草莓DNA甲基转移酶基因分离及表达探析

基于转基因技术发展,植物微繁殖技术的应用范围也进一步扩大,但微繁殖技术的不完善往往会使植物出现一些变异,目前这些遗传变异问题已成为植物转基因技术的重要研究方向。本文综述分析了草莓DNA甲基转移酶基因分离及表达研究和应用现状,DNA甲基化在植物基因组中普遍存在,它是一种表观修饰DNA的方法,对植物的生长发育起到促进的作用。DNA甲基转移酶有催化DNA甲基化过程的作用,DNA甲基转移酶对DNA甲基化过程及其植物的生长发育具有促进作用。

大鲵胸腺素β-4全长基因的分离与表达研究

【目的】对大鲵胸腺素β-4基因进行生物信息学分析和原核表达载体构建,为大鲵胸腺素β-4蛋白生理活性的深入研究奠定基础。【方法】从大鲵皮肤cDNA文库中分离获得了大鲵胸腺素β-4基因全长序列,采用多种生物软件对其进行生物信息学分析,用定量PCR研究胸腺素β-4基因在不同组织的表达情况,并以pET32a为载体构建该基因原核表达载体。【结果】胸腺素β-4基因全长共699bp,其中开放阅读框135bp,5′-UTR 87bp,3′-UTR为474bp,该基因编码45个氨基酸,表达蛋白的理论分子质量为5 141.59u,等电点为5.34;结构分析发现,大鲵胸腺素β-4蛋白氨基酸序列的第7～43位处有"THY"特征模体,没有跨膜螺旋区、细胞膜外在跨膜结构域。氨基酸序列进化关系表明,大鲵与人、大鼠、牛、猩猩、鸡、倭蛙等动物亲缘关系较近,氨基酸同源性高于85%;与猪和爪蟾的亲缘关系次之,氨基酸同源性均为83%;而与虹鳟、大菱鲆、斑马鱼等鱼类的亲缘关系较远,氨基酸同源性低于80%。荧光定量PCR检测结果表明,胸腺素β-4基因在大鲵肺中的相对表达量最高。SDS-PAGE电泳结果表明,构建的大鲵胸腺素β-4原核表达重组菌株pET32a-Tβ4,在37℃条件下,用终浓度1.0mmol/mL IPTG诱导4h后,大鲵胸腺素β-4基因获得了较高的表达。【结论】分离获得了大鲵胸腺素β-4基因全长cDNA序列,该基因氨基酸序列与人等高等动物的同源性最高,能在大鲵肺中及大肠杆菌中获得高效表达。

基于互信息特征分离表达的红外与可见光图像融合

针对红外与可见光图像融合方法存在的对源图像特征分离不充分、可解释性低且融合规则难以准确设计等问题,提出基于互信息特征分离表达的红外与可见光图像融合方法,有效分离特征的同时保留源图像的典型信息。首先,采用互信息约束的编码网络提取特征,最大化源图像与特征间互信息来保留源图像的特征表示,同时通过最小化私有和公有特征的互信息来达到分离表达的目的;其次,特征融合阶段设计了层级特征自适应融合模块来有效融合不同层级的特征信息,减小语义差距并调整感受野,增强网络对特征的学习能力;此外,损失函数采用软加权强度损失来平衡红外与可见光特征分布;最后,对比实验的主客观评价结果表明,所提方法能有效融合红外与可见光图像的重要信息,具有良好的视觉感知。

Java自动并行编译系统JAPS中表达式分离技术的研究

一、引言 1.1 自动并行编译的提出并行程序设计基本上有两种途径:显式途径和隐式途径。研究者设计了很多的并行程序设计语言,这就是所谓的“显式途径”。但是,并没有一种并行程序设计语言成为流行的语言,主要的问题在于并行程序的编制困难,对程序员的要求高。相比较而言,隐式途径,即自动并行编译技术就有许多的优势。用户使用串行语言编制程序。

