Identification and expression analysis of cellulose synthase family genes in Aquilaria sinensis / 药学学报

Cellulose synthase (CesA), one of the key enzymes in the biosynthesis of cellulose in plants, plays an important role in plant growth and plant resistance. In this study, a total of 21 AsCesA genes from Aquilaria sinensis were systematically identified and the physico-chemical characteristics were analyzed based on genome database and bioinformatical methods. The phylogenetic tree was constructed and the gene location on chromosome, cis-acting elements in the 2 000 basepairs upstream regulatory regions and conservative motifs were analyzed. The AsCesA proteins were mainly located on the plasma membrane. The number of amino acids of the proteins ranged from 390 to 1 261. The isoelectric point distributed from 5.67 to 8.86. All of the 21 AsCesA proteins possessed the transmembrane domains, the number of which was from 6 to 8. The genes were classified into 3 groups according to the phylogenetic relationship. Obvious differences were observed in motif composition in genes from different groups. However, motif2, motif6, motif7 and motif10 were observed in all of AsCesA proteins. Analysis of cis-acting elements indicated that AsCesA genes family has cis-acting elements related to plant hormones, abiotic stresses, and biological processes. Seven AsCesA genes with differential expression were selected according to the calli transcriptome data induced by NaCl at different times and their expression levels under different abiotic stresses were analyzed by quantitative real-time PCR. The results indicated that salt, low temperature, drought, and heavy metal stresses could affect the expression level of AsCesA genes, and the abundance of AsCesA1, AsCesA3 and AsCesA20 showed a significant change, implying their potential important roles to the abiotic stresses. The accumulation pattern of cellulose content under different abiotic stresses was similar to the expression trend of AsCesA genes. Our results provide valuable insights into the role of cellulose synthase in A.sinensis in plant defense.

LC-MS analysis of 2-(2-phenylethyl) chromones in sodium chloride-treated suspension cells of Aquilaria sinensis / 中国中药杂志

Qualitative and quantitative analysis of 2-(2-phenylethyl) chromones in sodium chloride(NaCl)-treated suspension cells of Aquilaria sinensis was conducted by UPLC-Q-Exactive-MS and UPLC-QQQ-MS/MS. Both analyses were performed on a Waters T3 column(2.1 mm×50 mm, 1.8 μm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as mobile phases at gradient elution. MS data were collected by electrospray ionization in positive ion mode. Forty-seven phenylethylchromones was identified from NaCl-treated suspension cell samples of A. sinensis using UPLC-Q-Exactive-MS, including 22 flindersia-type 2-(2-phenylethyl) chromones and their glycosides, 10 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and 15 mono-epoxy or diepoxy-5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones. Additionally, 25 phenylethylchromones were quantitated by UPLC-QQQ-MS/MS. Overall, the rapid and efficient qualitative and quantitative analysis of phenylethylchromones in NaCl-treated suspension cells of A. sinensis by two LC-MS techniques, provides an important reference for the yield of phenylethylchromones in Aquilariae Lignum Resinatum using in vitro culture and other biotechnologies.

Cloning and expression analysis of cytochrome P450 AsCYP71D1 from Aquilaria sinensis (Lour.) Gilg / 药学学报

Cytochrome P450 (CYP450) is a kind of superfamily oxidase containing heme, which is distributed in various aerobic organisms. They are widely involved in the biosynthesis of terpenoids, alkaloids, flavonoids, fatty acids, etc. In this study, the full-length cDNA sequence of a P450 was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) technology, with the specific primers that designed according to the sequence of a transcript annotated as P450 from the Aquilaria sinensis (Lour.) Gilg transcriptome database. The tissue expression and subcellular localization were also studied. The full-length cDNA of the cloned P450 gene is 1 920 bp, with 88 bp 5′-untranslated region (UTR), 344 bp 3′-UTR and a 21 bp polyA tail, and 1 488 bp open reading frame (ORF), encoding 495 amino acids. Sequence alignment revealed that the protein belonged to CYP71D family of cytochrome P450 family, and named AsCYP71D1. Tissue expression analysis indicated that AsCYP71D1 was mainly expressed in stem. Further subcellular localization of onion epidermis showed that AsCYP71D1 was expressed in cytoplasm, nucleus and cell membrane. This study will provide a foundation for further research on its function in agarwood sesquiterpene biosynthesis.

