Molecular characterization of plastidic phosphoserine aminotransferase in serine biosynthesis from Arabidopsis

Serine biosynthesis in plants proceeds by two pathways; a photorespiratory pathway which is associated with photorespiration and a pathway from phosphoglycerate. A cDNA encoding plastidic phosphoserine aminotransferase (PSAT) which catalyzes the formation of phosphoserine from phosphohydroxypyruvate has been isolated from Arabidopsis thaliana . Genomic DNA blot analysis indicated that this enzyme is most probably encoded by a single gene and is mapped on the lower arm of chromosome 4. The deduced protein contains an N-terminal extension exhibiting the general features of a plastidic transit peptide, which was confirmed by subcellular organelle localization using GFP (green flourescence protein). Northern analysis indicated preferential expression of PSAT in roots of light-grown plants, supporting the idea that the phosphorylated pathway may play an important role in supplying the serine requirement of plants in non-green tissues. In situ hybridization analysis of PSAT revealed that the gene is generally expressed in all types of cells with a significantly higher amount in the meristem tissue of root tips.     

柞蚕Kazal型丝氨酸蛋白酶抑制剂ApKTSPI基因克隆及表达特征分析

【目的】克隆柞蚕(Antheraea pernyi)Kazal型丝氨酸蛋白酶抑制剂(ApKTSPI)基因的cDNA序列并进行序列分析,研究ApKTSPI基因的组织表达分布及病原物免疫刺激后的表达模式,原核表达ApKTSPI。【方法】利用RACE-PCR方法扩增柞蚕ApKTSPI基因全长cDNA,生物信息学软件进行序列分析,利用实时定量PCR检测柞蚕ApKTSPI基因的组织分布及免疫刺激后的表达模式,利用pET-28a载体在大肠杆菌BL21中融合表达ApKTSPI。【结果】柞蚕ApKTSPI基因的cDNA全长568 bp,开放阅读框编码96个氨基酸,含一个Kazal结构域。ApKTSPI基因在柞蚕5龄幼虫脂肪体中特异性高表达,在核型多角体病毒、大肠杆菌和白僵菌免疫刺激后表达量都能上调,但上调的程度和时间都不同。ApKTSPI在大肠杆菌中成功诱导表达。【结论】获得了柞蚕ApKTSPI基因的cDNA全长,并研究了ApKTSPI基因的表达模式,为进一步研究其在柞蚕免疫中的功能及作用机理奠定了基础。     

Molecular characterization of the sans fille gene in Antheraea pernyi: A highly conserved gene during the evolution of animals

Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B″, and plays an important role in sex determination in Drosophila melanogaster. In this study, the snf gene from Antheraea pernyi (Lepidoptera: Saturniidae), an economically important insect, was isolated and characterized. The obtained 925 bp cDNA sequence contains an open reading frame of 669 bp encoding a polypeptide of 222 amino acids, showing 78% sequence identity to that from D. melanogaster. A database search revealed that SNF protein homologs are present in many animals, including invertebrates and vertebrates, with more than 70% amino acid sequence identities, suggesting that they were highly conserved during the evolution of animals. Phylogenetic analysis revealed that A. pernyi SNF was closely related to Bombyx mori SNF. Quantitative real-time PCR (qRT-PCR) analysis showed that the A. pernyi snf gene was transcribed during five larval developmental stages, and in six tested tissues (ovaries, testes, silk glands, fat body, integument, and hemolymph), with the most abundance determined in the gonads (ovaries or testes). Investigation of expression changes throughout embryonic development indicated that A. pernyi snf mRNA was expressed at a low level from days 0 to 4, and reached a maximum level at day 10, but decreased to a low level before hatching. These results suggest that the product of the snf gene may play important roles in the development of A. pernyi.     

柞蚕Septin基因的克隆及表达分析

Septin蛋白参与了细胞分裂、细胞内物质转运、细胞周期调控及细胞凋亡等生理反应,并且发现与肿瘤发生、神经功能障碍和病原物侵染的过程直接相关。研究克隆了柞蚕septin基因全长,序列分析表明septin基因全长1904 bp,开放阅读框长度为1137 bp,编码378个氨基酸。多重序列比对分析表明柞蚕septin与家蚕及黑脉金斑蝶septin相似性最高,聚为一类。实时定量PCR结果表明柞蚕septin基因在各个组织中都有表达,但在血细胞中表达量最高,其次是表皮,而在丝腺中表达量最低。通过构建原核表达载体,柞蚕septin蛋白在大肠杆菌中成功诱导表达。     

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein from the Chinese oak Silkmoth, Antheraea pernyi.

