Journal of Henan Agricultural Sciences

Research Progress on Plant DNA Methylation under Heavy Metal Stress

GUO Tianjiao, AN Penghu, WEI Zhuangzhuang, JIA Guotao, XU Xiaobo, WEI Gang, HUANG Wuxing

2021, 50(10): 1-9. DOI: 10.15933/j.cnki.1004-3268.2021.10.001

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DNA methylation often occurs at the fifth carbon position of cytosine，which is widespread in plants responding to heavy metal stress.The research progresses in plant DNA methylation mechanisms，the effects of different heavy metal stresses on DNA methylation，DNA methylation related research techniques，and the heritability of DNA methylation were reviewed，with a view to breeding of new varieties with stronger stress resistance.

Screening and Evaluation of Maize Varieties with High Yield and Nitrogen Use Efficiency in Huang‑Huai‑Hai Region

2021, 50(10): 10-17. DOI: 10.15933/j.cnki.1004-3268.2021.10.002

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Forty‑one summer maize varieties，which are composed of the main varieties and the varieties that will be spread in the future in maize production in Huang‑Huai‑Hai region，were selected as experimental materials.Two nitrogen（N）rates of 225 kg/ha（normal N rate）and 180 kg/ha（reducing 20% of normal N rate）were set up.The grain yields and N efficiencies of different maize varieties were analyzed under different N rates，maize varieties were divided into different types according to yield and N efficiencies，and maize varieties with high yield and N use efficiency were selected，so as to lay the foundation for reducing N fertilizer and increasing N use efficiency and protecting environment during maize production.The results indicated that Jingke 999（JK999）got the highest yield under the two N rates，and Zhongyu 303（ZY303）had higher yield. Shengyuan 96（SY96）got the lowest yield of 8.74 t/ha under normal N rate，while Longdan 639（LD639）got the lowest yield of 7.68 t/ha under reducing 20% of normal N rate.Cluster analysis showed that JK999 and ZY303 were the high‑yield varieties. The variation trend of N efficiency was consistent with yield of maize varieties under different N rates. The nitrogen efficiency classification result showed that，17 varieties including JK999 and ZY303 were high N efficiency type under both low and high N rates，5 varieties including Denghai 717（DH1717）and Youdi 919（YD919）were high N efficiency type under high N rate，5 varieties including Zhengdan 958（ZD958） and Jingnongke 728（JNK728）were high N efficiency type under low N rate. By considering the nitrogen efficiency and yield，JK999 and ZY303 could be used in large area as high‑yield and ‑N use efficiency varieties.

Validation of Molecular Markers Related to Pre‑harvest Sprouting Resistance in High‑Quality Wheat Varieties

LI Yaqing, ZHANG Nan, PENG Yifeng, ZHANG Shichang, LI Mengjun

2021, 50(10): 18-26. DOI: 10.15933/j.cnki.1004-3268.2021.10.003

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The molecular markers of six wheat(Triticum aestivum)genes[Tamyb10 ,TaDFR（dihydroflavone reductase），TaVp‑1(Viviparous‑1)，TaSdr(seed dormancy),TaPM19 ‑A1(plasma membrane 19‑A1）and TaMFT（mother of FT and TFL1)］related to pre‑harvest sprouting resistance was validated using seed germination indices of 42 high‑quality wheat varieties，so as to lay a foundation for the selection and breeding of high quality wheat varieties resistant to panicle germination. The results showed that two allelic variations for all the six genes(Tamyb10 ，TaDFR，TaVp‑1 ，TaSdr，TaPM19 ‑A1 and TaMFT)was identified in 42 high‑quality wheat varieties，and the proportion of favourite alleles was obviously different，which ranged from 4.8% to 78.6%，but the Vp‑1 Bb allele was not detected.The correlation analysis between allelic variations and germination indices showed that myb10D，MFT‑3A and MFT‑A2 were significantly correlated with pre‑harvest sprouting resistance，but the correlations between DFR‑B，Vp1B3，Sdr2A，Sdr2B，PM19‑A1 and pre‑harvest sprouting resistance were not significant.The STS marker myb10D could be applied in pre‑harvest sprouting resistance screening of red‑grain high‑quality wheat varieties，while the CAPS marker MFT‑3A and the STS marker MFT‑A2 could be applied in pre‑harvest sprouting resistance screening of white‑grain high‑quality wheat varieties.TaMFT maybe plays an important role in pre‑harvest sprouting resistance mechanism of high‑quality wheat varieties.

