Scientia Agricultura Sinica

SSR Sequences and Development of PCR Markers Based on Transcriptome of Dasypyrum villosum No.1026

CHEN JingNan,MA XiaoLan,WANG Zhen,LI ShiJin,XIE Hao,YE XingGuo,LIN ZhiShan

Scientia Agricultura Sinica. 2019, 52(1): 1-10. doi:10.3864/j.issn.0578-1752.2019.01.001

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【Objective】The aim of this study is to explore the characteristics of the EST-SSR sequences of a Dasypyrum villosum accession No.1026 (Dv#4) introduced from the former Soviet Union, and their distributions on chromosomes and polymorphism in different D. villosum accessions and between Dv#4 and common wheat. 【Method】Transcriptome sequences of Dv#4 plants were obtained by Illumina HiSeq sequencing and used to search and analyze the SSR sequences using MISA software and design primers by Primer 3. In total, 238 pairs of primers were selected randomly for synthesis and used to amplify the genomic DNAs of wheat Chinese Spring (CS) and the two different D. villosum accessions. The polymorphisms of the PCR products on agarose gel were evaluated. Further, the features of their chromosome distributions in Dv#4 were studied by using a set of wheat- D. villosum alien chromosome lines (including disomic addition lines and disomic substitution lines). 【Result】 A total sequence length of 62.76 Mb was detected and 10 497 SSR loci were found in the transcriptome data. They are involved in 8 735 unigenes. Repeated unit of mono-, di-, tri- nucleotides are the main type, holding 95.85% of all loci among 1-6 nucleotides repeats, among which tri-nucleotide is the richest component that makes up of 50.33% and contains CCG/CGG motif by 41.66%. The next component is mono-nucleotide tandem repeat, and its occurrence frequency is 18.39%, and A/T repeats occupy 74.58% in this type. Di-nucleotide ranks the 3rd, and it holds 18.39% in the total SSR loci. Among 238 pairs of randomly synthetized EST-SSR primers, 88 pairs amplified polymorphic fragments between CS and D. villosum (including Dv#2 and Dv#4); 8 pairs only had amplifications in D. villosum and some single alien chromosome lines; 4 pairs could specifically amplify bands in D. villosum and multiple alien chromosome lines. But, many primers which had amplification in both D. villosum accessions had no amplification in any alien chromosome lines. Therefore, it can be inferred that variations on the flanking conserved sequences of SSR might occur during the transferring of the exogenous genome or chromosomes into wheat. Additionally, 47 pairs of primers (19.74%) showed polymorphisms between Dv#2 and Dv#4. By using a pair of EST-SSR primer and a PCR marker, respectively, polymorphic amplicons were detected in 48 D. villosum plants, indicating that polymorphic SSR primer can be used to detect the heterogeneity of D. villosum effectively.【Conclusion】 D. villosum Dv#4 has abundant SSR sequences in its transcriptome, of which, CCG/CGG, A/T, AG/CT and other tri-, mono-, and di-nucleotide are the main tandem repeats. The flanking conserved sequences of partial EST-SSR in Dv#4 are associated with a single or several chromosomes specifically, which provides an ideal sequence resource for the development of specific molecular markers to track and detect D. villosum chromosomes or chromosome fragments in wheat background. Some of the EST-SSR polymorphism between Dv#4 and Dv#2 indicates that there is a certain degree of genetic diversity in some of the expressed sequences between the two different origin D. villosum accessions. Therefore, Dv#4 is valuable to be further investigated.

Inheritance and QTL Mapping for α-Tocopherol in Soybean

LIANG HuiZhen,YU YongLiang,XU LanJie,YANG HongQi,DONG Wei,TAN ZhengWei,LI Lei,PEI XinYong,LIU XinMei

Scientia Agricultura Sinica. 2019, 52(1): 11-20. doi:10.3864/j.issn.0578-1752.2019.01.002

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【Objective】 Inheritance and main QTL for α-tocopherol were detected by genetic analysis and QTL mapping. The results lay a genetic foundation for the selection of soybean varieties with high α-tocopherol content in soybean.【Method】The 447 RILs were derived from a cross between Jindou23 of commercial cultivar as the female parent and Huibuzhi of farm variety from Shanxi Province (ZDD02315) as the male parent that construct SSR genetic linkage map. The parent lines and the RILs were cultivated in summer at Yuanyang testing ground of Academy of Agricultural Sciences, and in Winter at Sanya of Hainan province in 2011, 2012, 2015. A complete random design with two replications was used in this study. Each plot of a single genotype provided 15.00 g big-plump seeds with same size in six environmental conditions. α-tocopherol content was detected quantitatively and qualitatively by High Performance Liquid Chromatography (HPLC). Major gene plus polygene mixed inheritance and QTL mapping for α-tocopherol were detected by major gene plus polygene mixed inheritance analysis and composite interval mapping with WinQTLCart 2.5.【Result】The results showed that α-tocopherol was controlled by four pairs of main genes by major gene plus polygene mixed inheritance analysis. and the four pairs of main genes distributed in two parents. Three main genes shared the same direction with positive additive effect and involved novel alleles from the same parent, Jindou23; one main gene has negative additive effects and donated by Huibuzhi of black beans. Three pairs of genes shared the different direction of positive or negative epistatic effects shared the different direction to α-tocopherol contribution. The phenotypic variation explained by QTL by environment interaction ranged from 0.13 to 4.05%, and indicated that α-tocopherol was significantly affected by four pairs of main genes, more than by environment. Seventeen QTLs for α-tocopherol were mapped on 8 chromosomes 1, 2, 5, 6, 8, 14, 16, and 17, separately; the variation accounted for by each of these seventeen QTLs ranged from 8.35% to 35.78%; and QTL showed additive effect. qα-D1a-1 was all located in marker intervals between Satt320-Satt254 (19.79 cM) on chromosomes 1 in four environmental conditions of 2011 at Yuanyang, 2012 at Yuanyang and Sanya, and 2015 at Yuanyang. and explained 12.55%, 12.01%, 11.89%, 12.61% of the phenotypic variation. It had an additive effect of 0.119–0.132 donated by Jinbean23. qα-A2-1 was all located in marker intervals between Sat_129-Satt377 (44.53 cM) on chromosomes 8 in three environmental conditions of 2011 at Yuanyang and Sanya, 2015 at Yuanyang. and explained 23.18%, 22.56%, 23.01% of the phenotypic variation. It had an additive effect of -0.195–-0.180 donated by Huibuzhi. qα-D1a-1 and qα-A2-1 can be stably expressed in different genetic backgrounds.【Conclusion】α-tocopherol was controlled by four pairs of additive epistatic effect major genes genetic model (4MG-AI), and it less affected by environmental factor. The two stable main QTL of Satt320-Satt254 and Sat_129-Satt377 were co-localization marker intervals in soybean.

