Yong,YAO MiaoAi,LÜ HuaiZhong,HU PeiHong,DONG Jing

Scientia Agricultura Sinica. 2020, 53(24):  5104-5114.  doi:10.3864/j.issn.0578-1752.2020.24.013

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【Objective】 As a novel non-thermal sterilization technology, the cold plasma has been widely applied in food industry. The aim of this study was to explore the antibacteral mechanism of cold plasma against bacteria at cytomembrane level, thus providing a foundation for the application of the cold plasma in food industry. 【Method】 In this study, the Listeria monocytogenes (LM) was selected as the test strain. After cold plasma treatment, the changes of morphology and intracellular materials were detected to reveal the integrity of LM cytomembranes. The changes of cell membrane fatty acid content and type, as well as the 8-aniline-1-naphthalene sulfonic acid (ANS) fluorescence intensity were also measured to detect the fluidity change of cytomembranes after cold plasma treatment. The changes of cytomembrane permeabilization were detected by propidium iodide (PI) fluorescence, conductivity and β-galactosidase activity. Finally, in order to demonstrate the oxidative damage of LM cytomembrane caused by cold plasma treatment, the changes of reactive oxygen species (ROS) and reactive oxygen species related genes were detected as well. 【Result】After cold plasma treatment, damaged and deformed structures were observed on the surface of the LM cytomembrane, the contents of proteins and DNA were decreased by 68 mg?mL ^-1 and 14 μg?mL ^-1, respectively, which showed that cold plasma destroyed the integrity of cytomembrane. After cold plasma treatment, the content of unsaturated fatty acids in LM cytomembrane increased from 40.17% to 53.91%, along with the decrease of saturated fatty acids from 53.68% to 41.57%. The ANS fluorescence intensity decreased from 8.99 to 3.73, indicating the increase of cytomembrane fluidity caused by cold plasma action. In addition, cold plasma treatment resulted in the penetration of PI and reacted with DNA to emit red fluorescence. By analyzing the changes of β-galactosidase activity and conductivity (increased from 0.15 mS?cm ^-1 to 0.33 mS?cm^-1), it was concluded that the cold plasma enhanced the permeabilization of LM cytomembrane. The changes of ROS fluorescence intensity indicated that cold plasma stimulated the generation of ROS on cytomembrane, thus exerting oxidative damage to cytomembrane. Finally, the results of qRT-PCR showed that cold plasma down-regulated the expression of perR and recA genes by 43.29% and 52.71%, while up-regulated the expression of sigB gene by 89.42%, which disclosed the regulatory mechanism of oxidative stress in microorganism at genic level.【Conclusion】Through the action on cell membrane of the active groups of cold plasma, the viability of LM was inhibited and eventually died.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

Association of Rumen Histomorphology of Sheep with Different Feed Efficiencies

ZHANG DeYin,ZHANG XiaoXue,LI FaDi,LI Chong,LI GuoZe,ZHANG YuKun,LI XiaoLong,SONG QiZhi,ZHAO Yuan,LIU XiaoQing,MA LiangQiang,WANG WeiMin

Scientia Agricultura Sinica. 2020, 53(24):  5115-5124.  doi:10.3864/j.issn.0578-1752.2020.24.014