In our research to identify gene involved in the cuticle protein, we cloned a novel cuticle protein gene, ApCP13, from the Chinese oak silkmoth, Antheraea pernyi, larvae cDNA library. The ApCP13 gene encodes a 120 amino acid polypeptide with a predicted molecular mass of 13 kDa and a pI of 4.01, and is intron-less gene. The ApCP13 contained a type-specific consensus sequence identifiable in other insect cuticle proteins and the deduced amino acid sequence of the ApCP13 cDNA is most homologous to another wild silkmoth, A. yamamai CP12 (86% protein sequence identity), followed by Bombyx mori LCP18 (35% protein sequence identity). Northern blot analysis revealed that the ApCP13 showed the epidermis-specific expression. This is the first report of cuticle protein gene in the wild silkmoth, A. pernyi.     

Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus polyhedrin gene and its variant forms

The segments 10 (S10) of the 11 double stranded RNA genomes from Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) encoding a novel polyhedrin polypeptide was converted to cDNA, cloned, and sequenced. Three cDNA clones consisting of 1502 (AmCPV10-1), 1120 (AmCPV10-2), and 1415 (AmCPV10-3) nucleotides encoding polyhedrin of 254, 339, and 319 amino acids with molecular masses of 29, 39, and 37 kDa, respectively, were obtained, and verified by Northern analysis. These clones showed 70-94% sequence identity among them but none with any sequences in databases. The expression of AmCPV10-1 cDNA encoded polyhedrin in Sf-9 cells was detected by immunoblot analysis and formation of polyhedra by electron microscopy, as observed in AmCPV-infected gut cells, but no expression of AmCPV10-2 or AmCPV10-3 cDNA was detected, indicating that during AmCPV replication, along with functional S10 RNA, some defective variant forms of S10 RNAs are packaged in virion particles.     

Molecular characterization of a 14-3-3 zeta gene from Plodia interpunctella: A potential marker for phylogenetic inference

The 14-3-3 proteins are a large family of approximately 30 kDa acidic proteins and acting in the regulation of many biological processes. In this study, a 14-3-3 zeta (Pi14-3-3z) gene from the Indian meal moth, Plodia interpunctella (Lepidoptera, Pyralidae) was isolated and characterized. The full-length cDNA of Pi14-3-3z is 1382 bp, including a 5'-untranslated region (UTR) of 141 bp, 3′-UTR of 497 bp and an open reading frame (ORF) of 744 bp encoding a polypeptide of 247 amino acids which contains a 14-3-3 homologues domain (PF00244). The deduced Pi14-3-3z protein sequence has 81%–100% identity with the homologues in comparison to with other individuals. qPCR analysis revealed that Pi14-3-3z was expressed at the four developmental stages and in all tissues tested. Based on the amino acid of 14-3-3z, phylogenetic analysis demonstrated a similar topology with the traditional classification, suggesting 14-3-3z protein has the potential value in phylogenetic inference.     

Accounting for gene rate heterogeneity in phylogenetic inference

Traditionally, phylogenetic analyses over many genes combine data into a contiguous block. Under this concatenated model, all genes are assumed to evolve at the same rate. However, it is clear that genes evolve at very different rates and that accounting for this rate heterogeneity is important if we are to accurately infer phylogenies from heterogeneous multigene data sets. There remain open questions regarding how best to incorporate gene rate parameters into phylogenetic models and which properties of real data correlate with improved fit over the concatenated model. In this study, two methods of accounting for gene rate heterogeneity are compared: the n-parameter method, which allows for each of the n gene partitions to have a gene rate parameter, and the alpha-parameter method, which fits a distribution to the gene rates. Results demonstrate that the n-parameter method is both computationally faster and in general provides a better fit over the concatenated model than the alpha-parameter method. Furthermore, improved model fit over the concatenated model is highly correlated with the presence of a gene with a slow relative rate of evolution.     

