Progress in circular RNAs of plants / 生物工程学报

With the development of high-throughput sequencing technology, circular RNAs (circRNAs) have gradually become a hotspot in the research on non-coding RNA. CircRNAs are produced by the covalent circularization of a downstream 3' splice donor and an upstream 5' splice acceptor through backsplicing, and they are pervasive in eukaryotic cells. CircRNAs used to be considered byproducts of false splicing, whereas an explosion of related studies in recent years has disproved this misconception. Compared with the rich studies of circRNAs in animals, the study of circRNAs in plants is still in its infancy. In this review, we introduced the discovery of plant circRNAs, the discovery of plant circRNAs, the circularization feature, expression specificity, conservation, and stability of plant circRNAs and expounded the identification tools, main types, and biogenesis mechanisms of circRNAs. Furthermore, we summarized the potential roles of plant circRNAs as microRNA (miRNA) sponges and translation templates and in response to biotic/abiotic stress, and briefed the degradation and localization of plant circRNAs. Finally, we discussed the challenges and proposed the future directions in the research on plant circRNAs.

CircPlant: An Integrated Tool for circRNA Detection and Functional Prediction in Plants / 基因组蛋白质组与生物信息学报·英文版

The recent discovery of circular RNAs (circRNAs) and characterization of their functional roles have opened a new avenue for understanding the biology of genomes. circRNAs have been implicated to play important roles in a variety of biological processes, but their precise functions remain largely elusive. Currently, a few approaches are available for novel circRNA prediction, but almost all these methods are intended for animal genomes. Considering that the major differences between the organization of plant and mammal genomes cannot be neglected, a plant-specific method is needed to enhance the validity of plant circRNA identification. In this study, we present CircPlant, an integrated tool for the exploration of plant circRNAs, potentially acting as competing endogenous RNAs (ceRNAs), and their potential functions. With the incorporation of several unique plant-specific criteria, CircPlant can accurately detect plant circRNAs from high-throughput RNA-seq data. Based on comparison tests on simulated and real RNA-seq datasets from Arabidopsis thaliana and Oryza sativa, we show that CircPlant outperforms all evaluated competing tools in both accuracy and efficiency. CircPlant is freely available at http://bis.zju.edu.cn/circplant.

Identification and differential expression of miRNA related to seed dormancy of Paris polyphylla var. chinensis / 中国中药杂志

The purpose of this study was to explore the expression pattern of miRNA in the process of embryo dormancy and provide a reference for the mechanism of regulating seed dormancy and germination by miRNA. We used high-throughput sequencing technology, bioinformatics analysis and real-time fluorescent quantitative PCR(qPCR) technology to sequence, screen and identify miRNAs of dormant and dormant embryos. The results showed that there were 23 811 977, 24 276 695, 20 611 876 and 20 601 811 unique sequences in the four sample libraries during the period of dormancy and dormancy release. MiRNAs are mainly distributed between 21 and 24 nt, among which the length of 24 nt occurred most frequently. A total of 31 known miRNAs were identified, belonging to 13 different families. 93 new miRNAs were predicted by bioinformatics software. Ten miRNAs(mir156 a-5 p, mir160 a-5 p, mir160 h-1, mir169 a-5 p, mir157 d, mir159 a-1, mir395-3, mir156 f-5 p, mir156-2 and mir171 a-3 p) were screened out. In this study, 10 miRNAs related to seed dormancy release were identified. The target genes mainly involved carbohydrate metabolism, plant hormone signal transduction, cell division and growth. The results of qRT-PCR showed that the sequencing results were consistent with the actual results.

Screening of long non-coding RNA related to CYP450s involved in biosynthesis of tanshinones / 中国中药杂志

Tanshinones are abietane-type norditerpenoid quinones that make up the main bioactive ingredients of traditional Chinese medicine Salvia miltiorrhiza. Cytochrome CYP450 plays an important role in the post-structural modification of tanshinone biosynthesis pathway. Long non-coding RNA( lncRNA) have been defined as transcripts longer than 200 nucleotides,which have been functionally characterized in regulating the growth and development,secondary metabolism and stress of medicinal plants. In this study,we perform a comprehensive identification of lncRNAs in response to tanshinone metabolism induced by yeast extract( YE) and Ag~+ S. miltiorrhiza hairy roots. Deep RNA sequencing was used to identify a set of different 8 942 lncRNAs,of which 6 755 were intergenic lncRNAs. We predicted a total of 1 115 814 lncRNA-coding gene pairs,including 122 lncRNA-coding gene as cis pairs. The correlation analysis between lncRNA and CYP450 related to tanshinone biosynthesis was carried out and a total of 16 249 lncRNA-CYP450 target gene pairs were identified. Further analysis with functional known CYP76 AH1,CYP76 AH3 and CYP76 AK1 involved in tanshinone biosynthesis,we also identified a set of 216 target genes. These candidate genes will be the important target in the downstream regulation mechanism analysis of the tanshinone biosynthesis pathway.

