绿色杜氏藻转录组分析

为了深入了解绿色杜氏藻(Dunaliella viridis)基因信息及功能、耐盐相关通路(甘油脂代谢)及关键酶,本文首次通过Illumina HiSeq^TM 2000高通量测序技术对绿色杜氏藻转录组进行测序,利用Trinity软件将数据组装形成转录本,对所有转录本进行COG(Clusters of Orthologous Groups)、GO(Gene Ontology)和KEGG(Kyoto Encyclopedia of Genes and Genomes)分类和功能注释、Pathway注释以及蛋白编码区(Opening reading fragment,ORF)的预测,并对甘油脂代谢通路关键酶基因进行了分析。转录组测序共获得81 593个转录本,其中ORF共有77 117条,约占所有转录本的94.50%。COG分类结果表明,16 569条转录本被分为24个类别。GO分类结果表明,76 436条转录本被注释。在所有注释分类中,生物学过程转录本数量最多,为30 678条,占总转录本数的40.14%。KEGG分析结果表明,317个标准途径中包含26 428条转录本,含转录本最多的类别是代谢,为9949条(37.65%)。与代谢有关的途径为131条,占所有注释途径的41.32%。在甘油脂代谢通路中仅发现1条关键酶转录本(二羟丙酮激酶),该酶可能与绿色杜氏藻耐盐胁迫中甘油的合成有较大关系。本研究进一步完善了绿色杜氏藻的基因信息,为绿色杜氏藻代谢途径研究奠定了坚实的基础。     

枫香叶片变色期全长转录组测序及分析

枫香因其树形优美,入秋后叶色红艳或橙黄,极具观赏价值,是优良的景观生态树种。为了解枫香叶片变色及其次级代谢过程的遗传基础,该文以枫香5个叶片变色期叶片混合样品为材料,利用单分子实时测序技术(PacBio平台)对其进行全长转录组测序。结果表明:(1)全长转录组测序共获得41.04 Gb的高质量数据,从中鉴定出全长非嵌合序列563 180条,通过聚类和去冗余,获得27 269条高质量全长转录本。在27 269条全长转录本中预测到2 035条长链非编码RNA(lncRNA),并检测出14 892个简单重复序列(SSR)位点和1 856个转录因子。(2)基因注释结果表明,NR、GO、COG、KEGG 等8个数据库共注释了24 857条转录本,KEGG数据库共获得了124个条代谢途径,主要有核糖体、碳代谢、氨基酸生物合成等,在类黄酮和叶绿素代谢途径中分别有49和71个转录本参与。上述结果初步揭示了枫香叶片变色期转录组信息以及功能特性,为后续研究枫香叶片变色分子机制、色素代谢合成途径和调控、相关功能基因克隆以及叶色改良提供基础数据。     

黄秋葵花和果荚转录组测序及类黄酮代谢差异表达分析

采用Illumina Hi seq 2500转录组高通量测序,构建黄秋葵花和果荚转录组文库,并利用测序评估、差异基因功能注释等生物信息学方法进行分析。研究结果表明：(1)分别获得黄秋葵花和果荚有效数据7.12 Gb和8.14 Gb,碱基百分比(Q30)均达到91.0%以上。(2)获得差异表达基因(DEGs)1 336个,其中上调基因319个,下调基因1 017个。(3)获得功能注释的基因有1 131个,GO将455注释基因归成41个功能小类,主要涉及代谢过程、催化活性、单一生物过程和细胞过程等过程;KOG注释了472个DEGs,涉及23个功能分类,其中与次生代谢直接相关过程O和Q类别获得111个注释结果;KEGG将372个DEGs注释到80条代谢通路中,获得F3H、F3′5′H、DFR、ANR、ANS、GT、LAR共10个关键差异基因,其中F3H、DFR在黄秋葵花中表现上调效应,F3′5′H、ANR、LAR在黄秋葵果荚中表现显著上调效应,ANS、GT则分别在花和果荚中均有上调或下调效应。(4)实时定量PCR分析显示,其中7个差异表达基因得到的相对表达量与转录组表达谱分析趋势完全一致。(5)类黄酮代谢途径分析表明,黄秋葵花通过F3H、DFR、ANS、GT途径将NAR催化为生成花青素苷;果荚则将NAR通过F3′5′H将催化为DHM,后在FLS催化下生成黄酮醇类物质等;部分NAR在F3H、 DFR催化下,生成无色飞燕草素苷元,再分别在ANS、LAR作用下,进入花青素苷元和原花青素合成途径。该研究结果丰富了黄秋葵转录组信息,为黄秋葵类黄酮物质纯化和功能利用提供参考依据。     

