植物耐盐性:演化和盐基因组学

由于社会的发展需要使用盐地产出植物,因此植物耐盐性的研究变得日益重要。植物在长期演化中主要形成嗜盐植物和非嗜盐植物两类,在细胞乃至群体处理盐的机制方面发生了广泛和深度的遗传变异。鉴于发掘植物耐盐、使植物耐盐和改变植物盐环境的各领域都取得了重大进展,结合植物的演化扩展对植物耐盐的认识,从细胞机制、个体机制扩展到群体机制乃至多基因组参与的生态互作,进行以盐基因组学为视角的研究就显得非常重要。本文根据该领域的新近进展,对植物耐盐性进行演化和组学视角的回顾和分析,以期对今后的植物耐盐研究提供参考。

利用人类全基因组亚硫酸氢盐测序数据检测CNVs的研究

DNA甲基化异常可能导致拷贝数变异(copy number variants,CNVs)的发生,而CNVs的发生又可能改变DNA甲基化水平。全基因组亚硫酸氢盐测序(whole genome bisulfite sequencing,WGBS)技术能够获得DNA水平的测序数据,具有挖掘CNVs的潜力和优势,但利用WGBS数据挖掘CNVs的效果尚不清楚。本研究选取了5款检测CNVs不同策略的软件(BreakDancer、cn.mops、CNVnator、DELLY、Pindel),基于人类的真实(2.62 billion reads)和模拟(12.35 billion reads)测序数据,进行150次CNVs检测,评估CNVs检出数量、精确率、召回率、相对检出能力、内存占用和运行时间等指标,旨在讨论利用WGBS数据检测CNVs的最佳方案。基于真实WGBS数据,Pindel检出缺失型和重复型CNVs的数量最多,CNVnator对缺失型CNVs的检测精确率最高,cn.mops对重复型CNVs的检测精确率最高,Pindel对缺失型CNVs的召回率最高,cn.mops对重复型CNVs的召回率最高。基于模拟WGBS数据,BreakDancer检出缺失型CNVs数量最多,cn.mops检出重复型CNVs数量最多,CNVnator对缺失型和重复型CNVs的检测精确率和召回率均为最高。与全基因组测序数据相比,CNVnator在真实和模拟WGBS数据中检出CNVs的能力与之相当。此外,DELLY和BreakDancer的内存占用峰值和CPU运行时间最小,CNVnator的内存占用峰值和CPU运行时间最大。结果表明,利用WGBS数据检测CNVs具有可行性,使用CNVnator和cn.mops在WGBS数据上检测CNVs的准确率较高,这些工作为利用WGBS数据深入研究CNVs和DNA甲基化之间的相互关系提供一定的参考和帮助。

亚硫酸氢盐-基因组测序法在检测基因异常甲基化中的应用

目的:介绍一种准确、敏感的检测多个标本、多个CG二核苷酸甲基化状态的方法。即亚硫酸氢盐-基因组测序法(BisulfiteGenomicSequencing,BGS)。方法:单链DNA的胞嘧啶可被亚硫酸氢盐修饰转变成尿嘧啶,而5'甲基胞嘧啶仍保持不变。这种修饰处理可在甲基化和非甲基化基因组DNA间产生DNA序列差异,而这种DNA序列差异可通过BGS法较为灵敏地筛查出。利用该原理检测肝癌细胞AFP基因启动子区CG二核苷酸甲基化状态。结果:在癌细胞中发现两种异常甲基化,即高度甲基化及部分甲基化。结论:BGS法在基因甲基化检测中将有很大的应用潜能,为探讨肿瘤发生机制提供了新的分子分析方向。

Screening an Na^+/H^+ Antiporter Gene from the Halophiles Colonizing in the Dagong Ancient Brine Well of Zigong City,China

[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the protein encoded by this gene. [Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na＋/H＋ antiporter activity in antiporter-defi- cient E. coil KNabc strain by functional complementation. Then the start codon, stop codon, ORF, -35 region, -10 region and SD sequence of Na~/H＋ antiporter gene, as well as the molecular weight, isoelectric point, hydrophobic region, transmembrane domain, phyletic evolution and salt resistance of protein encoded by the gene were investigated. [Result] A new Na＋/H＋ antiporter gene m-nha was obtained, which ,ren- dered the antiporter-negative mutant E. coil KNabc cells with both the resistance to Na＋ and the ability to grow under alkaline conditions. [Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na＋/H~ antiporters, and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na＋/H＋ antiporter gene. This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource, but also in exploring new salt-tolerant genes.