Study on Changes of Microorganisms and Volatile Components in Aquilariae Lignum Resinatum with Different Aging Years / 中国实验方剂学杂志

ObjectiveThis study aims to investigate the changes in fungal community diversity and volatile components during the aging process of Aquilariae Lignum Resinatum and explore the internal relationship between them. MethodAquilariae Lignum Resinatum samples with different aging years were collected. High-throughput sequencing was employed to analyze the fungal diversity and abundance， and α and β diversity indicators were calculated to reveal the composition and dynamic changes of the fungal community. In addition， the essential oils of Aquilariae Lignum Resinatum with different aging years were extracted， separated， and identified by two-dimensional gas chromatography-high resolution time-of-flight mass spectrometry. ResultA total of 61 compounds were identified from the volatile components of five groups of Aquilariae Lignum Resinatum samples， including 2 monoterpenes， 24 sesquiterpenoids， 1 diterpene， 13 aromatic hydrocarbons， 9 alkanes， and 12 other compounds. Among them， the volatile compounds isolated from the sample aged for 1 year had the largest number， and those from the sample aged for 2 years accounted for the largest proportion of the total components. The internal transcribed spacer（ITS） amplicon sequencing revealed that the fungi in the five groups of samples belonged to 162 genera. Kirschsteiniothelia， Aspergillus， Lasiodiplodia， Phaeoacremonium， and Trichoderma were the dominant fungal genera. The fungal diversity in the Aquilariae Lignum Resinatum sample aged for 4 years was significantly higher than that in the samples aged for 0 to 3 years. ConclusionThe volatile component content and composition of Aquilariae Lignum Resinatum altered dramatically during aging. The aging of Aquilariae Lignum Resinatum was accompanied by the increasing fungal diversity， decreasing relative content of aromatic hydrocarbons， and increasing relative content of sesquiterpenoids. In general， aging was beneficial to the transformation of sesquiterpenoids and the enrichment of fungi.

Cloning, expression and functional identification of cytochrome P450 reductase gene in Aquilaria sinensis / 药学学报

Cytochrome P450 reductase (CPR) is essential for the electron transport chain of cytochrome P450s, playing an indispensable role in electron transfer in vivo. In this study, one cDNA encoding cytochrome P450 reductase (Ascpr1) was identified from the callus of Aquilaria sinensis. Ascpr1 contains an open reading frame of 2 124 bp. The deduced protein is composed of 707 amino acids, with a predicted molecular weight of 78.82 kD. Phylogenetic analysis revealed that AsCPR1 is a type Ⅱ CPR protein closely related to the CPR from Theobroma cacao. Transmembrane prediction using TMHMM 2.0 indicated that the amino acids 52-71 of AsCPR1 comprise a transmembrane region. After truncating of 67 amino acid residues from N-terminal, the truncated AsCPR1 was successfully expressed in E. coli Transetta (DE3). Further purification of the recombinant AsCPR1 by affinity chromatography and determination of the enzymatic activity allowed the reducing ability of AsCPR1 to cytochrome C in vitro. The results pave the way for further study on the synthesis of defensive chemicals involved in P450s and the functions of CPR in self-defense of A. sinensis.

Cloning and expression analysis of chalcone isomerase from Aquilaria sinensis / 药学学报

Chalcone isomerases (CHIs) play an essential role in the biosynthesis of flavonoids important in plant self-defense. Based on the transcriptome data of Aquilaria sinensis Calli, a full-length cDNA sequence of CHI1 (termed as AsCHI1) was cloned by reverse transcription PCR. AsCHI1 contains a complete open frame (ORF) of 654 bp. The deduced protein is composed of 217 amino acids, with a predicted molecular weight of 23.11 kDa. The sequence alignment and phylogenetic analysis revealed that AsCHI1 has conserved most of the active site residues in type I CHIs, indicating a close relationship with the CHI from Gossypium hirsutum. The recombinant AsCHI1 protein was obtained by heterologous expression of AsCHI1 in E. coli BL21(DE3). The purified AsCHI1 protein exhibited CHI activity by catalyzing the production of naringenin from naringenin chalcone. Remarkably, AsCHI1 expression in A. sinensis Calli treated with various abiotic stresses including salt, mannitol, cold, and heavy metals could be markedly increased, and plant hormones such as abscisic acid (ABA), gibberellin (GA3), and salicylic acid (SA) could also increase the expression of AsCHI1, suggesting that AsCHI1 might play an important role in plant self-defense. The results expand our understanding of the biosynthesis of flavonoids in A. sinensis and give further insight into the defensive responses of A. sinensis to abiotic and biotic stresses.

