野生与笼养绿孔雀种群的随机扩增多态DNA研究

利用随机扩增多态DNA（RAPD）技术对野生14只和笼养18只绿孔雀（Pavo muticus）个体进行了种群遗传多样性分析。用23个随机引物,野生与笼养绿孔雀分别获得161和166个扩增片段,计算发现野生与笼养绿孔雀的种群内平均相对遗传距离分别是0.0555和0.1355,两种群间的为0.1635;两种群的Shannon多样性指数平均分别是0.4348和1.0163,有显著性差异。以上分析都显示野生绿孔雀的遗传多样性很低。用UPGMA法聚类显示两个种群都是分别来源于两个家系,可据此进行繁育管理。 Abstract:Random-amplified polymorphic DNA(RAPD) was used to investigate the genetic diversity of the population of 14 wild green peafowl and 18 captive green peafowl(pavo muticus).Total of 161 and 166 bands were obtained respectively,and 23 random primers were used to amplify the genomic DNA of the wild and captive green peafowls.The average relative hereditary distance of the wild and captive green peafowls is 0.0555 and 0.1355 respectively;and the Shannon diversity index is 0.4348 and 1.0163 respectively.There is a prominent differentia between the two populations by T-Test of HO.All the analyses above show that the genetic diversity is very low in wild green peafowl.It tells us that the two populations come from two families by using UPGMA,which can be useful in the breeding management in the future.     

cDNA cloning of two novel T-superfamily conotoxins from Conus leopardus

The full-length cDNAs of two novel T-superfamily conotoxins,Lp5.1 and Lp5.2,were clonedfrom a vermivorous cone snail Conus leopardus using 3'/5'-rapid amplification of cDNA ends.The cDNA ofLp5.1 encodes a precursor of 65 residues,including a 22-residue signal peptide,a 28-residue propeptide anda 15-residue mature peptide.Lp5.1 is processed at the common signal site -X-Arg- immediately before themature peptide sequences.In the case of Lp5.2,the precursor includes a 25-residue signal peptide anda 43-residue sequence comprising the propeptide and mature peptide,which is probably cleaved to yield a29-residue propeptide and a 14-residue mature toxin.Although these two conotoxins share a similar signalsequence and a conserved disulfide pattern with the known T-superfamily,the pro-region and mature peptidesare of low identity,especially Lp5.2 with an identity as low as 10.7% compared with the reference Mr5.1a.The elucidated cDNAs of these two toxins will facilitate a better understanding of the species distribution,the sequence diversity of T-superfamily conotoxins,the special gene structure and the evolution of thesepeptides.     

Expressing an (E)-β-farnesene synthase in the chloroplast of tobacco affects the preference of green peach aphid and its parasitoid

(E)-β-Famesene(EβF) synthase catalyses the production of EβF,which for many aphids is the main or only component of the alarm pheromone causing the repellence of aphids and also functions as a kairomone for aphids' natural enemies.Many plants possess EβF synthase genes and can release EβF to repel aphids.In order to effectively recruit the plant-derived EβF synthase genes for aphid control,by using chloroplast transit peptide(CTP) of the small subunit of Rubisco(rbcS) from wheat(Triticum aestivum L.),we targeted AαβFS1,an EβF synthase gene from sweet wormwood(Artemisia annua L),to the chloroplast of tobacco to generate CTP + AαβFS1 transgenic lines.The CTP +AαβFS1 transgenic tobacco plants could emit EβF at a level up to 19.25 ng/day per g fresh tissues,4-12 fold higher than the AαβFS1 transgenic lines without chloroplast targeting.Furthermore,aphid/parasitoid behavioral bioassays demonstrated that the CTP + AαβFS1 transgenic tobacco showed enhanced repellence to green peach aphid(Myzus persicae) and attracted response of its parasitoid Diaeretiella rapae,thus affecting aphid infestation at two trophic levels.These data suggest that the chloroplast is an ideal subcellular compartment for metabolic engineering of plant-derived EβF synthase genes to generate a novel type of transgenic plant emitting an alarm pheromone for aphid control.     

