Investigation on the Interface between Exons and Introns in the Genomic DNA of Arabidopsis thaliana in the Phase Space

In this study,three weight vectors L1,L2 and L3 were set.After calculating the probability of three bases in the exons or introns in the genomic DNA of Arabidopsis thaliana,64-dimensional vector P was obtained.Dot products of P vector and three weight vectors were the feature coordinates for the exons and introns in 3-dimensional phase space.The expression for the interface between the exons and the introns in the genomic DNA of Arabidopsis thaliana in 3-dimensional phase space was established,which could be used to distinguish the exons and the introns in the genomic DNA of Arabidopsis thaliana with an accuracy higher than85%in 3-dimensional phase space.

A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana

The newly developed CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas(CRISPR-associated) system has emerged as an efficient tool for genome-editing, providing an alternative to classical mutagenesis and transgenic methods to study gene function and improve crop traits. CRISPR/Cas facilitates targeted gene editing through RNA-guided DNA cleavage followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. Here we describe a detailed procedure for our previously developed and highly efficient CRISPR/Cas9 method that allows the generation of heritable-targeted gene mutations andcorrections in Arabidopsis. This protocol describes the strategies and steps for the selection of targets, design of single-guide RNA(sg RNA), vector construction and analysis of transgenic lines. We also offer a method to target two loci simultaneously using vectors containing two different sg RNAs. The principles described in this protocol can be applied to other plant species to generate stably inherited DNA modifications.

Effort and Contribution of T-DNA Insertion Mutant Library for Rice Functional Genomics Research in China:Review and Perspective

With the completion of the rice （Oryza sativa L.） genome-sequencing project, the rice research community proposed to characterize the func- tion of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.

Boosting wheat functional genomics via an indexed EMS mutant library of KN9204

A better understanding of wheat functional genomics can improve targeted breeding for better agronomic traits and environmental adaptation.However,the lack of gene-indexed mutants and the low transformation efficiency of wheat limit in-depth gene functional studies and genetic manipulation for breeding.In this study,we created a library for KN9204,a popular wheat variety in northern China,with a reference genome,transcriptome,and epigenome of different tissues,using ethyl methyl sulfonate(EMS)mutagenesis.This library contains a vast developmental diversity of critical tissues and transition stages.Exome capture sequencing of 2090 mutant lines using KN9204 genome-designed probes revealed that 98.79%of coding genes had mutations,and each line had an average of 1383 EMS-type SNPs.We identified new allelic variations for crucial agronomic trait-related genes such as Rht-D1,Q,TaTB1,and WFZP.We tested 100 lines with severemutations in 80 NAC transcription factors(TFs)under drought and salinity stress and identified 13 lines with altered sensitivity.Further analysis of three lines using transcriptome and chromatin accessibility data revealed hundreds of direct NAC targets with altered transcription patterns under salt or drought stress,including SNAC1,DREB2B,CML16,and ZFP182,factors known to respond to abiotic stress.Thus,we have generated and indexed a KN9204 EMS mutant library that can facilitate functional genomics research and offer resources for genetic manipulation of wheat.

Genome-wide mining, characterization, and development of microsatellite markers in Marsupenaeus japonicus by genome survey sequencing

The kuruma prawn, Marsupenaeus japonicus, is one of the most cultivated and consumed species of shrimp. However, very few molecular genetic/genomic resources are publically available for it. Thus, the characterization and distribution of simple sequence repeats(SSRs) remains ambiguous and the use of SSR markers in genomic studies and marker-assisted selection is limited. The goal of this study is to characterize and develop genome-wide SSR markers in M. japonicus by genome survey sequencing for application in comparative genomics and breeding. A total of 326 945 perfect SSRs were identified, among which dinucleotide repeats were the most frequent class(44.08%), followed by mononucleotides(29.67%), trinucleotides(18.96%), tetranucleotides(5.66%), hexanucleotides(1.07%), and pentanucleotides(0.56%). In total, 151 541 SSR loci primers were successfully designed. A subset of 30 SSR primer pairs were synthesized and tested in 42 individuals from a wild population, of which 27 loci(90.0%) were successfully amplified with specific products and 24(80.0%) were polymorphic. For the amplified polymorphic loci, the alleles ranged from 5 to 17(with an average of 9.63), and the average PIC value was 0.796. A total of 58 256 SSR-containing sequences had significant Gene Ontology annotation; these are good functional molecular marker candidates for association studies and comparative genomic analysis. The newly identified SSRs significantly contribute to the M. japonicus genomic resources and will facilitate a number of genetic and genomic studies, including high density linkage mapping, genome-wide association analysis, marker-aided selection, comparative genomics analysis, population genetics, and evolution.

