Objective The aim was to provide basis for molecular marker assisted selection and resistance breeding of Langya chicken. Method The genetic polymorphism of Hae III site of Mx gene 3＇ sequence in Langya chicken was ...Objective The aim was to provide basis for molecular marker assisted selection and resistance breeding of Langya chicken. Method The genetic polymorphism of Hae III site of Mx gene 3＇ sequence in Langya chicken was analyzed by PCR-RFLP. Result The results showed that Hae III site controlled by allele A and B were polymorphic in Langya chicken breeds and the allele frequencies of A and B were 0.562 and 0.438 respectively. The genotype distribution of Hae III site was significantly not in Hardy-Weinberg equilibrium （ P 〈0.01 ）. The polymorphic fragments were cloned and sequenced, and the results revealed that the fragment size was 357 bp and a deletion of 31 bp occurred in variation sequences. Conclusion It was found that Hae III-RFLP exists in Mx gene 3＇ sequence in Langya chicken breeds of Shandong Province.展开更多
The duck circovirus （DuCV） infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province Bas...The duck circovirus （DuCV） infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%-97.5% nucleotide sequence identity to all the four Taiwan isolates （TC1/2002, TC2/2002, TC3/2002, TC4/2002）, 82.9% identity to the America （33753-52） isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however, showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.展开更多
A technique of direct sequence analysis of β-globin gene with the products of amplifi-cation by polymerase chain reaction (PCR) was reported and a case of β-thalassemia with therare mutation in Chinese,‘codon 14/15...A technique of direct sequence analysis of β-globin gene with the products of amplifi-cation by polymerase chain reaction (PCR) was reported and a case of β-thalassemia with therare mutation in Chinese,‘codon 14/15 (+G)’ was detected by this method.After the se-quence of the mutation site was determined,an analysis of the restriction map of the gene anddot blot hybridization with radioactive allele specific oligonucleotide probe was designed to con-firm the result of DNA sequencing.展开更多
[Objective] Torque teno virus （TIT） is a novel virus with negative single-strand DNA discovered in recent years, which is ubiquitous and nonpatho- genie. Torque teno sus virus （TrsuV） is widely prevalent in swine ...[Objective] Torque teno virus （TIT） is a novel virus with negative single-strand DNA discovered in recent years, which is ubiquitous and nonpatho- genie. Torque teno sus virus （TrsuV） is widely prevalent in swine populations, which is considered to be associated with some diseases such as post-weaning multi- systemic wasting syndrome （PMWS）. This study aimed to provided data for epidemiology of Tl＇suV in Guangdong Province. [Method] PCR primers were synthe- sized based on untranslated region （UTR） segment of TYsuV genome, to conduct PCR detection of 14 swine serum samples from two swine farms in Guangdong Province. A total of four PCR products of TrsuV1 and TrsuV2 from two swine farms were selected for cloning, sequencing and analysis. [ Result] Ten TrsuVl pos- itive samples （71% ） and eight TrsuV2 positive samples （57%） were obtained by PCR, including five double-positive samples （36%）. Sequence analysis of PCR products and reference strains showed that the UTR segments of samples GDTI-1 and GDT1-2 were both 305 bp, sharing 90.2% -95.1% similarity with Tl＇suVl reference strain, and the UTR segments of samples GDT2-1 and GDT2-2 were respectively 259 bp and 248 bp, sharing 67.3% - 100% similarity with TrsuV2 refer- ence strain. [ Conclusion] These results conformed that there are at least two types of Tl＇suV in Guangdong Province, with relatively high detection rates in some swine farms. Despite the putative harmlessness of Tl＇suV, the public health significance of TTsuV is noticeable due to its potential pathogenicity.展开更多
[Objective] The research aimed to isolate flanking sequences adjacent to the transgenic T-DNA in Brassica napus by an improved inverse PCR method.[Method] Using single clone of transgenic FS4 in Brassica napus as the ...[Objective] The research aimed to isolate flanking sequences adjacent to the transgenic T-DNA in Brassica napus by an improved inverse PCR method.[Method] Using single clone of transgenic FS4 in Brassica napus as the research materials,total DNA was extracted from transgenic Brassica napus by using modified CTAB method.After enzyme digestion and purification,self-joining was made.Two circles of nested PCR and the sequence alignment were carried out.[Result] A fragement with the size of 4.0 kb was amplified ...展开更多
A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 （Pseudomonas sp.） is described, whereby a lipase gene （lip） was clon...A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 （Pseudomonas sp.） is described, whereby a lipase gene （lip） was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.展开更多