家蝇防御素基因的cDNA克隆及序列分析

Defensin is a kind of cationic.inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms.with their broad antimicrobial spectrum and strong pharmaceutical effects.antimicrobial peptides,including defensins,represent a source of novel antibiotic agents.A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDnA library of houseflies(Musca domestica) that had been challenged by E.coli and staphylococcus taincd an NH2-terminal signal sequence(1-22)followed by a propeptide and the mature peptide(53-92),The sequence identity with other insect defensin is between 51% and 73%.The mature peptide,with a predicted molecular weight of 4.0kDa,and pI of 8.69,has 1 negative charged amino acid and 4 positice ones,the putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds,Cys1-Cys4,Cys2-Cys5 and Cys3-Cys6,These results suggest that the novel full-length cDNA of the defensin gene.Denominated Mdde,has been successfully cloned from houseflies.     

Physiological evidence of integrin-antibody reactive proteins influencing the innate cellular immune responses of larval Galleria mellonella hemocytes

Larval Galleria melonella(L.)hemocytes form microaggregates in response to stimulation by Gram-positive bacteria Hemocyte adhesion to foreign materials is mediated by the CAMP/protein kinase A pathway and the B-subunit of cholera toxin using a cAMP-independent mechanism.Cholera toxin-induced microaggregation was inhibited by the integrin inhibitory RGDS peptide,implying integrins may be part of the mechanism.Based on the types of mammalian integrin-antibody reactive proteins affecting hemocyte adhesion and bacterial-induced responses ars,ory,Ai,and B3 subunits occred on both granular cell and plasmatocyte hemocyte subtypes.A fluorescent band representing the binding of rabbit as-integrin subunit antibodies occurred between adhering heterotypic hemocytes.The frequency of the bands was increased by cholera toxin.The as andβrabbit integrin subunit antibodies inhibited removal of Bacillus subtilis(Cohn)from the hemolymph in vivo,A as ir-specific synthetic peptide blocker similarly diminished hemocyte function whereas the 0v Bs-specific inhibitory peptide and the corresponding integrin subunit antibodies did not influence nonself hemocyte activities.Western blots revealed several proteins reacting with a given integrin-antibody subtype.Thus integrin-antibody reactive proteins(which may include integrins)with possible as and B epitopes modulate immediate hemocyte function.Confocal microscopy established plasmatocyte adhesion to and rosetting over substrata followved by granular cell microaggregate adhesion to plasmatocytes during early stage nodulation.     

Characterization of a novel α4/4-conotoxin,Qcl.2, from vermivorous Conus quercinus

As part of continuing studies of the identification of gene organization and cloning of novel α-conotoxins, the first α4/4-conotoxin identified in a vermivorous Conus species, designated Qcl.2, was originally obtained by cDNA and genomic DNA cloning from Conus quercinus collected in the South China Sea. The predicted mature toxin of Qc1.2 contains 14 amino acid residues with two disulfide bonds （Ⅰ-Ⅲ, Ⅱ-Ⅳ connectivity） in a native globular configuration. The mature peptide of Qcl.2 is supposed to contain an N-terminal post-translationally processed pyroglutamate residue and a free carboxyl C-terminus. This peptide was chemically synthesized and refolded for further characterization of its functional properties. The synthetic Qcl.2 has two interconvertible conformations in aqueous solution, which may be due to the cis-trans isomerization of the two successive Pro residues in its first Cys loop. Using the Xenopus oocyte heterologous expression system, Qcl.2 was shown to selectively inhibit both rat neuronal α3β2 and α3β4 subtypes of nicotinic acetylcholine receptors with low potency. A block of -63% and 37% of the ACh-evoked currents was observed, respectively, and the toxin dissociated rapidly from the receptors. Compared with other characterized α-conotoxin members, the unusual structural features in Qcl.2 that confer to its receptor recognition profile are addressed.     