Effects of Planting Pattern and Sowing Date on Nitrogen Translocation and Yield of Winter Wheat

YU Meng, GU Xiaobo, LI Yuannong, CHEN Pengpeng, YANG Jinyu, LI Yupeng

2021, 50(10): 27-36. DOI: 10.15933/j.cnki.1004-3268.2021.10.004

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The field experiments were conducted to explore the effect of different sowing dates［normal sowing（D0：October 14），postponed sowing（D1：October 21；D2：October 28）］and planting patterns［ridge‑furrow with full film mulching（RM），flat planting with full film mulching（FM）and flat planting without mulching（NM）］on the dry weight，nitrogen accumulation，nitrogen transport rate，the contribution rate of nitrogen to grains and yield of winter wheat，so as to provide theoretical basis for the proper arrangement of sowing date in response to tight farming time，delayed autumn rain and climate change and realizing high and stable yield of winter wheat. The results showed that as a whole，proper postponed sowing date（D1）increased the dry weight of leaves and stems at flowering stage，nitrogen accumulation in leaves at flowering and mature stages，nitrogen transport rate in leaves（55.4%）and the contribution rate of nitrogen to grains（104.2%），but decreased the number of grains per spike，effective spikes number and yield. At the same sowing date，compared with NM treatment，RM and FM treatments generally increased the dry weight of winter wheat at all stages，improved the ear traits and yields，and the increase of yield of RM treatment was greater than that of FM treatment. RM treatment mainly increased the nitrogen accumulation in leaves，FM treatment increased the nitrogen accumulation in stems，and both increased the number of effective spikes and yields. The number of grains per spike of RMD0 treatment was the most，and the greatest 1 000‑grain weight was obtained in NMD0 treatment. The effective spikes number of FMD0 treatment was the highest，which was 54. 4% higher than that of NMD0 treatment.Compared with NMD0 treatment，the yield of all treatments under RM and FM increased significantly，FMD0 treatment increased by 74. 9%，RMD0，RMD2 and RMD1 treatments increased by 61. 3%，46.2% and 44.6% respectively.In conclusion，ridge‑furrow with full film mulching under postponed sowing date and flat planting with full film mulching under normal sowing date could ensure the high and stable yield of winter wheat.

Detection of Important Traits Genes with KASP Markers in Yumai 158 and Its Hard Variant Line

ZHAO Yongtao, ZHANG Feng, ZHANG Zhongzhou, YUAN Qian, ZHEN Shicong, WANG Junsen

2021, 50(10): 37-43. DOI: 10.15933/j.cnki.1004-3268.2021.10.005

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Yumai 158（national certification number：2014004）is a semi‑winter wheat cultivar，and it has outstanding performance with relatively large grains and better processing quality.A natural mutation line of Yumai 158 was found in field experiment，and it was designated as Yumai 158‑42. Compared with Yumai 158，Yumai 158‑42 exhibited increased grain hardness and improved processing quality，reaching the standard for high‑quality strong gluten wheat. To investigate the genetic basis of the improved traits of Yumai158‑42，genotyping was performed with KASP（kompetitive allele specific PCR） markers for important genes/loci related to plant height，photoperiod，vernalization，grain weight，stress resistance and quality.The results showed that，Yumai 158 and Yumai 158‑42 had the same genotypes for all the detected genes/loci except for the seed hardness genes and the major‑effect genes related to yellow pigment content. Yumai 158 had the hardness genes Pinb‑ D1 a，Pina‑ D1 a and Pinb2，while Yumai 158‑42 lost Pinb‑ D1 a and Pina‑ D1 a，only had Pinb2 ，and showed increased grain hardness compared with Yumai 158.In addition，Yumai 158 had the low yellow pigment content related gene Psy‑ D1 a，while Yumai 158‑42 had the high yellow pigment content related gene Psy‑ D1g.Yumai 158 and Yumai 158‑42 are valuable for researches on grain hardness and quality.

Effect of Floating Seedling Tray with Different Sizes on Seedlings Quality of Cigar and Yield and Quality of Tobacco Transplanted above Film

GAO Huajun, LIN Beisen, HE Yuan, CAI Bin, MA Xinghua, LI Fangyou, XIA Changjian, GENG Zhaoliang, XIANG Xiaohua, CHEN Dexin, LÜ Hongkun

2021, 50(10): 44-50. DOI: 10.15933/j.cnki.1004-3268.2021.10.006

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In order to select the floating seedling tray which can cultivate high‑quality cigar tobacco seedlings suitable for transplanting on film，the effects of seedling tray treatments of 162 pores（CK），136 pores and 78 pores on seedling quality，field growth，leaf yield，and economic benefit were studied.The results showed that the dry weight of root and whole plant，morphological index of root and strong seedling index of 78‑pores seedling tray were significantly higher than those of 162‑pores seedling tray，and the dry weights of root and whole plant were 79.55% and 61.84% higher than those of 162‑pores seedling tray，with 162 and 136 pores treatments. The 78 pores treatment could improve the survival rate and growth potential of transplanted tobacco plants in the field，also increase the input cost of seedlings，but the economic benefit was the highest，which was 17.55% higher than that of 162‑pores treatment.The seedling quality of 136‑pores seedling tray was relatively moderate，the field growth was better after transplanting，the input cost of seedlings was relatively lower，and the economic benefit was 1.57% higher than that of 162‑pores treatment. The root system of 162‑pores seedling tray was weak，and the index of strong seedling was the lowest.In general，78‑pores and 136‑pores floating seedling trays can be recommended for Hainan cigar tobacco seedling followed by transplanting on film.

