金龟子绿僵菌酸性海藻糖酶基因的克隆及其序列特征分析

根据酸性海藻糖酶的蛋白质序列设计引物,PCR扩增出CQMa102 ATM1的cDNA和DNA序列,登录号分别为:DQ237957,EF190950.序列分析表明,ATM1 DNA序列含有3个内含子,其开放阅读框(ORF)编码1个含1 073个氨基酸的蛋白质,具有1个20个氨基酸的信号肽序列和含有30个可能的N-糖基化位点(Asn-Xaa-Ser/Thr).NCBI序列比对(Blastn)显示该蛋白与Aspergillus fumigatus的alpha、alpha-trehalose glucohydrolase,Aspergillus nidulans的酸性海藻糖酶前体和Talaromyces emrsonii的酸性海藻糖酶分别有62%、59%和57%的氨基酸相似性,与另外两个真菌Saccharomyces cerevisiae(Ath1p)和Candida albi-cans(Atc1p)的酸性海藻糖酶也具有约25%的氨基酸相似性.Southern杂交表明,ATM1基因在CQMa102基因组中为单拷贝.     

PRRSV福建流行毒株结构蛋白基因之克隆分析

对福建省流行的PRRS病毒FJ-1的结构蛋白基因进行了克隆、测序.FJ-1结构蛋白基因序列长3 188个核苷酸,包含7个开放阅读框(ORF).将FJ-1结构蛋白基因与国内外已发表的18个报道全长结构蛋白基因的PRRSV毒株进行核苷酸序列和推定的氨基酸序列比较,发现:其与17个美洲型毒株核苷酸同源性达到89.7%～92.4%,推定各个ORF编码氨基酸的同源性在85.6%～98.6%之间;而与欧洲型毒株Lelystad核苷酸同源性为54.9%,推定各个ORF编码氨基酸同源性为53.2%～78.2%.遗传进化树分析表明FJ-1与美洲型毒株进化距离近,而与欧洲型毒株进化距离远.从分子水平上证明了福建省流行的PRRS病毒属于美洲型毒株.     

大熊猫核糖体蛋白L30亚基基因(RPL30)的cDNA克隆及序列分析

RPL30是核糖体大亚基60S的组成部分,由RPL30基因所编码,主要存在于真核生物中.根据已报道的部分哺乳动物核糖体蛋白L30亚基基因(RPL30)的相关信息设计引物,运用RT-PCR技术,以大熊猫(Ailuropoda melanoleuca)的肌肉组织为材料,成功地克隆了核糖体蛋白L30亚基RPL30基因,并对其进行了测序及初步分析.结果表明:大熊猫L30亚基基因的表达序列长为388bp,开放阅读框(ORF)为348 bp,编码115个氨基酸的蛋白质,并含有6个功能位点.进一步分析发现,该基因的表达序列及其编码的氨基酸序列与已报道的人、牛、褐家鼠、小家鼠有很高的相似性,其表达序列同源性分别为93.97%,96.26%,89.66%和89.94%,其编码的氨基酸序列同源性分别为99.13%,98.26%,99.13%,99.13%,且其蛋白质的高级结构相似性也很高.     

沙田柚S-RNase基因的克隆及序列分析

利用高通量测序技术对沙田柚自交和异交花柱进行转录组测序。通过差异分析得到沙田柚S-RNase基因序列。该基因全长为1 238bp(GenBank登录号为KP172529),开放阅读框(ORF)全长为834bp,共编码278个氨基酸,编码的蛋白质的相对分子质量为31.402kDa,理论等电点为5.30。S-RNase蛋白为亲水性蛋白,共有17个可能的磷酸化位点。氨基酸序列分析表明,其编码的氨基酸与柚Citrus maxima、沙糖桔Citrus reticulata和甜橙Citrus sinensis的同源性分别为99%、98%和96%。系统进化树显示沙田柚S-RNase基因与柚、沙糖桔和甜橙亲缘关系很近,属于同一进化分支。     