Through exploring the microsatellite primers from the random genome sequences of Chinese shrimp （Fenneropenaeus chinensis）, some microsatellite primers were obtained with rich polymorphic genetic information, and a ...Through exploring the microsatellite primers from the random genome sequences of Chinese shrimp （Fenneropenaeus chinensis）, some microsatellite primers were obtained with rich polymorphic genetic information, and a triplex PCR was established using three primers （RS1101, RS0683 and H081 primers）. By adjusting the final concentration of Mg^2＋, dNTP and primers, and using a touch-town PCR program, the optimum amplification parameters of PCR system were obtained, which could successfully amplify the three primers in a PCR reaction. In the denatured PAGE gel, the amplified DNA fragments of three primers RS1 101,RS0683 and H081 could be easily identified each other. For the triplex PCR system, the PPE （probabilities of paternity exclusion） is 0.967 9,and the DP （discrimination power） is 0.999 327.Using the triplex PCR to test ten individuals of a parentage and their parents, an individual was excluded from the parentage in all of the three microsatellite loci, which might be mixed into the parentage for some unknown reason such as factitious misplay. The triplex PCR will be of great practical value in identifying the parentages of F. chinensis.展开更多
[Objectives]This study was conducted to detect and analyze tomato leaf curl New Delhi virus(ToLCNDV).[Methods]Through PCR detection,sequence analysis,and pathogenicity verification,tomato leaf curl New Delhi virus(ToL...[Objectives]This study was conducted to detect and analyze tomato leaf curl New Delhi virus(ToLCNDV).[Methods]Through PCR detection,sequence analysis,and pathogenicity verification,tomato leaf curl New Delhi virus(ToLCNDV)was identified in zucchini exhibiting systemic disease symptoms during a 2024 outbreak in Qingzhou City,Shandong Province,and was designated as ToLCNDV-SD.[Results]Specific primer amplification showed that all eight diseased samples produced bands of 504 bp(DNA-A)and 892 bp(DNA-B).Sequencing analysis revealed that ToLCNDV-SD DNA-A shared 96.10%homology with an Indonesian melon isolate(LC421834.1),while DNA-B showed 88.31%homology with a Malaysian bitter gourd isolate(MW248678.1).Phylogenetic analysis indicated its closest relationship with Southeast Asian cucurbit-infecting isolates.Friction transmission tests confirmed that the virus could spread mechanically,inducing typical symptoms 14 d after inoculation with positive PCR detection.[Conclusions]This study provides important insights for understanding the epidemic mechanisms and control strategies of ToLCNDV in China.展开更多
[Objective]The aim was to study homology between Ran gene in Allium cepa,Allium sativum and Brassica napus and Ran2 gene in Arabidopsis in order to determine whether three kinds of plant material as substitute for Ara...[Objective]The aim was to study homology between Ran gene in Allium cepa,Allium sativum and Brassica napus and Ran2 gene in Arabidopsis in order to determine whether three kinds of plant material as substitute for Arabidopsis. [Method]By using RT-PCR method,homology gene was cloned from totoal RNA which extracted from splinter cells of Allium cepa,Allium sativum and Brassica napus with Arabidopsis Ran2 primer,then,carrying out sequence and comparative analysis. [Result]The results showed that the open reading frames of Ran genes in Allium cepa,Allium sativum and Brassica napus were 666,663,666 bp,coding 221,220 and 221 amino acids respectively,with the molecular weight of 24.3 kDa. The sequence analysis showed that the amino acid homology of Ran genes between Allium cepa,Allium sativum,Brassica napus and Arabidopsis Ran2 were respectively 99.1 %,100 % （except an Asp D at Allium sativum C terminal）,96.4 %. The phylogenetic tree indicated that Ran genes from Allium cepa and Allium sativum had closer evolutionary relationship with Arabidopsis Ran2. [Conclusion]The research laid a foundation for further study on the biological function of plant Ran gene.展开更多
In this study, identification and rDNA-ITS sequence analysis of an anthracnose pathogen on Dracaena fragrans were carried out. [Method] D. fra-grans leaves with lesions were used as experimental materials to isolate a...In this study, identification and rDNA-ITS sequence analysis of an anthracnose pathogen on Dracaena fragrans were carried out. [Method] D. fra-grans leaves with lesions were used as experimental materials to isolate anthrac-nose pathogen. Morphological observation, rDNA-ITS amplification and sequence analysis were performed to identify the pathogen strain. [Result] Caonidia of the iso-lated anthracnose pathogen were straight or curved, el iptic to crescent, with 2-5 oil droplets, 7.5-20 × 4.5-5 μm. According to molecular phylogenetic analysis, the iso-lated pathogen strain was identified as a new species, which was named Col-letotrichum dracaena-fragrantis sp. nov. [Conclusion] This study provided theoretical basis for the prevention and control of anthracnose.展开更多
