芝麻香型高温大曲制曲过程中理化生化与细菌DGGE指纹图谱的探究

采用16S rRNA、PCR-变性梯度凝胶电泳(PCR-DGGE)技术,在芝麻香型高温大曲培曲及前期储存期间进行采样,并分别对高温大曲的各项理化生化指标变化规律及细菌DGGE指纹图谱进行综合分析,揭示了在高温制曲及贮存进程中,各项理化生化指标的变化规律及细菌种类具有多样性及相似性,该探究为指导高温大曲功能微生物筛选应用、优化提升大曲制作工艺提供参考。

CloneIRD:面向代码溯源的克隆代码继承关系判定方法

随着开源软件的广泛使用,代码溯源成为管理软件源代码、降低潜在风险的重要技术手段。基于代码克隆检测的大规模代码溯源分析,从其检测结果中鉴别代码克隆对之间的继承关系,对代码来源追踪、组件依赖关系分析、软件脆弱性分析以及代码缺陷修复等具有重要意义。目前,已有方法在原始代码片段存在微小修改的情况下,会产生许多误判,并且检测克隆对的效率也有待提高。针对上述问题,提出了代码溯源中克隆代码继承关系的判定方法CloneIRD,包括一个基于自研快速分布式克隆检测工具FastDCF的代码溯源分析框架,以及该框架的核心算法——基于代码演化信息的克隆代码继承关系判定算法EIHR。为验证框架和算法的有效性,首先设计并实现了CloneIRD方法,并在Linux内核V4.9和V4.12的开源代码上进行了实验。实验结果表明,CloneIRD方法能够有效判定代码溯源结果中克隆对的继承关系,且基于FastDCF的溯源分析框架能够胜任大规模代码的溯源分析任务。

基于PCR-DGGE技术分析浓香白酒窖泥梭菌多样性

窖泥是浓香型白酒酿制过程中最主要的微生物源,窖泥中微生物的类型、丰度、新陈代谢活动等均对浓香型白酒品质产生很大的影响。为探究窖泥中梭菌微生物的多样性,利用窖泥理化结合聚合酶链式反应-变性梯度凝胶电泳技术对10个窖泥样品中的梭菌群落及变化规律进行研究。结果表明,所选10个窖泥的理化参数均符合优质窖泥指标要求;在微生物层面,窖泥中检测到的梭菌在属水平上有:嗜碱菌属、丁酸弧菌属、梭菌属、喜热菌属、瘤胃梭菌属、粪球菌属、沉淀杆菌属(Sedimentibacter)、钙原杆菌属(Caldicoprobacter)、温带菌(Tepidimicrobium)、梯氏菌(Tissierella)、孢子菌(Sporanaerobacter)、硫酸盐还原菌属、鲁替孢菌属和梭状芽胞杆菌(Clostridiisalibacter)等,这些菌是优质窖泥的重要指示菌,可知窖泥中含有极其丰富的酿酒功能菌。揭示了可能在白酒酿造中起关键作用的梭菌菌群,在分子水平上为研究浓香型白酒提供了理论依据。

Cost-Effective Method of Gene Synthesis by Sequencing from Microchip-Derived Oligos for Droplet Cloning

Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthesis has been increasing for the past few decades, yet available methods remain expensive. A solution to this problem involves microchip-derived oligonucleotides (oligos), an oligo pool with a substantial number of oligo fragments. Microchips have been proposed as a tool for gene synthesis, but this approach has been criticized for its high error rate during sequencing. This study tests a possible cost-effective method for gene synthesis utilizing fragment assembly and golden gate assembly, which can be employed for quicker manufacturing and efficient execution of genes in the near future. The droplet method was tested in two trials to determine the viability of the method through the accuracy of the oligos sequenced. A preliminary research experiment was performed to determine the efficacy of oligo lengths ranging from two to four overlapping oligos through Gibson assembly. Of the three oligo lengths tested, only two fragment oligos were correctly sequenced. Two fragment oligos were used for the second experiment, which determined the efficacy of the droplet method in reducing gene synthesis cost and speed. The first trial utilized a high-fidelity polymerase and resulted in 3% correctly sequenced oligos, so the second trial utilized a non-high-fidelity polymerase, resulting in 8% correctly sequenced oligos. After calculating, the cost of gene synthesis lowers down to 0.8 cents/base. The final calculated cost of 0.8 cents/base is significantly cheaper than other manufacturing costs of 7 - 30 cents/base. Reducing the cost of gene synthesis provides new insight into the cost-effectiveness of present technologies and protocols and has the potential to benefit the fields of bioengineering and gene therapy.

