2021 Vol. 36, No. 2
Display Method:
2021, 36(2): 135-140.
doi: 10.19303/j.issn.1008-0384.2021.02.001
Abstract:
Objective In order to determine the function of SU protein of enzootic nasal tumor virus of goats (ENTV-2), Method RT-PCR method was used to amplify the SU gene fragment from ENTV-2 FJ and then the SU gene was cloned into pMD-19T Simple Vector; After sequencing, the cloning vector was subcloned into PET-32a (+), the recombinant plasmid was transformed into RosettagamiB (DE3) competent cells. SDS-PAGE, Western-blot and ELISA were used for identification and antigenicity analysis of recombinant proteins. Result The result showed that the expressed recombinant protein was about 64.38 kD, and the best expression condition was induced at 37 ℃ for 4 h at a final IPTG concentration of 0.4 mmol/L. The purified ENTV-2 virus was used for SDS-PAGE, and the mice anti-SU polyclonal antibody was used as primary antibody for Western-blot. The Western-blot analysis showed the mice anti-SU polyclonal antibody could react with ENTV-2 antigen specifically. It is proved that the expressed SU recombinant protein has better antigenicity. Conclusion The results proved that the expressed SU recombinant protein has better antigenicity, and provided a basis for the preparation of ENTV-2 specific monoclonal antibody and the establishment of ENTV-2 specific serological method.
2021, 36(2): 141-147.
doi: 10.19303/j.issn.1008-0384.2021.02.002
Abstract:
Objective To determine a previously unknown pathogen that caused an outbreak of highly lethal disease in a geese farm. Method In August 2019, a disease characterized by gout broke out in a goose farm in Nanping City, Fujian Province. Three clinical samples from the diseased geese, which showed symptoms of the infectious disease characterized by gout with an incidence rate as high as 40% that resulted in a mortality rate of 80% on 5- to 20-d-old geese, were collected and tested in laboratory for pathogen identification. Result The PCR results confirmed the existence of novel goose astrovirus in all three samples, but no bacteria were isolated from medium culture. Inoculating the virus into the 11-d-old goose embryos did not induce death but showed dotted hemorrhage on the body skin and hemorrhage on the kidneys and lungs. The allantoic fluid and tissue specimens collected for PCR were tested positive as novel goose astrovirus, not other viruses commonly known to infect geese. The isolated virus was named FJ-NP, and its partial ORF2 genes showed a close relationship with the strains found in Anhui (i.e., GD AHAU2, AHAU3, and AHAU5), in Heilongjiang (i.e., AstV-Goose-2018-HLJ01) and in Henan (i.e., AstV-HN02-Goose-1119-18, AstV-AH02-Goose-0715-18, AstV-HB02-Goose-0310-19, and AstV-HN03-Goose-0402-19) but far from the Hubei, Beijing or Fujian strains. Conclusion This study successfully isolated a strain of novel goose astrovirus, and performed a genetic evolution analysis on its partial ORF2
2021, 36(2): 148-156.
doi: 10.19303/j.issn.1008-0384.2021.02.003
Abstract:
Objective Sequence of the gene associated with the key enzyme in sucrose metabolism pathway of potatoes, sucrose synthase (SuSy), was determined for studying the biological function of the enzyme. Method The core fragment (PGSC0003DMG400002895, SuSy 4) of SuSy was screened from the transcriptome database on the plantlets of Alpine potatoes (Solanum tuberosum L. cv. Huaiyushan). The gene was cloned by RT-PCR, and its sequence analyzed using the bioinformatics method. Result The total length of cDNA of SuSy gene was 2 418 bp containing 45.08% of G+C. The hydrophilic protein had 805 amino acids with a molecular weight of 92 471.33 Da and an isoelectric point of 5.87. Its secondary structure was 45.84% of alpha helix, 15.16% of β -extended strands, and 39.01% of random coils. The β-lamellae and α-helix were found throughout the entire protein but appeared as irregular, curl, extension chains at the C- and N-terminals. The tetrameric gene mainly existed in the cytoplasm, mitochondria, and chloroplast. It closely related to those of S. lycopersicum, S. pennellii, S. chilense, S. tuberosum, Capsicum annuum, and C. baccatum in evolution, especially, S. tuberosum. Conclusion The SuSy gene in the Alpine potato of Huaiyushan had the characteristic structure of a typical sucrose synthase. The amino acid and nucleic acid sequences were highly conserved and homologous to the similar species. The findings would be of great significance for further understanding on the biological function of the enzyme.