In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower...In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower (Helianthus annuus L.) inbred lines. Results indicated that factors for a successful multiplex PCR assay were related to the cycling touchdown annealing temperature, the balance of primer concentration at the various loci, the concentration of PCR buffer and the Taq DNA polymerase. Based on the optimization, a tailed primer strategy was outlined, and the effective ways were proposed to overcome the troubleshootings commonly encountered in the multiplex PCR and the multiplex gel electrophoresis.展开更多
[ Objective ] The aim of this study is to clone and analyze the actin gene from Rehmannia glutinosa. [ Method ] Degenerate primers were designed according to the conserved regions of actin sequences of Rehmannia gluti...[ Objective ] The aim of this study is to clone and analyze the actin gene from Rehmannia glutinosa. [ Method ] Degenerate primers were designed according to the conserved regions of actin sequences of Rehmannia glutinosa and its similar species, RT-PCR was next conducted to amplify the actin gene from Rehmannia glutinosa. [ Result] The amplified fragment is 724 bp and correspondingly 240 amino acids. The BLAST results indicate that the homology between the amplified fragment and other higher plants for aetin gene sequences and amino acid are more than 80% and 90%, respectively, suggesting that the amplified fragment is the actin gene of Rehmannia glutinosa. [ Conclusion] Phylogenetic analysis shows that the actin gene of Rehmannia glutinosa has an intimate genetic relationship with actin7 gene of Nicotiana tabacum.展开更多
[ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the ...[ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the biological function and application of chitinasa gene. [ Method] cDNA library of Leymus chinensis leaves were constructed, and its DNA sequence was determined or analyzed, while the homology of chitinasa gene and amino acid sequence was compared with that in GenBank. [ Result] One full length cDNA fragment with length of 996 bp was cloned from cDNA library of Leymus chinensis leaves. The length of ORF was 768 bp encoding 225 amino acids （GenBank accession number： EU344908）. The encoding products lacked CBD and C-terminal extension region from the view of structure, but had structural characters of Class Ⅱ chitinase gene, which indicated that amino acid sequence had high homology compared with Class Ⅱ chitinase gene of rye and wheat. The constructed recombinant vector pQE-LcChi2 could express a protein of 27 kD through induction, which was consistent with the deduced encoding product of pQE-LcChi2 gene. [ Conclusion] LcChl2 gene is an expression gene, which can express in E. coll.展开更多
There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical ...There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.展开更多
[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing...[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.展开更多
[Objective] The aim was to lay a foundation for research of contraceptive rodenticide with LDH-C4 as target protein. [ Method] EST sequence of LDH-C gene from black-lipped pika was cloned by PCR with degenerate primer...[Objective] The aim was to lay a foundation for research of contraceptive rodenticide with LDH-C4 as target protein. [ Method] EST sequence of LDH-C gene from black-lipped pika was cloned by PCR with degenerate primers; then the full length open reading frame （ORF） and 3＇UTR sequence were cloned by RACE technique. [ Result] The full length cDNA was 1 498 bp containing an ORF of 996 bp and a 3＇UTR of 486 bp. The ORF encoded a polypeptide of 332 amino acids. The alignment of LDH-C gene ORF nucleotide sequences from different species showed that the gene was conserved even between large taxons. The phylogenic tree showed that black-lipped pika LDH-C was closer to prima- tes and artiodactyla than to rodents. [Conclusion] cDNA sequence of LDH-C gene from black-lipped pika was cloned successfully.展开更多
基金Supported by Shandong Project of Agricultural Improved VarietyEngineering~~
文摘Objective The aim was to provide basis for molecular marker assisted selection and resistance breeding of Langya chicken. Method The genetic polymorphism of Hae III site of Mx gene 3＇ sequence in Langya chicken was analyzed by PCR-RFLP. Result The results showed that Hae III site controlled by allele A and B were polymorphic in Langya chicken breeds and the allele frequencies of A and B were 0.562 and 0.438 respectively. The genotype distribution of Hae III site was significantly not in Hardy-Weinberg equilibrium （ P 〈0.01 ）. The polymorphic fragments were cloned and sequenced, and the results revealed that the fragment size was 357 bp and a deletion of 31 bp occurred in variation sequences. Conclusion It was found that Hae III-RFLP exists in Mx gene 3＇ sequence in Langya chicken breeds of Shandong Province.