斜带石斑鱼神经坏死病毒基因组RNA1和RNA2序列测定及分析

根据GenBank数据库公布的鱼类神经坏死病毒(nervous necrosis virus,NNV)同源序列设计了7对特异性引物,采用RT-PCR方法扩增出目的片断,将PCR产物测序和分析.斜带石斑鱼Epinephelus coioides神经坏死病毒(orange-spotted NNV,OGNNV)基因组由两个片断(RNA1和RNA2)组成,RNA1由3 103个核苷酸组成,含有一个开放阅读框,编码982个氨基酸;RNA2由1 433个核苷酸组成,含有一个开放阅读框,编码338个氨基酸.OGNNV基因组与新加坡GGNNV(greasy grouper NNV)的基因组有高度的相似性.分析病毒的RNA2序列发现:OGNNV与DGNNV(dragon grouper NNV)、RGNNV(redspotted NNV)和GGNNV的亲缘关系很近,并且具有相同的中和位点;分析病毒的RNA1序列,发现在OGNNV的RNA1序列中同样可以找到依赖RNA的RNA聚合酶的6个模序(motif).根据同源性比较和系统进化分析,OGNNV属于RGNNV血清型的成员.     

大熊猫核糖体蛋白RPL5基因的克隆、表达与比较分析

为了解大熊猫(Ailurophilic melanosome)核糖体蛋白亚基RPL5基因的结构特点及其与已报道的人和其他哺乳动物核糖体蛋白亚基RPL5基因的异同,运用RT-PCR技术,从大熊猫的肌肉组织总RNA中和DNA中分别对核糖体蛋白亚基RPL5基因的表达序列和其结构基因进行了克隆、测序;采用ORF finder软件对表达序列的开放阅读框(ORF)进行了查找和氨基酸序列的推定;采用Gen scan对结构基因进行了分析;采用DNAMAN Version 6对基因序列和氨基酸序列进行了同源性比较;采用ExPASy软件进行蛋白质功能位点和生化特性进行了预测分析;对大熊猫核糖体蛋白亚基RPL5基因进行了超表达实验.结果表明:大熊猫RPL5结构基因长为8 633bp,具有8个外显子和7个内含子;mRNA长为918bp,ORF为894bp,编码295个氨基酸,该蛋白的相对分子质量为34 402.6,等电点为9.73,含有1个依赖于AMP和GMP的蛋白激酶磷酸化位点,4个蛋白激酶C磷酸化位点,2个酪蛋白激酶Ⅱ磷酸化位点,1个酪氨酸激酶磷酸化位点,8个十四(烷)酰化位点.分析表明,大熊猫RPL5基因的表达序列及其编码的氨基酸序列与已报道的部分哺乳动物包括人(Homo sapiens)、牛(Bos Taurus)、猪(Sus scrofula)、小家鼠(Mus altocumulus)和褐家鼠(Rat-hauser nonstrategic)具有很高的相似性,大熊猫RPL5核苷酸序列与这些物种的相似性分别为94.52%、92.51%、91.95%、91.05%和89.15%,而氨基酸序列相似性分别为99.33%、98.65%、98.65%、98.32%和98.65%.超表达实验结果显示:大熊猫RPL5基因能在大肠杆菌BL21中有效表达,且在2h时达到表达高峰.运用分子生物学原理与相应的技术手段,成功地扩增出大熊猫RPL5基因的表达序列,并对其编码的蛋白进行了初步分析,丰富和完善了哺乳动物RPL5基因资料库,同时也为深入研究大熊猫RPL5基因的功能提供了相关基础数据.     

中华绒螯蟹内源性白斑综合症病毒基因EjsWSSV的克隆与鉴定(英文)

基于已获得的表达序列标签序列,应用RACE技术从中华绒螯蟹基因组中获得了一个白斑综合症病毒基因(EjsWSSV)的全长cDNA序列,全长为3 864 bp并包含一个3 732 bp的开放阅读框,编码一个138.25 kDa的含1 243个氨基酸的多肽,编码的氨基酸序列包含一个VWA结构域.通过序列比对、序列结构比较和一些生物信息学预测分析,表明EjsWSSV蛋白序列与白斑综合症病毒基因组的ORF16具有高同源性,它不含有信号肽和跨膜区,为非分泌型和α-螺旋型蛋白,可能定位于细胞质.通过PCR检测验证,EjsWSSV基因可能为内源性病毒基因.     

