Mutation rates and dominance levels of genes affecting total fitness in two angiosperm species

Theories about the evolution of sex and the effects of inbreeding depend on knowledge of the mutation rate and dominance level of deleterious alleles affecting total fitness. In two species of largely self-fertilizing annual plants, minimal estimates of such mutation rates were found to be 0.24 to 0.87 per sporophyte genome per generation, but confidence intervals exceeded 1.0 in each of the four populations. Dominance levels were near zero in one species and intermediate (0.28 to 0.35) in the other. These results suggest that the detrimental effects of inbreeding are a result of new partially recessive mutations rather than overdominance.     

Adaptive mutations in bacteria: high rate and small effects

Evolution by natural selection is driven by the continuous generation of adaptive mutations. We measured the genomic mutation rate that generates beneficial mutations and their effects on fitness in Escherichia coli under conditions in which the effect of competition between lineages carrying different beneficial mutations is minimized. We found a rate on the order of 10(-5) per genome per generation, which is 1000 times as high as previous estimates, and a mean selective advantage of 1%. Such a high rate of adaptive evolution has implications for the evolution of antibiotic resistance and pathogenicity.     

High deleterious genomic mutation rate in stationary phase of Escherichia coli

In natural habitats, bacteria spend most of their time in some form of growth arrest. Little is known about deleterious mutations in such stages, and consequently there is limited understanding of what evolutionary events occur. In a deleterious mutation accumulation experiment in prolonged stationary phase of Escherichia coli, about 0.03 slightly deleterious mutations were observed per genome per day. This is over an order of magnitude higher than extrapolations from fast-growing cells, but in line with inferences from observations in adaptive stationary phase mutation experiments. These findings may affect understanding of bacterial evolution and the emergence of bacterial pathogenicity.     

Costs and benefits of high mutation rates: adaptive evolution of bacteria in the mouse gut

We have shown that bacterial mutation rates change during the experimental colonization of the mouse gut. A high mutation rate was initially beneficial because it allowed faster adaptation, but this benefit disappeared once adaptation was achieved. Mutator bacteria accumulated mutations that, although neutral in the mouse gut, are often deleterious in secondary environments. Consistently, the competitiveness of mutator bacteria is reduced during transmission to and re-colonization of similar hosts. The short-term advantages and long-term disadvantages of mutator bacteria could account for their frequency in nature.     

利用CRISPR/Cas9基因组编辑技术定向降低水稻落粒性

【目的】易落粒既不利于稻谷收获,也不适宜于水稻机械化生产。利用现阶段前沿的分子育种手段--CRISPR/Cas9技术对水稻落粒性主效基因qSH1进行定点编辑,并调查分析易落粒性状的改良效果,为创制稳产和适合机械化生产的水稻新种质奠定材料基础和探索新途径。【方法】以qSH1为靶标基因,根据CRISPR/Cas9技术原理设计靶标位点。将所设计的靶点序列在水稻参考基因组中比对分析以排除非特异性靶位点,最终筛选出qSH1-T1和qSH1-T5靶标位点。化学合成靶位点寡核苷酸序列,退火后分别与pYLgRNA-U3、pYLgRNA-U6a载体连接构建U3-qSH1T5-gRNA、U6a-qSH1T1-gRNA表达盒,最后将2个gRNA表达盒同时连接至pYLCRISPR/Cas9表达载体中,构建pYLCRISPR/Cas9-qSH1-T51表达载体。利用农杆菌介导转化易落粒的籼稻品种HR1128,以潮霉素抗性为筛选标记筛选获得T₀代转基因阳性植株。利用靶位点扩增测序法判断T₀代转基因植株在预期靶标位点是否发生突变,并进一步分析突变类型及基因型。将靶点序列在水稻参考基因组中比对,选择与靶点序列匹配度大于或等于15 bp且3′端具有NGG的位点作为潜在脱靶位点进行脱靶效应评估。利用潮霉素基因及靶点检测进一步筛选无T-DNA成分的qsh1突变植株并进一步构建qsh1突变系,并分析qsh1突变系的落粒性、qSH1的表达量及预测编码氨基酸序列。【结果】pYLCRISPR/Cas9-qSH1-T51载体成功地实现了对qSH1靶标位点的定点编辑。在T₀代转基因阳性植株中获得7个突变单株,其中qSH1-T1和qSH1-T5靶点的突变频率分别为54.55%和63.64%,突变基因型包括纯合突变、杂合突变、双等位突变和嵌合突变,突变类型包括碱基插入、碱基缺失及碱基突变。通过对T₁代植株进行潮霉素基因筛选、靶位点扩增测序,结果表明,在T₁代植株中Cas9载体骨架和qSH1突变位点都发生了分离,获得2种不含T-DNA成分的qsh1纯合突变株,并以此构建2个T₂代qsh1纯合突变系群体（17SZ01和17SZ02）。对46株转基因阳性植株进行脱靶效应分析,发现3个潜在脱靶位点均未发生突变,表明所设计的靶标位点具有较高的特异性。通过落粒性测定分析表明,与野生型对照相比,2个qsh1纯合突变系的落粒性显著降低。进一步分析发现,2个突变系氨基酸翻译均发生改变并提前终止,同时17SZ01突变系qSH1的表达量显著降低。【结论】利用CRISPR/Cas9技术对水稻基因组进行定点编辑定向改良水稻品种落粒性,是一条高效、安全的分子改良育种策略。     