2021, 36(2): 157-167.
doi: 10.19303/j.issn.1008-0384.2021.02.004
Abstract:
Objective The diversified phenotypic characteristics of Blumea balsamifera (L.) DC. germplasms were classified to facilitate the breeding program. Method Nine quantifiable and 13 quality phenotypic traits of the plant were used to describe the 159 germplasm samples of B. balsamifera. Genetic diversity, correlation, principal component, and cluster analyses were applied for the study. Result The genetic diversity on phenotypic characteristics of the collected germplasms was rich. Among the quantifiable traits, plant height had the highest genetic diversity index of 2.072; and, number of flowering branches topped the coefficient of variation at 32.76%. The crown breadth of the plants was found significantly correlated with the height, leaf width, petiole length, length of flower branch, and opening angle of flower branch (P＜0.01). On the quality traits, the highest genetic diversity index of 1.201 was the leaf shape. And, the color intensity of anthocyanin in leaf vein and that in main stem, as well as, those in leaf edge and petiole were significantly correlated. The cumulative contribution rate of the first eight principal components reached 64.32%. They included the factors associated with yield, color-intensity, leaf smoothness, leaf edge, leaf shape, leaf greenness, and flower-branch angle. Based on the phenotypic traits, the sum of squares deviations method using the genetic distance of 10 on the germplasms divided the 159 varieties into Group Ⅰ of 39 that accounted for 24.53% of the total, Group Ⅱ of 38 that accounted for 23.90% of the total, and Group Ⅲ of 82 that accounted for 51.57% of the total. Conclusion Genetically, the phenotypic characteristics of the 159 B. balsamifera germplasms appeared richly diversified. The leaf width of the plants could be the most outstanding trait for breeding selection of high-yield B. balsamifera varieties.
2021, 36(2): 168-175.
doi: 10.19303/j.issn.1008-0384.2021.02.005
Abstract:
Objective Role of SlSIP1L12, an SIP1 subfamily gene of trihelix transcription factors, played in tomato seed germination was investigated. Method Expressions under and responses to phytohormone and abiotic stress of SlSIP1L12 were tested by RT-PCR. The RNAi technology was used to suppress the SlSIP1L12 expression to reveal its function on the seed germination. ELISA was applied to detect the endogenous ABA contents in the seeds. Result (1) The length of SlSIP1L12 was 1 125bp in AC++ that encoded 374 amino acids. The phylogenetic analysis confirmed that SIP1 genes had indeed evolved in AC++. (2) SlSIP1L12 was primarily expressed in the stems and mature leaves, and secondly, in the flowers and fruits. (3) The suppressed expression of SlSIP1L12 could be artificially induced by ABA or dehydration, which implied a close relationship between the gene and ABA. (4) The germination of the SlSIP1L12 transgenic seeds was faster and grew to longer radicles than control. (5) ABA contents were significantly reduced in the transgenic seeds 7d after germination. Conclusion The downregulated SlSIP1L12 that stimulated germination of tomato seeds was postulated to be closely associated with the ABA response of the plant.
2021, 36(2): 176-181.
doi: 10.19303/j.issn.1008-0384.2021.02.006
Abstract:
Objective Two new protein phosphatase genes, MiSTPP1 and MiSTPP4, from macadamia (Macadamia integrifolia) were cloned for structure and function analyses by bioinformatics. Method The genes were cloned from M. integrifolia using transcriptome sequencing and RT-PCR technique. Homology, phylogenetic evolution, physicochemical properties, phosphorylation sites, subcellular localization, transmembrane domains, and signal peptides of the genes were analyzed. Result The cloned MiSTPP1 and MiSTPP4 from M. integrifolia were assigned with the GenBank accession numbers MT374548 and MT374551, respectively. The amino acid sequences of the genes had a similarity to other plant PP1 proteins containing the same typical structural domain MPP_PP1_PPKL. The evolutionary tree analysis showed that they closely related to PP1 family proteins. The basic physicochemical properties of MiSTPP1 indicated it be an unstable hydrophilic protein, and those of MiSTPP4 a stable hydrophilic protein. Phosphorylation sites on the genes were principally serine and threonine. The predicted subcellular localization, transmembrane domain, and signal peptide suggested that MiSTPP1 and MiSTPP4, as non-secretory and non-transmembrane proteins, were most likely located in the cytoplasm. The secondary and tertiary structures of the genes contained mainly α-helixes and random coils. Conclusion MiSTPP1 and MiSTPP4 from macadamia belonged to the protein phosphatase PP1 gene family and might play a role in response to stress and signal transduction as well as other physiological and biochemical processes of the plant.