Identification and analysis of NAC family in ginger(Zingiber officinale) based on RNA-seq data / 中国中药杂志

The NAC family is an important transcription factor which regulate plant growth and development， signal transduction， and stress response.In this study， the protein identification， subfamily classification， the determination of physical and chemical properties， protein structure， and expression pattern of NAC family were performed using bioinformatic methods based on the RNA-seq data of ginger. The results showed that a total of 72 NAC transcription factors were identified in 271.1 Mb total nucleotides， and they could be clustered into 13 subfamilies according to the phylogenetic tree.The physical and chemical properties， structure analysis revealed that the amino acid number and isoelectric point were different among 13 NAC subfamilies; the secondary structure of NACs transcription factors mainly consist of random coil， and the tertiary structure is similar.In addition，the expression patterns of genes under different soil moisture and Ralstonia solanacearum infection showed that 23 NACs were differentially expressed， which were mainly distributed in Ⅷ，Ⅶ， and ⅩⅤ subfamilies related to plant senescence， hormone metabolism and cell wall metabolism.The results provide some valuable information for the research and development of NAC transcription factors in ginger.

Transcriptomic analysis identifies differentially expressed genes (DEGs) associated with bolting and flowering in Saposhnikovia divaricata / 中国天然药物

Saposhnikovia divaricata is a valuable Chinese medicinal herb; the transformation from vegetative growth to reproductive growth may lead to the decrease of its pharmacological activities. Therefore, the study of bolting and flowering for Saposhnikovia divaricata is warranted. The present study aimed to reveal differentially expressed genes (DEGs) and regularity of expression during the bolting and flowering process, and the results of this study might provide a theoretical foundation for the suppression of early bolting for future research and practical application. Three sample groups, early flowering, flower bud differentiation, and late flowering (groups A, B, and C, respectively) were selected. Transcriptomic analysis identified 67, 010 annotated unigenes, among which 50, 165 were differentially expressed including 16, 108 in A vs B, and 17, 459 in B vs C, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional classification analysis were performed on these differentially expressed genes, and five important pathways were significantly impacted (P ≤ 0.01): plant circadian rhythm, other glycan degradation, oxidative phosphorylation, plant hormone signal transduction, and starch and sucrose metabolism. Plant hormone signal transduction might play an important role in the bolting and flowering process. The differentially expressed indole-3-acetic acid (IAA) gene showed significant down-regulation during bolting and flowering, while the transport inhibitor response 1 (TIR1) gene showed no significant change during the bolting process. The expression of flowering related genes FLC, LYF, and AP1 also showed a greater difference at different development stages. In conclusion, we speculate that the decrease in auxin concentration is not caused by the degrading effect of TIR1 but by an alternative mechanism.

Transcriptomic analysis identifies differentially expressed genes (DEGs) associated with bolting and flowering in Saposhnikovia divaricata / 中国天然药物

Saposhnikovia divaricata is a valuable Chinese medicinal herb; the transformation from vegetative growth to reproductive growth may lead to the decrease of its pharmacological activities. Therefore, the study of bolting and flowering for Saposhnikovia divaricata is warranted. The present study aimed to reveal differentially expressed genes (DEGs) and regularity of expression during the bolting and flowering process, and the results of this study might provide a theoretical foundation for the suppression of early bolting for future research and practical application. Three sample groups, early flowering, flower bud differentiation, and late flowering (groups A, B, and C, respectively) were selected. Transcriptomic analysis identified 67, 010 annotated unigenes, among which 50, 165 were differentially expressed including 16, 108 in A vs B, and 17, 459 in B vs C, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional classification analysis were performed on these differentially expressed genes, and five important pathways were significantly impacted (P ≤ 0.01): plant circadian rhythm, other glycan degradation, oxidative phosphorylation, plant hormone signal transduction, and starch and sucrose metabolism. Plant hormone signal transduction might play an important role in the bolting and flowering process. The differentially expressed indole-3-acetic acid (IAA) gene showed significant down-regulation during bolting and flowering, while the transport inhibitor response 1 (TIR1) gene showed no significant change during the bolting process. The expression of flowering related genes FLC, LYF, and AP1 also showed a greater difference at different development stages. In conclusion, we speculate that the decrease in auxin concentration is not caused by the degrading effect of TIR1 but by an alternative mechanism.