Experimental genomics:The application of DNA microarrays in cellular and molecular biology studies

The genome sequence information in combination with DNA microarrays promises to revolutionize the way of cellu-lar and molecular biological research by allowing complex mixtures of RNA and DNA to interrogated in a parallel and quantita-tive fashion. DNA microarrays can be used to measure levels of gene expression for tens of thousands of gene simultane-ously and take advantage of all available sequence information for experimental design and data interpretation in pursuit of biological understanding. Recent progress in experimental genomics allows DNA microarrays not simply to provide a cata-logue of all the genes and information about their function, but to understand how the components work together to comprise functioning cells and organisms. This brief review gives a survey of DNA microarrays technology and its applications in ge-nome and gene function analysis, gene expression studies, biological signal and defense system, cell cycle regulation, mechanism of transcriptional regulation, proteomics, and the functionality of food component.

从品种特性到功能基因组:双峰驼基因组学研究进展

双峰驼是亚洲中部戈壁沙漠地区重要的畜种之一,具有耐寒、耐热、耐饥饿、免疫系统较强等独特的生物学特性,受到国内外学者的广泛关注。近年来,随着生物测序技术的快速发展,双峰驼全基因组测序及其功能基因组的研究相继发布。该文归纳了不同家养双峰驼品种特性,总结了双峰驼全基因组研究现状,回顾了双峰驼起源驯化历程,并重点介绍了双峰驼沙漠适应能力和免疫相关功能基因的研究进展,为全面了解双峰驼独特的生物学特性和遗传改良提供理论参考。

桑树基因组学研究进展

栽桑养蚕的历史对整个人类文明产生了重要的影响。桑树是多年生木本植物,具有重要的经济价值、药用价值和生态价值。自2013年桑树基因组测序项目实施以来,桑树的研究进入了基因组学时代,为桑树的分子育种奠定了基础。本文从桑树基因组测序的实施开始,系统总结了桑树基因组学研究的最新进展,包括了测序技术、拼接策略、组装质量以及基于全基因组重测序应用等方面。未来,随着技术的不断创新,桑树基因组学研究将迎来更广阔的发展前景。本文旨在推动桑树产业的发展并为林木基因组学的研究提供理论与技术参考。

利用转录组测序挖掘奶牛基因多态位点的准确性研究

转录组测序(RNA-Seq)不仅用于揭示基因表达谱,同时还可以检测基因多态位点,是挖掘奶牛蛋白编码基因功能突变的有效策略。本研究对20头中国荷斯坦牛进行全基因组测序(DNA-Seq)与转录组测序(RNA-Seq),以DNA-Seq检出的单核苷酸多态位点(SNP)为参考基准,评估RNA-Seq检出SNP的准确性及其影响因素。结果显示,同一个体DNA-Seq和RNA-Seq的SNP位点一致性为0.65~0.75,而不同个体间的一致性仅为0.28~0.42。通过优化RNA-Seq检测SNP的筛选标准,包括针对连续SNP(SnpCluster)、单一连续碱基(Homopolymer)、测序深度(DP<5)、位点基因型频率(P<0.13)等4个过滤参数的优化,检出的高准确性SNP占比由71.07%提升至95.27%。最后,经功能注释筛选出10号染色体上E1BHN1基因一个起始密码子突变(BTA10:25435325 T/A),该突变导致免疫球蛋白结构域类似物蛋白从异四聚体变成异二聚体,推测该位点可能为奶牛隐性致死突变位点。

L. plantarum SGJ-24 全基因组测序及活性 相关基因的挖掘和分析

为全面了解植物乳杆菌SGJ-24的遗传信息,深入解析该菌与耐受酒体“恶劣”环境、生物降酸、生物胺降解活性有关的功能基因,本文通过二代Illumina HiSeq联合三代PacBio RS II测序技术,对该菌株开展全基因组测序和分析。结果表明,该菌株的基因组序列全长为3451191 bp,编码基因数量为3361,注释到营养物(糖类、氨基酸等)转运和代谢、蛋白质合成、核酸代谢等功能的基因数量较多。此外,对该菌株的功能基因挖掘过程中,发现其含有与苹果酸乳酸发酵活性(MLE等)、耐酸性(gadB等)、耐乙醇(PFK、GK等)和降解生物胺(GAPDH)功能有关的基因。研究结果深入揭示了其遗传信息特征,为进一步开发利用该菌株提供了理论依据。

