Journal of Henan Agricultural Sciences

Application Advance of Virus-Induced Gene Silencing Technology in Dicotyledons

ZHANG Qinqin, JI Zhaolin, ZHU Feng

2020, 49(2): 1-8. DOI: 10.15933/j.cnki.1004-3268.2020.02.001

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Virus-induced gene silencing(VIGS) is one of the most important methods that can quickly identify gene function,and it is a kind of post-transcriptional gene silencing phenomenon at the level of RNA.Because of its lots of benefits,such as short experimental period,simple operation method,low cost,high efficiency,fast phenotype and high throughput,it has been widely used by researchers for researching plant gene function.This paper further explains the mechanism,application,problems and prospects of VIGS technology in dicotyledons including Solanaceae,Leguminosae,Rosaceae,Cucurbitaceae,Malvaceae and Compositae,which will provide a reference for further expanding the application of VIGS.

Effect of Nitrogen of Organic Manure Replacing Chemical Nitrogenous Fertilizer on Growth,Nitrogen Use Efficiency and Yield of Maize in Hetao Irrigation Area

YANG Yang, CUI Chao, MA Guangquan, HANG Guoqing

2020, 49(2): 9-16. DOI: 10.15933/j.cnki.1004-3268.2020.02.002

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Aiming to the Hetao irrigation area’ s current situation of long-term excessive application of chemical fertilizer and less application of organic fertilizer,this paper discussed the effect of replacing chemical fertilizer nitrogen with organic fertilizer nitrogen on the growth and development,nitrogen efficiency and yield of spring maize,which provided the theoretical basis for exploring the feasibility of combining chemimal fertilizer nitrogen reduction and organic fertilizer nitrogen application in Hetao irrigation area and the proportion of fertilizer reduction.The effect of replacing chemical fertilizer nitrogen with organic fertilizer nitrogen on the growth and development,nitrogen efficiency and yield of maize in Hetao irrigation area of Inner Mongolia was discussed by using field experiments,and by setting up five fertilization models of no nitrogen fertilization(CK),farmers’ conventional fertilization(CF),reduced fertilization(TS),replacing 30% fertilizer nitrogen with organic fertilizer nitrogen on the basis of TS(MF),and replacing 50% fertilizer nitrogen with organic fertilizer nitrogen on the basis of TS(OF).The result revealed that the nitrogen fertilizer application could speed up the maize growth,and the method of replacing fertilizer nitrogen with organic fertilizer nitrogen(MF,OF) was more effective in promoting the growth of maize. In tasseling stage,the leaf area indexes of MF and OF treatments were the highest,which were 60.4%,11.1%,35.6%,and 60.1%,10.9%,35.3% higher than that of CK,CF and TS respectively.In mature period,the OF treatment’s dry matter accumulation was the highest,which was 2.6 times of CK,and was 45.1%,43.0% and 57.7% higher than that of CF,TS and MF treatments respectively.Under the condition of same N application,the nitrogen efficiency and yield of maize could be improved more by replacing chemical fertilizer nitrogen with organic fertilizer nitrogen than only applying chemical fertilizer nitrogen(TS treatment). In terms of photosynthetic characteristics of maize,the effect of MF treatment was more obvious.The yields of MF and OF treatments were not significantly different from that of CF treatment.But the outputs of MF and OF treatments were 162.1% and 161.7% higher than that of CK treatment,32.6% and 32.4% higher than that of TS treatment respectively.The results of comprehensive analysis showed that the growth rate,leaf area index,photosynthetic characteristics,nitrogen efficiency and yield of maize could be significantly improved by replacing part of chemical fertilizer nitrogen with organic fertilizer nitrogen under the same amount of nitrogen application.Replacing 50% chemical fertilizer nitrogen with organic fertilizer nitrogen was a reasonable way of increasing the yield of spring maize and increasing the efficiency of nitrogen fertilizer in Hetao irrigation area of Inner Mongolia.

Cloning and Genetic Transformation in Arabidopsis thaliana of Sulfate Assimilation Reductase Gene APR2 in a Purple-leaf Rapeseed Line