Effects of Different Harvest Time on Yield and Quality of Cigar Leaves

LU Ruilin, LIU Lulu, HU Xi, YE Keyuan, ZHONG Qiu, ZOU Yuhang, SHI Xiangdong

2021, 50(10): 51-59. DOI: 10.15933/j.cnki.1004-3268.2021.10.007

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In order to explore the effects of different harvest periods on the yield and quality of cigar leaves，Dexue No.3 was used as the material to study the differences in water content，physical characteristics，conventional chemical composition content，neutral aroma components content after fermentation and economic traits of cigar leaves harvested in three harvest periods（The middle and upper leaves were harvested for the first time at 70 d and 77 d after transplanting，and then harvested every 10 d，a total of three times）through field experiments and laboratory tests. The results showed that the later harvest，the faster water loss rate of cigar leaves during modulation. When the middle leaves were harvested at 80 d after transplanting，the physical properties such as tensile force and thickness of the fermented leaves were better.In addition，the total nitrogen content decreased by 0.180 percentage points compared with that harvested at 70 d and the nicotine content increased by 0. 450 percentage points compared with that harvested at 90 d after transplanting. At this time，the coordination of chemical components of tobacco leaves was best，and the total amount of aroma substances in tobacco leaves was 17.68% and 5.58% higher than that of 70 d and 90 d after transplanting，respectively.When the upper leaves were harvested at 87 d after transplanting，the physical properties were better after fermentation.The nitrogen‑alkali ratio was 4.926，and the coordination of chemical components of tobacco leaves was best.Moreover，the total amount of aroma components was 16.23% and 1. 88% higher than that of 77 d and 97 d after transplanting，respectively. In addition，the output value of the middle leaves harvested at 80 d after transplanting was 16. 85% and 12.10% higher than that at 70 d and 90 d after transplanting，respectively. The output value of the upper leaves harvested at 87 d after transplanting was 13.57% and 16.52% higher than that at 77 d and 97 d after transplanting，respectively.Therefore，when the Dexue No.3 plants have been transplanted for about 80 d and 87 d，the middle and upper leaves should be harvested respectively to get the best physical characteristics and chemical composition.The cigar leaves also have adequate aroma amount，better sensory quality and the highest output value.

Effect of Stropharia rugosoannulata‑Maize Rotation on Straw Degradation，Soil Physico‑chemical Properties，Yield and Economic Benefit of Crops

LIU Gaoyuan, HE Ailing, DU Jun, XUE Guangyuan, YANG Huanhuan, YANG Zhanping, PAN Xiuyan, XU Jidong, ZHANG Yuting

2021, 50(10): 60-68. DOI: 10.15933/j.cnki.1004-3268.2021.10.008

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With the wheat‑maize rotation［WM，3 t/ha（WM3），6 t/ha（WM6）and 9 t/ha（WM9）maize straws were returned to the field］as control，the effects of Stropharia rugosoannulata‑maize rotation（SM，total maize straw input was 120 t/ha）on straw degradation rate，straw lignocellulosic components，soil physico‑chemical properties，crop yields and economic benefits were studied，so as to provide new ideasfor promoting straw conversion and improving soil fertility. The results showed that，the straw degradation rate of SM treatment was 73.1%，which significantly increased by 35.4—46.7 percentage points compared with WM treatments.Compared with WM treatments，the contents of cellulose，hemicellulose and lignin of residual straws of SM treatment significantly decreased by 40.7%—57.1%，29.9%—50.2% and 12.1%—17.3%，respectively.The soil water content of SM treatment significantly increased by 5.4—8.7 percentage points compared with WM treatments，soil bulk density of SM treatment significantly decreased by 9.4% and 6.5% compared with WM3 and WM6 treatments，and soil porosity of SM treatment significantly increased by 5.7 and 4.0 percentage points compared with WM3 and WM6 treatments.The contents of soil organic matter，available phosphorus and available potassium of SM treatment significantly increased by 17.7%—31.1%，42.2%—65.9% and 14.2%—24.5% compared with WM treatments，and soil alkali hydrolysable nitrogen content of SM treatment significantly increased by 18.5% compared with WM3 treatment. The yields of Stropharia rugosoannulata and maize of SM treatment were 33 543.1 kg/ha and 10 266.9 kg/ha，and maize yield of SM treatment significantly increased by 13.9%—22.9% compared with WM treatments，but maize yields were not significantly different among WM3，WM6 and WM9 treatments.The economic benefit of SM treatment increased by 96 600—99 100 Yuan/ha compared with WM treatments.In conclusion，Stropharia rugosoannulata‑maize rotation can not only promote straw degradation and improve soil fertility，but also increase economic benefit，so it is an effective measure to realize straw resources utilization and soil fertility improvement.

