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The Application of Nicotiana benthamiana as a Transient Expression Host to Clone the Coding Sequences of Plant Genes
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作者 Jianzhong Huang Peng Jia +3 位作者 Xiaoju Zhong Xiuying Guan Hongbin Zhang Honglei Ruan 《American Journal of Molecular Biology》 CAS 2024年第2期54-65,共12页
Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using co... Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using complementary DNA (cDNA) derived from messenger RNA (mRNA) extracted from plant tissues and generated by reverse transcription. However, some CDS are difficult to acquire through this process as they are expressed at extremely low levels or have specific spatial and/or temporal expression patterns in vivo. These challenges require the development of alternative CDS cloning technologies. In this study, we found that the genomic intron-containing gene coding sequences (gDNA) from Arabidopsis thaliana, Oryza sativa, Brassica napus, and Glycine max can be correctly transcribed and spliced into mRNA in Nicotiana benthamiana. In contrast, gDNAs from Triticum aestivum and Sorghum bicolor did not function correctly. In transient expression experiments, the target DNA sequence is driven by a constitutive promoter. Theoretically, a sufficient amount of mRNA can be extracted from the N. benthamiana leaves, making it conducive to the cloning of CDS target genes. Our data demonstrate that N. benthamiana can be used as an effective host for the cloning CDS of plant genes. 展开更多
关键词 Coding sequence Genomic sequence Nicotiana benthamiana Plant genes
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Full-length transcriptome sequence and SSR marker development for genetic diversity research in yellowfin seabream Acanthopagrus latus
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作者 Jin ZHANG Jinmei LIU +6 位作者 Chong HAN Cheng PENG Yong LI Junhong XIA Yong ZHANG Shuisheng LI Haoran LIN 《Journal of Oceanology and Limnology》 SCIE CAS CSCD 2023年第3期1073-1083,共11页
Yellowfin seabream Acanthopagrus latus is an important economic fish in Chinese coastal areas.Given its narrow distribution and overfishing,the genetic diversity of yellowfin seabream has been restricted for artificia... Yellowfin seabream Acanthopagrus latus is an important economic fish in Chinese coastal areas.Given its narrow distribution and overfishing,the genetic diversity of yellowfin seabream has been restricted for artificial breeding and reproduction.We performed full-length transcriptome sequencing and assembly of the genome of yellowfin seabream.A total of 68086 unigenes were obtained,with an N50 of 3391 bp on average length of 2933 bp.A total number of 50593 expressed sequence tags linked to simple sequence repeats(EST-SSR)were identified,among them dinucleotide repeats(40.6%)and AC/GT motifs(38.5%)were the most frequent.Of the 190 EST-SSRs for which PCR primer pairs were designed,150 primer pairs successfully amplified target loci and 15 SSRs showed high polymorphism.The alleles per locus ranged 6-50 on average of 25.3.The expected and observed heterozygosity varied from 0.632 to 0.969 and from 0.519 to 0.953,respectively.The polymorphic index content(PIC)values of each locus ranged 0.587-0.966 on average of 0.851.Among six yellowfin seabream population samples preliminarily tested for genetic diversity and differentiation,the Fangchenggang(FCG)population in Guangxi Province had the highest mean observed heterozygosity(H_(o))value(0.786),whereas the Zhangzhou(ZZ)population in Fujian Province had the lowest(0.678).The pairwise fixation index(Fst)values indicated significant population differentiation among six yellowfin seabream populations.This study provided evidence for the usefulness of the transcriptomic resource information and EST-SSR markers for natural resource conservation,population genetics,and breeding studies of yellowfin seabream in South China. 展开更多
关键词 yellowfin seabream Acanthopagrus latus full-length transcriptome expressed sequence tags linked to simple sequence repeats(EST-SSR)marker genetic diversity
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Detection of Novel BEST1 Variations in Autosomal Recessive Bestrophinopathy Using Third-generation Sequencing
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作者 Jia-xun LI Ling-rui MENG +6 位作者 Bao-ke HOU Xiao-lu HAO Da-jiang WANG Ling-hui QU Zhao-hui LI Lei ZHANG Xin JIN 《Current Medical Science》 SCIE CAS 2024年第2期419-425,共7页
