全基因组测序技术研究及其在木本植物中的应用

基因组序列是开展遗传研究重要的信息基础,随着测序技术飞速发展至第3代长片段测序方法,测序读长历经从几十到数万个碱基的提升,对进一步提升基因组组装的完整度以及准确性提供了极大的裨益。现已完成了大量植物种全基因组测序工作,其中木本植物有40多个,还有更多树种的全基因组测序正在进行之中。针对各类测序技术的基因组组装及后续分析,研究人员也开发了大量的生物信息学工具。笔者从测序技术、基因组装技术和全基因组测序生物信息学分析等方面,罗列了目前已完成全基因组测序的木本植物,介绍了全基因组测序技术的发展与应用,以及适用于第3代数据基因组组装的生物学分析软件,为林木基因组研究者提供一定的借鉴。     

我国桉树遗传育种研究进展

桃金娘科(Myrtaceae)桉属(Eucalyptus)、杯果木属(Angophora)和伞房属(Corymbia)树种统称桉树,引入我国已有130余年的历史,是重要的工业用材林树种。我国桉树遗传育种研究始于20世纪60年代的种子园建设和70年代后期的种源试验,一些技术显著促进了其进程,主要有:①早期的种子园技术促进了无性繁殖困难树种的有性扩繁;②20世纪90年代兴起的扦插和组织培养技术推动了优良无性系选育与应用;③20世纪90年代末分子标记技术开启了我国桉树分子育种研究的新纪元;④21世纪初转基因技术为品种创制提供了崭新的手段;⑤刚尝试的基因组编辑技术展现了巨大的应用潜力。桉树育种策略和种质资源是其遗传育种研究的基础,已对一些树种制定了育种策略和育种计划,兼顾纯种内轮回选择和杂种无性系的选育,主要经济性状包括材积生长、木材密度、抗病虫和抗风等;累计已收集了近200个树种3 000余个家系的种质资源。我国桉树遗传育种研究已取得显著进展,主要包括:①几个主要树种的轮回选择和世代改良,仅尾叶桉(E. urophylla)进入了第3个世代;②杂交育种的成效显著,培育了目前仍主栽的DH32-29和DH33-27等优良杂种无性系;③无性系育种结合无性繁殖技术(尤其是组织培养)的研发,极大地推动了无性系林业的发展;④开发了多种分子标记,包括基于新一代测序技术的标记,并基于分子标记利用连锁作图和关联分析的方法,在尾叶桉等6个树种中检测了与生长、材性和/或抗逆等性状相关的基因组位点;⑤已对逆境响应、激素和木材形成等相关的功能基因进行了克隆和表达分析,一些功能基因显示了较好的育种应用潜力;⑥已优化遗传转化体系,获得了转基因植株,并尝试了基因组编辑的可行性。但是,我国桉树遗传育种研究仍面临诸多挑战,如基因型×环境互作的复杂性和高质量基因组/泛基因组的缺乏,种质资源流失,新无性系缺乏,尚待从头克隆和鉴定具有育种价值的优异基因,基因组选择实用性有待探索,遗传转化率需进一步提高等。桉树遗传育种研究对促进我国林业生产的意义是显著的,将有望在高世代改良、种质资源的长期评价、杂种优势的机制与利用、基因组选择的有效应用和转基因与基因组编辑技术等方面取得突破。     

现代林木育种关键核心技术研究现状与展望

良种是提升人工林生产力和增强其碳汇能力的重要基础。“林业种质资源培育与质量提升”是国家“十四五”重点研发任务之一。突破制约林木育种效率和遗传增益提升的瓶颈对于保障国家木材安全和实现“双碳”目标具有十分重要的战略意义。由于传统林木育种周期长、效率低、表型选择精度差,以分子育种为代表的现代育种技术可以显著缩短林木育种周期,精准改良目标性状,成为实现高效林木遗传改良的关键途径。笔者分析了限制林木遗传改良进程的主要问题,阐述了全基因组选择育种等现代林木育种关键核心技术的发展现状及应用情况,并对这些技术未来发展方向进行了展望,为加速林木遗传改良的重大技术创新提供参考。     