Isolation and Identification of Soil Bacterial Strains Metabolizing Propionic Acid

DU Cuiying, LU Jiayi, HU Lang, YAO Guoxin, DAI Yujun, LI Jing, ZHENG Cao

2021, 50(10): 69-75. DOI: 10.15933/j.cnki.1004-3268.2021.10.009

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Isolation and identification of soil bacterial strains that metabolize propionic acid can enrich the scientific understanding of the bacterial types which are able to metabolize propionic acid in nature.Through soil sample pre‑culture，solid plate culture by using propionic acid as the sole carbon source，aswell as morphological observation and the 16S rDNA sequences homologous analysis methods，the targetbacterial strains that were capable of metabolizing propionic acid were isolated and identified.Further，the key enzyme genes of 2‑methylcitrate cycle，the propionic acid metabolizing pathway，in the obtained target strains were analyzed by bioinformatics method.A total of 10 target bacterial strains were isolated and successfully classified：3 strains of Bacillus genus，2 strains of Rhodococcus genus，and 1 strain each for Arthrobacter genus，Streptomyces genus，Microbacterium genus，Isoptericola genus，and Xylanimicrobium genus.Besides，the ability of these bacteria to metabolize propionic acid was significantly different.Bioinformatics analysis showed that most of the strains contained the key enzyme genes of 2‑methylcitrate cycle，and the arrangement of the key enzyme genes was not consistent in different types of bacteria.

Biological Control of Sharp Eyespot and Growth Promotion in Wheat by Bacillus velezensis YB‑145

XIA Mingcong, DENG Xiaoxu, QI Hongzhi, XIE Xia, XU Wen, ZHANG Jie, SUN Runhong, PAN Yamei, WU Chao, YANG Lirong

2021, 50(10): 76-83. DOI: 10.15933/j.cnki.1004-3268.2021.10.010

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The aim was to find an excellent strain with growth‑promoting effect on wheat and efficient control of wheat sharp eyespot. Firstly，the inhibitory effect of strain YB‑145 on mycelial growth of six plant pathogens，including Rhizoctonia cerealis，Fusarium oxysporum f.sp.niveum，Bipolaris sorokinana，Alternaria solani，Fusarium moniliforme and Fusarium graminearum PH‑1 was studied in dual cultures on PDA plates. Furthermore，the strain was identified according to its morphological，physiological and biochemical characteristics and 16S rRNA gene sequencing. The growth‑promoting effect of strain YB‑145 on wheat and its biological control effect on sharp eyespot were determined by Petri dish，pot and field experiments. The results showed that strain YB‑145 was identified as Bacillus velezensis，which could produce IAA（indole acetic acid）and siderophores and had extracellular protease and β‑1，3‑glucanase activities. In dual cultures，Bacillus velezensis YB‑145 significantly inhibited mycelial growth of 6 plant pathogens，especially R. cerealis.Bacillus velezensis YB‑145 at doses of 1×107 cfu/mL and 1×108 cfu/mL significantly promoted growth of wheat seedlings with more fresh weight of roots，fresh weight of shoots and height of shoots.Moreover，the efficacy of Bacillus velezensis YB‑145 at a doses of 1×108 cfu/mL in controlling wheat sharp eyespot reached 73.31%. Field investigation showed that the control effect of strain YB‑145 on sharp eyespot was up to 63.37% and the yield of wheat increased by 8.2%. The control effect（73.65%）and yield‑increasing rate of wheat（5.8%）of the treatment with 6% tebuconazole suspension had no significant difference with YB‑145. The above results demonstrate that Bacillus velezensis YB‑145 is a promising biological control agent to both promote wheat growth and suppress wheat sharp eyespot.

Control Effect of Pyroxasulfone Mixed with Cyanazine on Weeds in Wheat Field

WU Renhai, XU Hongle, LI Huilong, SUN Lanlan, SU Wangcang, XUE Fei, WANG Qiang, LU Chuantao

2021, 50(10): 84-91. DOI: 10.15933/j.cnki.1004-3268.2021.10.011

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To illustrate the control effect of the mixture of pyroxasulfone and cyanazine on weeds in wheat field，indoor activity determination and field plot experiment were set up.The results of indoor test showed that the inhibition medium dose（GR₅₀）of pyroxasulfone and cyanazine on Alopecurus myosuroides were 1.14 g/ha and 108.43 g/ha，respectively. As mixing with the ratio of 1∶2. 5，1∶5，1∶7. 5，1∶10，the values of cotoxicity coefficient（CTC）were 128.74，127.42，146.52，172.71，which showed that the combination of the two herbicides had synergistic effect at the ratio of 1∶2. 5，1∶5，1∶7. 5，1∶10. When pyroxasutfone was mixed with cyanazine at the ratio of 1∶5，1∶7. 5 and 1∶10，the selectivity indexes against wheat and A.myosuroides were 8. 38，1. 30 and 1. 53 respectively.The results of field plot experiment demonstrated that at 100 days after treatment，the combined use of pyroxasulfone and cyanazine with the ratio of 1∶5 at 540，720，900，1 440 g/ha in field showed above 94% of plant number and fresh weight controt effects to Capsella bursa‑ pastoris and Geranium carolinianum，and 87%—93% to Alopecurus myosuroides，and the wheat yield increased by 14%—21%.In conclusion，the mixture of pyroxasulfone and cycazine at the ratio of 1∶5 is an efficient，broad spectrum and safe herbicide in wheat field.