Analysis of bacteria distribution characteristics in different layers of agarwood based on Hiseq sequencing / 中国中药杂志

To explore the diversity of bacterial community structure between different layers of agarwood, Hiseq(high-throughput sequencing) was used to analyze the bacterial community structure of samples from different layers of agarwood. Our results showed that 1 150 096 optimized sequences and 9 690 OTUs were obtained from 15 samples of 5 layers of agarwood, which belonged to 28 bacterial phyla, 61 classes, 110 orders, 212 families and 384 genera. Further analysis revealed that the normal layer(NL) had the lowest bacterial species richness and the smallest number of OTUs. And the total number of OTUs of the agarwood layer(AL) and NL was zero, which was quite different.At the same time, there were significant differences in bacterial community structure and species diversity between NL and the other four layers. While there were some common dominant bacterial genera in both transition layer(TL) and NL. The similarity of bacterial distribution in 4 non-NL layers was relatively high, which had four common genera, such as Acidibacter, Bradyrhizobium, Acidothemus and Sphingomonas. While Acidibacter, Bradyrhizobium and Acidothemus were the dominant bacterial genus of DA and AL, and all of these layers contained volatile oil. In addition, the Bradyrhizobium was the most abundant in agarwood layer. Our results showed that bacterial community diversity and abundance were decreasing from DL to AL, and different layers showed significant differences in bacterial enrichment. It provided the clues to investigate how bacteria participate in the formation of agarwood.

Gene cloning, subcellular localization and expression analysis of the AsERF1 gene from Aquilaria sinensis / 药学学报

Ethylene-response factors, which are a subfamily of the AP2/ERF family, play an important role in ethylene signal transduction, plant growth and plant resistant. In this study, a full-length cDNA of the AsERF1 gene was cloned from Aquilaria sinensis. Sequence analysis, prokaryotic expression and purification, subcellular localization, tissue-specific analysis and expression analysis under different abiotic stresses was performed. The open reading frame (ORF) of the AsERF1 gene was 691 bp, encoding a protein of 229 amino acids with a predicted molecular mass of 25.36 kD. The AsERF1 protein contained the conserved AP2 sequence of ERF protein. A phylogenetic analysis indicated that the AsERF1 protein showed greatest sequence similarity with ERF2 from Populus trichocarpa. The recombinant AsERF1 protein was expressed in Escherichia coli BL21(DE3) cells using the prokaryotic expression vector pET28a-AsERF1 and the recombinant AsERF1 protein was purified. Agrobacterium-mediated protein expression experiments demonstrated that AsERF1 mainly localized to the nucleus. Expression analysis indicated that AsERF1 was primarily observed in leaves. The AsERF1 expression level was induced by salt, drought, low temperature and CdCl2 treatment, while the abundance of AsERF1 was most significantly induced by drought stress. These results provide valuable insights into the role of AsERF1 in plant defense and the mechanism of agarwood formation.

Study on sesquiterpenoids from Aquilaria sinensis / 中草药

Objective: To investigate the sesquiterpenoid constituents from Aquilaria sinensis. Methods: The chemical constituents were isolated and purified by various separation techniques such as silica gel, ODS, Sephadex LH-20, and preparative high- performance liquid chromatography, and their structures were determined according to their physicochemical properties, MS, 1D, and 2D NMR. The antibacterial activity of the obtained compounds against methicillin-resistant Staphylococcus aureus was tested by 96-well plate microdilution method. Results: Seven sesquiterpenoids were obtained from 95% ethanol aqueous extract of Aquilaria sinensis and their structures were identified as (+)-4a,5-dimethyl-3-(prop-1-en-2yl)-octahydronaphthalene-2β,8a-diol (1), baimuxinic acid (2), baimuxinol (3), vetaspira-2(11),6-dien-14-al (4), baimuxinal (5), (-)-10-epi-γ-eudesmol (6), and 9β-hydroxyl-α-agarofuran (7). The minimum inhibitory concentration (MIC) of compound 1 against methicillin-resistant Staphylococcus aureus was 210 μmol/L. Conclusion: Compound 1 is a new compound named as 2β,8aα-dihydroxy-11-en-eremophilane, which has a good inhibitory effect against methicillin-resistant Staphylococcus aureus.

