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。     

沙冬青根瘤菌固氮酶基因nifH PCR-RFLP分析

利用固氮基因nifH PCR扩增,nifH PCR-RFLP及扩增产物序列分析方法对分离自宁夏和内蒙古部分地区的沙冬青根瘤菌代表菌株的nifH 进行了遗传多样性和系统发育分析.结果表明,在80%相似水平上42株根瘤菌nifH PCR-RFLP基因图谱中有5个基因型聚类群.对不同基因型代表菌株的nifH PCR产物进行序列测定和系统发育关系分析,测试菌株在系统发育地位上与中慢生根瘤菌和中华根瘤菌相近,测试菌株与温带中慢生根瘤菌(Mesorhizobium temperatum isolate HAMBI 2583)序列相似性范围为95.6%～96.9%,与草木樨中华根瘤菌(Sinorhizobium meliloti strain CCBAU10062)基因序列相似性范围为99.8%～100%,基因序列同源性较高.本研究中nifH基因所揭示的沙冬青根瘤菌遗传多样性和系统发育关系说明沙冬青根瘤菌受染色体基因背景、地理环境以及菌株个体的进化而存在一定的差异.     

江苏连云港沿海蛤蜊科贝类18S-ITS1序列分析

利用PCR技术扩增了江苏连云港海域3种贝类(西施舌、中国蛤蜊,四角蛤蜊)核糖体RNA基因18S-ITS1区域序列.用DNAStar和MEGA 3.1软件分析序列的碱基组成、进行序列比对,构建系统发育树.结果显示,西施舌、中国蛤蜊、四角蛤蜊18S-ITS1部分序列长度分别为1 052 bp、1 017 bp、1 013 bp,ITS1部分序列长度分别为910bp、875 bp、871 bp,G+C含量为58.90%～60.23%;18S区序列保守,种间仅存一个碱基变异位点.序列分析显示,中国蛤蜊与四角蛤蜊的ITS1同源性为70.1%～71.1%.西施舌与中国蛤蜊、四角蛤蜊的同源性为44.7%～46.3%;18S-ITS1序列适于蛤蜊科种间特别是蛤蜊属内种间的分子系统发育分析.     

柑桔黄龙病亚洲种病原的PCR-SSCP分析

应用PCR—SSCP技术对采自我国柑桔主产区、泰国和法国留尼湾等8个不同地方的柑桔黄龙病亚洲种的病原(Candidatus Liberibacter asiaticus)进行研究。对引物P1、P2扩增的PCR产物进行SSCP分析，得到两条清晰且泳动速率一致的条带，表明不同地方柑桔黄龙病亚洲种病原扩增的DNA片段之间没有差异。     

茎瘤芥酸性核糖体蛋白P1基因的克隆与序列分析

以茎瘤芥幼苗叶片总RNA为模板,通过SMART RACE技术进行反转录和PCR扩增,克隆到瘤芥酸性核糖体蛋白P1 基因(命名为BjARPP1,GenBank登录号:JX282179),并利用ProtParam、DNAMAN等生物信息学工具对其核酸序列和蛋白序列进行分析.结果表明,茎瘤芥酸性核糖体蛋白P1基因开放阅读框为342 bp,编码113个氨基酸,等电点为4.27,相对分子质量为11.25 ku:二级结构预测显示α-螺旋占53.98％、不规则卷曲占33.68％、延伸链占12.39％,还含有5个磷酸化位点;同源进化树预测分析其与拟南芥的同源性为96％,与其他物种同源性低.茎瘤芥酸性核糖体蛋白P1基因为首次克隆,为进一步研究该基因的结构、功能、遗传变异规律以及其与生产性能的关系提供科学依据.     

