Loading...

Table of Content

    16 September 2021, Volume 54 Issue 18
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Transforming and Upgrading Off-Season Breeding in Hainan Through Molecular Plant Breeding
    ZHANG Xingping,QIAN Qian,ZHANG JiaNan,DENG XingWang,WAN JianMin,XU Yunbi
    Scientia Agricultura Sinica. 2021, 54(18):  3789-3804.  doi:10.3864/j.issn.0578-1752.2021.18.001
    Abstract ( 1189 )   HTML ( 140 )   PDF (659KB) ( 951 )   Save
    Figures and Tables | References | Related Articles | Metrics

    Generation advancement at non-target environments is a cost-effective and efficient approach to accelerating plant breeding. Hainan, as a southernmost province of China where crops can grow all year round, has been used as a core off-season breeding station, largely for seed increasing and an annually added crop generation advancement, while its favorable climate and environments are not fully exploited. Molecular biotechnologies such as marker-assisted selection, when integrated with other modern breeding technologies, will facilitate the transformation and upgrading of the Hainan off-season breeding. The transition will be made from a simple generation advancement to a full pipeline breeding including germplasm introduction and evaluation, selection, purity testing, germplasm exchange and variety right protection, and from one added winter breeding season to breeding all year round. Geographical and ecological advantages of Hainan as an off-season breeding station should be transitioned into a breeding pipeline by integration of generation advancement with functional breeding system to accelerate breeding procedures, improve breeding efficiency and promote advanced breeding industry. Considering the geographical and ecological advantages of Hainan and current status of off-season breeding, following issues are discussed in this article on transformation and upgrading of Hainan off-season breeding enterprise: necessity, possibility, challenges and opportunities. Such transition depends on the change of mind-set on non-target environment selection, favorable government policy regulation, breeding platform development, biosafety containment, well-established variety right protection, and resource-sharing and exchanging regulation. Breeding theories required for the transition include quantitative and population genetics, genotype by environment interaction, and molecular design and big- data management. Molecular design should be implemented at both macro-scale involving individuals, populations and species and micro-scale involving genes, metabolisms and networks. The breeding platform needs to be established for high-throughput precision phenotyping, envirotyping, information management and networking, and decision support system. As a key support technology for molecular breeding, genotyping by target sequencing and liquid chip (GBTS-LC) has been developed, by which GenoPlexs can be used to multiplex up to 5 000 target primers while GenoBaits can capture in solution up to 40K target loci each with multiple SNPs. Compared to other genotyping platforms, GBTS-LC has several advantages to make it a replacement of solid SNP chips, including wide platform suitability, high marker flexibility, high genotyping efficiency, easy information accumulation, less-demanding support system, wide application fields. For breeding in Hainan but testing across China, an integrative breeding system can be established with high-efficient breeding facilities supported by speeding breeding, gene transfer, genome editing, DH technology, and genomic selection. Other proposals are also suggested, including establishment of open-source breeding programs for resource-sharing, development of methodologies, technologies and platforms, implementation of germplasm introduction, monitoring and evaluation, construction of germplasm fingerprinting, and intensification of variety right protection, seed quality ensurance and see purity control. It is expected that this article will stimulate public awareness and relevant government policy development for the transformation and upgrading of breeding in Hainan, and thus enhance the scientific and technological progress and advancement of breeding and seed industry as a whole.

    Generation of ospin9 Mutants in Rice by CRISPR/Cas9 Genome Editing Technology
    WU ShiYang,YANG XiaoYi,ZHANG YanWen,HOU DianYun,XU HuaWei
    Scientia Agricultura Sinica. 2021, 54(18):  3805-3817.  doi:10.3864/j.issn.0578-1752.2021.18.002
    Abstract ( 763 )   HTML ( 69 )   PDF (2333KB) ( 485 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】Auxin efflux protein family PIN-FORMED (PIN) is a key protein family in controlling polar auxin transport (PAT). OsPIN9 is one of the monocot-specific PIN genes in rice, while its biological function still needs to be further elucidated. In this study, OsPIN9 was edited and ospin9 homozygous mutants were obtained using CRISPR/Cas9 genome editing technology. The resultant ospin9 mutant lines could provide a basis for further research on the function of OsPIN9.【Method】The specific target sequence was designed according to OsPIN9 genome sequence and OsPIN9 genome editing vector was constructed. Nippobare (Oryza sativa japonica) was used as the material and the hygromycin-resistant rice was obtained by Agrobacterium-mediated transformation. The positive transgenic lines were screened by PCR. The mutation sites were confirmed by the combination of PCR and subsequent analysis of sequencing results, the homozygous mutants were obtained and the difference of amino acid sequence and tertiary structure of OsPIN9 protein was analyzed between WT and ospin9 mutants. The expression of OsPINs genes in mutant roots was performed by quantitative real-time PCR (qRT-PCR), and the phenotype of ospin9 mutants was analyzed at the seedling stage. The effects of 1-naphthaleneacetic acid (NAA) treatment on seedling development were also analyzed under 0.05 μmol·L -1 NAA for 7 d.【Result】The target site sequence was designed based on the sequence of exon1 of OsPIN9 and, subsequently, the OsPIN9 genome editing recombinant vector was constructed. A total of 18 independent transgenic lines were obtained by transformation. Sequencing analysis revealed that three different mutation types were present in 7 T0 generation lines, including 3 lines with T insertion, 3 lines with G insertion and 1 line with C insertion, and all the mutation sites happened at the 18 th base of the target sequence. Two homozygous mutation lines were further identified in the T1 generation. BLAST analysis showed that the two types of OsPIN9 mutations caused frame-shift mutation and premature termination of translation, and the mutation protein was shortened from 426 aa in WT to 172 aa, thus leading to the complete disappearance of the transmembrane helices. qPCR analysis indicated that the transcription abundance of OsPIN9 significantly decreased in ospin9 mutants compared with WT, OsPIN1a and OsPIN5b were up-regulated, while OsPIN5a was down-regulated in ospin9 mutants. Both the shoot height and the number of adventitious roots of ospin9 mutants were reduced significantly than that of WT, while its root length was comparable to that of WT. The plant growth was inhibited and the adventitious root number was still less than that of WT under NAA treatment, but no significant difference was found between ospin9 mutants and WT plants. 【Conclusion】 Auxin efflux carrier OsPIN9 was directionally edited by using CRISPR/Cas9 technology, and two transgene-free homozygous ospin9 mutants were obtained. The mutation of OsPIN9 affected the expression level of other OsPINs genes, the shoot and root development were inhibited in ospin9 mutants at the seedling stage and NAA treatment partially rescued the development of adventitious roots in ospin9 mutants.