ZHANG Hong, LI Gang, HAN Fengying, ZHAO Fuyong

2020, 49(2): 17-26. DOI: 10.15933/j.cnki.1004-3268.2020.02.003

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The purple-leaf line XY-PL,an advanced recombinant inbred line,was screened out from the progenies of interspecific hybridization between Brassica napus and Brassica juncea,had performance of stable purple leaf phenotype,high anthocyanin content and good agronomic traits and could be potentially used as intermediates for high anthocyanin content breeding programs.To explore the relationship between the purple-leaf trait of XY-PL and its APR2,a key enzyme for sulfur assimilation and metabolism,applying homologous cloning method and XY-PL and green leaf Zhongshuang No.11 (ZS11) as materials three APR2 homologs were successfully isolated,designated as PL-APR2,GL-APR 2.1 and GL-APR2.2 respectively.Results of sequence analyses showed that they encompassed four exons and three introns,the encoded proteins each had a typical chloroplast transport peptide domain and shared 98.2%—99.1% identities among them,and had the closest phylogenetic relationship with APR2 from cruciferous plants.Compared with GL-APR2.1 and GL-APR2.2,PL-APR2 had three amino acid residues (51SLK53) deletion in the chloroplast transport peptide transporter domain,which would affect the transmembrane transport of its precursor proteins and the efficiency of sulfur assimilation and utilization.Overexpression of PL-APR2 in Arabidopsis thaliana demonstrated that all transgenic lines did not exhibit purple-leaf phenotype from generation T0 to T2 perhaps because of the presence of wild-type AtAPR2 in transformation receptor material.Next,the Arabidopsis apr2 mutant will be used as genetic transformation receptor for further function verification and the transport function of chloroplast transport peptide of PL-APR2 will be analyzed deeply.

Effect of Clipping Stage and Stubble Height on Quality and Yield of Herbage and Grain Yield of Ratoon Rice

PENG Ting, WANG Tongtong, LI Lingzi, CHEN Yalan, JING Lili, WANG Jiaqi, WANG Chengzhang, ZHANG Jing, TENG Yongzhong, ZHAO Quanzhi

2020, 49(2): 27-33. DOI: 10.15933/j.cnki.1004-3268.2020.02.004

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In order to alleviate the serious situation of land used for growing food and herbage crops,and develop the planting model of rice for both grain and feed,split plot design was carried out,clipping stage(heading-blooming stage,filling stage and wax maturity stage) was set as the main plot,stubble height(25 cm,35 cm and 45 cm) was set as the sub-plot,and the effect of clipping stage and stubble height on the yield,quality of dry herbage and silage of first-season rice and the grain yield of ratoon rice was studied.The results showed that the yield of dry herbage and silage of first-season rice increased,and the yield of ratoon rice decreased gradually with the delay of clipping time. Regarding to the stubble height,the yield of dry herbage and silage of the first-season rice decreased gradually with the increase of stubble height,while higher yield of ratoon rice was harvested when the lower of the stubble height in the headingblooming stage and higher of the stubble height in the filling stage and the wax maturity stage.The quality of dry herbage clipped at heading-blooming stage and silage clipped at wax maturity stage was the best,both reaching the first grade according to the standard.Taken together,two candidate cultivation technologies of rice served as food and herbage crops are recommended.First,ratoon rice is main,and high quality dry herbage is as a supplement.In detail,when first-season rice is clipped at heading-blooming stage with 25 cm stubble height,10.51 t/ha high-quality first-grade dry herbage and 10.58 t/ha ratoon rice will be harvested.Second,high quality silage is main,and ratoon rice is as a supplement.In detail,wihen firstseason rice is clipped at wax maturity stage with 25 cm stubble height,34.66 t/ha high-quality first-grade silage and 4.21 t/ha ratoon rice will be harvested.The above two cultivation technologies are new ways to realize the organic integration of planting industry and breeding industry.

Genome-Wide Identification and Expression Analysis of NBS-LRR Gene Family in Setaria italica

LI Renjian, SHEN Zheyuan, LI Xukai, HAN Yuanhuai, ZHANG Baojun

2020, 49(2): 34-43. DOI: 10.15933/j.cnki.1004-3268.2020.02.005

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The NBS-LRR family members of foxtail millet(Setaria italica)were identified,and their chromosome location,gene structure,conserved motif,phylogeny and expression level were analyzed,which laid a foundation for the application of NBS-LRR family genes in molecular breeding of disease resistance in Setaria italica.The results showed that 411 genes of NBS-LRR family were obtained,including 376 genes with CN(CC-NBS) structure,33 genes with CNL(CC-NBS-LRR) structure,1 gene with TIR(Toll interleukin-1 receptor) structure,and only 1 gene with NBS structure.Most of the NBS-LRR family genes were located on chromosome 8.There were four conserved sequences in the NBS domain of Setaria italica,and the family was divided into three groups in phylogeny.The gene structure was diverse,and Motif1 was the most conserved.Twenty-three NBS-LRR genes of Setaria italica were collinear with those of Setaria viridis,21 NBS-LRR genes of Setaria italica were collinear with those of Oryza sativa,1 NBS-LRR gene of Setaria italica was collinear with those of Arabidopsis thaliana.Among them, Seita. 8G088500 and Seita. 8G088400 were identified as high homologous genes to rice blast resistance genes.The Ka/Ks values of collinear genes between Setaria italica and Oryza sativa,Arabidopsis thaliana were less than 1,but among the collinear genes between Setaria italica and Setaria viridis,Ka/Ks values of Seita.6G014500 and Seita. 6G023500 were greater than 1.Expression profile analysis showed that the number of NBS-LRR genes highly expressed in the roots and panicles of foxtail millet was more,followed by the leaves,and the least in the shoots,indicating that the family genes may play an important role in the process of disease resistance in the roots and panicles.