产细菌素贝莱斯芽孢杆菌CM7-4全基因组测序及抑菌活性研究

【目的】贝莱斯芽孢杆菌(Bacillus velezensis)CM7-4是从海水中分离的具有良好抗菌活性的芽孢杆菌。本试验旨在探究贝莱斯芽孢杆菌CM7-4的抗菌机理,并对其全基因组序列进行分析,以期挖掘其抗菌等功能特性基因。【方法】通过酸沉淀法得到贝莱斯芽孢杆菌CM7-4的细菌素粗提物,通过固体琼脂打孔法测定蛋白酶K对其抑菌效果的影响;基于MGI DNBSEQ-T7平台对贝莱斯芽孢杆菌CM7-4进行全基因组测序分析,通过序列组装、基因预测和功能注释分析其产细菌素的潜质。【结果】贝莱斯芽孢杆菌CM7-4的细菌素粗提物对大肠杆菌、沙门菌、粪肠球菌、单增李斯特菌等8种不同的革兰阴性和阳性菌具有良好的抑菌效果;与对照组相比,经蛋白酶K处理后细菌素粗提物对8种指示菌的抑菌效果均极显著下降(P<0.01);全基因组测序显示,贝莱斯芽孢杆菌CM7-4的基因组大小为3953124 bp,GC含量为47.04%;共预测到3870个蛋白编码基因、9个rRNA、81个tRNA、1个ncRNA;基因组中含有11个与细菌素合成相关的基因、10个与抗氧化活性相关基因,以及4个与调控免疫和炎症信号通路相关的基因。【结论】贝莱斯芽孢杆菌CM7-4是一株潜在的产细菌素菌株,为进一步研究其抗菌特性提供了理论基础。

产脂肪酶木糖葡萄球菌YCC3全基因组测序及序列分析

木糖葡萄球菌(Staphylococcus xylosus,S.xylosus)YCC3是从发酵肉制品中分离筛选到的1株产脂肪酶活性高的菌株,为研究该菌株在香肠发酵过程中的代谢机理和功能,采用PromethION和Illumina HiSeq测序平台对S.xylosus YCC3进行完成图测序分析。结果表明,S.xylosus YCC3基因组为1套含有3个质粒的环状分子,基因组序列总长度为2773035 bp,GC含量(鸟嘌呤和胞嘧啶占基因组序列的比率)为32.88%。基因组中预测到2540个编码基因,编码基因总长度为2742136 bp,平均长度为1079.58 bp,占基因组的83.24%。S.xylosus YCC3的编码基因通过GO(Gene Ontology)数据库注释,预测到与抗氧化活性相关的基因3个;COG(Cluster of Orthologous Groups of Proteins)数据库注释到与脂质运输和代谢相关的基因中有8个含有脂肪酸合成基因、4个含有脂肪水解酶基因;KEGG(Kyoto Encyclopedia of Genes and Genomes)数据库注释到与脂肪酸合成相关的基因16个,与脂肪酸降解相关的基因10个,与不饱和脂肪酸合成相关的基因3个。S.xylosus菌株的基因组基本功能注释和代谢通路基因信息注释旨在为菌株作为发酵剂在香肠发酵中的应用提供一定的理论依据。

Amycolatopsis sp.的全基因组测序及香兰素合成途径分析

拟无枝酸菌(Amycolatopsis sp.)CCTCC NO:M2011265是一株可以转化阿魏酸生成香兰素菌株,该研究利用Illumina Hiseq测序平台对拟无枝酸菌进行全基因组测序、拼接、基因预测及功能注释,并从拟无枝酸菌的全基因组中筛选和鉴定参与香兰素合成的功能基因。结果表明,总共组装得到64个scaffolds,整个基因组组装大小约为8425551 bp,总GC含量为71.89%。通过生物信息学分析发现了香兰素合成关键基因ech、fcs和ech2基因,及香兰素分解代谢基因vdh基因,其中ech和fcs为一个基因簇,并进一步构建过表达ech-fcs-ech2菌株,其摇瓶发酵表明转化阿魏酸生成香兰素速率加快,发酵时间显著缩短。该研究获得的拟无枝酸菌的全基因组信息为解析其转化阿魏酸生成香兰素发酵过程中的代谢机理提供遗传信息基础,也为通过代谢工程获得高产香兰素菌株的研究提供理论支持。