    Evaluation and Screening of Nitrogen Efficiency of Wheat Germplasm Resources at Mature Stage
    ZHAO Rui,ZHANG XuHui,ZHANG ChengYang,GUO JingLei,WANG Yu,LI HongXia
    Scientia Agricultura Sinica. 2021, 54(18):  3818-3833.  doi:10.3864/j.issn.0578-1752.2021.18.003
    Abstract ( 505 )   HTML ( 90 )   PDF (3812KB) ( 466 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】 In order to provide theoretical foundation for the study of physiological mechanism of nitrogen efficiency in wheat and material basis for the breeding of nitrogen efficiency varieties, the evaluation method for nitrogen efficiency of wheat at mature stage was established to explore and screen nitrogen efficiency germplasm resources. 【Method】 In the field experiment, 108 different genotype wheat varieties were used for the treatment of 4 nitrogen application(0, 180, 240, 360 kg·hm-2). Eleven indexes of major agronomic traits, including plant height, spike length, flag leaf length, flag leaf width, stem diameter, fertile spikelet number, grain number per spike, thousand kernel weight, grain length, grain width and grain weight per spike were measured for two consecutive years. The nitrogen tolerance and genotype difference of wheat varieties were assessed by the method of Fuzzy membership function, principal component analysis and cluster analysis. 【Result】 The results of two-year data show that plant height, spike length, flag leaf length, flag leaf width, stem diameter, fertile spikelet number, spike grain number and grain weight per spike under low nitrogen stress were all inhibited to varying degrees, among which flag leaf length was the most sensitive to nitrogen stress. Four principal components were extracted by principal component analysis, and the contribution rate was 39.766%, 16.661%, 9.361% and 9.275%, respectively. The cumulative contribution rate reached 75.064%. According to the comprehensive evaluation D value of low nitrogen tolerance, the tested wheat varieties were divided into 5 types: strong low nitrogen tolerance, low nitrogen tolerance, intermediate, low nitrogen light sensitive and low nitrogen sensitive. Five low-nitrogen tolerant wheats including Wenmai19, Xinong 529, Shi 4185, Longmai 212 and Fengkang 2 and two strong low-nitrogen tolerant wheats (Zhongmai 875 and Xinong 158) were selected. Different from low nitrogen stress, high nitrogen stress only inhibited 5 traits including stem diameter, thousand kernel weight, grain length, grain width and grain weight per spike, and 6 traits including plant height, spike length, flag leaf length, flag leaf width, fertile spikelet number and spike grain number increased with the increase of nitrogen application. Principal component analysis extracts 4 principal components, the contribution rates are 31.348%, 20.387%, 12.452% and 9.850%, respectively, and the cumulative contribution rate is 74.037%. Based on the D value of comprehensive evaluation of high nitrogen tolerance, the tested wheat varieties were divided into four categories: high nitrogen tolerance type, intermediate type, high nitrogen light sensitive type and high nitrogen sensitive type. Nine wheat varieties with high nitrogen tolerance were identified, including Lankao Aizao 8, Liangxing 99, Nongda 179, Yunnong 9901, Lankao 926 and Zhengnong 46. Based on the D value of comprehensive evaluation of nitrogen and grain yield, 108 wheat varieties were divided into four nitrogen efficiency types, dual-efficiency type (Xinong 158 and Longmai 212 et al.), low-nitrogen efficiency type (Xinong 585 and Shi 4185 et al.), high-nitrogen efficiency type (Changfeng 1 and Zhongsimai 10 et al.) and dual-inefficiency type (Jinfeng 7183 and Fanmai 5 et al.). 【Conclusion】 Nitrogen supply level has a significant influence on wheat yield-related traits and indexes. The nitrogen efficiency of wheat germplasm resources at maturity might be accurately evaluated combined with three evaluation methods, based on the difference of nitrogen absorption and utilization efficiency between wheat germplasm.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Combined Effects of Shade and Waterlogging on Yield and Photosynthetic Characteristics of Summer Maize
    YU WeiZhen,ZHANG XiaoChi,HU Juan,SHAO JingYi,LIU Peng,ZHAO Bin,REN BaiZhao
    Scientia Agricultura Sinica. 2021, 54(18):  3834-3846.  doi:10.3864/j.issn.0578-1752.2021.18.004
    Abstract ( 484 )   HTML ( 4 )   PDF (649KB) ( 192 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】 The purpose of this study was to investigate the physiological mechanism of shade and waterlogging combined treatment on photosynthetic characteristics of summer maize during different growth stages. 【Method】 DH605 was selected as the test variety. Shading treatment, waterlogging treatment and shading + waterlogging treatment were set in different growth periods under field conditions, including waterlogging at the third leaf stage (V3-W), the sixth leaf stage (V6-W), and flowering stage (VT-W); shading at V3 (V3-S), V6 (V6-S), and VT (VT-S); waterlogging and shading combined treatment at V3 (V3-S-W), V6 (V6-S-W), and VT stage (VT-S-W). The effects of shading and waterlogging compound treatment on yield, photosynthetic characteristics and dry matter accumulation and distribution of summer maize were studied, and the differences of photosynthetic characteristics and yield between single treatment and compound treatment were compared. 【Result】 After shading and waterlogging, the LAI, relative chlorophyll content, net photosynthetic rate (Pn), Transpiration rate (Tr), and stomatal conductance (Gs) of summer maize were significantly decreased. Compared with CK, the Pn under V3-S-W, V6-S-W and VT-S-W was significantly decreased by 43.7%, 41.1% and 35.1%, respectively. Moreover, the net photosynthetic rate of the combined treatment of shading and waterlogging was decreased more than that of shading or waterlogging treatment. The combined treatment of shading and waterlogging resulted in the decrease of photosynthetic performance of summer maize, leading to the decrease of dry matter accumulation ability and grain distribution ratio of summer maize, and thus resulting in a significant decrease of maize yield. Compared with CK, the yield under V3-S-W, V6-S-W and VT-S-W were reduced by 32.5%, 28.3% and 24.7%, respectively. Compared with single shading or flooding treatment, the yield under V3-S-W was 26.3% and 13.1% lower than that under V3-S and V3-W, respectively. Compared with V6-S and V6-W, the grain yield under V6-S-W was decreased by 14.9% and 6.2%, respectively. Compared with VT-S and VT-W, the yield of VT-S-W was reduced by 2.9% and 15.0%, respectively. 【Conclusion】 Combined treatment with shading and waterlogging resulted in significantly reduced LAI, decreased relative chlorophyll content and inhibited photosynthetic performance of summer maize, which led to hindered accumulation and distribution of photosynthetic compounds in summer maize, and thus resulting in the decrease of maize yield. Combined treatment with shading and waterlogging at V3 stage had the greatest effect on photosynthetic characteristics and yield of summer maize, followed by V6 and VT stage. At the same time, the photosynthetic characteristics and yield decreased by combined treatment with shading and waterlogging were higher than either single treatment.

    The Possible Effects of Global Warming on Cropping Systems in China ⅫⅠ. Precipitation Limitation on Adjusting Maturity Cultivars of Spring Maize and Its Possible Influence on Yield in Three Provinces of Northeastern China
    LI E,ZHAO Jin,YE Qing,GAO JiQing,YANG XiaoGuang
    Scientia Agricultura Sinica. 2021, 54(18):  3847-3859.  doi:10.3864/j.issn.0578-1752.2021.18.005
    Abstract ( 673 )   HTML ( 2 )   PDF (2289KB) ( 187 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】Under the background of climate change, this study focused on the changing precipitation and its possible influence on yield in the sensitive areas with adjusting spring maize maturity cultivars in the three provinces in Northeastern China, which could provide a scientific reference for the local cultivars’ adjustment of spring maize. 【Method】 Taking 1985 as the time node, the period of 1961-2017 was split into two sub-periods (1961-1985 and 1986-2017) in this study. Based on the observed meteorological data in 24 meteorological stations and observed maize growth stages in 16 agrometeorological experimental stations in the sensitive area, the changing characteristics of water conditions in the maize growth stages was analyzed, and the influence of precipitation on the climatic potential yield was adjusted by the ‘correction step by step’ method. 【Result】(1)During 1961-2017, the actual sowing dates in the sensitive area showed an advanced trend, while the maturity dates showed a delayed trend, which indicated the adjustment of maturity cultivars in the actual practice and the actual growth stage was prolonged. (2)The adjustment of maturity cultivars in sensitive areas increased the water demand in the early (sowing to jointing) and late (flowering to maturity) stages, but decreased the water demand in the middle (jointing to flowering) stage. Meanwhile, the effective precipitation in the early stage showed an increasing trend, while a decreasing trend was found in the middle and late stages. (3)After the adjustment of maturity cultivars, the satisfaction rate of effective precipitation in the middle stage was the lowest. (4)With the adjustment of maturity cultivars, the climatic potential yield showed a decreasing trend in Kuandian and Tongyu stations in the south and west of area 5, where those middle-late-maturing cultivars were adjusted into late-maturing ones. However, the climatic potential yield showed an increasing trend and decreased in the north of area 1 where especial-early-maturing cultivars were adjusted into early-maturing cultivars, and area 3 where early-maturing cultivars were adjusted into middle-maturing cultivars. 【Conclusion】 Under the background of climate change, the effective precipitation satisfaction rate, K value in sensitive areas of Northeast China decreased in the middle and late growth stages, and the climatic potential yield decreased in the west and south of the study area, while increased in the east with high instability. Therefore, in the eastern, western, and southern parts of sensitive areas, more attention should be paid to the selection of mature cultivars of spring maize, and irrigation should be carried out in time in the middle and late growth stages of spring maize.

