Journal of Henan Agricultural Sciences

Research Progress on Molecular Mechanism of Long Non-coding RNA

GUO Dongguang, CHEN Mingyan, LI Wenming, WANG Feng, GUO Zanying, SUN Guopeng, LI Peng, YUE Feng, ZHU Yanping, WANG Xuannian

2021, 50(4): 1-7. DOI: 10.15933/j.cnki.1004-3268.2021.04.001

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Long non-coding RNAs(lncRNAs) are non-coding transcripts that lack an open reading frame and are more than 200 nt in length.They are large in absolute quantity and variety and have obvious cell specificity in expression patterns.They play a variety of biological functions through base pairing with DNA or RNA or binding to proteins via the advanced structure of RNA,thus constituting a complex and delicate molecular regulatory network.In this paper,combined with the research findings of lncRNA in recent years,based on expounding the research progress of molecular mechanism of lncRNA,the related research of lncRNA in animal production performance and animal growth and development was briefly described,to provide a reference for further research on the biological regulation function of lncRNA,and also provide the scientific basis for the application of lncRNA in animal husbandry.

New Herbicide-Resistant Japonica Rice Germplasms Created by EMS Mutagenesis

WANG Fuhua, LI Zichao, WANG Ya, FU Jing, YANG Wenbo, YIN Haiqing, WANG Shengxuan, WANG Yuetao, BAI Tao, ZHANG Zhen

2021, 50(4): 8-16. DOI: 10.15933/j.cnki.1004-3268.2021.04.002

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In order to create new herbicide-resistant rice germplasms,the dry seeds of Zhengdao 19 were soaked in ethylmethane sulfonate( EMS) solution.At three-leaf stage,M₂ seedlings were sprayed with imazapic(112.5 g/ha)and nicosulfuron(60 g/ha).The herbicide-resistant mutants were screened after 14 days.The ALS (acetolactate synthase) gene sequences of mutants were analyzed to find out the mutation sites.The results showed that the herbicide-resistant plants grew normally after spraying imazapic,the leaf kept green,while the leaves of sensitive lines yellowed,and their growth were inhibited;after spraying nicosulfuron,the growth of herbicide-resistant plants were inhibited in the early stage,but could recover slowly.A total of 63 000 M₂lines were screened with imazapic,and 6 imazapic-resistant mutants (HF1—HF6) were obtained,including two types of ALS mutations,Ser-627-Asn and Gly-628-Glu.A total of 30 000 M₂ lines were screened with nicosulfuron, and 2 herbicide-resistant mutants(HF7—HF8) were obtained,including two types of ALS mutations, Pro-171-Ser and Ala-179-Val. To
obtain a specific ALS nucleotide mutation,an average of 23 250 M₂ lines were needed to screen.The fresh weight reduction rates of imazapic-resistant mutants HF1 ( Gly-628-Glu) and HF2 ( Ser-627-Asn) were 13.09% and 20.03% respectively after spraying 1 time the conventional herbicide dose(112. 5 g/ha),which were significantly less than that of Zhengdao 19 (P<0.05); the mutants could still grow normally after spraying 8 times the conventional herbicide dose in the field,showing strong resistance to imazapic herbicides.The fresh weight reduction rates of mutants HF1 and HF2 were 24.81% and 13.13% after spraying 30 g/ha nicosulfuron,and were 34.13% and 28.01% after spraying 60 g/ha nicosulfuron,respectively,which were significantly less than those of Zhengdao 19 (P<0.05), showing certain nicosulfuron resistance. In summary, 8 herbicide-resistant mutant lines were obtained, 2 lines were resistant to nicosulfuron,6 lines were resistant to imazapic,and two of the imazapic-resistant mutants exhibited nicotinsulfuron resistance.The screened herbicide-resistant mutants could be used to breed non-GMO herbicide-resistant varieties,and also be used as a basic germplasm for the multi-site mutation of ALS,so as to further strengthen the herbicide resistance of mutants.

Optimization of Identification of Gene Editing Progeny by CRISPR-Cas9 System in Arabidopsis thaliana

LI Ziwen, LIU Yan, LÜ Mengyang, GAO Kaili, ZHANG Hairong

2021, 50(4): 17-21. DOI: 10.15933/j.cnki.1004-3268.2021.04.003

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Nowadays, the technology for site-specific editing of plants using the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system has been matured,but the identification of the progeny of the edited plant is still time-consuming and labor-intensive.In order to optimize the identification method,we took Arabidopsis sugar-dependent 1(SDP1)gene as the editing object.Sequence analysis showed that there was protospacer adjacent motif(PAM)in exons of SDP1 gene,and there were four suitable restriction sites in upstream of PAM sequences.We screened these restriction sites for their positions and performance,selected the restriction site Sac Ⅰ in the second exon of SDP1 gene as the editing target,and constructed the CRISPR-SDP1-Cas9 vector.Through enzyme digestion identification and sequencing analysis of T1 seedlings screened with hygromycin,the heterozygous edited lines with three bands were identified.Among 71 randomly selected T1 plants,24 heterozygous edited lines were found,the editing efficiency was 16.9%.Among 48 T2 plants,5 homozygous edited lines were screened out,and the editing sites were all insertions at the Sac Ⅰ restriction site.In summary, the appropriate restriction sites near the PAM sequence of the gene to be edited were selected as editing targets,and the progeny plants were identified through the restriction enzyme digestion and sequencing analysis.The traditional method identifing the progeny of CRISPR-Cas9 transgenic plants is extracting DNA from the progeny plants,amplifing the target site by PCR and connecting it to the T vector for sequencing several times.This method is simple,fast,economical and practical compared with the traditional method,providing a simple and quick method for CRISPR-Cas9 gene-edited progeny identification.

