马MxA基因第13外显子的多态性研究(英文)

[Objective] To investigate the polymorphism of the thirteenth exon of MxA gene in 4 species of horse. [Method] The thirteenth exon of MxA gene fragments were amplified from genomic DNA of Sanhe horse, Xinihe horse, Wushen horse and Baerhu horse with the primers designed according to the MxA sequence announced in GenBank; the polymorphism of MxA gene was detected by PCR-SSCP and the products were sequenced. [Result] The polymorphism of the thirteenth exon of MxA gene appeared only in Wushen horse, the 2 081 nt of which mutated from guanine (G) to adenine (A) and the corresponding amino acid of which changed from glutamate (Glu) to alanine (Ala). [Conclusion] The study provided a basis for exploring the antiviral effect of MxA protein.     

Genetic Polymorphism of Kappa Casein （K-CN）in Iraqi Buffalo Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

This study was carried out the animal production department, genetic engineering lab, college of agriculture, （UoB）, Iraq. The aim of this study was to use the polymerase chain reaction restriction fragment length polymorphism （PCR-RFLP） as a fast, efficient and low cost method to detect the genetic variants of kappa-casein gene （k-CN） in Iraqi buffalo using three different primers specific for bovine k-CN to amplify the gene segment, followed by digestion using restriction enzyme （Hind III） for genotyping. DNA from 50 Iraqi buffaloes was extracted by phenol chloroform method. PCR was carried out in a final reaction volume of 25 μL and the reaction mixture was subjected to standard PCR protocol. The results of this work show that among the examined 50 Iraqi Buffalo were homozygous for the K-CN and genotyped as BB for all three primers but gave different bands. Thus PCR-RFLP using Hind III revealed all the samples to be monomorphic for this locus. The restriction digestion analysis of 397 bp PCR product of k-CN indicates the presence of two fragments of 154 bp and 225 bp for BB-genotype. A 437 bp fragment of the bovine genomic K-CN gene was amplified. One Hind III restriction site is found in position 346 of the amplified fragment of allele k-CN B, yielded 91 bp and 346 bp. Amplified products from Iraqi buffalo （530）, after being digested with Hind III, yielded two separate DNA fragments of different sizes i.e., 160 bp and 370 bp. For the first time completed research such specifications in Iraq, for the first time using molecular biology in genetic identification. Our objectives of this study have been to aid in understanding domestication, Buffalo origin and their history and evolution, to identify genetically unique breeds, to provide an objective basis for conservation decisions and to aid the formulation of breeding plans.     

Rapid and Sensitive Identification of Horse and Donkey Meat in Iraqi Markets Using SSR and PCR-RFLP Based on Mitochondrial DNA Cytochrome B Gene

This study was carried out at the Genetic Engineering Lab, College of Agriculture, University of Basrah. DNA was extracted from samples containing meat of commercial products in the Basrah markets. The products subjected to analysis using polymerase chain reaction （PCR） with species specific repeat （SSR） and polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） techniques based on the sequence of the mitochondrial DNA cytochrome b gene （mtDNA cyt b gene）. Then digestion with the Alu l restriction enzyme to establish a differential diagnosis detects and discriminates between meat species and adulteration in the products. SSR primers were applied, has been detected amplification of the encoded gene product, generated 221 bp in some imported minced and canned meat samples. The results show that SSR analysis produced a pattern that allowed a direct identification of horse and donkey meats in some imported minced and canned meat samples （Hana, Monde and Bavaria）. The amplified 359 bp gene of mtDNA cyt b gene from samples in different product was cut using Alu 1 restriction enzyme resulting in restriction fragment length polymorphism （RFLP）. Alu 1 was used to distinguish between the animals meat that belong to the family or one species. The digestion of the PCR product showed differences between products. Where the fragment length generated were 74, 76 and 189 bp. It belonged to horse meat. The fraud was detected in Hana, Monde and Bavaria products available in Basrah markets showing the presence of horse meat in these products that labeled as beef meats 100%. This revealed mtDNA cyt b gene as highly conserved and consequently a good molecular marker for diagnostic studies. Thus, this technique can be applied to food authentication for the identification of different species of animals in food products.     