AiiA蛋白的可溶性表达及其抗菌活性研究

AHLs是革兰氏阴性细菌在增殖过程中产生的一类信号分子,与其致病性密切相关。AiiA蛋白作为一种胞内解酯酶。能水解致病菌产生的AHLs分子,使内酯环开环后不能再激活某些胞外酶的表达,从而极大地减弱了细菌的致病性。本研究从苏云金芽孢杆菌LLB15中分离编码aiiA基因的质粒DNA,用PCR方法克隆AiiA基因,并利用pET载体构建6-His融合表达质粒pET29a-aiiA,转化E.coli BL21(DE3)菌株,并筛选得到E.coli BL21(DE3)-pET29a-aiiA工程菌。在20℃的低温和0.8 mmol/L IPTG条件下,经25 h的诱导表达,获得了54.4μg/mL可溶性AiiA蛋白。通过镍柱亲和层析,在国内外首次纯化了带6-His标记的AiiA蛋白。水解活性和抗病性检测表明。该蛋白能水解AHLs分子。对胡萝卜欧文氏软腐病菌具有较强的抗病作用。

cDNA Cloning, Expression and Characterization of Taxadiene Synthase, a Diterpene Cyclase from Taxus chinensis

Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthase of Taxus chinensis (Pilg.) Rehd. was cloned by homology-based PCR and cDNA library screening. The 5′-terminal 611 bp cDNA fragment of taxadiene synthase was isolated by PCR. The two fragments were ligated together and gave a 2*!712 bp cDNA fragment with a 2*!586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial transit peptide. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia (97% identity). Heterologous overexpression of 2.5 kb cDNA fragment from T. chinensis was obtained using a fusion expression vector pET-32a and the Escherichia coli strain BL21trxB. The expressed proteins from E. coli BL21trxB were present as inclusion bodies. After the inclusion bodies were denatured, renatured and refolded, the recombinant enzyme was purified by a single step with a His-binding metal affinity column. The catalytic product of taxadiene synthase of T. chinensis was detected by capillary gas chromatography-mass spectrometry (GC-MS) and identified as taxa-4(5),11(12)-diene.

Cloning and Differential Expression of a 1-Aminocyclopropane-1-Carboxylate Synthase cDNA from Peach

The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach Prunus persica (L.) Batsch) fruit ripening, we cloned a full_length cDNA of ACC synthase pacs from peach using 5′/3′ RACE PCR. The nucleic acid sequence of pacs was 1 848 bp, containing 177 bp of 5′untranslated sequence, 1 449 bp of an open reading frame, and 219 bp of 3′untranslated sequence (excluding the stop codon TAA). The pacs open reading frame encoded a 483_amino acid polypeptide with a predicted size of 54 kD and a calculated PI of 6.43. The deduced protein from ACC synthase cDNA pacs had 65%, 70%, 75%, and 90% homology with the other deduced proteins from tomato (S19677), plum (AB031026), papaya (U68216) and apple (AB034993), which contained the active site of ACC synthase SLSKDMGFPGFR conserved among these plant ACC synthases. RNA_based PCR amplification combined with hybridization analysis with pacs and another ACC synthase cDNApacs12 (AF467782) cloned by us before as probes, indicated that expression patterns of both clones were very similar. mRNAs of both clones expressed in the alabastrum and petal, and were induced after ethylene treatment. Wounding and IAA treatments could induce ACC synthase expression of both clones in the leaves. However, the wounding treatment of leaves has induced more abundant pacs ACC synthase expression than that ofpacs12. Pacs mRNA expressed in both green mature and ripening fruit, whilepacs12mRNA was little or undetectable in green mature fruit, but apparent in ripening fruit. Both clone mRNAs accumulated more in leaves (following wounding and IAA treatments) and flowers than in fruits.

基因克隆技术的研究进展

为能快速而准确地克隆目的基因,综述了一些基因克隆常用技术,包括差异表达基因分离技术、转座子标签技术、图位克隆技术、同源序列技术、表达序列标签技术的原理、应用及应用潜力,并对其作了简要的评价.这些技术有利有弊,应根据不同的实验目的和水平来选择相应的技术.