Effect of Pre- and Post-Anthesis High-Temperature Stress on Agronomic Traits and Yield of Different Maize Hybrid Combinations

2021, 50(4): 22-30. DOI: 10.15933/j.cnki.1004-3268.2021.04.004

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In order to explore the effect of pre- and post-anthesis high-temperature stress on growth,silking and anthesis characteristics and yield, 25 advanced cross combinations with separate genetic background were used as materials,42℃ was imposed through automatic intelligent heating system from 10 d before anthesis to 10 d after anthesis,with the suitable temperature as control.The results showed that after high temperature treatment,silking duration was prolonged as much as 7 days compared with the control; the anthesis duration was shorter, and anthesis-silking interval was elongated. After high temperature treatment,the SPAD values of all cross combinations decreased by 4.1%—12.9%,plant height increased by 1.1%—35.4%,the dry weight of the shoots all decreased by more than 10%,and dry root weight,ear height and stem diameter of all cross combinations were all affected,but the change trends were inconsistent. High temperature treatment before and after anthesis reduced grain yield by 1.8%—11.3%.According to the heat resistance coefficient and membership function value, the cross combinations 22 and 14 were relatively heat resistant.According to the results of principal component analysis,9 traits including duration of silking and anthesis,silking-anthesis interval,grain yield per ear,dry shoot weight and root weight, plant height, stem diameter and root-shoot ratio could be used as indicators for heat resistance screening of hybrid combinations.

Effects of Deep Tillage and Fertilization on Plant Growth,Photosynthetic Characteristics and Yield of Summer Maize in Lime Concretion Black Soil

GUAN Yanxia, WANG Qun, ZHAO Hanmei, JIA Xucun, YAN Zhenhua, LIU Dongyao, DONG Pengfei, LI Chaohai

2021, 50(4): 31-39. DOI: 10.15933/j.cnki.1004-3268.2021.04.005

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To explore the effect of different patterns of deep tillage and fertilization on maize production,five treatments were designed,including no tillage and fertilization in single layer(CK),deep tillage on side position+fertilization in single layer(T1),deep tillage on side position+fertilization in three layers (T2),deep tillage on direct position+fertilization in three layers(T3)and deep tillage on direct position+fertilization in single layer (T4),and the differences of soil water content,seedling emergence quality,photosynthetic characteristics and yield among different treatments were studied.The results showed that,compared with CK,deep tillage increased the soil water content and dry shoot biomass,the effect of deep tillage on side position was greater than that of deep tillage on direct position,and T2 treatment had the highest value. Deep tillage increased the seedling emergence rate, plant height uniformity, net photosynthetic rate, stomatal conductance and transpiration rate of leaves, decreased the intercellular carbon dioxide concentration,the effect of deep tillage on side position was greater than that of deep tillage on direct position,the effect of fertilization in three layers was greater than that of fertilization in singlelayer,and T2 treatment was the best. Compared with CK,deep tillage increased grain yield by 9.03%,and the yield of deep tillage on side position was greater than that of deep tillage on direct position with the increase rate of 6.84%; the yield of fertilization in three layers showed no significant difference with fertilization in single layer; the order of yield among treatments was T2>T1>T3>T4>CK,and the yield of T2 treatment increased by 13.24% compared with CK.In summary,deep tillage on side position could promote the growth and yield of summer maize more than deep tillage on direct position,and deep tillage on side position+fertilization in three layers obtained the best effect.

Comparison of the Contents of Primary Secondary Metabolites of Flue-cured Tobacco Leaves in Different Production Areas in Henan

ZHANG Xi, WANG Huifang, XU Shixiao, XUE Gang, YANG Tiezhao

2021, 50(4): 40-47. DOI: 10.15933/j.cnki.1004-3268.2021.04.006

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In order to explore the differences in the content of main secondary metabolites of flue-cured tobacco leaves in different producing areas in Henan,the contents of organic acids,alkaloids,polyphenols and sterols in the tobacco leaves of the C3F and B2F grades of the main flue-cured tobacco varieties in seven different producing areas in Henan were determined by GC/MS-SIM,GC-MS,HPLC and UPLCMS,and the test results were analyzed. Through descriptive statistical analysis,malic acid,citric acid and oxalic acid were the main components of flue-cured tobacco organic acids; nicotine, nornicotine,pseudoequiine and neonicotine were the main components of flue-cured tobacco alkaloids;chlorogenic acid and rutin were the main components of flue-cured tobacco polyphenols;stigmasterol and β-sitosterol were the main components of flue-cured tobacco sterols. The organic acid content of flue-cured tobacco leaves in Henan tobacco area was 120.01—216.01 mg/ g,with an average of 151.77 mg/ g;the alkaloid content was 18.85—29.97 mg/ g, with an average of 23.13 mg/g; the content of polyphenols was 16.85—27.92 mg/ g,and the average was 21.74 mg/ g;the sterol content was 2.11—2.87 mg/ g,and the average was 2.54 mg/g.There were differences in the total amount of secondary metabolites in tobacco leaves from different producing areas,which showed Fangcheng>Luoning>Biyang>Lushi>Jiaxian>Xiangxian> Linying.Fangcheng flue-cured tobacco had the highest organic acid and alkaloid content;Xiangxian and Jiaxian flue-cured tobacco had the highest polyphenol content;Luoning flue-cured tobacco had the highest sterol content.

