Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 （HSC70） in Fenneropenaeus chinensis

The cDNA encoding hsc70 of Chinese shrimp Fenneropenaeus chinensis was cloned from hepatopancreas by RT-PCR based on its EST sequence. The full length cDNA of 2090 bp contained an open reading frame of 1956 nucleotides and partial 5‘- and 3‘-untranslated region(5‘- and 3‘-UTR). PCR amplification and sequencing analysis showed the existence of introns in the region of 1--547 bp, but they did not exist in the region of 548--2090 bp of hsc70 cDNA. When the deduced 652 amino acid sequence of HSC70 was compared with the members of HSP70 family from other organisms, the results showed 85.9% similarity with HSC71 from Oncorhynchus mykiss and HSC70 from Homo sapiens. It also exhibited 85.8% similarity with HSP70 from Mus musculu and 85.4% with HSC70 from Manduca sexta. Expression analysis showed that hsc70 mRNA was espressed constitutively in hepatopancreas, muscle, eyestalks, haemocytes, heart, ovary, intestine and gills in Fenneropenaeus chinensis. No difference could be detected on hsc70 mRNA level in muscle between heat-shocked and control animals.     

Tissue expression, association analysis between three novel SNPs of the RXRα gene and growth traits in Chinese indigenous cattle

Retinoid X receptor(RXR) α is a member of the nuclear hormone receptor superfamily that mediates the biological effects on several hormones,vitamins,and regulates lipid,glucose and energy metabolism.In this study,the tissue expression profiles of the bovine RXRαgene and association analysis of single nucleotide polymorphisms(SNPs) with growth traits were carried out in 413 Chinese native cattle.The expression profile was analysed in ten Jiaxian cattle tissues by real-time PCR,and the results showed that RXRαgene was abundantly expressed in adipose tissue and spleen,moderately expressed in heart,liver,lung,kidney,muscle and testis.Meanwhile,three SNPs(T27919A,T28139C and G28142A) and five haplotypes were identified.Haplotype with TTG was dominant with frequency of 69.1%.Chi-square test showed all populations were in Hardy-Weinberg equilibrium(P>0.05) at the three sites except Jiaxian cattle at G28142A site and Qinchuan cattle at T27919A site.Statistical analysis of combined sites showed that the individuals with TTGA genotype had significantly higher heart girth than those with TAGG genotype(P<0.05) and the animals with AAGA genotype had higher body weight than those with TAGG genotype(P<0.05) in T27919A-G28142A site.Heart girth,abdominal circumference and body weight of individuals with TCAG genotype were exceedingly higher than those with TTGG(P<0.01),TTGA and TCGG(P<0.05) in T28139C-G28142A site.For T27919A-T28139C site,the individuals of TCTA and TCTT genotype had significantly higher heart girth and lower height at hip cross than those with TTTA(P<0.05),and the body weight of TCAA and TCTT genotype individuals was higher than those with TTTA(P<0.05).In conclusion,these results provided evidence that the polymorphisms of RXRαgene were associated with growth traits and might apply to Chinese indigenous yellow cattle breeding program as a possible candidate for marker-assisted selection(MAS).     

Alternative splicing and expression of the insulin-like growth factor （IGF-1） gene in osteoblasts under mechanical stretch

Insulin-like growth factor 1 (IGF-1) promotes osteoblasts differentiation and bone formation, and its expression is induced by mechanical stretch, thus IGF-1 has been considered an effector molecule that links mechanical stimulation and local tissue responses. In this study, a mechanical stretching device was designed to apply physiological level static or cyclic stretching stimulation to osteoblasts. Different isoforms of IGF-1 mRNA were amplified by RT-PCR from the cells using respective primers and these amplified products were sequenced. An iso-form of IGF-1 splicing product was found to be selectively produced by osteoblasts under stretching stimulation. This IGF-1 isoform had identical sequence with the mechano growth factor (MGF) which was originally identified in muscle cells. Regulations of the expression of the liver-type IGF (L.IGF-1) and MGF in osteoblasts under stretch stimulation were further studied using semi-quantitative RT-PCR. Stretch stimulation was found to promot the expression of IGF-1 (L.IGF-1 and MGF), and for both isoforms expression was more effectively stimulated by cyclic stretch than static stretch. MGF was detected only in osteoblasts subjected to mechanical stretch, suggesting MGF was a stretch sensitive growth factor. Expression of MGF peaked earlier than that of L.IGF-1, which was similar to their regulation in muscle and suggested similar roles of MGF and L.IGF-1 in bone as in muscle cells. The functions of MGF and LIGF-1 in osteoblasts shall be established by further experimental studies.     