A low genomic number of recessive lethals in natural populations of bluefin killifish and zebrafish

Despite the importance of selection against deleterious mutations in natural populations, reliable estimates of the genomic numbers of mutant alleles in wild populations are scarce. We found that, in wild-caught bluefin killifish Lucania goodei (Fundulidae) and wild-caught zebrafish Danio rerio (Cyprinidae), the average numbers of recessive lethal alleles per individual are 1.9 (95% confidence limits 1.3 to 2.6) and 1.4 (95% confidence limits 1.0 to 2.0), respectively. These results, together with data on several Drosophila species and on Xenopus laevis, show that phylogenetically distant animals with different genome sizes and numbers of genes carry similar numbers of lethal mutations.     

Nucleosomes suppress spontaneous mutations base-specifically in eukaryotes

It is unknown how the composition and structure of DNA within the cell affect spontaneous mutations. Theory suggests that in eukaryotic genomes, nucleosomal DNA undergoes fewer C→T mutations because of suppressed cytosine hydrolytic deamination relative to nucleosome-depleted DNA. Comparative genomic analyses and a mutation accumulation experiment showed that nucleosome occupancy nearly eliminated cytosine deamination, resulting in an ~50% decrease of the C→T mutation rate in nucleosomal DNA. Furthermore, the rates of G→T and A→T mutations were also about twofold suppressed by nucleosomes. On the basis of these results, we conclude that nucleosome-dependent mutation spectra affect eukaryotic genome structure and evolution and may have implications for understanding the origin of mutations in cancers and in induced pluripotent stem cells.     

An abundance of rare functional variants in 202 drug target genes sequenced in 14,002 people

Rare genetic variants contribute to complex disease risk; however, the abundance of rare variants in human populations remains unknown. We explored this spectrum of variation by sequencing 202 genes encoding drug targets in 14,002 individuals. We find rare variants are abundant (1 every 17 bases) and geographically localized, so that even with large sample sizes, rare variant catalogs will be largely incomplete. We used the observed patterns of variation to estimate population growth parameters, the proportion of variants in a given frequency class that are putatively deleterious, and mutation rates for each gene. We conclude that because of rapid population growth and weak purifying selection, human populations harbor an abundance of rare variants, many of which are deleterious and have relevance to understanding disease risk.     

High direct estimate of the mutation rate in the mitochondrial genome of Caenorhabditis elegans

Mutations in the mitochondrial genome have been implicated in numerous human genetic disorders and offer important data for phylogenetic, forensic, and population genetic studies. Using a long-term series of Caenorhabditis elegans mutation accumulation lines, we performed a wide-scale screen for mutations in the mitochondrial genome that revealed a mutation rate that is two orders of magnitude higher than previous indirect estimates, a highly biased mutational spectrum, multiple mutations affecting coding function, as well as mutational hotspots at homopolymeric nucleotide stretches.     

利用CRISPR/Cas9技术定向编辑水稻OsIAA11

[目的]OsIAA11参与的生长素信号途径在水稻生长发育阶段和环境因子响应中起重要作用,并影响水稻生育后期的产量形成过程.利用CRISPR/Cas9编辑技术对粳稻中花11 (ZH11)的OsIAA11序列进行编辑,获得OsIAA11突变植株,通过对突变植株的农艺性状指标开展田间调查分析,以期探索OsIAA11突变对水稻...     