Difference of Mineral Elements and Main Medicinal Components in Different Phenotypes of Artemisia argyi

LI Chao, YANG Chengmin, GAO Li, CUI Zhanhu, ZHANG Chaoyun, HUANG Xianzhang

2021, 50(4): 48-55. DOI: 10.15933/j.cnki.1004-3268.2021.04.007

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In order to study the phenotypic diversity of germplasm resources,and clarify the differences of mineral elements and main medicinal components in different phenotypes of Artemisia argyi,in the present study,inductively coupled plasma mass spectrometry( ICP-MS) was used to detect the contents of 35 mineral elements, including Be, Na, Mg, Al, K, etc.in 4 different phenotypes of A.argyi, and then comprehensive evaluation was carried out by principal component analysis and factor analysis.In addition,high-performance liquid chromatography(HPLC) was used to detect the contents of isotendin,chlorogenic acid and isochlorogenic acid B.The results showed that the contents of 32 mineral elements were significantly different in 4 phenotypes of A.argyi(P<0.05).With the content of each element as a variable,a total of 4 principal components were extracted, and the variance contribution rates were 70.092%,11.055%,9.485%,and 3.743%,respectively.According to the calculation rule of adding the product of the load of each principal component (F₁—F₄) and the variance contribution rate, the comprehensive evaluation function was obtained as F=0.700 9F₁+0.110 6F₂+0.094 9F₃+0.037 4F₄.Accordingly,the phenotype 1 and phenotype 2 had the highest scores.The content of eupatilin in phenotype 1 was the highest(3. 01 mg/g),which was significantly higher than that of phenotype 2 and phenotype 3,but the difference between phenotype 4 and phenotype 1 was not significant;the contents of chlorogenic acid in phenotype 1 and phenotype 3(3.56 mg/g and 4.59 mg/g, respectively)were significantly higher than those in phenotype 2 and 4;The similar result was found for isochlorogenic acid B in phenotype 1 and phenotype 3 ( 4.91 mg/ g and 5.10 mg/g, respectively) when compared with phenotype 2,but not with phenotype 4. These results indicate that phenotype 1 can be the priority in the selection of 4 cultivars based on the comprehensive consideration of the content of mineral elements and
medicinal components.

Study on Physical and Chemical Properties and Microbial Community Composition in Root Soil of Rheum tanguticum

LIU Hancheng, KONG Zhaoxia, LONG Xueping, LI Baocheng, MA Xiaomei, ZHA Xizongji, LIU Jing

2021, 50(4): 56-65. DOI: 10.15933/j.cnki.1004-3268.2021.04.008

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In order to explore the change rule of soil physical and chemical properties and microbial community composition in different growing years of Rheum tanguticum,the farmlands without planting Rheum tanguticum was used as control (site 1),and the farmlands with growing Rheum tanguticum for 1 to 3 years (site 2,site 3 and site 4)were designed as planting years treatments.The soil physical and chemical properties and microbial community structure of the root of Rheum tanguticum in different growing years were studied by conventional determination method and plate method.The results showed that except for site 1,the content of water,organic matter and total potassium increased gradually with the increase of planting years of Rheum tanguticum,while pH value decreased with the increase of planting years of Rheum tanguticum.The total phosphorus content was the highest in site 4,and the total nitrogen content was the highest in site 3.Comparing the physical and chemical indexes of different soil layers in the same point,it can be seen that the soil water content of the third layer (30—45 cm) of site 1 and site 3 was significantly higher than that of the first layer (0—15 cm) and the second layer (15—30 cm),but there was no significant difference in other sites.The pH value in the second layer of site 1 was significantly higher than that in the third layer,but there was no significant difference with that in the first layer,and there was no significant difference in other sites.The content of organic matter in the second layer of site 2 was the highest,and the content of organic matter in the first layer was significantly higher than that in second and third layers in other sites.The contents of total nitrogen,total phosphorus and total potassium were different in different sites and there was no obvious rule.The number of soil bacteria was the most,followed by actinomycetes and fungi.The number of bacteria and actinomycetes was significantly negatively correlated with the growing years of Rheum tanguticum,and significantly positively correlated with the content of organic matter and total potassium,and the number of fungi was significantly positively correlated with the growing years of Rheum tanguticum and the content of total phosphorus.It showed that long-term cultivation of Rheum tanguticum could inhibit the communities of bacteria and actinomycetes in soil,but promote the growth of fungi.