藏茵陈基源植物皱边喉毛花的全长转录组信息分析

皱边喉毛花为藏药藏茵陈基源植物之一,其包含丰富的药用成分。为进一步了解皱边喉毛花转录组,丰富其基因注释、代谢通路等遗传信息,该研究利用PacBio测序平台对皱边喉毛花叶片进行全长转录组测序。结果表明:(1)全长转录组测序共获得17 Gb的高质量数据,对795 698 个CCS序列进行聚类和去冗余,最终获得87 814 条高质量的全长转录本。(2)与7个数据库比对后,共有277 451 条转录本注释成功,其中注释到NR数据库的转录本最多,有39 214 条。26 396 条转录本成功注释到KOG数据库中,共有26 个子类。39 104 条转录本注释到KEGG数据库中,涉及6 个主要通路和40 个子通路。39 102 条转录本注释到GO数据库中,按分子功能、生物学过程和细胞成分3大类对注释成功的转录本进行分类。(3)SSR分析共鉴定到22 861 个SSR,其中单碱基重复最为丰富; 共检测到1 874 个转录因子和15 166 个长非编码RNA(LncRNA),而注释到转录本最多的转录因子家族是C3H。(4)筛选出55 条与单萜类及黄酮类化合物合成相关的转录本。该研究结果丰富了皱边喉毛花的转录组信息,为进一步筛选皱边喉毛花药用成分合成相关的关键基因提供了重要的遗传资源。     

不同品种桂花转录组分析及桂花精油成分差异的初步探讨

为丰富桂花转录组数据信息,阐明不同品种桂花精油合成的分子差异,本研究选择三种不同品种桂花,通过Illumina Hiseq2000平台测序,总计产出14.4G数据,三个品种的桂花样品共获得70029个Unigene序列,平均长度762 nt,N50达到1183 nt。通过Unigene的表达差异分析与通路富集分析,发现与桂花精油物质合成途径有关的差异基因主要注释在二萜合成、萜类骨干化合物合成、单萜合成、倍半萜与三萜合成、苯丙烷类合成、苯丙酸类代谢、亚麻酸代谢以及亚油酸代谢8个通路中。而三种桂花差异基因均显著富集的通路则注释在苯丙烷类合成、苯丙酸类代谢、二萜合成以及萜类骨干化合物合成通路,共获得650个编码精油物质合成途径中54个差异酶的差异基因。本研究建立了以金球桂、白洁和日香桂为代表的桂花转录组数据库。通过转录组分析,确定了不同品种桂花精油物质合成差异的表达途径,初步分析了转录组数据库中与精油物质合成途径相关的差异基因。本研究将为桂花精油成分代谢调控及体外表达提供依据。     

朝仓花椒幼芽转录组测序数据组装及基因功能注释

采用2代Illumina Hi-Seq测序技术对朝仓花椒雌株结果期幼芽的转录组进行测序,构建了转录组数据库,获得49 672 080条Clean Reads数据,包含总长度为4 967 208 000 nt序列数据信息;经拼接组装,获得转录组基因信息长达55 902 243 nt的176 407个Contig片段,再通过进一步拼接,共获得平均长度为878 nt的96 475个Unigene片段。与Nt、Nr、Swiss-Prot、COG、GO、KEGG等数据库进行BLAST信息比对(E-value艽10-5),共获得68 315个注释基因,以及62 150个表达序列标签(EST)。与NR数据库比对,发现花椒转录组基因序列与同科柑橘属的甜橙(Citrus sinensis)和克莱门柚(Citrus clementina)具有较高的同源性,分别为43.96%及41.86%,与其他物种的同源性较低,均不足10%;可将花椒转录组的Unigene的功能通过与COG数据库进行注释比对划分为25类;根据GO数据库的注释,可将其分为生物过程、细胞组分和分子功能3大类共55分支,根据与KEGG数据库的比较分析发现朝仓花椒的转录组数据中含有代谢通路相关基因128类,其中包括多种化合物的代谢途径和次生代谢产物的生物合成途径,其中有较多的次生物质代谢途径,如萜类化合物生物合成途径、黄酮类生物合成代谢途径以及花青素的生物合成途径等。     