林麝、马麝及梅花鹿活化素基因βA亚基成熟肽序列的克隆和分析

活化素(Activin)对睾丸和卵巢产生多重调节作用,对动物的繁育非常关键。本文参考已克隆的其它物种的活化素基因βA亚基成熟肽序列,设计一对简并引物,通过PCR方法从林麝(Moschus berezovskii)、马麝(Moschus chrysogaster)和梅花鹿(Cervus nippon)的基因组DNA中直接扩增目的基因片段。将目的片段分别克隆到pMD1l8-T载体中,并进行序列测定。DNA序列测定及分析表明,林麝、马麝和梅花鹿的活化素基因βA亚基成熟肽序列长345bp,三物种核苷酸序列同源性在98％以上,氨基酸序列同源性在99％以上,核酸限制性酶切图谱也高度相似。将它们与GenBank中已公布的其它物种的活化素序列进行比较,发现该片段在不同进化程度的物种间高度保守。这是首次从麝科动物中成功克隆与生殖相关的核基因,对麝的人工繁育及麝资源的保护利用提供了相关基础资料。     

中华眼镜蛇短链神经毒素cDNA的克隆及在大肠杆菌中的表达

利用RT-PCR 技术从中华眼镜蛇毒腺组织中成功地克隆了短链神经毒素cDNA。测序结果表明,该基因开放阅读框架编码83个氨基酸残基,其中21个为信号肽,成熟肽为62个氨基酸残基。该基因与GenBank 报道的相同物种的神经毒素基因有相当的同源性,不同物种之间的信号肽序列十分保守。将短链神经毒素cDNA再经PCR扩增除去信号肽序列,克隆到pT7ZZ表达质粒中,转化E. coli BL21(DE3)后,经IPTG诱导可高效表达分子量为23kDa②左右的融合蛋白。表达产物占菌体总蛋白的25%左右。 Abstract:A novel short-chain neurotoxin cDNA was cloned from Chinese cobra venom by RT-PCR. The cDNA was cloned into the pGEM-T vector and sequenced. It has a ORF encoding 83 amino acid residues and a 21 residues signal peptide. This neurotoxin gene of Chinese cobra was highly homogeneous to the short-chain neurotoxin gene of similar species reported in GenBank. Among the genes of neurotoxin from different species, the signal peptides were very conserved. The cDNA encoding the mature peptide was amplified by PCR and was cloned into pT7ZZ vector. The recombinant vector was transformed into E. coliBL2(DE3). The E. coli highly expressed the fusion protein whose mollecular weight is 23kDa, after induced by 0.1 mol/L IPTG. The expressed protein was accumulated up to more than 25% of total bacterial protein.     

蚕豆豆类胰岛素基因的cDNA克隆及序列分析

为探明豆科植物中豆类胰岛素基因的结构特征与进化关系,在已获得大豆豆类胰岛素基因的基础上,以蚕豆种子胚根mRNA为材料,采用RT-PCR技术,克隆了蚕豆豆类胰岛素基因的cDNA序列,编码的前体多肽包括信号肽、成熟型豆类胰岛素及另一多肽的45个氨基酸残基。DNA序列分析表明,克隆片段与大豆和豌豆的同源性分别为62.5%和58.7%。在氨基酸水平上分别具有44.2%和43.6%的同源性,其中存在着高度保守的半胱氨酸位点,它们在维持豆类胰岛素的空间结构与生理功能方面,可能具有重要的作用。 Abstract:In order to elucidate the relationship between the structural features of leginsulin gene in legume plants and their phylogenetic significance,we have cloned the cDNA sequence of leginsulin gene from radicles of broad bean (Vicia faba) via RT-PCR techniques according to the leginsulin gene sequence we previously obtained from soybean (Glycine max).The cloned cDNA encoded for a precursor protein consisting of the signal peptide,mature leginsulin and an additional 45 amino acids of another polypeptide.A sequence search for homology comparison revealed the cloned leginsulin cDNA fragment shares 62.5% and 58.7% similarity to soybean and pea,respectively.The results also shown that leginsulin cDNA from broad bean presents 44.2% and 43.6% amino acid sequence homology with soybean and pea (Pisum sativum),respectively,and that there exists highly conserved cysteine sites among the leginsulin cDNAs,which may play a crucial role in maintaining the three-dimensional structure and the physiological functions of leginsulin.     