Gene Cloning and Bioinformatics Analysis of phoR Gene from Vibrio alginolyticus HY9901

PhoR is a histidine kinase in a two-component regulatory system that regulates phosphorus metabolic pathways and undertakes the key mission of information transmission in pathogenic bacteria.The full-length phoR gene was successfully cloned from the Vibrio alginolyticus HY9901 strain.A comprehensive analysis of the cloned gene was conducted using bioinformatics.Sequence analysis revealed that the total length of the phoR gene(GenBank accession No.:KJ958404.1)is 1299 bp,with the coding region containing a total of 432 amino acid residues.The phylogenetic tree of PhoR revealed that it belongs to the same subclade as V.diabolicus.The SMART program was employed for the purpose of functional domain prediction,which revealed that PhoR possesses three major functional domains:PAS(amino acids 98-166),HisKA(amino acids 205-272),and HATPase_c(amino acids 317-429).

Molecular Cloning and Bioinformatics Analysis of msrA Gene from Vibrio alginolyticus Strain HY9901

[Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With Vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of msrA,and its bioinformatics analysis was carried out.[Results]The full length of msrA gene was 639 bp,encoding 212 amino acids,and its theoretical molecular weight was about 23729.60 Da.The protein had a stable structure,and it was hydrophobic overall.The structure of signal peptides at the N terminal of the amino acid sequence was predicted,and it was found that there was no signal peptide cleavage site and no transmembrane region.The amino acid sequence of MsrA contained multiple signal binding sites.Protein subcellular localization showed that MsrA protein was most likely located in the cytoplasm.Homology analysis showed that MsrA of V.alginolyticus had high homology with other Vibrio species,and the highest homology with V.alginolyticus.In the prediction of functional domains,MsrA had the function of methionine sulfoxide reduction.In secondary structure prediction,MsrA contained random coils at a proportion of 46.70%,which was the highest.The similarity between the tertiary structure model of MsrA and template Q87SW6.1.A was 89.15%.PTM analysis showed that MsrA protein had many PTM modification sites such as phosphorylation and glycosylation sites.[Conclusions]This study provides some reference value for further study on the role of MsrA in bacterial antioxidant stress.

Molecular Cloning of sodB Gene from Vibrio alginolyticus HY9901 and Its Bioinformatics Analysis

Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino acids.The predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of 4.95.Upon prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane region.The amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal site.The probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the protein.The amino acid sequence of the sodB gene of V.alginolyticus exhibited 98%-100%homology to other Vibrio species,clustering into the same subfamily with V.parahaem,indicating a relatively close relationship between them.In the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,respectively.The similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.

采用FISH、DGGE和Cloning对短程脱氮系统中硝化菌群的比较分析

针对4种不同的实际污水短程生物脱氮系统（SBR大型中试反应器、UASB-A/O小型反应器、A/O中试反应器和SBR小型反应器）,采用Fish、PCR-DGGE和PCR-Cloning-Sequencing分子生物学方法对系统中硝化菌群AOB和NOB进行定性与定量化分析.Fish结果表明,在4种短程脱氮系统中,AOB相比于NOB已成为明显的优势菌群,占总菌群的3%～12%;在SBR中试和小试反应器中没有检测出NOB;A/O中试反应器中存在极少量的Nitrospira（＜0.2%）,而UASB-A/O小型反应器中存在极少量的Nitrobacteria（＜0.2%）.PCR-DGGE结果表明SBR中试、A/O和UASB-A/O 3种短程脱氮系统中的AOB均以Nitrosomonas-like为主.SBR大型中试反应器中污泥样品的PCR-Cloning-Sequencing结果表明,所有的克隆相似于Nitrosomonas,其中60%以上的克隆相似于Nitrosomonas europaea.