Tolerance and Enrichment Characteristics of Cordyceps militaris to Cu²⁺

MENG Jiangling, HE De, LI Cuixin

2021, 50(4): 66-71. DOI: 10.15933/j.cnki.1004-3268.2021.04.009

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To study the effects of Cu²⁺ enrichment on growth of Cordyceps militaris and human health,different concentration of Cu²⁺was added into culture medium to observe the effects of Cu²⁺on the growth of mycelia and fruiting bodies of C.militaris,the content of polysaccharides and Cu²⁺enrichment.The results showed that Cu²⁺inhibited the growth of C.militaris,because the addition of Cu²⁺at the minimum concentration(1.6 mg/L) led to degenerative changes in the fruiting bodies of C.militaris,such as dwarfing and bifurcation.The polysaccharide content of C.militaris increased with the increase of Cu²⁺addition. When the Cu²⁺addition was 3.2 mg/L,the Cu²⁺enrichment rate of mycelium and fruiting body was the highest,54.41% and 191.01% respectively.Therefore,by comparing the above factors,it is found that the optimum Cu²⁺addition is 1.6—3.2 mg/L.With the minimum copper ion addition of 1.6 mg/L, 29 g dry or 116 g fresh fruiting bodies are needed daily to supplement the amount of Cu²⁺needed for people.

Study on Screening and Biological Characteristics of Endogenous Manganese-tolerant Bacteria in Conyza canadensis

TENG Zheng, YANG Cuifeng, JIA Guikang, LAN Zhaoyun, GU Lili

2021, 50(4): 72-78. DOI: 10.15933/j.cnki.1004-3268.2021.04.010

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In order to broaden the bioremediation of heavy metal contaminated soil and enrich the resource library of functional strains,six strains of manganese-resistant endophytic bacteria were isolated from the leaves of Conyza canadensis,named XFP-01—06,with the tolerance to 800—2 500 mg/L Mn²⁺.Among them,four strains also showed high tolerance to Pb²⁺ and Cr⁶⁺,of which XFP-04 strain was the most outstanding,and the highest tolerable contents of Mn²⁺,Pb²⁺,Cr⁶⁺were 2 500,800,and 400 mg/L,respectively.It also had the promotion characteristics of IAA production,iron carrier production,nitrogen fixation,and phosphorus dissolution,and also could withstand high osmotic pressure,so it was a potential dominant strain for bioremediation of heavy metal contaminated soil.Through the determination of physiological and biochemical characteristics and 16S rDNA sequence analysis,the strain XFP-04 was identified as Bacillus cereus.

Evaluation on Farmland Soil Fertility in Danjiangkou Reservoir Area Based on Principal Component Analysis and Hierarchical Cluster Analysis:A Case Study of Xichuan County

ZHANG Jun, CAI Debao, YANG Shuqiong, CHEN Xiuwen, CHEN Jibao

2021, 50(4): 79-87. DOI: 10.15933/j.cnki.1004-3268.2021.04.011

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In order to apply fertilizer rationally and reduce environmental pollution,Xichuan County in Danjiangkou Reservoir area was used as the research area to evaluate the nutrient indexes of 160 farmland surface soils by using principal component analysis and cluster analysis.The results showed that most of farmland soils in Xichuan County was acidic;the average values of the soil organic matter content,total nitrogen content,total phosphorus content,and total potassium content were 16.96,1. 22,0.50,and 16.21 g/kg,respectively.The overall fertility of the soil was at a medium level.The content of alkalihydrolyzable nitrogen and available phosphorus was low,belonging to lack or low-medium level.Through principal component analysis, the cumulative contribution rate of the two principal components was 53.289%.The main factors affecting the comprehensive evaluation of soil fertility were organic matter content,total nitrogen content, alkali-hydrolyzable nitrogen content and pH value, and through further division of soil fertility level,the comprehensive level of soil fertility quality was divided into categories Ⅰ,Ⅱ and Ⅲ,which represented high,medium and low soil fertility levels respectively.Among them,the soils in the Ⅱ and Ⅲ grades accounted for 56.25% and 38.75% of the total samples respectively.The soil fertility quality of farmland in Xichuan County was low.

Effect of Mechanical Stirring Intensity on Anaerobic Co-digestion of Cattle Manure and Corn Straw

ZHU Jiaoning, LI Yongping, PANG Zhenpeng, TANG Yun

2021, 50(4): 88-95. DOI: 10.15933/j.cnki.1004-3268.2021.04.012

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To investigate effects of mechanical stirring intensity on the biogas production characteristics and fermentation stability of cattle manure and corn straw anaerobic co-digestion and determine the optimum stirring intensity,experiments with cattle manure and corn straw(volatile solid ratio of 7∶3) as fermentation materials at different stirring intensity of 0(the control treatment),30,60,90 r/min were conducted under the conditions of 7% volatile solid and (35±1)℃ .The results showed that the biogas production and potential volatile solid methane production were increased firstly and then decreased with the increase of stirring intensity. When the stirring intensity was 60 r/min, the cumulative biogas production,total methane production and volatile solid methane production were higher than other treatments,reaching 120.67,71.44 L and 194.39 mL/g,which were 10.0%,9.9%,9.9% higher than that of the control treatment,respectively.No significant differences were observed in methane content (60%—63%) among different stirring intensity treatments.The pH value maintained between 6.60—7.30 and the ratio of volatile fatty and alkalinity was below 0.4 for all treatments,showing the stability of the anaerobic fermentation systems.Lower ratios of volatile fatty and alkalinity for stirring treatments than the control treatment showed that stirring could reduce the ratio of volatile fatty and alkalinity of biogas slurry and enhance the stability of anaerobic fermentation systems. Based on comprehensive consideration of biogas production and stability of anaerobic fermentation system,the optimal stirring intensity was 60 r/min.