Molecular cloning,characterization and expression of cDNA encoding phosphoserine aminotransferase involved in phosphorylated pathway of serine biosynthesis from spinach

Phosphoserine aminotransferase (PSA) catalyzes the conversion of phosphohydroxypyruvate to phosphoserine in the phosphorylated pathway of serine biosynthesis. A cDNA clone encoding PSA was isolated from the cDNA library of spinach (Spinacia oleracea L.) green leaves. Determination of the nucleotide sequence revealed the presence of an open reading frame encoding 430 amino acids, exhibiting 38-50% homology with the amino acid sequences of bacterial, yeast and animal PSA. It contains an N-terminal extension of ca. 60 amino acids in addition to the sequences from other organisms. The general features of plastidic transit peptide are observed in this N-terminal sequence, suggesting the plastid localization of the PSA protein encoded by this cDNA. The bacterial expression of the cDNA could functionally rescue the auxotrophy of serine in the serC- mutant, Escherichia coli KL282. The enzymatic activity of PSA was demonstrated in vitro in the extracts of E. coli over-expressing the cDNA. Southern blot analysis indicated the presence of a couple of related genes (Psa) in the spinach genome. RNA blot hybridization suggested the preferential expression of the Psa gene in the roots of green seedlings and in the suspension cells cultured under a dark condition.     

微量添食对柞蚕蚁蚕抗逆性的影响

通过卵面微量添食葡萄糖、有益微生物 (EM)及CaCl2 3种物质 ,以半数致死时间 (LT50 )为指标 ,研究添食不同物质对柞蚕初孵蚁蚕抗逆性的影响。结果表明 ,卵面添食这 3种物质均能增强蚁蚕的抗低温能力 ,延长其在低温条件下的半数致死时间 (LT50 )。添食葡萄糖能显著延长蚁蚕对饥饿的LT50 ;添食EM能极显著延长蚁蚕对饥饿的LT50 ;添食CaCl2 对蚁蚕耐饥饿能力的影响不显著。各种添食物质对蚁蚕抗逆性的影响均存在浓度效应     

Three pheromone-binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyi.

Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone-binding proteins (PBPs) could be identified in pheromone-sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI-TOF MS of sensillum lymph droplets from pheromone-sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant-binding proteins, immunoreactivity was found only with an anti-(Apol PBP) serum. Free-flow IEF, ion-exchange chromatography and Western blot analyses revealed at least three anti-(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N-Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K. , Krieger, J. & Breer, H. (1989) FEBS Lett. 256, 2215-2218] and a new PBP having only 57% identity with this amino-acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N-terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin-labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta 1088, 277-284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)-6, 11-hexadecadienyl acetate (AC1) and the (E,Z)-6,11-hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H-labelled acetate, whereas no binding of the 3H-labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively.     

Interspecific hybrids of Antheraea roylei and A. pernyi — a cytogenetic reassessment

Summary A rare case of interspecific hybridization between the Indian oak feeding silkworm Antheraea roylei (n=31) and Chinese oak feeding silkworm A. pernyi (n=49) yielding fertile and vigorous offspring is reported. The F₁ and the backcross (A. roylei X A. pernyi X A. pernyi male individuals of the above cross and the F₂₃ and F₃₂ male offspring derived from an earlier cross between another race of A. roylei (n=30) and A. pernyi (n=49) were cytogenetically analysed in order to study their chromosome dynamics. The F₁ hybrids showed 18 trivalents and 13 bivalents in the first meiotic prophase and metaphase. The backcross individuals possessed either 9 trivalents and 31 bivalents or 49 bivalents, in Metaphase I cells. The F₂₃ and F₃₂ individuals were karyotypically alike and exhibited 49 bivalents. The following conclusions were drawn from the above observations: (a) in spite of allopatry and karyotypic divergence in number, a high degree of homology exists between the chromosomal complements of the two species; (b) A. pernyi possibly evolved from A. roylei, during the course of which 18 chromosomes of the latter underwent fission to give rise to the 36 chromosomes of the former. This is demonstrated by trivalent formation and pairing affinities in F₁ hybrids; (c) selection has favoured the elimination of large A. roylei chromosomes which participated in trivalent formation in successive generations of inbred hybrids to establish a stable Karyotype like that of A. pernyi.     

Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution

Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, ¹³C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol–gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were -helix and random coil in solution. There also existed a little β-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no -helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol–gel transformation. The velocity increased obviously when the temperature was above 30 °C. During the sol–gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to β-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.