Effects of Different Nitrogen Application Rate on Dry Matter Accumulation Characteristic and Yield of Potato

CUI Liang, KRISTIINA Laanemets, LIU Guanqiu, TERJE Tähtjärv, MATI Koppel, ZHOU Huanan, PAN Jiaquan, WAN Bo, MENG Lingwen

2020, 49(2): 44-51. DOI: 10.15933/j.cnki.1004-3268.2020.02.006

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Liaoshu No. 6 was used as material to investigate the effects of topdressing rate of N(N₁ :0 kg/ha;N₂ :60 kg/ha;N₃:105 kg/ha;N₄ :180 kg/ha;N₅ :225 kg/ha)on dry matter accumulation characteristics and yield of potato based on 750 kg/ha NPK (15∶15∶15) compound fertilizer as the base fertilizer through film mulched drip irrigation.The results showed that the dry matter accumulation and distribution ratio of potato leaf and stem increased with the increase of nitrogen application rate,and N₅ treatment had the highest values;the dry matter accumulation and distribution ratio of potato tuber increased first and then decreased with the increase of nitrogen application rate,and N₃ treatment had the highest values.The translocation amount and ratio of dry matter,average tuber weight,tuber weight per plant and yield of N₃treatment were the highest.The yield of N₃ treatment was 46.5 t/ha,which increased by 92.9%,78.2%,9.7%,18.6% compared with N₁,N₂,N₄,N₅ treatments.Correlation analysis showed that there were significantly positive relationships between dry matter accumulation,dry matter accumulation rate,dry matter translocation amount,dry matter translocation rate and average tuber weight,tuber weight per plant,yield.The above results indicated that the optimum N topdressing rate was 105 kg/ha.

Application of Simple Drying in Extraction of Large Amount of DNA from Rice Leaves

TIAN Mengxiang, GONG Yanlong, ZHANG Shilong, LEI Yue, HE Youxun, LI Jiali, YU Benxun, ZHANG Dashuang, YE Yongyin, YAN Zhiqiang

2020, 49(2): 52-57. DOI: 10.15933/j.cnki.1004-3268.2020.02.007

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In order to explore the feasibility of drying method instead of liquid nitrogen freezing method to grind rice leaves to extract DNA,firstly the amplification effects of DNA extracted from grinded rice leaves by low temperature drying under 50℃ and liquid nitrogen freezing were compared,and then the amplification effects of DNA extracted from leaf grinded by drying method under different temperature (50,60,70,80,90℃ ) were further analyzed to screen the best drying temperature.The results showed that the DNA extracted from leaf grinded by drying method under 50—70℃ could be effectively amplified,the effect was the best under 50℃ ,and there was no significant difference between the amplification effects of the DNA extracted from leaf grinded by drying method and liquid nitrogen freezing method.The drying method of rice leaves had many advantages,such as simple operation,low cost and strong practicability,which could replace the liquid nitrogen freezing method to prepare the leaves in the large-scale extraction of DNA,and had good popularization and application value.

Effects of Basal/topdressing Ratio and Application Time of Organic Fertilizer on Yield and Quality of Upper Tobacco Leaf and Root Growth

HE Xiaolei, GOU Zhenggui, LIU Daxue, YANG Shengyu, ZHAO Qichao, YANG Shenghui, WU Guangbin, XIONG Maorong

2020, 49(2): 58-67. DOI: 10.15933/j.cnki.1004-3268.2020.02.008

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To alleviate the problem of soil nutrients imbalance and lower yield and quality of the upper tobacco leaves caused by tobacco continuous cropping in Pingtang County,the reasonable application time and basal/topdressing ratio of organic fertilizer were explored. Field experiments were carried out using Yunyan 87,the main cultivar as material,to investigate the effects of application time(20 and 30 days after transplanting) and basal/topdressing ratio levels(4∶1 and 3∶2) on root dry matter,root volume,enzyme activities of soil and the yield and quality of upper leaves,with 100% organic fertilizer as base fertilizer (applied when ridging) as control (CK) in 2016 and 2017.The results showed that the enzyme activity of soil was significantly increased 70 days after transplanting in the T1 treatment(20% organic fertilizer application at 20 days after transplanting)compared with control.In detail,the urease activity of soil was increased by 16.59%(2016) and 14.17%(2017) respectively,while the increase rate was 1.77% (2016) and 1.73%(2017) for phosphatase activity.Besides,the tobacco root development index of T1treatment was significantly higher than that of control.The vitality of tobacco adventitious root was increased by 11.37%(2016),9.76%(2017) respectively,the weight of root dry matter was increased by 30.02% (2016) and 19.96% (2017) respectively, and the increase rate was 32. 84% (2016) and 32.69%(2017) for root volume.The score of appearance quality and smoking quality,yield and output were maximal in T1 treatment,and there was significant difference between T1 treatment and control.In conclusion,the T1 treatment had the best effect on improving soil enzyme vitality and promoting the development of tobacco plants,and the yield and quality of upper leaves were significantly better than that of control.Thus,the T1 treatment,20% organic fertilizer application at 20 days after transplanting,contributed to improvement of the yield and quality of Yunyan 87 upper tobacco leaves in Pingtang County.