Separation and Identification of Differentially Expressed Proteins in Pistillate Flowers between Different Mulberry Cultivars

[Objective] This study aimed to explore the proteins related to pistillate flower development in different mulberry cultivars. [Method] The total proteins of the pistillate flowers of two mulberry cultivars Dal0 （Morus atropurpurea Roxb.） and SG01 （Morus muIticaulis Perr.） were extracted, separated and detected through two- dimensional electrophoresis （2-DE） and mass spectrometry. [Result] There was sig- nificant difference in the expression of proteins from the pistillate flowers of different mulberry cultivars. From the 2-DE images of Dal0 and SG01, 445_＋17 and 425_＋12 protein spots were respectively detected. The expression levels of 75 protein spots differed significantly. Thirteen spots those were expressed at high levels and well separated were analyzed by mass spectrometry, and nine of them were identified successfully. The nine proteins are involved in the glycometabolism, protein and amino acid metabolism and defense responses during the development of mulberry pistillate flower after they were pollinated. [Conclusion] The findings will provide reference for further study on the molecular mechanism of mulberry pistillate flower de- velopment.

Isolation of side population cells from gallbladder carcinoma of human being and the expression of ABCG2 gene

Objective: To investigate whether the side population cells (SP cells) exist in human gallbladder carcinoma cell line and the differences of drug resistance gene ABCG2 expression in SP cells, non-SP cells and GBC-SD cell lines. Methods: Fluorescence activated cell sorter (FACS) was used to sort the SP and non-SP cells from GBC-SD cell line of gallbladder carcinoma of human being. Then, the sorting cells were cultured and detected the expression of ABCG2 gene among the SP cells, non-SP cells and GBC-SD cell lines by using reverse transcription polymerase chain reaction (RT-PCR), immunofluo-rescence chemistry, western blot and flow cytometry techniques. Results: A very small fraction cells (0.64 ± 0.08%) were iso-lated through FACS analysis which had the potency of stem cells and highly expressed ABCG2 gene (89.56 ± 3.86%). On the contrary, there were nearly no expression in non-SP cells (1.32 ± 0.49%) and lower expression in GBC-SD cell line (12.37 ± 1.61%). Conclusion: The side population cells that had the potency of stem cells existed in human gallbladder carcinoma cell line and over-expressed the drug resistance gene ABCG2. They may be play an important role in drug resistance of tumor.

Isolation of 24 novel cDNA fragments from microdissected human chromosome band

The strategy of isolating the band-specific expression fragments from a probe pool generated by humanchromosome microdissection was reported. A cliromosome 14q24.3 band-specific single copy DNA pool was constructed based on this probe pool. Using total DNA of the pool as probe to hybridize the human marrow cDNA library, 68 primary positive clones were selected from 5 ×105 cDNA clones. Among these primary clones, 32 secondary clones were obtained after second-round screening and designed as cFD14-1～32. Finally, 24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization. Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but cann’t hybridize to 17q11～12 DNA. Partial sequences of 13 fragments of them were sequenced and identified as novel cDNA sequences , and these sequences were proved to have some homology with known genes in NCBI database. Analysis of expression spectrum of cFD14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3region.

Isolation and Expression Analysis of FTZ-F1 Encoding Gene of Black Rock Fish (Sebastes schlegelii)

Sex related FTZ-F1 is a transcriptional factor regulating the expression of fushi tarazu (a member of the orphan nuclear receptors) gene. In this study, FTZ-F1 gene (FTZ-F1) was isolated from the testis of black rockfish (Sebastes schlegeli) by homology cloning. The full-length cDNA of S. schlegeli FTZ-F1 (ssFTZ-F1) contained a 232bp 5′UTR, a 1449bp ORF encoding FTZ-F1 (482 amino acid residules in length) with an estimated molecular weight of 5.4kD and a 105bp 3′UTR. Sequence, tissue distribution and phylogenic analysis showed that ssFTZ-F1 belonged to FTZ group, holding highly conserved regions including Ⅰ, Ⅱ and Ⅲ FTZ-F1 boxes and an AF-2 hexamer. Relatively high expression was observed at different larva stages. In juveniles (105 days old), the transcript of ssFTZ-F1 can be detected in all tissues and the abuncance of the gene transcript in testis, ovary, spleen and brain was higher than that in other tissues. In mature fish, the abundance of gene transcript was higher in testis, ovary, spleen and brain than that in liver (trace amount), and the gene was not transcribed in other tissues. The highest abundance of gene transcript was always observed in gonads of both juvenile and mature fish. In addition, the abundance of gene transcript in male tissues were higher than that in female tissue counterparts (P<0.05).