Objective:Autosomal recessive bestrophinopathy(ARB),a retinal degenerative disease,is characterized by central visual loss,yellowish multifocal diffuse subretinal deposits,and a dramatic decrease in the light peak on ... Objective:Autosomal recessive bestrophinopathy(ARB),a retinal degenerative disease,is characterized by central visual loss,yellowish multifocal diffuse subretinal deposits,and a dramatic decrease in the light peak on electrooculogram.The potential pathogenic mechanism involves mutations in the BEST1 gene,which encodes Ca2+-activated Cl−channels in the retinal pigment epithelium(RPE),resulting in degeneration of RPE and photoreceptor.In this study,the complete clinical characteristics of two Chinese ARB families were summarized.Methods:Pacific Biosciences(PacBio)single-molecule real-time(SMRT)sequencing was performed on the probands to screen for disease-causing gene mutations,and Sanger sequencing was applied to validate variants in the patients and their family members.Results:Two novel mutations,c.202T>C(chr11:61722628,p.Y68H)and c.867+97G>A,in the BEST1 gene were identified in the two Chinese ARB families.The novel missense mutation BEST1 c.202T>C(p.Y68H)resulted in the substitution of tyrosine with histidine in the N-terminal region of transmembrane domain 2 of bestrophin-1.Another novel variant,BEST1 c.867+97G>A(chr11:61725867),located in intron 7,might be considered a regulatory variant that changes allele-specific binding affinity based on motifs of important transcriptional regulators.Conclusion:Our findings represent the first use of third-generation sequencing(TGS)to identify novel BEST1 mutations in patients with ARB,indicating that TGS can be a more accurate and efficient tool for identifying mutations in specific genes.The novel variants identified further broaden the mutation spectrum of BEST1 in the Chinese population. 展开更多
关键词 autosomal recessive bestrophinopathy BEST1 gene third-generation sequencing MUTATION
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To Analyze the Sensitivity of RT-PCR Assays Employing S Gene Target Failure with Whole Genome Sequencing Data during Third Wave by SARS-CoV-2 Omicron Variant
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作者 Pooja Patel Yogita Mistry +1 位作者 Monika Patel Summaiya Mullan 《Advances in Microbiology》 CAS 2024年第5期247-255,共9页
Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the... Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community. 展开更多
关键词 SARS-CoV-2 S gene Target Failure Whole Genome sequencing Omicron
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The Influence of Aerial Exposure on Sea Anemones Aulactinia veratra Mucin Genes Expression Using the RNA Sequencing
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作者 Alaa Haridi 《Open Journal of Marine Science》 2024年第1期1-18,共18页
Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air duri... Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air during the low tide and produces large quantities of mucus as an external covering. The relation between low tide and mucus secretion is still unclear, and what is the role of mucin during arial exposure is not yet investigated. This study hypothesised that the mucin genes in A. veratra would have significantly high expression in response to aerial exposure. Therefore, the aim of current study was to examine and analyses the response of A. veratra mucins in response to an experiment involving three hours of aerial exposure. To achieve this, aim the RNA-sequencing and bioinformatics analyses were used to examine the expression profile of A. veratra mucin genes in response to aerial exposure. The generated results have shown that, Mucin4-like and mucin5B-like were up-regulated in response to the three hours of aerial exposure in A. veratra. This finding shows a significant role of mucin5B-like and mucin4-like genes in response to air stress at low tide. The data generated from this study could be used in conjunction with future mucin gene studies of sea anemones and other cnidarians to compare A. veratra mucin gene expression results across time, and to extend our understanding of mucin stress response in this phylum. 展开更多
关键词 Aulactinia veratra Mucin4-Like Mucin5B-Like CNIDARIA Mucin gene Expression RNA-sequencing Sea Anemones MUCUS Aerial Exposure
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Generation and Analysis of Expressed Sequence Tags(ESTs) from Muscle Full-Length cDNA Library of Wujin Pig 被引量:2
