Major histocompatibility complex class IIA and IIB genes of Nile tilapia Oreochromis niloticus: Genomic structure,molecular polymorphism and expression patterns

Major histocompatibility complex (MHC) is a large genomic region characterized by extremely high polymorphism, and it plays an important role in the immune response of vertebrates. In the present study, we isolated MHC class II genes from Nile tilapia in order to investigate the immune mechanism in tilapia and develop better strategies for disease prevention. Moreover, we cloned the full-length cDNA sequences of MHC IIA and IIB from Nile tilapia by the RACE approach. In addition, the genomic structure, molecular polymorphism and expression patterns of MHC II genes in Nile tilapia were also examined. Compared with that of other teleosts, Nile tilapia MHC class IIA contained four exons and three introns. The deduced amino acid sequence of the MHC IIA molecule shared 25.4–64.5% similarity with those of other teleosts and mammals. Six exons and five introns were identified from Nile tilapia MHC IIB, and the deduced amino acid sequence shared 26.9–74.7% similarity with those of other teleosts and mammals. All the characteristic features of MHC class II chain structure could be identified in the deduced sequences of MHC IIA and IIB molecules, including the leader peptide, α1/β1 and α2/β2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. A total of 12 MHC IIA alleles were identified from six individuals. Four alleles originating from a single individual suggested that at least four MHC IIA loci existed. Moreover, 10 MHC IIB alleles were identified, among which four were detected in a single individual, suggesting that at least four MHC IIB loci existed. The expression of MHC IIA and IIB at the mRNA level in 10 types of normal tissues was determined using quantitative real-time PCR analysis. The highest expression level was detected in stomach and gill, whereas the lowest expression was detected in muscle and brain. Furthermore, MHC IIA and IIB were probably two candidate immune molecules involved in the resistance against streptococcosis, because their expression was significantly up-regulated in gill, kidney, intestine and spleen after the intraperitoneal injection of Streptococcus agalactiae.     

Characterization of the major histocompatibility complex class II genes in miiuy croaker

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. In order to study the molecular function and genomic characteristic of class II genes in teleost, the full lengths of MHC class IIA and IIB cDNA and genomic sequence were cloned from miiuy croaker (Miichthys miiuy). As in other teleost, four exons and three introns were identified in miiuy croaker class IIA gene; but the difference is that six exons and five introns were identified in the miiuy croaker class IIB gene. The deduced amino acid sequence of class IIA and class IIB had 26.3-85.7% and 11.0-88.8% identity with those of mammal and teleost, respectively. Real-time quantitative RT-PCR demonstrated that the MHC class IIA and IIB were ubiquitously expressed in ten normal tissues; expression levels of MHC genes were found first upregulated and then downregulated, and finally by a recovery to normal level throughout the pathogenic bacteria infection process. In addition, we report on the underlying mechanism that maintains sequences diversity among many fish species. A series of site-model tests implemented in the CODEML program revealed that positive Darwinian selection is likely the cause of the molecular evolution in the fish MHC class II genes.     

Cloning and Characterization of Full-length Triticin cDNA and Genes from Wheat Varieties K-68 and Chinese Spring

Full-length cDNA and genes for triticin protein were cloned and characterized from wheat varieties K-68 and Chinese Spring differing considerably in grain colour, total protein content, grain hardness, milling behaviour and baking characteristics. Wheat variety K-68 possesses excellent chapatti (unleavened bread) making quality in contrast to Chinese Spring. The nucleotide and deduced amino acid sequences of the full-length triticin cDNA and genes were compared with those of other legumin genes. Although minor variations in the nucleotide sequences were observed when compared with the published sequence of the partial triticin cDNA clone λTri-25, the deduced amino acid sequence of the full-length triticin cDNA clone (Tri-cK68) revealed large variation in the Hyper Variable Region. The deduced amino acid sequence of the full-length triticin cDNA clone Tri-cK68 revealed two lysine-rich regions in the triticin protein. Comparative analysis of the nucleotide sequences of the triticin genes with the cDNA clone λTri-25 revealed the presence of a stretch of 31 nucleotides in the 5′ UTR of λTri-25 having exact complementarity with a stretch of nucleotides of the same length in the 3′ UTR of the full length triticin genes cloned from the wheat varieties K-68 (Tri-gK68) and Chinese Spring (Tri-gCS). Analysis of the nucleotide sequence of triticin promoter (Tri-pK68) revealed the presence of several elements responsible for seed-specific expression and responsiveness to light.     

