2023 Vol. 38, No. 8
Display Method:
2023, 38(8): 889-893.
doi: 10.19303/j.issn.1008-0384.2023.08.001
Abstract:
Objective A polyclonal antibody specific to the carboxy-terminus subfragment of Rep protein of the new-genotype Muscovy duck parvovirus (N-MDPV) was prepared. Methods Based on the protein sequence, the C-terminus subfragment region 487–627 aa of Rep of N-MDPV was synthesized to add His-tag. Using seamless cloning method, the segment was introduced into pET-28a(+) vector, transformed into Rosetta (DE3) Escherichia coli, and induced to express the recombinant protein. The recombinant protein was then purified by nickel column affinity chromatography for immunization on New Zealand white rabbits to produce the polyclonal antibody. Results The prokaryotic expression plasmid pET-28a-Rep-487-627 was constructed, purified, and expressed. The recombinant protein was approximately 24 kDa and mainly expressed in a soluble form. The indirect immunofluorescence and western blot showed that the prepared Rep polyclonal antibody could react specifically with the overexpressed N-MDPV in cells. Conclusion The prepared Rep polyclonal antibody exhibited a reaction specificity that recognized the conformational epitopes and linear epitopes of Rep protein.
2023, 38(8): 894-900.
doi: 10.19303/j.issn.1008-0384.2023.08.002
Abstract:
Objective To facilitate studies on the functions of the porcine circovirus type 2 Rep protein and its interaction with the host c-Myc protein, a polyclonal antibody against the virus was prepared. Method According to the complete sequence of porcine c-Myc (GenBank No. NM_001005154), primers were designed to amplify the CDS sequence of the gene by reverse transcription PCR. The CDS was double-digested by Nde I/Xho I enzymes to construct the prokaryotic expression vector pET-30a(+) and transformed into bacterium Arctic-ExpressTM (Escherichia coli) for induction and expression. The obtained products were denatured, re-natured, and affinity purified by His-band Ni+ chromatography as the antigen to prepare polyclonal antibody by immunizing New Zealand white rabbits. The resulting antibody was purified by the antigen affinity purification chromatography, and the titer and specificity determined by indirect ELISA and western blot. Result The cloned CDS-sequence of porcine c-Myc was 1359 bp. The recombinant protein of c-Myc-His was mainly in the form of inclusion bodies with a molecular weight of approximately 63 kDa. The titer of the polyclonal antibody prepared from the protein was greater than 1∶1093500. It could specifically recognize the target proteins including recombinant c-Myc-His and porcine c-Myc proteins. Conclusion The target polyclonal antibody against the porcine c-Myc protein was successfully obtained for further studies on the functions of the protein as well as its interactions with the PCV2 Rep protein.
2023, 38(8): 901-909.
doi: 10.19303/j.issn.1008-0384.2023.08.003
Abstract:
Objective Bioinformatics and expressions under stresses of glycerol-3-phosphate acyltransferase (GPAT) gene family member, ZmGPAT13, in maize were studied. Method Sequences of GPAT family members in Arabidopsis thaliana and Zea mays L. were obtained from the TAIR and MaizeGDB databases. Physicochemical properties, protein structure, subcellular localization, conserved motifs, phylogenetic evolution, phosphorylation sites, and cis-acting elements of promoter sequence of the GPATs were analyzed by bioinformatic methods. Expressions of ZmGPAT13under the stresses of mannitol, NaCl, abscisic acid (ABA), high temperature at 42 ℃, and low temperature at 4 ℃ were examined by qRT-PCR. Result A transmembrane protein, ZmGPAT13 localized in mitochondria, shared the same 15 conserved motifs with other GPAT family members, and had the highest homology with AtGPAT1 in arabidopsis. It contained 62 phosphorylation sites and might interact with multiple GPATs of maize. The promoters of ZmGPAT13consisted of numerous cis-acting elements associated with the responses to low temperature and ABA stresses. The gene expressed most highly in the coleoptile, followed by the roots and the lowest in the leaves. And, relatively, the expressions were significantly elevated under the stress of mannitol, NaCl, ABA or high or low temperature. Conclusion A high evolutionary conservation seemed existed in ZmGPAT13. It was postulated that, at transcription level, ZmGPAT13 might be involved in the responses of maize plants to abiotic stresses such as drought, salt, and extreme temperatures.