Field Wheat Ears Counting Based on Superpixel Segmentation Method

DU Ying,CAI YiCheng,TAN ChangWei,LI ZhenHai,YANG GuiJun,FENG HaiKuan,HAN Dong

Scientia Agricultura Sinica. 2019, 52(1): 21-33. doi:10.3864/j.issn.0578-1752.2019.01.003

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【Objective】Wheat ears number is a major factor of yield composition and plays an important role in yield estimation and genetic improvement. Using image processing technology to accurately identify and count wheat ears in the field, a new method for obtaining agricultural information was proposed in this paper, which provided a reliable reference for the yield estimation and crop growth monitoring. 【Method】In this paper, wheat with different growth conditions after treatment with nitrogen fertilizer gradient was the research object. Firstly, the simple linear iterative cluster (SLIC) was used to segment the wheat image, and the unit of an image was transformed from pixels to superpixel block. After analyzing the color histograms, the classifiers were trained to identify wheat ears. Then, we performed a simple morphological treatment on the classification results to segment the wheat ears and performed binarization. We obtained the main body of wheat and conducted regional statistics through a series of morphological calculations, such as corrosion and expansion. The wheat ear skeleton was extracted to detect the skeleton corner points to calculate the number of wheat ears. Lastly, the results of counting wheat ears under different nitrogen levels (no nitrogen, low nitrogen, normal nitrogen, high nitrogen) were verified by linear regression analysis. 【Result】 (1) Super green value (Eg) and normalized red green index (Dgr) could be used as classification features to effectively identify wheat ears after color histogram analysis. (2) Compared to directly identifying the field image, the main body of wheat identified after superpixel segmentation was more explicit and complete in shape. (3) By comparison, wheat with better growth situation had better ear counting results, which approached 94.4% in high nitrogen level. However, the wheat ear count accuracy was low at a poor nitrogen level, which was only 81.9%. After eliminating the nitrogen-free condition, the overall growth of the mixed sample was uniform, and the wheat ear count accuracy reached 92.9%. The accuracy was improved by 8.3% compared to mixed samples with large differences in growth.【Conclusion】In the general environment, the automatic counting method of wheat ears using superpixels and color features could quickly and accurately calculate the number of wheat ears in field. While the growth vigor was too weak and the difference was too large, this method was not recommended. The research results provided a new reference for wheat field estimation.

Effects of Soil Water and Nitrogen on Plant Growth, Root Morphology and Spatial Distribution of Maize at the Seedling Stage

ZHANG XinYue,WANG Yin,CHEN Jian,CHEN AnJi,WANG LiYing,GUO XiaoYing,NIU YaLi,ZHANG XingYu,CHEN LiDong,GAO Qiang

Scientia Agricultura Sinica. 2019, 52(1): 34-44. doi:10.3864/j.issn.0578-1752.2019.01.004

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【Objective】 The frequent spring drought has severely negative impacts on seed emergence and seedling growth in the maize production of Northeast China. It is necessary to understand the coupling effects of soil water condition and nitrogen (N) rate on maize plant and root growth at the seedling stage, and further to provide reference for optimizing water and N management in maize production of Northeast China. 【Method】In this study, two pot experiments were conducted in 2016 and 2017, with a two factor factorial design of soil water and N rates. The soil water condition included 30%, 50%, 70% and 90% of field capacity, respectively, representing severe water-stress (W0), moderate water-stress (W1), well-watered (W2) and over-watered (W3), respectively. The N rates included 0, 0.12 and 0.24 g·kg ^-1 soil, representing N-omission (N0), low N (N1) and high N (N2), respectively. 【Result】 Soil water and N rate had significant individual effects on maize plant and root growth at the seedling stage, and showed interactive effects on dry matter (DM), root morphology, N uptake, and N fertilizer use efficiency (NUE). Both soil water deficit and excess had negative impacts on maize plant growth, DM accumulation, root development, and N uptake at the seedling stage, and was especially serious under W0 treatment. Compared with W2 treatment, on average in two years, shoot and root DM and plant N uptake under W0 treatment decreased by 55.5%, 60.1% and 45.8%, respectively, NUE decreased by 7.8 percentage points. And root length (RL) and root surface area (RSA) decreased by 58.2% and 59.5%, respectively. The N fertilization improved significantly maize plant growth and N uptake but reduced root/shoot ratio at the seedling stage. Moreover, the plant and root growth responses of N fertilizer differed obviously with the different soil water conditions. The N fertilization improved root growth in terms of higher RL, RSA and root volume (RV) under W2 treatment, and therefore showed the highest plant DM and N uptake. However, N fertilization limited root growth and decreased significantly RL and RSA under W0 and W1 treatments. The N fertilization also improved root growth under W3 treatment, but the N fertilizer response was still lower than that under W2 treatment. Across all the soil water conditions, maize plants showed higher RL and RSA under N1 treatments than that under N2 treatments, but the RV was equal or smaller, indicating that low N supply induced fine root development at the seedling stage. Soil water and N rate not only affected significantly maize root morphology, but also had great effects on root system spatial distribution. The water-stress induced deeper root growth and RL distribution in subsoil. Compared with W2 treatment, on average, the distribution ratio of RL in 0-12 cm soil layer decreased by 11.0 percentage points under W0 treatment and 8.3 percentage points under W1 treatment, but their distribution ratio in 24-36 cm soil layer increased by 9.5 and 6.9 percentage points, respectively. In contrast to soil water-stress condition, maize root system showed a concentrated trend in topsoil under over-watered condition. The N fertilization improved significantly root distribution in topsoil. Compared with N0 treatment, the RL distribution ratio increased by 16.3 and 13.7 percentage points higher in 0-12 cm soil layer under N1 and N2 treatments, respectively, and the distribution ratio decreased by 11.5 and 12.5 percentage points lower in 24-36 cm soil layer, respectively. Across all the soil water-N treatments, maize root system showed the more balanced spatial distribution under the W1N1 treatment.【Conclusion】Soil water condition and N rate had significant coupling effects on maize seedling growth and root development. The appropriate soil water and N management could optimize root morphology and spatial distribution, and improve plant DM accumulation and N uptake. Therefore, we suggested reducing basal N rate to stimulate deeper root growth with more fine root by inducing the water-N coupling effect, and further to enhance plant resistance to drought stress and to improve NUE in spring maize production of Northeast China.

