[目的]筛选红豆杉NAC转录因子家族中影响根系形成的关键基因,探索红豆杉根系生长发育分子机理.[方法]利用曼地亚红豆杉全长转录组数据,通过生物信息学方法鉴定NAC转录因子,并对筛选出的NAC转录因子进行蛋白结构及基因组织表达谱等分析.[结果]共鉴定出44个NAC转录因子,其在N端均具有典型的保守NAC结构域,分别聚类到拟南芥的7个亚家族中,蛋白三级结构较为相似,且成员大多含有5个保守亚结构域.组织表达谱显示TmNAC15、16、18、21、22、29、39、40、41和44在根中表达水平高于茎和叶.[结论]从曼地亚红豆杉中共鉴定出44个NAC转录因子,其结构较为保守,且聚类为7个亚家族,其中成员TmNAC21、22、39、40和44极有可能参与红豆杉根系生长发育.

N6-methyladenosine (m6A) RNA modification is a conserved mechanism to regulate gene expression that plays vital roles in the development of plants. However, the m6A RNA modification in forest trees remains limited. Here, we performed a complete analysis of m6A writers, erasers and readers in Poplar 84K, including gene location, gene structures, conserved motifs, phylogenetic relationships, promoter analysis, expression profiles and the homology modeling. We have identified 61 m6A pathway genes in Poplar 84K (Populus alba × Populus glandulosa), including 14 m6A writers, 14 m6A erasers and 33 m6A readers. Phylogenetic analysis indicated that the m6A writers and erasers were clustered into four groups and m6A readers were clustered into two groups. Promoter analysis showed that m6A pathway genes were mainly responsive to low oxygen followed by ABA and ethylene. The expression of the identified m6A pathway genes showed tissue-specific expression patterns in leaves, xylem, phloem and roots. Moreover, 17 genes were significantly up-regulated and 13 genes were significantly down-regulated in poplar overexpressing the transcription factor LBD15. Homology modeling and molecular docking results suggested that PagFIP37b was most likely to be regulated by LBD15, and the qPCRshowed that PagFIP37b were up-regulated in the LBD15-oe plants. The results provide insights that aid in the future elucidation of the functions of these m6A pathway genes and the epigenetic regulation mechanism of these genes in Poplar 84K.

The MYB transcription factor family is one of the largest gene families playing regulatory roles in plant growth and development. The MYB family has been studied in a variety of plant species but has not been reported in Taxus chinensis. Here we identified 72 putative R2R3-MYB genes in T. chinensis using a comprehensive analysis. Sequence features, conversed domains and motifs were characterized. The phylogenetic analysis showed TcMYBs and AtMYBs were clustered into 36 subgroups, of which 24 subgroups included members from T. chinensis and Arabidopsis thaliana, while 12 subgroups were specific to one species. This suggests the conservation and specificity in structure and function of plant R2R3-MYBs. The expression of TcMYBs in various tissues and different ages of xylem were investigated. Additionally, miRNA-mediated posttranscriptional regulation analysis revealed that TcMYBs were the targets of miR858, miR159 and miR828, suggesting the posttranscriptional regulation of MYBs is highly conserved in plants. The results provide a basis for further study the role of TcMYBs in the regulation of secondary metabolites of T. chinensis.

Poplar 84K (Populus alba x P. tremula var. glandulosa) is a fast-growing poplar hybrid. Originated in South Korea, this hybrid has been extensively cultivated in northern China. Due to the economic and ecological importance of this hybrid and high transformability, we now report the de novo sequencing and assembly of a male individual of poplar 84K using PacBio and Hi-C technologies. The final reference nuclear genome (747.5 Mb) has a contig N50 size of 1.99 Mb and a scaffold N50 size of 19.6 Mb. Complete chloroplast and mitochondrial genomes were also assembled from the sequencing data. Based on similarities to the genomes of P. alba var. pyramidalis and P. tremula, we were able to identify two subgenomes, representing 356 Mb from P. alba (subgenome A) and 354 Mb from P. tremula var. glandulosa (subgenome G). The phased assembly allowed us to detect the transcriptional bias between the two subgenomes, and we found that the subgenome from P. tremula displayed dominant expression in both 84K and another widely used hybrid, P. tremula x P. alba. This high-quality poplar 84K genome will be a valuable resource for poplar breeding and for molecular biology studies.