2021, 36(2): 182-187.
doi: 10.19303/j.issn.1008-0384.2021.02.007
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Objective Mycelial growth of Agaricus bisporus W192 in a shaking flask was monitored to determine viability of the culture method for scale-up application. Method Physiological and biochemical properties of A. bisporus mycelia biomass in the liquid culture including the number and diameter of mycelium pellets, pH of fermentation broth, content of reducing sugar and amino nitrogen as well as the activity of extracellular carboxymethyl cellulase, amylase, acid protease, and laccase were monitored for the analysis. Result On the 8th day of culture, the fungal biomass reached a maximum at 9.7 mg·ml−1, the greatest number of mycelium pellets of 880 CFU·mL−1, the largest average diameter of mycelium at 0.809 mm, the peak reducing sugar and amino nitrogen contents at 5.228 and 0.079 mg·ml−1, respectively, and the highest activities of carboxymethyl cellulase, amylase, and laccase of 0.69 , 2.11 , and 15.02 U, respectively. The acid protease activity of 2.93U maxed on the 10th day. By transferring the mycelia in a simulated liquid fermentation tank after 8d of culture, 115 CFU·mL−1 of mycelium pellets and 7.56mg·mL−1 in biomass were obtained. Conclusion The mycelial growth of A. bisporus in the liquid shaking flask culture correlated with some of selected physiological and biochemical indices. The established method was deemed applicable for tank fermentation in propagating A. bisporus for large scale production of the mushroom.
2021, 36(2): 188-194.
doi: 10.19303/j.issn.1008-0384.2021.02.008
Abstract:
Objective Effects of the symbiosis between Epulorhiza sp. and Dendrobium officinale in a liquid culture medium on the growth and nutrient content of the plant were analyzed. Method In a potted experimentation, the sterile D. officinale seedlings were irrigated with a liquid medium as control or one containing Epulorhiza sp. for the treatment. Agronomic characteristics and nutrient contents of the stems and leaves from one- and 2-year-old D. officinale plantlets were monitored. Result Showing dark green leaves and robust stems, the treatment plantlets grew more vigorously than control. In the presence of Epulorhiza sp., on average the girths of the productive stems of one- and 2-year-old plantlets significantly increased by 65.67% and 74.25%, respectively, and the single stem weight by 55.29% and 51.45%, respectively (P＜0.05). The increases meant improved crop yield. Meanwhile, the contents of polysaccharides, dendrobine, crude protein, and amino acids increased as well. For the one-year-old plantlets, the crude polysaccharides rose significantly by 30.39%, and the crude protein 18.7% (P＜0.05). For both one- and 2-year-old plantlets, the total amino acids significantly increased by 27% and 30.25%, respectively (P＜0.05), while the crude fiber and ash significantly decreased by 17.76% and 36.36%, respectively (P＜0.05). Conclusion Both crop yield and nutrient content of D. officinale could be significantly improved by the presence of Epulorhiza sp. in medium. It suggested the potential benefits of organic fungi-containing manure utilization, artificial greenhouse operation, and harvest time management for the cultivation of D. officinale.
2021, 36(2): 195-201.
doi: 10.19303/j.issn.1008-0384.2021.02.009
Abstract:
Objective A subcritical water extraction method was optimized by response surface experiments to extract polysaccharides from pitaya stems. Method On the basis of a single factor test, pitaya stems were extracted using phenol-sulfuric acid to determine the polysaccharide content. To optimize the subcritical water extraction process assisted by ultrasonic pretreatment, water temperature, extraction time, liquid-to-material ratio, and pH were used against polysaccharide extraction rate in a response surface experiment. Result The optimized processing conditions included the applications of water at pH 5.9 and 144 ℃ with a liquid-to-material ratio of 31:1 (mL:g) to extract for 19 m. A polysaccharide extraction rate of 26.47% was achieved. Conclusion The established method appeared adequate for polysaccharide extraction from pitaya stems, and the mathematical model obtained be used to analyze and predict the process parameters.