Effects of the Interference of Key Magnetic Response Genes on the Longevity of Brown Planthopper (Nilaparvata lugens) Under Near-Zero Magnetic Field

HE JingLan,ZHANG Ming,LIU RuiYing,WAN GuiJun,PAN WeiDong,CHEN FaJun

Scientia Agricultura Sinica. 2019, 52(1): 45-55. doi:10.3864/j.issn.0578-1752.2019.01.005

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【Objective】Cryptochrome (Cry) and iron-sulfur cluster protein IscA (iron-sulfur cluster assembly, MagR) are potential magnetic receptor proteins in organisms. In this study, key magnetic response genes of the brown planthopper (Nilaparvata lugens) were knocked-down by RNA interference (RNAi), including NlCry1, NlCry2 and NlMagR. The objective of this study is to investigate the role of these three magnetic response genes in the longevity mediation of N. lugens in near-zero magnetic field (NZMF). Thus, the response of these three genes to magnetic field could be studied indirectly.【Method】Newly emerged brachypterous female and male adults of N. lugens fed in the lab magnetic field were chosen as the experimental material, and RNAi technology was used to inhibit the key magnetic response genes’ (NlCry1, NlCry2 and NlMagR) expression by injection of double stranded RNA, respectively. Then the RNAi treated adults were immediately transformed into the geomagnetic field (GMF) and NZMF respectively to observe their longevity. The total RNA of the RNAi treated adults under GMF was extracted by using the RNAiso Plus method on the 1st, 2nd and 3rd day after the microinjection, respectively. And then the gene expressions of NlCry1, NlCry2 and NlMagR were measured by using the RT-qPCR (real-time quantitative polymerase chain reaction) after the reverse transcription synthesis of first strand DNA in order to test the efficiency of RNAi. 【Result】There was no significant difference in the longevity of female and male adults after the injection of dsNlCry1 between the treatments of NZMF and GMF, while after the injection of dsNlCry2, the longevity of female (27.78%) and male (50.04%) adults under NZMF was significantly longer than that of the individuals under GMF, respectively. Moreover, the longevity of female adults injected with dsNlCry2 was shorter under GMF while longer under NZMF than that of individuals injected with dsGFP, even if the difference was not significant. The longevity of male adults injected with dsNlCry2 was shorter than that of individuals injected with dsGFP under NZMF (25.41%) and GMF (10.73%), respectively, and the difference under GMF reached the significant level. Furthermore, the longevity of female adults injected with dsNlMagR was significantly shorter (16.48%) than that of individuals injected with dsGFP under the NZMF. 【Conclusion】There is a difference in the regulation of the key genes of magnetic susceptibility (NlCry1, NlCry2 and NlMagR) on the female and male longevity for N. lugens under the change of magnetific field. Hereinto, the NlCry2 susceptibly responses to the changes of magnetic fields, which shows that the gene knock-down and its interaction with magnetic field changes can significantly influence the longevity of female and male adults, and characterized as “sexual dimorphism”. Similarly, the NlMagR (IscA) also sensitively responds to magnetic field changes, but just for the female adults of N. lugens under the NZMF in contrast to the GMF. However, there is no response of NlCry1 to magnetic field changes, and this gene may not be involved in the regulation of female and male longevity for N. lugens.

RNA Interference of Vitellogenin Receptor Gene in Beet Armyworm (Spodoptera exigua)

ZHAO Jing, TAO Rong, HAO DeJun, XIAO LiuBin, TAN YongAn

Scientia Agricultura Sinica. 2019, 52(1): 56-64. doi:10.3864/j.issn.0578-1752.2019.01.006

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【Objective】Vitellogenin receptor (VgR) is the main receptor that mediates the endocytosis of insect vitellin. The objective of this study is to clarify the function of VgR of beet armyworm (Spodoptera exigua) through RNA interference (RNAi) method, and to provide a basis for further understanding the molecular mechanism of reproductive physiology and developing effective new methods for prevention and control.【Method】The fragment of VgR was amplified from the cDNA of female adults abdomen tissues of S. exigua by PCR which included the ligand-binding domain region. The green fluorescent protein gene (GFP) fragment was amplified from the GFP plasmid stored in the laboratory by specific primers. The fragment of VgR and GFP was then inserted into the pMD-19T for sequencing. The nucleic acid sequence was analyzed by DNAMAN software. The correct plasmid confirmed by sequencing acted as the DNA template. PCR amplification was performed using primers with T7 promoter. VgR and GFP dsRNA were synthesized with T7 RiboMAX ^TM Express RNAi System synthesis kit. The abdomens of S. exigua female pupae on 2nd and 6th day were injected with 3 μL double RNA by 10 μL microsyringe (2 μg·μL ^-1). RT-qPCR was used to detect the changes of VgR expression in 0-, 24-, 48-hour-old female adults. Meanwhile, eclosion rate and eggs per female were evaluated in control groups (blank control, GFP-dsRNA injection) and treatment group (VgR-dsRNA injection). 【Result】 The VgR and GFP gene fragments obtained by amplification were 327 and 417 bp, respectively. The VgR expression level of 0-, 24-, 48-hour-old female adults in the VgR-dsRNA group decreased by 79.35%, 84.22% and 67.68% compared with the GFP-dsRNA group, respectively. Through anatomical observation of the ovary of 0-, 24-, 48-hour-old female adults, it was found that compared with the GFP-dsRNA group, the ovary development of the VgR-dsRNA group was significantly delayed. Compared with the GFP-dsRNA group, the length of the ovary tube in the VgR-dsRNA group decreased by 23.92% for the 24-hour-old female adults. The GFP-dsRNA group has more mature eggs in the ovary with larger average diameter of (0.46±0.05) mm while the number of mature eggs in the VgR-dsRNA group was small with an average diameter of (0.23±0.02) mm. There was no significant difference of eclosion rate between GFP-dsRNA group and VgR-dsRNA group. In the VgR-dsRNA group, the average number of eggs per female was 170, while in the control groups (blank group, GFP-dsRNA group), the average number of eggs per female was 451 and 420, respectively. There was a significant difference in the amount of oviposition between control groups and treatment group. 【Conclusion】 The function of VgR was studied by dsRNA injection in vitro, which could significantly reduce the expression of VgR. VgR plays an irreplaceable role in the reproduction of S. exigua, which directly affects the ovary development and spawning capacity, and can be used as a potential target for controlling S. exigua.