2023, 38(8): 910-916.
doi: 10.19303/j.issn.1008-0384.2023.08.004
Abstract:
Objective Mechanism of red or blue light in regulating the root development of flue-cured tobacco seedlings was studied. Method Effects of red or blue LED light exposure on the root growth and the synthesis and accumulation of endogenous gas signal molecule hydrogen sulfide (H2S) in CB-1 tobacco seedlings were monitored. Results After 7 days of the exposure to red or blue LED in comparison to white light, (1) the biomasses of aboveground parts and roots of the seedlings treated by red light increased by 74.62% and 15.64%, respectively, while the roots of the seedlings exposed to the blue LED decreased by 7.00%; (2) the H2S content on the red light-treated plants increased by 61.72%, but reduced on the blue light-treated counterparts; (3) the red LED increased the activities of DES and CS by 24.28% and 25.61%, respectively, but the blue LED decreased them by 32.10% and 18.30%, respectively; (4) the NtDES expression in the red light-treated seedlings were 8.8-fold of that in the plants treated by white light, whereas the blue light resulted in merely 13.82% of what white light did. Conclusion In contrast to blue LED, the exposure of the tobacco seedlings to red LED rose H2S in the plants through increased expression of the key enzyme coding genes in the biosynthesis inducing a thriving generation of lateral roots.
2023, 38(8): 917-923.
doi: 10.19303/j.issn.1008-0384.2023.08.005
Abstract:
Objective Factors contributing to the distinctively different texture of the fruits of two pear cultivars were investigated. Method Cellular microstructures of the pulp of Yuluxiangli and Daguoshuijingli pears were observed under an electron microscope SU810 . Textural firmness and brittleness, as well as contents of cell wall-modifying enzymes and relative expressions of the associated genes, of the fruits during develop stages were measured to correlate with the microstructure observations. Result ①The firmness of the pears of both varieties gradually declined during the fruit development. The force required to break into the fruit decreased 60d after flowering as the fruits were ripening. In comparison, Yuluxiangli pears were significantly softer and less brittle than Daguoshuijingli. ②At 400× magnification, the cells of Yuluxiangli pear pulp appeared larger than those of Daguoshuijingli at young stage. As the fruits expanded and matured, the cells of both varieties enlarged significantly but those of Yuluxiangli became smaller than those of Daguoshuijingli. And upon cutting, the edge of fractured Yuluxiangli pears were clean and smooth, while that of Daguoshuijingli rough and irregular. ③ In the matured fruits, the pectin methylesterase (PME) activity in Yuluxiangli pears were extremely significantly lower than that in Daguoshuijingli, whereas the polygalacturonase (PG) and β-galactosidase (β-Gal) activities extremely significantly higher. ④ The relative expressions of PME, PG, and β-Gal in Yuluxiangli were extremely significantly lower than those in Daguoshuijingli during the fruit development. And that of β-Gal was significantly positively related to the textural firmness, but that of PG to the brittleness, of the fruits for both pear varieties. Conclusion The unique texture of Yuluxiangli and Daguoshuijingli pears could not only be differentiated by their pulp cell ultrastructural differences but also closely related to the cell wall-modifying enzymes, such as, PG and β-Gal, in the fruits. Hence, the genes associated with those enzymes likely played a critical role in the texture of the pears.
2023, 38(8): 924-931.
doi: 10.19303/j.issn.1008-0384.2023.08.006
Abstract:
Objective Transcriptomes of Acorus gramineus tissues were applied to molecularly differentiating flavonoid synthesis genes in the organs. Methods The high-throughput sequencing technology was employed to obtain the transcriptomes of the genes in 7 organs of A, gramineus. Thereby, the functions of unigenes were annotated, the flavonoid biosynthesis pathway deciphered, the differentially expressed genes (DEGs) in the pathway screened, and an expression analysis performed. Results A total of 39.91 to 42.97 M of high-quality data were secured with 5.98–6.45 Gb bases, more than 94.05% Q30 bases, and a GC content of 47.81%–50.32%. From the GO database, 62506 were annotated to classify the 18616 unigenes into 3 functional groups that included cellular components, molecular functions, and biological processes corresponding to 6, 14, and 21 subcategories, respectively. Numerous genes were distributed in the subcategories, such as cellular anatomical entities, connections, catalytic activities, and cellular processes. There were 10124 unigenes enriched in the 5 major categories and 19 subclasses of the KEGG database. From which, 4 flavonoid biosynthesis pathways, 14 key enzymes, and 63 DEGs were screened. The transcriptomes in 7 tissues of A. gramineus obtained in this study led to the identification of 63 DEGs involved in 4 flavonoid biosynthesis pathways. These genes differed in expressions in the organs indicating their diversified roles in the flavonoid biosynthesis in A. gramineus Conclusion The research results have enriched the genetic information of A. gramineus and provided a reference for further elucidating the functional genes involved in the biosynthesis of flavonoids in A. gramineus.