A mouse model of mitochondrial disease reveals germline selection against severe mtDNA mutations

The majority of mitochondrial DNA (mtDNA) mutations that cause human disease are mild to moderately deleterious, yet many random mtDNA mutations would be expected to be severe. To determine the fate of the more severe mtDNA mutations, we introduced mtDNAs containing two mutations that affect oxidative phosphorylation into the female mouse germ line. The severe ND6 mutation was selectively eliminated during oogenesis within four generations, whereas the milder COI mutation was retained throughout multiple generations even though the offspring consistently developed mitochondrial myopathy and cardiomyopathy. Thus, severe mtDNA mutations appear to be selectively eliminated from the female germ line, thereby minimizing their impact on population fitness.     

基因组编辑系统CRISPR-Cas9研究进展及其在猪研究中的应用

规律成簇的间隔短回文重复(CRISPR)及其相关的蛋白(Cas)原本是细菌抵御病毒的获得免疫系统,人们很快发现其应用潜力,即RNA引导的核酸酶Cas9对靶DNA进行基因组编辑:敲除、敲入、敲降。CRISPR-Cas9是继ZNF、TALENS技术之后的第三代基因组编辑技术,具有突变效率高、成本低、制作简单、能够诱导多位点同时突变等特点。除了基因工程功能外,CRISPR-Cas9系统还具有基因调控、基因组标记功能、大片段删除、全基因组扫描与编辑RNA等,且发展到CRISPR3.0,并继续开发其新应用。从2012年底开始,CRISPR的一系列创新性应用开启了整个基因组编辑研究的革命,成为生命科学领域的技术明星。文章对其最新进展进行了综述,并对其在猪中的应用及潜力进行了展望。     

Hereditary early-onset Parkinson's disease caused by mutations in PINK1

Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra. We previously mapped a locus for a rare familial form of PD to chromosome 1p36 (PARK6). Here we show that mutations in PINK1 (PTEN-induced kinase 1) are associated with PARK6. We have identified two homozygous mutations affecting the PINK1 kinase domain in three consanguineous PARK6 families: a truncating nonsense mutation and a missense mutation at a highly conserved amino acid. Cell culture studies suggest that PINK1 is mitochondrially located and may exert a protective effect on the cell that is abrogated by the mutations, resulting in increased susceptibility to cellular stress. These data provide a direct molecular link between mitochondria and the pathogenesis of PD.     

Chromosomal effects of rapid gene evolution in Drosophila melanogaster

Rapid adaptive fixation of a new favorable mutation is expected to affect neighboring genes along the chromosome. Evolutionary theory predicts that the chromosomal region would show a reduced level of genetic variation and an excess of rare alleles. We have confirmed these predictions in a region of the X chromosome of Drosophila melanogaster that contains a newly evolved gene for a component of the sperm axoneme. In D. simulans, where the novel gene does not exist, the pattern of genetic variation is consistent with selection against recurrent deleterious mutations. These findings imply that the pattern of genetic variation along a chromosome may be useful for inferring its evolutionary history and for revealing regions in which recent adaptive fixations have taken place.     

利用CRISPR/Cas9基因编辑技术构建水稻ospin9突变体

【目的】生长素输出载体蛋白(PIN-FORMED,PIN)是控制生长素极性运输的关键蛋白,水稻OsPIN9是单子叶植物特有的PIN基因,但其生物学功能仍有待研究。利用CRISPR/Cas9基因编辑技术对OsPIN9进行编辑,获得OsPIN9发生突变的基因编辑株系,对进一步深入研究OsPIN9功能提供依据。【方法】根据OsPIN9序列设计特异性编辑位点,构建OsPIN9编辑载体,以日本晴愈伤组织为受体,通过农杆菌介导法获得抗性植株,通过PCR鉴定转基因植株。转基因植株通过PCR和测序明确OsPIN9的突变类型,获得ospin9纯合突变体并分析突变蛋白与野生型蛋白的差异。qRT-PCR分析突变体幼苗根部OsPINs的表达,进一步明确突变体与野生型对照植株之间的表型差异。以0.05μmol·L^-1的萘乙酸(1-naphthaleneacetic acid,NAA)处理幼苗7 d,分析NAA对植株表型的影响。【结果】在水稻OsPIN9第1外显子处设计靶点并构建表达载体,通过遗传转化成功获得18株T₀代转基因植株,测序分析发现转基因株系中有3种不同的突...     