Identification of Pathogens Causing Salvia miltiorrhiza Root Rot Disease in Henan Province

YANG Jin, WEN Yi, GAO Suxia, LIU Yuxia, LU Chuantao, WANG Fei, LIU Hongyan

2021, 50(10): 92-98. DOI: 10.15933/j.cnki.1004-3268.2021.10.012

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To clarify the pathogenic species and distribution of root rot of Salvia miltiorrhiza in Henan Province，the incidence of root rot on S. miltiorrhiza in Yuzhou，Mianchi，Fangcheng and Songxian of Henan Province was investigated. In addition，the pathogens were isolated from S.miltiorrhiza root rot by tissue isolation and culture，and 30 pathogenic strains（No. F1—F30）were obtained and identified based on morphological and molecular biological methods. The pathogenicity of different pathogens was further tested by pot inoculation.The results showed that the incidence of root rot of S. miltiorrhiza in Henan Province ranged from 3.5% to 85.0%，with the highest incidence in continuous cropping fields.Moreover，F.usarium solani，F.proliferatum and F.oxysporum could systematically infect the roots of S.miltiorrhiza and caused root rot. The results of pot inoculation tests showed that the pathogenicity of different strains differed，with the strain F21 as a strong pathogenic strain，the strains F2 and F17 as intermediate pathogenic strains，the strains F1，F5，F8，F14 and F15 as weak pathogenic strains，and the strain F25 as a conditional pathogen. In summary，the main pathogens causing root rot disease on S.miltiorrhiza in Henan Province were F.solani，F.proliferatum and F.oxysporum，and the distribution and pathogenicity of the pathogens differed in various production areas

Effects of Different Foliar Fertilizers on Growth，Yield and Quality of Ligularia schmidtii

WANG Lei, ZHANG Mingyue, DONG Ran, LI Panpan

2021, 50(10): 99-108. DOI: 10.15933/j.cnki.1004-3268.2021.10.013

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With Ligularia schmidtii as experimental material，the effects of different foliar fertilizers［urea（CK），high‑carbon organic foliar fertilizer（T1），amino acid foliar fertilizer（T2），mixed fertilization of high‑carbon organic foliar fertilizer and amino acid foliar fertilizer with ratio of 1：1（T3）］on the growth and yield of Ligularia schmidtii，were comprehensively compared and analyzed to determine the optimal treatment，and the quality indexes of the optimal treatment were determined，so as to provide theoretical reference for the establishment of fertilization program for high yield and quality. The results showed that compared with CK，T1 and T3 treatments could significantly increase the leaf length，leaf width and plant height of Ligularia schmidtii；T1，T2，and T3 treatments significantly increase the yield of fresh leaves of Ligularia schmidtii by 25.39%，44.86% and 59.13% respectively. In general，T3 treatment had the highest SOD activity and chlorophyll content. The contents of soluble protein，soluble sugar，vitamin C, carotenoids and vitamin B2 in leaves of T3 treatment increased by 17. 26%，34.04%，28.13%，41.03% and 38.98% respectively compared with CK；the contents of calcium（Ca），manganese（Mn） and strontium（Sr）increased by 36.33%，12.55% and 17.48% compared with CK；the contents of 15 kinds of amino acids of T3 treatment were higher than those of CK，of which aspartic acid content was the highest，followed by glutamic acid and proline，which increased by 12.55%，9.68% and 5.26% respectively compared with CK. By comprehensive comparison，mixed fertilization of high‑carbon organic foliar fertilizer and amino acid foliar fertilizer with ratio of 1∶1 could promote the growth of Ligularia schmidtii，increase the yield of fresh leaves，and improve the quality of Ligularia schmidtii.

