Comprehensive integration of single-cell transcriptomic data illuminates the regulatory network architecture of plant cell fate specification

Plant morphogenesis relies on precise gene expression programs at the proper time and position which is orchestrated by transcription factors(TFs)in intricate regulatory networks in a cell-type specific manner.Here we introduced a comprehensive single-cell transcriptomic atlas of Arabidopsis seedlings.This atlas is the result of meticulous integration of 63 previously published scRNA-seq datasets,addressing batch effects and conserving biological variance.This integration spans a broad spectrum of tissues,including both below-and above-ground parts.Utilizing a rigorous approach for cell type annotation,we identified 47 distinct cell types or states,largely expanding our current view of plant cell compositions.We systematically constructed cell-type specific gene regulatory networks and uncovered key regulators that act in a coordinated manner to control cell-type specific gene expression.Taken together,our study not only offers extensive plant cell atlas exploration that serves as a valuable resource,but also provides molecular insights into gene-regulatory programs that varies from different cell types.

Multi‑omics integration identifies regulatory factors underlying bovine subclinical mastitis

Background Mastitis caused by multiple factors remains one of the most common and costly disease of the dairy industry.Multi-omics approaches enable the comprehensive investigation of the complex interactions between mul-tiple layers of information to provide a more holistic view of disease pathogenesis.Therefore,this study investigated the genomic and epigenomic signatures and the possible regulatory mechanisms underlying subclinical mastitis by integrating RNA sequencing data(mRNA and lncRNA),small RNA sequencing data(miRNA)and DNA methylation sequencing data of milk somatic cells from 10 healthy cows and 20 cows with naturally occurring subclinical mastitis caused by Staphylococcus aureus or Staphylococcus chromogenes.Results Functional investigation of the data sets through gene set analysis uncovered 3458 biological process GO terms and 170 KEGG pathways with altered activities during subclinical mastitis,provided further insights into subclin-ical mastitis and revealed the involvement of multi-omics signatures in the altered immune responses and impaired mammary gland productivity during subclinical mastitis.The abundant genomic and epigenomic signatures with sig-nificant alterations related to subclinical mastitis were observed,including 30,846,2552,1276 and 57 differential methylation haplotype blocks(dMHBs),differentially expressed genes(DEGs),lncRNAs(DELs)and miRNAs(DEMs),respectively.Next,5 factors presenting the principal variation of differential multi-omics signatures were identified.The important roles of Factor 1(DEG,DEM and DEL)and Factor 2(dMHB and DEM),in the regulation of immune defense and impaired mammary gland functions during subclinical mastitis were revealed.Each of the omics within Factors 1 and 2 explained about 20%of the source of variation in subclinical mastitis.Also,networks of impor-tant functional gene sets with the involvement of multi-omics signatures were demonstrated,which contributed to a comprehensive view of the possible regulatory mechanisms underlying subclinical mastitis.Furthermore,multi-omics integration enabled the association of the epigenomic regulatory factors(dMHBs,DELs and DEMs)of altered genes in important pathways,such as‘Staphylococcus aureus infection pathway’and‘natural killer cell mediated cyto-toxicity pathway’,etc.,which provides further insights into mastitis regulatory mechanisms.Moreover,few multi-omics signatures(14 dMHBs,25 DEGs,18 DELs and 5 DEMs)were identified as candidate discriminant signatures with capac-ity of distinguishing subclinical mastitis cows from healthy cows.Conclusion The integration of genomic and epigenomic data by multi-omics approaches in this study provided a better understanding of the molecular mechanisms underlying subclinical mastitis and identified multi-omics candidate discriminant signatures for subclinical mastitis,which may ultimately lead to the development of more effective mastitis control and management strategies.

Integrative Analysis of Transcriptome and Phenolic Compounds Profile Provides Insights into the Quality of Soursop (Annona muricata L.) Fruit