Effects of Biochar Addition in Soil on the Growth of Mung Bean under Salt-Alkali Stress

ZHANG Shengye, FU Dongbo, LIU Huimin, WANG Bai, LIAO Meifang, LI Zuotong, ZHAO Changjiang

2020, 49(2): 68-73. DOI: 10.15933/j.cnki.1004-3268.2020.02.009

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In the study,mung bean cultivar Lüfeng 2 was used as test material,and no biochar treatment was regarded as control group (0%,CK). And the other two were 10% and 20% biochar treatments.Three different kinds of sodium bicarbonate solution (0,100,200 mmol/L) were used for root treatment,and then the growth and physical and chemical indicators of mung bean were determined at flowering and pod-forming stage,in order to study the effects of different proportions of biochar on the growth of mung bean seedlings under salt-alkali stress,which would provide a basis for the restoration,exploitation and utilization of saline-alkali land and the high-quality and high-yield cultivation of mung bean in salinealkali land.The results indicated that the mung bean biomass,plant height,leaf area,chlorophyll content,Fv/Fm and root morphological indexes decreased significantly in CK group under salt-alkali stress,and the Na⁺content in mung bean plants increased markedly while the contents of K⁺, Ca²⁺and Mg²⁺significantly reduced.The addition of biochar had a positive effect on the growth and ion accumulation of mung bean plants under salt-alkali stress,especially the 20% biochar treatment in which the root dry mass,aboveground dry weight,plant height and leaf area increased by 75.00%,19.72%,54.82% and 123.10%,and the root length,total root surface area and root tip number increased by 57.85%,488.50%and 227.83% under 200 mmol/L sodium bicarbonate solution,while the average diameter of the roots did not change significantly.In addition,the addition of biochar increased the chlorophyll content and Fv/Fm value of mung bean leaves,and increased the contents of K⁺,Ca²⁺and Mg²⁺ ,and decreased the Na⁺content.Obviously,the addition of biochar can improve the morphology of the roots,shoots and leaves of mung bean plants,improve the leaf photosynthesis and root functions,and promote the accumulation of mung bean biomass,which will lay a foundation for later mung bean yield under saline-alkali stress.Overall,20% biochar treatment was better than 10% biochar treatment.

The Final Residues and Degradation Dynamics of 1.5% Matrine·Osthol AS in Tomato and Soil

CHEN Yan

2020, 49(2): 74-80. DOI: 10.15933/j.cnki.1004-3268.2020.02.010

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The difference of degradation dynamics and final residues of 1.5%matrine·osthol AS in tomato and soil under open field and greenhouse conditions was studied,in order to provide theoretical references for pesticide usage in tomato and to improve safety procedure measures.The degradation dynamics and final residues were determined by HPLC with PSA solid phase extraction after 1.5%matrine·osthol AS was applied on tomato and soil in open field and greenhouse conditions. The results showed that,in two cultivation patterns the half-lives of degradation of matrine were 5.18—6.70 d in tomato and 7.45—8.08 d in soil,the half-lives of osthol were 1.70—1.99 d in tomato and 2.30—2.67 d in soil.The sampling time at 1,3,7 d after last pesticide application,the final residues of matrine in open field and greenhouse were ND—0.427 4 mg/kg and 0.010 2—0.522 8 mg/kg in tomato,and the final residues of osthol in open field and greenhouse were ND—0.388 7 mg/kg and ND—0.395 2 mg/kg in tomato,respectively.Matrine and osthol are easily degradable pesticides.The half-lives of degradation of matrine and osthol in greenhouse were longer than those in open field.With few exceptions,the final residues in tomato and soil in greenhouse were generally higher than in open field under the same application dosage,times and sampling interval.

Screening and Identification of High-efficiency Potassium-solubilizing Bacteria and Their Growth-promoting Effects on Plants

XI Beibei , YE Jianren

2020, 49(2): 81-88. DOI: 10.15933/j.cnki.1004-3268.2020.02.011

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In order to screen bacteria with strong potassium-dissolving activity, and to alleviate soil potassium deficiency, five strains of potassium bacteria ( SK1-7, SK2-3, SK2-7, SK3-3, SK3-10) with strong potassium-dissolving activity were isolated and screened from forest soil. SK1-7,SK2-3,SK2-7,SK3-3, SK3-10 were identified as Bacillus aryabhattai, Ensifer adhaerens, Klebsiella singaporensis,Microbacterium maritypicum and Raoultella ornithinolytica respectively by morphological,physiological and biochemical experiments and 16S rRNA gene sequence analysis. Potassium ion contents in fermentation filtrate of the 5 strains of potassium-solubilizing bacteria were 42.70,50.40,49.70,41.50,45.23 μg/mL,respectively after 7 days of incubation in the medium,and the pH values of fermentation filtrate were also significantly reduced. The growth-promoting test of 5 strains of potassium-solubilizing bacteria on tomato showed that,the fermentation filtrate of SK1-7,SK2-3,SK2-7 could improve the germination rate of tomato seeds significantly or extreme significantyl.Tomato plant height,fresh weight and dry weight increased after treatment of tomato plants with SK1-7,SK2-3,SK2-7 for 40 d.Biological activity difference tests showed that 5 strains of potassium-solubilizing bacteria could secrete IAA;the fermentation filtrate of SK2-7 and SK3-10 had antibacterial effects;SK1-7 exhibited stronger ability of secreting protease.