甘薯时空特异性高效表达载体的构建

根据已知的甘薯块根贮藏蛋白基因序列,设计和合成了两对PCR引物.用PCR方法,从南薯88基因组中分别扩增出两种DNA片段:一种含有贮藏蛋白基因启动子和核糖体结合序列(SP2);第二种不仅含有第一种DNA片段的全部序列,而且还有贮藏蛋白的信号肽编码序列(SP1).核苷酸序列分析表明,这两种DNA片段的启动子和核糖体结合区虽然具有很高的同源性(82%),但它们并不相同.在SP1和SP2的基因启动子区都存在几种典型的保守序列,如受糖诱导调控的蔗糖盒(Suc-box),CAAT盒和TATA盒.用这两种DNA片段分别构建了可调控的甘薯高效表达载体.     

猪瘟流行野毒E~(rns)基因的序列分析

 利用反转录 ( RT)和 PCR技术完成了 5株近期 ( 1 997～ 1 998年 )猪瘟流行毒和 1株猪瘟 C-株弱毒疫苗毒 (兰州 ) Erns(或称 E0 )基因的核酸序列测定。通过序列分析发现 ,这 5株流行毒与 C-株疫苗毒的核酸序列同源性为 83%～ 84%;推导的氨基酸序列同源性为 88%～ 91 %,我国 50～ 60年代流行的中国石门株 ( SM)的核酸序列同源性为 85%;氨基酸序列同源性为 89%～ 91 %。而 5株流行毒间的核酸序列同源性为 91 %～ 98%;氨基酸序列同源性为 94%～ 98%。     

猪传染性胸膜肺炎放线杆菌血清5a型ApxⅣA全基因的序列测序及其分子特征

根据GenBank上公布的猪传染性胸膜肺炎放线杆菌(APP)血清型1型和血清型3型ApxⅣA基因全序列,设计了7对引物,对APP血清5a型毒株263株ApxⅣA基因全序列进行了分段PCR扩增和克隆。序列测定结果表明:该基因核酸序列全长5856 bp,比GenBank上公布的血清1型和3型的相应基因核酸序列分别长438 bp和1287 bp,与其核酸和氨基酸序列同源率均分别为95.5%和87.6%;与GenBank刚公布的血清5b型毒株L20株的核酸和氨基酸序列的同源性均为98.2%。生物信息学分析表明,该基因编码的蛋白具有较强的亲水性,共有66个抗原决定簇,存在较多的可能性功能位点。     

Decreasing detection frequency of MITE(MCLas-A) in the population of ‘Candidatus Liberibacter asiaticus' recently collected in southern China

An active miniature inverted-repeat transposable element(MITE), MCLas-A, was previously identified from ‘Candidatus Liberibacter asiaticus' known to be associated with citrus Huanglongbing(HLB, yellow shoot disease). To explore the recent transposition status of MCLas-A, 389 ‘Ca. L. asiaticus' strains collected from nine regions in China were amplified using a specific primer set and three representative ‘Ca. L. asiaticus' strains were analyzed by next-generation sequencing(NGS) approach. PCR and genomic analysis showed that the entire MCLas-A was only present in 1.80%(7/389) and the jumping-out type of the MITE was predominant(81.23%) in samples tested, suggesting high frequency transposition occurred in ‘Ca. L. asiaticus' strains recently collected from China. Biological roles of transposition of the active MITE remain to be determined.     

中国柑橘黄龙病病原菌两个原噬菌体超变异基因遗传多样性

【目的】通过原噬菌体区域高度变异的基因位点研究柑橘黄龙病病原菌亚洲种（‘Candidatus Liberibacter asiaticus’）的种群分化,探讨病原菌种群遗传多样性水平和遗传结构。【方法】基于2种原噬菌体类型（SC1和SC2）对应的超变异基因区域设计2对引物（Lap-TJ-F/Lap-TJ-R1和Lap-TJ-F/Lap-TJ-R2）,对中国不同柑橘产区的224个‘Ca. L. asiaticus’株系进行PCR检测和序列分析。【结果】PCR扩增的条带类型呈多态性,具有4种条带类型（SC1-1、SC1-2、SC2-1和SC2-2）,西南地区以SC1-1型为主,广东、广西地区以SC2-1型为主,福建、江西、浙江地区没有明显优势的扩增型。分析SC1-1和SC1-2对应序列表明,其差异系由于132 bp的卫星序列和24 bp的小卫星序列2种串联重复序列数不同引起,而SC2-1和SC2-2的差异系由原噬菌体内部基因重排引起。【结论】中国不同地理来源病原菌株系在原噬菌体区域具有较丰富的多态性,对该基因区域研究将有助于揭示中国‘Ca. L. asiaticus’种群的遗传多样性。     