Isolation of Antagonistic Endophytes against Myrica rubra Wilt and Their Antagonistic Mechanism

CAO Pengfei, LIU Qing’e

2021, 50(4): 96-105. DOI: 10.15933/j.cnki.1004-3268.2021.04.013

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In order to isolate and screen antagonistic endophytes for the control of M.rubra wilt disease,healthy M.rubra, Houttuynia cordata, phoenix and honeysuckle were used as materials to screen the antagonistic endophytic strains.The changes of cell membrane,cell wall,respiratory metabolism,RNA content and topoisomerase Ⅰ activity were determined to study the antagonistic mechanism.The results showed that 14 strains of antagonistic endophytes against M.rubra wilt were isolated from four plants,of which the FWB1J strain and YMB1J strain had the best antibacterial effect.And these two strains were
identified as Trichoderma sp.and Candida sp.respectively. Their MICs were 0.400 0 g/mL,their EC₅₀ were 0.034 7 g/mL and 0.029 0 g/mL respectively,their EC₉₀ were 0.315 5 g/mL and 0.254 6 g/mL,respectively.After being treated with the extracellular metabolites of two dominant endophytic strains,the content of N-acetylglucosamine and chitinase activity of pathogen were significantly higher than those of the control,and reached the maximum at 36 h,among them,the content of N-acetylglucosamine in the EC₉₀ group of YMB1J strain was 176. 700 μg/ g higher than that of the control.The leakage of extracellular soluble sugar and soluble protein increased gradually,and reached the maximum at 10 h.The content of ergosterol was significantly lower than that of the control group,and the EC₉₀ group of FWB1J strain was 82.460 μg/g lower than the control.The content of RNA in pathogen was always lower than that of the control group,and the RNA content of the EC90 group of FWB1J strain was the lowest,which was 16.450 μg/g lower than that of the control group at 60 h.The activities of succinate dehydrogenase and malate dehydrogenase were significantly lower than that of the control group,and the activities of the two enzymes in the EC₉₀group of FWB1J strain were decreased by 2.750 U/mg and 318.330 U/mg at 72 h,respectively.Theses indicated that FWB1J and YMB1J strains could destroy the integrity of cell wall, increase the permeability of cell membrane, inhibit nucleic acid synthesis and respiratory metabolism,and then inhibit the growth of the pathogen and even promote their death.

Prediction of Suitable Area of Eriosoma lanigerum in China under Future Climate Conditions

HOU Qinwen, BAI Haiyan, LI Yunling

2021, 50(4): 106-114. DOI: 10.15933/j.cnki.1004-3268.2021.04.014

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Eriosoma lanigerum(Homoptera:Aphididae) is an important quarantine pest in the main apple producing areas in China.Predicting of the suitable areas of E.lanigerum in China under future climatic conditions can provide a basis for the quarantine supervision and scientific control of E.lanigerum. In this study, principal component analysis, correlation analysis and Jackknife method were used to screen 20 environmental variables of 294 distribution points of E.lanigerum and 7 dominant variables were obtained.With the current climate, scenario in 2050, and scenario in 2070 as input variables,the maximum entropy model was run to simulate the suitability of E.lanigerum.The ArcGIS software was used to classify the most suitable simulation results,and the distribution map of the suitable areas for E.lanigerum was obtained.The results showed that under current climatic conditions, the suitable areas for E. lanigerum in China were mainly concentrated in North China,East China,Southwest China,north of Central China,south of Northeast China,and east of Northwest China.The total area of suitable areas was 576.5×10⁴km² , accounting for about 60.1% of China’s land area. Among them,the area of highly suitable area was 102.2×10⁴ km²,the area of moderately suitable area was 126.7×104 km²,and the area of lowly suitable area was 347.6×104 km².The prediction of suitable areas in the future (in 2050 and 2070) showed that the total suitable areas of the two efflux scenarios of RCP2.6 and RCP8.5 were both smaller than the total suitable area under the current climate,but the total area of highly suitable areas increased significantly. Under the RCP2.6 scenario,the total area of highly suitable areas for E.lanigerum in 2050 was the largest,accounting for 12.0% of China’s land area,which was 13.0× 10⁴ km² more than the predicted area under current climate. The areas of Shaanxi and Henan provinces would increase significantly.