基金Shandong Provincial Excellent YoungScientist Fund (2004BS06002)Innovation Fund ofShandong Agricultural University
文摘The duck circovirus （DuCV） infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%-97.5% nucleotide sequence identity to all the four Taiwan isolates （TC1/2002, TC2/2002, TC3/2002, TC4/2002）, 82.9% identity to the America （33753-52） isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however, showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.
文摘A technique of direct sequence analysis of β-globin gene with the products of amplifi-cation by polymerase chain reaction (PCR) was reported and a case of β-thalassemia with therare mutation in Chinese,‘codon 14/15 (+G)’ was detected by this method.After the se-quence of the mutation site was determined,an analysis of the restriction map of the gene anddot blot hybridization with radioactive allele specific oligonucleotide probe was designed to con-firm the result of DNA sequencing.
基金Supported by Funding Project of Guangdong Science and Technology Department(No.2011B010500023)Funding Project of Guangzhou Science and Information Technology Department(No.12A64071507)
文摘[Objective] Torque teno virus （TIT） is a novel virus with negative single-strand DNA discovered in recent years, which is ubiquitous and nonpatho- genie. Torque teno sus virus （TrsuV） is widely prevalent in swine populations, which is considered to be associated with some diseases such as post-weaning multi- systemic wasting syndrome （PMWS）. This study aimed to provided data for epidemiology of Tl＇suV in Guangdong Province. [Method] PCR primers were synthe- sized based on untranslated region （UTR） segment of TYsuV genome, to conduct PCR detection of 14 swine serum samples from two swine farms in Guangdong Province. A total of four PCR products of TrsuV1 and TrsuV2 from two swine farms were selected for cloning, sequencing and analysis. [ Result] Ten TrsuVl pos- itive samples （71% ） and eight TrsuV2 positive samples （57%） were obtained by PCR, including five double-positive samples （36%）. Sequence analysis of PCR products and reference strains showed that the UTR segments of samples GDTI-1 and GDT1-2 were both 305 bp, sharing 90.2% -95.1% similarity with Tl＇suVl reference strain, and the UTR segments of samples GDT2-1 and GDT2-2 were respectively 259 bp and 248 bp, sharing 67.3% - 100% similarity with TrsuV2 refer- ence strain. [ Conclusion] These results conformed that there are at least two types of Tl＇suV in Guangdong Province, with relatively high detection rates in some swine farms. Despite the putative harmlessness of Tl＇suV, the public health significance of TTsuV is noticeable due to its potential pathogenicity.
基金Supported by National 863 Program of China(2006AA10A113)Natural Science foundation of Zhejiang Province(Y306097)~~
文摘[Objective] The research aimed to isolate flanking sequences adjacent to the transgenic T-DNA in Brassica napus by an improved inverse PCR method.[Method] Using single clone of transgenic FS4 in Brassica napus as the research materials,total DNA was extracted from transgenic Brassica napus by using modified CTAB method.After enzyme digestion and purification,self-joining was made.Two circles of nested PCR and the sequence alignment were carried out.[Result] A fragement with the size of 4.0 kb was amplified ...
基金The article was a part of the research program financed by the Science and Technology Bureau of Hebei Province, China (06220106D)
文摘A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 （Pseudomonas sp.） is described, whereby a lipase gene （lip） was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.