马IGF-Ⅰ基因3′端侧翼区PCR-SSCP多态性分析(英文)

[Objective] To explore the polymorphism of the 3′ flank region of equine IGF-Ⅰ gene. [Method] The 3′ flank region sequences of IGF-Ⅰ gene were amplified from genomic DNA of 270 horses, which included 4 types of Mongolian horse, Sanhe horse and Thoroughbred, and then analyzed by PCR-SSCP. [Result] Three genotypes (AA, BB and AB) were detected by PCR-SSCP and the distribution of genotypes of all research objects except Xinihe horse and Baerhu horse were in line with the "Hardy-Weinberg Law". [Conclusion] There was a polymorphic locus in the 3′ flank region of IGF-Ⅰ gene, which might affect the equine growth and development mechanism. The study is of important theoretical and practical significance to improve the performance and to develop equine industry.     

华山松疱锈病的重寄生真菌(深绿木霉)中几丁质酶基因cDNA片段的克隆(英文)

[Objective] The aim of this study was to isolate chitinase gene from Trichoderma atroviride strain SS003. [Method] With the aeciospore wall of armandii pine blister rust as inducer, chitinase gene was induced to express in Trichoderma atroviride cells. The cDNA fragment of chitinase gene was cloned by RT-PCR approach. [Result] The activity of chitinase induced reached 40.17 μg/10 min; and the specific fragment amplified was 834 bp in length and proved to be the fragment of chitinase gene by sequencing and sequence analysis. [Conclusion] The result showed the feasibility of isolating the full length of chitinase gene and its transformation, and further producing chitinase.     

金黄色葡萄球菌FnBP配体结合区基因的克隆及其原核表达(英文)

[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.     

拮抗链霉菌AFLP分析技术体系的研究(英文)

[Objective] The aim of this study was to investigate the preparation method and amplification system of antagonistic streptomyces DNA templates based on AFLP assays, and also provide a basis for the application of AFLP technology in the analysis of streptomyces or even actinomyces. [Method] The DNAs were extracted by the modified CTAB method and amplified by the Pst Ⅰ/Mse Ⅰ AFLP kit and its reaction system. The amplified products were analyzed by the denatured polyacrylamide gel electrophoresis. [Result] The genomic DNAs of ten antagonistic strains of Streptomyces were extracted and tested. The result of 0.8% agarose gel electrophoresis showed that the major DNA bands were clear without degradation and RNA residue, with the fragment sizes ranging from 37.64 to 40.86 Kb. By ultraviolet spectrophotometry, the OD260/OD280 values varying from 1.625 to 1.833 were obtained. Furthermore, the agarose gel electrophoresis of DNA products digested by Pst Ⅰ/Mse Ⅰ presented the dispersed fluorescent long band, which indicated that the enzymatic hydrolysis was fully carried out. The amplified bands of DNA templates by the screened three pairs of primers were clear with rich polymorphism. [Conclusion] The preparation method and amplification system of DNA template established in this study can be used in the AFLP analysis of Streptomyces.     

雄性育性基因RNA干扰载体的构建与鉴定(英文)

cDNA fragment of fertility gene MS2 from cotton was cloned by RT-PCR approach, it was highly homologous with relevant genes of Brassica napus and Arabidopsis thaliana. According to the principles of constructing RNAi vector, sense and antisense fragments of MS2 gene carrying restriction endonuclease recognition sites were amplified via PCR technique, ligated with the first intron of upland cotton chinase gene, then inserted into artificially modified plant expression vector pBI121, yielding RNAi vector pBGP12MSIn. The results showed that RNAi vector pBGP12MSIn harboring MS2 gene driven by anther specific promoter BGP was successfully constructed. Our results laid a foundation for studying the function of this gene and genetic transformation of plant male sterile lines.     

