牛分支杆菌ESAT-6基因的克隆与序列分析

从患结核病鹿的肺组织中粗分离牛分支杆菌制备DNA模板,用PCR对牛分支杆菌ESAT-6基因进行了扩增,将扩增的产物连接于测序载体pGEM-TE上克隆,经测序确定无误;并使用分子生物学软件对ESAT-6基因进行了分析。结果显示:ESAT-6基因编码的蛋白有较好的抗原性;ESAT-6基因在结核分支杆菌和牛分支杆菌各基因型中具有高度保守性。本研究为下—步研制重组标记疫苗奠定了基础。     

玉米SAMS基因的克隆与序列分析

腺苷甲硫氨酸合成酶（SAMS）是植物代谢中的1个关键酶,它催化ATP和L一甲硫氨酸合成腺苷甲硫氨酸（SAM）。以玉米为材料,用Trizol一步法提取总RNA,根据已报道的水稻和小麦等的SAMS基因设计1对引物．通过RT-PCR方法获得1条约1．2kb特异片段,连接T载体测序,其全长共1191bp,编码496个氨基酸残基。利用生物信息学分析,结果表明：SAMS蛋白疏水性最大值为1．73,最小值为-2．1;无跨膜域;二级结构主要以无规卷曲为主。     

野桑蚕中肠组织cDNA文库的构建及丝氨酸蛋白酶基因片段的克隆与序列分析(英文)

[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.     

伪狂犬病病毒Fa株gE基因的克隆与序列的比较分析

根据已发表的伪狂犬病病毒（ＰＲＶ）的基因序列,在ＰＲＶｇＥ基因阅读框外的上、下分别设计一对引物Ｐ１和Ｐ２,在引物的 ５’端分别加上 ＫｐｎＩ和 ＥｃｏＲＩ位点。以 ＰＲＶ Ｆａ株的 ＤＮＡ为模板成功地扩增得到一条长的 １．７ｋｂ的片段,经酶切鉴定,初步确定为ｇＥ基因。将此ＰＣＲ产物经ＫｐｎＩ、ＥｃｏＲＩ消化后与同样酶切的ＰＵＣ１８质粒连接、转化,得到了明显大于载体的重组质粒ＰＰｇＥ。重组质粒ＰＰｇＥ经酶切鉴定确定为含有ＰＲＶ 的 ｇＥ基因,测序ＰｐｇＥ得到完整的ｇＥ基因片段,并与４株不同来源的毒株进行了比较分析。在对 ＰＲＶ Ｆａ（来自福建）、Ｒｉｃｅ（来自俄国）、ＴＮＬ（来自台湾）、Ｅａ（来自武汉）、ＳＨ（来自上海）的ｇＥ同源性比较分析发现毒株间同源性最低也高达９７％,这一定程度上也体现ｇＥ基因组的保守性。分析还表明Ｆａ与ＴＮＬ同源。同时９９％的高同源性也显示 Ｆａ、Ｅａ、ＳＨ可能是同一毒株。本实验认为 ｇＥ序列在 １０４０－ １４１０碱基的高同源性区域作为ＰＣＲ检测或核酸探针是非常有用的。     

白菜型油菜春化基因BrFLCsBrFLC序列变异分析

为寻找与冬春性关系密切相关的变异,为开发冬春性品种的鉴定开发分子辅助标记提供理论依据。本研究,采用巢式和半巢式扩增法对感温性和抗寒性强弱不同的5个白菜型油菜品种的BrFLCsBrFLC基因的第4外显子至第7外显子进行了特异性扩增。除BrFLC5无扩增结果外,其余三个基因均得到扩增序列。序列比对结果发现,3个BrFLCsBrFLC在第4外显子至第7外显子均存在有不同程度的等位变异,发生的主要等位变异有SNP和Indel两种类型。BrFLC1主要变异与冬春性相关性不大,与冬春性关系密切的变异主要发生在BrFLC2和 BrFLC3上。BrFLC2中检测到1个Indel类型和5个SNP类型变异位点与冬春性有关。BrFLC3中检测到1个Indel类型和16个SNP类型变异位点与冬春性有关。       

多花水仙HDS基因cDNA全长克隆与序列分析

以‘黄花水仙2号’和‘金盏银台’为材料,水仙各自花瓣的cDNA为模板,采用RT-PCR和RACE技术克隆出2个HDS基因,分别命名为NtHDSY和NtHDSJ,测序结果表明,NtHDSY和NtHDSJ基因全长分别为2 592 bp和2 599 bp,2个基因均含有1个2 178 bp的开放阅读框（ORF）,编码745个氨基酸。同源性分析表明,黄花水仙2号与金盏银台、铁皮石斛、长春花、大豆、葡萄和苹果的相似系数分别为：97.77％、79.29％、75.29％、75.51％、75.02％和74.40％。Real-time PCR分析表明：NtHDS基因在花瓣和副冠内的表达量在开花过程的花蕾期与盛花期存在明显差异,推测该基因可能与多花水仙香气物质的合成有关。     