基金This study was supported by the National High-Tech Research and Development Program of China(863)under contract No.2005AA603210the National"948"Foundation of China under contract No.2006-G55(B)+2 种基金the National Natural Science Foundation of China under contract No.30500378the 0pen-end Funds of Jiangsu Key Laboratory of Marine Biotechnology,the Huaihai Institute of Technology under contract No.2006HS004the 0pen-end Funds of Key Laboratory of Aquatic Genetic Resources and Aquacultural Ecosystem of the Ministry of Agriculture of China,Shanghai Fisheries University under contract No.KFT2006-6.
文摘Through exploring the microsatellite primers from the random genome sequences of Chinese shrimp （Fenneropenaeus chinensis）, some microsatellite primers were obtained with rich polymorphic genetic information, and a triplex PCR was established using three primers （RS1101, RS0683 and H081 primers）. By adjusting the final concentration of Mg^2＋, dNTP and primers, and using a touch-town PCR program, the optimum amplification parameters of PCR system were obtained, which could successfully amplify the three primers in a PCR reaction. In the denatured PAGE gel, the amplified DNA fragments of three primers RS1 101,RS0683 and H081 could be easily identified each other. For the triplex PCR system, the PPE （probabilities of paternity exclusion） is 0.967 9,and the DP （discrimination power） is 0.999 327.Using the triplex PCR to test ten individuals of a parentage and their parents, an individual was excluded from the parentage in all of the three microsatellite loci, which might be mixed into the parentage for some unknown reason such as factitious misplay. The triplex PCR will be of great practical value in identifying the parentages of F. chinensis.
基金Supported by Taishan Industry Leading Talent Program in Shandong Province(tscx202306156)Weifang Science and Technology Development Program(2024GX073).
文摘[Objectives]This study was conducted to detect and analyze tomato leaf curl New Delhi virus(ToLCNDV).[Methods]Through PCR detection,sequence analysis,and pathogenicity verification,tomato leaf curl New Delhi virus(ToLCNDV)was identified in zucchini exhibiting systemic disease symptoms during a 2024 outbreak in Qingzhou City,Shandong Province,and was designated as ToLCNDV-SD.[Results]Specific primer amplification showed that all eight diseased samples produced bands of 504 bp(DNA-A)and 892 bp(DNA-B).Sequencing analysis revealed that ToLCNDV-SD DNA-A shared 96.10%homology with an Indonesian melon isolate(LC421834.1),while DNA-B showed 88.31%homology with a Malaysian bitter gourd isolate(MW248678.1).Phylogenetic analysis indicated its closest relationship with Southeast Asian cucurbit-infecting isolates.Friction transmission tests confirmed that the virus could spread mechanically,inducing typical symptoms 14 d after inoculation with positive PCR detection.[Conclusions]This study provides important insights for understanding the epidemic mechanisms and control strategies of ToLCNDV in China.
基金Supported by National Natural Science Foundation of China(30070370)~~
文摘[Objective]The aim was to study homology between Ran gene in Allium cepa,Allium sativum and Brassica napus and Ran2 gene in Arabidopsis in order to determine whether three kinds of plant material as substitute for Arabidopsis. [Method]By using RT-PCR method,homology gene was cloned from totoal RNA which extracted from splinter cells of Allium cepa,Allium sativum and Brassica napus with Arabidopsis Ran2 primer,then,carrying out sequence and comparative analysis. [Result]The results showed that the open reading frames of Ran genes in Allium cepa,Allium sativum and Brassica napus were 666,663,666 bp,coding 221,220 and 221 amino acids respectively,with the molecular weight of 24.3 kDa. The sequence analysis showed that the amino acid homology of Ran genes between Allium cepa,Allium sativum,Brassica napus and Arabidopsis Ran2 were respectively 99.1 %,100 % （except an Asp D at Allium sativum C terminal）,96.4 %. The phylogenetic tree indicated that Ran genes from Allium cepa and Allium sativum had closer evolutionary relationship with Arabidopsis Ran2. [Conclusion]The research laid a foundation for further study on the biological function of plant Ran gene.