Background The major medicinal alkaloids isolated from Uncaria rhynchophylla (gouteng in chinese) capsules are rhynchophylline (RIN) and isorhynchophylline (IRN). Extracts containing these terpene indole alkaloids (TIAs) can inhibit the formation and destabilize preformed fibrils of amyloid β protein (a pathological marker of Alzheimer’s disease), and have been shown to improve the cognitive function of mice with Alzheimer-like symptoms. The biosynthetic pathways of RIN and IRN are largely unknown. Results In this study, RNA-sequencing of pooled Uncaria capsules RNA samples taken at three developmental stages that accumulate different amount of RIN and IRN was performed. More than 50 million high-quality reads from a cDNA library were generated and de novo assembled. Sequences for all of the known enzymes involved in TIAs synthesis were identified. Additionally, 193 cytochrome P450 (CYP450), 280 methyltransferase and 144 isomerase genes were identified, that are potential candidates for enzymes involved in RIN and IRN synthesis. Digital gene expression profile (DGE) analysis was performed on the three capsule developmental stages, and based on genes possessing expression profiles consistent with RIN and IRN levels; four CYP450s, three methyltransferases and three isomerases were identified as the candidates most likely to be involved in the later steps of RIN and IRN biosynthesis. Conclusion A combination of de novo transcriptome assembly and DGE analysis was shown to be a powerful method for identifying genes encoding enzymes potentially involved in the biosynthesis of important secondary metabolites in a non-model plant. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the capsule extract from Uncaria , and provides information that may aid in metabolic engineering to increase yields of these important alkaloids.

Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC-MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.

BACKGROUND:Plant cell culture represents an alternative source for producing high-value secondary metabolites including paclitaxel (Taxol®), which is mainly produced in Taxus and has been widely used in cancer chemotherapy. The phytohormone methyl jasmonate (MeJA) can significantly increase the production of paclitaxel, which is induced in plants as a secondary metabolite possibly in defense against herbivores and pathogens. In cell culture, MeJA also elicits the accumulation of paclitaxel; however, the mechanism is still largely unknown.METHODOLOGY/PRINCIPAL FINDINGS:To obtain insight into the global regulation mechanism of MeJA in the steady state of paclitaxel production (7 days after MeJA addition), especially on paclitaxel biosynthesis, we sequenced the transcriptomes of MeJA-treated and untreated Taxus × media cells and obtained ∼ 32.5 M high quality reads, from which 40,348 unique sequences were obtained by de novo assembly. Expression level analysis indicated that a large number of genes were associated with transcriptional regulation, DNA and histone modification, and MeJA signaling network. All the 29 known genes involved in the biosynthesis of terpenoid backbone and paclitaxel were found with 18 genes showing increased transcript abundance following elicitation of MeJA. The significantly up-regulated changes of 9 genes in paclitaxel biosynthesis were validated by qRT-PCR assays. According to the expression changes and the previously proposed enzyme functions, multiple candidates for the unknown steps in paclitaxel biosynthesis were identified. We also found some genes putatively involved in the transport and degradation of paclitaxel. Potential target prediction of miRNAs indicated that miRNAs may play an important role in the gene expression regulation following the elicitation of MeJA.CONCLUSIONS/SIGNIFICANCE:Our results shed new light on the global regulation mechanism by which MeJA regulates the physiology of Taxus cells and is helpful to understand how MeJA elicits other plant species besides Taxus.

To sequence the transcriptome of Ginkgo biloba L.cells,Illumina Genome Analyzer IIx was used.One purpose is to discover candidate genes involved in ginkgolide biosynthesis and new hydroxylases in Ginkgo biloba cells such as taxoid 9-alpha hydroxylase,which will complete the unknown hydroxylation steps in taxol biosynthesis pathway in Taxus sp.A total of 69 286 contigs,56 387 scaffolds and 32 032 unigenes with average length of 636bp were generated.Unigene qualities from several aspects like gap distribution,GC content,gene coverage were assessed.The results indicate that the sequencing data is good with high quality and reliability.Analyzed the information of unigene expression and functional annotation,we found that 66 unigenes belong to CYP450 gene family,726 relate to secondary metabolism among which 59 involved in terpenoid metabolism and 17 involved in diterpenoid biosynthesis.At last,15 hydroxylase candidates were selected by bioinformatics analysis our transcriptome data of Ginkgo biloba L.cells and the Michigan State University transcriptome data of Ginkgo biloba L.tissues.These candidate genes selection work set foundation for the further research.