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作者 ZHAO Su-mei LIU Yong-gang +4 位作者 PAN Hong-bing ZHANG Xi GE Chang-rong JIA Jun-jing GAO Shi-zheng 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2014年第2期378-386,共9页
Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle ... Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identified in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1 076 bp, and the cDNA fullness ratio was 86.2%. A total of 1 058 unique sequences with 342 contigs (32.3%) and 716 singleton (67.7%) expressed sequence tags (EST) were obtained by clustering and assembling. Meanwhile, 826 (78.1%) ESTs were categorized as known genes, and 232 (21.9%) ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index of Sus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank (accession numbers: EU650784-EU650788, GE843306, GH228978-GH229100). The abundantly expressed genes in porcine muscle tissue were related to muscle fiber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs. 展开更多
关键词 muscle tissue full-length cDNA library expressed sequence tag PIG
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Molecular Cloning and Sequence Analysis of Class Ⅱ Chitinase Gene in Leymus chinensis 被引量:5
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作者 金华 安晓雯 姜国斌 《Agricultural Science & Technology》 CAS 2009年第4期96-100,共5页
[ 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
关键词 Leymus chinensis Chitinase gene CLONING sequence analysis
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Sequence Analysis of HA Genes from Three H9N2 Subtype Avian Influenza Viruses 被引量:2
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作者 韩春华 林健 +3 位作者 刘月焕 潘洁 马明 刘永宏 《Animal Husbandry and Feed Science》 CAS 2009年第1期32-35,共4页
[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu... [ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% ( Dk/HK/Y439/97 ) -98.0% ( Ck/GX17/00 ), 85.1% (Dk/HK/Y439/97) - 99.1% ( Ck/GXl 7/00), 90.7% ( Ck/BJ/3/01 ) - 99.1% (Ck/GX17/00) with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L ( Leu), suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin. 展开更多
关键词 H9N2 subtype avian influenza virus HA gene sequence analysis
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Complete Sequence and Gene Organization of the Mitochondrial Genome of Tokay (Gekko gecko) 被引量:8
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作者 韩德民 周开亚 《Zoological Research》 CAS CSCD 北大核心 2005年第2期123-128,共6页
Long-PCR amplification, clone and primer-walking sequencing methods were employed in determine the complete sequence of mitochondrial genome of tokay (Gekko gecko). The genome is 16 435 bp in size, contains 13 protein... Long-PCR amplification, clone and primer-walking sequencing methods were employed in determine the complete sequence of mitochondrial genome of tokay (Gekko gecko). The genome is 16 435 bp in size, contains 13 protein-coding, 2 ribosomal and 22 transfer RNA genes. The mt genome of Gekko is similar to most of the vertebrates in gene components, order, orientation, tRNA structures, low percentage of guanine and high percentage of thymine, and skews of base GC and AT. Base A was preferred at third codon positions for protein genes is similar to amphibians and fishes rather than amnion vertebrates. The standard stop codes (TAA) present only in three protein genes, less than those of most vertebrates. Transfer RNA genes range in length from 63 to 76 nt, their planar structure present characteristic clover leaf, except for tRNA-Cys and tRNA-Ser (AGY) because of lacking the D arm. 展开更多
关键词 Gekko gecko SQUAMATE Complete sequence of mitochondrial genome gene organization
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Determination and Analysis of Mitochondrial ND2 Gene Sequence of Anas platyrhynchos 被引量:4
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作者 涂剑锋 司方方 +1 位作者 邢秀梅 杨福合 《Agricultural Science & Technology》 CAS 2009年第3期46-49,共4页
[Objective] The study was to analyze the phylogenesis of Anas platyrhynchos. [Method] Complete sequence of mitochondrial ND2 gene of 4 Anas platyrhynchos was determined by direct DNA sequencing based on PCR products. ... [Objective] The study was to analyze the phylogenesis of Anas platyrhynchos. [Method] Complete sequence of mitochondrial ND2 gene of 4 Anas platyrhynchos was determined by direct DNA sequencing based on PCR products. Combined with ND2 gene sequences of the Anas Linnaeus accessed in GenBank, phylogenetic tree was constructed by Neighbor-joining and maximum parsimony methods. [Result] The ND2 gene sequences of 4 Anas platyrhynchos were identical(1 041 bp in length; the nucleotide contents of A, G, T, and C were 28.91%, 13.35%, 20.75% and 36.98% respectively; A+T content approximated to that of C+G). Sequences of ND2 gene of mallard were same as spotbill duck, and had high homology with others. The phylogenetic trees indicated mallard and spotbilled duck were close in genetic relationship, both shared a haplotype; then Philippine duck, green-winged teal and northern pintail fell into branch ''A". [Conclusion] The domestic duck may be domesticated from mallard and spotbilled duck. 展开更多