Ultrastructure observations on antennal sensilla of Heortia vitessoides the most serious pest of Aquilaria sinensis / 中国中药杂志

Heortia vitessoides is the most serious pest of Aquilaria sinensis,which is an economically important evergreen tree native to China and is the principal source of Chinese agarwood. In severe infestations,the insects completely eat up the leaves of A. sinensis,causing severe economic losses. In a more recent study,we found that the antennal sensilla of adult play important roles in the host location,mating and oviposition of H. vitessoides. Here,the external morphology of the antennal sensilla of H. vitessoides were examined using scanning electron microscopy. The result showed that the antennae of both sexes of H. vitessoides were filiform in shape,which consist of the scape,pedicel and about 64 segments of flagellomeres. Eight morphological sensilla types were recorded in both sexes,including sensilla trichodea,sensilla chaetica,sensilla basiconica,sensilla coeloconica,sensilla styloconica,sensilla auricillica,sensilla squamiformia and böhm bristle. Major differences were recorded in the distribution and quantity of different sensilla types in each segment of antenna. The sensillas are almost confined to the ventral and lateral surfaces rather than the back side of antennae. Antennal flagella contained the most sensilla while the scape and pedicel segments only contained böhm bristles and sensilla squamiformias. Sensilla trichodea Ⅲ were only found on male antennae. These results are discussed in relation to the possible roles of the sensilla types in the host location,mating and oviposition selection behavior of H. vitessoides.

Teratogenic and Mutagenic Toxicity of Agarwood Extracts Produced by Agar-Wit / 中国药学杂志

OBJECTIVE: To research the teratogenic and mutagenic toxicity of agarwood extracts produced by agar-wit technique for functional food development (slices dose 0.60 g•d-1). METHODS: Pregnant rats of 6 to 15 d were given with agarwood extracts at 45, 105, 150 mg•kg-1 by gastric perfusion directly every day, then killed and anatomized after being pregnant for 20 d. During the whole course of treatment, the activity and body weight of pregnant rats, fetal survival rate, absorption number, malformation type and number of fetus were observed and recorded. RESULTS: The agarwood extracts had no effect on the body weight of pregnant rats at 45, 105, 150 mg•kg-1 dosages, and the numbers of implantation, live fetuses, fetuses and stillbirths of fetus, malformation type and number had no significant difference compared with the control group (P>0.05). CONCLUSION: Agarwood extracts have no teratogenic and mutagenic toxicity for rat fetuses.

Chronic Toxicity of Agarwood Extracts Produced by Agar-Wit on SD Rats / 中国药学杂志

OBJECTIVE: To assess the chronic toxicity of agarwood extracts produced by agar-wit. METHODS: Agarwood usage in Chinese Pharmacopoeia was referred. SD rats were used as the experimental animal and fed with agarwood extracts at pharmaceutical dosages (1 262.4, 631.2 and 157.8 mg•kg-1) and functional food dosages (150, 105 and 45 mg•kg-1) for 90 d. The weight and food utilization were recorded. The rats were killed on the 90th day, blood and urine were collected for routine blood, serum biochemical indicators and urine index tests, and the bodies were dissected for histological examination. RESULTS: The two groups of rats showed same results. The body weight, food utilization, routine blood, serum biochemical indicators and urine index were not significantly changed compared with the control group (P>0.05). Histological examination showed that the male and female rats both had some lesions, but the group of high dose extracts had no significant difference compared with the control group, also indicating that the lesions had no relationship with agarwood extracts. CONCLUSION: The agarwood extracts produced by agar-wit have no chronic toxicity for mammal in regards of body weight, biochemical indicators and pathology at both pharmaceutical dosages and functional food dosages.

