凡纳滨对虾繁育相关基因Allatostatin-AR的克隆与表达分析

咽侧体抑制素主要抑制昆虫咽侧体保幼激素的分泌,是昆虫体内重要的神经肽,在甲壳动物体内可能参与繁殖发育的调节。本研究利用RACE PCR技术对凡纳滨对虾(Litopenaeus vannamei)A型咽侧体抑制素受体(Allatostatin-A receptor,AST-AR)基因进行克隆,获得全长为2 650 bp的cDNA,包括1 425 bp的开放阅读框,1 170 bp 5''非编码区,55 bp 3''非编码区,编码474个氨基酸,形成具有7个跨膜区的不稳定亲水性蛋白。同源性和系统发育分析结果表明凡纳滨对虾AST-AR基因与斑节对虾(Penaeus monodon)、美洲龙虾(Homarus americanus)同源性最高,亲缘关系最近。氨基酸多重序列比对结果显示AST-AR氨基酸TM7后C末端精氨酸在不同物种之间高度保守。实时荧光定量PCR结果显示,AST-AR基因在雌、雄凡纳滨对虾多个组织(眼柄、脑、胃、肝胰腺、鳃、性腺、肠、肌肉)中均有表达,除脑组织外AST-AR基因在雌性凡纳滨对虾中相对表达量高于雄性。在幼体发育中,AST-AR基因的相对表达量随发育阶段上升,同时该基因在不同群体仔虾生长对比实验中表达量差异显著。据此推测AST-AR基因参与凡纳滨对虾的繁殖、幼体发育、幼虾生长到成虾,基因表达基本贯穿凡纳滨对虾的一生。     

甜荞花同源异型基因FeMADS1的克隆和序列结构分析

采用同源克隆方法,并结合RACE技术,从甜荞花芽分离得到1个A类MADS-box基因FeMADS1的cDNA全长,GenBank登录号为KM386627,其cDNA全长1 107bp,包括1个编码234个氨基酸、长为705bp的开放阅读框。序列同源比对和分子系统发生分析表明,其蛋白与拟南芥AGL8(FUL)的相似性最高,属A类MADS-box基因亚家族中的euFUL进化系,含MADS、I、K和C末端4个明显的结构域,并且K结构域包含K1、K2和K3共3个保守的富含疏水氨基酸残基的亚结构域,C末端结构域含FUL型基因2个特有的模体:FUL motif和paleoAP1motfi。     

陕西茶树地方种质资源特征性生化成分分析

为了发掘新的茶树种质资源,对陕西省10个地区88份茶树资源的特征性生化成分进行了测定评价和遗传多样性分析。结果表明,88份茶树种质资源的生化成分变异幅度大,氨基酸、茶多酚、咖啡碱和水浸出物含量及酚氨比平均变异系数为28.00%,遗传多样性指数（H''）变幅为1.96～2.08,均值为2.01。聚类分析发现,当欧氏距离为15时,88份资源可分为4大类复合组,无独立组存在。根据生化成分含量,可以从中初步筛选出高咖啡碱资源1份、低咖啡碱资源1份和低茶多酚资源1份,为今后的生产和育种提供利用。     

稀有白甲鱼西江种群mtDNA D环区的结构及遗传多样性

采用PCR扩增和DNA直接测序技术,测定分析了采自珠江水系西江的30尾稀有白甲鱼(Onychostoma rara)mtDNA D环约467 bp的序列。结果显示,该序列的长度为467～470 bp,共计发现了16个变异位点,占分析总位点数的3.40%,其碱基组成A+T的平均含量(67.6%)高于C+T(32.3%)。稀有白甲鱼西江种群mtDNA D环区在结构上可分为终止序列区、中央保守区和保守序列区,存在着与终止相关的序列ETAS以及保守序列CSB-F、CSB-E、CSB-D和CSB1。定义了11种单倍型,单倍型多样性指数(Hd)为0.733 3,单倍型之间平均遗传距离(P)为0.010 2,核苷酸多样性(Pi)为0.005 79,平均核苷酸差异数(K)为2.705 75。11个单倍型构建的非加权分组平均法(UPGMA)系统树聚为2个分支。该研究表明稀有白甲鱼西江种群存在着较丰富的遗传多样性。研究亮点:(1)有关濒危鱼类稀有白甲鱼西江种群mtDNA D环测序及遗传多样性研究尚未见报道;(2)本研究揭示了稀有白甲鱼西江种群尚保存有较丰富的遗传多样性;(3)对稀有白甲鱼西江种群遗传学研究具有理论价值,并对开展该种群的保护和资源利用具有现实指导意义。     