Structure,organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3′ and 5′ RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5′-terminal untranslated region (UTR), a 355 bp 3′-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74–96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1-k long genomic DNA of carp NKEF-B containing six exons and five introns. Real-time RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity.     

黄鳝Hprt基因的克隆及表达分析

次黄嘌呤鸟嘌呤磷酸核糖转移酶(Hprt)参与嘌呤核苷酸的补救合成。采用RACE技术克隆了黄鳝的次黄嘌呤鸟嘌呤磷酸核糖转移酶基因,它的全长cDNA 为1 452 bp,预测编码218个氨基酸,与人类、小鼠、鸡和斑马鱼等脊椎动物Hprt氨基酸序列之间的同源性超过76.7％。基于该基因氨基酸序列构建了进化树,显示与斑马鱼Hprt基因更同源。RT-PCR表明黄鳝Hprt基因在多种组织中广谱表达,表明黄鳝该基因在功能和进化上的保守性。      

Cloning and characterization of ribonuclease T2 gene (RNHe30) from the basidiomycete,Hericium erinaceum

A gene encoding a ribonuclease T2 (RNase T2) family enzyme, RNHe30, was cloned from Hericium erinaceum by PCR. The deduced amino acid sequence from the complimentary DNA (cDNA) (1074 bp) encodes a 302-aa protein (RNase He30) that has the consensus amino acid sequences of RNase T2 family enzymes including the putative signal peptide. The presence of five introns in the genomic DNA was confirmed by comparison of the cDNA and genomic DNA sequences. The promoter region contains a putative CAAT box and a consensus TATA box. Genes coding homologous enzymes were also identified in various other basidiomycetes. A phylogenetic tree of RNase T2s from these fungi was constructed from a multiple alignment of the deduced amino acid sequences. The tree showed that the enzymes were divided into two main groups.     

Selection, trans-species polymorphism, and locus identification of major histocompatibility complex class IIβ alleles of New World ranid frogs

Genes encoded by the major histocompatibility complex (MHC) play key roles in the vertebrate immune system. However, our understanding of the evolutionary processes and underlying genetic mechanisms shaping these genes is limited in many taxa, including amphibians, a group currently impacted by emerging infectious diseases. To further elucidate the evolution of the MHC in frogs (anurans) and develop tools for population genetics, we surveyed allelic diversity of the MHC class II β1 domain in both genomic and complementary DNA of seven New World species in the genus Rana (Lithobates). To assign locus affiliation to our alleles, we used a “gene walking” technique to obtain intron 2 sequences that flanked MHC class IIβ exon 2. Two distinct intron sequences were recovered, suggesting the presence of at least two class IIβ loci in Rana. We designed a primer pair that successfully amplified an orthologous locus from all seven Rana species. In total, we recovered 13 alleles and documented trans-species polymorphism for four of the alleles. We also found quantitative evidence of selection acting on amino acid residues that are putatively involved in peptide binding and structural stability of the β1 domain of anurans. Our results indicated that primer mismatch can result in polymerase chain reaction (PCR) bias, which influences the number of alleles that are recovered. Using a single locus may minimize PCR bias caused by primer mismatch, and the gene walking technique was an effective approach for generating single-copy orthologous markers necessary for future studies of MHC allelic variation in natural amphibian populations.     

cDNA cloning, characterization and expression analysis of peroxiredoxin 5 gene in the ridgetail white prawn Exopalaemon carinicauda