Soursop(Annona muricata L.)is a tropical fruit highly valued for its uniqueflavor,nutritional value,and health-promoting properties.The ripening process of soursop involves complex changes in gene expression and metabo-lite accumulation,which have been studied using various omics technologies.Transcriptome analysis has provided insights into the regulation of key genes involved in ripening,while metabolic compound analysis has revealed the presence of numerous bioactive compounds with potential health benefits.However,the integration of transcrip-tome and metabolite compound data has not been extensively explored in soursop.Therefore,in this paper,we present a comprehensive analysis of the transcriptome and phenolic compound profiles of soursop during ripen-ing.The integration analysis showed that the genes and phenolic compounds were mainly involved in the starch and sucrose metabolism pathways during soursop ripening.Further,the phenolic compounds Kaempferol 3-Q-galactoside,Procyanidin C1,Procyanidin trimmer C1,and m-Coumaric,as well as the genes Ubiquitin-like protein 5(UBL5_ARATH),ATP-dependent zinc metalloprotease FTSH8(FTSH8_ORYSJ),Zinc transporter 4(ZIP4_AR-ATH),Thioredoxin-like 3-1(TRL31_ORYSJ),Mitogen-activated protein kinase YODA(YODA_ARATH),R-man-delonitrile lyase-like(MGL_ARATH),26s protease regulatory subunit 6A homolog(PRS6_SOLLC),Cytochrome P45072A13(C7A13ARATH),Cytochrome P45084A1(C84A1_ARATH)and Homoserine O-trans-acetylase(MET2-ORYSJ)were correlated and differentially accumulated and expressed,respectively.Our study provides new insights into the molecular mechanisms underlying soursop ripening and may contribute to the development of strategies for improving the nutritional quality and shelf life of this important fruit.

CONSTRUCTION OF A NEW GENE INTEGRATION PLATFORM SYSTEM FOR THE SYNECHOCOCCUS SP.PCC7942

According to the known sequence of iron stress-induced gene (isiAB operon), wecloned its 1. 5 kb fragment by PCR, and used this Fragment as integration homologous fragment. Afterseveral steps of subcloning donor DNA into the isiAB fragment, a donor plasmid pZL which could be inte-grated into the chromosomal DNA of Synechococcus sp. PCC7942 was constructed. In order to express theheterologous gene at a high level through the integration platform system, we constructed the donor DNAby the following steps. We cloned the strong promoter (240 bp) of heat shock gene groESL operon fromSynechococcus sp. PCC7942 by PCR. Then subcloned the multiple cloning sites (MCS), rbcS polyA intothe downstream of the groESL promoter. The kanamycin resistance gene, as the marker gene, was alsosubcloned into the donor DNA. Thus, in the donor plasmid pZL, the integration homologous fragment andseveral expression elements, such as groESL promoter, MCS, rbcS polyA teminator and kanamycin re-sistance gene, were all included.After naturally transformed and introduced the donor plasmid pZL into Synechococcus sp. PCC7942,as in the pZL, the donor DNA sequence is flanked by two DNA fragments (0.4 kb and 0.7 kb) homolo-gous to the isiAB fragment of Synechococcus sp. PCC7942, the homologous DNA can recombine with thechromosomal DNA. After screening by kanamycin, the transformants which integrated the heterologousDNA were selected. The efficiency of transformation is about 1×10-6. By southem blot analysis, it wasconfirmed that the donor DNA had been integrated into the chromosomal DNA of Synechococcus sp.PCC7942, located on the site of the isiAB gene, and can be replicated with the chromosomal DNA.

Homologous recombination mediates stable Fah gene integration and phenotypic correction in tyrosinaemia mouse-model

AIM To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout(Fah-/-) mice by homologous-recombination-mediated targeted addition of the Fah gene.METHODS C57 BL/6 Fah?exon5 mice served as an animal model for human tyrosinaemia type 1 in our study. The vector was created by amplifying human Fah c DNA including the TTR promoter from a lentivirus plasmid as described. The Fah expression cassette was flanked by homologous arms(620 bp and 749 bp long) of the Rosa26 gene locus. Mice were injected with 2.1 × 108 VP of this vector(r AAV8-ROSA26.HAL-TTR.FahROSA26.HAR) via the tail vein. Mice in the control group were injected with 2.1 × 108 VP of a similar vector but missing the homologous arms(r AAV8-TTR.Fah). Primary hepatocytes from Fah-/-recipient mice, treated with our vectors, were isolated and 1 × 106 hepatocytes were transplanted into secondary Fah-/-recipient mice by injection into the spleen. Upon either vector application or hepatocyte transplantation NTBC treatment was stopped in recipient mice. RESULTS Here, we report successful HR-mediated genome editing by integration of a Fah gene expression cassette into the "safe harbour locus" Rosa26 by recombinant AAV8. Both groups of mice showed long-term survival, weight gain and FAH positive clusters as determined by immunohistochemistry analysis of liver sections in the absence of NTBC treatment. In the group of C57 BL/6 Fah?exon5 mice, which have been transplanted with hepatocytes from a mouse injected with r AAV8-ROSA26.HAL-TTR.Fah-ROSA26.HAR 156 d before, 6 out of 6 mice showed long-term survival, weight gain and FAH positive clusters without need for NTBC treatment. In contrast only 1 out 5 mice, who received hepatocytes from r AAV8-TTR.Fah treated mice, survived and showed few and smaller FAH positive clusters. These results demonstrate that homologous recombinationmediated Fah gene transfer corrects the phenotype in a mouse model of human tyrosinaemia type 1(Fah-/-mice) and is long lasting in a proliferating state of the liver as shown by withdrawal of NTBC treatment and serial transplantation of isolated hepatocytes from primary Fah-/-recipient mice into secondary Fah-/-recipient mice. This long term therapeutic efficacy is clearly superior to our control mice treated with episomal r AAV8 gene therapy approach.CONCLUSION HR-mediated r AAV8 gene therapy provides targeted transgene integration and phenotypic correction in Fah-/-mice with superior long-term efficacy compared to episomal r AAV8 therapy in proliferating livers.