普通小麦济麦21的LOX1基因cDNA片段克隆及分析

以普通小麦品种济麦21为材料,根据GenBank中已发表的LOX1基因序列设计引物,利用RTPCR技术获得一个小麦LOX1的基因片段,并对该片段进行测序和生物信息学分析。结果表明:克隆到的基因片段为384bp(GenBank登录号JX126806),GC含量为64.84%,推导氨基酸残基为127个,分子质量为11.15ku,等电点(pI)为4.93,与杜伦小麦、大麦、高粱、水稻的同源性分别为99.7%、94.8%、73.7%、56.2%。进化树分析表明,六倍体普通小麦LOX1基因与杜伦小麦LOX1基因(GenBank登录号HM126468)亲缘关系较近,先聚为一支,然后再与大麦的LOX1基因(GenBank登录号L35931.1)、高粱的LOX1基因(GenBank登录号GQ369443)聚为一类,最终与亲缘关系较远的水稻LOX1基因(GenBank登录号EU267789)聚类,这与物种亲缘关系的远近基本一致,表明所得LOX1基因片段可作为研究物种亲缘关系或遗传距离的参考标记之一。     

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.     

橡胶树水通道蛋白基因HbPIP1;2和HbPIP2;2的克隆及序列分析

根据GenBank中已经公布的不同植物水通道蛋白(AQPs)的保守氨基酸序列设计简并引物,结合RT-PCR和RACE技术,从橡胶树(Hevea brasiliensis)中克隆了2个AQP基因的全长cDNA序列,并对其进行生物信息学分析。结果获得橡胶树2个AQPs基因的全长,分别命名为HbPIP1;2和HbPIP2;2。HbPIP1;2编码273个氨基酸,理论分子量为29.15kD,等电点为8.55;HbPIP2;2编码278个氨基酸,理论分子量为29.78kD,等电点为8.20。HbPIP1;2和HbPIP2;2均具有6个跨膜区和MIP家族信号序列SGXHXNPAVT,为疏水性蛋白;HbPIP1;2和HbPIP2;2与同科蓖麻水通道蛋白基因RcPIP1-3和RcPIP2-1的同源性分别为91%和89%,并具有相同功能域。因此,推测Hb-PIP1;2和HbPIP2;2都属于水通道蛋白基因家族的新成员。     

锦江河3种鳜的遗传变异及其多样性评价

为探究锦江河国家级水产种质资源保护区内鳜类遗传变异特征及其多样性水平,基于PCR扩增与测序技术对大眼鳜、斑鳜和中国少鳞鳜3个群体97尾鱼的线粒体控制区序列进行比较分析。结果显示,大眼鳜序列长度为843 bp,无变异位点,只有1种单倍型,斑鳜有852 bp和856 bp两种序列,28个多态位点和11个单倍型,中国少鳞鳜也有845 bp和846 bp两种长度,5个多态位点和5个单倍型;大眼鳜、斑鳜和中国少鳞鳜3种鳜鱼群体内的遗传距离分别为0. 000 0、0. 008 7±0. 002 0和0. 002 0±0. 000 9,单倍型多样度和核苷酸多态性分别为0. 000 0、0. 859 9、0. 736 7和0. 000 0、0. 008 7、0. 002 0。分析表明,3种鳜鱼在终止序列区和保守序列区存在种或属间差异,锦江斑鳜是一个遗传变异大,多样性丰富的稳定种群;中国少鳞鳜虽然单倍型多样度丰富,但遭受过瓶颈效应,导致其核苷酸多样性偏低;大眼鳜遗传组成单一,多样性十分贫乏,推测可能有入侵的养殖群体。锦江系梵净山国家级自然保护区内最大的河流,历史上鳜类资源丰富,但已遭严重的破坏,加强其鳜类遗传资源的研究与保护十分必要。     