Evolution and Expression Divergence of the Chitinase Gene Family against Leptosphaeria maculans and Sclerotinia sclerotiorum Infection in Brassica napus

XU Wen, ZHOU Tengsheng, LU Dandan, AN Sufang, HOU Jinna, LI Genyi

2020, 49(2): 89-103. DOI: 10.15933/j.cnki.1004-3268.2020.02.012

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Blackleg and sclerotinia stem rot caused by Leptosphaeria maculans and Sclerotinia sclerotiorum respectively are two major diseases in rapeseed worldwide,which cause serious yield losses. Chitinases are pathogenesis-related proteins and play important roles in host resistance to various pathogens and abioticstress responses.However,a systematic investigation of the chitinase gene family and its expression profile against L.maculans and S.sclerotiorum infection in rapeseed remains elusive. In this study,68 chitinase genes were identified in Brassica napus genome.These genes were divided into five different classes and distributed among 15 chromosomes.Evolutionary analysis indicated that the expansion of the chitinase gene family was mainly attributed to segmental and tandem duplication.Moreover,the expression profile of the chitinase gene family was investigated using RNA sequencing (RNA-Seq) and the results revealed that some chitinase genes were both induced while the other members exhibited distinct expression in response to L.maculans and S.sclerotiorum infection.Thus,the functions of different chitinase gene family members have diverged during long-term evolution and might exhibit different roles against different disease stresses in Brassica napus.

Identification and Optimization of Fermentation Conditions of an Antagonistic Endophytic Fungus from Mountain-cultivated Ginseng

ZHOU Chunyuan, XU Shiquan, YAN Meixia, CUI Lili, HUA Shuang, WANG Yingping

2020, 49(2): 104-110. DOI: 10.15933/j.cnki.1004-3268.2020.02.013

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The antagonistic endophytic fungi were isolated and screened from healthy mountain-cultivated ginseng leaves,to provide good strains for the biological control of ginseng black disease.Tissue isolation method was used to isolate the endophytic fungi.The antagonistic fungi was identified based on morphological characteristics and phylogenetic analyses of 18S rDNA.The single factor experiment and orthogonal experiment were adopted to optimize the liquid fermentation medium and conditions.The results showed that endophytic fungi were isolated from healthy mountain-cultivated ginseng leaves.Among them,strain FS-01 was found to have strong antagonistic activity against Alternaria panax.Strain FS-01 was identified as Chaetomium globosum.The optimal medium was composed of 1.50% starch,1.00% yeast extract powder,0.05% dipotassium phosphate,0.10% magnesium sulfate and the optimal culture conditions were inoculation size of 6.0% and cultivated for 5 d at 30℃ with initial pH of 7.0.Strain FS-01 has strong antagonistic activity against Alternaria panax,and could be a biocontrol strain for developing against ginseng black disease.

Effects of Mulching with Solanum photeinocarpum Straw on Cadmium Accumulation of Cyphomandra beracea Seedlings

LIAN Huashan, LI Hongyan, LIN Lijin

2020, 49(2): 111-115. DOI: 10.15933/j.cnki.1004-3268.2020.02.014

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To study the effects of mulching with Solanum photeinocarpum straw on cadmium (Cd)accumulation of Cyphomandra beracea seedlings,the pot experiment was carried out by mulching roots,stems and leaves straw of S.photeinocarpum on soil under Cd stress.The results showed that mulching straw on soil increased the biomass and photosynthetic pigment content of C.beracea seedlings,decreased the Cd contents in roots and shoots of C.beracea seedlings.Compared with the control,mulching with the root,stem and leaf straws of S.photeinocarpum decreased the Cd contents in roots of C.beracea seedlings by 25.32%,15.71% and 21.73%,respectively,and decreased the Cd contents in shoots by 13.73%,10.50% and 11.76%,respectively.In addition,mulching straw also increased the Cd translocation factor of C.beracea seedlings,the soil pH value increased significantly,but the exchangeable Cd content decreased.Therefore,mulching S.photeinocarpum straw on soil promoted the growth of C.beracea seedlings,increased the photosynthetic pigment content,and reduced the Cd contents in roots and shoots,and the root straw was the best.