    PLANT PROTECTION
    Construction and Verification of Fusarium Head Blight Prediction Model in Haihe Plain Based on Boosted Regression Tree
    TAO Bu, QI YongZhi, QU Yun, CAO ZhiYan, ZHAO XuSheng, ZHEN WenChao
    Scientia Agricultura Sinica. 2021, 54(18):  3860-3870.  doi:10.3864/j.issn.0578-1752.2021.18.006
    Abstract ( 426 )   HTML ( 31 )   PDF (1008KB) ( 244 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Background】 Since 1995, Fusarium head blight (FHB) has gradually spread and risen from a secondary disease to a major disease in Haihe Plain, from sporadic occurrence to continuous occurrence, showing the characteristics of rapid outbreak, large area and heavy loss in epidemic years. To realize effective prevention and control of FHB, accurate forecasting technology is an important prerequisite for controlling the occurrence and development of FHB. 【Objective】According to the occurrence of FHB in Haihe Plain, the prediction model of FHB suitable for Haihe Plain was established to provide technical supports for scientific prevention and control of FHB.【Method】Based on the data about spike rate of FHB and meteorological factors of key growth stage of wheat in 21 counties of Haihe Plain from 2001 to 2016, the key meteorological factors which have significant influences on the FHB occurrence in Haihe Plain were screened by stepwise regression analysis, and the prediction models of FHB occurrence based on multiple linear regression model and boosted regression tree model were constructed, respectively.【Result】When the learning efficiency (lr) of the boosted regression tree model was 0.005 and the complexity (tc) of the tree was 6, the prediction deviation of the model was the lowest, and the residual standard error was 0.006311. Eight key meteorological factors, including MRH15, Rain-35, MRH-55, SD15, LT-65, MWS-55, MT-25 and DRain15, which had a significant impact on the occurrence of FHB in Haihe Plain, were screened out, and a multiple linear regression model with eight predictive variables was established (R2=0.8158, corrected R 2=0.8018, P<2.2×10 -16). Meanwhile, the importance of each key meteorological factor was evaluated by using the boosted regression tree model, with the values of 69.62%, 14.08%, 4.89%, 4.34%, 3.35%, 2.02%, 1.20% and 0.50%, respectively. According to the key predictive variables, the prediction model was further simplified, and a multiple linear regression model with four predictive variables was constructed (y=-19.45376+0.11689MRH15+0.17346Rain-35+0.04185SD15+0.26592MRH-55, R2=0.7575, corrected R 2=0.7468, P<2.2×10 -16). When the prediction variables was reduced from 8 to 4, the prediction accuracy of the multiple linear regression model decreased from 88.43% to 85.90%, but the prediction accuracy on the disease spike rate of the boosted regression tree model increased from 87.72% to 91.23%, which was verified by using the historical data of Anxin, Dingzhou and Guantao, etc in 2008, 2010 and 2012. The prediction accuracy on the disease spike rate of the multiple linear regression model and the boosted regression tree model changed from 87.53% to 87.42% and from 89.20% to 89.21%, respectively, but there was no significant difference between the multiple linear regression model and the boosted regression tree model, when they were verified with the historical data of Zhengding and Luancheng from 2001 to 2016. In a word, the prediction accuracy of multiple linear regression model showed a downward trend, while the prediction accuracy of boosted regression tree model showed an upward trend.【Conclusion】In this study, the boosted regression tree model with four predictive variables was constructed, with the prediction accuracy of 89.21%. At the same time, the disease spike rate predicted by the boosted regression tree model was basically consistent with the observed fluctuation trend, indicating that the boosted regression tree model had a good application prospect in the prediction of FHB in Haihe Plain.

    Analysis of Resistance Mechanism of CiNPR4 Transgenic Plants to Citrus Canker
    ZHANG JingYun,LIU YuNuo,WANG ZhaoHao,PENG AiHong,CHEN ShanChun,HE YongRui
    Scientia Agricultura Sinica. 2021, 54(18):  3871-3880.  doi:10.3864/j.issn.0578-1752.2021.18.007
    Abstract ( 333 )   HTML ( 25 )   PDF (1402KB) ( 251 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】Non-expressor of pathogenesis-related genes 1 (NPR1) is an important transcription factor in salicylic acid (SA)-mediated signal transduction of systemic acquired resistance and plays a key role in regulating plant disease resistance. The objective of this study is to evaluate the resistance of CiNPR4, a NPR1-like gene from Huanglongbing-tolerant ‘Jackson’ grapefruit (Citrus paradisi), to citrus canker and preliminarily explore the resistance mechanism of CiNPR4 transgenic plants to citrus canker.【Method】The fully mature leaves from Huanglongbing-resistant CiNPR4 transgenic citrus plants were selected to challenge the Xanthomonas citri subsp. citri (Xcc) pathogen via in vitro pin-prick inoculation. Then, the CiNPR4 transgenic plants with enhanced resistance to citrus canker were inoculated with Xcc in vitro by the pin-prick method and then counted the number of Xcc at 0, 1, 3, 5, 7 and 9 d after inoculation. The leaves of CiNPR4 transgenic plants with enhanced resistance to citrus canker were inoculated with Xcc by injection and collected at 0, 3 and 5 d after inoculation. The contents of SA and jasmonic acid (JA) in the leaves were measured. Meanwhile, the expression level of defense-related genes CsPR1 and CsPDF1.2 mediated by SA and JA, respectively, was analyzed by quantitative real-time PCR. According to the interaction network of CiNPR4 protein and TGA transcription factor, it is predicted that Ciclev10005080m and Ciclev10001081m can interact with CiNPR4, respectively, these two genes were analyzed by blastx on the website https://www.citrusgenomedb.org/ to predict the candidate proteins that interact with CiNPR4 in the sweet orange genome. Then, the cDNA sequences of CiNPR4 and candidate protein genes were cloned, and inserted into the pGBKT7 and pGADT7 plasmid to construct the bait and prey vectors by homologous recombination method, respectively. After that, the bait and prey plasmids were co-transformed into yeast strain Y2HGold for point-to-point yeast two-hybridization analysis.【Result】The disease resistance evaluation in vitro showed that overexpression of CiNPR4 alleviated the symptoms of citrus canker on transgenic citrus plants. Xcc grew slowly in the leaves of CiNPR4 transgenic plants with enhanced resistance to citrus canker during the entire observation period, and the number of Xcc in the transgenic plants was significantly lower than that of wild-type (WT) plants at 9 d after inoculation. The SA contents among the transgenic citrus lines and WT plants were not significantly different at 0 d. However, with the prolongation of Xcc induction time, the SA content in CiNPR4 transgenic plants significantly increased. The JA content in WT plants was significantly higher than that in CiNPR4 transgenic plants at 0 d after inoculation, after that, the JA content in CiNPR4 transgenic plants gradually increased and was significantly higher than that in WT plants at 5 d after Xcc induction. However, the SA and JA contents in WT plants were not significantly changed during the entire induction period. The expression level of CsPR1 was significantly increased in the transgenic plants with increased canker resistance than that in WT plants after Xcc induction for 3 d, and the expression level of CsPDF1.2 in the above-mentioned transgenic plants increased at 5 d after inoculation. However, WT plants exhibited no significantly different expression of CsPR1 after Xcc induction, but had an increased expression level of CsPDF1.2 at 5 d after inoculation, which was significantly higher in WT plants than that in the CiNPR4 plants. The interaction between CiNPR4 and CsTGA2 was confirmed by the yeast two-hybrid analysis.【Conclusion】These results indicated that CiNPR4 interacts with CsTGA2 to regulate the resistance of transgenic plants to citrus canker by promoting SA- and inhibiting JA-mediated defense response.