Seasonal variation of gut Cyanophyta contents and liver GST expression of mud carp （Cirrhina molitorella） and Nile tilapia （Oreochromis niloticus） in the tropical Xiangang Reservoir （Huizhou, China）

Liver glutathione S-transferase(GST) plays a major role in the detoxification of microcystins(MCs) via conjugation to glutathione(GSH).We evaluated the relationship between seasonal variation in fish gut contents and the expression of GST isoforms in mud carp(Cirrhina molitorella) and Nile tilapia(Oreochromis niloticus).We quantified the abundance and diversity of plankton in the water column and foregut of mud carp and Nile tilapia in the tropical Xiangang Reservoir between October 2007 and July 2008.The mRNA expression of 7 liver GST isoforms was determined by real-time RT-PCR.The gut contents of both species were dependent on the amount and type of phytoplankton and zooplankton in the water.The expression of liver GST genes in Nile tilapia and mud carp was positively and negatively correlated,respectively,with the abundance of toxic cyanobacteria in the fore-gut.The expression of liver GST mRNA was correlated to the abundance of toxic cyanobacteria in the gut contents of both species,suggesting that mRNA expression of GST isoforms could be used as a biomarker in Nile tilapia and mud carp to monitor cyanobacteria blooms in reservoirs.     

Inclusion of the Eastern Asia endemic genus Sorolepidium in Polystichum （Dryopteridaceae）： Evidence from the chloroplast rbcL gene and morphological characteristics

The taxonomic status of the Eastern Asia endemic Sorolepidium is controversial. Some authors accept it as member of the large diverse genus Polystichum, whereas others suggest that it is an independent genus separated from the later by the exindusiate sorus and the absence of aristate teeth at the pinnae margins. Here we infer phylogenetic relationship of Sorolepidium using DNA sequences of the chloro-plast rbcL gene. Phylogenies were inferred using maximum-parsimony, maximum-likelihood, and Bayesian inference methods. Molecular data establish that Sorolepidium is deeply nested within the large genus Polystichum and has a close relationship with P. duthiei and P. lachenense in the model-based analyses. The Kimura 2-parameter distances of the rbcL sequences between S. glaciale and P. duthiei and P. lachenense were 0.1 and 0.2%, respectively. Furthermore, S. glaciale differed from P. duthiei by a single nucleotide in their rbcL sequences. Close relationships between S. glaciale and P. duthiei and P. lachenense are also supported by the shared spore ornamentation with echinate fenes-trate folds.     

The composition and distribution of metal clusters in the MoFe protein from a nifZ deletion strain （DJ 194） of Azotobacter vinelandii

Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (△nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194).The results of Western blotting after anoxic native electrophoresis and SDS-PAGE showed that △nifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Furthermore, △nifZ Avl was also similar to OP Av1 at the molybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (△ε) of circular dichroism (CD)at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the △nifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and substrate (C2H2, H^ and N2)-reduction activity of △nifZ Av1 were 74% and 46%-50% of those of OP Av1, respectively. Furthermore, the △ε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between △nifZ Avl and OP Av1 resulted from P-cluster rather than FeMoco, and from the half number of P-cluster in △nifZ Av1, but the composition or redoxstate of P-cluster in △nifZ Av1 were not changed. Thus it could propose that △nifZ Av1 is composed of two different αβsubunit pairs. One is a FeMoco-and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-containing pair. Since the deletion of nifZ gene leads to the deficiency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.     

高原鼢鼠肌肉脂肪酸分析

采用气相色谱和质谱联用分析高原鼢鼠肌肉脂溶性物质,共确定31种脂肪酸。其主要成分有异十六碳酸(23．4％),十六碳酸(21．69％),十八碳二烯(9,12)酸(14．97％),十八碳烯(9)酸(12．58％),十八碳烯(11)酸(8．7％),十八碳酸(8．83％)等。另外,高原鼢鼠肌肉脂溶性物质含有一些特殊脂肪酸成分,其特殊性在于奇数碳原子脂肪酸和异构脂肪酸种类较多,反式油酸比例很高(21．28％),并含有十八碳二烯(8,11)酸(0．18％)和十八碳二烯(9,11)酸(0．07％)等。     