Casbene synthase may play an important role in biosynthesis of tigliane type terpenoid.Casbene synthase gene,named as EfCS(GenBank accession number JN862821),was cloned from the root of Euphorbia fischeriana Steud.through RACE-PCR.The fragment was 1 969 bp in length and contained an open reading frame coding a polypeptide of 602 amino acids.This CS protein from E.fischeriana has a typical terpenoid synthase gene structure.Through biochemical analysis of the amino acid sequence coding by CS gene,we found the theoretical isoelectric point of CS is 5.36 and the molecular weight is 69.364 8 kD.By hydrophobic analysis,we found the overall performance of CS is hydrophilic.The lead peptide analysis revealed that the peptide has a chloroplast guide sequence.Secondary structure prediction showed that this CS protein is mainly composed with α-helix,extended chain,β-turn and random coil,which accounts for 64.95%,6.64%,3.16% and 25.25% respectively.Protein tertiary structure prediction showed that our casbene synthase belongs to class Ⅰ terpene synthase.

The enzyme taxoid 14 beta-hydroxylase (14OH) directs a side-route of taxol pathway to 14 beta-hydroxy taxoids. Suppression of this side-route could increase the production of taxol. To suppress taxoid 14 beta-hydroxylase gene (14OH) expression in the Taxus x media TM3 cell line, antisense RNA inhibition approach was used in this study. Following the construction of an antisense RNA expression vector of 14OH from Taxus chinensis, the antisense 14OH cDNA (as14OH) was introduced into TM3 cells by Agrobacterium tumefaciens-mediated transformation. Southern blot analysis of hygromycin phosphotransferase gene (HYG) revealed that this selection gene was integrated successfully into the genome of Taxus x media cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the 14OH mRNA level in transgenic cells dropped dramatically, suggesting that the expression of endogenous14OH gene was significantly suppressed by the exogenous as14OH gene. Correspondingly, the total yield of three major C-14 oxygenated taxoids (yunnanxane, taxuyunnanine C, sinenxan C) was markedly reduced in the silenced cell lines when compared with those of the non-transgenic controls. These results indicated that the antisense RNA strategy is a useful tool in suppressing the expression of genes in Taxus and this method could be used to silence other important genes that divert Taxol pathway to side-route metabolites.

In order to reveal the differentially-expressed genes involved in triterpenoid saponin biosynthesis of Siraitia grosvenorii,50 DAF(days after flowering)and 70 DAF fruits from different developmental stages were employed to construct subtractive cDNA library by suppression subtractive hybridization(SSH)in this study.Total 641 positive clones were selected randomly and sequenced from the forward-subtracted cDNA library(70 DAF as the tester and 50 DAF as the driver),and 622 high quality sequences were obtained.The rate of the recombination fraction was above 96%.The distribution of inserted fragments ranged from 101 bp and 934 bp,and the average fragment size was 500 bp.Followed by BLASTN and BLASTX for sequences dates,201 ESTs showed no significant matches to any other sequences in NCBI nucleotide sequence database,they were probably novel genes.The other 421 ESTs carried with remarkable identity to proteins with known function in the database,which fell into several functional categories including energy and secondary metabolism,transcription factors,ripening,senescence and pathogen-resistance.Further results indicated that the cDNA library quality were conformed to SSH library standard and could be used in further studies,also would provide reference for studying the differentially expressed genes,exploring molecular mechanism of triterpenoid saponin biosynthesis,and increasing productivity and quality of mogrosides in S.grosvenorii.

An experiment was conducted to induce the in vitro shoots of Oplopanax elatus Nakai. into dormancy by the method of promoting dormancy. The suitable concentrations of ABA and KT supplemented into the medium for in vitro germplasm conservation were determined by uniform design. Results showed that N-68 basic medium with 2.37mg/L ABA and 0.60mg/L KT was fit for in vitro germplasm preservation,with a dormancy rate of 87.8%. The conservation period for germplasm of O. elatus could last for 37 months at normal temperature and natural light condition by the method of promoting dormancy. The dormant shoots could germinate and grow rapidly after the dormancy was relieved.

In this study,the adventitious buds induced from the tender stems of Rosa rugosa was used as explant,the main factors with different levels for germplasm in vitro conservation were investigated through uniform design experiments,and to screen the appropriate medium for in vitro conservation the adventitious buds of Rosa rugosa.The results showed that B 5+phloridzin 2.80 mg/L+KT 0.40 mg/L was suitable for in vitro conservation of the adventitious buds,the rate of regeneration was 0.83 % in 40 months.The contrast test results of rooting of before and after in vitro conservation of the adventitious buds,shoots take root quickly after in vitro conservation,the rate of rooting was more than 99.8 %,which is higher rooting rate than 98.5 % before in vitro conservation.The experiment results proved that in vitro conservation the adventitious buds of Rosa rugosa is feasible new method by deferring growth.