    Cloning, Expression and Functional Analysis of SeDuox from Spodoptera exigua
    FU ChaoRan, LI YaZi, WU Han, ZHAO Dan, GUO Wei, GUO XiaoChang
    Scientia Agricultura Sinica. 2021, 54(18):  3881-3891.  doi:10.3864/j.issn.0578-1752.2021.18.008
    Abstract ( 313 )   HTML ( 35 )   PDF (2222KB) ( 281 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】The production of reactive oxygen species (ROS) mediated by dual oxidase (Duox) is an important immune mechanism to regulate the dynamic balance of insect intestinal microorganisms. The objective of this study is to analyze the sequence characteristics and expression patterns of SeDuox from Spodoptera exigua, explore the effect of SeDuox silencing on the intestinal bacterial load of host by RNAi and analyze the immune response of SeDuox to Bacillus thuringiensis (Bt), so as to clarify the role of SeDuox in the intestinal immune regulation of insects, and provide new ideas and targets for the comprehensive control of S. exigua.【Method】The SeDuox was cloned by reverse transcription PCR (RT-PCR), and the sequence characteristics were analyzed by bioinformatics. The phylogenetic tree was constructed by ClustalX and MEGA_X software. The extramembrane fragment of SeDuox (91-1 767 bp) named SeDuox-OM was cloned by PCR. The Bacmid-SeDuox-OM was constructed using the Bac to Bac expression system and transfected into the Sf9 insect cells by liposome transfection to express SeDuox-OM. Real-time quantitative PCR (RT-qPCR) was used to analyze the expression level of SeDuox in different tissues (peritrophic membrane, hemolymph, fat body, Malpighian tubules, midgut, epidermis) and developmental stages (egg, 1st- to 5th-instar larvae, female pupa, male pupa). The 4th instar larvae were injected with dsSeDuox using RNAi technology for functional analysis, with dsGFP as control group. The efficiency of gene silence and intestinal bacterial load changes were detected at 48 h and 72 h after injection. In addition, Bt was fed to S. exigua larvae to detect relative expression of SeDuox and related immune genes by RT-qPCR.【Result】The length of open reading frame (ORF) of SeDuox is 4 497 bp, encoding 1 498 amino acids, and the deduced molecular weight of protein is 171.62 kD. SMART analysis showed that SeDuox domain is similar to that of Spodoptera litura, Spodoptera frugiperda, including peroxidase domain, nicotinamide adenine dinucleotide binding domain (NAD), N-terminal calcium binding domain (EFH), iron reductase binding domain (Ferric) and flavin adenie dinucleotide binding domain (FAD). TMHMM analysis showed that there are seven transmembrane regions. The transmembrane fragment named SeDuox-OM was successfully expressed about 80 kD recombinant protein in insect cells. The results of RT-qPCR showed that SeDuox was expressed in different tissues of the 4th instar larvae of S. exigua, and the expression level was higher in peritrophic membrane and midgut. It was expressed in each developmental stage, and highly expressed at the egg stage. Compared with dsGFP injection, dsSeDuox injection significantly reduced the expression of SeDuox, which was reduced by 62.08% and 74.94% at 48 h and 72 h, respectively, and the load of intestinal microbial in S. exigua larvae was significantly increased. Compared with the control group, the expression level of SeDuox was significantly increased at 48 h after being fed on Bt GS36. With the extension of infection time, the expression level of SeDuox was increased at first, then decreased and turned to stabilize finally.【Conclusion】SeDuox contains conserved domains, which is highly expressed in the midgut and perinogphic matrix. SeDuox plays an important role in the regulation of host intestinal immunity, it probably has synergistic effect with antimicrobial peptide gene against Bt infection.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Win-Win Strategy for National Food Security and Agricultural Double-Carbon Goals
    ZHANG WeiJian,CYAN ShengJi,CZHANG Jun,CJIANG Yu,CDENG Aixing
    Scientia Agricultura Sinica. 2021, 54(18):  3892-3902.  doi:10.3864/j.issn.0578-1752.2021.18.009
    Abstract ( 1543 )   HTML ( 80 )   PDF (436KB) ( 688 )   Save
    References | Related Articles | Metrics

    Food security is of vital importance for national safety, and carbon peaking and neutralization have become China’s long-term strategies. China is a large country in grain consumption, and more than 95% of China’s demand of grain and important agricultural products depend on domestic agricultural production. Meanwhile, the agriculture sector is one of the main sources of carbon emission and also the important pool of carbon sequestration. Given the great potential of carbon sequestration and emission mitigation, the agriculture sector can play a great role in achieving both national food security and double-carbon goals. Based on literature review and the authors’ research summary, therefore, the authors systematically discussed the inter-linkages and win-win strategy between food security and double-carbon goals. The results showed that there were a lot of tradeoffs and synergies between food security and double-carbon goals, especially between yield enhancement and carbon sequestration, yield enhancement and carbon emission mitigation, and carbon sequestration and emission mitigation, as well as between agricultural institutes and their supporting policies. The effects of the synergies were greater than those of the tradeoffs. Moreover, double-carbon goals of agricultural sectors and green high-quality development were highly consistent in their directions and also complementary in their targets. Research practices and technology demonstration further proved that agricultural double-carbon goal and national food security could achieve win-win situation through technological innovation and supporting policy creation. According to the new trends of China’s agriculture development, the carbon emission of crop production sector has almost reached its peak. Thus, the peak value and peaking time of agricultural carbon emission under the background of food security would depend on the requirements of animal husbandry development and the improvement of people's living standards. And the achievement of agricultural carbon neutral would mainly depend on the emission reduction of non-CO2 greenhouse gases, such as CH4, and the efforts of comprehensive carbon sequestration in agricultural ecosystem. Although agricultural carbon emission would reach to peak in the near future, the setting of carbon peak goal should not affect national food security and the improvement of people’s lives. However, agricultural carbon neutral is very difficult to achieve, both carbon sequestration and emission reduction should be implemented together, and the emission reduction of non-CO2 greenhouse gases, such as agricultural CH4, should be given a priority. In summary, it is a systematic project to achieve agricultural double-carbon goals under food security, which needs a great efforts and comprehensive support of scientific and technological innovation of agricultural carbon sequestration and emission reduction, the foundation of agricultural carbon monitoring and evaluation methods, and the creation of new coordinating mechanisms and supporting policies of government agencies related to agriculture. Our research can provide important references for national and local governments to set the goals and action plans of agricultural carbon peak and carbon neutral, as well as new ideas for scientific and technological innovation and supporting policy creation of agricultural carbon sequestration and emission reduction.