氨肽酶生产菌株Bacillus subtilis LBJ4-5的全基因组测序及基因功能分析

本研究从郫县豆瓣中分离获得一株高产氨肽酶菌株B. subtilis LBJ4-5,通过溶血性试验及抗生素敏感性试验评估了其安全性。同时,采用Illumina二代测序技术和第三代高通量Pacbio测序平台对B. subtilis LBJ4-5进行全基因组测序,获得其基因组特征信息,并通过COG、GO、KEGG、CAZY、CARD和VFDB等数据库进行基因预测及功能注释,以及评价其氨肽酶生产性能和安全性。结果显示,B. subtilis LBJ4-5经48 h摇瓶培养后发酵上清中的氨肽酶活达到179.23 U/mL,菌株无溶血活性且对头孢唑林、万古霉素、青霉素G、头孢氨苄等10种抗生素均高度敏感(直径≥20 mm),显示出良好的安全性。B. subtilis LBJ4-5的基因组由一条环状闭合DNA及一个环状质粒组成,大小为4053926 bp,GC含量为43.66%,共预测到4285个蛋白质编码基因、85个tRNA基因、33个rRNA基因、119个sRNA。在COG、GO、KEGG、CAZY、CARD和VFDB数据库中分别注释到3386、2581、2552、144、256和401个功能基因。进一步的信息挖掘分析表明,B. subtilis LBJ4-5的基因组中含有31个氨肽酶编码基因。而毒力基因与耐药性基因的预测分析,进一步证实了B. subtilis LBJ4-5菌株的安全性。综上,B.subtilis LBJ4-5可用作发酵食品加工的发酵菌剂。

一株羊口疮病毒的全基因组测序及功能分析

为了更好了解羊口疮病毒(Orf virus,ORFV)基因组的数据库信息及功能分析,本试验通过Illumina TruSeq^(TM)Nano DNA Sample Prep Kit方法构建文库对分离株ORFV-Q进行全基因组测序,进行NR注释、GO注释、KEGG注释等进行生物信息学分析。试验结果显示,ORFV-Q株基因组大小为127160 bp,GC含量分布未见异常,预测到123个基因。根据NR数据库注释对比结果,有116个基因得到注释;根据GO数据库注释,其中参与生物学途径基因有30个,细胞组分基因有33个,分子功能基因有8个;通过与KEGG代谢通路数据库进行比对,有5个基因可以对应;Swiss-Prot数据库注释共有75个基因的蛋白序列功能被注释。本研究通过对ORFV-Q株进行全基因组测序,并对其进行基因组组分分析和基因功能注释,不仅为ORFV基因组的研究提供数据支持,也为后续疫苗的开发研究提供了理论依据。

27株关中黑猪肠道食淀粉乳杆菌基因组分析

研究从基因组水平解析27株关中黑猪肠道食淀粉乳杆菌的遗传背景和功能基因特征,采用Illumina测序技术完成27株食淀粉乳杆菌的全基因组测序、框架图组装,通过平均核苷酸一致性分析、系统进化分析、泛基因组分析等比较基因组学方法分析菌株间的同源性和分子进化规律,使用eggNOG、CAZy、ResFinder、VFDB等数据库注释功能基因。结果显示,27株食淀粉乳杆菌平均基因组序列大小为1881796 bp,平均GC含量为38.10%,菌株间具有高度同源性和遗传多样性。功能基因注释显示,这些菌株不仅具备良好的生存能力和安全性,还具有环境应激、氧化应激、核黄素合成、胞外多糖分泌和群体感应等潜在益生基因。研究表明,分离自关中黑猪肠道的27株食淀粉乳杆菌适合作为候选益生菌株。

西藏牦牛源牛支原体T10株全基因组测序及其序列分析

旨在进一步完善牛支原体全基因组数据库,阐明其生物学功能和遗传进化关系,对西藏牦牛源牛支原体T10株进行了全基因组测序及其序列分析。使用Nanopore和Illumina PE150测序平台对T10株进行全基因组序列测定,利用生物信息学对其进行基因组特征分析和利用基因系统进化树与国内外分离株进行遗传进化关系比对。基因测序结果显示,T10株全基因组大小为987 812 bp, GC含量为29.27%,预测到822个编码基因,编码基因总长度为709 129 bp;通过功能数据库注释结果显示,注释到与膜转运蛋白相关基因22个,碳水化合物酶相关基因7个、糖基转移酶类相关基因4个,分泌蛋白基因相关43个、T3SS效应蛋白相关基因51个,病原与宿主互作相关基因28个,细菌毒力相关基因14个,抗性相关基因10个;通过比较基因组学分析结果显示,T10与08M、CQ-W70、Hubei-1、Ningxia-1和PG45均存在氨基酸突变和基因片段的插入或缺失,以及基因家族数量的差异,其中基因家族数量差异在8~32个。通过遗传进化关系分析结果显示:T10与HB0801、16M、Hubei-1、CQ-W70、NM2012、Ningxia-1、08M、JF4278、KG4397和PG45均在同一分支,但与PG1、PG2处于不同分支,其中与HB0801亲缘关系最近,与PG45亲缘关系较远。本研究不仅完善了牛支原体全基因组数据库,详细阐述了与致病性相关基因,还充分阐明了T10株以及与国内外其他牛支原体之间的遗传进化关系,明确了T10株基本基因组信息以及与国内外菌株亲缘关系,为后续进行致病机制以及疫苗研究提供理论依据。