暗纹东方鲀sox9基因的克隆和组织表达分析

为初步探究暗纹东方鲀sox9基因在性腺发育和性别分化过程中的作用,通过设计兼并引物、RACE及荧光定量PCR技术,成功克隆出暗纹东方鲀sox9基因的cDNA全长序列,并分析其相应的生物信息学特征及其在雌雄个体的组织表达水平。结果表明:sox9a基因cDNA全长为1 248 bp(NCBI登录号:MH218818),包括684 bp的ORF,编码227个氨基酸,297 bp的5′UTR和267 bp的3′UTR。sox9b基因全长为1 941 bp(NCBI登录号:MH218819),包括1 470 bp的ORF,编码489个氨基酸,306 bp的5′UTR和165 bp的3′UTR。同源性和系统发育分析结果表明暗纹东方鲀sox9基因与红鳍东方鲀同源性最高,亲缘关系最近。氨基酸多重序列比对结果显示两个Sox9氨基酸序列的HMG box结构域在哺乳动物和鱼类中均高度保守。荧光定量PCR分析显示两个sox9基因普遍存在于雌鱼和雄鱼的各个组织中,且均在雌鱼下丘脑中表达量最高,在精巢中有少量表达,卵巢中几乎不表达。总体来说,除少数组织外,两个sox9基因在雌鱼组织中的表达量普遍高于雄鱼。本研究为了解暗纹东方鲀sox9的遗传特性及相关的生理功能,探索性别分化与性腺发育的分子调控机制提供理论依据。     

基于线粒体COX1基因的蚕蛾类系统发育分析

基于生物信息学的方法,以16种蚕蛾类昆虫作为研究对象,利用线粒体基因COX1的碱基序列作为分子标记对其系统发育信息进行分析。结果表明,COX1基因序列具有明显的AT偏倚性,编码氨基酸的密码子有26个具有明显的使用偏好性,种群间的校正遗传距离在0.003～0.177之间,表明蚕蛾类昆虫的亲缘关系近。利用MEGA6软件,分别采用邻接法（NJ）和最大似然法( ML)构建系统发育树,其发育树的分支明显,大部分的节点支持率都很高,进一步说明了蚕蛾类昆虫的亲缘关系较近。本研究补充了对蚕蛾类传统分类的认识,也为蚕蛾类昆虫的分类提供依据。     

牦牛应激型 HSPA1A和 HSPA2基因的克隆及序列分析

为了解牦牛应激型 HSPA1A和 HSPA2基因的特点,根据GenBank已公布的牛应激型 HSPA1A和 HSPA2基因序列设计4对引物,每个基因分两段扩增,测序并拼接,首次克隆牦牛应激型 HSPA1A和 HSPA2基因。序列分析表明,扩增到的牦牛应激型 HSPA1A基因全长2 134 bp,开放阅读框全长1 926 bp,编码641个氨基酸,分子质量为70.26 ku; HSPA2基因全长1 911 bp,开放阅读框全长1 911 bp,编码636个氨基酸,分子质量为69.85 ku。将 HSPA1A和 HSPA2基因开放阅读框编码的氨基酸序列进行ScanProsite分析,均得到3个HSP70蛋白家族标记。核苷酸序列同源性分析表明, HSPA1A和 HSPA2基因具有较高的保守性,牦牛与牛的 HSPA1A和 HSPA2基因核苷酸序列同源性最高,分别为99.8%和99.1%。遗传进化关系分析表明,牦牛与牛的应激型 HSPA1A和 HSPA2基因亲缘关系最近。     

Sequence variations of <Emphasis Type="Italic">PDHA1</Emphasis> gene in Triticeae species allow for identifying wheat-alien introgression lines

Abstract In order to develop a molecular marker for the detection of alien chromatin, an allele-specific primer targeting pyruvate dehydrogenase (PDHA1) gene was used to analyze 12 taxa representing different basic genomes in Triticeae. Ampli....cation products with different sizes were generated among species. The sequence alignments indicated that the PDHA1 genes contained some deletions/insertions of Miniature Inverted-repeat Terminal Elements (MITE) and simple sequence repeats (SSRs), thus suggesting that the Triticeae genomes have been rapidly evolving during speciation. The genome-specific amplicons and chromosomal location of PDHA1 gene on Triticeae genomes can be used to trace the corresponding alien chromatins from Aegilops, Secale and Dasypyrum species in wheat background.     