香蕉CRISPR/Cas9基因编辑技术体系的建立

【目的】建立香蕉CRISPR/Cas9基因编辑技术体系,为在香蕉上利用CRISPR/CAS9技术开展香蕉基因功能研究和香蕉育种工作开辟新的路径。【方法】根据香蕉A基因组八氢番茄红素脱氢酶(phytoene dehydrogenase,PDS)基因组序列,利用在线工具ZiFiT Targeter Version 4.2确定合适的CRISPR/Cas9靶标序列,选择其中一个位点作为靶标位点,设计包含靶标基因MaPDS序列的sgRNA。利用一套改良的CRISPR/Cas9多靶点载体系统,以pYLg RNA-Lac Z-U6a质粒为模版,Overlapping PCR法构建U6a-sgRNA表达盒,再利用Golden Gate Cloning法将U6a-sgRNA表达盒克隆到pYLCRISPR/Cas9载体中,构建以MaPDS为靶标基因的pYLCRISPR/Cas9-sgRNA载体。构建的质粒含Cas9p和sgRNA表达盒,其中Cas9p由P_(Ubi)启动子驱动,sgRNA由水稻来源的RNA启动子U6a驱动。将构建好的载体转入农杆菌EHA105,转化香蕉主栽品种巴西蕉胚性细胞悬浮系,获得抗性再生植株。设计PCR引物扩增包含靶标序列的MaPDS序列片段,检测和分析再生植株MaPDS被编辑的情况。【结果】试验选择MaPDS作为CRISPR/Cas9靶标基因,设计一个靶标位点,利用Overlapping PCR法获得了U6a-sgRNA表达盒,利用Golden Gate Cloning法将其克隆到pYLCRISPR/Cas9的Bsa I位点,成功构建了针对MaPDS的pYLCRISPR/Cas9-sgRNA载体。经过农杆菌浸染、抗性筛选、抗性胚诱导、萌发及生根,最终获得抗性独立转化株系129个。其中,71个株系出现白化表型,产生白化表型的几率达55%。失绿突变体的出现意味着MaPDS蛋白功能丧失。随机取转化株系中的白化表型株系33个和正常表型株系14个,提取其叶片基因组DNA,扩增含有MaPDS的靶位点片段,序列分析结果表明,白化表型株系的MaPDS靶位点序列发生了基因编辑。主要是在靶位点附近增加1个碱基T或A,或是在靶位点附近或下游发生碱基颠换或转换,出现非靶标位点突变。这些突变形式均能导致MaPDS蛋白翻译错误,从而使MaPDS蛋白丧失功能,表现为白化。转化株系中表型正常植株的MaPDS靶位点序列与野生型一致,未检测到变异。【结论】成功在香蕉体内实现了对内源MaPDS的定点敲除,获得了基因定点敲除的突变体株系,为进一步利用基因编辑技术在香蕉上的应用奠定了基础。     

一种CRISPR-Cas9介导的拟南芥高效基因编辑系统的构建与应用

【背景】近些年兴起的CRISPR-Cas9基因编辑技术在多种植物中实现了高效的基因打靶,为基因功能研究提供了一种高效快速的方法,但一些CRISPR-Cas9载体编辑效率很低。【目的】通过构建一种由RIBOSOMAL PROTEIN S5 A（RPS5A）启动子启动Cas9并带有红色荧光蛋白筛选标记的CRISPR-Cas9载体,提高拟南芥CRISPR-Cas9编辑效率,并利用这套系统对拟南芥木葡聚糖内糖基转移/水解酶基因TOUCH4（TCH4）进行编辑。【方法】在pKSE401载体的基础上,以从胚胎发育早期就表现出高转录活性的RPS5A启动子替换35S启动子、以DsRed2替换潮霉素抗性基因,构建拟南芥中使用的CRISPR载体pRSE-WH;以AtTCH4为靶基因,使用CRISPR-P 2.0（http://crispr.hzau.edu.cn）设计靶位点,将所设计的靶点序列在拟南芥参考基因组中比对分析以排除非特异性靶位点,最终筛选出2个靶位点target 1和target 2。化学合成带有接头的靶位点寡核苷酸序列,退火后分别与pRSE-WH载体连接,构建TCR1和TCR2表达载体,采用农杆菌介导的沾花法侵染野生型拟南芥Col-0,以红色荧光蛋白为标记筛选获得T₁代转基因阳性植株。通过靶位点扩增测序法判断T₁代转基因植株在预期靶位点是否发生编辑,根据测序结果的峰图对编辑情况进行解码,进一步分析突变类型及基因型。【结果】构建了一个在拟南芥中高效编辑的CRISPR载体pRSE-WH。TCR1和TCR2成功地实现了对TCH4的定点编辑,靶点一的编辑效率为80%,靶点二的编辑效率为100%,总编辑效率为86%。根据测序结果的峰图解码了T₁代植株的突变结果,纯合编辑、杂合编辑、双等位编辑均有出现。对不同的编辑类型进行统计发现,59株T₁代阳性植株中,无编辑8株,占比13.56%,纯合编辑9株,占比15.25%,双等位编辑40株,占比67.80%,杂合编辑2株,占比3.39%。在T₁代发生纯合编辑以及双等位编辑的株系中选择了无红光种子进行繁种,并对T₂代植株编辑情况进行测序检测,结果发现T₁代中的突变成功遗传到了T₂代无Cas9株系中。【结论】pRSE-WH在拟南芥中展现了极高的编辑效率,并且通过对种子进行红色荧光筛选,能够简便地获得无Cas9且稳定遗传的T₃代突变体。     