The genome of a motile marine Synechococcus

Marine unicellular cyanobacteria are responsible for an estimated 20-40% of chlorophyll biomass and carbon fixation in the oceans. Here we have sequenced and analysed the 2.4-megabase genome of Synechococcus sp. strain WH8102, revealing some of the ways that these organisms have adapted to their largely oligotrophic environment. WH8102 uses organic nitrogen and phosphorus sources and more sodium-dependent transporters than a model freshwater cyanobacterium. Furthermore, it seems to have adopted strategies for conserving limited iron stores by using nickel and cobalt in some enzymes, has reduced its regulatory machinery (consistent with the fact that the open ocean constitutes a far more constant and buffered environment than fresh water), and has evolved a unique type of swimming motility. The genome of WH8102 seems to have been greatly influenced by horizontal gene transfer, partially through phages. The genetic material contributed by horizontal gene transfer includes genes involved in the modification of the cell surface and in swimming motility. On the basis of its genome, WH8102 is more of a generalist than two related marine cyanobacteria.     

<Emphasis Type="Italic">ftsZ</Emphasis> gene and plastid division

As the important cellular organelles in plants, plas-tids comprise one of the primary features that distinguish plant cells from those of other eukaryotes. Seen from the origin, plastids derive from endosymbiotic photosynthetic bacteria. Subsequently, plastids have evolved to become essential components for plant cell function. Besides the important role of chloroplasts in photosynthesis, some water-soluble proteins that involved in biosynthesis of starch, fatty acids, amino acids, nucleic aci…     

Human chromosome 11 DNA sequence and analysis including novel gene identification

Chromosome 11, although average in size, is one of the most gene- and disease-rich chromosomes in the human genome. Initial gene annotation indicates an average gene density of 11.6 genes per megabase, including 1,524 protein-coding genes, some of which were identified using novel methods, and 765 pseudogenes. One-quarter of the protein-coding genes shows overlap with other genes. Of the 856 olfactory receptor genes in the human genome, more than 40% are located in 28 single- and multi-gene clusters along this chromosome. Out of the 171 disorders currently attributed to the chromosome, 86 remain for which the underlying molecular basis is not yet known, including several mendelian traits, cancer and susceptibility loci. The high-quality data presented here--nearly 134.5 million base pairs representing 99.8% coverage of the euchromatic sequence--provide scientists with a solid foundation for understanding the genetic basis of these disorders and other biological phenomena.     

Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene

PRIMARY hypertension is a polygenic condition in which blood pressure is enigmatically elevated; it remains a leading cause of cardiovascular disease and death due to cerebral haemorrhage, cardiac failure and kidney disease. The genes for several of the proteins involved in blood pressure homeostasis have been cloned and characterized, including those of the renin-angiotensin system, which plays a central part in blood pressure control. Here we describe the introduction of the mouse Ren-2 renin gene into the genome of the rat and demonstrate that expression of this gene causes severe hypertension. These transgenic animals represent a model for hypertension in which the genetic basis for the disease is known. Further, as the transgenic animals do not overexpress active renin in the kidney and have low levels of active renin in their plasma, they also provide a new model for low-renin hypertension.     

哺乳动物克隆的现状和研究进展

 哺乳动物细胞克隆是20世纪末生命科学领域最引人注目的高新技术,该技术对于优良种畜的复制、减少试验用动物数目、动物遗传多样性保存及濒危动物挽救、转基因动物培育等方面具有重要意义。近年来克隆技术发展迅速,多种哺乳动物相继克隆成功,但也存在克隆效率太低、克隆动物表型正常而实质异常的问题。本文详细阐述了克隆效率太低、克隆动物表型正常而实质异常问题,介绍了当前动物克隆技术的发展现状,并对动物克隆涉及的技术进行了总结和概括,着重介绍了卵母细胞的去核方法和重组胚的构建方法。     

基因编辑婴儿的伦理、法律和社会蕴含

 同试管婴儿、克隆人、商业化代孕、干细胞治疗等医疗新技术引发的伦理争议相比，贺建奎基因编辑婴儿事件引发的关注更大、社会反响更为猛烈。探讨贺建奎基因编辑婴儿事件引发国际国内热议的原因，讨论了基因编辑婴儿的伦理、法律和社会蕴含。建议明确界定基因编辑技术的适用对象和范围，建立健全新型医疗技术临床应用的伦理审查和监管体系，提升全社会的伦理意识。     