利用DGGE-cloning技术分析农肥和化肥施用对黑土细菌多样性的影响

本试验以东北黑土为研究对象,采用DGGE-cloning测序技术,研究了不同施肥制度和肥料用量对黑土土壤细菌多样性的影响。本实验共设六个处理,分别为:对照,施用农肥低量,施用农肥高量,施用化肥低量,施用化肥高量和农肥和化肥1:1的处理。试验得出的DGGE图谱分析表明,施用较高量的农肥处理中土壤细菌16SrDNA条带数、多样性指数及均匀度指数均要高于其他处理,聚类分析显示,施用化肥的土壤与其他处理相比土壤细菌群落结构差异较大,相似性只有53%,说明施用化肥与施用农肥比较,可以显著的改变土壤细菌的群落结构。从DGGE凝胶上切取9条带进行DGGE-cloning测序,结果表明,施用农肥和化肥均可对土壤的细菌群落结构产生影响,且施肥制度对细菌群落结果的影响要大于施肥量,施用农肥可以提高细菌多样性。

Effect of methamidophos on soil fungi community in microcosms by plate count,DGGE and clone library analysis

Methamidophos was widely used a pesticide in northern China. The potential influences of methamidophos on soil fungal community in black soil were assessed by plate count, 28S rDNA-PCR-DGGE, and clone library analysis. Three methamidophos levels （50, 150, and 250 mg/kg） were tested in soil microcosms. Results from plate count during a 60-d microcosm experiment showed that high concentrations of methamidophos （250 mg/kg） could significantly stimulate fungal populations. DGGE （denaturing gradient gel electrophoresis） fingerprinting patterns showed a significant difference between the responses of culturable and total fungi communities under the stress of methamidophos. Shannon diversity indices calculated from DGGE profiles indicated that culturable fungi in all microcosms with methamidophos treatment increased after 1 week of incubation. However, the diversity indices of total fungi decreased in the first week, as compared to the stimulation of culturable fungi. At the 8th week, however, all the microcosms treated by methamidophos were similar to the control microcosms in community structure as suggested by the Shannon diversity indices for both culturable and total fungi. In contrast, after 1 week the fungal structure of culturable and unculturable both were disturbed to different extent under the stresses of methamidophos by clustering analysis. Clone sequencing analysis indicated the stimulation of pathogenic and unculturable fungal populations by methamidophos treatment, suggetsing potential risks of plant disease outbreak.

Economical phase-covariant cloning with multiclones

This paper presents a very simple method to derive the explicit transformations of the optimal economical 1 to M phase-covariant cloning. The fidelity of clones reaches the theoretic bound [D＇Ar]ano G M and Macchiavello C 2003 Phys. Rev. A 67 042306]. The derived transformations cover the previous contributions [Delgado Y, Lamata Let al, 2007 Phys. Rev. Lett. 98 150502] in which M must be odd.

Superovulation of the Cloned Cattle Derived from Somatic Cells and the Transfer of the Vitrified-Thawed Embryos of the Cloning Cattle

In this experiment, it was designed to carry out superovulation on the two cloned cattles, vitrification and transfer of the embryos recovered from them. First of all, it was carried out vitrification on embryos obtained by IVF. Results showed that there were no significant differences between the blastocysts (obtained by IVF) vitrified in EPS10 and these in EPS20 on the resuscitative rate and the developmental rate. The hatched rate of the blastocysts vitrified in EPS10 (31.3%, 35/112) was significantly higher than that in EPS20 (12.2%, 13/107)(P<0.01), so EPS20 was selected as the vitrification solution to freeze the embryos recovered from the cloned cattle. After superovulation, six (four usable embryos) and ten (nine usable embryos) embryos were respectively recovered from Kangkang and Shuanghuang. Two embryos were selected from the recovered embryos of each cloned cattle to freeze in EPS20, subsequently thawed and transferred into luteal ipsilateral uterine horns of 4 Holstein recipient cows after synchronization of estrus, respectively. At last, one recipient cow (No. 9908) became pregnant and delivered one healthy calf (descendant of the cloned cattle-Shuangshuang). The results of this experi- ment show that the cloned cattle as well as common cattle had better response to the exotic FSH and better ability to multiovulation, the embryos recovered from the cloned cattle can be vitrificated.