Change of Growth and Physiological Characteristics of Daylily under Mixed Salt Stress

HAN Zhiping, ZHANG Haixia, ZHOU Guiling, LI Xia

2020, 49(2): 116-122. DOI: 10.15933/j.cnki.1004-3268.2020.02.015

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To reveal the physiological response of daylily plant to salinity,the experiment studied the change of growth and some physiological indicators of Datong daylily under the mixed salt stress of isoconcentration of NaCl and Ca(NO₃)₂ in sand culture by irrigating the nurtrient solution.The results showed that the morphological indexes,biomass and leaf water content of daylily decreased obviously,root water content was relatively stable with the increase of salt concentration(0—250 mmol/L),and some plants were dead under 200 and 250 mmol/L mixed salt stress on the 20th day after treatment.With the increase of salt concentration, the plasma membrane permeability and proline content increased significantly during treatment, the AsA content decreased gradually on the 5th day of treatment, and showed the change of increase-decrease after 10 d.The content of soluble sugar decreased gradually on the 5th day of treatment,decreased significantly after 10 d,while the content of soluble protein was almost unchanged.The research illustrated that the membrane lipid peroxidation aggravated, and the water content in the aerial part decreased under mixed salt stress,which inhibited the growth of daylily plant.But the salt tolerance of daylily plant was strong,and the damage caused by salt stress could be partly alleviated by promoting the synthesis of antioxidants and organic osmolytes.Daylily could still grow normally under 150 mmol/L mixed salt stress, and the plant life was threatened beyond this concentration,and 200 mmol/L was the lethal salt concentration for daylily.

Effects of Nitrogen Reduction under Alternate Root-Zone Irrigation on Grape Growth,Yield and Fruit Quality

CHEN Linan, , LIU Xiuchun , HAN Xiaori , SUN Zhanxiang , RONG Chuansheng

2020, 49(2): 123-129. DOI: 10.15933/j.cnki.1004-3268.2020.02.016

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The purpose of this study was to discuss the effect on yield and fruit quality of grape by coupling of alternate irrigation method and nitrogen reduction. 4 years old Liaofeng grape(Vitis vinifera ‘Liaofeng’)trees were taken as experimental material.The experiment was conducted with two irrigation methods:alternate partial root-zone drip irrigation(AI) and conventional irrigation(CI),three N applied rates:no N fertilizer applied(NN,0 kg/ ha N),recommended N rate(RN,100 kg/ha N) and farmer practice N rate(FN,200 kg/ha N).New shoots growth,redundant growth,leaf photosynthetic characteristics were observed.The results showed that there was a significant interaction between the irrigation methods and N applied rates.The new shoot length of CIFN treatment was the highest,which was 11.9% and 6.8% higher than that of AIRN and AIFN, respectively.At the same irrigation mode,the pruning amount of new shoots of two N applied treatments was higher than NN treatment(P<0.01).The total pruning amount of AI(average of all N treatments) was significantly decreased by 21.1% compared to CI.Pn of AI increased compared to CI,Tr of AI was significantly lower than CI.iWUE of AI increased significantly.And iWUE of AI coupling with RN was highest,which increased by 17.6% and 34.0% compared to CIRN and CIFN.The yield,soluble sugar and Vc contents increased of AI.And sugar-acid ratio of AI coupling with RN was significantly increased.In conclusion,the synergistic effect of alternate root-zone irrigation and recommended nitrogen rate reduced the redundant growth of grape,created good photosynthetic characters, and promoted grape yield and quality.

Study on Salt Tolerance Identification Method of Tulip

LI Ronghua, WANG Yajun, FANG Zheng, ZHAO Bin

2020, 49(2): 130-135. DOI: 10.15933/j.cnki.1004-3268.2020.02.017

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In order to select suitable methods for salt tolerance identification of tulip,9 tulip varieties were identified by cultivation method and leaf disc method respectively.The results showed that the salt solution was Hoagland+0.6 mol/L NaCl by cultivation method,and the identification time was 18 days.The salt solution was MS+0.6 mol/L NaCl by leaf disc method,and the identification time was 4 days.Under the treatment of the two methods,the varieties with strong salt tolerance of tulips were both AD Rem and Ha Kuhn,while the varieties with weak salt tolerance were Word Favourite and Judith.The order of relative salt tolerance of 9 tulip varieties is basically the same by the two methods,among which cultivation method takes long time,high cost and requires a lot of manpower and material resources.Leaf disc method has the characteristics of short identification time,simple operation and low cost, and can be applied to the determination of large quantities of samples.

Secretory Expression of CSFV E^rns Protein in Baculovirus Expression System and Its Activity Analysis