微藻(Chlorella sorokiniana)的转录组分析:油脂生物合成相关的途径解析和基因挖掘

【目的】为了构建和改造产油微藻(Chloralla sorokiniana),需要从基因组水平解析脂肪酸、三酰甘油、淀粉等生物大分子的合成和降解途径,并分析和确定其中的重要基因。【方法】本研究选取了初始氮浓度分别为KNO3∶8g/L和KNO3∶2 g/L的培养条件培养微藻C.sorokiniana,培养至84h收集藻细胞进行Illumina Hiseq2000双端测序,利用Trinity进行de novo拼接,得到的转录本通过Nr数据库、UniProtKB/Swiss-Prot数据库、COG数据库进行功能注释及分类,通过KEGG数据库进行相关代谢途径注释。最后利用RSEM计算每个转录本的RPKM值,并对相关代谢途径中的转录本的表达差异进行了初步分析。【结果】Illumina Hiseq2000双端测序共获得49M 100 bp的raw reads,de novo拼接得到49885个转录本,其长度范围在300bp到14100bp之间,N50为1941 bp。其中26479个转录本成功注释到功能,2357个转录本注释到了EC编号,利用这些转录本注释到207条代谢途径。在此基础上构建了C.sorokiniana的脂肪酸、三酰甘油、淀粉等生物大分子的合成及降解途径。并初步确定了代谢途径中基因的上调及下调水平。【结论】通过RNA-seq分析实现了对非模式藻株C.sorokiniana的基因组解析,在此基础上构建了脂肪酸、三酰甘油、淀粉的生物合成和降解途径及重要基因,所构建的代谢途径与模式藻株莱氏衣藻是一致的,并比较了相关途径中的基因的表达差异,这些信息有助于将来对微藻进行遗传改造提高其产油能力。     

扶桑绵粉蚧转录组分析

【目的】建立扶桑绵粉蚧Phenacoccus solenopsis Tinsley的转录组数据库,揭示扶桑绵粉蚧转录组的整体表达特征。【方法】采用Illumina Hi SeqTM40`00测序平台开展对扶桑绵粉蚧雌成虫转录组测定,对原始数据进行过滤和组装。【结果】共获得58 322 258条序列读取片段(reads),共9.48 Gb(Gen Bank登录号:SAMN06130426)57 422 032条有效转录组数据。进一步组装拼接后,共获得94 475个单基因簇(unigene),平均长度为700 bp。将unigene与数据库中的序列进行BLASTX比对,成功注释20 949个unigenes,其中,Nr注释的unigenes与豌豆蚜Acyrthosiphon pisum unigenes同源性最高,达18.69%。扶桑绵粉蚧转录组unigenes根据GO功能注释大致可分为细胞组分、分子功能和生物过程三大类55个分支,与结合活性、催化活性、细胞进程和代谢进程相关的unigenes较多。此外,本研究还筛选到20条与脂类代谢相关的途径和与性信息素代谢相关的序列。并通过与Nr和Swiss-prot蛋白质数据库比对,获得了15 037条编码序列(CDS)片段。【结论】本研究初步阐明扶桑绵粉蚧雌成虫转录组的整体表达模式,为进一步研究扶桑绵粉蚧的基因功能及性信息素的代谢途径奠定了基础。     