2021, 36(2): 202-208.
doi: 10.19303/j.issn.1008-0384.2021.02.010
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Objective To better understand the pathogenicity of Didymella bryoniae on melons, this study aimed to establish an efficient means of preparing and regenerating the fungal protoplasts. Method Driselase at 20 g·L−1 and a lysing enzyme at 8 g·L−1 were combined and used for the protoplast preparations. A single-factor experiment was conducted to analyze the effects of age of mycelia, time and temperature of enzymatic digestion, rotational speed of culture vessel, type and concentration of osmotic stabilizer, and pH of medium on releasing of the protoplasts. Conditions for the subsequent protoplast regeneration in medium were also optimized. Result The highest yield of protoplasts of 9.65×107 cells·mL−1 was achieved using the mycelia cultured for 36 h to be enzymatically digested in a solution containing NaCl 0.7 mol ·L−1 as the osmotic stabilizer at 30 ℃ and pH 7.0 with 140 r·min−1 constant rotation for 4 h. The protoplast regeneration rate could reach up to 22.53% in a 0.5% agar SR culture medium but declined rapidly in 10 h and then leveled off in storage. Conclusion The protoplasmic preparation and regeneration methods were efficient and could materially aid the establishment and research on the genetic transformation of D. bryoniae.
2021, 36(2): 209-214.
doi: 10.19303/j.issn.1008-0384.2021.02.011
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Objective Pathogenicity and molecular mechanisms of tobacco mosaic virus (TMV), a typical member belonging to Tobamovirus of Vigaviridae that infect more than 400 species in 36 families of plants causing 20%-30% reduction or complete loss on crop yield, were studied. Methods Sequence of the 1 425 bp TMV specific P54 protein was amplified by RT-PCR from the cDNA of Nicotiana tabacum var. Samsun NN infected with TMV and cloned into prokaryotic expressing plasmid, pEASY®-Blunt E1, followed by expressing in E. coli BL21 (DE3) by IPTG induction. Subsequently, the expression products were retrieved and purified by Ni-NTA chromatography and confirmed by western-blotting identification. Results The genome of TMV was a positive-sense single-stranded RNA of 6 400 bp that encoded one structural protein and two nonstructural proteins. The amplified 1 425 bp P54 was inserted in the virus replication associated P183 gene. The prokaryotic expressed recombinant P54 was insoluble. It was retrieved and purified to show a molecular weight of approximately 60 kDa and verified by means of western blot. Conclusions TMV P54 protein was successfully expressed and purified for further study on the devastating diseases on plants caused by TMV.
2021, 36(2): 215-220.
doi: 10.19303/j.issn.1008-0384.2021.02.012
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Objective A rapid method to detect Bactrocera tau (Walker) was established for inspection on produce to prevent potential introduction and spread of the invasive pest. Method Based on the species-specific PCR (SS-PCR) technology, B. tau as control and 20 common fruit flies of similar morphology were used to extract the genomic DNA templates, and the homology of the specimens checked with the COI mt DNA sequence. A rapid and accurate identification method applying the B. tau-specific primers NF404 and NR610 was designed and verified by PCR amplification and electrophoresis analysis. Result The newly developed SS-PCR detection method clearly amplified a single 207 bp band only on the target B. tau, not on any other fruit flies. Comparisons on the test results on the insects or body parts of all fruit flies with homogenous band sequence to B. tau validated the new methodology. Conclusion The newly established SS-PCR method exhibited high repeatability, specificity, and accuracy in detecting B. tau. It was considered applicable for the entry/exit and quarantine inspection on the invasive insect, larvae or dead body parts found on vegetables and fruits.
2021, 36(2): 221-227.
doi: 10.19303/j.issn.1008-0384.2021.02.013
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Objective Host factors in Nicotiana benthamiana that interact with the N protein of tomato spotted wilt virus (TSWV), a representative member of plant negative stranded RNA viruses, were identified in preparation for further analysis on the regulation mechanism and in search for effective prevention and control of TSWV-induced disease on plants. Method Using the yeast two-hybrid (Y2H) method, proteins in N. benthamiana that interacted with the bait N protein of TSWV were screened. Result Fifteen host proteins were identified. Conclusion These identified proteins are known to be associated with pigment biosynthesis, thylakoid membrane assembly, plant defense response, ribosome biogenesis, lipid metabolism, and cellular functions in plants. Being upregulated in N. benthamiana infected by TSWV, these proteins might also act as auxiliary proteins in translating the regulation playing an important role in the development and response to abiotic stress of the plant.