Effects of Applying Different Barrier Materials on the Yield,Quality and Nitrogen and Phosphorus Absorption of Chinese Cabbage

ZHANG Yunhong, LUO Xiaosheng, KOU Changlin, GUO Zhanling

2021, 50(4): 115-123. DOI: 10.15933/j.cnki.1004-3268.2021.04.015

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Barrier materials are some materials which can reduce soil nitrogen and phosphorus leaching.To clarify the biological effects of fly ash(A),sepiolite (B)and zeolite (C)as barrier materials,pot experiments were conducted to study the effects of different barrier materials and their combinations on the yield,quality, nitrogen and phosphorus absorption of Chinese cabbage (Brassica campestris L.ssp.chinensis).Six treatments were set up in the experiment,including control (without barrier material added), fly ash, sepiolite, zeolite, composite material Ⅰ (A1B2C2 ), and composite material Ⅱ(A2B2C1).The amounts of these barrier materials added for each treatment were 10 g/kg.The amounts of fly ash,sepiolite and zeolite in composite material Ⅰ were 2 g/kg,4 g/kg and 4 g/kg respectively,and those of composite material Ⅱ were 4 g/kg,4 g/kg and 2 g/kg respectively.These barrier materials were applied before planting.The results showed that compared with the control（without barrier materials added),the yields of the Chinese cabbage under the treatments added with fly ash,sepiolite and zeolite were increased by 37.1%,15.7% and 42.8%,respectively.The contents of total chlorophyll and soluble sugar of the zeolite treatment were significantly increased by 8.0% and 6.9%,but the nitrate nitrogen (NO₃^--N) content was significantly decreased by 12.8%.The soluble protein content of the fly ash treatment was significantly increased by 7.9%,but the contents of soluble sugar,vitamin C and NO₃^--N were significantly decreased.The contents of total chlorophyll, soluble protein and NO₃^--N of the sepiolite treatment were significantly decreased.The treatments of zeolite and fly ash improved nitrogen accumulation amount by 26.7% and 22.7% in Chinese cabbage respectively,but reduced phosphorus accumulation amount by 11.8% and 21.7%,and soil available phosphorus content were increased by 19.8% and 18.9%.The accumulation amount of nitrogen and phosphorus and soil ammonium nitrogen content under the sepiolite treatment were significantly decreased by 16.3%,12.4%,and 63.2%,respectively.There was no significant increase in the yield of Chinese cabbage under the two composite materials treatments; moreover, nitrogen and phosphorus accumulation amount under the A2B2C1 treatment was significantly reduced,but the NO₃^--N content was significantly increased.Taken together,these results indicated that the treatments of zeolite and fly ash increased the yield of Chinese cabbage,and improved the quality to a certain extent.These two barrier materials also promoted nitrogen absorption by Chinese cabbage,but inhibited phosphorus absorption.The sepiolite treatment also promoted the yield of Chinese cabbage,but had an inhibitory action on nitrogen and phosphorus absorption.Two composite materials treatments had no good application effects on Chinese cabbage.

Effects of Ridging Cultivation on Yield,Disease,Soil Microenvironment and Mechanized Harvesting of Carrot

YI Zehui, WANG Shengwu, JIANG Haofu, ZHAO Jing, LIU Zhao, MAO Liping

2021, 50(4): 124-130. DOI: 10.15933/j.cnki.1004-3268.2021.04.016

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In order to improve the benefit of carrot cultivation,Hongying No.2 was used as experimental material,and two treatments of flat cultivation and ridging cultivation were set up to analyze the effects of ridging cultivation on yield,benefit,irrigation water utilization ratio,irrigation water economic benefit,disease,soil micro-environment and mechanized harvesting of carrot.The results showed that ridging cultivation could obviously promote the growth of carrot plants.Compared with the flat cultivation,the total yield,first-grade yield,irrigation water utilization ratio and irrigation water economic benefit of ridging cultivation were significantly improved by 12.72%, 55.75%, 12.70% and 23.69% (P<0.05),respectively, and the increase in income reached 20 873.3 Yuan/ha.Ridging cultivation could significantly reduce the occurrence of carrot diseases, and the total incidence rate was significantly reduced by 15.18 percentage points compared with flat cultivation.The microenvironment of rhizosphere soil could be improved by ridging cultivation model.The soil water content,total nitrogen content, total phosphorus content, invertase activity, urease activity and phosphatase activity were significantly increased by 8.82%,48.72%,15.09%,69.67%,29.57% and 58.62% (P<0.05);theamount of soil microorganisms increased significantly by 5.65 times and the value of bacteria/ fungi and actinomycetes/fungi were increased significantly by 66.67% and 185.71%,respectively(P<0.05);soil bulk density and organic matter content of carrot rhizosphere soil were significantly reduced by 10.37%and 13.13%, respectively (P<0.05).Ridging cultivation was more suitable than flat cultivatiom for mechanized harvesting of carrots. Compared with the flat cultivation,the harvest rate of ridging cultivation was significantly increased by 13.89%,and the rate of damaged carrot due to mechanized harvesting was significantly reduced by 0.87 percentage points(P<0.05). In summary,ridging cultivation could improve soil micro-ecological environment,increase carrot yield,commercialization,irrigation water utilization rate and reduce disease occurrence, which should be widely promoted as a high-yield, high-quality, highefficiency and mechanized cultivation model of carrot.

Morphological Characteristics of Pollen of Five Ornamental Plant Cultivars of Salvia L.