PCR-DGGE技术分析韩式大酱与中式大酱中微生物多样性

微生物在大酱发酵过程中起到至关重要的作用,并与大酱的风味与质量密切相关,因此研究大酱中微生物的多样性有重要意义。该研究选择加工工艺不同的韩式大酱与中式大酱为对象,采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术,通过PCR扩增、切胶回收、PCR测序等分析不同大酱中的微生物多样性。结果表明,大酱中的微生物由于制作工艺的不同存在明显差异。在韩式大酱中,细菌如芽孢杆菌属(Bacillus)、不动杆菌属(Acinetobacter)、四联球菌属(Tetragenococcus)、嗜盐单胞菌属(Halomonas),真菌如根毛霉属(Rhizomucor)、青霉菌属(Penicillium)、毛霉属(Mucor)、外瓶霉属(Exophiala)、曲霉属(Aspergillus)等分布广泛。在中式大酱中,乳酸菌如片球菌属(Pediococcus)、乳杆菌属(Lactobacillus)、明串珠菌属(Leucanostoc)、肠杆菌属(Enterobacter),酵母如鲁氏接合酵母(Zygosaccharomyces rouxii)分布较多。与中式大酱相比,韩式大酱中的真菌种类更丰富。研究结果为进一步探讨传统发酵大酱品质提供了理论依据。

An integrated microfluidics platform with high-throughput single-cell cloning array and concentration gradient generator for efficient cancer drug effect screening

Background:Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment.Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput way is thus of great value for developing precision therapy.However,current techniques suffer from limitations in dynamically characterizing the responses of thousands of single cells or cell clones presented to multiple drug conditions.Methods:We developed a new microfluidics-based“SMART”platform that is Simple to operate,able to generate a Massive single-cell array and Multiplex drug concentrations,capable of keeping cells Alive,Retainable and Trackable in the microchambers.These features are achieved by integrating a Microfluidic chamber Array(4320 units)and a sixConcentration gradient generator(MAC),which enables highly efficient analysis of leukemia drug effects on single cells and cell clones in a high-throughput way.Results:A simple procedure produces 6 on-chip drug gradients to treat more than 3000 single cells or single-cell derived clones and thus allows an efficient and precise analysis of cell heterogeneity.The statistic results reveal that Imatinib(Ima)and Resveratrol(Res)combination treatment on single cells or clones is much more efficient than Ima or Res single drug treatment,indicated by the markedly reduced half maximal inhibitory concentration(IC50).Additionally,single-cell derived clones demonstrate a higher IC_(50) in each drug treatment compared to single cells.Moreover,primary cells isolated from two leukemia patients are also found with apparent heterogeneity upon drug treatment on MAC.Conclusions:This microfluidics-based“SMART”platform allows high-throughput single-cell capture and culture,dynamic drug-gradient treatment and cell response monitoring,which represents a new approach to efficiently investigate anticancer drug effects and should benefit drug discovery for leukemia and other cancers.

Cloning of PsMYB62 and analysis of cadmium resistant in Potentilla sericea

Potentilla sericea is a heavy metal hyperaccumulator landscaping plant.MYB transcription factors play an important role in regulating plant stress response to adversity.However,there are few studies on MYB transcription factors in stress tolerance in Potentilla sericea.In this study,the PsMYB62 gene was successfully cloned from Potentilla sericea.Methods:Bioinformatic analysis and real-time quantitative PCR(qPCR)methods were used to evaluate this gene.The transgenic A.thaliana were obtained by flower dipping and the gene function was identified by determining physiological indicators under cadmium stress.Results:The open reading frame of PsMYB62 is 942 bp,which encodes 313 amino acids(aa)and belongs to the R2R3 MYB transcription factor.The plant overexpression vector PBI121-PsMYB62-GFP was constructed and successfully transferred into A.thaliana.The relative expression level of PsMYB62 was significantly increased by CdCl_(2),NaCl,ABA,and mannitol treatments.The germination rate of transgenic seeds was higher than those of wild type(WT)and empty vector(EV)under different concentrations of cadmium treatment.Upon treatment with 100μmol·L^(−1)of CdCl_(2)·2.5H_(2)O,the activities of superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT)in the transgenic plants were significantly higher than those in the WT and EV.The contents of H_(2)O_(2),O_(2)·−and malondialdehyde(MDA)in transgenic lines were increased,but lower than those in WT and EV.The expression levels of AtGSH,AtPCS,and AtNAS4 that were related to the regulation of cadmium were increased,but the expression levels of transgenic lines were higher than those of WT and EV.Conclusion:The above results showed that PsMYB62 could be induced by cadmium and could improve the cadmium resistance of plants.