Slow-Release Synergistic Effect of Humic Acid on Low Concentration Chlorothalonil

WEI ShiPing,WU Meng,LI GuiLong,JIANG ChunYu,LIU Ming,CHEN RuiRui,LI ZhongPei

Scientia Agricultura Sinica. 2019, 52(1): 65-72. doi:10.3864/j.issn.0578-1752.2019.01.007

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【Objective】The objective of this study is to clarify the slow-release synergistic effect of humic acid on low concentration (5 mg·L ^-1) of chlorothalonil, provide a new scientific idea for reducing pesticide use and prolonging pesticide efficacy, and to provide a theoretical basis for selecting new fungicide synergists. 【Method】Fusarium oxysporum was cultured in vitro solid and liquid medium including low concentrations of chlorothalonil with or without 50 mg·L ^-1 humic acid addition. The inhibition rate (IR) and spore number were determined through mycelium growth test and blood counting chamber. The heat released during the growth and metabolism process of F. oxysporum was measured by isothermal microcalorimetry technology. 【Result】The IR of humic acid-chlorothalonil combined treatment significantly increased by 10.29% (P<0.05) when IR of control was set to 0. The relative synergism was 25.33%. During liquid shake culture, there was no significant difference in spore number between the chlorothalonil treatment and humic acid-chlorothalonil combined treatment on the 3rd cultured day. On the 7th cultured day, the spore number of chlorothalonil treatment was significantly lower than that of other treatments (P<0.01). On the 14th cultured day, the spore number of humic acid-chlorothalonil combined treatment was significantly lower than other treatments (P<0.05), while there was no significant difference in spore number between chlorothalonil treatment and control. It showed that humic acid extended the inhibitory effect of low concentration chlorothalonil on the increase of spore number. The heat flow-time curve of each treatment showed that no heat release peak was detected in the humic acid-chlorothalonil combined treatment within 72 hours of monitoring, while that in the chlorothalonil treatment was found in later monitoring, and the heat flow-time curve of humic acid treatment was close to the control. P_max (peak power) of the chlorothalonil treatment was significantly lower than that of the control and humic acid treatment (P<0.05), while the T_max (peak power time) was significantly higher than that of the control and humic acid treatment (P<0.05). P_max, Q (total heat evolution), and k (microbial growth rate constant) of the humic acid-chlorothalonil combined treatment were all significantly lower than other treatments (P<0.05), which indicated that the pathogen metabolic activity of the combinated treatment was significantly lower than that in other treatments. That is, the growth and metabolism of F. oxysporum were most inhibited. There was no significant difference in P_max, T_max, Q, and k between the humic acid treatment and control, that is, the inhibition effect of humic acid treatment on pathogen was not monitored, and which could verify that the inhibition effect of combined treatment was irrelevant to humic acid itself.【Conclusion】The addition of humic acid can significantly enhance the ability of low concentration chlorothalonil to inhibit the growth of mycelia, increase of spore number and heat emission during growth and metabolism process of F. oxysporum. Using humic acid as a fungicide synergist of chlorothalonil is an effective measure to reduce the amount of chlorothalonil and extend the efficacy.

Optimization of Phosphorus Rate and Soil Available Phosphorus Based on Grain Yield and Nutrient Contents in Dryland Wheat Production

MA QingXia,WANG ZhaoHui,HUI XiaoLi,ZHANG Xiang,ZHANG YueYue,HOU SaiBin,HUANG Ning,LUO LaiChao,ZHANG ShiJun,DANG HaiYan

Scientia Agricultura Sinica. 2019, 52(1): 73-85. doi:10.3864/j.issn.0578-1752.2019.01.008

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【Objective】It is of great importance to explore the wheat grain yield, soil available phosphorus (P) and grain nutrient contents under a long term P application at different rates, for the purpose of appropriate P application, wheat yield increase and improvement of nutritional quality in drylands.【Method】Field experiments were conducted to investigate the effects of different phosphorus (P) rates on wheat yield, biomass, yield components, grain nitrogen (N)-, P- and potassium (K)-contents, soil available P content, and P absorption and utilization, based on the long-term fixed field experiment which was initiated in 2004 in the Loess Plateau. Soil and plant samples were collected in the consecutive experimental years of 2014-2015, 2015-2016 and 2016-2017. 【Result】 The three-year averaged results showed that long-term application of P increased wheat yield, biomass, spike number and grains per spike by 67%, 58%, 64% and 8%, respectively, while 1000-grain weight was decreased by 7% compared with no P application. The wheat yield and biomass were quadratically correlated with the P rate, and the maximum wheat yield was 6 465 kg·hm ^-2 at P rate of 144 kg P₂O₅·hm ^-2. The P and K content of grain increased with the P rate increasing, while the N content showed an opposite trend. There was a significant positive correlation between the soil available P content and the P rate. The soil available P was 16.9 mg·kg ^-1at sowing and 20.4 mg·kg ^-1at harvest when the maximum yield was occurred. The P absorption and utilization efficiency decreased with the increased of P rate. For each 50 kg P₂O₅·hm ^-2 increment, the P requirement increased by 0.4 g·kg ^-1 for the grain yield formation, while the P harvest index and the P physiological efficiency decreased by 1.3% and 45.1 kg·kg ^-1, respectively. 【Conclusion】 By balancing the wheat grain yield and key nutrient contents, the target grain yield should be 95% of the maximum yield in drylands of the experimental area, and the corresponding P application rate should be kept at 94 kg P₂O₅·hm ^-2, the available P at 12.0 and 13.8 mg·kg ^-1 at sowing and harvest, respectively.

Effects of Different Water and Fertilizer Coupling on Yield and Quality of Cucumber and Partial Factor Productivity of Fertilizer

JIANG JingJing,QU Feng,SU ChunJie,YANG JianFeng,YU Jian,HU XiaoHui

Scientia Agricultura Sinica. 2019, 52(1): 86-97. doi:10.3864/j.issn.0578-1752.2019.01.009

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【Objective】Substrate cultivation is one of the effective ways to solve the adverse influences of soil texture deterioration on crop production which were cultivated in protected environment. With lacking standard of water and fertilizer management for substrate cultivation, in order to ensure the standard of irrigation and fertilizer coupling on yield, dry weight, quality, partial factor productivity of fertilizer and water use efficiency of cucumber cultivated in substrate in spring, the irrigation and fertilizer supply systems for high yield, quality, and efficient production of cucumber substrate were investigated.【Method】‘ChunyouNo.1’ cucumber was chosen as the materials, and the experiment was subjected to three irrigation water levels (W1 (75% crop evapo-transpiration, ETc), W2 (100% ETc) and W3 (125% ETc)) and three Yamazaki cucumber nutrient solution formula concentrations (F1 (75%), F2 (100%), F3 (125%)). The study analyzed the effects of different water and fertilizer coupling on cucumber yield, dry weight, quality, partial factor productivity of fertilizer and water use efficiency. The multivariate regression analysis and spatial analysis methods were used to obtain the best combination of irrigation and fertilizer for efficient cultivation of spring cucumber in plastic greenhouses. 【Result】The increase of irrigation was beneficial to the growth of cucumber yield and partial factor productivity of fertilizer (PFP). The yield (7 667.3 kg/667m ²) and PFP (205.67 kg·kg ^-1) of W3F1 treatment were the largest during the 60 days of the harvest period. Under the same fertilization conditions, PFP was increasing with the increase of irrigation volume. Under the condition of F1, the net photosynthetic rate of W3 treatment was lower than W1, but its leaf area index was larger, therefore higher assimilation amount and yield were obtained. Under the same irrigation condition, the Vitamin C and reducing sugar performance of cucumber fruit under W1 level was the best, while the soluble solids and soluble protein of cucumber fruit under W3 had the best value. Through the multivariate regression analysis and the spatial analysis methods to evaluate yield, quality and partial factor productivity of fertilizer, the results showed that the fertilization and irrigation amount obtained was about 36.0-42.2 kg/667m ² and 198.0-219.8 m ³/667m ², or 42.2-44.6 kg/667m ² and 206.3-219.8 m ³/667m ². 【Conclusion】Irrigation level and fertilization level significantly affected growth, yield, quality, WUE and PFP. The best strategy of fertilization and irrigation for the production of drip-irrigated cultivated cucumber grown in substrate bags in Spring is level which yield, nitrate content and PFP of cucumber were ±10% of optimal value.