NaCl胁迫下黑果枸杞转录组测序分析

研究黑果枸杞在不同浓度盐胁迫下基因表达谱变化情况,为进一步研究黑果枸杞抗盐分子机制奠定研究基础。对0(CK)、50、250 mmol/L NaCl溶液胁迫的黑果枸杞组培苗的根和叶在胁迫时间为0、1、12 h时分别取样,采用转录组测序(RNASeq)技术进行测序分析。结果表明,转录组测序共产生222.49 Gb原始数据,拼接出Unigenes 86 037条,注释到7大功能数据库(GO、KEGG、KOG、NR、Pfam、Swiss-Prot和egg NOG)上的Unigenes总数为46 594个,占总Unigenes的54.76%,还有38 929个Unigenes在这些数据库中没有得到注释。通过GO分类和KEGG Pathway富集性分析,分别归于51个GO类别和211条代谢途径。差异表达基因分析显示,黑果枸杞叶片和根的上调基因和下调基因数随着NaCl浓度的增大和处理时间的延长均呈增加趋势,叶片中的上调基因数(7 514)小于下调基因数(9 032),根中的上调基因数(12 347)大于下调基因数(11 559)。在黑果枸杞盐胁迫下转录组中发现28 325个SSR位点,最多的为单核苷酸SSR,占70.47%。综合分析表明,黑果枸杞对盐胁迫的反应是一个多基因参与、多个生物过程协同调控的过程,基因表达量的变化可能是基因调控的主要方式。     

络石藤与地瓜藤抑制NO活性及差异性化学成分研究

络石藤为药典记载的常用中药，而地瓜藤在民间部分地区用作络石藤。为明确两种药材抗炎活性及相关化学成分间的差异，初步探讨其可能的药效物质基础，该研究通过检测不同浓度络石藤与地瓜藤醇提物对RAW264.7细胞存活率的影响，采用Griess试剂法测定LPS诱导的RAW264.7细胞上清液NO释放量，利用UPLC-Q-TOF-MS/MS技术对络石藤与地瓜藤醇提物中的化学成分进行分析，并结合多元统计学中的OPLS-DA模型鉴定两种药材的差异性化学成分。结果表明：(1)两种药材皆具有一定的NO抑制作用，络石藤粗提物的抑制作用稍强于地瓜藤。(2)络石藤与地瓜藤的化学成分存在明显差异，络石藤中的21个差异性成分主要为木脂素类，而地瓜藤中的10个差异性成分主要为黄酮类。综上认为，差异性化学成分可能是造成这两种药材抗炎活性差异的主要活性物质。该研究结果为络石藤与地瓜藤的抗炎药效物质基础和质量控制提供了理论依据。     

High-throughput sequencing uncover Ficus tikoua Bur. chloroplast genome

Ficus tikoua Bur., called “di guo”, is a very important economic plant. However, we know little on its molecular mechanisms for a long time. In this study, three F. tikoua chloroplast genomes were first obtained through Illumina sequencing. The F. tikoua Bur. cp genomes exhibited a typical circular structure, including a pair of inverted repeats, a small single copy sequence, and a long single copy sequence. More than 100 genes and about 70 ncRNAs (rRNAs, tRNAs, snRNA, snoRNA, etc.) were identified in each cp genomes. Some repeats, including DNA element, long terminal repeat, small RNA, simple repeat and low complexity, were examined in the three complete cp genomes. In addition, phylogeny tree from six Ficus. species and five Morus. species in family Moracea were constructed for clarifying taxonomy. This data can help further the understanding of evolution and phylogenetic relationships in F. tikoua Bur.

     

云南莲瓣兰主栽品种SSR指纹图谱的构建和遗传差异分析

为了解云南莲瓣兰(Cymbidium tortisepalum)的遗传多样性,利用SSR技术对32个莲瓣兰主栽品种进行遗传变异分析,并构建莲瓣兰栽培品种的指纹图谱。结果表明,筛选出的12对多态性高、稳定性好的引物共检测到95个等位基因,每对引物检测到4~18个等位基因,有效等位基因数(N E)为61.489,平均有效等位基因数(NA)为5.124,Shannon信息指数(I)和多态性信息含量(PIC)分别为0.806~2.624和0.789~0.953。12对引物中,以引物SSR03的等位基因数、NE、观测杂合度、I和PIC最高。32个品种在12对引物上都具有不同的特异性条带,可以彼此区别。从12对引物中筛选出3对核心引物SSR02、SSR03和SSR12构建了莲瓣兰主栽品种SSR分子指纹图谱,这3对核心引物组合即可鉴定32个莲瓣兰栽培品种。这为莲瓣兰的品种鉴定、遗传多样性分析和分子育种研究提供理论基础和技术支持。     