    Response of Wheat Yield and Protein Concentration to Soil Nitrate in Northern Wheat Production Region of China
    MA Yue,TIAN Yi,YUAN AiJing,WANG HaoLin,LI YongHua,HUANG TingMiao,HUANG Ning,LI Chao,DANG HaiYan,QIU WeiHong,HE Gang,WANG ZhaoHui,SHI Mei
    Scientia Agricultura Sinica. 2021, 54(18):  3903-3918.  doi:10.3864/j.issn.0578-1752.2021.18.010
    Abstract ( 337 )   HTML ( 32 )   PDF (735KB) ( 315 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】The changes of wheat grain yield and protein concentration caused by reduced nitrogen (N) fertilizer at different soil nitrate N residue levels were clarified, for the purpose of N fertilizer reduction and soil residual nitrate N decline in northern wheat production region of China.【Method】In our research, 43-site field experiments from 2018 to 2019 were conducted in northern wheat region of China, to investigate the effects of reduced N fertilizer on grain yield, protein concentration, yield components, and N uptake and utilization of wheat at different soil nitrate N residue levels. 【Result】The results showed that the recommended fertilizer application based on soil nitrate test (RF) could reduce N rates by 55 kg·hm-2, correspond 26% of farmers’ fertilizer application (FF). Compared with the FF, the grain yield (5 885 kg·hm-2) of RF was increased by 3.1%, while no significant difference was observed for grain protein concentration between the two treatments. For the minimum of grain yield was found to be 4 252 kg·hm-2 when the soil nitrate N residue was less than 55 kg·hm-2, and the maximum was 7 186 kg·hm-2 at the soil nitrate N residue level of 55-100 kg·hm-2. Higher soil nitrate N residue was not capable to increase the grain yield sustainability. For recommended fertilizer application based on soil nitrate test without N added (RF-N), the grain yield was significantly decreased with the corresponding soil nitrate N residue less than 100 kg·hm-2, while no obvious reduction for grain yield for the RF regardless of nitrate N residue levels in soils. The grain protein concentration reached the highest when the soil nitrate-N residue was higher than 300 kg·hm-2. The grain protein concentration was significantly decreased in RF-N, when the soil nitrate N residue was less than 200 kg·hm-2, but it was not influenced obviously by soil nitrate N residue in the RF treatment. With soil nitrate N residue ranging from 55 to 100 kg·hm-2, the grain protein concentration of FF and RF was 124.5 and 123.1 g·kg-1, respectively. For RF, the N fertilizer uptake efficiency and N partial factor productivity were 1.36 and 45.7 kg·kg-1, respectively; which were significantly increased by 61.5% and 57.1%, respectively, compared with the FF. 【Conclusion】For maintaining the higher grain yield and protein concentration of wheat, the residual nitrate N in 0-100 cm soil at harvest should be kept at 55-100 kg·hm-2 in northern wheat production region. Determination of reasonable N fertilizer rates, based on combination of target grain yield, grain protein concentration and soil nitrate testing, played important roles in achieving N fertilizer reduction and agricultural green production in wheat fields.

    Source Analysis and Control Strategies of Non-Point Source Pollution in Typical Agricultural Small Watershed: A Case Study of Danjiangkou Water Conservation Area
    GONG ShiFei,DING WuHan,JU XueHai,XIAO NengWu,YE QingSong,HUANG Jin,LI Hu
    Scientia Agricultura Sinica. 2021, 54(18):  3919-3931.  doi:10.3864/j.issn.0578-1752.2021.18.011
    Abstract ( 349 )   HTML ( 34 )   PDF (527KB) ( 193 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】The investigation and analysis of agricultural non-point source pollution status in typical small watershed of Danjiangkou Reservoir Area were conducted to parse the pollution load and pollution sources of non-point agricultural sources in this area, so as to provide the reference for formulating targeted prevention and promoting the green development of agriculture. 【Method】Field visits were carried out through questionnaire survey in the Tanjiawan watershed, and the pollution sources, such as planting, breeding and living, were classified in two villages. Meanwhile, the pollution load was estimated according to the export coefficient method approach and the equivalent standard pollution load method. 【Result】The results showed that: the actual load of agricultural non-point source pollution in Tanjiaban watershed decreased from 162.32 t in 2015 to 27.79 t in 2020, the total equivalent standard pollution load decreased from 62.44 m3 to 21.14 m3, and the dominant source of pollution changed from land utilization to livestock and poultry breeding. The total annual loads of TN, TP and COD of agricultural non-point source pollution in the watershed were 5.56, 0.86 and 21.37 t, respectively. The contribution of different pollution sources were found in the following order: livestock and poultry breeding > land utilization > rural life. TN, TP and COD load of pig breeding accounted for 50.91%, 64.20% and 46.66% of the total load of the watershed, respectively, making it the most important source of pollution in the region. TN was the most important non-point agricultural source in the watershed, and its equivalent standard pollution load accounted for 52.6% of the total load, followed by TP, with a pollution load rate of 40.7%, and the minimum equivalent standard pollution load rate of COD was 6.7%. According to the result of environmental pollution risk assessment, the alarm value of livestock and poultry manure load in Tanjiawan watershed was 0.489 and the risk level wasⅡ, indicating the environmental pollution was "slightly". There was still room for expansion of 10 815 pig’s equivalent on the basis of the current total amount of cultivation in the watershed. Since 2015, the intensity of non-point agricultural source pollution in Tanjiawan watershed has been reduced significantly. 【Conclusion】It was of great significance to keep a reasonable amount of livestock and poultry breeding, and to take some effective measures to promote the sustainable emission reduction of non-point agricultural source pollution in the typical watershed of the Danjiangkou Reservoir area.

    HORTICULTURE
    Identification of Candidate Genes for Waterlogging Tolerance in Apple Rootstock by Using SLAF-seq Technique
    SONG ChunHui,CHEN XiaoFei,WANG MeiGe,ZHENG XianBo,SONG ShangWei,JIAO Jian,WANG MiaoMiao,MA FengWang,BAI TuanHui
    Scientia Agricultura Sinica. 2021, 54(18):  3932-3944.  doi:10.3864/j.issn.0578-1752.2021.18.012
    Abstract ( 289 )   HTML ( 22 )   PDF (2317KB) ( 234 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Background】 Apple (Malus×domestica Borkh) is one of the most cultivated fruit crops in China. While apple trees frequently encounter waterlogging stress mainly due to excess rainfall and poor soil drainage, resulting in yellowing leaf, declined fruit quality and yield in summer and autumn. 【Objective】 The aim of this study was to identify waterlogging-tolerant genes of apple, so as to provide a basis for waterlogging tolerance molecular marker assisted breeding, high-quality and high-yield cultivation of apple.【Method】In this study, 50 waterlogging-tolerant plants and 50 waterlogging-sensitive plants were selected to construct two bulked DNA pools from the 495 F1 population, which were derived from a cross of the waterlogging-tolerant apple rootstock G41 and the waterlogging-tolerant M. sieverii (Ledeb) Roem. Specific-locus amplified fragment (SLAF) labels and single nucleotide polymorphism (SNP) markers were developed by SLAF-seq technique. The mapping and candidate gene prediction of waterlogging tolerance in apple were carried out by combining apple genome and genetic association analysis, and the relative expression of the candidate gene was also analyzed in different waterlogging tolerance plants under waterlogging stress.【Result】A total of 119 072 SLAF labels were obtained, of which, 11 133 were polymorphic between both parents. A total 16 237 071 SNPs were identified by sequence analysis, including 170 617 SNPs with polymorphic. By association analysis with Eudidean distance (ED) and SNP-index, a candidate locus was found to be strongly associated with waterlogging tolerance, which was a region of 1.94-3.25 Mb on apple chromosome 10. The associated region was 1.31 Mb that contained 120 genes, and functional annotation of the genes in this region revealed a gene MD10G1014500, alcohol dehydrogenase gene (ADH1) related to respiratory metabolism. The expression level of ADH1 gene in waterlogging tolerant plants was significantly higher than that in waterlogging sensitive plants at 1, 2, 4 and 6 days after waterlogging treatment. 【Conclusion】 One gene (MD10G1014500) was identified as a potential candidate gene involved waterlogging tolerance of apple, which located in a 1.94-3.25 Mb interval on chromosome 10. These findings laid the foundation for gene cloning and functional analysis for waterlogging tolerance of apple.