交链格孢菌EST-SSR信息分析与分子标记通用性评价

为了弄清交链格孢菌(Alternaria alternata) EST数据库中的SSR信息资源,并开发可用于研究近缘种细极链格孢(Alternaria tenuissima)遗传多样性的EST-SSR分子标记。从NCBI数据库下载A.alternata表达序列标签(Expressed sequence tag,EST)共727条,经过研究分析发现,A.alternata EST中SSR含量非常丰富,每5.343 kb含有1个SSR位点。根据SSR两端保守序列,设计合成13对EST-SSR引物,对来自新疆不同地区不同寄主的38株A.tenuissima进行通用性检测。检测结果显示共有5对引物能扩增出多态性条带,扩增多态率为55.56%。因此,基于A.alternata EST数据库开发的SSR分子标记,在开展近缘种A.tenuissima遗传多样性研究时具有较好的通用性。     

Do the tropical freshwater fishes feed on aquatic fungi?

Consumption and processing of allochthonous plant litter by fishes is more common in tropical than temperate streams and rivers. Therefore, aquatic hyphomycetes in water (filtration), leaf litter (bubble chamber incubation), and fecal pellets (direct observation and inoculation to sterile leaf litter) of three dominant fishes belonging to the family Cyprinidae (Aplocheilus lineatus, Puntius filamentosus, and Rasbora daniconius) in two locations of the River Kali of the Western Ghats, India, were evaluated during postmonsoon season. Spores of 14 and 9 species of aquatic hyphomycetes were recovered on filtering water samples of Kaiga stream and Kadra dam with equal number of spores (32 spores · 100 mL⁻¹) and high Shannon diversity in Kaiga stream. In a bubble chamber incubation of leaf litter, 16 and 9 species were recovered from Kaiga stream and Kadra dam with high spore output (1122 versus 324 spores per mg dry mass) and high Shannon diversity in Kaiga stream. Both direct and indirect methods of examination of fecal pellets of fishes revealed more species in Kaiga stream than Kadra dam (4–7 versus 1–4 species). The spore release in leaf litter incubated with fecal pellets ranged from 2.3 to 98 spores·mg⁻¹·d⁻¹ with the highest Aplocheilus lineatus in Kaiga stream; while in Kadra dam, it was from 0.02 to 22.9 spores·mg⁻¹·d⁻¹ with the highest in Puntius filamentosus. The Shannon diversity of aquatic hyphomycetes was high in fecal pellets of Aplocheilus lineatus of Kaiga stream and Rasbora daniconius of Kadra dam. The top-ranked five species of aquatic hyphomycetes differed in water, leaf litter, and fish fecal pellets; however, Triscelophorus konajensis was common for all. All five top-ranked species of aquatic hyphomycetes in feces produced multicelled spores; thus, they were likely to have a better chance of viability through gut passage than single-celled spores. Preferential feeding, fungi in gut and feces, and survival and dissemination of spores by invertebrates and fishes with reference to aquatic hyphomycetes were discussed.     

Identification of a novel enhancin-like gene from Bacillus thuringiensis

An enhancin-like gene was cloned from Bacillus thuringiensis (Bt) strain GS8 isolated from soil samples in china. The sequence analysis revealed that an open reading frame (ORF) of 2202 nucleotides encoding a protein containing 733 amino acids with a molecular mass of 84 kDa. The enhancin-like protein showed 100% identity to Bel protein (FJ644935) and 23%–41% identity to viral enhancin proteins; in the 252 to 261 amino-acid sequence of enhancin-like protein, a conserved metal binding motif (HEIAH) similar to that in the reported bacterial enhancin-like proteins was found (HEXXH in viral enhancin protein), which indicated that the enhancin-like protein belongs to metalloprotease. The purified enhancin-like protein was fed together with Cry9Ea to Spodopera exigua and Trichoplusia ni larvae, but no significant increase in toxicity was observed.     