枸杞抗坏血酸氧化酶基因的克隆与表达分析

以枸杞为材料,利用同源克隆技术,克隆了枸杞抗坏血酸氧化酶(AO)的全长cDNA序列,命名为LcAO基因(GenBank:KP712033),该基因开放阅读框(ORF)大小为1737bp,编码578个氨基酸,与西红柿AO蛋白的同源性达到90%.LcAO编码的氨基酸序列包含3个多铜氧化酶结构域和跨膜信号序列.采用实时荧光定量PCR分析显示,LcAO基因在枸杞的花和果实中表达量最高,成熟叶中表达量最少.     

大熊猫核糖体蛋白S26亚基基因(rps26)的cDNA克隆及序列分析

为了解大熊猫核糖体蛋白亚基rps26基因的结构特点及其与已报道的人和其他哺乳动物核糖体蛋白亚基基因rps26的异同,以大熊猫的肌肉组织为材料,根据已报道的部分哺乳动物核糖体蛋白S26亚基基因(rps26)的相关信息设计引物,运用RT-PCR技术,成功地克隆了核糖体蛋白亚基基因rps26的表达序列,并对其进行了测序及初步分析.结果表明:大熊猫rps26亚基基因的表达序列长为413 bp,开放阅读框(ORF)为348 bp,编码115个氨基酸的蛋白质,该蛋白的相对分子质量为13.025 2×103,等电点为11.61.拓扑预测显示该蛋白含有3个功能位点:1个N-糖基化位点,1个酪蛋白激酶Ⅱ蛄姿峄?位点和1个核糖体蛋白S26e signature位点.进一步分析发现,大熊猫rps26基因与已报道的人、西藏黄牛、野猪、褐家鼠和小家鼠5个哺乳动物物种的表达序列其编码的氨基酸序列具有很高的相似性:编码序列同源性分别为90.23%、91.67%、92.82%、87.36%和86.78%;氨基酸序列同源性均为99.86%.     

五种蓝舌病毒的分子系统(英文)

对蓝舌病毒(BTV)血清型2、11和13的双链RNA中L3基因的全序列进行了测定,该基因编码这种病毒的重要内壳蛋白质(VP3),应用前人测定的血清型10和17的BTV的L3基因序列,共5种美国存在的BTV的L3基因。该病毒的每种L3基因片断都有2772个核苷酸,包含一个开放读框(ORF)。这个开放读框(ORF)编码由901个氨基酸组成的蛋白质,VP3(分子量103kD),其等电点为6 0。用这5种蓝舌病毒血清型的核苷酸和氨基酸序列进行分子系统学分析发现,BTV-2血清型与BTV-10、11、13和17的距离较远,美国的五种血清型BTV可分为两个不同的类群。     

Molecular characterization of<Emphasis Type="Italic">Tomato yellow leaf curl China</Emphasis> virus and its satellite DNA isolated from tobacco

Virus isolates, Y8, Y36 and Y38, were obtained from tobacco plants showing leaf curl symptoms in Honghe,Yunnan Province. In reactions with 14 monoclonal antibodies raised against Begomoviras particles, Y8, Y36 and Y38 had similar antigenic reaction in TAS-ELISA as Tomato yellow leaf carl China virus (TYLCCNV). The complete DNA-A nueleotide sequences of Y8, Y36 and Y38 were determined and they contain 2727, 2730 and 2730 nueleotides, respectively. Each of the DNA-A sequences has a typical Begomovirus genome organization encoding 60RFs with 20RFs[AVI(CP) and AV2] in virion-sense DNA and 40RFs (AC1 to AC4) in complementary-sense DNA. Comparisons with total DNA-A, intergenie region and deduced amino acid sequences of individual ORFs show that Y8, Y36 and Y38 are isolates of TYLCCNV. Satellite DNA molecules (DNAβ) were found to be associated with Y8, Y36 and Y38, which consist of 1338, 1339 and 1338 nucleotides, respectively. Comparisons show that these DNAβ molecules share 98%--99% sequence identities on nucleotide level and have a common ORF (designated C1) encoding 126 amino acids on the complementary strand.     