Influence of Saline Stress on Ionic Balance of Wheat （Triticum Aestivum） and Its Wild Congeners

Purpose of this work was to study changes in the ion balance under salt stress in roots and leaves of seedlings of T. aestivum L. and its wild congeners with different genomic composition. Laboratory assessment of salt tolerance in seedlings was carried out and contents of ions K^＋, Na^＋ and Ca^2＋ in roots and leaves were defined. Considerable change of ionic balance in roots and leaves of studied forms of wheat against salt stress is shown. It is noticed that the parity K^＋/Na^＋ decreases in process of strengthening of salt stress. Authentic negative correlation between a relative gain of a biomass of leaves and roots in the stress conditions and accumulation of ions Na^＋ and between a relative gain of a biomass of leaves in the stress conditions and a parity in them of ions K^＋/Na^＋ is revealed. The research results also show that the stability characteristics of indicators of changes in ionic composition or balance in the leaves of young plants are more informative than in the roots and allow to reveal the form, characterized by relatively high of K^＋/Na^＋ ratio and the relatively low accumulation of Ca2^＋ as the most resistant to salt stress.

尖音库蚊复合体蚊虫DNA甲基化水平分析

目的分析比较尖音库蚊复合体中淡色库蚊、致倦库蚊、骚扰库蚊的DNA甲基化水平以及致倦库蚊吸血前后DNA甲基化水平。方法采集羽化后5 d且未吸血的3个库蚊亚种和吸血3 d的致倦库蚊,提取DNA,超声切割为约250 bp的片段后进行测序,测序数据与致倦库蚊基因组序列(Taxonomy:ID7176)进行比对,获取全基因组胞嘧啶碱基甲基化信息。从基因组、染色体和染色体元件水平分析3个库蚊亚种的甲基化水平(甲基化水平高于3%定义为高甲基化)。比较未吸血的3个库蚊亚种间以及吸血前后致倦库蚊的甲基化水平差异,P<0.001且甲基化差异绝对值>5的位点记为差异甲基化位点;Q<0.05且甲基化差异绝对值>3的区域记为差异甲基化区域(DMR)。将距离DMR最近的转录起始位点(TSS)所在基因记为DMR相关基因,对其进行基因本体论(GO)富集分析。结果淡色库蚊、骚扰库蚊和致倦库蚊全基因组甲基化水平分别为0.454%~0.672%、0.491%~0.649%和0.499%~0.655%(均低于3%),CHH位点甲基化水平分别为0.631%、0.618%和0.624%,均高于CHG位点的0.567%、0.559%、0.559%(t=7.14、83.43、6.87,均P<0.05)和CG/CpG位点的0.508%、0.505%、0.505%(t=10.59、12.52、13.33,均P<0.05);3个库蚊亚种共有的高甲基化位点有56个、高甲基化区域有11个。淡色库蚊、骚扰库蚊和致倦库蚊之间全基因组甲基化水平差异无统计学意义(F=0.07,P>0.05),1号染色体(0.568%、0.562%、0.565%)、2号染色体(0.573%、0.564%、0.566%)、3号染色体(0.575%、0.566%、0.569%)的甲基化水平差异均无统计学意义(F=0.05、0.11、0.13,均P>0.05),启动子(0.567%、0.552%、0.556%)、外显子(0.562%、0.556%、0.558%)、内含子(0.561%、0.550%、0.555%)和TSS(0.579%、0.506%、0.621%)的甲基化水平差异均无统计学意义(F=0.37、0.06、0.06、0.16,均P>0.05)。淡色库蚊和骚扰库蚊间筛选出178个差异甲基化位点、4个DMR,淡色库蚊和致倦库蚊间筛选出209个差异甲基化位点、8个DMR,骚扰库蚊和致倦库蚊间筛选出215个差异甲基化位点、11个DMR。GO富集结果显示,DMR相关基因主要富集于对辐射反应、对光刺激反应和对非生物刺激反应等生物过程。致倦库蚊吸血后全基因组甲基化水平从0.602%升高至0.617%,但差异无统计学意义(t=1.21,P>0.05);吸血前1、2、3号染色体的甲基化水平分别为0.569%、0.569%和0.572%,吸血后上升为0.596%、0.597%和0.600%,但差异均无统计学意义(t=1.31、1.33、1.30,均P>0.05);吸血前启动子、外显子、内含子和TSS的甲基化水平分别为0.557%、0.561%、0.560%、0.552%,吸血后上升为0.585%、0.584%、0.584%、0.594%,但差异均无统计学意义(t=1.48、1.35、1.20、1.69,均P>0.05)。致倦库蚊吸血前后基因组间有6个DMR。GO富集结果显示,DMR相关基因在细胞组分上主要富集于内体、囊泡等,在分子功能上主要富集于蛋白结合、小GTP酶结合等。结论淡色库蚊、致倦库蚊、骚扰库蚊全基因组甲基化水平较低,两两亚种间的DMR相关基因主要与对非生物刺激反应生物过程相关。吸血后的致倦库蚊甲基化水平略有升高,吸血前后的DMR相关基因主要和蛋白结合相关。