WEI Qiang, BAI Yilin, CHAI Shujun, LIU Yunchao, SONG Yapeng, ZHANG Gaiping

2020, 49(2): 136-141. DOI: 10.15933/j.cnki.1004-3268.2020.02.018

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In order to obtain the CSFV E^rns protein for the application in ELISA,the gene sequence of the E^rns protein was optimized according to the codon bias of Spodoptera frugiperda cells.Firstly,the gene was inserted into the recombinant vector pFastBacl-gp67,which contains the gp67 signal peptide sequence.Secondly,the resulting plasmid pFastBacl-gp67-E^rns was transformed into the DH10Bac competent cells to get the recombinant bacmid through blue-white screening.Thirdly,the recombinant Bacmid-E^rns was transfected into the sf21 cells to generate the recombinant baculovirus.Finally,the sf21 cells were infected with the recombinant baculovirus.At three days post infection, culture supernatant was collected.The E^rns protein was purified by nickel-affinity chromatography.The expression level and purity of the protein were identified using SDS-PAGE and Western blot analysis.The glycosylation pattern of the purified Erns protein was analyzed using PNGase F.And the indirect ELISA was carried out to analyze the antigenicity of the protein.Consequently,the recombinant pFastBacl-gp67-E^rns plasmid was generated.And the Bacmid-E^rns was obtained using blue-white screening.The recombinant baculovirus was used for large-scale expression of E^rns.The secretory expression of Erns was achieved using signal peptide.It was shown that the purified E^rns was of high purity.And the Erns was glycosylated properly.The purified E^rns protein could be specifically recognized by the CSFV positive serum,indicating that the purified protein was of high activity.

Effects of Coptidis Powder for Detoxification and Flavomycin on Ileal Microflora in Broilers of Different Ages

YUAN Lin, LI Wanli, DU Chenhong, WANG Zhixiang, YI Baodi, WANG Mingfa

2020, 49(2): 142-149. DOI: 10.15933/j.cnki.1004-3268.2020.02.019

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In order to study the effect of coptidis antidotal powder (CPD) and flavomycin (FLA) on the structure of intestinal microflora in the ileum of broilers,one hundred and eighty one-day-old AA broilers were randomly divided into three groups:basal diet (CK),basal diet with 5 mg/kg FLA (FLA group),basal diet with 1.25 g/kg CPD (CPD group).The results showed that the diversity of ileal microflora decreased with the increase of age, and the abundance of operational taxonomic unit (OTU) in FLA and CPD groups at 35 days of age was significantly lower than that at 15 and 25 days of age (P<0.05).Compared with the control group,dietary FLA significantly decreased the relative content of 10 genera in ileal flora of 35-day-old broilers,such as Flavobacterium,Streptococcus and Amycolatopsis (P<0.05),and CPD significantly decreased the relative content of 6 genera in ileal flora of 35-day-old broilers,such as Klebsiella,Flavobacterium and Amycolatopsis (P<0.05).There were also significant differences in the metabolic pathway and protein function of ileum microbial community among different treatment groups.At 35 days of age,compared with FLA group,the signalling molecules and interaction function of ileal flora in CPD group increased significantly (P<0.05),while transcription and cytoskeleton decreased significantly(P<0.05).It could be seen that CPD supplement in broiler diet could regulate the structure of ileum microbiota, similar to FLA.The metabolic pathways of the functional genes in different microbial communities were found to vary with diet.

Effects of Hawthorn and Astragalus on Blood Biochemical and Immunological Indexes of Dairy Cows during Perinatal Period

LIU Guolin, TAO Jinzhong, BAI Lingrong, LI Xianglong, CHEN Yonghong, ZHANG Meng, LUO Fang, LI Yachao, GUO Yansheng

2020, 49(2): 150-158. DOI: 10.15933/j.cnki.1004-3268.2020.02.020

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To study the effects of hawthorn and Astragalus on the negative energy balance and immune function of perinatal dairy cows,blood biochemical and immune parameters of cows were measured after adding different doses of hawthorn and Astragalus.45 Holstein cows in 2—4 parity and healthy perinatal period(21 d before delivery—21 d after delivery) were chosed,and divided into group A,group B and group C randomly. Group A was fed a basic diet,groups B and C were added 100 g/(head·d) and 300 g/(head·d)hawthorn and Astragalus(hawthorn and Astragalus 1∶1),respectively.Blood was collected from the tail vein on 21 d (-21 d) before and 7 d (-7 d) before delivery,on the day of delivery(0 d),on 7 d(+7 d) and 21 d(+21 d) after delivery,and blood biochemical and immune parameters of cows in each group were measured.The results showed as followed:compared with group A,in terms of protein indicators,the total protein content of group B increased significantly by 44.10%,and globulin content increased significantly by 66.16% at +21 d.In terms of calcium and phosphorus indicators,the calcium content in group B increased significantly by 47.28% at +7 d.Calcium content increased significantly by 41.67% and phosphorus content increased significantly by 33.57% at +21 d.In terms of fat indicators,the content of non-esterified fatty acids in group C decreased by 27.16% at +7 d,and group B decreased significantly by 34.38% at +21 d.In terms of energy indicators,the glucose contents of group B and C increased significantly by 24.49% and 23.72% at 0 d,respectively.Ketone body content of group B decreased significantly by 25.93% at +21 d.In terms of immune indicators,the levels of immunoglobulin G in group B and C were significantly or extremely significantly increased(P<0.05 or P<0. 01),and the content of tumor necrosis factor alpha in group B was significantly or extremely significantly increased(P<0.05 or P<0.01) at -7 d,0 d,+7 d,and +21 d.In summary,the addition of 100 g/(head·d)hawthorn and Astragalus to perinatal dairy cow diets can regulate lipid, protein and calcium and phosphorus metabolism in postpartum dairy cows,reduce postpartum negative energy balance, reduce postnatal ketone body levels,and improve cow immunity.