iCEMIGE: Integration of CEll-morphometrics, MIcrobiome, and GEne biomarker signatures for risk stratification in breast cancers

BACKGROUND The development of precision medicine is essential for personalized treatment and improved clinical outcome,whereas biomarkers are critical for the success of precision therapies.AIM To investigate whether iCEMIGE(integration of CEll-morphometrics,MIcro-biome,and GEne biomarker signatures)improves risk stratification of breast cancer(BC)patients.METHODS We used our recently developed machine learning technique to identify cellular morphometric biomarkers(CMBs)from the whole histological slide images in The Cancer Genome Atlas(TCGA)breast cancer(TCGA-BRCA)cohort.Multivariate Cox regression was used to assess whether cell-morphometrics prognosis score(CMPS)and our previously reported 12-gene expression prognosis score(GEPS)and 15-microbe abundance prognosis score(MAPS)were independent prognostic factors.iCEMIGE was built upon the sparse representation learning technique.The iCEMIGE scoring model performance was measured by the area under the receiver operating characteristic curve compared to CMPS,GEPS,or MAPS alone.Nomogram models were created to predict overall survival(OS)and progress-free survival(PFS)rates at 5-and 10-year in the TCGA-BRCA cohort.RESULTS We identified 39 CMBs that were used to create a CMPS system in BCs.CMPS,GEPS,and MAPS were found to be significantly independently associated with OS.We then established an iCEMIGE scoring system for risk stratification of BC patients.The iGEMIGE score has a significant prognostic value for OS and PFS independent of clinical factors(age,stage,and estrogen and progesterone receptor status)and PAM50-based molecular subtype.Importantly,the iCEMIGE score significantly increased the power to predict OS and PFS compared to CMPS,GEPS,or MAPS alone.CONCLUSION Our study demonstrates a novel and generic artificial intelligence framework for multimodal data integration toward improving prognosis risk stratification of BC patients,which can be extended to other types of cancer.

Integrated Expression of the Oenococcus oeni mleA Gene in Saccharomyces cerevisiae

Malolactic enzyme is the function enzyme which catalyses the reaction for L-malate converting to L-lactic during malolactic fermentation （MLF）. In this paper, researches concerning the malolactic enzyme gene mleA cloned from a patent strain Oenococcus oeni SD-2a screened in Chinese wine and integrated expressing in Saccharomyces cerevisiae were performed in order to carry out both alcoholic fermentation （AF） and malolactic fermentation （MLF） during winemaking, with a view to achieving a better control of MLF in enology. To construct the expression plasmid named pYILmleA, cloned mleA gene, PGK1 promoter, and ADH1 terminator were ligated and inserted into integrating vector YIp5. Yeast transformants were screened on SD/-Ura and identified by auxotrophic test, mating type test, and colony PCR. Target protein was detected by SDS-PAGE and the targeted gene integrated to the chromosome was detected by dot bloting hybridization. After the transformant was cultured in SD/-Ura adding glucose （10%） and L-malate （5 648 mg L-1） for 4 d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC. 1 278-1 312 mg L-1 L-lactic acids were detected, while the comparative drop rates of L-malate were 20.18-20.85%. L-malate and L-lactic contents of the transformants showed extra significant difference and significant difference with the control ones by t-test respectively. The result indicated that the functional expression was achieved in recombinants S. cerevisiae.