Conversion of the high-yield salinomycin producer Streptomyces albus BK3-25 into a surrogate host for polyketide production

An ideal surrogate host for heterologous production of various natural products is expected to have efficient nutrient utilization,fast growth,abundant precursors and energy supply,and a pronounced gene expression.Streptomyces albus BK3-25 is a high-yield industrial strain producing type-Ⅰ polyketide sahnomycin,with a unique ability of bean oil utilization.Its potential of being a surrogate host for heterologous production of PKS was engineered and evaluated herein.Firstly,introduction of a three-gene cassette for the biosynthesis of ethylmalonyl-CoA resulted in accumulation of ethylmalonyl-CoA precursor and sahnomycin,and subsequent deletion of the sahnomycin biosynthetic gene cluster resulted in a host with rich supplies of common polyketide precursors,including malonyl-CoA,methylmalonyl-CoA,and ethylmalonyl-CoA.Secondly,the energy and reducing force were measured,and the improved accumulation of ATP and NADPH was observed in the mutant.Furthermore,the strength of a series of selected endogenous promoters based on microarray data was assessed at different growth phases,and a strong constitutive promoter was identified,providing a useful tool for further engineered gene expression.Finally,the potential of the BK3-25 derived host ZXJ-6 was evaluated with the introduction of the actinorhodin biosynthetic gene cluster from Streptomyces coelicolor,and the heterologous production of actinorhodin was obtained.This work clearly indicated the potential of the high-yield sahnomycin producer as a surrogate host for heterologous production of polyketides,although more genetic manipulation should be conducted to streamline its performance.

Nitrate assimilation by marine heterotrophic bacteria

Nitrate assimilation is a process where bacteria utilize nitrate as a nitrogen source and synthesize it into organic nitrogen. We found that nitrate-assimilating bacteria(NAB) are widely distributed in various marine environments, from surface to the deep ocean and sediment, which indicates that NAB are significant to the oceanic nitrogen cycle. Comparative genomic analysis revealed nitrate-assimilating genes(nas A) in these marine heterotrophic NAB showed different gene arrangements and diverse regulation systems. Summary on recent findings will contribute to understanding the process of nitrate assimilation in NAB and their ecological significance in the nitrogen cycle. A systematic analysis of a number of studies on bacterial nitrate assimilation in marine ecological systems was conducted to clarify directions for future research.