FENG Qi, TIAN Lin, ZHAO Yixuan, ZHANG Jiapei, LIU Dongyun

2021, 50(4): 131-136. DOI: 10.15933/j.cnki.1004-3268.2021.04.017

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In order to clarify the pollen morphological characteristics of some ornamental plants of the Salvia L.genus, and explore the degree of evolution and the relationship between them, the pollen morphology of 5 species (Salvia microphylla‘Hot Lips’,Salvia leucantha,Salvia greggii,Salvia elegans and Salvia splendens‘Vista Purple’) of Salvia L.was observed by scanning electron microscopy and according to the principal component scores of polar axis length, equatorial axis length and germinal groove width cluster analysis was conducted.The results showed that the pollen of five species of Salvia L.was single-grain,the polar axis length of pollen was 65.70—120.71 μm,the range was large, the largest pollen grain was Salvia splendens‘Vista Purple’,and the smallest was Salvia leucantha.The P/E values of five pollens were between 1.15 and 1.23,all of which belonged to oblong pollens.The pollen of the genus Salvia L. had 6 germination grooves,and the outer wall was decorated with large and small meshes or three sets of reticulation,and the shape of mesh was mostly irregular polygon.The ridges were thick.According to the evolution rule of pollen surface decoration and pollen volume size,Salvia splendens ‘Vista Purple’ was far from the other four species,and the degree of evolution was high,while Salvia leucantha and other species were more primitive species. At the genetic distance of 2.0,5 species of Salvia L.were divided into three groups.Salvia microphylla‘Hot Lips’,Salvia elegans and Salvia greggii were divided into one group,while Salvia leucantha and Salvia splendens‘Vista Purple’were separately divided into one group.

Prediction of the Distribution of Taihangia in China Based on MaxEnt Model and Climate Change Scenario

LI An, GAO Mengmeng, CHEN Xi, ZHANG Xiaohua, LI Jingbo, LI Liangtao

2021, 50(4): 137-146. DOI: 10.15933/j.cnki.1004-3268.2021.04.018

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In order to clarify the introduction and propagation range of Taihangia, and find the appropriate threshold of environment factors for Taihangia growth,this paper based on MaxEnt model and ArcGIS geographic information system simulated the distribution of Taihangia in the current and future climate conditions.The results showed that the suitable areas of Taihangia were roughtly E112.8°—115.3°,N33.2°—37.8°,of which the high suitable area was 800 km² ,mainly distributed at the border of three provinces of Hebei, Henan and Shanxi.Precipitation was the main factor that dominated the distribution of Taihangia.The model had an AUC value of 0.996.The model fitted well and the prediction results had high credibility.We should pay close attention to high-suitability areas and carry out appropriate protection and breeding work based on this.In the future climate conditions,the suitable area of Taihangia will increase first and then decrease.It is suggested that relevant departments should take protective measures as soon as possible.

Expression and Purification of CRISPR/Cas13a and Establishment of Its Collateral RNase Activity Assay

WANG Xun, ZHANG Yuhang, SUN Yaning, LI Qingmei, LI Ge, WANG Li, GUO Junqing, DENG Ruiguang, ZHANG Gaiping

2021, 50(4): 147-153. DOI: 10.15933/j.cnki.1004-3268.2021.04.019

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To estabish LwaCas13a molecular detection platform,LwaCas13a was expressed in E.coli and its collateral RNase activity was determined.The recombinant plasmid pET His6-TwinStrep-SUMOLwaCas13a was transformed into Rosetta(DE3) competent cells to induce expression.The results of SDSPAGE electrophoresis and Western blot analysis showed that the soluble LwaCas13a recombinant protein was successfully expressed. Optimization of inducing temperature and time showed that maximal production of LwaCas13a was obtained at 15 h after induction at 16℃ .The expressed LwaCas13a recombinant protein was purified by nickel column affinity chromatography to obtain a single target band with ideal purification effect.In addition,the collateral RNase assay was also established with in vitro synthesized CRISPR RNA and target RNA.The assay could be performed within 30 min at constant 37℃ ,with the detection limit of 31.2 nmol/L.To sum up,the LwaCas13a was successfully expressed in E.coli Rosetta (DE3).Purified LwaCas13a exhibited ideal collateral RNase activity,and the collateral RNase assay for LwaCas13a was optimized and established.

Establishment and Evaluation of E^rns/E2 Fusion Protein-Based Indirect ELISA for Detection of Classical Swine Fever Virus Antibody

WANG Li, FENG Lili, ZHANG Yuhang, SUN Yaning, YANG Jifei, ZHAO Dong, WANG Ruining, LI Xuewu, GUO Junqing, ZHANG Gaiping

2021, 50(4): 154-161. DOI: 10.15933/j.cnki.1004-3268.2021.04.020

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In order to establish a method for the detection of serum antibody against classical swine fever virus(CSFV),the nucleic acid sequence encoding the major antigenic region of co-expressed CSFV E^rnsand E2 was cloned into plasmid pET-32a,the recombinant bacteria were obtained by transforming Rosetta TM2(DE3) with positive recombinant plasmid.SDS-AGE,Western blot and solubility analysis indicated that the molecular weight of expressed Erns/E2 protein was about 43 ku,could be recognized by CSFV positive serum and was mainly in the form of inclusion bodies.The purified inclusion bodies protein was refolded by rapid dilution method,and the recovery efficiency was more than 40%.The purified and refolded protein of E^rns/E2 was used as the coating antigen to develop an indirect ELISA ( E^rns/E2-ELISA)for detecting CSFV antibody after optimization of reaction conditions. Results from the specificity,sensitivity and reproducibility tests showed that the Erns / E2-ELISA were specific, sensitive and roproducible.A total of 121 serum samples were detected by the Erns/E2-ELISA and IDEXX CSFV antibody test kit,the E^rns/E2-ELISA coincidence rate,sensitivity and specificity were 86.78%,92.11% and 77.78%,respectively.Four 30-day-old healthy piglets were immunized with C strain vaccine and the fluctuation dynamics of antibody was evaluated by the E^rns/E2-ELISA and IDEXX CSFV antibody test kit.The antibody can be detected as early as 7 days and 14 days respectively,reaching the highest level on the 35th—42th days and finally stabilizing on the 49th—56th days.