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species. In this study, a full-length of peroxiredoxin 5 (designated EcPrx5) cDNA was cloned from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of the EcPrx5 was of 827 bp, containing a 5′ untranslated region (UTR) of 14 bp, a 3′ UTR of 228 bp with a poly (A) tail, and an open reading frame of 585 bp encoding a polypeptide of 194 amino acids with the predicted molecular weight of 20.83 kDa and estimated isoelectric point of 7.62. BLAST analysis revealed that amino acids of EcPrx5 shared 89, 68, 66, 65, 53 and 51 % identity with that of Macrobrachium rosenbergii, Megachile rotundata, Harpegnathos saltator, Acromyrmex echinatior, Danio rerio, and Homo sapiens counterparts, respectively. The conserved Prx domain and the signature of peroxiredoxin catalytic center identified in EcPrx5 suggested that EcPrx5 belonged to the atypical 2-Cys Prx subgroup. Real time quantitative RT-PCR analysis indicated that EcPrx5 could be detected in all the tested tissues with highest expression level in hepatopancreas. As time progressed, the expression level of EcPrx5 both in hemocytes and hepatopancreas increased in the first 6 h after Vibrio anguillarum and white spot syndrome virus challenge, and showed different expression profiles. The results indicated that EcPrx5 involved in immune response against bacterial and viral infection in E. carinicauda.     

凹耳蛙MHCII类B基因第二外显子多态性分析

两栖类正经历全球范围内的种群衰退,很多两栖动物集群灭绝事件与环境病原体(如壶菌(Batrachochytrium dendrobatidis)的侵扰有关。MHC基因的表达产物在有颌脊椎动物免疫应答过程中起关键作用,其多态性通常与动物对疾病的抗性或易感性密切相关,因而被认为是研究动物适应性进化的最佳候选基因之一。本文对中国特有的无尾两栖动物凹耳蛙(Odorrana tormota)MHC II类B基因多态性进行初步研究。首先,利用1对通用引物扩增出凹耳蛙MHC II类B基因exon2长约180bp的DNA片段。在此基础上,利用ligation-mediated PCR进一步获取侧翼未知序列,序列拼接后长2,030bp,包含exon2以及intron1和intron2的部分序列。基于上述序列设计出凹耳蛙B基因exon2特异性引物(IIQ1BU/IIQ1BD),对该物种黄山种群32个样品进行PCR扩增和克隆测序,共获得34个不同的等位基因,等位基因序列核苷酸和氨基酸变异位点的比例分别为16.17%(33/204)和26.87%(18/67),大多数氨基酸变异位点位于推测的抗原结合位点(antigen binding sites,ABS)。每个样品包含2-5个等位基因,结合等位基因序列特征以及cDNA表达分析结果,推测凹耳蛙至少拥有3个可表达的B基因座位。与文献报道的蛙科其他物种比较后发现,尽管凹耳蛙目前的分布区非常狭窄,但其MHC II类B基因多态性明显高于蛙科其他动物。等位基因碱基替换模式提示凹耳蛙MHC II类B基因曾经历过强烈的正选择作用,ABS区的dN值显著大于dS(P<0.05),PAML软件包CODEML程序中不同模型的似然比检测(likelihood rate test)结果同样支持上述推论,贝叶斯经验贝叶斯路径(Bayesian Em-pirical Bayes)共检测出5个显著受正选择作用的氨基酸位点。贝叶斯系统树的拓扑结构显示,无尾两栖类不同科的等位基因分别形成单系群,但蛙科不同属的等位基因未能形成单系群,蛙属绿池蛙(Rana clamitans)的1个等位基因与臭蛙属凹耳蛙的部分等位基因享有共同的谱系关系,提示蛙科不同属间的B基因存在跨种多态性。     

Cloning, characterization and impact of up- and down-regulating subabul cinnamyl alcohol dehydrogenase (CAD) gene on plant growth and lignin profiles in transgenic tobacco