关键词 Anas platyrhynchos ND2 gene sequence analysis Phylogenetic tree
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Cloning and Sequence Analysis of 16S rRNA and COI Gene in Mitochondrial DNA of Scortum barcoo 被引量:2
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作者 张龙岗 安丽 +2 位作者 董学飒 孟庆磊 付佩胜 《Agricultural Science & Technology》 CAS 2010年第7期176-178,182,共4页
[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
关键词 Scortum barcoo 16S rRNA and COI gene sequence analysis
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Cloning and Sequence Analysis of Lactate Dehydrogenase C(LDH-C)Gene from Black-lipped Pika in Western Sichuan Plateau 被引量:1
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作者 贺庆华 徐亚欧 +2 位作者 郑玉才 陈锋 张文磊 《Agricultural Science & Technology》 CAS 2009年第6期63-66,共4页
[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
关键词 LDH-C gene RACE EST sequence analysis
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Molecular Cloning, Sequence Analysis and Prokaryotic Expression of Ovine Activin Receptor Type IIB(ActRIIB) Gene 被引量:1
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作者 张雪梅 安静 +1 位作者 张宁 刘明军 《Agricultural Science & Technology》 CAS 2014年第10期1644-1648,共5页
Objective] This study aimed to clone ovine activin receptor type llB (Ac-tRIIB) gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verif... Objective] This study aimed to clone ovine activin receptor type llB (Ac-tRIIB) gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verification. [Method] The template cDNA which was reversely transcribed from total RNA of sheep liver tissue, was subjected to polymerase chain reaction (PCR) using specific primers of ActRIIB. The ful-length cDNA of ovine ActRIIB was obtained by pMD18-T cloning and sequencing for bioinformatics analysis. Ovine ActRIIB encoding sequence was subcloned into prokaryotic expression vector pET41a with restriction sites BamHl/Notl, and then transformed into BL21 (DE3). The induced products by lPTG were analyzed with SDS-PAGE and Western Blot. [Result] The amplified ful-length cDNA of ovine Ac-tRllB gene was 1 564 bp in length (Genbank accession number: JX422071.1) with an open reading frame of 1 539 bp, encoding 512 amino acides. Ovine ActRllB shared the highest homology (99.6%) with bovine ActRllB. ActRllB had highly ho-mologous C-terminal domains and belonged to the TGFβ family. After prokaryotic expression, an approximately 92 kD His-tagged ActRllB recombinant protein was obtained, which was consistent with the excepted result. [Conclusion] Cloning and successful expression of ovine ActRIIB laid solid foundation for further investigation of its biological function. 展开更多
关键词 SHEEP ActRIIB gene sequence analysis Prokaryotic expression
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Cloning and Sequence Analysis of HN and F Protein Genes from a Strain of Goose Paramyxovirus 被引量:2
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作者 易春华 潘杰 +3 位作者 付薇 颜健华 徐贤坤 熊毅 《Agricultural Science & Technology》 CAS 2009年第4期75-78,共4页
[ Objective] The study was to clone HN and F genes from GX1 strain of goose paramyxovirus and analyze their sequences. [ Method] According to the full nucleotide sequence of GPV-SF02 strain of goose paramyxovirus, two... [ Objective] The study was to clone HN and F genes from GX1 strain of goose paramyxovirus and analyze their sequences. [ Method] According to the full nucleotide sequence of GPV-SF02 strain of goose paramyxovirus, two pairs of pdmers were designed to amplify the HN and F genes from GX1 strain of goose paramyxovirus isolated from diseased goose in Guangxi Zhuang Autonomous Region; the amplified products were ligated into pMD18-T vector and sequenced. [ Result ] HN and F genes of this strain tested were 1 716 and 1 662 bp in full nucleotide length, respectively; both showed the homologues of about 97.3% with GPV- SF02 strain, of 80.3% -97.5% with strains LaSota, F48E9 and JS, of just 84.8% with Miyadera strain. [ Conclusion] The results show that isolated strain BX1 matches to virulent APMV-1 strain, belonging to genotype Ⅶ of APMV-1 strain. 展开更多