Effects of Exogenous Nitric Oxide on Seed Germination，Seedling Growth and Antioxidant Characteristics of Lettuce under Cadmium Stress

XIONG Mingguo

2021, 50(10): 109-115. DOI: 10.15933/j.cnki.1004-3268.2021.10.014

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In order to explore the mitigation effects of exogenous NO on seed germination and seedling growth of lettuce under cadmium（Cd）stress，the effects of exogenous sodium nitroprusside（SNP，NO donor）with different concentrations（100，150，200，250 μmol/L）on seed germination，seedling growth and antioxidant properties of lettuce under 100 μmol/L Cd stress were studied. The results showed that exogenous NO had no significant effect on lettuce seed germination rate and germination potential under Cd stress，but generally significantly improved lettuce seedling growth，chlorophyll content and activities of peroxidase（POD），catalase（CAT）and superoxide dismutase（SOD），and significantly decreased malondialdehyde（MDA）content. The treatment with 250 μmol/L SNP had the best effect under Cd stress，and POD，CAT，SOD activities and chlorophyll content significantly increased by 85.00%，81.20%，63.27% and 155.63% respectively. Overall，exogenous NO could mitigate the damage of lettuce caused by Cd，and 250 μmol/L SNP had the best effect.

Construction and Biological Characterization of an Attenuated Salmonella typhimurium Type Ⅲ Secretion System Carrying NDV HN Gene

2021, 50(10): 116-123. DOI: 10.15933/j.cnki.1004-3268.2021.10.015

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In order to construct a recombinant attenuated Salmonella typhimurium type Ⅲ secretion system which carries the hemagglutinin‑neuraminidase（HN） protein gene of Newcastle disease virus（NDV），the HN gene was amplified from the plasmind pMD18‑T‑HN and cloned into the vector pYA3493‑sopE_Nt100.Then the recombinant vector pYA3493‑sopE_Nt100‑HN was transformed into attenuated Salmonella typhimurium strain Δ crpΔ asd SL1344 by electroporation，and the growth characteristics，genetic stability，expression characteristics and virulence of the recombinant strain Δ crpΔ asd SL1344（pYA3493‑sopE_Nt100‑HN）were analyzed. The identification results of PCR and enzyme digestion showed that the recombinant attenuated Salmonella typhimurium strain Δ crp Δ asd SL1344（pYA3493‑sopE_Nt100‑HN）was successfully constructed. The growth curve of the recombinant strain was similar to that of the complementary strainΔ crpΔ asd SL1344（pYA3493‑sopE_Nt100）and the deletion strain Δ crpΔ asd SL1344，which were significantly different from their parent strain SL1344.The HN gene was stably inherited in the recombinant strain.The HN protein was presented by the attenuated Salmonella typhimurium Ⅲ type secretion system and expressed in chicken embryo fibroblast（CEF）.The LD50 of the recombinant strain was not significantly different from the complementary strain and the deletion strain，but significantly different from the parent strain SL1344. The above results indicated that the recombinant attenuated Salmonella typhimurium Ⅲ secretion system carrying the NDV HN gene was successfully constructed，which had high safety，genetic stability，and expression efficacy of the NDV HN gene.

Tissue Expression and Polymorphism of Jiangquhai Pig FoxO3 Gene and Their Associations with Meat Quality

ZHU Shubin, NI Ligang, CHEN Zhangyan, ZHANG Junsheng, ZHOU Chunbao, ZHAO Xiuling

2021, 50(10): 124-131. DOI: 10.15933/j.cnki.1004-3268.2021.10.016

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To define the expression and polymorphism of FoxO3 gene and their correlations with meatquality in Jiangquhai pig，PCR‑SSP molecular marker technique was used in the study and the genetic variation of FoxO3 gene exon 2 in 112 Jiangquhai pigs was analyzed，and then the expression of FoxO3 in heart，liver，spleen，kidney，abdominal fat，subcutaneous fat，longissimus dorsi，soleus，gastrocnemius and semitendinosus in Jiangquhai pig was detected by RT‑qPCR. The results indicated that there was polymorphism in exon 2 with three genotypes（TT/TC/CC），and the frequency of allele T was 0.424，showing middle polymorphism（PIC=0. 369）.The gene frequencies and genotype frequencies of pigs tested reached the Hardy‑Weinberg equilibrium（P>0. 05）.Analysis of the effect of different gengtypes on meat quality showed that there was significant correlation between polymorphism of FoxO3 gene and intramuscular fat（IMF）content，shear stress.FoxO3 gene was expressed in all ten tissues of Jiangquhai pig.FoxO3 gene was negatively correlated with muscle fiber diameter and intramuscular fat content，but positively correlated with muscle fiber density and shear force. The correlation coefficient between FoxO3 gene and gastrocnemius myofibril diameter was -0. 937，and the correlation coefficient between FoxO3 gene and longissimus dorsi was -0.874. The results showed that FoxO3 gene had a certain effect on the development of meat quality traits in Jiangquhai pig，which could be used as a candidate gene affecting the meat quality in Jiangquhai pig.

