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。

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.

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.

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.

马铃薯野生种烯酰水合酶超家族基因ScDHNS的克隆与功能分析

【目的】1,4-二氢氧-2-石脑-CoA合成酶(1,4-dihydroxy-2-naphthoyl-CoA synthase,DHNS)基因是茄科植物糖苷生物碱合成代谢的潜在重要基因,开展马铃薯DHNS基因功能研究与验证,为低糖苷生物碱马铃薯品种(系)的选育提供基因和材料来源。【方法】利用RACE方法克隆得到马铃薯野生种恰柯薯(Solanum chacoense)ScDHNS基因,对其进行生物信息学分析和亚细胞定位,通过构建过表达载体pBWA(V)HS-DHNS转化马铃薯栽培种进行功能验证。【结果】ScDHNS cDNA序列开放阅读框1023 bp,编码340个氨基酸,分子量为37.34 kD,等电点pI为8.592,具有典型的ECH保守结构域,属于烯酰水合酶超家族成员,在二穗短柄草(Brachypodium distachyon)、蒺藜苜蓿(Medicago truncatula)等植物基因组中都有其同源基因,且存在基因扩张和收缩事件。过表达ScDHNS基因后发现转化株ScDHNS和SGT1基因表达量显著上调,且表达量显著高于马铃薯WT植株。且对应转化植株的总糖苷生物碱含量显著高于马铃薯WT植株,最高可达到364.3 mg/kg,是对照的2.4倍。亚细胞定位结果显示ScDHNS定位于过氧化物酶体。【结论】马铃薯ScDHNS基因可能参与调控糖苷生物碱合成关键基因SGT1的表达,通过β-氧化途径和甲羟戊酸通路协同影响糖苷生物碱的合成,该基因与糖苷生物碱在亚细胞水平上的区室化有重要关系,对于培育低糖苷生物碱的马铃薯品种(系)具有重要的应用价值。

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。

考虑行为克隆的深度强化学习股票交易策略

为提高股票投资的收益并降低风险,将模仿学习中的行为克隆思想引入深度强化学习框架中设计股票交易策略。在策略设计过程中,将对决DQN深度强化学习算法和行为克隆进行结合,使智能体在自主探索的同时模仿事先构造的投资专家的决策。选择不同行业的股票进行数值实验,说明了所设计的交易策略在年化收益率、夏普比率和卡玛比率等收益与风险指标上优于对比策略。研究结果表明:将模仿学习与深度强化学习相结合可以使智能体同时具有探索和模仿能力,从而提高模型的泛化能力和策略的适用性。

洋葱AcSCL4的克隆及其功能分析

【目的】初步探讨AcSCL4在洋葱成花中的功能,为解析洋葱成花分子调控机制提供理论基础。【方法】以长日生态型洋葱品系SB2为试验材料,根据已有的洋葱转录组数据,通过qRT-PCR筛选目的基因AcSCL4后进行基因克隆,并对其进行生物信息学分析;构建AcSCL4基因的过表达载体pB221-3300-AcSCL4并转化农杆菌,采用花序浸染法浸染拟南芥,并对T_(3)代纯系转基因植株进行表型分析和开花相关基因表达量分析。【结果】洋葱AcSCL4基因CDS长1515 bp,编码504个氨基酸;AcSCL4蛋白N端高度变异,C端有GRAS结构域。聚类分析发现,AcSCL4与石刁柏和棉花的SCL4蛋白亲缘关系较近。与野生型拟南芥相比,AcSCL4过表达植株表现晚花表型。荧光定量PCR结果表明,与野生型拟南芥相比,AcSCL4过表达植株开花调控基因CO和FT的表达量极显著下降。【结论】AcSCL4通过调控CO及FT基因表达来抑制洋葱开花。