Both cDNA including 5′UTR and 3′UTR and genomic clones of cinnamyl alcohol dehydrogenase (CAD) were isolated and characterized from a pulp-yielding leguminous tree Leucaena leucocephala (LlCAD1). The deduced amino acid sequence shared high identity with orthologous sequences of Acacia mangium?×?Acacia auriculiformis (83%), Medicago sativa (83%), Nicotiana tabaccum (83%) and Aralia cordata (81%). Full length cDNA contained 78 bases of 5′UTR and 283 bases of 3′UTR, while the genomic clone contained 5 exons and 4 introns. Western blot analysis revealed elevated expression of LlCAD1 in seedling roots and shoots compared to leaves. Sense and antisense CAD tobacco transgenics showed increased and reduced CAD activity accompanied by a change in monomeric lignin composition. Histochemical staining of lignin in down-regulated plants suggested an increase in aldehyde units and a decrease in S/G ratio. Down-regulation of CAD resulted in accumulation of syringic, ferulic, p-coumaric and sinapic acids compared to untransformed controls. These observations were validated by anatomical studies of down-regulated transgenic stems which showed thin walled, elongated phloem and xylem fibres, accompanied by a reduction in the density of vessel elements and amount of secondary xylem when compared to untransformed plants. Furthermore, Klason lignin analysis of CAD antisense transgenics showed 7–32% reduced lignin and normal phenotype as compared to untransformed plants. Such a reduction was not noticed in up-regulated transgenics. These results demonstrate a unique opportunity to explore the significant role that down-regulation of CAD gene plays in reducing lignin content thereby offering potential benefits to the pulp and paper industry.     

中华鲟促性腺激素释放激素受体基因cDNA序列的克隆及表达分析

为了研究中华鲟(Acipenser sinensis)促性腺激素释放激素受体(Gonadotropin-releasing hormone receptor, GnRH-R)基因在中华鲟中的组织表达特征, 为中华鲟生长发育调控研究提供基础数据, 通过构建中华鲟(Acipenser sinensis)垂体的SMART cDNA质粒文库, 采用cDNA末端快速扩增(RACE), 克隆得到了中华鲟GnRH-R基因的cDNA全长序列。该序列全长1530 bp, 有478 bp的5′非翻译区, 579 bp的开放阅读框和473 bp的3′非翻译区, 共编码192个氨基酸, 其成熟多肽含有5个Ｎ连糖基化位点。通过和已知其他鱼类的GnRH-R基因进行氨基酸序列多重比对, 发现其与真鲷(Pagrus major)的同源性最高, 为76%, 与米氏叶吻银鲛(Callorhinchus milii)的同源性最低, 为39%。采用实时荧光定量PCR (Real time PCR)方法, 检测了GnRH-R的mRNA在中华鲟心、肝、脾、肾、肠道、精巢、肌肉及脑组织中的表达状况, 发现其在精巢中大量转录, 而在其他组织中则表达微弱。以上结果表明中华鲟GnRH-R基因在性腺发育特别是精子发生过程中可能起重要作用。     

Comprehensive analysis of medaka major histocompatibility complex (MHC) class II genes: Implications for evolution in teleosts

The major histocompatibility complex (MHC) class II molecules play central roles in adaptive immunity by regulating immune response via the activation of CD4 T cells. The full complement of the MHC class II genes has been elucidated only in mammalian species to date. To understand the evolution of these genes, we performed their first comprehensive analysis in nonmammalian species using a teleost, medaka (Oryzias latipes). Based on a database search, cDNA cloning, and genomic PCR, medaka was shown to possess five pairs of expressed class II genes, comprising one IIA and one IIB gene. Each pair was located on a different chromosome and was not linked to the class I genes. Only one pair showed a high degree of polymorphism and was considered to be classical class II genes, whereas the other four pairs were nonclassical. Phylogenetic analysis of all medaka class II genes and most reported teleost class II genes revealed that the IIA and IIB genes formed separate clades, each containing three well-corresponding lineages. One lineage contained three medaka genes and all known classical class II genes of Ostariophysi and Euteleostei and was presumed to be an original lineage of the teleost MHC class II genes. The other two lineages contained one nonclassical medaka gene each and some Euteleostei genes. These results indicate that multiple lineages of the teleost MHC class II genes have been conserved for hundreds of millions of years and that the tightly linked IIA and IIB genes have undergone concerted evolution.