Chemical Composition and Antioxidant Activity in Stems and Leaves of Paeonia Intersubgeneric Hybrids
2023, 38(8): 932-943.
doi: 10.19303/j.issn.1008-0384.2023.08.007
Abstract:
Objective Pharmaceutical property of the stems and leaves of Paeonia intersubgeneric hybrids (PIHs) was evaluated. Method Secondary metabolites in 16 PIHs were extracted from the stems and leaves and analyzed by the high-performance liquid chromatography with a diode array detector (HPLC-DAD) and the high-performance liquid chromatography quadrupole time-of-fight mass spectrometry (HPLC-Q-TOF-MS). Total phenolic content and in vitro antioxidant activity of the extract were measured. Results (1) Nineteen metabolites were identified from the extracts that included 5 monoterpene glycosides, 6 flavonoids, 4 tannins, and 4 phenols. (2) The compositions of all tested hybrids were essentially identical. (3) Except for phenols, the content of individual group or total amount of compounds in the 3 groups were significantly higher in the leaves than in the stems. (4) The in vitro antioxidant activity was tested significantly greater in the leaves than in the stems as well. (5) The contents of paeoniflorin in the leaves of 5 varieties were above the minimum 18 mg·g−1 as stipulated by the pharmacopoeia. (6) A cluster analysis grouped the 16 cultivars into 3 categories with one that contained two varieties, which scored the highest on all indicators except being moderate on monoterpene glycosides. Conclusion The stems and leaves of “Unique” and “Lollipop” in the 16 tested PIHs appeared to have pharmaceutical values.
2023, 38(8): 944-952.
doi: 10.19303/j.issn.1008-0384.2023.08.008
Abstract:
Objective Applicability of the guidelines recently released by the International Union for the Protection of New Varieties of Plants (UPOV) for quantitative DUS traits determination and classification of Antirrhinum majus L. in China was examined. Method An experimental planting of 40 germplasms of A. majus L. was conducted following the guidelines TG/221/1 issued by the UPOV. Thirteen DUS traits of the plants, leaves, flowers, and other parts were collected to statistically analyze their variations, classes, and principal components using SPSS for the application on the plant varieties in China. Result The variation coefficients that met the selection criteria set by the guidelines were 16.91%-65.87% among different species and 5.29%-12.18% within a same species on 11 traits, which were specified by the guidelines, as well as two additional ones identified by this study. The expressions of these 13 traits differentiated by least significant difference could all be applied to adequately identify the germplasms and for the DUS guideline development on A. majus L in China. In addition to the 4 factors listed in the UPOV guidelines, the principal component analysis suggested 2 new criteria for the plant classification. Conclusion The current UPOV guidelines provide quantitative DUS traits of A. majus L. germplasms for the species classification. A close examination with a specially designed experimentation revealed additional criteria on inflorescence length, plant height, and number of primary branches on the plant for establishing guidelines applicable in China.
2023, 38(8): 953-959.
doi: 10.19303/j.issn.1008-0384.2023.08.009
Abstract:
Objective Means utilizing molecular markers to differentiate rapidly and accurately male and female Populus yunnanensis plants at young stages was investigated. Method Using simple sequence repeats (SSR), molecular markers were screened for those might associate with the sex of P. yunnanensis at seedling stages and before flowering. Result Fifteen pairs of SSR primers were selected from the reported literatures. One of them, coded BPCA90, showed clear bands and stable reaction in agarose gel detection. After rescreening the primer by polyacrylamide gel electrophoresis, a specific band of approximately 680 bp appeared in the PCR amplification of male P. yunnanensis. Subsequent testing on 30 plants using these SSR primers showed significantly more males than females in a ratio of 13 to 2. Conclusion Short of a visible morphological sign for the determination, the SSR primers screened by this study appeared to suffice the general purpose of differentiating the sex of P. yunnanensis at early growth stage.