狼毒大戟为大戟科多年生草本植物.本研究以狼毒大戟(Euphorbia fischoriana)为试验材料,构建狼毒大戟cDNA文库及EST测序.研究结果表明,我们成功地构建了狼毒大戟的cDNA文库,并获得了2099个EST的序列.进一步对这些ESTs数据进行了Unigene的拼接、ORF预测和相应的功能注释及分类,发现了一些可能参与Prostratin生物合成的候选结构基因Transketolase,NADPH-cytochrome P450 reductase,Cytochrome P450,S-adenosylmethionine synthetase,acetyltransferas,l-deoxy-D-xylulose-5-phosphate synthase (DXP)和候选调节基因AP2类ERF转录因子(APE-like ethylene-responsive transcription factor,AP2-like ERF),此研究为今后开展抗HIV次生代谢物Prostratin的代谢工程奠定了基础.

MicroRNAs (miRNAs) are important regulators of gene expression that are increasing being implicated in controlling plant development and its interaction with the environment. The advent of new high-throughput sequencing technologies has enabled both the discovery and quantification of miRNAs from a diverse range of species. In this study, we employed high throughput Illumina sequencing to identify miRNAs from Taxus chinensis (T. chinensis) cells to investigate the effect of the taxoid elicitor methyl jasmonate (MJ) on miRNA expression. In a dataset of approximately 6.6 million sequences, a total of 58 miRNAs, belonging to 25 families were identified. A majority of them are conserved between angiosperms and gymnosperms. However, two miRNAs (miR1310 and miR1314) appear gymnosperm-specific, with miR1314 likely to exist as a cluster. MJ treatment significantly affected the expression of specific miRNAs; 14 miRNAs from 7 different families (miR156, miR168, miR169, miR172, miR396, miR480 and mir1310) were down regulated whereas 3 miRNAs from 2 families (miR164 and miR390) were up regulated.

Taxoid 14β-hydroxylase directs a side-route of taxol pathway that leading to a large number of 14β-hydroxy taxoid side products. A RNAi vector was used to suppress the expression of taxoid 14β-hydroxylase (14OH) gene in Taxus×media suspension cells. The dsRNA expression unit was introduced into Taxus×media cells via Agrobacterium tumefaciens-mediated transformation approach. PCR-Southern blot analysis of transgenic cells showed that the dsRNA unit was integrated into the genome of Taxus×media. The levels of transcripts for 14OH gene in transformed and the control cell lines were determined by using RT-PCR method. In addition,the yield of taxoids were analyzed by HPLC. The transcription of 14OH gene was suppressed and the total amount of three C-14 oxygenated taxoids (yunnanxane,sinenxan A,sinenxan C) in Taxus×media transgenic cell lines reduced significantly as compared to those in the non-transgenic control. Inhibition of the 14OH gene via RNAi might provide a new way to enhance the production of Taxol in Taxus cells.

丹参是唇彤科覃本植物丹参Salvia miltiorrhizaBge.的根及根茎,具有活血祛瘀,凉血清心,养血安神的功效,是临床常用的中药.毛状根是农杆菌中Ri质粒的一段DNA嵌入植物基因组中并表达的结果,许多植物受发根农杆菌感染后在受伤部位能长出大量毛状根.由于其生长速度快,不需外源植物激素,合成次生代谢物质能力强而且稳定.毛状根作为一种转基因的器官,一种新的药用原料,越来越被人们所重视.

In response to gravitational stresses, angiosperm trees form tension wood in the upper sides of branches and leaning stems in which cellulose content is higher, microfibrils are typically aligned closely with the fibre axis and the fibres often have a thick inner gelatinous cell wall layer (G-layer).Gene expression was studied in Eucalyptus nitens branches oriented at 45 degrees using microarrays containing 4900 xylem cDNAs, and wood fibre characteristics revealed by X-ray diffraction, chemical and histochemical methods.Xylem fibres in tension wood (upper branch) had a low microfibril angle, contained few fibres with G-layers and had higher cellulose and decreased Klason lignin compared with lower branch wood. Expression of two closely related fasciclin-like arabinogalactan proteins and a beta-tubulin was inversely correlated with microfibril angle in upper and lower xylem from branches.Structural and chemical modifications throughout the secondary cell walls of fibres sufficient to resist tension forces in branches can occur in the absence of G-layer enriched fibres and some important genes involved in responses to gravitational stress in eucalypt xylem are identified.