    Genetic Analysis and Gene Mapping of Canary Yellow in Watermelon Flesh
    DIAO WeiNan,YUAN PingLi,GONG ChengSheng,ZHAO ShengJie,ZHU HongJu,LU XuQiang,HE Nan,YANG DongDong,LIU WenGe
    Scientia Agricultura Sinica. 2021, 54(18):  3945-3958.  doi:10.3864/j.issn.0578-1752.2021.18.013
    Abstract ( 384 )   HTML ( 27 )   PDF (2220KB) ( 234 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】In order to find out the formation mechanism of watermelon canary yellow flesh color, the pigment composition, pigment contents, and the inheritance of white and canary yellow flesh watermelon color were studied in this study, and the candidate genes related to canary yellow flesh were predicted by BSA-seq. 【Method】Two six generations populations were constructed by crossing four materials, including Bingtangcui (white flesh) and Xihua (canary yellow flesh), as well as Sashengnaiyougua (white flesh) and Xinjinlanxuan (canary yellow flesh). High performance liquid chromatography (HPLC) was used to determine the carotenoid composition and content in flesh of four parents at four different development stages. Two BSA-seq populations (BSA-seq I and BSA-seq II) were initially located by using the bulked sergeant analysis (BSA), and then the candidate genes were screened according to the annotation information of watermelon reference genome ‘97103’ V2, and verified by real-time quantitative polymerase chain reaction (qRT-PCR).【Result】During the development of watermelon fruit, violaxanthin and lutein accumulated differently in both parents, and the content of violaxanthin was higher in canary yellow flesh than in white flesh. The content of violaxanthin in white and canary yellow flesh watermelon fruits were (10.96±4) μg·g -1DW and (22.84±2) μg·g-1DW, respectively. The content of lutein in white and canary yellow flesh watermelon fruits were (2.23±1) μg·g-1DW and (3.97±1) μg·g-1DW, respectively. The content of violaxanthin was about 7 times of lutein. Two six generations segregation populations were analyzed, and the results showed that the flesh color of F1 and Ⅰ BC1P1 and Ⅱ BC1P1 populations were non-canary yellow, the segregation ratio of non-canary yellow and canary yellow in F2 population were consistent with the Mendelian segregation ratio of 3:1, and the segregation ratio of non-canary yellow and canary yellow inⅠ F1, Ⅱ F1, Ⅰ BC1P2 and Ⅱ BC1P2 backcross populations were 1:1, so it could be concluded canary yellow was recessive to white in watermelon flesh. Through SNP and InDel association analysis of BSA-seq Ⅰ and BSA-seq Ⅱ data, the major locus which regulated canary yellow flesh color in watermelon fruit was mapped on chromosome 6 within a physical distance between 24.00 to 24.61 Mb, and there were 70 genes in this region. Combined with reference genomic annotation and qRT-PCR experiment, five genes that maybe related to canary yellow flesh color trait in watermelon fruit were obtained. Among them, Cla97C06G121680, Cla97C06G121700 and Cla97C06G121890 were all related to chloroplast biogenesis and chloroplast structure size. These three genes might affect fruit flesh color by affecting the formation of chromoplast; Cla97C06G121910 was an AP2 transcription factor in response to ethylene synthesis which related to watermelon fruit ripening, which might affect the accumulation of carotenoids in flesh by affecting fruit ripening and Cla97C06G122090 was described as a transmembrane transport effect, which played a role in the transmembrane transport of carotenoids.【Conclusion】The main pigments of white and canary yellow flesh watermelon were violaxanthin and lutein, and the pigment accumulation in canary yellow flesh was significantly higher than white flesh watermelon. Genetic analysis showed that canary yellow was recessive to white in watermelon flesh color. Based on BSA-seq analysis, a major locus was located in the region of 24.00 Mb to 24.61 Mb on chromosome 6, while Cla97C06G121680, Cla97C06G121700, Cla97C06G121890, Cla97C06G122090 and Cla97C06G121910 were predicted as candidate genes which related to canary yellow formation in watermelon flesh.

    Effects of Tea-Litsea Cubeba Intrercropping on Soil Microbial Community Structure in Tea Plantation
    HAO HaiPing,BAI HongTong,XIA Fei,HAO YuanPeng,LI Hui,CUI HongXia,XIE XiaoMing,SHI Lei
    Scientia Agricultura Sinica. 2021, 54(18):  3959-3969.  doi:10.3864/j.issn.0578-1752.2021.18.014
    Abstract ( 320 )   HTML ( 44 )   PDF (856KB) ( 284 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】 The aim of this study was to analyze the changes of soil microbial community structure and its driving factors, so as to provide data support for soil ecological effect evaluation of Tea-Litsea cubeba intercropping. 【Method】GS-MS, ICP-MS, 16S and ITS sequence analysis were used to determine the soil extract composition, soil mineral element content, soil bacteria and soil fungi community structure.【Result】 The soil microbial community structure was significantly affected by the model of Tea-Litsea cubeba intercropping. The abundance of soil functional bacteria related to N, P and Mn transformation increased significantly, while the abundance of plant pathogenic bacteria and fungi was significantly decreased. In the root distribution layer, the abundance of Burkholderia paraburkholderia was 86 times compared to control, and the abundance of Fusarium graminearum was 73.13% lower than that of the control. Tea-Litsea cubeba intercropping increased the P content of tea plantation soil. In the root distribution layer, P content increased by 76.42%. The soil of Tea-Litsea cubeba intercropping contained some antibacterial substance, including 12.80% camphor, 6.72% Alpha-terpineol, and 12.65% citronellol. Redundancy analysis (RDA) showed that the soil extraction and P in the intercropping area were the main environmental factors affecting the community structure of soil microbes.【Conclusion】 Tea-Litsea cubeba intercropping changed the abundance and community structure of soil microbes, and the camphor, Alpha-terpineol, citronellol and P were the main ecological factors that affected the soil microbial community. The selection of intercropping plants in tea plantations and other farmland systems should pay attention to the secondary metabolites that enter the soil, especially those with killing or antibacterial functional components.

    FOOD SCIENCE AND ENGINEERING
    Application of Ultrasound-Assisted Thawing in the Role of Maintaining Physicochemical Properties and Reducing Protein Loss in Mutton During Multiple Freeze-Thaw Cycles
    GU MingHui,YANG ZeSha,MA Ping,GE XinYu,LIU YongFeng
    Scientia Agricultura Sinica. 2021, 54(18):  3970-3983.  doi:10.3864/j.issn.0578-1752.2021.18.015
    Abstract ( 270 )   HTML ( 44 )   PDF (2133KB) ( 289 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】In this study, it was expected to provide a theoretical reference for actual production by investigating the beneficial effects of ultrasound-assisted thawing with different powers on reducing the quality loss of mutton during multiple freeze-thaw cycles.【Method】The longissimus dorsi muscle in the Shanbei white cashmere goat was used as the test material, of which the refrigerated thawing treatment (LT) and hydrostatic thawing treatment (WT) were commonly used as controls. With the help of 180 W (UT180), 320 W (UT320) and 400 W (UT400) power ultrasound, the meat quality was analyzed by exploring the changes of the physicochemical properties (pH, drip loss, texture characteristics, color, and fat oxidation), microstructure and amino acids of freeze thawed mutton during multiple freeze-thaw cycles. 【Result】The results showed that ultrasound could increase the mutton drip loss and decrease the pH compared with the control, but the UT180 group treatment could significantly delay this trend (P<0.05). The mutton L* value of the ultrasound group was lower than that of the control group, especially the L* value of UT320 group was the lowest in the experiment (P<0.05), and its a* value was the highest during 3 to 7 freeze-thaw cycles. The b* value increase rate of the mutton in the UT320 group was the lowest during freeze-thaw process, and the b* values were significantly lower than those in the control group (P<0.05). The h° values of the LT group and the UT180 group, was the highest during freeze-thaw process (P<0.05), while the h° value of the UT320 group was the opposite. In addition, ultrasound-assisted thawing could significantly affect the color of frozen-thawed mutton, and the ΔE value of the UT320 group was the highest in the experimental (P<0.05), followed by UT180 group and UT400 group. Ultrasound reduced mutton hardness and springiness compared with control, especially, the mutton of UT400 group was the lowest during freeze-thaw (P<0.05). Compared with the control group, the ultrasound treatment of the UT320 and UT180 groups was beneficial to the stability of mutton resilience, and there was no significant difference between the two groups (P>0.05). Meanwhile, the mutton TBARS values of the UT320 and UT180 groups were significantly lower than that of the control group after freeze-thaw 7 times, and their PUFA content was higher than that of LT control group during freeze-thaw (P<0.05). The PCA of the mutton physicochemical properties showed that the main components of the UT320 group with the same freeze-thaw cycles were distributed at the top, while which of the LT group were distributed at the bottom. Compared with the microstructure of the control group, ultrasound treatment in the UT320 group was more conducive to maintaining the integrity of the mutton fiber structure. The amino acid indexes of mutton samples were analyzed by clustering, and the results showed that the initial sample and the 3 times freeze-thaw ultrasound-assisted thawing treatment group were the first category, with the highest amino acid nutritional quality, and the initial sample was closer to the UT320 group, followed by the mutton of the UT320 and UT180 groups whose composition and ratio of essential amino acids were kept stable in the freeze-thaw cycle 7 times. 【Conclusion】The results of mutton physicochemical properties showed that muscle-assisted thawing at 320W had excellent quality retention ability, which could slow down the oxidation of fat, maintain the stability of color and recovery, especially it could improve mutton tenderness. At the same time, 320 W power ultrasound-assisted thawing had the advantage of maintaining the structural integrity of mutton muscle fiber and reducing protein loss to maintain mutton amino acid quality.