林木分子育种研究的基因组学信息资源述评

分子育种是指利用与性状相关的DNA标记进行选育,也称标记辅助选择或标记辅助育种,广义上还包括基因工程育种和基因组学辅助育种。林木分子育种为早期选择和加速育种提供了极具潜力的高效手段。笔者对林木分子育种研究的基因组学信息资源进行了进展综述和前景展望。近30年来,林木分子标记技术从早期的低通量方法发展到目前基于微阵列芯片和新一代测序的高通量技术,如测序分型、转录组测序、重测序、扩增子测序和外显子组测序等,并广泛用于连锁作图、关联分析和基因组选择等林木性状相关的DNA变异检测研究。随着2006年毛果杨基因组序列的发表,已有50余个树种完成了基因组测序。基于连锁作图和关联研究检测了林木10余个属生长、材性和抗逆及非木质产品品质等性状相关的大量基因组位点,主要趋势表现为：① 表型广泛,涵盖经济性状、生理指标和代谢成分等;②标记数量成千上万甚至上百万,覆盖全基因组;③转录组和降解组等多组学的分子变异开始应用;④ 利用大群体以提高位点检测的精度;⑤ 重视环境的影响,大田试验设置多个地点,解析QTL与环境、年份的互作效应;⑥ 结合参考基因组序列和/或转录组差异表达基因进一步挖掘性状相关的候选基因,建立了桉属、松属和云杉属等主要造林树种的基因组选择模型。此外,积累了泛基因组、相关软件和算法、功能基因、基因组编辑技术及网站和数据库等其他信息资源。林木分子育种面临的挑战主要包括：① 如何获得稳定性好的性状相关基因组位点和基因组选择（GS）模型;② 缺乏自动化、无损和高通量的表型测定技术;③对大基因组的针叶树和一些多倍体树种,仍难获得高质量的基因组序列;④ 标记辅助选择增加了常规育种之外的费用,且存在不确定性;⑤多数树种的加速育种仍较困难。后基因组时代的林木分子育种将有效结合到常规育种程序中,显著促进遗传增益的提高。     

Perspectives for vascular genomics

Diseases of the vascular system result from a complex mixture of genetic and environmental factors. Data sets, technologies and strategies emanating from the human genome programme have been applied to the analysis of both rare single-gene and common multigenic vascular disorders. Genomic approaches including inter- and intraspecies sequence comparisons, genotyping with dense marker sets spanning the genome, large-scale mutagenesis screens of model organisms, and genome-wide expression profiling have all begun to contribute to the identification of new genes and mechanisms that are central to cardiovascular disease processes.     

Detection of Grass Carp Hemorrhage Virus （GCHV） from Vietnam and Comparison with GCHV Strain from China

Grass carp plays an important role in small-scale aquaculture in Vietnam. However, a severe disease, known in Vietnam as “Red Spot Disease“, is causing significant economic loss in grass carp aquaculture. In this study, the tissue samples isolated from the grass carp with Red Spot Disease in Vietnam are investigated and eomparied with the control GCHV isolated in China by experimental infection, culture cell infection, serological cross reactivity, and RT-PCR amplifica-tion. Infected grass carp exhibits hemorrhage symptoms about 5 days after experimental injection with GCHV-V (Vietnam) strain. The symptoms and lethality induced by the GCHV-V strain are identical to that induced by the Chinese GCHV-9014 strain. The Chinese GCHV-873 strain in-duces typical cytopathogenic effects in 4 cell lines, such as CIK, CAB, FHM and GCO, from the6 fish cell lines examined. No cytopathogenic effects are observed in all the 6 examined cell lines,including CAB, FHM, CIK, EPC, CCO and G(X), infected by the GCHV-V strain and GCHV-9014 strain. Immunodiffusion assays demonstrate an obvious cross-reactivity among three GCHVstrains. Precipitin lines are clearly observed not only between the anti-GCHV-873 serum and thetwo strains GCHV-873 and GCHV-9014, but also between the anti-GCHV-873 serum and the GCHV-V strain. GCHV can be detected by immunodiffusion assays after three generations of blind propagations in the cell lines inoculated by GCHV-V strain. This implicates that GCHV-Vviruses have been replicated and amplified despite there being no cytopathogenic eifects observed in these examined cell lines. Three genome segments of GCHV, including S8, S9 and S10, are amplified by three sets of PCR primers designed according to the segment sequences published re-cently. The Q8fp and Q8rp primer set specific for genome segment S8 amplifies a 955 bp frag-ment from the extracted sample of diseased fish with Red Spot Disease, and the fragment size is i-dentical to that amplified by the same primer set from control GCHV-873 strain. Simultaneously,the Q9fp and Q9rp primer set specific for genome segment S9 generates a same 635 bp product,and the Q10fp and Q10rp primer set specific for genome segment S10 produces a same 697 bp fragment from both template samples of diseased fish with Red Spot Disease and control GCHV-873 strain. The RT-PCR amplification and corresponding size comparison data indicate that the three GCHV-V genome segments extracted from the diseased grass carp with Red Spot Disease in Vietnam should be identical to that in control GCHV-873 strain from China. The data confirm that the causative agent of grass carp Red Spot Disease in Vietnam is a virus, and the virus is closely similar to GCHV strain in China.     