Exciting Forces for a Wave Energy Device Consisting of a Pair of Coaxial Cylinders in Water of Finite Depth

Two coaxial vertical cylinders-one is a riding hollow cylinder and the other a solid cylinder of greater radius at some distance above an impermeable horizontal bottom,were considered.This problem of diffraction by these two cylinders,which were considered as idealization of a buoy and a circular plate,can be considered as a wave energy device.The wave energy that is created and transferred by this device can be appropriately used in many applications in lieu of conventional energy.Method of separation of variables was used to obtain the analytical expressions for the diffracted potentials in four clearly identified regions.By applying the appropriate matching conditions along the three virtual boundaries between the regions,a system of linear equations was obtained,which was solved for the unknown coefficients.The potentials allowed us to obtain the exciting forces acting on both cylinders.Sets of exciting forces were obtained for different radii of the cylinders and for different gaps between the cylinders.It was observed that changes in radius and the gap had significant effect on the forces.It was found that mostly the exciting forces were significant only at lower frequencies.The exciting forces almost vanished at higher frequencies.The problem was also investigated for the base case of no plate arrangement,i.e.,the case having only the floating cylinder tethered to the sea-bed.Comparison of forces for both arrangements was carried out.In order to take care of the radiation of the cylinders due to surge motion,the corresponding added mass and the damping coefficients for both cylinders were also computed.All the results were depicted graphically and compared with available results.

ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSED IN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

Objective.To investigate the differentiation process of the human glioblastoma cells. Methods.Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325.Routine method of cDNA library screening was performed to clone full-length cDNA. Results.Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered,amplified and cloned.Of them,46 ESTs were sequenced and delivered into the GenBank.The homology comparison using BLAST algorithm revealed that 22ESTs are highly homologous with the known genes and many of them play important roles in the cell differentiation progress.A dot-blot hybridization was conducted to certify the differentiation expression.The result showed that 27 EST clones are expressed at different level in control and all-trans retinoic acid treated BT-325 cells.A full-length cDNA was cloned using the EST-HGBB098. Conclusion.DDRT-PCR was a simple and effective method to serially analyze the differentially expressed genes.

Isolation of Immune-Relating 185/333-1 Gene from Sea Urchin(Strongylocentrotus intermedius) and Its Expression Analysis

The 185/333 gene family involved in the immune response of sea urchin.One 185/333 c DNA was isolated from Strongylocentrotus intermedius,and named as Si185/333-1.Its full-length c DNA was 1246 bp in length with a 906 bp open reading frame encoding a protein of 301 aa.The molecular weight of the deduced protein was approximately 33.1 k D with an estimated PI of p H 6.26.Si185/333-1 had high identities(70%–86%) to most of Sp185/333.An extraordinary identity of 92% was found between Si185/333-1 and Sp185/333 C5 alpha(ABR22474).Moderate identities(63%–64%) were displayed between Si185/333-1 and He185/333.Si185/333-1 had similar structure to Sp185/333.A signal-peptide,a gly-rich region and a his-rich region were found in its secondary structure.RGD motif was found in gly-rich region at position 116–118aa.There was no transmembrane region in Si185/333-1.The element pattern of Si185/333-1 is different from any available pattern that identified in Sp185/333.Si185/333-1 clustered together with pattern C Sp185/333 in phylogenetic tree.The Si185/333-1 m RNA could be detected in tissues including peristomial membrane,coelomocytes,muscle of Aristotles lantern,gut and tube feet,with the highest expression level detected in peristomial membrane and a relatively low expression in ovary and testis.The temporal expression of Si185/333-1 in peristomial membrane and coelomocytes were up-regulated after bacterial,β-D-glucan and ds RNA challenges,reaching the maximum at 12 h post-stimulation.The up-regulation was more obvious in coelomocytes,and bacterial challenge triggered the highest response.These results proved that 185/333-1 gene was involved in the immune defense of S.intermedius,while more studies were necessary for its function in S.intermedius immunity.