Acute Toxicity and Genetic Toxicity Evaluation of Agarwood Extracts Produced by Agar-Wit / 中国药学杂志

OBJECTIVE: To evaluate the acute toxicity and genotoxicity of agarwood extracts produced by agar-wit technology for functional food development (slices dose 0.60 g•d-1). METHODS: SD rats were used as animal model, the total dose of 10 g•kg-1 was administered to the rats twice within 24 h, activity and poisoning of rats were observed and recorded in the next 14 d, and Ames test, mammalian erythrocyte micronucleus test and in vitro mammalian cells chromosome aberration test were used to research the genotoxicity comprehensively. RESULTS: The rats showed normal activity and no poisoning symptoms appeared during acute toxicity test at 10 g•kg-1 dose. In the three genotoxicity test, extracts had no significant difference compared with the negative control (P>0.05), but significant difference compared with positive control (P<0.01). CONCLUSION: Under the condition of this study, the agarwood extracts have neither acute toxicity nor genotoxicity on rats.

Anti-inflammatory Chromones from Agarwood Produced Via Whole-Tree Agarwood-Inducing Technique of Aquilaria sinensis / 中国药学杂志

OBJECTIVE: To investigate the chromones and anti-inflammation effect of agarwood produced via whole-tree agarwood-inducing technique (Agar-Wit) from A. sinensis. METHODS: The constituents were isolated and purified by chromatographic technique and semi-preparation HPLC. RAW264.7 cell model induced by lipopolysaccharide was used to evaluate the anti-inflammatory activity of some compounds, and the secretion level of TNF-α was detected by ELISA. RESULTS: A new 2-(2-phenylethyl) chromone, (7'R)-2-[2-(7'-hydroxyphenyl)ethyl] chromone (1), along with nine analogues (2-10) were isolated from the agarwood produced by Agar-Wit, their structures were identified on the basis of physicochemical characteristics and spectroscopic data analysis as 6,7-dimethoxy-2-[2-(7'-hydroxyphenyl)ethyl] chromone (2), 6-hydroxy-2-[2-(7'-hydroxyphenyl)ethyl] chromone (3), 8-chloro-6-hydroxy-2-(2-phenylethyl)chromen-4-one (4), 8-chloro-6-hydroxy-2-[2-(4'-methoxyphenyl)ethyl] chromone (5), 8-chloro-6-hydroxy-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl] chromone (6), 7-hydroxy-6-methoxy-2-(2-phenylethyl) chromone (7), 6-hydroxy-7-dimethoxy-2-[2-(4'-hydroxyethyl)] chromone (8), 6-hydroxy-2-[2-(3'-methoxy-4'-hydroxy)ethyl] chromone (9), 6-hydroxy-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl] chromone (10). The results showed that compounds 1, 4, 6, 9 significantly inhibited the release of TNF-α inflammatory factors in RAW264.7 cells induced by LPS, with IC50 values of 13.30, 8.53, 7.72, and 10.87 μg•mL-1, respectively. CONCLUSION: Compound 1 is a new compound. The crystal structure of compound 4 is reported for the first time. Compounds 1, 4, 6, 9 show significant anti-inflammation activities.

ROS Synthesis and Accumulation during Wound-induced Sesquiterpene Biosynthesis in Aquilaria sinensis Callus / 中国药学杂志

OBJECTIVE: To explore the response intensity of reactive oxygen species(ROS) components including H2O2, O-2 and OH- in the course of wound-induced sesquiterpenes formation in Aquilaria sinensis callus. METHODS: The synthesis of sesquiterpenes in wounded callus was analyzed by using GG-MS technique. Total ROS distribution in cells was observed by fluorescence microscope after H2DCF fluorescent probe staining. The H2O2, O-2 and OH- contents were measured according to biochemical reaction principle. RESULTS: Two sesquitterpenes were synthesized and accumulated in the wounded cells. As a signal molecule, ROS burst and accumulated in the process of wound-induced sesquiterpenes formation, DCF fluorescence was observed to increase gradually. DCF fluorescence reached peak at 3 d and then began to decrease. The response of H2O2 and O-2 to injury took place earlier than those of OH-, H2O2 and O-2 reached the maximum value at 3 d and then decreased, while OH- reached the maximum value at 5 d. CONCLUSION: All ROS components respond to injury stimulation as signaling molecules, and the difference in response intensity of each component can synergistically regulate defense response to promote the synthesis of sesquiterpenes in A.sinensis callus.