高原鼢鼠油脂中油酸含量定量分析

用气象色谱和气象色谱-质谱联用方法对高原鼢鼠油脂中油酸含量进行了定量分析,并对方法的准确度进行了检验。结果发现,高原鼢鼠油脂中油酸含量为18.57%±0.43%,皂化和酯化最佳时间分别为15min和7min,油酸甲酯标准曲线回归系数r=0.9999,油酸甲酯色谱峰面积的相对偏差为0.0171,样品中油酸甲酯回收率为94.08%。     

高原鼢鼠肌肉脂溶性物质对大鼠组织脂肪酸的影响

为了探讨高原鼢鼠肌肉脂溶性物质的抗缺氧机制,将20只SD大鼠随机分为高剂量组、低剂量组和对照组,分别给予10%和1%高原鼢鼠肌肉脂溶性物质以及蒸馏水20g/(kg·d),连续15d。试验结束后,取各组动物的骨骼肌、心肌和肝组织,提取其中脂肪酸,用气相色谱和气相色谱-质谱联用分析各组织脂肪酸含量。结果发现,高原鼢鼠肌肉脂溶性物质能显著提高SD大鼠骨骼肌和肝组织中不饱和脂肪酸的含量,但对心肌组织中不饱和脂肪酸具有降低作用。     

高原鼢鼠油脂中亚油酸含量定量分析

用气象色谱方法对高原鼢鼠油脂中亚油酸含量进行了定量分析,并对方法的准确度进行了检验。结果发现:高原鼢鼠油脂中亚油酸含量为10.84%±0.38%,皂化和酯化最佳时间分别为15min和7min,亚油酸甲酯标准曲线回归系数r=0.9999,亚油酸甲酯色谱峰面积的相对偏差为0.0124,样品亚油酸甲酯回收率为95.08%。     

Molecular cloning of a full-length cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1-25 amino acid sequence is a predicted signal peptide and the other 26-216 amino acid sequence is a mature peptide. The 26-45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca2+- dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

cDNA cloning and expression of earthworm fibrinolytic enzyme component A

Earthworm fibrinolytic enzyme component A(EFEa),a protein with dual fibrinolytic activity ,is one of the major therapeutically important earthworm fibrinoltic enzyme components .The cDNA fragment encoded the mature protein was cloned from earthworm (Eisenia fetida )by the RT-PCR technique,The deduced amino acid sequence of the EFE component A show high homology with some members of serine proteases trypsin family,and the amino acid residues constituting the active sites are conserved in the EFEa as compared with the other proteins of the trypsin family ,The cDNA fragment was subcloned into the expression vector pQE31 and pMAL-c2X of E.coli.The resulting expression plasmids,pQE-efea and pMAL-efea ,were used to transform the E.coli strain M15.Recombinant protein bands corresponding with calcuated molecular witht were induced .The induced His6-EFEa fusion protein with pQE-efea was accumulated into inclusion body ,while the induced MBP-EFEa fusion protein with pMAL-efea was soluble and showed fibrinoloytic activities.     

高原鼢鼠心肌和骨骼肌肌红蛋白含量的季节变化

为了研究高原鼢鼠肌红蛋白在不同季节的变化,用分光光度法测定了肌红蛋白含量。结果显示:在春季、夏季和秋季,高原鼢鼠心肌肌红蛋白含量分别为1 001.25±103.25(nmol/g),726.50±54.70(nmol/g),767.33±88.73(nmol/g);骨骼肌肌红蛋白含量分别为781.85±100.98(nmol/g),672.15±108.88(nmol/g),676.80±77.31(nmol/g)。春季高原鼢鼠心肌肌红蛋白和骨骼肌肌红蛋白含量显著高于夏季和秋季(P<0.05)。说明随着洞道氧气和二氧化碳浓度的季节性变化,高原鼢鼠通过改变肌红蛋白含量,来维持机体的氧平衡。     

Cloning and expression analysis of MBLL cDNA

The mbl (muscleblind) gene of Drosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation of mbl gene will disturb the differentiation of all the Drosophila's photoreceptors. Primers have been designed according to human EST086139, which is highly homologous to mbl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designated MBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology to Drosophila's mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show that MBLL is a widely expressed gene, but the expression amounts differ in these tissues.