文摘In this study, identification and rDNA-ITS sequence analysis of an anthracnose pathogen on Dracaena fragrans were carried out. [Method] D. fra-grans leaves with lesions were used as experimental materials to isolate anthrac-nose pathogen. Morphological observation, rDNA-ITS amplification and sequence analysis were performed to identify the pathogen strain. [Result] Caonidia of the iso-lated anthracnose pathogen were straight or curved, el iptic to crescent, with 2-5 oil droplets, 7.5-20 × 4.5-5 μm. According to molecular phylogenetic analysis, the iso-lated pathogen strain was identified as a new species, which was named Col-letotrichum dracaena-fragrantis sp. nov. [Conclusion] This study provided theoretical basis for the prevention and control of anthracnose.
文摘In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower (Helianthus annuus L.) inbred lines. Results indicated that factors for a successful multiplex PCR assay were related to the cycling touchdown annealing temperature, the balance of primer concentration at the various loci, the concentration of PCR buffer and the Taq DNA polymerase. Based on the optimization, a tailed primer strategy was outlined, and the effective ways were proposed to overcome the troubleshootings commonly encountered in the multiplex PCR and the multiplex gel electrophoresis.
基金National Natural Science Foundation of China (No 30472155)Beijing Natural Science Foundation (No 5062035)~~
文摘[ Objective ] The aim of this study is to clone and analyze the actin gene from Rehmannia glutinosa. [ Method ] Degenerate primers were designed according to the conserved regions of actin sequences of Rehmannia glutinosa and its similar species, RT-PCR was next conducted to amplify the actin gene from Rehmannia glutinosa. [ Result] The amplified fragment is 724 bp and correspondingly 240 amino acids. The BLAST results indicate that the homology between the amplified fragment and other higher plants for aetin gene sequences and amino acid are more than 80% and 90%, respectively, suggesting that the amplified fragment is the actin gene of Rehmannia glutinosa. [ Conclusion] Phylogenetic analysis shows that the actin gene of Rehmannia glutinosa has an intimate genetic relationship with actin7 gene of Nicotiana tabacum.
基金Supported by Science and Technology Research Project of Education Department of Liaoning Province(2008120)IntroducedTalent Start-up Fund Project of Dalian Nationalities University(20056209)~~
文摘[ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the biological function and application of chitinasa gene. [ Method] cDNA library of Leymus chinensis leaves were constructed, and its DNA sequence was determined or analyzed, while the homology of chitinasa gene and amino acid sequence was compared with that in GenBank. [ Result] One full length cDNA fragment with length of 996 bp was cloned from cDNA library of Leymus chinensis leaves. The length of ORF was 768 bp encoding 225 amino acids （GenBank accession number： EU344908）. The encoding products lacked CBD and C-terminal extension region from the view of structure, but had structural characters of Class Ⅱ chitinase gene, which indicated that amino acid sequence had high homology compared with Class Ⅱ chitinase gene of rye and wheat. The constructed recombinant vector pQE-LcChi2 could express a protein of 27 kD through induction, which was consistent with the deduced encoding product of pQE-LcChi2 gene. [ Conclusion] LcChl2 gene is an expression gene, which can express in E. coll.
基金supported by the Shandong Seed Projectthe National Natural Science Foundation of China(No.31372524)Science and Technology Development Plan of Shandong Province,China(No.2014GHY 115002)
文摘There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.
基金Supported by Agricultural Seed Project of Shandong Province~~
文摘[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.
基金Supported by State Ethnic Affairs Commission of P.R.C.(08XN04)Applicable and Fundamental Research Funds of SichuanProvince(2008JY0068)Academic Culture and TechnologyLeaders in Sichuan Province Foundation~~
文摘[Objective] The aim was to lay a foundation for research of contraceptive rodenticide with LDH-C4 as target protein. [ Method] EST sequence of LDH-C gene from black-lipped pika was cloned by PCR with degenerate primers; then the full length open reading frame （ORF） and 3＇UTR sequence were cloned by RACE technique. [ Result] The full length cDNA was 1 498 bp containing an ORF of 996 bp and a 3＇UTR of 486 bp. The ORF encoded a polypeptide of 332 amino acids. The alignment of LDH-C gene ORF nucleotide sequences from different species showed that the gene was conserved even between large taxons. The phylogenic tree showed that black-lipped pika LDH-C was closer to prima- tes and artiodactyla than to rodents. [Conclusion] cDNA sequence of LDH-C gene from black-lipped pika was cloned successfully.