铜绿假单胞菌脂肪酶Lipase基因的原核表达(英文)

[Objective] The aim of this study was to investigate the prokaryotic expression of pseudomonas aeruginosa Lipase gene.[Method]Lipase gene was amplified by PCR from the genome DNA of pseudomonas aeruginosa,and its nucleotide sequence was determined.The prokaryotic expression vector of Lipase gene was constructed by the gene recombination technique.The protein expression was induced for 4 hours by IPTG with the final concentration of 1.0 mmol/L,and then SDS-PAGE electrophoresis was analyzed.[Result]The sequence of mature peptides in Lipase gene cloned from pseudomonas aeruginosa had a 99.36% homology with that of pseudomonas aeruginosa lipase submitted in NCBI,so the prokaryotic expression vector of Lipase gene pET32a-Lip was successfully constructed.Furthermore,the results of SDS-PAGE electrophoresis showed that the target gene was expressed highly and effectively.[Conclusion]The cloned pseudomonas aeruginosa lipase with its signal peptide could be normally expressed in E.coli and also used for further study.     

马MxA基因第12外显子多态性检测

MxA蛋白是Ⅰ型干扰素（IFNα／β）所诱导产生的蛋白之一,属于大分子（70～80kD）GTP酶,能抵抗各种RNA病毒和部分DNA病毒。MxA蛋白结构与其抗病毒活性有直接的关系。有报道表明,MxA蛋白活性部位的一个氨基酸序列的变换,会导致其抗病毒活性的改变。MxA基因多态性分析是研究马MxA蛋白基因抗病毒作用和寻找马MxA蛋白抗性基因的基础。     

马MxA基因第12外显子多态性检测研究

[目的]建立检测马MxA基因第12外显子多态性的快速、准确方法。[方法]采用错配聚合酶链反应（mismatchPCR）对马MxA基因第12外显子进行扩增,对PCR产物进行限制性片段长度多态性分析（RFLP）,鉴定MxA基因cDNA第1790位核苷酸点突变,并对PCR产物进行核苷酸序列分析。[结果]马MxA基因第12外显子区域存在AA、AB、BB3个基因型;位于cDNA序列第l790位点的碱基发生变异（由汕c）,引起了MxA蛋白编码区第562氨基酸由色氨酸变成半胱氨酸的变异;使用mismatchPCR—RFLP法所得PCR产物特异性序列,与RFLP分析结果相符。[结论]采用mismatch PCR—RFLP对马MxA基因12外显子的多态性进行检测操作简单,结果准确。     

马MxA基因第13外显子的多态性研究

[目的]研究4个品种马MxA基因第13外显子的多态性。[方法]根据GenBank中已公布的MxA基因序列设计引物,利用PCR技术从三河马、锡尼河马、乌审马和巴尔虎马血液基因组DNA中扩增MxA第13外显子基因片段,利用PCR-SSCP方法检测该基因片段多态性并进行测序。[结果]只有乌审马MxA基因第13外显子存在多态性,第2 018位点发生了突变,鸟嘌呤（G）→腺嘌呤（A）,由此导致氨基酸发生改变,由谷氨酸（G lu）→丙氨酸（A la）。[结论]该研究为探讨马MxA蛋白基因抗病毒作用奠定了基础。     

猪CSTB基因的克隆及多态性分析

[目的]利用RT-PCR方法克隆猪CSTB基因exon2序列,对所得序列进行多态性分析。[方法]以大白猪cDNA为模板,采用RT-PCR克隆方法,首次获得猪exon2序列,提交GenBank收录(GenBank登录号:DQ534493)。[结果]通过RT-PCR方法,克隆测序得到的猪CSTB基因exon2序列与人、鼠的CSTB基因exon2序列同源性分别为80.39%与72.55%。对所得猪exon2序列进行多态性分析,发现第26和78个碱基位置发生突变,分别为A→T和C→T,其中第26个碱基为错意突变,导致氨基酸的改变。[结论]CSTB基因exon2序列在猪、人和鼠上相对保守。     