Analysis of Fungi Diversity in Agarwood Wood from Hainan Province and Guangdong Province / 中国药学杂志

OBJECTIVE: To characterize the fungal communities in agarwood wood from Hainan province and Guangdong province. METHODS: Fungal ITS genes in the agarwood wood were analyzed by high-throughput pyrosequencing using Illumina HiSeq system and bioinformatically. RESULTS: The fungal microbiomes exhibited much similarity among the samples from different regions in each province. And there were notable differences between Guangdong province and Hainan province. Moreover, the fungal communities in the agarwood wood from Hainan Province showed higher diversity and richness than that in the agarwood wood from Guangdong province. At the phylum level, Ascomycota most dominated in the agarwood wood, followed by Basidiomycota. Ascomycota dominated in the agarwood wood from Haikou, Wanning, Ledong, Danzhou and Dongguan regions. Basidiomycota dominated in the agarwood wood from Huazhou region. At the class level, agaricomycetes dominated in the agarwood wood from Huazhou region. Sordariomycetes dominated in the agarwood wood from Dondguan and Danzhou regions, Dothideomycetes dominated in the agarwood wood from Haikou, Ledong and Wanning regions. At the order level, Pleosporales dominated in the agarwood wood from Haikou, Ledong, Wanning and Dondguan regions. Trechisporales and Polyporales dominated in the agarwood wood from Huazhou and Danzhou regions, respectively. At the genus level, Lignosphaeria dominated in the agarwood wood from Haikou and Wanning regions, Perenniporia and Pyrigemmula dominated in the agarwood wood from Ledong and Danzhou regions, respectively. Phaeoacremonium dominated in the agarwood wood from Huazhou and Dondguan regions. Agarwood wood from different regions contained different dominate fungal populations. CONCLUSION: The fungi in the agarwood wood from Hainan province has higher diversity and richness than that in the agawood wood from Guangdong province. Agarwood wood from different regions has distinct fungal communities.

Molecular Cloning and Expression Analysis of the Transcription Factor AsWRKY25 in Aquilaria sinensis (Lour.) Gilg / 中国药学杂志

OBJECTIVE: To clone AsWRKY25, a transcription factor gene of Aquilaria sinensis, for bioinformatic analysis and tissue expression analysis, and express its protein in prokaryotic cells, thus to lay a foundation for further study of the biological function of AsWRKY25. METHODS: With the cDNA isolated from A. sinensis callus as template, the full-length coding sequence (CDS) of AsWRKY25 was amplified using PCR method. The recombinant vector pET-28a-AsWRKY25 was transformed into Escherichia coli BL21 (DE3) for prokaryotic expression. The physiochemical properties and bioinformatic characters of AsWRKY25 were calculated by a series of bioinformatics tools and softwares. The expression patterns in different tissues were detected by RT-PCR. RESULTS: The full-length coding sequence of AsWRKY25 transcription factor was cloned from the callus of A. sinensis. The CDS of AsWRKY25 was 1 728 bp in length and contained a 1 728 bp open reading frame (ORF) encoding 575 amino acids. The CDS sequence was codon-optimized and then ligated into the pET-28a expression vector. The optimal induction condition of recombinant pET-28a-AsWRKY25 was 1 mmol•L-1 IPTG at 37℃ for 6 h in E. coli BL21 (DE3). The result of tissue expression analysis showed that AsWRKY25 had the highest expression level in the agarwood layer and the lowest level in roots, stems and branches. CONCLUSION: The WRKY transcription factor AsWRKY25 is successfully cloned from A. sinensis and expressed in E. coil. It is speculated that AsWRKY25 may be involved in the wound-induced agarwood-formation process in A. sinensis.

Differential expression of transcription factors in three generations of excised roots of Aquilaria sinensis under salt stress / 中草药