The Changes in the Contents of Ascorbic Acid and the Activities of Related Enzymes in Black Currant Fruits During the Process of Its Growth and Development

SUN XiaoJuan,LIU QingShuai,YUN AngRan,ZHANG Yan,HUO JunWei,QIN Dong,JIANG Ting

Scientia Agricultura Sinica. 2019, 52(1): 98-110. doi:10.3864/j.issn.0578-1752.2019.01.010

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【Objective】 The changes in the ascorbic acid (AsA) contents and the enzymatic activities during the anabolic process of different cultivars and growth stages of black currant fruits were studied to determine the relationship between AsA contents and anabolic enzymes during the growth and development of fruits, so as to provide a theoretical basis for comprehensively revealing the accumulation rule of AsA in black currant fruits. 【Method】 Three different cultivars of black currant fruits (Adelinia, Brodtrop and Heifeng) were studied and determined the contents of reduced AsA, oxidized ascorbic acid (DHA), reduced glutathione (GSH) and oxidized glutathione (GSSG) and anabolic enzymatic activities of young, expansion, half-veraison, veraison and maturity stages. 【Result】 There were significant diversities in fruit sizes, AsA contents and AsA metabolites of different cultivars of black currant fruits. The Adelinia had the largest weight of single fruit (1.97 g). During the growth and development process of fruits, the changes in the total ascorbic acid (T-AsA) and AsA contents of fruits were consistent among the three cultivars, and the young fruits had the highest contents. The AsA content of young Adelinia fruit was the highest (83.17 μmol?g ^-1 FW) and then sharply decreased rapidly to the maturity stage with the growth of the fruit, which decreased to 21.28 μmol?g ^-1 FW at maturity stage. The contents of GSH and T-GSH in the three cultivars increased with the development of fruits, but the different cultivars increased in different stages and degrees. The content of GSSG was quite different among different cultivars. For the mature fruits, the GSSG content of Heifeng was the lowest, which was 0.008 μmol?g ^-1 FW and only accounted for 10.2% of Adelinia. In AsA-GSH recycling regeneration metabolism, the activities of dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) showed the highest level at expansion period, and finally decreased to the lowest level at maturity stage. The DHAR and MDHAR activities of Brodtrop fruits showed slightly higher than those of Adelinia and Heifeng fruits. The activity of glutathione reductase (GR) was the highest level at young stage. The GR activity of Adelinia young fruits was the highest (0.06 μmol?min ^-1?g ^-1 FW), and then decreased with the growth of fruits. The changes in the activities of ascorbate peroxidase (APX) were similar to the changes in the activities of GR. The changes in the activities of L-galactose-1,4-lactone dehydrogenase (GalLDH), a key enzyme of L-galactose pathway, were consistent with the changes in AsA contents. The GalLDH activity of Adelinia young and mature fruits showed higher than that of Heifeng and Brodtrop young and mature fruits, respectively. According to the correlation analysis, the GalLDH activity showed a highly significant positive correlationship with T-AsA, AsA, DHA, DHAR and MDHAR. The correlation coefficient was above 0.91. The higher GalLDH activity was found in the fruits, the higher AsA contents of fruits also was found. There was a highly significant positive correlationship between DHAR and MDHAR, T-AsA and AsA. The APX had a high correlation with T-GSH and GSH. 【Conclusion】 The AsA content of black currant young fruits was the highest and there were significant differences among the three cultivars. The GalLDH, MDHAR and DHAR might be the key enzymes for AsA anabolism in black currant fruits. The accumulation of AsA content of black currant fruits resulted from the activity of GalLDH, which indicated that the anabolic pathway played a more important role and were found to be a dominant position. The related enzymes of AsA-GSH recycling regeneration pathway also contributed to the AsA anabolism. The accumulation of high AsA content in black currant fruits resulted from the combined effects of anabolic and recycling pathways.

Identification of the Fruit Characteristic Organic Acids of Chaenomeles speciosa from Qijiang, Chongqing by GC-MS and Their Dynamic Change Researching During Its Fruit Developing Period

LIU ShiYao,RAN Hui,MAO YunZhi,CHEN XinYu

Scientia Agricultura Sinica. 2019, 52(1): 111-128. doi:10.3864/j.issn.0578-1752.2019.01.011