A fast and efficient protocol for small RNA extraction in Japanese plum and other Prunus species

Background: Small ribonucleic acids represent an important repertoire of mobile molecules that exert key roles in several cell processes including antiviral defense. Small RNA based repertoire includes both small interfering RNA (siRNA) and microRNA (miRNA) molecules. In the Prunus genus, sharka disease, caused by the Plum pox virus (PPV), first occurred on European plum (Prunus domestica) and then spread over among all species in this genus and thus classified as quarantine pathogen. Next-generation sequencing (NGS) was used for the study of siRNA/miRNA molecules; however, NGS relies on adequate extraction protocols. Currently, knowledge of PPV-Prunus interactions in terms of siRNA populations and miRNA species is still scarce, and siRNA/miRNA extraction protocols are limited to species such as peach, almond, and sweet cherry. Results: We describe a reliable procedure for siRNA/miRNA purification from Prunus salicina trees, in which previously used protocols did not allow adequate purification. The procedure was based on a combination of commercially available RNA purification kits and specific steps that yielded high quality purifications. The resulting molecules were adequate for library construction and NGS, leading to the development of a pipeline for analysis of both siRNAs and miRNAs in the PPV­P. salicina interactions. Results showed that PPV infection led to altered siRNA profiles in Japanese plum as characterized by decreased 24-nt and increased 21- and 22-nt siRNAs. Infections showed miR164 and miR160 generation and increased miR166, miR171, miR168, miR319, miR157, and miR159. Conclusion: We propose this protocol as a reliable and reproducible small RNA isolation procedure for P. salicina and other Prunus species.

Diversity and capacity to promote maize growth of bacteria isolated from the Amazon region / Diversidade e capacidade de promoção de crescimento de milho de bactérias isoladas da região Amazônica

Maize plants can establish beneficial associations with plant growth-promoting bacteria. However, few studies have been conducted on the characterization and inoculation of these bacteria in the Amazon region. This study aimed to characterize endophytic bacteria isolated from maize in the Amazon region and to assess their capacity to promote plant growth. Fifty-five bacterial isolates were obtained from maize grown in two types of ecosystems, i.e., a cerrado (savanna) and a forest area. The isolates were characterized by the presence of the nifH gene, their ability to synthesize indole-3-acetic acid (IAA) and solubilize calcium phosphate (CaHPO4), and 16S rRNA partial gene sequencing. Twenty-four bacteria contained the nifH gene, of which seven were isolated from maize plants cultivated in a cerrado area and seventeen from a forest area. Fourteen samples showed the capacity to synthesize IAA and only four solubilized calcium phosphate. The following genera were found among these isolates: Pseudomonas; Acinetobacter; Enterobacter; Pantoea; Burkholderia and Bacillus. In addition, eight isolates with plant growth-promoting capacity were selected for a glasshouse experiment involving the inoculation of two maize genotypes (a hybrid and a variety) grown in pots containing soil. Inoculation promoted the development of the maize plants but no significant interaction between maize cultivar and bacterial inoculation was found. A high diversity of endophytic bacteria is present in the Amazon region and these bacteria have potential to promote the development of maize plants.