植原体翠菊黄化组分类研究进展

本文介绍了植原体翠菊黄化组分类研究概况及最新进展,四个遗传进化参数16S rRNA、rp、tuf、secY基因应用于翠菊黄化组植原体的分类,基于16S rRNA、rp、tuf、secY序列的RFLP分析,分别可将翠菊黄化组植原体划分为15个、8个、10个、8个亚组,国际比较菌原体学研究计划署(IRPCM)提出将暂定种‘CandidatusPhytop lasm a asteris’作为翠菊黄化植原体的分类参考标准。     

低氧胁迫下斑马鱼鳃中核糖体蛋白基因家族的表达分析

为研究斑马鱼核糖体蛋白应对低氧胁迫的生物学功能，对低氧胁迫和常氧条件下斑马鱼（Danio rerio）鳃组织进行转录组分析，利用高通量测序检测了低氧胁迫与常氧条件下斑马鱼鳃转录组中核糖体蛋白家族基因的表达差异。结果表明：在两个不同浓度的低氧胁迫下，斑马鱼鳃组织中60个核糖体蛋白基因的表达量显著上调。其中大亚基核糖体蛋白基因35个，小亚基核糖体蛋白基因25个。在低氧胁迫下斑马鱼鳃中显著差异表达基因GO富集的前15条通路中，均包括核糖体蛋白基因，且其中的5条通路与核糖体蛋白组装合成相关。在富集到“translation”GO通路中，富集到44个核糖体蛋白基因。另外，利用前期筛选出的低氧条件下斑马鱼鳃中显著低表达的2个miRNAs，针对低氧下表达量显著上调的60个核糖体蛋白基因进行靶基因预测，结果表明：斑马鱼miR-455-3p可以同时靶向核糖体蛋白基因rpl13和rplp1来调控斑马鱼对低氧环境的适应。     

香蕉枯萎病菌4个生理小种核糖体DNA内转录间隔区的RFLP分析

【目的】明确香蕉枯萎病菌Fusarium oxysporum f.sp.cubense 4个生理小种的系统发育关系。【方法】利用PCR扩增该病菌4个生理小种的核糖体DNA内转录间隔区(Internal transcribed spacer,ITS),利用AIu I、Hpa II和Taq I 3种限制性内切酶对这些小种的ITS产物进行RFLP分析。【结果】PCR扩增结果表明:来自4个生理小种的8个菌株均可扩增得到568 bp的ITS单一片段,但这些生理小种间的片段没有明显的多态性;对这些生理小种ITS产物进行RFLP分析发现,不同生理小种之间的差异很小。【结论】PCR-RFLP技术不适用香蕉枯萎病菌生理小种的分析鉴定。     

Preliminary Studies on CPG/Hinf Ⅰ RFLP Groups of Citrus tristeza virus Infected Sweet Oranges in China

Citrus tristeza virus (CTV) causes economically important losses to the citrus industry worldwide. Mild strain cross protection (MSCP) against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces (Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi) were tested by direct tissue blot immuno-assay (DTBIA), and 158 of them were tested positively, which therefore were subjected to coat protein gene (CPG)/Hinf Ⅰ restriction fragment length polymorphism (RFLP) analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.     