Advances in the study of helminth mitochondrial genomes and their associated applications

Helminths, including flatworms and roundworms, are abundant organisms that have a variety of life histories. Of these, the genera Schistosoma, Echinococcus, Trichinella are notable parasites of veterinary and medical importance, and cause substantial socio- economic losses throughout China and the rest of the world. Genetic markers in the mitochondrial (mt) genome have proven use- ful for systematic, ecological, evolutionary and population studies, and the growth of mt genomic research has increased in the last two decades. Technological improvements, such as the long-polymerase chain reaction method and high-throughput se- quencing have allowed minute amounts of DNA from single worms, biopsy samples or microscopic organisms to be used for whole mt genome characterization. To facilitate the retrieval, annotation and analyses of mitochondrial features, multiple data- bases and specific software have also been designed and established. This review focuses on current progress, applications and perspectives regarding helminth mt genomics. To date, the complete mt genomes for 93 species of helminths have been sequenced and analyzed. Analyses of the mt genes, including gene content, arrangement, composition and variation have revealed unique features among the helminths when compared with other metazoans. This provides important data concerning their functional and comparative mitochondrial genomics, molecular taxonomy and characterization, population genetics and systematics, and evolu- tionary history. Moreover, mt genome data for parasitic helminths are important for diagnosis, epidemiology and ecology of in- fections. Mitochondrial genome data offer a rich source of markers for the systematics and population genetics of socioeconomi- cally important parasitic helminths of humans and other animals.     

The complete genome of an individual by massively parallel DNA sequencing

The association of genetic variation with disease and drug response, and improvements in nucleic acid technologies, have given great optimism for the impact of 'genomic medicine'. However, the formidable size of the diploid human genome, approximately 6 gigabases, has prevented the routine application of sequencing methods to deciphering complete individual human genomes. To realize the full potential of genomics for human health, this limitation must be overcome. Here we report the DNA sequence of a diploid genome of a single individual, James D. Watson, sequenced to 7.4-fold redundancy in two months using massively parallel sequencing in picolitre-size reaction vessels. This sequence was completed in two months at approximately one-hundredth of the cost of traditional capillary electrophoresis methods. Comparison of the sequence to the reference genome led to the identification of 3.3 million single nucleotide polymorphisms, of which 10,654 cause amino-acid substitution within the coding sequence. In addition, we accurately identified small-scale (2-40,000 base pair (bp)) insertion and deletion polymorphism as well as copy number variation resulting in the large-scale gain and loss of chromosomal segments ranging from 26,000 to 1.5 million base pairs. Overall, these results agree well with recent results of sequencing of a single individual by traditional methods. However, in addition to being faster and significantly less expensive, this sequencing technology avoids the arbitrary loss of genomic sequences inherent in random shotgun sequencing by bacterial cloning because it amplifies DNA in a cell-free system. As a result, we further demonstrate the acquisition of novel human sequence, including novel genes not previously identified by traditional genomic sequencing. This is the first genome sequenced by next-generation technologies. Therefore it is a pilot for the future challenges of 'personalized genome sequencing'.     

Translating insights from the cancer genome into clinical practice

Cancer cells have diverse biological capabilities that are conferred by numerous genetic aberrations and epigenetic modifications. Today's powerful technologies are enabling these changes to the genome to be catalogued in detail. Tomorrow is likely to bring a complete atlas of the reversible and irreversible alterations that occur in individual cancers. The challenge now is to work out which molecular abnormalities contribute to cancer and which are simply 'noise' at the genomic and epigenomic levels. Distinguishing between these will aid in understanding how the aberrations in a cancer cell collaborate to drive pathophysiology. Past successes in converting information from genomic discoveries into clinical tools provide valuable lessons to guide the translation of emerging insights from the genome into clinical end points that can affect the practice of cancer medicine.     