Plantas de milho podem estabelecer associações benéficas com bactérias promotoras do crescimento vegetal. No entanto, poucos estudos de caracterização e inoculação com essas bactérias foram realizados na região Amazônica. O objetivo deste estudo foi caracterizar bactérias endofíticas isoladas de milho na região Amazônica e avaliar sua capacidade de promover o crescimento. Cinquenta e cinco isolados bacterianos foram obtidos de plantas de milho cultivadas em diferentes tipos de ecossistemas, cerrado (savana) e área de floresta. Estes foram caracterizados pela presença do gene nifH, capacidade de sintetizar ácido indol-3-acético (AIA), solubilizar fosfato de cálcio (CaHPO4) e sequenciamento parcial do gene 16S rRNA. Vinte e quatro bactérias apresentaram o gene nifH. Destas, sete são oriundas de plantas cultivadas em uma área de cerrado e 17 em área de floresta. Quatorze bactérias demonstraram a capacidade de solubilizar AIA e quatro de solubilizar fosfato de cálcio. Entre estas bactérias, foram encontrados os seguintes gêneros: Pseudomonas; Acinetobacter; Enterobacter; Pantoea; Burkholderia e Bacillus. Oito isolados com capacidade de promoção do crescimento vegetal foram selecionados para experimento em casa de vegetação com a inoculação em dois diferentes genótipos de milho (um híbrido e uma variedade) em vasos com solo. Quanto à promoção de crescimento vegetal, a inoculação promoveu o desenvolvimento de plantas de milho, mas não foi encontrada interação significativa entre a cultivar de milho e a inoculação bacteriana. Na região Amazônica há uma diversidade de bactérias endofíticas e estas possuem potencial para promover o desenvolvimento de plantas de milho.

Religión y salud: la intervención pública de agentes religiosos católicos formados en bioética en el debate parlamentario sobre la muerte digna en la Argentina / Religion and health: the public intervention of Catholic religious agents trained in bioethics in the parliamentary debate on death with dignity in Argentina

Desde una perspectiva sociológica, este trabajo aborda una de las aristas de la intervención pública de ciertos sectores del catolicismo en la elaboración y sanción de leyes de salud. En particular se hace foco en el debate en comisiones parlamentarias sobre la llamada ley de "muerte digna" (Ley 26742) en el cual se convocó a un grupo de expertos en bioética para asesorar a los senadores sobre los alcances y límites de la ley. La mayoría de los expertos invitados pregonan la perspectiva de la bioética personalista, un desarrollo teológico de la bioética del catolicismo contemporáneo. En el debate no participaron representantes de otros credos consolidando la ampliamente estudiada imbricación entre el catolicismo y lo político en Argentina.

This paper discusses from a sociological perspective one of Catholicism's fronts of public intervention in the development and enactment of health legislation. In particular we analyze the debate in parliamentary committees on the so-called "death with dignity" law (No. 26742), for which a group of bioethics experts was convened to counsel senators regarding the scope and limits of the law. The majority of the invited experts advocated a personalist bioethics perspective, which is a theological bioethics development of contemporary Catholicism. In the debate no representatives of other faiths were present, reinforcing the widely studied overlap between Catholicism and politics in Argentina.

Transcriptome analysis of bioenergy plant Miscanthus sinensis Anderss by RNA-Seq / 生物工程学报

Miscanthus sinensis Anderss is a perennial C4-grass. It is a promising bioenergy plant, which has been proposed as general feedstock for biomass and lignocellulosic biofuel production. In this study, the flower and leaf buds transcriptomes of Miscanthus sinensis Anderss were sequenced by the platform of Illumina HiSeq 2000. In total 98 326 Unigenes were generated by de novo assembly with an average length of 822 bp and N50 of 1 023 bp. Based on the NR, NT, Swiss-Prot, KEGG, GO and COG databases (Evalue < le-5), 74 134 (75.40%) Unigenes were annotated. A total of 45 507 Unigenes were mapped into different GO terms. In KEGG pathways identification, 36 710 sequences were assigned to 128 KEGG pathways. Sorghum bicolor (37 731, 60.86%), Zea mays (16 258, 26.22%), and Oryza sativa (3 065, 4.94%) showed high similarity to Miscanthus sinensis Anderss. And 24 photosynthesis-related enzyme genes were identified. The result provides a foundation for further characterizing the functional genes in Miscanthus sinensis Anderss.

Construction of cDNA expression library of watermelon for isolation of ClWRKY1 transcription factors gene involved in resistance to Fusarium wilt.

Full-length cDNAs are very important for genome annotation and functional analysis of genes. The number of full-length cDNAs from watermelon remains limited. Here we report first the construction of a full-length enriched cDNA library from Fusarium wilt stressed watermelon (Citrullus lanatus Thunb.) cultivar PI296341 root tissues using the SMART method. The titer of primary cDNA library and amplified library was 2.21 × 106 and 2.0 × 1010 pfu/ml, respectively and the rate of recombinant was above 85%. The size of insert fragment ranged from 0.3 to 2.0 kb. In this study, we first cloned a gene named ClWRKY1, which was 1981 bp long and encoded a protein consisting of 394 amino acids. It contained two characteristic WRKY domains and two zinc finger motifs. Quantitative real-time PCR showed that ClWRKY1 expression levels reached maximum level at 12 h after inoculation with Fusarium oxysporum f. sp. niveum. The full-length cDNA library of watermelon root tissues is not only essential for the cloning of genes which are known, but also an initial key for the screening and cloning of new genes that might be involved in resistance to Fusarium wilt.