Factor Analysis and Path Analysis of Body Size and Slaughter Characters in Qingyuan Partridge Chickens

DONG Jingchao, GAN Jiankang, LI Hua, ZHANG Zhengfen, KANG Huimin

2021, 50(4): 162-168. DOI: 10.15933/j.cnki.1004-3268.2021.04.021

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To reveal the relationship between body measurements and slaughter traits, and further investigate the direct and indirect effect sizes of body measurements on slaughter traits in Qingyuan partridge chickens, 125-day-old Qingyuan partridge chickens were slaughtered and their body measurements (crown height, shank length, shank circumference, body length and keel length) and slaughter traits(body mass,carcass mass and eviscerated mass) were determined,including 98 roosters and 62 hens. The results of factor analyses showed that three relatively independent factors extracted from five body measurements and three slaughter traits were closely related to mass,height and width, named weight factor,longitudinal body structure factor and transverse body structure factor.The contributions of each factor for roosters were 44.027%, 19.256% and 10.778%, respectively, and for hens were 47.641%,20.685% and 10.617%,respectively.The results of path analyses showed that hank girth had the largest effect on the body mass,carcass mass and eviscerated mass of roosters,with the direct effeck sizes of 0.492,0.383 and 0.281,respectively.In hens, shank circumference had the largest effect on body mass and carcass mass,with the direct effects of 0. 473 and 0. 472,respectively. Shank length had the largest effect on eviscerated mass,with the direct effect size of 0.397.The results of this study provide a new perspective and theoretical basis for precise breeding and rational development of Qingyuan partridge chickens.

Effect of Feeding Phoxim on the Alkaline Phosphatase Activity in Different Tissues of Bombyx mori

YANG Weike, TANG Fenfen, YANG Hai

2021, 50(4): 169-173. DOI: 10.15933/j.cnki.1004-3268.2021.04.022

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In order to investigate the effect of phoxim on alkaline phosphatase(ALP) activity in different tissues of silkworm,the spectrophotometry was used to detect the changes of ALP activities in fat body,hemolymph,midgut and silk gland of Bombyx mori after feeding phoxim(in the control group sterile water was fed).The results showed that the midgut ALP activity was gradually increased at 6 h after feeding phoxim and reached a maximum value at 15 h,but it decreased sharply and was significantly lower than that of the control group at 24 h.The ALP activities in hemolymph and fat body were increased at 12 h and 15 h respectively after feeding phoxim and both of them reached higher level at 18 h.However,the ALP activity was inhibited at 21 h and 24 h and significantly lower than that of the control group.In silk gland,the ALP activity began to decline at 9 h and continued to decrease with the extension of treatment time.The enzyme activity decreased to 13% of the control group at 24 h and the difference between phoxim treatment group and control group reached an extremely significant level. The above results indicated that the ALP activity was inhibited to a certain extent in the silk gland after treatment with phoxim,while it showed a trend of increasing first and then decreasing sharply in the fat body,hemolymph and midgut of silkworm.It suggests that the ALP in fat body,hemolymph and midgut of silkworm may play an important role in the metabolizing of phoxim.

Image Recognition of Wheat Leaf Diseases Based on Lightweight Convolutional Neural Network and Transfer Learning

FENG Xiao, LI Dandan, WANG Wenjun, ZHENG Guoqing, LIU Haijiao, SUN Yongsheng, LIANG Shan, YANG Ying, ZANG Hecang, ZHANG Hui,

2021, 50(4): 174-180. DOI: 10.15933/j.cnki.1004-3268.2021.04.023

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In order to realize the convenient recognition of wheat leaf disease image on mobile terminal,image recognition model of wheat leaf disease was established based on lightweight convolutional neural network (CNN) and transfer learning.Firstly,a sample data set of 224 pixel×224 pixel was established,which was composed of images of wheat powdery mildew,stripe rust and leaf rust. Secondly,the deep learning framework Tensorflow 2.0 was used to build the wheat leaf disease image recognition model based on MobileNetV2, and the trained parameters on ImageNet data set were used as the initial parameters of the model. Finally,the effects of transfer learning method,sample size,adding Dropout layer before global average pooling and initial learning rate setting on the model performance were analyzed.The results showed that the average recognition accuracy of the images of three wheat diseases was as high as 99.96% by setting all layers of the model as trainable,selecting suitable data enhancement method to increase the sample size, adding Dropout layer before global average pooling and setting the initial learning rate of 0.000 01.Based on MobileNetV2 and transfer learning, a wheat leaf disease image recognition model with high recognition accuracy and strong generalization ability could be constructed for mobile terminal.

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