基于CRISPR/Cas9系统的水稻光敏色素互作因子OsPIL15基因编辑

【目的】光作为一种环境信号,可影响植物的基因表达、酶活性和形态建成。光敏色素互作因子在光信号传导过程中起着重要作用。本研究旨在构建水稻光敏色素互作因子OsPIL15的CRISPR/Cas9表达载体,创制OsPIL15突变体,挖掘水稻功能基因,丰富和完善水稻光信号调控分子机制。【方法】依据CRISPR/Cas9技术原理,设计OsPIL15突变靶点。将所设计靶序列在水稻基因组中进行比对,排除非特异性靶位点,同时使该靶序列含有常用酶切位点,方便后期突变体鉴定。化学合成靶位点寡核苷酸序列并与载体pBUN411连接构建CRISPR/Cas9表达载体,利用农杆菌介导法导入粳稻品种日本晴,以除草剂抗性标记筛选获得阳性转基因植株。利用酶切法判断T0代转基因植株是否发生突变,结合测序结果分析突变单株的突变基因型。将靶点序列在水稻全基因组中进行比对分析,选择5个与靶序列同源性较高且错配在4 bp以内的位点作为潜在脱靶位点进行脱靶效应评估,分析所设计靶序列特异性。【结果】所构建表达载体成功实现了对OsPIL15的定向编辑,酶切显示在选取的25株T0代转基因植株中获得15株突变体,其中包括5株纯合突变体、6株双等位突变体和4株杂合突变体,共10种不同突变基因型和11个突变株系。突变类型以单碱基插入或缺失为主,同时也得到2种56和66 bp较大片段缺失株系。对部分纯合突变、双等位突变和杂合突变体的T1代植株进行分析,结果表明,T0代产生的突变基因型绝大部分能稳定遗传给下一代。T0代纯合突变体后代为纯合突变单株,仅在株系14纯合突变体后代中检测到1株未突变单株;T0代双等位突变体后代可得到2种纯合突变型和1种双等位突变型;T0代杂合突变体后代则可得到纯合、杂合及未突变3种类型。对T0代未突变植株的后继世代酶切分析显示,62株T1代转基因植株均未发生突变,表明CRISPR/Cas9在T1代转基因阳性植株中未重新发挥基因编辑作用。对20株突变体的5个潜在脱靶位点进行分析,5个潜在脱靶位点均未检测出脱靶效应,表明所设计靶序列具有较高特异性。对选取的3组不同基因型ospil15 T1代突变体表型进行初步观察,结果表明,突变体生育期和分蘖数未出现明显变化,株高极显著下降,籽粒粒长极显著增加,最大增幅达5.69%。【结论】CRISPR/Cas9系统能对OsPIL15进行定向编辑,获得的10种不同突变基因型的ospil15突变体与野生型相比株高极显著降低、籽粒粒长极显著增大。     

水稻籼粳杂交产生两系核不育株的初步研究

在水稻(Oryza sativa L.)两系核不育研究过程中,用S1325(母本,籼粳杂交偏籼材料)与9663-1(父本,粳型材料)杂交,在其杂交后代F_4中出现不育株,不育株花粉镜检为典败、套袋自交结实率为0,与同株系可育株杂交,其F_1可育株与不育株出现1∶1分离,与三系保持系杂交,其F_1结实正常,结实性表现完全恢复,不育株再生苗经21℃冷水处理,其花粉育性恢复正常并可正常结实,由此确定该不育株为两系不育株;又对双亲及其后代不育株进行6 K水稻全基因组芯片分析,排除不育株是来自外来花粉串粉的可能性,由此推测籼粳杂交后代可以产生新的两系核不育株。这是关于籼粳杂交可以创造新两系不育系的首次报道。