Evaluation of Parkia pendula lectin mRNA differentially expressed in seedlings / Avaliação de mRNA da lectina de Parkia pendula diferencialmente expressa em plântulas

Parkia pendula (Willd.) Walp. (Fabaceae) is a neotropical species of the genus Parkia more abundantly distributed in Central to South America. From the seeds of P. pendula a glucose/mannose specific lectin (PpeL) was isolated that has been characterised and used as a biotechnological tool but until now this is the first manuscript to analyse P. pendula mRNA expression in seedlings. For this porpoise a Differential display reverse transcription polimerase chain reaction (DDRT-PCR) was used to evaluate the expression of P. pendula lectin mRNAs in non-rooted seedlings. No bands were observed in the agarose gel, indicating the absence of mRNA of PpeL seedlings. our findings confirm that lectins mRNAs are differently regulated among species even if they are grouped in the same class.

Parkia pendula (Willd.) Walp. (Fabaceae) é a espécie neotropical do gênero Parkia mais abundantemente distribuída na América Central a do Sul. Das sementes de P. pendula foi isolada uma lectina glicose/manose específica (Ppel) que foi caracterizada e usada como ferramenta biotecnológica, porém até o momento esse é o primeiro artigo a analisar a expressão do mRNA nas plântulas de P. pendula. Para esse propósito uma reação de PCR diferencial de transcriptase reversa (DDRT-PCR) foi utilizada para avaliar a expressão do mRNA da lectina de P. pendula em plântulas não enraizadas. Nenhuma banda foi observada no gel de agarose, indicando a ausência de mRNA das plântulas de PpeL. Nossos achados confirmam que os mRNAs de lectinas são regulados de forma diferentes entre as espécies, mesmo que sejam agrupadas na mesma classe.

Extraction of total RNA and cloning of sgDHAR gene from Siraitia grosvenorii / 药学学报

Total RNA was isolated from Siraitia grosvenorii fruit by the method of modified Trizol, according to S. grosvenorii fruit characteristics of rich phenols, polysaccharide, oil and proteins. The OD260/280, OD260/230, RNA integrity (RIN) and yield of the total RNA with this method were 2.01, 2.02, 9.50 and 260 mirog.g-1, respectively. The open reading frame (ORF) of dehydroascorbate reductase (DHAR), named as SgDHAR, was cloned by rapid amplification of cDNA ends (RACE) and RT-PCR method from S. grosvenorii. The GenBank accession number for this gene is KC907731. The SgDHAR gene contains a full-length cDNA of 1,252 bp including ORF of 819 bp and encodes a predicted protein of 272 amino acids. The molecular mass is 30.217 7 kD and the isoelectric point is 8.76. Homology comparison showed that it shared 87% nucleotide sequence homology with Cucumis sativus. Expression patterns using qRT-PCR analysis showed that SgDHAR was mainly expressed in fruit and stem, followed by flower, and was lowest in root, while the expression level was 6.83 times in triploid. T than that in diploid. Therefore, SgDHAR gene may be involved in abortion of triploid seedless S. grosvenorii.

Efficient transient expression to analyze miRNA targets in rice protoplasts / 生物工程学报

Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice.

Cloning and bioinformatics analysis of chalcone synthase (AsCHS1) gene in Aquilaria sinensis / 中国中药杂志

<p><b>OBJECTIVE</b>The study aimed to clone the open reading frame of chalcone synthase (CHS) from Aquilaria sinensis and analyze the bioinformatics and expression of the gene.</p><p><b>METHOD</b>One unique sequence containing CHS domain was discovered in our previous reported wound transcriptome dataset of A. sinensis. The open reading frame of CHS was cloned by RT-PCR strategy with the template of mixed RNA extracted from A. sinensis stem which treated by different wound time. The bioinformatic analysis of this gene and its corresponding protein was performed. The AsCHS1 expression in calli was analyzed with histone gene as an internal control gene under wound condition by qRT-PCR technique.</p><p><b>RESULT</b>One unique sequence of CHS, named as AsCHS1, was cloned from A. sinensis. The full length of AsCHS1 cDNA was containing a 1 192 bp ORF that encoded 397 amino acids. The result of qRT-PCR displayed that the highest expression level was at 12 h, which indicated that it was possibly involved in early-stage response to wound.</p><p><b>CONCLUSION</b>Cloning and analyzing AsCHS1 gene from A. sinensis provided basic information for study the function and expression regulation of AsCHS1 in the flavonoids biosynthesis.</p>