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【Objective】 The study was carried out to identify characteristic organic acids and disclosing their dynamic changing law of Chaenomeles speciosa fruit from Qijiang, Chongqing during its fruit developing period, so as to provide the basic data for the C. speciosa fruit organic acids metabolic research. 【Method】 By methanol extracting, methyl derivating and GC-MS detecting, the organic acids composition and their content of 8 developing period C. speciosa cv. Daluo fruit samples of Qijiang was measured. The 2012 chromatographic similarity analysis software of Chinese Pharmacopoeia Commission was used to match their common constituent peaks. The dynamic law of the total organic acids, strong-sour and weak-sour organic acids were showed in SigmaPlots10.0 software and the PCA and HCA of all the samples was processed separately by Simca-P 11.5 and SPSS 20.0 softwares. 【Result】 The total 41 common characteristic constituents, including 10 short-chain carboxylic acids, 21 long-chain fatty acids, 5 aromatic-organic acids, 3 monobasic-phenol acids and 2 amino-acids, were successfully matched among 8 TICs of different period fruit samples C. speciosa cv. Daluo from Qijiang. The baseline of all the TICs were smooth and stable, the organic acid derivatives peaks' distribution were well-distributed, and the separating-degree of samples’ peaks was high. All target components were well separated. The total organic acids content of Qijiang C. speciosa cv. Daluo fruit from 90 ^th day after its full-bloom stage to 160 ^th days showed the changing on reversed ‘Z’ that including the sharply decreasing, slowly increasing and then decreasing again. The total organic acids content was significant positive correlated with the strong sour (r=0.970) and the weak sour taste organic acids (r=0.998). At the same time, the strong sour taste organic acids were significant positive correlated with short-chain carboxylic acids (r=0.999) and positive correlated with monobasic phenol acids (r=0.747). The weak sour taste organic acids were significant positive correlated with long chain fatty acids (r=0.999). The malic acid, laevulic acid and citric acid, whose relative content sum was more than 90%, were the key ingredients of strong sour organic acids. The malic acid content had experienced a reverse ‘Z’ trend that decreased firstly (90 ^thd-120 ^thd), increased slightly (120 ^thd-130 ^thd) and decreased finally (130 ^thd-160 ^thd). The citric acid had a similar process, but the levulic acid had an obviously reverse process of changing with malic acid. Actually the levulic acid showed the increasing progress gradually. The correlation analysis showed that the strong sour taste organic acids had significant positive correlation with malate and citrate, but weakly negative correlation with levulic acid, isocitric acid, and salicylic acid. The analyzing result showed that most of the weak sour organic acids including 9-octadecenoic acid, 9,12-octadecadienoic acid, hexadecanoic acid and 10-hydroxy-hexadecanoic acid experienced the similar accumulating progress which was rapidly decreasing firstly, then slowly increased and decreased in the final stage. However, nonadecanoic acid showed an opposite process that was gradually increasing. Correlation analysis showed that the total weak sour organic acids were positively related to most of them such as 9-Octadecenoic acid and 9, 12-Octadecadienoic acid but negative to nonadecanoic acid. The PCA result by Simca-p 11.5 disclosed that PC1 and PC2 separately distributed 40.00% and 23.20% of the total variance contribution rate. The main component score of each sample showed that: S1 and S2 clustered together which decided by α-Ketoglutaric acid, malic acid, quinic acid, shikimic acid, hexadecanoic acid and linoleic acid. The S3, S4 and S5 got into one branch that mainly because of oleinic acid and 10-hydroxy-Hexadecanoic acid. The S6 was closer to S7 largely because of malonic acid, levulic acid, isocitric acid, salicyluric acid. The S8 formed a unit alone that mostly decided by succinic acid, nonadecanoic acid, tetracosanoic acid. This result was very similar to the other one that clustered by Ward's method with Squared Euclidean Distance in SPSS. 【Conclusion】 C. speciosa cv. Daluo in Qijiang,Chongqing is a typical malate-accumulating fruit. During its developing period of C. speciosa fruit, the accumulating pattern of organic acids changed from Malate-Citrate accumulating type on 90 ^th days to Levulinic acid-Malic acid - Citric acid accumulating type on 160 ^th days after its flower-blooming. The change of acid accumulation pattern played a key role in the determination of acidity and flavor quality of C. speciosa fruit in Chongqing.

Suitability Evaluation of Apple for Chips-Processing Based on BP Artificial Neural Network

ZHANG Biao,LIU Xuan,BI JinFeng,WU XinYe,JIN Xin,LI Xuan,LI Xiao

Scientia Agricultura Sinica. 2019, 52(1): 129-142. doi:10.3864/j.issn.0578-1752.2019.01.012

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【Objective】The aim of the paper was to establish suitability evaluation model for apple chips-processing from different cultivars and to achieve the quality prediction of apple chips based on raw material indicators.【Method】34 fresh apple samples of 21 apple varieties from 7 major growing regions were selected as research objects. Factor analysis (FA) and analytic hierarchy process (AHP) were used to establish comprehensive quality evaluation model for chips, and Error Back Propagation (BP) artificial neural network was used to establish chips-processing suitability evaluation model for apple fruits. (1) Chips were prepared by instant controlled pressure drop (DIC, French for détente instantannée controlee, also known as explosion puffing) and 17 indicators were measured. The core indexes of chips were selected by FA and correlation analysis. The weights of the core indexes were determined by AHP, and then the comprehensive quality evaluation scores of chips were calculated. (2) 22 indicators of 34 fruit samples with different cultivars and regions were measured. Then the characteristic indicators of apple fruits related to chip qualities were screened out by correlation analysis between data groups of apple fruit indicators and chip core indexes. Learning model with input of fruit characteristic indicators and output of chip comprehensive evaluation scores was established by database of 29 apple samples. 5 apple samples were chosen as test samples to verify the prediction accuracy of the learning model. Modified leaning models from different sample groups were compared by prediction accuracy, which could be the evidence to evaluate rationality and stability for application of BP neural network in the present research.【Result】The results showed that L* value, brittleness, puffing degree, titratable acid, soluble sugar and crude protein of apple chip were determined as the core indexes which the weights were 0.3724, 0.2665, 0.1583, 0.0890, 0.0569 and 0.0569, respectively. The comprehensive quality scores of chips from 34 apple samples ranged from 0.2069 to 0.7933, indicating significant variation. The top 3 apple samples with high scores were Liaoning Huahong, Liaoning Huajin and Shandong Yanfu 6, and the final ranking for Shanxi Qinguan. Correlation analysis was performed between core indexes of chips and quality indicators of apple raw materials to achieve characteristic indicators of apple fruits, including the fruit shape index, a* value (pulp), pH value, titratable acid content, Vc content, proportion of core, protein content, b* value (pulp), density, soluble solids content, crude fiber content and total sugar content. Therefore, learning models were established with input layer of the characteristic indicators value of fruit and output layer of the comprehensive quality score of apple chip, which could predict the comprehensive quality of apple chips from indicators of raw materials. Moreover, the model showed high prediction accuracy. The relative errors between the predicted and actual values of the three learning models groups did not exceed 10%, and the coefficients of determination R ² of linear fitting were higher than 0.95.【Conclusion】Suitability evaluation of apple fruit for chips-processing could be evaluated by fruit shape index, a* value (pulp), pH value, titratable acid content, Vc content, proportion of core, protein content, b* value (pulp), density, soluble solids content, crude fiber content and total sugar content. The established model could be used to quantitatively predict apple fruit suitability for chips-processing based on the indicators of raw fruits.