斑节对虾谷氨酰胺合成酶基因的克隆及氨氮胁迫对其时空表达的影响

根据本实验室构建的斑节对虾(Penaeus monodon)c DNA文库得到的EST序列,利用RACE技术获得了斑节对虾谷氨酰胺合成酶基因(Pm GS)的c DNA全长序列,进行了相关生物信息学分析,在此基础上采用荧光定量的方法研究了Pm GS基因在斑节对虾不同组织、氨氮胁迫过程中差异表达情况。该序列全长1 420bp,开放阅读框(ORF)为1 086 bp,3'非编码区(UTR)为294 bp,包括含有27个碱基的poly(A)尾,5'非编码区(UTR)为40 bp。ORF可编码361个氨基酸,预测分子量为40.423 ku,理论等电点为6.19。序列含有一个谷氨酰胺结合结构域(Gln-synt_C),8个磷酸化位点,2个糖基化位点。斑节对虾和中国明对虾的GS基因的相似性最高,达99%。Pm GS-mRNA在斑节对虾各组织中都有表达,在淋巴组织中表达量最高,其次为鳃组织,在胸神经中的表达量最低。96 h高浓度氨氮胁迫后,Pm GS-mRNA在肝胰腺中的表达水平显著高于对照组,但在鳃中的表达水平显著低于对照组(P0.05)。以上结果暗示,Pm GS在氨氮代谢方面具有重要的作用,可能参与了斑节对虾机体的急性氨氮胁迫应答反应。     

Cloning and structure analysis of an NBS-LRR disease-resistant gene from Setaria italica Beauv

Degenerate PCR primers targeting conserved motifs of most NBS-LRR disease-resistant genes in plants were tested in Setaria italica Beauv. cultivar Shilixiang, which is resistant to Uromyces setariae-italicae. A sequence with a length of 2673 bp has been obtained by using Genomic Walking technology. The nucleotide sequence contained an open reading frame that encoded 891 amino acid residues with a calculated molecular mass of 101.44 kDa. It was named RUS1 (Resistance against Uromyces setariae-italicae, GenBank No. FJ467296). It contained an NB-ARC domain and three conserved motifs P-loop, kinase 2, and kinase 3, which had the characteristics of NBS-LRR type resistant gene of plant. Phylogenetic analysis indicated that it was similar to RPM1 and might belong to LZ-NBS-LRR type disease resistance gene. Southern blotting result displayed that there were at least three copies of RUS1 in the foxtail millet genome.     

沙棘WRI1转录因子基因的生物信息学分析

以构建的沙棘(Hippophae rhamnoides Linn.)不同组织转录组数据库为基础,分离得到1个沙棘WRI1转录因子编码基因,命名为Hr WRI1,利用生物信息学方法对其基因结构、理化性质、保守域、信号肽、亚细胞定位、磷酸化位点和高级结构等进行了预测和较为全面的分析。结果表明,该成员含有1 206 bp的完整开放阅读框,编码401个氨基酸组成的不稳定亲水蛋白,无信号肽和跨膜结构域,存在多个磷酸化位点,定位于细胞核中,高级结构以无规则卷曲为主。与不同植物中已报道的同源WRI1转录因子进行多重序列比对后,发现它们在核酸与氨基酸水平均高度同源,并且具有相同的结构域。     

犬弓首蛔虫abcg-5基因的克隆及序列分析

研究犬弓首蛔虫(Toxocara canis,T.canis)腺苷三磷酸结合盒转运蛋白(ATP-binding cassette transporters, ABC)亚家族G5基因(abcg-5)的分子特征.根据犬弓首蛔虫基因组数据库中的Tc-abcg-5基因序列设计引物,通过PCR技术克隆Tc-abcg-5全长基因,并进行多重序列比对和种系发育进化树分析.结果表明:该基因全长为1 902 bp,编码633个氨基酸.功能结构域分析发现TcABCG5蛋白包含1个ABC转运蛋白结构域和6个跨膜区,同时发现了高度保守的Walker A和Walker B模体. GO分析显示ABCG5具有ATP结合和ATP酶活性.种系发育进化树分析显示TcABCG5与猪蛔虫进化关系较近,与哺乳动物进化关系较远.     

牛巴贝斯虫DXR酶的结构特征分析

利用反转录PCR方法扩增牛巴贝斯虫陕县株DXR基因,对其推导氨基酸序列进行生物信息学分析。研究牛巴贝斯虫DXR酶的结构,并探讨其是否可以作为磷胺霉素的作用靶点。结果表明：该基因编码框长度为1 152 bp,编码383个氨基酸,与NCBI上公布的美国牛巴贝斯T2Bo株DXR的相似性为97.4%。同源模建表明,DXR酶的三维结构同恶性疟原虫的三维结构相似,与磷胺霉素结合位点氨基酸具有保守性。