    Changes in Lipid-Soluble Pigments in Fresh Tea Leaves Treated by Methyl Jasmonate and During Postharvest Oolong Tea Manufacturing
    SHI Jiang,WANG JiaTong,PENG QunHua,LÜ Haipeng,BALDERMANN Susanne,LIN Zhi
    Scientia Agricultura Sinica. 2021, 54(18):  3984-3997.  doi:10.3864/j.issn.0578-1752.2021.18.016
    Abstract ( 335 )   HTML ( 34 )   PDF (1228KB) ( 193 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】The dynamic changes of lipid-soluble pigments in tea under the dual stress of exogenous methyl jasmonate induction and mechanical damage trigged during "making green" stage were systematically studied. It could greatly clarify the mechanism of improvement in the content of carotenoids in making oolong teas prepared from the leaves treated by methyl jasmonate. This will offer the scientific guidance applying of exogenous elicitors improving fresh tea leaves quality and the made tea quality.【Method】The 3-4-years ‘Jinxuan’ tea cultivars in greenhouse were used as plant materials, which were applied 0.25% (v/v) methyl jasmonate solution by spraying on tea leaves homogenously until it was evenly dripped. Fresh leaves under different treated time duration (0, 24, 48, and 168 h) and the responding made oolong teas were prepared using the fresh leaves. In this study, the targeted metabolomics analysis was carried out utilizing UPLC-QToFMS aiming at analysis of changes in content of lutein, β-carotene, neoxanthin, zeaxanthin, α-carotene and chlorophyll a/b. At the same time, the dynamic changes of the lipid-soluble pigments during crucial procedure “making-green” and rolling stage were monitored. Furthermore, the sensory evaluation of made oolong teas focusing on the aroma quality was investigated.【Result】The quantitative results showed that the content of lutein reached (405.06±17.71) µg·g -1 in fresh leaves which was the highest of all other carotenoids, however, it decreased dramatically in made oolong teas (277.36±32.72) µg·g-1. Lipid-soluble pigments changed significantly in methyl jasmonate treated tea samples. In the first 48 h after methyl jasmonate inducement, chlorophyll a behaved significantly increased in content compared with control (0 h) in fresh leaves, successively decreased in 168 h samples. Chlorophyll b decreased in all methyl jasmonate induced samples. Lutein exhibited an apparently decreasing in 12 h samples, thus achieved improvement in content in 24 h samples. The content of β-Carotene was lower in methyl jasmonate induced samples than that in controls, especially in 12H samples, its content was 116.36 µg·g -1, decreased by 34.55%. In made oolong teas prepared from methyl jasmonate-induced tea leaves (12, 24 and 48 h), the content of chlorophyll a behaved significantly decrease compared with 0 h, whilst lutein prompted quite absolutely, especially in 48 h, whose content reached 377.82 µg·g -1. Aiming at a clear glance, the heatmaps of the content changes of the lipid-soluble pigments were prepared, and the results showed that the content of chlorophyll a decreased in all treated samples during “making-green” stage (W1-W3) comparing with the control samples. Meanwhile, chlorophyll b apparently increased after 12h duration of methyl jasmonate inducement. Besides, the changes of the carotenoids behaved more complicated. Lutein behaved a decreasing tendency in 12 h samples, however increased in 24 and 48 h samples. β-Carotene exhibited a lower content in all treated samples, thus, zeaxanthin and α-carotene maintained a higher content compared with the control samples. In the rolling stage, the above-mentioned lipid-soluble pigments changed significantly in content. With exception of β-carotene which decreased in all treated samples, lutein, neoxanthin, zeaxanthin, and α-carotene achieved a higher content in 12, 24 and 48 h samples. It was clear that chlorophyll a decreased in methyl jasmonate treated samples in rolling stage, whilst chlorophyll b increased after 12 h. Moreover, the sensory assessment confirmed the results of aroma quality promotion in made oolong teas prepared from methyl jasmonate treated fresh leaves, however with a loss of brightness and softness in the foliage fundus.【Conclusion】In conclusion, the content of carotenoids in fresh tea leaves and the made oolong tea were absolutely improved within 24 h time duration after methyl jasmonate treatment. It was confirmed that the aroma quality was improved in made oolong tea prepared from MeJA-treated fresh leaves after 12 h which was in rich of fragrancy flavor. Differential accumulation of lipid-soluble pigments was primed under the dual stress of exogenous methyl jasmonate induction and mechanical damage trigged during “making-green” stage.

    ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT
    Identification of circINHBB During Follicular Atresia and Its Effect on Granulosa Cell Apoptosis
    MA MengNan,WANG HuiMing,WANG MiaoMiao,YAO Wang,ZHANG JinBi,PAN ZengXiang
    Scientia Agricultura Sinica. 2021, 54(18):  3998-4007.  doi:10.3864/j.issn.0578-1752.2021.18.017
    Abstract ( 281 )   HTML ( 28 )   PDF (2129KB) ( 260 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Background】The follicular development is related to the reproductive capacity and production performance of mammals. Follicular development includes the growth and development of oocytes, the initiation and growth of the primordial follicles, and the development of primary follicles. In addition, these biological processes are closely related to the proliferation and apoptosis processes of follicle granulosa cells. The main inducement of follicular atresia is the apoptosis of granulosa cells, which is extremely complex and regulated by various cytokines. Previous studies have shown that non-coding RNAs (ncRNAs) are involved in various biological processes as regulators, including cell proliferation and apoptosis. Circular RNA (circRNA) is a new type of ncRNAs, widely existing in organisms. The circRNA is involved in the regulation of various physiological processes, but there are few studies in the field of livestock reproduction, especially, its expression, distribution and biological function in the pig ovary and follicle are rarely studied. Inhibin (INH) is a gonadal glycoprotein hormone, which is mainly produced by granulosa cells of ovarian follicles in female animals. It is an important factor to control ovulation in mammals. Previous studies have shown that the coding gene of the precursor of INH βsubunit in porcine follicles may form a circular RNA, i.e. circINHBB. 【Objective】In this study, the sequence structure and cell distribution of circINHBB in porcine follicular tissue were verified, and its expression difference in healthy and atresia follicles analyzed to explore the effect of circINHBB on cell apoptosis in porcine granulosa cells cultured in vitro, so as to broaden the research ideas of circRNAs in the field of livestock breeding, and provide reference for improving the reproductive capacity of livestock. 【Method】 Firstly, the porcine follicles were collected, amplified by PCR and sequenced by Sanger sequencing to verify the sequence structure of circINHBB. Secondly, the follicles were divided into two groups, including healthy and atretic, and the expression difference of circINHBB was detected by qRT PCR in each group. Then, the FISH experiment was used to verify the distribution of circINHBB in the granulosa cells. Finally, the porcine ovarian granulosa cells were cultured in vitro, circINHBB was knocked down by siRNA, and the effect of circINHBB on granulosa cell apoptosis was detected by flow cytometry. 【Result】Sanger sequencing confirmed the existence of circINHBB in porcine follicles, and it was a circular RNA formed by reverse splicing of the INHBB mRNA 5’UTR. qRT PCR results confirmed that the expression level of circINHBB was higher in healthy follicles, but significantly decreased in atresia follicles. FISH further verified the distribution of circINHBB in the cytoplasm of porcine granulosa cells. After siRNA knockdown, the expression of circINHBB was significantly decreased, and the apoptosis rate of granulosa cells increased significantly. 【Conclusion】The circular structure of circINHBB was verified in the porcine ovary, and its distribution in cytoplasm suggested that circINHBB might be involved in post-transcriptional regulation. The fact that the expression of circINHBB was lower in atresia follicles, and knockdown of circINHBB could significantly increase the level of apoptosis, indicating that circINHBB had a significant inhibitory effect on the apoptosis of porcine granulosa cells. Thus, it might be an active regulatory factor in the growth and development of follicles. This study explored the role of circRNA in follicular atresia and granulosa cell apoptosis, which was an important supplement to the regulatory mechanism of follicular atresia.