Molecular cloning,expression, and anti-tumor activity of a novel serine protease from Arenicola cristata

Arenicola cristata, a marine annelid, is a wellknown and prized traditional Chinese medicine. However, the serine protease gene of A. cristata has not been cloned yet. In this study, a novel protease ofA. cristata was cloned, sequenced, and expressed in Escherichia coli, and the functions of this recombinant protease were also investigated. The whole complementary DNA （cDNA） of this novel protease was of 980 bp in length and consisted of an open reading frame of 861 bp encoding 286 aa. Sequence analysis of the deduced amino acid sequence revealed that the protease belongs to the serine protease family. The active enzyme of the pro posed A. cristata protease is composed of a signal peptide, a propeptide, and a mature polypeptide. The molecular weight of the recombinant mature protein was 26 kDa after overexpression in E. coli. The recombinant pro tein significantly inhibited cell growth and induced cell apoptosis of esophageal squamous cell carcinoma （ESCC） in vitro, and reduced tumorigenicity in vivo. Furthermore, administration of the recombinant protein led to the activa tion of caspase9 as well as downregulation of Mcl1 and Bcl2. Taken together, our findings indicated that the recom binant serine protease ofA. cristata could inhibit ESCC cell growth by mitochondrial apoptotic pathway and might act as a potential pharmacological agent for ESCC therapy.     

Characterization and Fine Mapping of a Novel Rice Narrow Leaf Mutant nal9

A narrow leaf mutant was isolated from transgenic rice （Oryza sativa L.） lines carrying a T-DNA insertion. The mutant is characterized by narrow leaves during its whole growth period, and was named nal9 （narrow leaf 9）. The mutant also has other phenotypes, such as light green leaves at the seedling stage, reduced plant height, a small panicle and increased tillering. Genetic analysis revealed that the mutation is controlled by a single recessive gene. A hygromycin resistance assay showed that the mutation was not caused by T-DNA insertion, so a map-based cloning strategy was employed to isolate the nal9 gene. The mutant individuals from the F2 generations of a cross between the nal9mutant and Longtepu were used for mapping. With 24 F2 mutants, the nal9 gene was preliminarily mapped near the marker RM156 on the chromosome 3. New INDEL markers were then designed based on the sequence differences between japonica and indica at the region near RM156. The nal9 gene was finally located in a 69.3 kb region between the markers V239B and V239G within BAC OJ1212_C05 by chromosome walking. Sequence and expression analysis showed that an ATP-dependent CIp protease proteolytic subunit gene （CIpP） was most likely to be the nal9 gene. Furthermore, the nal9 mutation was rescued by transformation of the CIpP cDNA driven by the 35S promoter. Accordingly, the CIpP gene was identified as the NAL9 gene. Our results provide a basis for functional studies of NAL9 in future work.     

人载脂蛋白AⅠ基因的克隆表达及功能

To identify, clone ,sequence and highly express the mature peptide gene of ApoA Ⅰ, total RNA was prepared from human fetal liver tissue. cDNA fragment encoding human ApoA Ⅰ was amplified by RT-PCR using specific primers, and then was inserted in pGEM-T vector. DNA sequencing indicates that the fragment is 729 base pairs in length and has 100% nucleotide homology with that of reported ApoA Ⅰ cDNA gene previously. The ApoA Ⅰ gene was cloned into pGEX 5X-1.The recombinant protein was expressed in E.coli DH5α, purified by glutathione-Sepharose 4B affinity chromatography and confirmed by SDS-PAGE. It was shown that the recombinant ApoA Ⅰ was expressed in E.coli, and the target protein amounted to 36% of total bacteria proteins. Cholesteryl ester transfer experiment showed that the recombinant ApoA Ⅰ was capable of promoting transfer of CE from HDL to LDL. Western blotting showed that the protein could react specifically with anti-ApoA Ⅰ antibodies.     