Identification of metastasis-associated genes in colorectal cancer through an integrated genomic and transcriptomic analysis

Objective： Identification of colorectal cancer （CRC） metastasis genes is one of the most important issues in CRC research. For the purpose of mining CRC metastasis-associated genes, an integrated analysis of mJcroarray data was presented, by combined with evidence acquired from comparative genornic hybridization （CGH） data. Methods： Gene expression profile data of CRC samples were obtained at Gene Expression Omnibus （GEO） website. The 15 important chromosomal aberration sites detected by using CGH technology were used for integrated genomic and transcriptomic analysis. Significant Analysis of Microarray （SAM） was used to detect significantly differentially expressed genes across the whole genome. The overlapping genes were selected in their corresponding chromosomal aberration regions, and analyzed by using the Database for Annotation, Visualization and Integrated Discovery （DAVID）. Finally, SVM-T-RFE gene selection algorithm was applied to identify ted genes in CRC. Results： A minimum gene set was obtained with the minimum number [14] of genes, and the highest classification accuracy （100%） in both PRI and META datasets. A fraction of selected genes are associated with CRC or its metastasis. Conclusions- Our results demonstrated that integration analysis is an effective strategy for mining cancer- associated genes.

Identification of key pathways and genes from gastric precancerous lesions to gastric cancer by integrated bioinformatics analysis

Objective:This work aimed to illuminate the potential key genes and pathways in GC tumorigenesis based on bioinfOrmatics analysis.Methods:The differentially expressed genes(DEGs)between GPL tissue samples and GC tissue samples were investigated using the GSE55696 and GSE87666 microarray data from the Gene Expression Omnibus(GEO)database.DEGs were identified by an empirical Bayes method based on the Limma R package.Then,KEGG and GO enrichment analyses of DEGs were performed followed by protein-protein interaction(PPI)network construction.Finally,the overall survival(OS)analysis of key genes was performed by the Kaplan-Meier plotter online tool.Results:A total of 250 DEGs were obtained,of which 216 were up-regulated and 34 were down-regulated.KEGG pathways analysis showed that the up-regulated DEGs were enriched in cytokine-cytokine receptor interaction,chemokine signaling pathway,metabolic pathways,PI3K-Akt signaling pathway,NF-kappa B signaling pathway,and other signaling pathways about cancer,while no down-regulated pathways were enriched.A PPI network of DEGs was constructed with 117 nodes and 660 edges,and 20 genes were selected as hub genes owing to high degrees in the network.According to the Kaplan-Meier analysis,6 out of 20 hub genes including CCR7,FPR1,C3,CXCR5,GNB4,and PPBP with high mRNA expression were associated with poor OS for GC patients.Conclusion:The results of this study provide possible factors for the occurrence of GC,and the identification of the genes and pathways associated with the progression from GPL to GC provides valuable data for investigating the pathogenesis in future studies.

Bioorthogonal Engineered Virus‑Like Nanoparticles for Efficient Gene Therapy

Gene therapy offers potentially transformative strategies for major human diseases.However,one of the key challenges in gene therapy is developing an effective strategy that could deliver genes into the specific tissue.Here,we report a novel virus-like nanoparticle,the bioorthgonal engineered viruslike recombinant biosome(reBiosome),for efficient gene therapies of cancer and inflammatory diseases.The mutant virus-like biosome(mBiosome)is first prepared by site-specific codon mutation for displaying 4-azido-L-phenylalanine on vesicular stomatitis virus glycoprotein of eBiosome at a rational site,and the reBiosome is then prepared by clicking weak acid-responsive hydrophilic polymer onto the mBiosome via bioorthogonal chemistry.The results show that the reBiosome exhibits reduced virus-like immunogenicity,prolonged blood circulation time and enhanced gene delivery efficiency to weakly acidic foci(like tumor and arthritic tissue).Furthermore,reBiosome demonstrates robust therapeutic efficacy in breast cancer and arthritis by delivering gene editing and silencing systems,respectively.In conclusion,this study develops a universal,safe and efficient platform for gene therapies for cancer and inflammatory diseases.