Toxicity Analysis of the Extract of Yedahuang Powder

TIAN Qizhen, CAI Bingyan, CHENG Han, HAO Fuxing, HUANG Yinyun

2020, 49(2): 159-164. DOI: 10.15933/j.cnki.1004-3268.2020.02.021

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In order to explore the safety of Yedahuang powder extract in clinical application,Kunming mice were selected as the research object to carry out acute toxicity and subchronic toxicity tests.The median lethal dose(LD₅₀) and maximal tolerable dose(MTD) of Yedahuang powder extract were measured in acute toxicity test. 2,10 and 20 g/kg of Yedahuang powder extract were used in subchronic toxicity test,respectively. The mice were given extract by gavage for 35 days to observe the clinical manifestations,calculate the organindex,detect the blood routine, serum biochemical and immunological indexes,and observe the pathological changes of organ tissues to determine the toxicity.The results showed that the LD50 could not be determined in acute toxicity test,and the MTD was ≥120 g/kg.In the subchronic toxicity test,compared with the normal saline control group, the lymphocyte number,the secretion level of sIgA in respiratory tract and intestinal tract and the level of IgG in serum of mice in the 10 g/kg and 2 g/kg dose groups of Yedahuang powder extract were significantly increased(P<0.05),and there were no significant pathological changes in the heart, liver, spleen,kidney and other tissues of each group.The results show that the extract of Yedahuang powder is a kind of non-toxic substance, and it is a safe Chinese medicine preparation.

Changes of Alkaloids Content of Cigar Leaves with Different Maturities during Air-curing

ZHANG Jiawen, LU Shaohao, ZHAO Zhe, ZHONG Qiu, ZHANG Ruina, ZHAO Mingqin

2020, 49(2): 165-173. DOI: 10.15933/j.cnki.1004-3268.2020.02.022

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In order to investigate the changes of alkaloid content of cigar leaves with different maturities during air-curing,taking Shiyan No.1 as the test material,the changes of 9 alkaloids contents of cigar leaves with different maturities were studied,and the correlation analyses were carried out.The results showed that,the contents of nicotine,nornicotine,anatabine and cotinine first increased and then decreased during airing.The myosmine and 2,3′-bipyridine contents decreased first and then increased.The trend of changes in anabasine,N-methylanabasine contents was basically unchanged.In the dry tending stage,the contents of anatabine,myosmine and N-methylanabasine were the lowest in the unripe treatment,and the other alkaloids were the lowest in the ripe treatment.The nicotine conversion rate showed a upward first then downward trend in different maturity treatments during airing, and it showed overripe>unripe>ripe.Correlation analysis showed that the positive correlations were observed between nicotine,nornicotine,anatabine,anabasine and N-methylanabasine in different maturity leaves,and the ripe treatment was significantly correlated.The anatabine content was negatively correlated with myosmine but not significant.Cotinine content had a positive correlation with anatabine and β-nicotyrine contents, but was negatively correlated with N-methylanabasine content and not significant.

Mechanism of Extracting Wool Keratin by Molten Urea Method

SA Rula, ZHAO Xuemei, MIYAGAWA Tuyoshi, TANAHASHI Mitsuhiko

2020, 49(2): 174-180. DOI: 10.15933/j.cnki.1004-3268.2020.02.023

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In order to find out the principle of wool keratin dissolved in molten urea,SDS-PAGE method was used to determine the molecular weight of dissolved wool keratin firstly.Secondly,standard compounds such as vanillin,vanillyl alcohol,vanillic acid,acetyl vanillin,formyl vanillic acid,4-acetyl-3-formyl succinic acid,salicylanilide,3,3-dihydroxydiphenyl disulfide,aniline,tyramine acid were used to substitute the functional group and chemical bond in wool keratin,respectively,and react with urea at 150℃ for 20 min,separated 3 times with ethyl acetate,dehydrated and concentrated for the organic layer,dissolved with acetone,and analyzed by GC-MS.Aqueous layer was freeze-dried,ethylationed,concentrated for GC-MS analysis.The result showed that,the wool keratin with high molecular weight,75 ku,still maintained a high molecular state.The aldehyde group was detached,the ester bond was only deacetylated,the carboxyl group was detached or bound to urea,the alcoholic hydroxyl group and the amino group were combined with urea,and the peptide bond and the disulfide bond were not broken.Main approaches of the dissolution mechanism of wool in molten urea were hydrogen bond cleavage,functional group detachment,urea bonding to functional group,etc. The primary structure of wool keratin was not destroyed and still maintained a high molecular state.The amino and carboxyl groups of tyrosine reacted with urea to form a cyclized structure,which could be dissolved in an organic solvent and could be detected from the analysis of GC-MS.There were neither compounds that were converted from raw materials,nor the raw materials themselves in the aqueous layer of all standard compounds and urea reaction products,so improvement of experimental method was needed for further research.

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