Effects of Bovine LncRNA-133a on the Proliferation and Differentiation of Skeletal Muscle Satellite Cells

LI Yan,CHEN MingMing,ZHANG JunXing,ZHANG LinLin,LI Xin,GUO Hong,DING XiangBin,LIU XinFeng

Scientia Agricultura Sinica. 2019, 52(1): 143-153. doi:10.3864/j.issn.0578-1752.2019.01.013

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【Objective】The objective of this paper was to investigate the effects of long non-coding RNA LncRNA-133a on the proliferation and differentiation of bovine skeletal muscle satellite cells. 【Method】 This study used qRT-PCR to detect the expression level of LncRNA-133a in the skeletal muscle tissues of 3, 6 and 9 months old fetal cattle and 24 months old adult bovine skeletal muscle, and obtained the tissue temporal expression profile of LncRNA-133a. The in vitro induced myoblast differentiation model of bovine skeletal muscle satellite cells was constructed to simulate the growth and development of bovine skeletal muscle. The qRT-PCR was used to detect the cells temporal expression profiles of LncRNA-133a and myocyte differentiation markers MyoG and MHC. The bovine skeletal muscle satellite cells were transfected with LncRNA-133a overexpression vector (pCDNA3.1-EGFP- LncRNA-133a) or LncRNA-133a inhibitor (si-LncRNA 133a), and the transfection efficiency and the mRNA expression levels of LncRNA-133a, MyoD, MyoG and MHC were detected by qRT-PCR in each transfection treatment group, then the protein expression level of MHC gene was detected by western blotting. In addition, the cell proliferation of the bovine skeletal muscle satellite cells and the extent of myotube fusion at the differentiation stage were detected by EdU cell proliferation assay and immunofluorescence protein staining, respectively. 【Result】 Tissue expression profiling revealed that LncRNA-133a had the highest expression in the muscle tissue of 3 months old fetal bovine, followed by the 6-month-old fetal bovine muscle tissue, and the lowest expression in the 9-month-old fetus and adult bovine muscle tissue, which demonstrated that the time expression showed a downward trend. Cell-time expression profiles of LncRNA-133a, MyoG, and MHC were analyzed by a successfully constructed bovine skeletal muscle satellite cell differentiation model in vitro, and the results showed that the expression levels of myogenic differentiation markers MyoG and MHC gradually increased during the differentiation of bovine skeletal muscle satellite cells (D0-D3). The expression of LncRNA- 133a increased in the differentiation stage, and the expression level reached the highest at 48 h of differentiation (D2). The bovine skeletal muscle satellite cell model of overexpressing LncRNA-133a or inhibiting LncRNA-133a was constructed successfully, and in the proliferative phase (D0): the number of EdU positive cells in the overexpressed LncRNA-133a-treated group was significantly increased (P<0.01), and the number of EdU positive cells in the LncRNA-133a inhibition treatment group was significantly decreased (P<0.01), compared with the control group. At 48 h of differentiation (D2): compared with the control group, the results of LncRNA-133a overexpression treatment showed that mRNA expression levels of myocyte differentiation markers MyoD, MyoG and MHC were significantly increased (P<0.05). Western blotting showed that the expression of MHC protein was also significantly increased (P<0.01), and the immunofluorescence protein staining of MHC protein showed that the volume of fusion myotubes was larger. On the contrary, in the LncRNA-133a inhibition treatment group, the mRNA expression levels of MyoD, MyoG and MHC were decreased, and MyoG was significantly decreased (P<0.05). Meanwhile, the expression of MHC protein was significantly decreased (P<0.01), and the volume fraction of MHC protein fusion myotubes was also decreased. 【Conclusion】Thus, this study confirmed that LncRNA-133a promoted the proliferation and differentiation of bovine skeletal muscle satellite cells, which laid a foundation for further research on the regulatory network mechanism of LncRNA-133a regulating the proliferation and differentiation of bovine skeletal muscle satellite cells.

Effects of Diets with Energy-to-Nitrogen Rations on Growth Performance and Nutrients Apparent Digestibility in Growing Yanshan Cashmere Goat

ZHANG JiWei,GAO Kun,ZHANG YingJie,LIU YueQin,DUAN ChunHui

Scientia Agricultura Sinica. 2019, 52(1): 154-165. doi:10.3864/j.issn.0578-1752.2019.01.014

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【Objective】In this study, the growing male Yanshan cashmere goats were used as experimental animals, and the objective was to explore the proper energy level, protein level and energy-to-nitrogen ration according to effects of diets with different energy-to-nitrogen rations on growth performance and nutrients apparent digestibility in goat by feeding trial and digestion-metabolism trial.【Method】Eighty-one six-month-old growing male goats (24.96±2.95) kg were divided into 9 groups according to a 3×3 (energy × protein) completely random experiment design and offered 9 pellet total mixed ration (nine lambs per diet), in which metabolic energy (ME) were formulated at 9, 10 and 11 MJ/kg·DM, and digestible crude protein (DCP) were 8.5%, 9.5%, and 10.5%, respectively. There was a 10-day adaption period before the 50-day experimental period, and then 4 goats in each group were selected for digestion and metabolism test when the average body weight reached 30 kg. The total collection of feces and urine was conducted for 3 days after a 4-day adaptation, and residual feed, feces and urine were collected continuously.【Result】The results showed that: 1) The dry matter intake (DMI) and ratio of feed to gain (F/G) were decreased significantly (P<0.05) with the increase of dietary ME levels, while no extremely effect on ADG (P>0.05). ADG and DMI increased first and decreased afterwards with the increase of dietary DCP levels, and ADG and DMI in medium DCP groups were extremely higher than low and high DCP groups (P<0.05). The interactions of dietary ME×DCP had a remarkable effect on DMI (P<0.05). ADG of Group Ⅴ was highest (222 g·d ^-1), ADG of Group Ⅴ was significant higher than Group Ⅰ, Group Ⅳ and Group Ⅵ (P<0.05), and higher than the other 5 groups with no significant difference (P>0.05). 2) The fecal energy decreased significantly (P<0.05) with the increase of dietary ME levels, while digestible energy and gross energy (GE) apparent digestibility increased significantly (P<0.05). The dietary DCP levels and the interactions of dietary ME×DCP had no significant effect on energy digestion and metabolism. 3) The nitrogen (N) intake, fecal N decreased significantly (P<0.05) with the increase of dietary ME levels while digestible N had the trend to decrease, and N apparent digestibility in high ME groups were extremely higher than low and medium ME groups (P<0.05). N intake, urinary N, digestible N and N apparent digestibility increased significantly (P<0.05) with the increase of dietary DCP levels. The interactions of ME×DCP had a significant effect on N digestibility (P<0.05). 4) The apparent digestibility of dry matter (DM), organic matter (OM), ether extract (EE) and calcium (Ca) increased significantly (P<0.05) with the increase of dietary ME levels (P<0.05), while Ca digestibility in low DCP groups were extremely lower than medium and high DCP groups.【Conclusion】The trial results showed the most suitable diet (ME:10 MJ?kg ^-1, DCP:9.5%) with the highest ADG (222 g·d ^-1) and lower F/G.