    Effects of Dietary Zearalenone Adsorbent on the Distribution and Expression of LC3 and PCNA in the Uterus of Gilts
    HUANG LiBo,WANG JinQuan,GAO WenBo,CHEN HongJu,HOU YanMeng,YUAN XueJun,WANG ChunYang
    Scientia Agricultura Sinica. 2021, 54(18):  4008-4017.  doi:10.3864/j.issn.0578-1752.2021.18.018
    Abstract ( 253 )   HTML ( 27 )   PDF (5193KB) ( 215 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】 It has been proved that zearalenone (ZEA) can activate estrogen sensitive gene through estrogen receptor in animals, resulting in reproductive toxicity, and affecting endometrial cell growth, oocyte maturation and follicular granulosa cell proliferation. The purpose of this experiment was to investigate the effects of ZEA adsorbent (zeolite + montmorillonite combinations) on distribution and expression of LC3 and PCNA in the uterus of gilts, and to discuss the detoxification effect of the new ZEA adsorbent from the perspective of histochemistry. 【Method】 A total of 48 healthy gilts with a body weight of 30±2.11 kg were randomly divided into 6 groups (n = 8): control group with basal diet, the ZEA group with basal diet + 1.008 mg∙kg-1ZEA, the 0.1% ZEA adsorbent (ZEA 0.1) group with ZEA diet + 1.0 g∙kg-1 new adsorbent, the 0.25% ZEA adsorbent (ZEA 0.25) group with ZEA diet + 2.5 g∙kg-1 new adsorbent, the 0.5% ZEA adsorbent (ZEA 0.1) group with ZEA diet + 5.0 g∙kg-1 new adsorbent, and the ZEA montmorillonite (ZEA+M) group with ZEA diet + 2.5 g∙kg-1 montmorillonite. The preliminary trail period was 7 d, and the trial period was 21 d. 【Result】 ZEA increased the uterine organ index, and adding 0.25% and 0.5% adsorbents in the diet decreased the index of uterine organs obviously. The LC3 and PCNA positive cells were mainly distributed in the glandular epithelial cells, and the LC3 immunoreactivity of lumen epithelial cells were weaker than that of glandular epithelial cells. The immunoreactivity of LC3 in control group was stronger than that of ZEA group. The immunoreactivity and number of positive cells of LC3 in the ZEA new adsorbent group were significantly higher than those in the ZEA group, and there was a dose-dependent trend, but the effect of the ZEA-M group was increased slightly more than that of ZEA group. The results of PCNA in the luminal and glandular epithelium were contrary to that of LC3. The WB and qRT-PCR results also showed that 0.25% and 0.5% new adsorbents could promote the expression of LC3, increase autophagy, and decrease the PCNA immunoreactive reaction. These results indicated that ZEA inhibited the expression of LC3, inhibited autophagy and destroyed the homeostasis of endometrial cells and glandular epithelial cells. However, the new adsorbent could increase the expression of LC3 protein and decrease the expression of PCNA, which could promote autophagy and resist the abnormal proliferation of endometrial cells caused by ZEA. The new adsorbent had a good protective effect on uterine cells through bidirectional regulation.The experimental results provided theoretical basis for the further application of the new adsorbent. 【Conclusion】In this experiment, ZEA induced the uterus proliferation reaction, the new adsorbent (zeolite + montmorillonite combinations) resisted the negative effect of ZEA on the normal physiological function of uterus within a certain limits. As a result, 0.25% and 0.5% doses of this newadsorbents was suitable.

    RESEARCH NOTES
    Endophytic Bacterial Community Composition and PICRUSt Gene Functions in Different Pumpkin Varieties
    HUANG ZiYue,LIU WenJun,QIN RenLiu,PANG ShiChan,XIAO Jian,YANG ShangDong
    Scientia Agricultura Sinica. 2021, 54(18):  4018-4032.  doi:10.3864/j.issn.0578-1752.2021.18.019
    Abstract ( 389 )   HTML ( 40 )   PDF (1081KB) ( 379 )   Save
    Figures and Tables | References | Related Articles | Metrics

    【Objective】The aim of this study was to provide a theoretical basis for utilizing functional strains of endophytic bacteria in different varieties of pumpkins, as well as to explore new directions for pumpkin breeding. 【Method】The endophytic bacterial community composition and PICRUSt gene functions of 5 pumpkins varieties were analyzed based on MiSeq high-throughput sequencing results. The diversity and richness of endophytic bacterial community compositions in terms of 5 different pumpkin varieties were compared at phylum and genus levels.【Results】In total, 18 phyla, 30 classes, 101 orders, 199 families, 362 genera, 567 species and 863 OTUs could be obtained as the endophytic bacterial composition in 5 pumpkin varieties. There was no significant difference in endophytic bacteria diversity among pumpkin varieties. Significant differences in total numbers of endophytic bacteria were observed at different classification levels among pumpkin varieties. The proteobacteria was the dominant bacteria in terms of different 5 pumpkin varieties at the phylum level, but the abundance and proportion in every pumpkin variety were quite different. Moreover, Pseudomonas, Mycobacterium, Ensifer and Devosia were the dominant endophytic bacteria at genus level of different 5 pumpkin varieties, but their composition and abundance varied with pumpkin varieties. The composition and proportion of dominant endophytes at the phylum and genus level were similar between the hybrid and parent species. PICRUSt analysis also showed that endophytic bacteria inhabited in terms of different pumpkin varieties, mainly involved in 6 biological metabolic pathways and 46 sub-functions. However, there were no significant differences of endophytic bacterial genes in terms of different pumpkin varieties at the secondary functional layer. However, the numbers of gene functional copies varied with different varieties. The diversity and abundance of endophytic bacteria in terms of pumpkin were significantly affected by environmental factors. Moreover, the different endophytic bacterial community structures could be found in different pumpkin varieties. Meanwhile, a higher abundance of endophytic bacteria tended to enrich in terms of higher quality hybrids. Bacteria in 13 genera of bacteria, including Pseudomonas, Mycobacterium, Ensifer, and Devosia, etc, were the dominant endophytic bacterial genera in terms of 5 different pumpkin varieties. By contrast, Glycomyces, Candidatus_Rubidus, and Actinokineospora were the specific dominant endophytic bacterial genera in terms of Guifeng NO.7. In addition, 37 secondary functional layer functions were all the highest endophytic bacterial genes in terms of Guifeng NO.7 among the five pumpkin varieties, which involved the metabolisms of carbohydrate, amino acid, cofactors, and vitamins.【Conclusion】The higher dominant endophytic bacterial genus and the richer secondary functional genes were essential reasons for higher quality of Guifeng NO.7 than that of other four pumpkin varieties. Glycomyces, Candidatus_Rubidus, and Actinokineospora could be considered as the alternative benefit endophytic bacterial genera for improving pumpkin quality.