Inheritance of partial resistance to powdery mildew in spring wheat

Summary Four spring wheat (Triticum aestivum L.) cultivars exhibiting partial resistance to powdery mildew induced by Erysiphe graminis f.sp. tritici were crossed to a common susceptible cultivar to study the inheritance of resistance. The genetic parameters contributing to resistance were estimated by generation means analyses. Additive gene action was the most important genetic component of variation among generation means in all four crosses. Additive by additive effects were significant in one cross and both additive by additive and additive by dominance effects were significant in another. Dominance effects were not significant. The F2/F3 correlations in three crosses ranged from 0.27 to 0.43. Three additional crosses among resistant cultivars were employed to study the effectiveness of selection in improving resistance. By selecting the most resistant plants from the F2 and evaluating the progenies in the F4, increases in resistance ranging from 21% to 31% were obtained. In all crosses, there was transgressive segregation in both directions indicating that the genes conferring resistance to these cultivars differ and exhibit additive effects.     

AFLP analysis of nephthytis (Syngonium podophyllum Schott) selected from somaclonal variants

This study analyzed genetic differences of 19 cultivars selected from somaclonal variants of Syngonium podophyllum Schott along with their parents as well as seven additional Syngonium species and six other aroids using amplified fragment length polymorphism (AFLP) markers generated by 12 primer sets. Among the 19 somaclonal cultivars, ‘Pink Allusion’ was selected from ‘White Butterfly’. Tissue culture of ‘Pink Allusion’ through organogenesis resulted in the development of 13 additional cultivars. Self-pollination of ‘Pink Allusion’ obtained a cultivar, ‘Regina Red Allusion’, and tissue culture propagation of ‘Regina Red Allusion’ led to the release of five other cultivars. The 12 primer sets generated a total of 1,583 scorable fragments from all accessions, of which 1,284 were polymorphic (81.9%). The percentages of polymorphic fragments within ‘White Butterfly’ and ‘Regina Red Allusion’ groups, however, were only 1.2% and 0.4%, respectively. Jaccard's similarity coefficients among somaclonal cultivars derived from ‘White Butterfly’ and ‘Regina Red Allusion’, on average, were 0.98 and 0.99, respectively. Seven out of the 15 cultivars from the ‘White Butterfly’ group and three out of six from the ‘Regina Red Allusion’ group were clearly distinguished by AFLP analysis as unique fragments were associated with respective cultivars. The unsuccessful attempt to distinguish the remaining eight cultivars from the ‘White Butterfly’ group and three from the ‘Regina Red Allusion’ group was not attributed to experimental errors or the number of primer sets used; rather it is hypothesized to be caused by DNA methylation and/or some rare mutations. This study also calls for increased genetic diversity of cultivated Syngonium as they are largely derived from somaclonal variants.     

Subcellular distribution and chemical forms of antimony in Ficus tikoua

Ficus tikoua (F. tikoua) was a potential species for antimony (Sb) phytoremediation due to its wide growth in the mining area. However, little was known about its tolerance mechanisms toward Sb. The determination of the distribution and chemical speciation of Sb in F. tikoua is essential for understanding the mechanisms involved in Sb accumulation, transportation, and detoxification. The present study investigated the subcellular distribution and chemical forms of Sb in F. tikoua. The plant was exposed to different Sb concentrations (0, 30, 90, and 180 μmol/L) for 30 days. The results showed that F. tikoua possessed a marked ability to tolerate and accumulate Sb. The proportional Sb increased with increasing Sb concentration in the solution, and the highest Sb concentration occurred in roots (1274.5–1580.9 mg/kg), followed by stems (133.5–498.9 mg/kg) and leaves (4.1–15.7 mg/kg). In the subcellular sequestration of Sb in F. tikoua, the largest accumulation of Sb occurred in cell walls (72.4–87.5%) followed by cytoplasmic organelles (8.2–18.6%) and cytoplasmic supernatant. The results suggested that cell walls act as important protective barriers against Sb toxicity in F. tikoua. Although Sb in all plant tissues found primarily in the fractions extracted by ethanol and distilled water, the current study found that the Sb amounts in the HAc-extractable fraction, HCl-extractable fraction, and residue fraction increased at the highest Sb level (180 μmol/L) compared to that under lower Sb levels. These results indicate that excessive Sb accumulated in F. tikoua under Sb stress is bound to non-dissolved or low-bioavailable compounds, a biochemical mechanism that benefits F. tikoua because it helps alleviate Sb toxicity.     

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.