Differentially Expressed MicroRNAs and Their Regulation Networks in Apis mellifera ligustica Larval Gut During the Early Stage of Ascosphaera apis Infection

GUO Rui,DU Yu,TONG XinYu,XIONG CuiLing,ZHENG YanZhen,XU GuoJun,WANG HaiPeng,GENG SiHai,ZHOU DingDing,GUO YiLong,WU SuZhen,CHEN DaFu

Scientia Agricultura Sinica. 2019, 52(1): 166-180. doi:10.3864/j.issn.0578-1752.2019.01.015

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【Objective】MicroRNA (miRNA) is a kind of key gene expression regulator, which can affect the interactions between host and pathogen. Ascosphaera apis is a lethal fungal pathogen that specifically infects honeybee larvae. The objective of this study is to analyze the differentially expressed miRNAs (DEmiRNAs) and their target genes in the Apis mellifera ligustica larval gut during the early infection stage of A. apis, reveal DEmiRNA’ roles in the stress responses of host at the miRNA omics level, and to screen the key miRNAs related to host response by constructing regulation networks of significant DEmiRNAs. 【Method】Normal and A. apis-infected 4-day-old larval gut of A. m. ligustica (AmCK and AmT) were deep-sequenced using small RNA-seq (sRNA-seq) technology, followed by quality-control of raw data and then mapping of the filtered data with the reference genome of Apis mellifera. The mapped tags were compared to the miRBase database to identify the expression of known miRNAs. The expression of miRNAs in each sample was normalized by TPM (tags per million) algorithm and significant DEmiRNAs were gained according to the standard |log₂fold change|≥1 and P≤0.05. Target genes of significant DEmiRNAs were predicted utilizing TargetFinder, and then annotated to the GO and KEGG databases. Cytoscape was used to visualize the regulation networks between significant DEmiRNAs and target mRNAs. Finally, Stem-loop RT-PCR and qPCR were conducted to verify the reliability of the sequencing data.【Result】sRNA-seq of AmCK and AmT produced 13 553 302 and 10 777 534 raw reads, and after strict filtration, 13 186 921 and 10 480 913 clean reads were obtained, respectively. The Pearson correlation coefficients among different biological replicates in each sample were above 0.9822 and 0.9508. There were 10 significant DEmiRNAs including 4 up-regulated miRNAs and 6 down-regulated miRNAs, and the overall expression level of DEmiRNAs in AmT was lower than that in AmCK. In total, 10 significant DEmiRNAs could link 3 788 target genes. The 1 240 target genes of up-regulated miRNAs could be annotated to 39 GO terms, and the mostly enriched terms were binding, cellular processes, metabolic processes, and response to stimulus. The 749 target genes of down-regulated miRNAs could be annotated to 34 GO terms, and the mostly enriched terms were cellular processes, binding, metabolic processes, and response to stimulus. The result of KEGG database annotation suggested that the target genes of up- and down-regulated miRNAs were respectively annotated in 95 and 66 pathways, the most abundant pathways were Wnt signaling pathway, Hippo signaling pathway, phototransduction and endocytosis, phosphatidylinositol signaling system, as well as purine metabolism. For up- and down-regulated miRNAs, there were 31 and 52 target genes could be annotated to endocytosis, 15 and 7 target genes could be annotated to ubiquitin-mediated proteolysis, 11 and 5 target genes could be annotated to Jak-STAT signaling pathway, 1 and 3 target genes could be annotated to the MAPK signaling pathway, respectively. Complex regulation networks existed between significant DEmiRNAs and their target mRNAs, among them 7 significant DEmiRNAs targeted 96 mRNAs associated with Wnt signaling pathway, and 8 significant DEmiRNAs targeted 55 mRNAs involved in endocytosis. Finally, the results of Stem-loop RT-PCR and qPCR verified the reliability of the sequencing data.【Conclusion】A. m. ligustica larval gut’s DEmiRNAs and their target genes during the early infection stage of A. apis were predicted and analyzed. DEmiRNA-mRNA regulation networks in the host were constructed and investigated. The results provide the expression profile and differential expression information of host miRNAs, and reveal that these DEmiRNAs likely participate in the stress responses of host via regulating biological processes such as cellular activity, metabolism, and immune defense. miR-4331-y, miR-4968-y, miR-8440-y, novel-m0023-5p and novel-m0025-3p jointly regulate Wnt signaling pathway and endocytosis of host and can be used as potential molecular targets for chalkbrood control.

Expression, Purification and Immunologic Function of Integrin β2 in the Silkworm (Bombyx mori)

ZHANG Kui,LI ChongYang,SU JingJing,TAN Juan,XU Man,CUI HongJuan

Scientia Agricultura Sinica. 2019, 52(1): 181-190. doi:10.3864/j.issn.0578-1752.2019.01.016

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【Objective】The objective of this study is to analyze the gene sequence and structural characteristics of integrin β2 in silkworm (Bombyx mori), and its expression profile in hemocytes following the larval exposure to different bacterial pathogens, investigate the binding and agglutination properties of the recombinant integrin β2 protein to various pathogen-associated molecular patterns (PAMPs) and bacteria, which will lay a foundation for further exploring the protein function of integrin β2 in B. mori. 【Method】Bioinformatics tools were used to determine the sequence and structural characteristics of integrin β2, and real-time fluorescent quantitative PCR (RT-qPCR) assay was executed to evaluate expression profile in hemocytes after microbial (Escherichia coli and Staphylococcus aureus) challenge. The cDNA fragment of integrin β2 was amplified using PCR, and the fragment containing the extracellular domain was inserted into a prokaryotic expression vector (pET22b). The insertion was confirmed in the recombinant plasmid and transformed into E. coli Rosetta (DE3), and then induced by IPTG to produce recombinant protein. The recombinant protein was purified using Ni-NTA affinity chromatography and analyzed by SDS-PAGE and Western blot. ELISA and Western blot were executed to determine the binding abilities of the recombinant protein to PAMPs (LPS and PGN) and different bacteria. Moreover, the agglutination ability and bacterial clearance assay were performed to understand the specific biological roles of integrin β2 in immunity.【Result】B. mori integrin β2 contains typical integrin β subunits, which comprises a long extracellular domain, a single transmembrane region and a short cytoplasmic tail. Further, it has several conserved motifs such as the MIDAS, EGF domain, Cys-repeat sequences and NPxY motifs. The RT-qPCR analysis showed that the integrin β2 expression varied significantly in hemocytes after infection with bacteria. High purity recombinant protein was obtained by prokaryotic expression and protein purification. The results of SDS-PAGE and Western blot showed that the purified recombinant protein was of high purity and could be used in subsequent tests. ELISA assay indicated that the purified recombinant integrin β2 protein had a strong binding ability to PAMPs (LPS and PGN). The results of bacterial binding test showed that the recombinant protein could bind many bacteria, but the binding ability with Gram-positive bacteria was higher than that with Gram-negative bacteria. The agglutination assay showed that the recombinant protein had strong agglutination effects on S. aureus in the presence of ca ²⁺. Further, bacterial clearance assay suggested that the recombinant protein could effectively promote the cleaning of exogenous invading bacteria from B. mori. 【Conclusion】The integrin β2 has a typical structure of the β integrin family, and it can recognize PAMPs (LPS and PGN) and enhance the aggregation of invading microbial pathogens by directly binding to them. Taken together, integrin β2 may play an important biological role in the bacterial immune response of B. mori.

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