Genetic Diversity of Chinese Soybean mosaic virus Strains and Their Relationships with Other Plant Potyviruses Based on P3 Gene Sequences

Soybean mosaic virus （SMV）, a member of the genus Potyvirus, is a major pathogen of soybean plants in China, and 16 SMV strains have been identified nationwide based on a former detailed SMV classification system. As the P3 gene is thought to be involved in viral replication, systemic infection, pathogenicity, and overcoming resistance, knowledge of the P3 gene sequences of SMV and other potyviruses would be useful in efforts to know the genetic relationships among them and control the disease. P3 gene sequences were obtained from representative isolates of the above-mentioned 16 SMV strains and were compared with other SMV strains and 16 Potyvirus species from the National Center for Biotechnology GenBank database. The P3 genes from the 16 SMV isolates are composed of 1041 nucleotides, encoding 347 amino acids, and share 90.7-100% nucleotide （NT） sequence identities and 95.1-100% amino acid （AA） sequence identities. The P3 coding regions of the 16 SMV isolates share high identities （92.4-98.9% NT and 96.0-100% AA） with the reported Korean isolates, followed by the USA isolates （88.5-97.9% NT and 91.4-98.6% AA）, and share low identities （80.5-85.2% NT and 82.1-84.7% AA） with the reported HZ 1 and P isolates from Pinellia ternata. The sequence identities of the P3 genes between SMV and the 16 potyviruses varied from 44.4 to 81.9% in the NT sequences and from 21.4 to 85.3% in the AA sequences, respectively. Among them, SMV was closely related to Watermelon mosaic virus （WMV）, with 76.0-81.9% NT and 77.5-85.3% AA identities. In addition, the SMV isolates and potyvirus species were clustered into six distinct groups. All the SMV strains isolated from soybean were clustered in Group I, and the remaining species were clustered in other groups. A multiple sequence alignment analysis of the C-terminal regions indicated that the P3 genes within a species were highly conserved, whereas those among species were relatively variable.     

植物丝裂原活化蛋白激酶激酶的生物信息学分析

丝裂原活化蛋白激酶(MAPK)信号传导通路是植物细胞内重要的信号传导系统,在多种生物和非生物胁迫,激素、细胞分化和发育进程中发挥关键作用。丝裂原活化蛋白激酶激酶(MAPKK)是信号传导中的重要成员,执行整合上游信号到丝裂原活化蛋白激酶的重要功能。基于BLAST搜索,得到植物丝裂原活化蛋白激酶激酶基因序列。采用CLUTALW、SMART、SwissModel和MEGA3,1软件分析这些序列。多序列比对揭示了它们具有典型的蛋白激酶ATP结合域签名和丝氨酸、苏氨酸蛋白激酶激活位点签名。空间结构预测表明它们与人MAPKK的晶体结构相似。系统进化树中烟草和番茄的丝裂原活化蛋白激酶激酶、野生稻和印度栽培稻丝裂原活化蛋白激酶激酶形成独立的分支,说明它们分别具有较近的亲缘关系。     

家蚕细胞色素P450基因Bmcyp6A8的克隆及序列与表达分析

昆虫细胞色素P450第6亚家族(CYP6)氧化酶在对异源有毒物质的代谢中具有重要作用。采用生物信息学方法获得与果蝇CYP6亚家族基因cyp6A8同源的家蚕cyp6A8基因序列,预测该基因的开放阅读框(ORF)为1572bp,编码523个氨基酸,推定的蛋白分子质量为61.52kD,等电点为8.17。以家蚕5龄第3天幼虫头部cDNA为模板,用设计的特异引物PCR扩增出一条约1500bp的条带,大小与家蚕cyp6A8基因的ORF预测值接近,命名为Bmcyp6A8基因(GenBank登录号:GQ241737)。同源性分析结果:Bmcyp6A8基因与野桑蚕cyp6AE8基因的相似性为93%;与棉铃虫cyp6AE12基因的相似性为57%;与人cyp3A43基因的相似性为48%。芯片数据分析显示Bmcyp6A8基因在家蚕5龄第3天幼虫的头部、表皮与中部丝腺前部高量表达,与家蚕CYP6亚家族的其它横向同源基因不同的是,只有该基因在中部丝腺前部表达,推测其具有功能特异性。