草莓FaWRKY70基因克隆与表达模式分析

【目的】WRKY是植物特异性转录因子家族之一,参与植物多种生命活动,但在草莓中鲜见WRKY70相关报道。深入解析WRKY70同源基因在草莓响应胁迫过程中的作用,加快分子育种技术应用,培育新草莓种质资源。【方法】用同源克隆法从‘红颜’草莓果实中克隆得到FaWRKY70基因,用生物信息学分析其保守结构域、理化性质、蛋白质结构及进化关系等,并结合qRT-PCR数据进行表达模式分析。【结果】FaWRKY70基因全长1020 bp,编码339个氨基酸;同源基因比对发现,FaWRKY70与苹果、牡丹等同科物种氨基酸序列相似度较高,且同源基因多与植物对生物、非生物胁迫响应相关,暗示FaWRKY70可能参与草莓抵御胁迫的过程;FaWRKY70在草莓不同器官中均有表达,且差异显著,在花中表达量最高,在果实中最低;水杨酸处理后,FaWRKY70基因快速响应,表达量在3 h后达到最高,随后逐渐降低;茉莉酸甲酯处理后FaWRKY70基因受诱导响应程度小,整体呈下调趋势。【结论】FaWRKY70能通过不同响应方式参与草莓生命活动及激素信号转导过程。

甘薯U6启动子克隆及其转录活性分析

在CRISPR/Cas9基因编辑系统中,U6启动子可以通过驱动sgRNA的表达来编辑目的基因,因而其转录活性会影响基因编辑效率。尽管人们已经在拟南芥、玉米、大豆、棉花等植物中开展了U6启动子的克隆与应用研究,然而目前在甘薯中U6启动子的相关研究还未见报道。本研究利用拟南芥的AtU6 SnRNA的保守序列在三浅裂野牵牛(Ipomoea trifida)基因组数据库中搜索候选U6 RNA,然后在其上游搜索到2个不同的候选U6启动子,长度分别为526 bp(IbU6p-1)和532 bp(IbU6p-2);序列比对分析结果显示,甘薯的U6启动子具有上游序列元件(USE)以及TATA-box,其序列也与拟南芥高度相似。然后,利用获得的U6启动子核酸序列构建了能够驱动萤火虫荧光素酶基因(LUC)表达的重组框U6::LUC。最后,将含有上述重组载体的根癌农杆菌瞬时转化到本氏烟草叶片和甘薯愈伤组织中,并通过荧光成像技术分析荧光素酶活性。结果发现,在烟草及甘薯愈伤组织中2个甘薯U6启动子均能驱动LUC基因表达,具有转录活性。同时,IbU6p-2的转录活性无论是在烟草叶片中还是在甘薯愈伤组织中都显著高于拟南芥U6启动子。本研究结果为进一步发展甘薯基因编辑技术提供了参考。

燕麦AsSOS1基因克隆及盐胁迫下的表达分析

为探索燕麦主要耐盐基因AsSOS1(Na^(+)/H^(+)逆向转运蛋白)的功能,本研究以耐盐品种青永久195为供试材料,在种子实生苗沙培3周后克隆AsSOS1基因,对其蛋白序列进行同源比对并分析理化性质,预测蛋白的跨膜结构和亲/疏水性以及二级和三级结构,用qRT-PCR分析100 mmol/L NaCl胁迫下AsSOS1基因在燕麦根、茎、叶中的表达。结果表明,AsSOS1的开放阅读框长度为3411 bp,编码1137个氨基酸;AsSOS1蛋白分子量为126.088 KDa,等电点6.62,属于酸性蛋白,稳定系数45.14,疏水性蛋白;AsSOS1蛋白二级结构中包含48.20%的α-螺旋和33.77%的无规则卷曲,三级结构与二级结构预测结果一致,以α-螺旋为主;跨膜结构域预测结果显示,AsSOS1蛋白中包含胞外结构域、跨膜螺旋结构域和胞内结构域,其中氨基酸序列11~424是Na^(+)/H^(+)逆向转运蛋白的保守序列,预测该区域与植物耐盐性相关;进化分析发现,其与硬直黑麦草的亲缘关系最近,相似度高达96%。正常生长条件下,AsSOS1基因在燕麦根、茎、叶中均有表达;在100 mmol/L NaCl胁迫下,AsSOS1基因在茎和根中的表达量分别增加了171.4%和54.1%,说明AsSOS1受盐胁迫诱导,在燕麦耐盐过程中起重要作用。