Genome organization and phylogenetic tree analysis of Garlic virus E, a new member of genus Allexivirus

The complete sequence of an Allexivirus isolated from garlic plants in Yuhang City, Zhejiang Province, China had been determined. The single-strand, positive RNA genome was 8451 nucleotides in length excluding poly(A) tail. The genome organization of this virus was similar to that of the other Allexiviruses but only with 62.8%–64.8% nucleotide acid identities. The amino acid sequences of proteins encoded by ORF1-6 shared 67.6%–78.5%, 55.4%–66.2%, 56.7%–66.4%,40.3%–55.6%,66.3%–79.7%and 52.2%–68.8% identities with those of the others respectively. The homology range between it and the other Allexiviruses was similar to that between the other distinct species in this genus. A more comprehensive comparison using all available CP amino acid sequences showed that it shared only 63.9%–79.8% amino acids identical with the others. Therefore, it had been considered as a new member of the genus, named as garlic virus E (GarV-E). Phylogenetic analysis confirmed GarV-E as a distinct member and the correct names and classification of some members of genus Allexivirus were also discussed.     

哺乳动物基因非翻译区开放阅读框架的分析

在所分析的5'UTR序列中约19%的序列在阅读框1下含有AUG,约22%和24%的序列分别在阅读框 2 和阅读框3下含有 AUG,而在 3'UTR序列当中,无论是在哪个阅读框下,约71%含有AUG.这些AUG绝大多数都是以小ORF的形式出现的;分析小ORF序列以及与它们相应的IC序列(阅读框间序列)长度,发现虽然3'UTR序列远远长于5'UTR序列,但是它们所包含的小ORF长度几乎没有差距,只是平均来说每一条3'UTR序列所包含的小ORF数量明显多于每一条5'UTR序列所包含的小ORF数量,而且dIC(下游阅读框间序列)比uIC(上游阅读框间序列)长很多,平均长50个碱基;第二类IC序列(UTR区相邻的两个阅读框之间的序列)的平均长度比其它两类IC序列(UTR区最后一个阅读框之后的序列)的平均长度小,而且又含有相对较多的终止密码;比较uORF序列与dORF序列密码子使用偏好的CAI值发现,尽管相差不大,但是无论在哪个阅读框下,uORF序列的CAI值都显著高于dORF序列的CAI值.     

Malvastrum yellow vein virus,a new Begomovirus species associated with satellite DNA molecule

Virus isolate Y47 was obtained from Malvastrum coromandelianum showing yellow vein symptom in Honghe, Yunnan Province. The complete nudcotide sequence of DNA-A was determined, it contains 2731 nuclcotides,having typical genomic organiTation of a begomovirns, encoding 6ORFs with 2ORFs [AVI(CP) and AV2] in virionsense DNA and 40RFs (ACl-AC4) in complementary-sense DNA. Comparisons show that the total DNA-A of Y47 has the highest sequence identity (77%) with that of Okra yellow vein mosaic virus-[201] (AJ002451), while less than 76% identities are found when compared with other begomoviruses. The molecular data show that virus isolate Y47 is a distinct begomovirns species, for which the name Maivastrum yellow vein vorus is proposed. Satellite DNA molecule (Y47β) was found to be associated with Y47 using the primers (beta01 and beta02) specific for DNAβ Y47β consists of 1348 nuclcotides, with a functional ORF (CI) in complemen-tary-sense DNA.Y47β has 62%--67% sequence identity with DNAβ molecule associated with Cotton leaf curl Muitan virus or Cotton leaf curl Rajasthan virus, while lower than 46% sequence identities are found when compared with other reported DNA[~ molecules. Relationship dendrograms show that DNAβ molecules are co-evolved with their help begomoviruses.     

Genome organization and phylogenetic tree analysis of Garlic virus E, a new member of genus Allexivirus

The complete sequence of an Allexivirus isolated from garlic plants in Yuhang City, Zhejiang Province, China had been determined. The single-strand, positive RNA genome was 8451 nucleotides in length excluding poly(A) tail. The genome organization of this virus was similar to that of the other Allexiviruses but only with 62.8%-64.8% nucleotide acid identities. The amino acid sequences of proteins encoded by ORF1-6 shared 67.6%-78.5%, 55.4%-66.2%, 56.7%- 66.4%, 40.3%-55.6%, 66.3%-79.7% and 52.2%- 68.8% identities with those of the others respectively. The homology range between it and the other Allexiviruses was similar to that between the other distinct species in this genus. A more comprehensive comparison using all available CP amino acid sequences showed that it shared only 63.9%- 79.8% amino acids identical with the others. Therefore, it had been considered as a new member of the genus, named as garlic virus E (GarV-E). Phylogenetic analysis confirmed GarV-E as a distinct member and the correct names and classification of some members of genus Allexivirus were also discussed.     