广西巴马小型猪JAK2基因外显子14克隆及SNP位点分析

【目的】克隆广西巴马小型猪非受体型酪氨酸蛋白激酶（JAK2）基因外显子14并进行SNP位点分析,为探讨广西巴马小型猪JAK2基因多态性与有关临床疾病治疗奠定基础。【方法】参照GenBank已发表的猪JAK2基因编码序列设计引物,克隆出猪JAK2基因外显子14,经测序鉴定后,利用PCR-SSCP技术对其进行SNP位点分析。【结果】PCR扩增获得的巴马小型猪JAK2基因外显子14序列为205 bp,与猪的同源性为100.0%,与人类的同源性为94.2%;猪和人类在JAK2基因外显子14序列中有6个位点不同;PCR-SSCP分析结果表明,JAK2基因外显子14不存在多态性。【结论】广西巴马小型猪JAK2基因外显子14不存在多态性,其基因纯合度高,是进行育种研究和医学试验的理想实验动物模型。     

CACNA2D1基因第14内含子的多态性及其对猪肌肉pH_(45)的影响

［目的］检测猪CACNA2D1基因第14内含子的多态性,为猪肉质性状的分子标记辅助选择提供条件。［方法］采用PCR-SSCP检测猪CACNA2D1基因第14外显子和内含子在6个猪群中的DNA 多态性,并分析不同基因型对猪眼肌和腿臀肌肉pH45的遗传效应。［结果］猪CACNA2D1基因第14内含子有3 种基因型,即AA、AG 和GG,在其序列（GenBank接受号：FJ156361）的1 145位碱基存在1个多态位点（G→A）。χ^2 独立性检验结果表明,这3种基因型在各猪群间的分布差异显著（P＜0.05）。AA基因型金华×皮特兰F2资源家系猪与AG基因型猪在眼肌和腿臀肌肉pH45值上的差异显著（P＜0.05）。［结论］猪CACNA2D1基因第14内含子的多态性影响猪眼肌和腿臀肌pH45值。      

娟姗牛催乳素基因型与产奶量的关联性分析

[目的]研究娟姗牛催乳素（PRL）基因多态性与产奶量之间的关联性,为选育高产奶量娟姗牛新品系奠定基础.[方法]利用PCR-RFLP分析44头娟姗牛PRL基因外显子2和外显子4的多态性,统计不同基因型娟姗牛305 d的平均产奶量,然后分析娟姗牛PRL基因型与产奶量的关联性.[结果]娟姗牛PR基因外显子2和外显子4存在RsaI酶切多态性,外显子2有AA和AG两种基因型,外显子4有AA、AG和GG 3种基因型;外显子2和外显子4的AG型频率分别是0.7272和0.5454,为优势基因型.PRL基因两个外显子AA型个体的305 d平均产奶量均高于AG型和GG型（P＞0.05）;基因型组合效应分析发现,A2A2A4G4为优势组合基因型,但在305 d平均产奶量方面是A2G2A4A4组合基因型的产奶量显著高于A2A2A4A4、A2A2A4G4和A2A2G4G4组合基因型（P＜0.05）.[结论]娟姗牛PRL基因外显子2和外显子4的碱基突变与产奶量之间存在关联性,其中第8398位碱基A是娟珊牛高产奶量的有利等位基因.     

五指山小型猪生长激素释放激素受体基因的cDNA克隆及序列分析

1材料与方法 1．1材料随机采集五指山猪品种耳组织样共30份,将其迅速放入液氮带回实验室,-70℃保存备用。 1．2试剂RT—PCRKit、PCR产物纯化回收试剂盒、重组质粒抽提试剂盒,均购自天根生物工程有限公司;SalⅠ、BamHⅠ、pMD18-TVector试剂盒、Ecoli DH5α均购自TaKaRa公司。