2023, 38(8): 960-965.
doi: 10.19303/j.issn.1008-0384.2023.08.010
Abstract:
Objective To determine the control efficiency of six insecticides (emamectin benzoate, spinetoram, clorfenapyr, chlorantraniliprole, lufenuron and indoxacarb) on Spodoptera frugiperda and their toxicity to Trichogramma chilonis Ishii. Methods Foliar spray application was used to evaluate the control efficiency of those insecticides on Spodoptera frugiperda in the field, leaf dipping was adopted to examine the toxicity of those insecticides to the second-instar larvae of S. frugiperda in the lab, and residues contact was carried out to test the toxicity of those insecticides to T. chilonis. Results Those tested insecticides had acute and long-lasting toxicity to S. frugiperda. The control efficiency was 78.68%-95.75%, more than 90%, and 88.27%-96.3%, respectively, on the 1st, 3rd and 7th day after insecticide application. Emamectin benzoate, spinetoram, clorfenapyr and chlorantraniliprole were highly toxic to T. chilonis, with half of their field recommended dosage causing mortality rates of 100%, implying the application of those insecticides in the field is dangerous to T. chilonis. However, the toxicity of lufenuron and indoxacarb to T. chilonis was low. Conclusion Emamectin benzoate , spinetoram, clorfenapyr and chlorantraniliprole can be used first for emergency control of S. frugiperda. Lufenuron and indoxacarb can also be used in the integrated pest management of S. frugiperda.
2023, 38(8): 966-975.
doi: 10.19303/j.issn.1008-0384.2023.08.011
Abstract:
Objective This study aimed to optimize the extraction process of Litsea cubeba essential oil and investigate its antibacterial activity and mechanism of action against plant pathogenic fungi. Methods Response surface methodology was employed to optimize the extraction conditions of Litsea cubeba essential oil, including NaCl volume fraction, liquid-solid ratio, and distillation time. The inhibitory effect of Litsea cubeba essential oil on Pythium aphanidermatum was evaluated by the growth rate method, while its physiological and biochemical effects were assessed by measuring fungal mycelium dry weight, MDA content, reducing sugar content, and protective enzyme activities such as catalase, peroxidase, and superoxide dismutase. Results The optimal extraction conditions for Litsea cubeba essential oil were determined as NaCl volume fraction of 2.39%, liquid-solid ratio of 4.8:1 (mL·g−1), and distillation time of 4.94 hours, resulting in an extraction yield of 4.43%. Litsea cubeba essential oil exhibited a potent inhibitory effect on Pythium aphanidermatum, with an EC50 value of 224.4 μg·mL−1. Treatment with Litsea cubeba essential oil reduced fungal mycelium dry weight, increased cell membrane permeability, decreased reducing sugar content in the fungal body, and increased protective enzyme activities. Conclusion The optimized extraction process was stable, feasible, and cost-effective compared to other methods. Litsea cubeba essential oil exerted antibacterial activity by inhibiting mycelial growth, disrupting cell membrane structure, and decreasing protective enzyme activities in the mycelia.
2023, 38(8): 976-982.
doi: 10.19303/j.issn.1008-0384.2023.08.012
Abstract:
Objective The present paper aimed to identify the pathogen causing leaf spot on Pittosporum tobira, to study the biological characteristics of the pathogen, and screen out effective fungicides to control this disease. Method The pathogen was isolated from the leaves with typical symptoms by tissue separation method and verified by pathogenic test. The morphological and molecular biology methods were used to identify species of the pathogen. The mycelium growth in plate was used to investigate the biological characteristics and fungicides toxicity of the pathogen. Result The HT10 strain was isolated from the disease leaves and inoculated onto healthy P. tobira. After inoculation, the center of the disease spot was grayish brown, the edge was brown, and the periphery was yellow halo, which was consistent with the field’s symptoms. On PDA media, the fungal colonies displayed brown and black. Conidia were inverted rod, ovoid or nearly elliptic, with transverse and longitudinal septa. The phylogenetic analysis used multiple genes of ITS, GAPDH and RPB2 clustered the isolate with Alternaria longipes clade. The optimum growth condition on PDA were full light, 28 ℃ temperature and, 6.0 pH. The optimal carbon source or nitrogen source is sucrose or glycine, respectively. The lethal temperature is 41 ℃/15 min. Laboratory toxicity test showed that the 25% SC of Imazalil·Fludioxonil has the best inhibitory effect to pathogenic fungus with EC50 of 0.799 μg·mL−1 and the 400 g·L−1 SC of Captan·Tebuconazole has the worst inhibitory effect with EC50 of 370.457 μg·mL−1. Conclusion The pathogen causing the leaf spot disease on P. tobira is A. longipes, which is the first report of leaf spot disease on P. tobira caused by A.longipes. 25% SC Imazalil·Fludioxonil has a strong inhibitory effect on the growth of A. longipes.