Comparison of different methods for isolating total RNA from bulblet of Fritillaria anhuiensis / 中国中药杂志

<p><b>OBJECTIVE</b>To optimize a simple and effective method for total RNA extraction from bulblet of Fritillaria anhuiensis.</p><p><b>METHOD</b>Four methods, i. e. guanidine isothiocyanate, bentonite, modified SDS/phenol and the RNAiso plus, were used to extract total RNA from bulblet of F. anhuiensis. Then the results of the extraction were compared and analyzed by electrophoresis detection and RT-PCR verification.</p><p><b>RESULT</b>The total RNA extracted by bentonite method were clear and no dispersion, the integrity of the RNA was well, and there was no obvious contamination with DNA and other impurities, was suitable for RT-PCR test.</p><p><b>CONCLUSION</b>The bentonite method is quick, economic, and efficient for total RNA extraction from bulblet of F. anhuiensis.</p>

RNA silencing, the fundamental security strategy of genomic immunity: a review / 生物工程学报

The 21-24 nucleotides small RNA that generated from double strands RNA can trigger two types of epigenetic gene silencing in plants. One is Post-Transcriptional Gene Silencing (PTGS), characterized by cleavage of homologous mRNA in cytoplasm. Transcriptional Gene Silencing (TGS) is another one, in which transcription inhibition is obtained through small RNA-directed DNA methylation of homologous promoter region. Here we summarized the relationship and differences between PTGS and TGS, the current achievement in the study of RNA silencing spreading, as well as the discrepancy of exogenous and endogenous gene silencing, and discussed the underlying reasons in the end.

Effective method for isolation of total RNA from Fagopyrum cymosum callus / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a method for isolation of the total RNA from Fagopyrum cymosum callus.</p><p><b>METHOD</b>The improved method combining that of CTAB extraction with the LiCl precipitation was used to isolate the total RNA from the four F. cymosum callus. The quality of the RNA was detected by UV spectrophotometric analysis, 0.8% non-denaturing agarose gel electrophoresis and RNA reverse transcription.</p><p><b>RESULT</b>The bands of 28S and 18S could be seen clearly by agarose gel electrophoresis, and the value of A260/A280 was between 1.9 and 2.0. The cDNA which was reverse-transcribed by the total RNA showed a wide length rage of 500 bp-5 kb.</p><p><b>CONCLUSION</b>The RNA extracted by this method meets the requirement of reverse transcription-polymerase chain reaction (RT-PCR), construction of cDNA libraries, et al. This improved method can be used to isolate the total RNA from F. cymosum callus with the advantage of simpleness, efficiency and low cost.</p>

Cloning and bioinformatic analysis of PqERF1 gene in Panax quinquefolius / 药学学报

ERF family transcription factor (TF) represented ethylene-responsive protein which harbored a conserved AP2 domain. After searching the plant transcription factor database, a total of 75 unigenes was found which contained AP2 domain from the transcriptome dataset of Panax quinquefolius L. One unique sequence of ERF transcript, named as PqERF1, was cloned with entire open reading frame of 933 base pairs (bp). Protein prediction result indicated that the gene was localized in nucleus and had a conserved AP2 domain. PqERF1 gene could be induced by methyl jasmonate (MeJA) which was consistent to the inducing profile of triterpene ginsenosides. InterproScan prediction indicated that PqERF1 was probably a pathogenesis-related gene. Sequence alignment and phylogenetic analysis demonstrated PqERF1 was with high identity and had relative close relationship to the NtERF4 (Nicotiana tabacum), PhERF12 (Petunia x hybrida) and DcERF1 (Daucus carota) which was related to plant defense, regulation of secondary metabolism and the flower senescence respectively. Therefore, the gene was likely involved in regulation of secondary metabolism, plant defense and physical processes which would provide gene resource for further study on secondary metabolite synthesis and molecular breeding of P. quinquefolius.