家蚕卵线粒体DNA限制性内切酶长度多态性研究

利用29种限制性内切酶对34个不同品种家蚕卵mt DNA进行酶切分析,发现一化、二化品种与多化品种的HaeⅢ酶切图谱有差异,但不同的地理品种间没有出现酶切多态性现象。另外,一些家蚕品种的受精卵和非受精卵mt DNA在Bgl I和Pst I酶切焉,呈现不同的酶切带型。同时,绘制了较精确的家蚕mt DNA限制性内切酶酶切图谱。     

惠阳胡须鸡LPL、FASN基因多态性的研究

以惠阳胡须鸡为研究对象,分别以LPL、FASN基因为脂肪性状相关的候选基因,采用限制片段长度多态性(RFLP)检测这2个候选基因在惠阳胡须鸡中的多态性。研究结果表明,(1)用限制性内切酶HinfⅠ对LPL基因第8内含子进行RFLP分析,发现76.3%为已报道的BB型,另发现了两种新的基因型,分别占1.7%和22%,无AA型;(2)用限制性内切酶HaeⅢ对FASN基因进行RFLP分析,发现BB基因型比例为64.8%,BC型12.24%,CC型基因型比例23.4%。B等位基因频率为0.7041,C等位基因频率为0.2959,B等位基因频率明显高于C等位基因频率。     

Mammalian ribosomal protein: analysis by electrophoresis on polyacrylamide gel

Ribosomal protein from five mammalian tissues when analyzed by discontinuous electrophoresis on polyacrylamide gel at pH 4.5 yielded 24 bands. Densitometric tracings indicated that the patterns of the basic ribosomal proteins from the several tissues were qualitatively similar. Protein from Escherichia coli ribosomes analyzed at pH 4.5 gave 29 bands, and the pattern was different from that of mammalian ribosomal protein. No distinct band was found when mammalian ribosomal protein was analyzed at pH 8.3 (acidic proteins). Ribosomal protein from Escherichia coli gave eight bands at pH 8.3. Thus, the structure of the genes responsible for synthesis of ribosomal protein in several mammalian tissues is the same, and different genes direct synthesis of ribosomal protein in bacteria.     

Preliminary Studies on CPG/Hinf Ⅰ RFLP Groups of Citrus tristeza virus Infected Sweet Oranges in China

Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces （Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi） were tested by direct tissue blot immuno-assay （DTBIA）, and 158 of them were tested positively, which therefore were subjected to coat protein gene （CPG）/Hinf Ⅰ restriction fragment length polymorphism （RFLP） analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.     

绒山羊瘤胃甲烷菌mcrA基因的RFLP分析(英文)

[Objective] The aim of this study is to understand the population composition of methanogens in rumen fluid of grazing Inner Mongolian cashmere goat. [Method] Total DNAs of various bacteria in rumen fluid were isolated for PCR amplification using the specifically designed primers based on conservative mcrA sequence of methanogens; then mcrA specific clone library was accordingly established. The restriction fragment length polymorphism(RFLP) of the library was further analyzed by digestion of restriction enzyme Taq I. [Result] One hundred and five randomly selected specific colonies were classified into six RFLP types, among which the dominant type accounts for 38%, and other types account for 27%, 18%, 5.5% and 4.5%, respectively. [Conclusion] There are at least six different methanogens in rumen fluid of grazing Inner Mongolian cashmere goat.     

6S rDNA-RFLP法快速鉴定蒙古国传统乳制品中的乳酸菌

以17株标准菌株为参照,采用限制性片段长度多态性（RFLP）分析和16S rDNA序列分析相结合的技术,对分离自蒙古国5个地区传统乳制品中的55株乳酸菌进行了快速鉴定。结果表明,通过限制性内切酶AluⅠ、HaeⅢ、BsmaⅠ、TspRⅠ和HinfⅠ的指纹图谱分析,将49株乳杆菌和2株片球菌准确鉴定到种,其他4株肠球菌鉴定到属。采用16S rDNA-RFLP方法鉴定乳酸菌具有准确性,可靠性和高效性。