Eukaryotic Expression and Biological Characterization of leptin Gene in Buffalo

LI Gonghe, CHEN Qiuyu, WU Wende, ZHENG Xibang

2021, 50(10): 132-137. DOI: 10.15933/j.cnki.1004-3268.2021.10.017

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To study the function of Leptin in buffalo，a eukaryotic expression vector EGFP‑leptin was constructed，the expression of leptin in NIH/3T3 cells was detected and its biological activity was determined.The primers were designed based on the sequence of buffalo leptin gene in GenBank，and the restrictive endonuclease sequences of both Hind Ⅲ and BamH Ⅰ were added to the forward and the reverse primers respectively. By means of PCR，the leptin gene was amplified using pMD18‑T‑leptin vector as a template，and subcloned to the pEGFP‑N1 vector to construct the EGFP‑leptin vector，which was identified by double restrictive endonuclease digestion and DNA sequencing.Then the recombinant plasmid was transfected into NIH/3T3 cells by liposome 2000，and stable transfected cell lines were screened using G418. The expression of EGFP‑Leptin fusion protein was verified by RT‑PCR and Western blot analyses. Ine three groups of animal tests，EGFP‑leptin plasmid（1 μg in 100 μL saline）was administrated via tail intravenous injection in the treatment group，1 μg each，and the fasting group and control group were administrated with the same volume of saline in the same way. All mice were weighed and serum Leptin levels were determined by ELISA after three days. The results showed that the EGFP‑leptin was successfully constructed，green fluorescence was observed in NIH/3T3 cells transfected with the recombinant plasmid，and the expression of leptin was further verified through RT‑PCR and Western blot assays.Animal tests showed that the weight loss ratio in the treatment group and fasting group was significantly faster than in the control group（P<0. 05），and the serum Leptin level in the treatment group was significantly higher than in the control group（P<0. 05）.In conclusion，EGFP‑leptin was stably expressed in NIH/3T3 cells and the fusion protein was functional biologically.

iTRAQ Quantitative Proteomic Analysis of Mycoplasma bovis

LI Haili, XU Yindi, YOU Yi, ZHU Wenhao, ZHANG Qingxian, JIAO Wenqiang, WANG Zhifang, XU Feng, WANG Keling, LANG Limin, ZHANG Lixian, ZOU Jing

2021, 50(10): 138-145. DOI: 10.15933/j.cnki.1004-3268.2021.10.018

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To understand the differentially expressed proteins‑Bovine Mycoplasma bovis clinical strain（HN12）and standard strain（PG45），this study adopted the quantitative proteomics research strategies，to identify differentially expressed proteins in total proteins of the clinical strain（HN12）and standard strain（PG45）and carried out bioinformatics analysis. The results showed as follows：a total of 2 402 proteins were identified，of which 66 were significantly different. Among them，the numbers of up‑regulated and down‑regulated proteins were 25 and 41 respectively. Proteins identified mainly included membrane protein，transmembrane protein，lipoprotein，prolongation factor，membrane lipoprotein P81，chaperone，variable surface lipoprotein，and ABC transporter，et al.

Effects of 5‑Aminolevulinic Acid Treatment on Storage of Fuji Apple during Fruit Expansion Stage

NIU Jiajia, ZHANG Sipu, ZHANG Ke, HAN Lixin, QU Zhenfang, MIAO Jianyin

2021, 50(10): 146-153. DOI: 10.15933/j.cnki.1004-3268.2021.10.019

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In order to improve the storage quality of Fuji apple，the leaves were sprayed for three times at the fruit expansion stage with 0（water，CK），0.5，5，10 mg/L of exogenous 5‑aminolevulinic acid（ALA），respectively. The fruit hardness，total soluble solids（TSS） content，titratable acid（TA） content，malondialdehyde（MDA）content，the activities of peroxidase（POD）and polyphenol oxidase（PPO）of Fuji apple during cold storage（0—200 d）were investigated，and the shelf‑life quality of Fuji apple treated with the optimal mass concentration was analyzed.The results showed that spraying exogenous ALA on leaves could significantly promote apple fruit coloring，increase TSS content and decrease TA content in fruit flesh，and was beneficial to maintaining fruit hardness during cold storage，inhibiting the decrease of TSS content，and keeping higher POD activity，lower PPO activity and MDA content in apple fruit.Among them，10 mg/L ALA treatment had the best effect. After 200 days of cold storage，compared with CK，fruit hardness，TSS content，TA content and POD activity of 10 mg/L ALA treatment were increased by 7.5%，17.0%，45.5% and 69.5%，respectively，MDA content and PPO activity were decreased by 17.7% and 39.5%，respectively. At the same time，the fruit quality of Fuji apple stored at room temperature for 10 days after 200‑day refrigeration was maintained by this treatment，and the fruit hardness，TSS content and TA content were higher than those of the control. In conclusion，multiple spraying of exogenous ALA at 10 mg/L during fruit expansion stage could significantly improve the harvest quality and storage quality of Fuji apple fruit.