产褐藻胶裂解酶菌株S10的鉴定、全基因组测序及分析

为了详细解析从仿刺参肠道中分离出1株具有较高酶活力高产褐藻胶裂解酶菌株S10的基因组序列信息,进而深入挖掘与褐藻胶裂解酶相关的基因资源,本研究通过形态学观察和16S rRNA序列分析对菌株S10进行菌种鉴定,然后利用Illumina二代测序技术和第三代高通量PacBio测序平台对菌株S10进行全基因组测序,并使用相关软件对测序数据进行基因组组装、基因预测及基因功能注释等。此外,根据注释结果对菌株S10中所含的3组假定褐藻胶裂解酶基因序列进行生物信息学分析及结构预测。结果表明,菌株S10被鉴定为Vibrio alginolyticus,基因组总长度为5397046 bp,GC含量为44.59%,由2条染色体和1条质粒组成。预测共有4936个编码基因,127个tRNA基因和37个rRNA基因。在直系同源集、基因本体论、京都基因与基因组百科全书和碳水化合物活性酶数据库中分别注释到4039、3163、3104个和96个功能基因。此外,在菌株S10中发现了3组潜在的褐藻胶裂解酶基因alg4755、alg4756和alg4760。生物信息学分析结果表明,褐藻胶裂解酶Alg4755、Alg4756和Alg4760均属于多糖裂解酶家族7(polysaccharide lyases,PL7)蛋白,具有3个PL7家族高度保守的基序(R*E*R、Q(I/V)H、Y*KAG*Y*Q)。综上,S10菌株全基因组测序及分析对该菌的高效产酶机制研究和新型褐藻胶裂解酶的挖掘具有重要意义,可为后期酶的表达及工业化应用提供理论基础。

一株猪源德尔卑沙门氏菌的分离鉴定、生物学特性以及全基因组序列分析

沙门氏菌作为人畜共患的致病菌,更是极易产生耐药性,严重影响了人类与动物的健康。为了更好的了解动物源性沙门氏菌的生物学和分子特性。对安徽凤阳县某市场猪肉样本进行细菌分离纯化、血清型鉴定、药敏实验和毒力基因检测,同时通过全基因组测序结果进行毒力、耐药基因注释等分子生物学信息分析,并通过qPCR对毒力基因进行验证。分离鉴定出1株德尔卑沙门氏菌,分子分型为ST1498型,将其命名为G-1B,该菌株对环丙沙星、卡那霉素和复方新诺明敏感。全基因组长度为4 805 494 bp,预测出4 660个基因,其中包含559个毒力基因与37个耐药基因。该研究为了解猪源德尔卑沙门氏菌的遗传背景和生物学特性奠定了基础,为进一步探讨德尔卑沙门氏菌致病性、耐药机制和分子进化规律提供参考依据。

发酵乳杆菌GZHN2023LC的益生特性及其全基因组测序分析

为探究威宁黄牛源发酵乳杆菌的潜在益生特性和开发益生菌制剂的可行性,本研究进行了益生菌的分离鉴定、耐酸耐胆盐试验、抑菌试验和全基因组测序研究。结果:从35份样品中分离得到11株发酵乳杆菌,均对低pH值和胆盐有良好的抗性,尤其是发酵乳杆菌5;抑菌试验发现发酵乳杆菌5有显著的抑制大肠杆菌的效果,将其命名为GZHN2023LC。基因组测序发现该菌基因组全长达2077921 bp,共编码2011个蛋白;经京都基因与基因组百科全书(KEGG)、直系同源簇(COG)、基因本体论(GO)、碳水化合物活性酶(CAZy)等数据库比对,发现该基因组碳水化合物和氨基酸代谢能力强,同时转录和膜运输活跃,可能与其发酵多糖产生乳酸以及能够合成抗菌肽、胞外多糖有关。综上,发酵乳杆菌GZHN2023LC能够快速增殖,对酸和耐盐有良好的抗性,且有显著的抗菌特性,基因组测序也发现该菌有良好的益生潜能,具备用于益生菌制剂的潜力。