2021, 36(2): 228-235.
doi: 10.19303/j.issn.1008-0384.2021.02.014
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Objective To investigate the effect of biochar made from rice straws on cadmium (Cd) immobilization in different types of soil under flooding. Method An in-lab experiment on 3 Cd-added different types of soil with or without a 5% addition of biochar made from rice straws was conducted under varied durations of flooding. The types of soils used were yellow soil, paddy soil derived from quaternary red clay, and brown soil. Result The biochar reduced pH and redox potential (Eh) but increased conductivity of the soils on the first day after flooding. As the flooding persisted, Eh in the soils reduced continuously, but the rate declined in the presence of the biochar. During the initial stage of flooding, the Cd contents in the soils were highest in the yellow soil at 272.5 μg·L−1 followed by the paddy soil at 23.48 μg·L−1, while the brown soil at 1.44 μg·L−1 being the lowest. The Cd reductions by 31.66% in the yellow soil, 75.04% in the paddy soil, and 66.67% in the brown soil were attributed to the added biochar. Under prolonged flooding, the Cd in the yellow and paddy soils gradually decreased even without the biochar addition. In 30d, the reductions were 89.34% on the yellow soil and 76.53% on the paddy soil. In comparison, the addition of the biochar brought about 85.41% Cd reduction on the yellow soil and 37.03% on the paddy soil. After 30d of flooding, the biochar out-performed control with the CaCl2-Cd contents in the yellow, paddy, and brown soils lowered by 17.3%, 56.3%, and 12.4%, respectively. Conclusion By adding 5% of the rice straw biochar, the water-soluble Cd in the 3 different types of soil could be significantly reduced. Prolonged flooding made the effect less pronounced, and the biochar immobilization of Cd appeared more effective in the paddy soil than the other two soil types.
2021, 36(2): 236-242.
doi: 10.19303/j.issn.1008-0384.2021.02.015
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Objective A visual loop-mediated isothermal amplification (LAMP) technique for rapid detection of Salmonella in soil was developed to facilitate the prevention and control of the pathogens in vegetable fields. Method Based on the sequence of the invasion protein A (invA) of Salmonella, LAMP primers were designed and reaction system optimized. Specificity and sensitivity of the methodology were challenged by artificial inoculation and verified by the National Standard Detection Method for Salmonella Detection in Vegetable Cultivation Soil. Result The LAMP reacted positively on Salmonella but negatively on 6 non-Salmonella strains. It showed a minimum detection limit of 7 CFU·25 μL−1 and a sensitivity of 4×102CFU·g−1 on the artificially inoculated Salmonella. Compared with the standard method, the LAMP exhibited same testing accuracy. Conclusion The newly developed visual LAMP assay was rapid, accurate and sensitive for the detection of Salmonella in soil for vegetable farming.
2021, 36(2): 243-248.
doi: 10.19303/j.issn.1008-0384.2021.02.016
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Objective Microalgae capable of effectively removing nitrogen and phosphorus from wastewater were screened for potential applications on antipollution or effluence treatment at aquaculture farms. Method Three selected microalgae, Chlorella sp. JY-1, Chlorella sp. SY-4, and Desmodesmus sp. SH-1, were evaluated for their capability in removing nitrogen and phosphorus from the wastewater at a shrimp aquaculture farm. Result After 5d of cultivation, the microalgae grew to a cell density in the (7±1)‰ salinity medium at 1.56×107·ml−1 on JY-1, 1.47×107·ml−1 on SY-4, and 6.62×106·ml−1 on SH-1. The removal rate on total nitrogen was 50.36% by JY-1, 41.51% by SY-4, and 49.74% by SH-1; that on ammonia nitrogen, 96.29% by JY-1, 84.92% by SY-4, and 96.65% by SH-1; that on nitrate nitrogen, 15.84% by JY-1, 3.69% by SY-4, and 12.56% by SH-1; and, that on total phosphorus, 93.51% by JY-1, 82.38% by SY-4, and 94.25% by SH-1; but not significant on nitrite nitrogen by any of them. The microalgae appeared to grow normally in a culture medium of 5%, 10%, 20% or 30% salinity. Among them, Chlorella sp. JY-1 performed the best on growth as well as nitrogen and phosphorus removal in the test wastewater. Conclusion Chlorella sp. JY-1 appeared to offer a promising potential for application of purifying aquaculture effluence.