Effect of Curing Technology on the Quality of Upper Six Leaves of Strong Flavor Type Flue‑cured Tobacco Grown in Central Henan

MENG Zhiyong, LI Jianhua, MA Haobo, ZONG Shengjie, LIU Fang, SUN Xiaowei , GAO Xiangbin, CHEN Chu, ZHANG Dongfeng, LIN Kun

2021, 50(10): 154-162. DOI: 10.15933/j.cnki.1004-3268.2021.10.020

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In order to further improve the quality and industrial usability of the upper six leaves of flue‑cured tobacco in central Henan，we chose the upper six leaves of Zhongyan 100 as materials，and studied the effect of temperature stabilization time in the yellowing stage and the color fixing stage on the quality of flue‑cured leaves. The results were as belows：compared with the conventional curing technology（the temperature was stabilized at 38 ℃ for 30 h in the yellowing stage，and the temperature was stabilized at 54 ℃ for 12 h in the color fixing stage），the quality indexes of flued‑cured leaves including oil content，structure and chromaticity of upper tobacco leaves were effectively improved，while the contents of total sugar，reducing sugar，starch and chlorine were reduced，and the ratios of total sugar to reducing sugar，potassium to chlorine were improved by prolonging baking time during 38 ℃ in the yellowing stage and 54 ℃ in the color fixing stage. During the yellowing stage of 38 ℃，prolonging baking time for 12 h could significantly improve the content of plastid pigment degradation products，phenyalanine degradation products，other neutral aroma substances and the total amount of neutral aroma substances in the upper flue‑cured tobacco leaves，meanwhile significantly improved the sensory indexes such as aroma quality and quantity，smoke concentration and strength of the upper flue‑cured tobacco leaves.After prolonging baking time for 12 h during the color fixing stage of 54 ℃，the content of neutral aroma substances in the upper flue‑cured leaves was slightly reduced，and the two evaluated indexes of smoke concentration and strength for sensory quality were significantly improved. When 38 ℃ in yellowing stage and 54 ℃ in color fixing stage were both prolonged for 12 hours，the content of neutral aroma substances in the upper leaves after baking was increased，but the sensory indexes such as aroma quality，aroma quality，concentration，miscellaneous gas，irritation，aftertaste，flammability and ash color were worsened. It was concluded that prolonging for 12 h during the 38 ℃ yellowing stage could improve the appearance and sensory quality，chemical composition coordination of the upper six flue‑cured leaves.Also，they had high aroma quality，sufficient aroma，obvious sweetness，transparent aroma，and the content of neutral aroma substances was increased significantly.All these suggested that this curing technology could be applied in central Henan.

A Method for Monitoring the Critical Growth Stages of Winter Wheat by Using Near‑earth Remote Sensing

WEI Qingwei, ZHU Liming , WANG Fuzhou

2021, 50(10): 163-171. DOI: 10.15933/j.cnki.1004-3268.2021.10.021

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The aim is to explore the effective method for monitoring the critical growth stages of winter wheat.Firstly，the device for measuring normalized vegetation index（SRS‑NDVI）was used to monitor time series normalized difference vegetation index（NDVI）of winter wheat growing season in 2017—2018 and 2018—2019 at Hebi Agrometeorological Experimental Station. Then，the neighborhood difference analysis method was used to reconstruct time series normalized difference vegetation index，and the S‑G filtering method（Savitzky‑golay，S‑G）was used to smooth the noise in normalized difference vegetation index time series.Finally，according to the characteristics of normalized difference vegetation index time series，the generalized dynamic threshold method，curve rate method and extreme value method were used to extract the key growth stages of winter wheat. The results showed that the neighborhood difference analysis method could effectively remove obvious abnormal values in normalized difference vegetation index time series. Besides，the normalized difference vegetation index time series processed by S‑G filtering method was more in line with the normalized difference vegetation index change rule of winter wheat.In addition，the average error of the critical growth stages of winter wheat was 2. 5 days，and the accuracy was significantly higher than that extracted by using satellite remote sensing.

Construction of Statistical Yearbook Data Cleaning Model Based on Workflow

ZHANG Hui, WEI Dong, QIAO Lu, LI Danan, ZHANG Yuyao, ZHENG Guoqing, FENG Xiao

2021, 50(10): 172-180. DOI: 10.15933/j.cnki.1004-3268.2021.10.022

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In order to realize the data integration and comprehensive quick query of statistical yearbooks，the data characteristics of China Statistical Yearbook and statistical yearbooks of 31 provincial regions such as Henan Statistical Yearbook from 2000 to 2018 were analyzed in depth. Based on workflow technology，Alteryx Designer 2019.2（learning version）was used to construct the data cleaning model of statistical yearbooks by 7 steps：extracting directory and file，extracting forms from file，extracting the contents of tables in the form，data cleaning and specification，data standardization by six dimensions，data reorganization and data output.The results showed that on a laptop with 16 GB RAM，the model took4 to 5 hours to clean and integrate the yearbook data with 21 GB，including more than 330 thousand files and around 1. 2 million forms into a single standard dataset that contained more than 60 million indicator data.The data cleaning modeling method had the advantages of high efficiency and traceability.

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