Integration of genome scale data for identifying newplayers in colorectal cancer

Colorectal cancers(CRCs) display a wide variety of genomic aberrations that may be either causally linked to their development and progression, or might serve as biomarkers for their presence. Recent advances in rapid high-throughput genetic and genomic analysis have helped to identify a plethora of alterations that can potentially serve as new cancer biomarkers, and thus help to improve CRC diagnosis, prognosis, and treatment. Each distinct data type(copy number variations, gene and micro RNAs expression, Cp G island methylation) provides an investigator with a different, partially independent, and complementary view of the entire genome. However, elucidation of gene function will require more information than can be provided by analyzing a single type of data. The integration of knowledge obtained from different sources is becoming increasingly essential for obtaining an interdisciplinary view of large amounts of information, and also for cross-validating experimental results. The integration of numerous types of genetic and genomic data derived from public sources, and via the use of ad-hoc bioinformatics tools and statistical methods facilitates the discovery and validation of novel, informative biomarkers. This combinatory approach will also enable researchers to more accurately and comprehensively understand the associations between different biologic pathways, mechanisms, and phenomena, and gain new insights into the etiology of CRC.

基于乡愁文化基因解码的乡村文旅融合路径研究

基于乡村居民视觉系统解析乡愁文化内涵,并探讨乡村文旅深度融合发展路径,是留住乡愁、助力乡村振兴的有效途径。借鉴生物学基因遗传研究中的逆转录提取法,结合扎根理论提取出乡愁文化的八大基因,进一步运用共现分析和社会网络分析法,探究乡愁文化基因的层级结构。研究发现:(1)乡愁文化基因包括家庭牵绊、乡村生活、自然景观、建筑风貌、劳作场景、文化记忆、地方节庆、传统技艺;(2)乡愁文化基因按照其重要程度呈现四大层级结构,结合功能属性将八大乡愁文化基因划分为主体基因、附着基因、混合基因、变异基因;(3)基于乡愁文化基因及其层次结构特征,最终提出四大乡村旅游融合发展的路径:主体基因决定旅游发展主题、附着基因指导旅游景观设计、混合基因融入旅游文化空间建设、变异基因指导文创产品开发。

从“依形套式”到“循章衍法”:构形机理导引的历史城镇空间基因整体识别与活态传承

针对历史城镇空间基因识别、保护实践“有形无态、重式轻法”问题,提出构形机理导引的空间基因整体识别与活态传承方法。以“原型特征+构形机理”的显隐属性和“城镇格局、地块形态、街巷形式、建筑风貌”四重空间尺度为历史城镇空间基因要素解析框架,以“要素特征与结构特征同步提取”和“原型解构到显型建构机理延续”为操作路径,通过川渝地区历史城镇/街区实证研究,提出历史城镇格局结构要素与建构逻辑、地块图底肌理与聚合关系、街巷局域特征与系统节奏、建筑绝对形态与相对尺度同步提取的空间基因整体识别方法,以及城镇格局结构要素保控+基质空间重构、地块空间图底肌理修补+立体形态调节、街巷空间典型断面整饬+系统节奏保持、建筑空间异化风貌矫正+建构传统赓续的空间基因活态传承方法,以促进历史城镇空间基因保护实践从“依形套式”到“循章衍法”的路径转变。

Impact of Copy Number on Transgene Expression In Tobacco

对农杆菌介导法获得的转 β_葡糖醛酸酶 (β_glucuronidase ,GUS)基因 (uidA)烟草 (NicotianatabacumL .)进行GUS表达分析 ,发现部分转基因植株无GUS活性。进一步Southern杂交结果发现 ,GUS基因失活植株的基因组中整合了多个uidA拷贝 ,而GUS活性高的转基因植株多为uidA单拷贝整合 ,表明uidA基因失活与基因多拷贝整合有关。Northern杂交结果显示 ,失活植株无特异uidARNA杂交带 ,而GUS活性高的植株可检测到明显的杂交信号 ,说明多拷贝引起的基因失活发生在RNA水平。

Gene Ontology在生物数据整合中的应用

异构数据的高效整合,在生物数据呈爆炸性增长、生物数据库复杂度不断增加的今天,具有重要的理论价值和实际意义。该文基于BioDW——一个整合的生物信息学数据仓库平台,利用统一的GeneOntology语义模型,建立异构数据库之间的语义链接,在概念和联系层次上有效地解决了生物异构数据的整合问题,实现了对生物数据智能化的多重、复合和交叉检索,为生物信息的进一步研究奠定了坚实的基础。