Cloning and Functional Validation of Mung Bean VrPR Gene

For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids were encoded.Bioinformatics analysis showed that the VrPR protein belonged to the NADB Rossmann superfamily,which was one of the non-transmembrane hydrophilic proteins.VrPR was assumed to have 44 amino acid phosphorylation sites and be contained in chloroplasts.The VrPR secondary structure comprised of random coil,αhelix,βangle,and extended chain,all of which were quite compatible with the anticipated tertiary structure.Moreover,analysis of the phylogenetic tree indicated that the soybean PR(Glyma.12G222200)and VrPR were closely related.Furthermore,chlorophyll content in leaves is markedly increased in Arabidopsis when VrPR is overexpressed.Ourfindings will serve as a reference for more functional studies on the PR genes in mung bean.

Cloning and Bioinformatics Analysis of vscB Gene of T3SS Chaperone of Vibrio alginolyticus

[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.

The Clones’Struggle for Happiness under the Doomed Fate in Never Let Me Go

Japanese British writer Kazuo Ishiguro is one of the leading writers in contemporary British literature.He has always been committed to creating works with universal significance.Responsibility and destiny are themes that run through his works.His novel Never Let Me Go tells a story of a group of clones growing up in the Hailsham,who are given the mission to donate organs at birth.So,there is no doubt that they will inevitably end their lives in the process of donating organs to human beings again and again.The tragic life of clones is determined by the motivation of human to create them.

An Analysis on the Role Identification of Clones in Never Let Me Go

With the guidance of Erikson’s identity theory,the article analyzes the clones’identity exploration through role identification in Never Let Me Go.It interprets the clones’puzzlement about their identity,and the process of their identity quest as well as role identification.Through the specific analysis,it is concluded that the clones,represented by Kathy,Tommy and Ruth,have gained self-certainty and social identity by realizing the role identity as a“carer”and a“donor”,in the meantime,they have constructed their identity as social persons with souls like ordinary people.Furthermore,the findings shows that Never Let Me Go is actually a microcosm of human’s quest for identity.Ishiguro aims to express his meditation on human life in the novel:human life is a process of seeking self-identity and social roles,and then fulfilling the obligations of the roles,which confirms Ishiguro’s internationalism that he attempts to convey his contemplation on human existence through his works.

羽衣甘蓝BoLMI基因的克隆及时空表达分析

【目的】探究羽衣甘蓝裂叶表型形成的分子机制,研究BoLMI基因对裂叶形成的作用。【方法】以裂叶羽衣甘蓝DH系为试验材料,利用同源克隆技术,成功获取基因完整的cDNA序列,将其命名为BoLMI。【结果】序列分析发现,BoLMI基因全长531个碱基对,共编码176个氨基酸,其蛋白分子量为20 789.35 Da,理论等电点为7.24。根据Pfam保守结构域分析,BoLMI蛋白包含homeobox保守结构域。系统发育分析结果显示,羽衣甘蓝的BoLMI基因与甘蓝型油菜以及结球甘蓝的BoLMI基因同属于一个分支,亲缘关系较近。BoLMI蛋白是位于细胞核内的可溶性蛋白,包含1个跨膜结构域,无信号肽序列。qRT-PCR分析表明,BoLMI基因在裂叶羽衣甘蓝的幼苗期表达水平较高,在莲座期表达水平较低;在羽衣甘蓝莲座期,裂叶品种不同部位叶片BoLMI基因的表达水平均高于圆叶品种,其中在第1片叶的表达量最高。裂叶品种和圆叶品种不同叶片BoLMI基因的表达量差异显著。【结论】推测BoLMI基因在羽衣甘蓝裂叶形成中起重要作用,为加速羽衣甘蓝叶形育种提供了理论基础。