关键词 Goose paramyxovirus HN protein gene F protein gene CLONING sequence analysis
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Cloning and Expression of Conserved Sequences of cry Gene in E.coli Strain Rosetta(DE3) 被引量:1
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作者 罗翠平 李金华 +1 位作者 谢烨明 曾万勇 《Agricultural Science & Technology》 CAS 2012年第5期958-961,996,共5页
[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta (DE3). [Method] Specific primers were designed according to NCBI database information and the conserved sequences of c... [Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta (DE3). [Method] Specific primers were designed according to NCBI database information and the conserved sequences of cry gene were amplified by PCR from Bt transgenic cotton. Then recombinant plasmids were constructed and expressed in E. coil strain Rosetta (DE3). Finally, the effects of different concentrations and inducing time of IPTG on the expression level of protein were investigated. [Result] Two conserved sequences (304 and 853 bp respectively) of cry gene were amplified. The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-I-304 and pGEX-4t-1-853 could express fusion proteins by IPTG induction and the molecular weight of protein products was 39 and 62.4 kDa respectively, which was in accordance with predicted result. The optimal protein ex- pression conditions were confirmed as induction with 0.15 mmol/L IPTG for 7 h. [Conclusion] This study prepared the ground for the further detection of Bt transgenic crops. 展开更多
关键词 Bt gene Conserved sequence CLONING Prokaryotic expression
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Construction of Midgut Tissue-Specific cDNA Library of Bombyx mandarina M. and Isolation and Sequence Analysis of Serine Protease Gene Fragment
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作者 王燕红 李兵 +4 位作者 王东 朱莎 赵华强 卫正国 沈卫德 《Agricultural Science & Technology》 CAS 2008年第3期35-38,共4页
[Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed v... [Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion. 展开更多
关键词 Bombyx mandarina M. MIDGUT tissue cDNA LIBRARY SERINE PROTEASE gene sequence analysis
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Cloning and Sequence Analysis of Sheeppox Virus RPO30 Gene
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作者 赵志荀 吴国华 +3 位作者 颜新敏 李健 朱海霞 张强 《Agricultural Science & Technology》 CAS 2011年第11期1721-1723,1728,共4页
[Objective] The study aimed to clone RPO30 gene from Sheeppox virus (SPPV) and predict the structure and function of the sequence. [Method] RPO30 gene of SPPV was cloned with PCR, linked into pMD18-T simple vector a... [Objective] The study aimed to clone RPO30 gene from Sheeppox virus (SPPV) and predict the structure and function of the sequence. [Method] RPO30 gene of SPPV was cloned with PCR, linked into pMD18-T simple vector and then transformed into E. coli DH5a. In blue-white screen, the white colonies were selected to prepare plasmids. The positive plasmids were selected by double digestion and PCR, and then sequenced. Finally, the structure and function of the sequence obtained were predicted by bioinformatics methods. [Results] The RPO30 gene was successfully obtained; its ORF was 585 bp, encoding 193 amino acids and containing a recognition site for Hind III. Moreover, the SPPV RPO30 gene shared different homologies with the RPO30 gene sequences of other pox virus strains from GenBank database. Further analysis by biological software showed that in RPO30 protein, amino acids 4-12, 18-26, 50- 61, 68- 92 and 176-190 had a high possibility to form the active center, and acting to these regions was likely to inactivate the enzyme encoded by the sequence, thus to inhibit viral replication efficiently. [Conclusion] This study will lay foundation for further study on the structure and function of RPO30. 展开更多
关键词 Sheeppox virus RPO30 gene sequence analysis BIOINFORMATICS
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Sequence Comparison and Analysis of HA Gene of Four H9N2 Avian Influenza Virus Isolates
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作者 章振华 于博 +4 位作者 姜北宇 钱爱东 李林 景小冬 张建伟 《Agricultural Science & Technology》 CAS 2009年第5期55-58,64,共5页