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【Objective】The individual pens was used to determine individual feed efficiency-related traits and rumen morphology indexes, and the association of feed efficiency of sheep and rumen histomorphology was discussed, so as to provide fundamental data for analyzing the influencing factors of sheep feed efficiency traits. 【Method】One hundred and eighty-seven Hu lambs with the similar birthday age, good growth and available pedigrees were selected randomly, and all lambs were transferred to the housed indoors in individual pens after weaning at 56 days, the lambs were subjected to a 14 days adaptation period. The pre-test period was 10 days and the experimental period was 100 days, during which all lambs were fed pellet feed and had free access to food and fresh drinking water. Lambs were weighed before feeding in the morning at 80 and 100 days, and feed intake (FI) were measured during 80-180 days. Average daily gain (ADG), metabolic body weight (MBW), feed conversion rate (FCR) and residual feed intake (RFI) were calculated and the descriptive statistics were carried out. The lambs were slaughtered at 180 days, and rumen abdominal sac tissue was collected, and stored in 4% formaldehyde solution for making tissue sections. The length, width and muscle thickness of the rumen papilla were observed. Finally, correlation analysis and variance analysis were carried out for the traits related to feed efficiency.【Result】The variation coefficients of feed efficiency-related traits were all greater than 10%, and the difference of the individuals with the largest and smallest residual feed intake was 0.57 kg per day. Phenotypic correlation analysis of feed efficiency-related traits showed that RFI was very significantly positively correlated with FCR (r= 0.68) and FI (r= 0.48) (P<0.01), there was no significant correlation with initial body weight (r=0) final body weight (r= -0.01) and average daily gain (r= -0.02) (P>0.05). The correlation analysis between feed efficiency and rumen histomorphology was found that the length of the rumen papilla was significantly or very significantly positively correlated with average daily gain, feed intake, initial body weight and final body weight (P<0.05 or P<0.01), while there were no significant differences in length and width of rumen papilla (P>0.05). The feed intake and feed conversion rate of lambs in the High-RFI group were significantly higher than those in the Low-RFI group (P<0.01), and the muscle thickness was significantly higher than that of the Medium-RFI group (P<0.05). There were significantly or extremely significantly differences in residual feed intake, feed intake, ADG, initial body weight, final body weight and the length of rumen in different FCR groups (P<0.05 or P<0.01), there was no significant difference between the rumen muscular thickness and the width of rumen papilla (P>0.05). Among them, the residual feed intake, feed intake, ADG, initial body weight and final body weight of the lambs in the High-FCR group were significantly or extremely significantly higher than those in the Low-FCR group (P<0.05 or P<0.01), The length of the rumen papilla in the medium-FCR group was significantly longer than that in the Low-FCR group (P<0.05). There were significant or extremely significant differences in the above indexes of the lambs in different FI groups (P<0.05 or P<0.01), and the residual feed intake, feed conversion rate, ADG, initial body weight, final body weight, muscle thickness and the length of the rumen papilla in the High-FI group were significantly or extremely significantly higher than those in the Low-FI group (P<0.05 or P<0.01). There were significant or extremely significant differences in feed intake, feed conversion rate, initial body weight, final body weight and muscle thickness in different ADG groups (P<0.05 or P<0.01), there was no significant difference between the length and the width of the rumen papilla (P>0.05), the feed intake, residual feed intake, initial body weight, final body weight and muscle thickness of the High-ADG group were significantly or extremely significantly higher than those of the Low-ADG group, and the feed conversion rate was significantly lower than that of the Low-ADG group.【Conclusion】There was a significant positive correlation between residual feed intake and the traits related to feed efficiency such as feed intake and feed conversion rate, indicating that it could be used as a potential index to measure feed efficiency. There was no significant correlation between the residual feed intake and feed conversion rate and the rumen histopathology. Feed intake and average daily gain were significantly positively correlated with the length of the rumen papilla and the muscular thickness, indicating that the morphology of the rumen tissue had significant effects on feed intake and body weight gain. However, the mechanism of action remains to be further studied.

Optimized Promoter Regulating of Duck Tembusu Virus E Protein Expression Delivered by a Vectored Duck Enteritis Virus in vitro

CHEN Liu,NI Zheng,YU Bin,HUA JiongGang,YE WeiCheng,YUN Tao,LIU KeShu,ZHU YinChu,ZHANG Cun

Scientia Agricultura Sinica. 2020, 53(24):  5125-5134.  doi:10.3864/j.issn.0578-1752.2020.24.015

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【Background】 Duck enteritis virus (DEV) and duck Tembusu virus (DTMUV) are considered to be two of the important viruses that infected ducks. DEV is classified into the family Herpesviridae, which has the characterization of live viral vector. 【Objective】In our previous study, a recombinant DEV delivering optimized DTMUV E451 gene (E451-dk) referring to duck’s codon usage bias has been selected. In this study, the promoter regulating E451-dk (Es in short) in rDEV-EF1 was also evaluated for enhancing E451-dk expression level. 【Method】 The transfer vector pEP-BGH-pro-Es were constructed by separately substituted pCMV (cytomegalovirus major immediate-early promoter) on the vector pEP-BGH-Es with pCAG (human cytomegalovirus enhancer and chicken-actin promoter), pSV40 (the simian virus 40 promotor), pRSV(Rous sarcoma virus (RSV) promoter), pgB（MDV）(marek's disease virus (MDV) gB gene promoter) and p1.8k（MDV）(MDV 1.8k gene promoter). The recombinant DEV BAC clone pDEV-pro-Es carrying pro-Es genes were generated by two-step Red E/T recombination in E. coli. pDEV-pro-Es were constructed by inserting pro-Es expression cassette between DEV US7 and US8 genes on the infectious clone of DEV (pDEV-EF1). The recombinant virus rDEV-pro-Es (rDEV-pCAG-Es, rDEV-pSV40-Es, rDEV-pRSV-Es, rDEV-pgB(MDV)-Es and rDEV-p1.8k(MDV)-Es) were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. The plaque size and expression of DTMUV Es in recombinant virus-infected CEFs were analyzed. 【Result】 All viruses were successfully rescued from CEFs. Western blot analysis showed that the expression level of Es in rDEV-pRSV-Es -infected cells was increased 169.12% compared to that of rDEV-Es -infected cells. 【Conclusion】pRSV was the highest effective promoter chosen in this study which regulating Es expression on recombinant DEV genome backbone. These studies laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.