心血管疾病的基因治疗

基因治疗在先天遗传性以及后天获得性心血管疾病治疗中均具有广阔的发展前景. 对心血管疾病致病机理的深入认识和疾病基因组学研究的发展, 进一步促进了临床前基因治疗的研究进展. 但基因治疗过程中存在的机体细胞免疫反应、外源基因表达水平不足、在体基因转导效率低下等因素都成为基因治疗临床应用转化的瓶颈. 近年来, 基因导入载体和基因组编辑技术的发展为上述问题的改善和解决提供了新的思路. 目前成族规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)/Cas9 基因组编辑技术已经成功应用于动物模型的在体基因编辑, 达到了显著改善血脂指标的疗效. 进一步研究体内组织特异和高效的基因导入方式, 提高基因编辑的靶向效率和特异性, 并建立全面有效的安全评估实验体系, 将推动基因治疗向临床应用的转化. 针对心血管疾病基因治疗中基因导入载体的研究以及CRISPR/Cas9 基因组编辑技术的应用展开讨论.     

Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms.

The stable propagation of genetic information requires that the entire genome of an organism be faithfully replicated once and only once each cell cycle. In eukaryotes, this replication is initiated at hundreds to thousands of replication origins distributed over the genome, each of which must be prohibited from re-initiating DNA replication within every cell cycle. How cells prevent re-initiation has been a long-standing question in cell biology. In several eukaryotes, cyclin-dependent kinases (CDKs) have been implicated in promoting the block to re-initiation, but exactly how they perform this function is unclear. Here we show that B-type CDKs in Saccharomyces cerevisiae prevent re-initiation through multiple overlapping mechanisms, including phosphorylation of the origin recognition complex (ORC), downregulation of Cdc6 activity, and nuclear exclusion of the Mcm2-7 complex. Only when all three inhibitory pathways are disrupted do origins re-initiate DNA replication in G2/M cells. These studies show that each of these three independent mechanisms of regulation is functionally important.     

用基因芯片技术分析干旱胁迫下生殖生长期大麦的基因表达

耐旱性是干旱地区稳定和增加大麦产量的一个关键因素。鉴定出与耐旱性相关的功能基因,一方面可了解大麦的耐旱机理,同时还可以促进利用生物技术来改良大麦的耐旱性。在研究中,2个在耐旱性上具有明显差异的大麦品种Tadmor(耐旱)和WI2291(干旱敏感)被选作材料,采用22000个ESTs(基因表达序列标签)的Affymetrix大麦基因芯片Barley1来分析生殖生长期干旱胁迫下2个大麦材料的差异表达基因。研究结果表明,干旱胁迫下2个大麦材料中有77个共调节基因,其中部分基因已被报道过可能与抗旱性相关。这些基因中已有功能注释的基因按其生物学功能被分为14组,猜测它们是干旱胁迫的响应基因,在抗旱性上可能不起重要作用,或者是必需的但单独不足以提高大麦的抗旱性。进一步比较2个材料差异表达的基因,发现二材料之间有372个受干旱调节基因的差异。这些基因中有功能注释基因的生物学功能中可分为15组,其中一些已被认为与抗旱性相关;而对那些未知功能的基因,推测可能亦在大麦的抗旱性上扮演一定的角色。研究所得结果可为阐述生殖生长期大麦的耐旱性机理提供新的认识。     

实时定量聚合酶链反应的研究及应用(综述)

实时定量聚合酶链反应（real-time quantitative PCR,RQ-PCR）是综合PCR、分子杂交和酶动力学的一种新技术，它根据荧光共振能量转移（fluorescence resonance energy translation,FRET）的原理进行。目前已经成功地开发出TayManTM、molecular beacon probe、amplifluor technologies、LightCycler和single-labeled probe几种相关的技术，并广泛地应用于病毒学、遗传性疾病和肿瘤等方面的诊断检测。实验研究表明：RQ－RCR作为一种快速、准确的核酸检测方法，在临床及生物学研究方面均具有相当的实用价值。