<Emphasis Type="Italic">Malvastrum yellow vein virus</Emphasis>, a new<Emphasis Type="Italic">Begomovirus</Emphasis> species associated with satellite DNA molecule

Virus isolate Y47 was obtained fromMalvastrum coromandelianum showing yellow vein symptom in Honghe, Yunnan Province. The complete nucleotide sequence of DNA-A was determined, it contains 2731 nucleotides, having typical genomic organization of a begomovirus, encoding 6 ORFs with 2 ORFs [AV1(CP) and AV2] in virionsense DNA and 4 ORFs (AC1–AC4) in complementarysense DNA. Comparisons show that the total DNA-A of Y47 has the highest sequence identity (77%) with that ofOkra yellow vein mosaic virus- [201] (AJ002451), while less than 76% identities are found when compared with other begomoviruses. The molecular data show that virus isolate Y47 is a distinct begomovirus species, for which the nameMalvastrum yellow vein virus is proposed. Satellite DNA molecule (Y47β) was found to be associated with Y47 using the primers (beta01 and beta02) specific for DNAβ. Y47β consists of 1348 nucleotides, with a functional ORF (C1) in complementary-sense DNA. Y47β has 62%–67% sequence identity with DNAβ molecule associated withCotton leaf curl Multan virus orCotton leaf curl Rajasthan virus, while lower than 46% sequence identities are found when compared with other reported DNAβ molecules. Relationship dendrograms show that DNAβ molecules are co-evolved with their help begomoviruses.     

猪繁殖和呼吸综合征病毒ORF7基因的克隆与序列分析

根据猪繁殖和呼吸综合征病毒（PRRSV）已发表的核苷酸序列,设计了1对特异性引物P1／P2,用RT-PCR扩增了PRRSV福建毒株FJ-1和FJ-2的全长核衣壳蛋白基因（ORF7）,将扩增产物连接到pUCm-T载体并转化大肠杆菌DH10B,提取阳性重组质粒进行序列测定与分析．结果表明：FJ-1和FJ-2毒株ORF7基因均为372bp,编码123个氨基酸,与美洲型参考毒株VR-2332及欧洲型参考毒株LV的核苷酸同源性分别为95．16％,94．35％和61．40％,60．65％,其推导的氨基酸同源性分别为95．93％,95．12％和56．49％,55．73％．研究表明,福建流行的FJ-1和FJ-2属于美洲型PRRSV毒株．     

Tobacco curly shoot virus iso-lated in Yunnan is a distinct species of Begomovirus

Virus isolate Y1 was obtained from tobacco showing curly shoot symptoms in Baoshan, Yunnan Province. Whitefly transmission test and virion morphology observation showed that it is a begomovirus. In reactions with 14 monoclonal antibodies raised against begomoviruses, Y1 was readily differentiated from begomoviruses reported in China, Pakistan and India. The complete nucleotide sequence of DNA-A was determined, it contains 2746 nucleotides, with two ORFs in virion-sense DNA and four ORFs in complementary-sense DNA. Comparisons with total DNA-A, intergenic region and deduced amino acid sequences of individual ORFs showed that Y1 is a distinct Begomovirus species, for which the name Tobacco curly shoot virus (TCSV) is proposed. The total DNA-A of TCSV is most closely related to that of Tomato leaf curl virus from India (85% sequence identity). In contrast, the deduced coat protein of TCSV is most like that of Cotton leaf curl virus 72b isolate from Pakistan (98% amino acid sequence identity).     

随机DNA序列中ORF的分布

研究了组分约束下的随机 DNA序列中 ORF数目、ORF的长度与随机序列总长度和GC含量之间的关系 .结果表明 ,ORF数目的对数与 ORF的长度之间存在很好的线性关系 ;ORF的最大长度随序列长度的增加而变长 ,ORF的总数与序列长度成正比 ;ORF的总数目随着 GC含量的增加明显减少 ;随着 GC含量的增加 ,长度较短的 ORF数目相对减少 ,长度较长的 ORF数目相对增加 ;随着序列长度的增加 ,长度较短的 ORF相对增多 .