[ Objective] To determine the HA gene sequences of four H9N2 Avian influenza virus (AIV) strains and carry out comparative analysis so as to understand the difference and variation pattern of each strain from the an... [ Objective] To determine the HA gene sequences of four H9N2 Avian influenza virus (AIV) strains and carry out comparative analysis so as to understand the difference and variation pattern of each strain from the angle of molecular biology and to know the distribution and epidemic law of H9N2 AIV. [Method] One pair of primers was designed referring to HA gene sequences of H9N2 AIV. The HA genes of A/Chicken/Hebei/WD/98 (H9N2; WD98 for short), A/Chicken/Hebei/ZD/04 (H9N2; ZD04 for short)), A/Chicken/Beijing/MY/06 (H9N2; MY06 for short) ), and A/Chicken/Beijing/PG/08 (H9N2; PG08 for short)) were amplified, cloned and sequenced. Then the HA gene sequences of these strains were compared with that of 10 H9N2 AIV stains in GenBank. [Result] The ORF of HA genes of the four strains was 1 683 bp in size, encoding 516 amino acids. The HA gene sequences of the four strains, WD98, MY06, PG08, and ZD04, were 82.6% -95.1%, 83.0% -99.0%, 82.7% -95.5%, and 81.3% -95.7% homologous to that of the 10 H9N2 AIV stains, respectively. And the homology of amino acid was respectively 86.6% -96.3%, 86.6% -97.9%, 87.0% -97.1%, and 86.9% -97.3%. [ Conclusion] The HA gene has greatly high homology among different strains. 展开更多
关键词 Avian influenza H9N2 sequence analysis Hemagglutinin gene
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Cloning and Sequence Analysis on IGF-1 Gene of Hubei White Swine
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作者 乔宪凤 张立苹 +6 位作者 毕延震 刘西梅 华文君 华再东 肖红卫 周荆荣 郑新民 《Agricultural Science & Technology》 CAS 2011年第11期1569-1571,1593,共4页
[Objective] The study aimed at cloning and analyzing the insulin-like growth factor-1 (IGF-1) gene from liver of Hubei white swine. [Method] The total RNA was extracted by using Trizol from the liver of Hubei white ... [Objective] The study aimed at cloning and analyzing the insulin-like growth factor-1 (IGF-1) gene from liver of Hubei white swine. [Method] The total RNA was extracted by using Trizol from the liver of Hubei white swine and used as template to amplify IGF-1 gene cDNA by RT-PCR. The cDNA product was cloned into pCRII vector, screened with blue-white colonies, digested with double enzymes and sequenced. [Result] The sequencing result indicated that the IGF-1 gene consisted of 607 nucleotides, containing 5'-untranslated region at nucleotides 1-145, a complete ORF at nucleotides 146-538 encoding 130 amino acids, and 3'-untranslated region at nucleotides 539-607. It shared 100% homology with the porcine IGF-1 gene reported by Muller et al. [Conclusion] The successful cloning and sequencing of the Hubei white swine IGF-1 gene confirmed that IGF-I gene was highly conserved, which provided technical basis for the use of transgenic technology for breeding of Hubei white swine. 展开更多
关键词 Hubei white swine IGF-1 gene sequence analysis
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Cloning,Sequence Analysis and Amino Acid Structure Prediction of TLR9 Gene from Wild Ovis ammon in Xinjiang
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作者 马长宾 孙延鸣 +1 位作者 张银国 张辉 《Agricultural Science & Technology》 CAS 2011年第10期1531-1534,1546,共5页
[Objective] The study aimed at cloning and identifying the toll receptor gene 9(TLR9) of wild Ovis ammon in Xinjiang,and predicting its structure and function.[Method] The TLR9 complete sequence of wild Ovis ammon w... [Objective] The study aimed at cloning and identifying the toll receptor gene 9(TLR9) of wild Ovis ammon in Xinjiang,and predicting its structure and function.[Method] The TLR9 complete sequence of wild Ovis ammon was cloned from its peripheral blood by PCR technology.Then the PCR products were purified by agarose gel electrophoresis and then sequenced.Finally,the structure and function of TLR9 sequence were predicted by molecular biological software.[Result] The complete sequence of TLR9 gene was 3 192 bp in length,encoding 1 064 amino acids with a signal peptide composed of 30 amino acids;the leucine percentage reached as high as 18.5%.The TLR9 amino acid possibly contained three hydrophobic regions,at amino acids 455-475,740-760 and 780-800.The 3-D structure of TLR9 was constructed by the extracellular LRR domain and intracellular TIL domain(Toll/IL IR).[Conclusion] The characteristics of TLR9 provided theoretical basis for further study on the TLR9 gene of wild Ovis ammon in Xinjiang. 展开更多
关键词 Wild Ovis ammon TLR9 gene cloning sequence analysis
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