2023, 38(8): 983-988.
doi: 10.19303/j.issn.1008-0384.2023.08.013
Abstract:
Objective A SYBR Green I-based quantitative RT-PCR (qPCR) method was developed for detecting salmonella in irrigation water. Method For the methodology development a pair of primers were designed based on the sequences of the invasion protein A gene (invA) of Salmonella. qPCR reaction conditions were optimized, the assay tested for specificity and sensitivity, and a standard curve of amplification constructed. Test results on a specimen of contaminated irrigation water using the qPCR assay were compared for detection accuracy with those obtained from the national standard method. Result The newly developed qPCR assay showed a minimum detection limit of 1×10−1pg·μL−1 and free of interference from genomic nucleic acids of non-target microbes. The constructed linear standard curve between 2×100cfu·mL−1 and 2×105 cfu·mL−1 had a high correlation coefficient of 0.999 6. The assay demonstrated same accuracy as did the national standard method in detecting salmonella in irrigation water. Conclusion The newly established qPCR assay could be adequately applied for salmonella detection in agriculture irrigation water.
2023, 38(8): 989-1003.
doi: 10.19303/j.issn.1008-0384.2023.08.014
Abstract:
Objective A molecular biology identification method to detect common plant-derived functional ingredients in health food products using genetic barcodes was developed. Method By means of retrospective investigation, 5 common health food raw materials in China, i.e., American ginseng (Panax quinquefolius), Dendrobium candidum, rhodiola (Rhodiola nobilis), wolfberries (Lycium barbarum L.), and ginseng (Radix Ginseng), were firstly tested for functional ingredients using the best extraction method on the target DNA. A stable, efficient, and accurate PCR reaction system was constructed with primer sequence designed based on the conservative characteristics of ITS2 internal transcribed spacer and components of each substance adjusted and optimized. Molecular identification on health food products of various forms was tested using the developed method. Result The innovative application of a modified CTAB method afforded successful extraction of the target DNA in health food materials as well as products at commonly formulated dosages. The PCR amplified with the detection sensitivity on nucleic acids of American ginseng, D. candidum, and rhodiola reached 1 ng·μL−1, and of wolfberries and ginseng 0.1 ng·μL−1. Conclusion By using the ITS gene barcodes obtained through the DNA extraction and PCR amplification, the key functional ingredients from plant materials such as American ginseng, ginseng, D. candidum, wolfberries, and rhodiola rosea could be identified for authentication of health food products in the forms of pills, tablets, and concoctions.
2023, 38(8): 1004-1010.
doi: 10.19303/j.issn.1008-0384.2023.08.015
Abstract:
Porcine circovirus virus type 2 (PCV2) is a major pathogen that causes postweaning multisystemic wasting syndrome (PMWS) and other swine-related diseases in piglets. Replicase (Rep) is a functional replication initiation protein encoded by the ORF1 gene participating in the DNA rolling loop replication of PCV2 by interacting with Rep′, another replication-related protein encoded by ORF. As an essential functional protein of PCV2, Rep could become a new target that can lead to breakthroughs in the research on antiviral therapy for PCV2, as most of the existing studies on the pathogenesis of PCV2 and vaccine have focused on Cap. This article reviewed the latest reports published in the world to systematically analyze Rep, especially, the critical roles and mechanisms that 8 known kinds of intracellular proteins interacted with it may play in the replication of PCV2. Furthermore, other cellular proteins that are possibly associated with the PCV2 Rep but not yet identified by the scientific communities are also included in the discussion for future exploration on the PCV2 pathogenesis.
Porcine circovirus virus type 2 (PCV2) is a major pathogen that causes postweaning multisystemic wasting syndrome (PMWS) and other swine-related diseases in piglets. Replicase (Rep) is a functional replication initiation protein encoded by the ORF1 gene participating in the DNA rolling loop replication of PCV2 by interacting with Rep′, another replication-related protein encoded by ORF. As an essential functional protein of PCV2, Rep could become a new target that can lead to breakthroughs in the research on antiviral therapy for PCV2, as most of the existing studies on the pathogenesis of PCV2 and vaccine have focused on Cap. This article reviewed the latest reports published in the world to systematically analyze Rep, especially, the critical roles and mechanisms that 8 known kinds of intracellular proteins interacted with it may play in the replication of PCV2. Furthermore, other cellular proteins that are possibly associated with the PCV2 Rep but not yet identified by the scientific communities are also included in the discussion for future exploration on the PCV2 pathogenesis.