植物叶片蛋白质双向电泳技术的改进与优化

通过对传统的双向电泳方法进行改进与优化,得到了一种适合于分析植物叶 片蛋白质的双向电泳新方法。用改进后的方法对水稻不同时期成熟叶片蛋白质进行双向电泳分离,结果显示其稳定性和重复性好,分辨率较高,经考马斯亮蓝染色后可分辨出300多个蛋白质(肽)点。它们的等电点和分子量主要分布于pI4.1-8.2和10-100kDa之间。本文还就实验过程中出现的一些技术问题进行了讨论。 Abstract:Base on the method of O farrell,an improved procedure for the two-dimensional gel electrophoresis of leaf proteins is presented.The two-dimensional separation of proteins from the mature leaves of different developmental stages of rice provides distinct and steady results.The detected protein(peptide)spots stained by Coomassie brilliant blue R-250 are more than 300,with isoelectric points(pI)ranging from 4.1 to 8.2 and molecular weights(MW)from 10 kDa to 100 kDa.     

A ketoreductase gene from Streptomyces mycarofaciens 1748 DNA involved in biosynthesis of a spore pigment

An efficient plasmid transformation system for S. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S. mycarofaciens 1748, the gene disruption experiment was carried out. For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S. mycarofaciens 1748. This suggested that homologous recombination between plasmid-borne MKR gene sequence and the chromosome of S. mycarofaciens 1748 had occurred. A Southern hybridization experiment using α- P-labelled MKR gene as probe indicated that the desired integration event had occurred in the re-combinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S. mycarofa-ciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was in-cubated wit     

Reference genes identified in the silkworm Bombyx moil during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR

Gene expression quantification at mRNA level is very important for postgenomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real-time polymerase chain reaction （qRT-PCR）, require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome-wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT- PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 （represented by sw22934, sw14876, and sw13956） were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin （sw22671）, the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism.     

microRNAs in a multicellular green alga Volvox carteri

microRNAs(miRNAs)have emerged as key components in the eukaryotic gene regulatory network.We and others have previously identified many miRNAs in a unicellular green alga,Chlamydomonas reinhardtii.To investigate whether miRNA-mediated gene regulation is a general mechanism in green algae and how miRNAs have been evolved in the green algal lineage,we examined small RNAs in Volvox carteri,a multicellular species in the same family with Chlamydomonas reinhardtii.We identified 174 miRNAs in Volvox,with many of them being highly enriched in gonidia or somatic cells.The targets of the miRNAs were predicted and many of them were subjected to miRNA-mediated cleavage in vivo,suggesting that miRNAs play regulatory roles in the biology of green algae.Our catalog of miRNAs and their targets provides a resource for further studies on the evolution,biological functions,and genomic properties of miRNAs in green algae.     

The complete mitochondrial genome sequence of Geisha distinctissima （Hemiptera： Flatidae） and comparison with other hemipteran insects

The complete nucleotide sequence of the mitochondrial genome （mitogenome） of Geisha distinctissima （Hemiptera： Flatidae） has been determined in this study. The genome is a circular molecule of 15,971 bp with a total A＋T content of 75.1%. The gene content, order, and structure are consistent with the Drosophila yakuba genome structure and the hypothesized ancestral arthro- pod genome arrangement. All 13 protein-coding genes are observed to have a putative, inframe ATR methionine or ATT isoleucine codons as start signals. Canonical TAA and TAG termination codons are found in nine protein-coding genes, and the remaining four （cox1, atp6, cox3, and nad4） have incomplete termination codons. The anticodons of all transfer RNA （tRNAs） are identical to those observed in D. yakuba and Philaenus spumarius, and can be folded in the form of a typical clover-leaf structure except for tRNASer（AGN）. The major non-coding region （the A ＋ T-rich region or putative control region） between the small ribosomal subunit and the tRNAne gene includes two sets of repeat regions. The first repeat region consists of a direct 152-bp repetitive unit located near the srRNA gene end, and the second repeat region is composed of a direct repeat unit of 19 bp located toward tRNAIle gene. Comparisons of gene variability across the order suggest that the gene content and arrangement of G. distinctissima mitogenome are similar to other hemipteran insects.