酿酒酵母转座子介导外源基因的整合效率

为了比较分析酿酒酵母不同转座子对外源基因整合效率的影响,构建了转座子Ty1、Ty2和Ty4介导的G418表达盒及酿酒酵母BY4741重组菌BY-Ty1、BY-Ty2、BY-Ty4。研究发现,Ty2介导的整合型表达G418的BY4741重组菌转化平均菌落数为122,整合效率为93.33%,均高于Ty1和Ty4。Ty2介导的G418抗性重组菌BY-Ty2,比BY-Ty1和BY-Ty4对G418抗耐受性更强,基因拷贝数更高。结果表明,转座子Ty2更适合在酿酒酵母中介导外源基因整合。

HPV与宿主基因整合在宫颈病变发生发展中的作用机制研究进展

宫颈癌病死率较高,高危人乳头瘤病毒(HPV)持续感染是其主要致病原因,其中HPV与宿主基因整合在宫颈病变的发生发展中发挥关键作用。目前,以三代测序技术为代表的新兴技术已用于HPV整合位点及基因改变等的相关检测。HPV整合后可能通过基因扩增、染色体结构改变、改变DNA序列甲基化状态、形成染色体外DNA、导致宿主致癌基因激活和抑癌基因失活以及影响微RNA的表达等发挥促癌作用。因此,深入研究HPV与宿主基因整合在宫颈病变发生发展中的作用机制,可以为相关疾病的治疗提供新思路。

基于药理学分析巴戟天治疗阿尔茨海默病的机制

目的:基于网络药理学、分子对接和GEO数据分析巴戟天治疗阿尔茨海默病(Alzheimer's disease,AD)的潜在靶点和作用机制。方法:利用中药系统药理学数据库与分析平台(TCMSP),获取巴戟天的主要活性成分,用SwissTargetPrediction获取巴戟天全部作用靶点。从DrugBank、PathCard、Chemogenomic Database和PubChem数据库获得AD相关靶点。使用韦恩图取交集,得到巴戟天治疗AD的共同作用靶点。利用Cytoscape3.8.0构建靶点的“成分-靶点”网络图,并分析靶点的相互作用PPI网络图、基因本体论(GO)和KEGG信号通路等。使用Autodock对关键成分和靶点进行分子对接,并用Pymol和Discovery Studio展示对接结果。最后利用GEO数据库Alzdata分析关键靶点基因在AD中的表达。结果:预测得到巴戟天的50种主要活性成分,636个作用靶点,674个AD相关靶点,其中巴戟天治疗AD共同靶点124个。GO富集分析得到蛋白质磷酸化、磷酸化的正调控、细胞对氮化合物的反应、水解酶活性的调节、细胞对化学刺激的反应。KEGG富集分析显示阿尔茨海默病为最显著的通路。分子对接显示,巴戟天的5个核心成分2-羟基-1,5-二甲氧基-6-(甲氧基甲基)-9,10-蒽醌、1-羟基-3-甲氧基-9,10-蒽醌、大黄素-A、甲基异茜草素、甲基异茜草素-1-甲醚和3个核心靶点EGFR、PARP1、FTO的结合较好,并且Egfr在AD病人中显著(P<0.05)上调表达,Parp1和Fto在AD病人中显著(P<0.05)下调表达。结论:巴戟天可能通过多个成分、多个靶点、多个通路,参与调控AD疾病进程。

文化基因视角下福马同城生活圈建构研究

两岸融合发展是我国推进和平统一进程的重大战略,两岸融合发展一旦缺失文化力量,就可能迷失方向。为此,从文化基因视角探索福马同城生活圈的建构具有重要时代价值。面对福马文化的传承断裂化、影响脆弱化、供需失衡化等文化困境,必须通过充分发挥福马文化各类型的多元驱动力、文化产业+产业文化双融合创新等途径,完善福马同城生活圈的建构机制。

Construction of Bacillus subtilis GMP Reductase Gene(guaC)Mutant

An integrated expression vector pGT9GH was constructed by using guaC gene of receptor strain as guide sequence for homologous recombination.And then,by using the method of double crossover recombination between plasmid and chromosome,the linearized plasmid pGT9GH were integrated into B.subtilis GJ06 to get B.subtilis GJ07 with guaC gene mutant,in which,one copy of riboflavin operon was inserted between 5' and 3' end of guaC gene.The homologous recombination events were confirmed by PCR methods,and then,the riboflavin yield of mutant strain GJ07 was measured.The results of shake flask culture showed that the production of riboflavin of GJ07 was 24.5% higher than that of GJ06 in 60 h.