Objective: To study the expression patterns and levels of transcription factors (TFs) in three generations of excised roots of Aquilaria sinensis under salt stress, and analyze the variation of TFs family genes in each generation in response to salt stress. Methods The excised roots of A. sinensis were used as experimental material, using highthroughput sequencing technology (Illumina Hiseq4000), all the unigene sequences were compared with the plant transcription factor database (PlantTFDB), and the three generation differential expressed TFs between the treated and the control group were analyzed. Results:A total of 48 286 Unigenes were obtained by de novo splicing from three generation of excised roots of A. sinensis, containing 1 156 potential TFs distributed in 54 TF families. Among them, bHLH, ERF, and NAC were the three most enriched families. Totally, 290, 277, and 349 differentially expressed TFs were respectively screened in three successive generations, which were mainly down-regulated. The expression induced by salt stress were different in each TF family, the numbers of up-regulated DEGs increased in NAC, MYB, and WRKY families, and decreased in GRAS family with the increase of stressed generations. There were 70 common TFs differentially expressed in three generations, and the down-regulated expression multiples of eight genes increased with the increase of salt stress generations. Conclusion:The effect of salt stress on the expression of TFs was mainly down-regulatied. The number of differential expressed TFs in the treated and control group increased with the increase of salt stress generations. The expression of different TF family genes was different under salt stress, and some genes might be involved in the transmission of stressful memory. This study is helpful to understand the expression characteristics of TFs at the whole level and provide a reference for further study on the stressful memories and the molecular mechanism of the salt stress response.

Molecular cloning, prokaryotic expression and characterization of a transcription factor AsWRKY62 in Aquilaria sinensis / 中草药

Objective: The molecular cloning and prokaryotic expression of the transcription factor AsWRKY62 of Aquilaria sinensis were carried out, at the same time, the bioinformatics analysis and expression pattern analysis were also performed. The purpose of this study was to lay a foundation for further study on the role of AsWRKY62 in the growth and development of A. sinensis and the formation of agarwood. Methods: With the cDNA isolated from A. sinensis callus as template, the full-length coding sequence (CDS) of AsWRKY62 was amplified using RT-PCR and PCR method. The recombinant vector pET-21a-AsWRKY62 which was built and verified by gene recombination technique was transformed into E. coli BL21 (DE3) for prokaryotic expression and purification. The characteristics of physiochemical properties, conserved domains and subcellular localization of AsWRKY62 were calculated by a series of bioinformatics tools. The analyses of multiple sequence alignment of amino acid and phylogenetic tree were performed using DNAMAN and MEGA 5.0, respectively. The gene expression pattern in different tissues was detected by RNA-seq data. Results: The full length CDS of AsWRKY62 (GenBank accession MH925301) was 1581 bp, encoding a 526-aa protein which belongs to WRKY group I. The optimized induction conditions of recombinant pET-21a-AsWRKY62 were 0.5 mmol/L IPTG at 37 ℃ for 4 h. According to the tissue-specific expression pattern analysis, the AsWRKY62 gene in A. sinensis is mainly expressed in roots and stems, followed by agarwood and flowers. Conclusion: Cloning, expression and characterization of the AsWRKY62 gene for the first time indicated that it may be related to the formation of agarwood, which provided a theoretical basis for further study of its biological function.

Full-length cDNA cloning of AsJAZ1 gene and damage-induced expression analysis in Aquilaria sinensis callus / 中草药

Objective: To clone the full-length cDNA of jasmonate-zim-domain protein (JAZ) gene in Aquilaria sinensis to provide the basic information for further study on gene function in sesquiterpenes biosynthesis pathway. Methods: With the total RNA as template, the full-length cDNA of JAZ in A. sinensis was cloned through rapid amplification of cDNA ends (RACE) technique and reverse transcription PCR (qRT-PCR) method. The bioinformatics of the JAZ gene was analyzed as well. The expression of this gene was detected by qRT-PCR method with MeJA and mechanical wounding treatment in A. sinensis callus. Results: The full-length cDNA (1 507 bp) of JAZ gene was named AsJAZ1; GenBank registration number was KP677281. AsJAZ1 was obtained with an open reading frame (ORF) of 990 bp and encoding 330 amino acids. The relative molecular mass of AsJAZ1 calculated was 34 280, and the isoelecric point was 6.89. Real time PCR results indicated that both MeJA treatment and mechanical wounding could stimulate the increase of mRNA expression of AsJAZ1; There was a sharp rise at 0.5 h with about 27 times higher than the control (without MeJA treatment) with MeJA treatment, then dropped significantly. In mechanical wounding treatment, the highest peak presented in 2 h about 17 times compared to the control, then dropped significantly too. The expression of AsJAZ1 gene returned to be normal in 24 h. Conclusion: We have obtained the full-length cDNA sequence of AsJAZ1 gene firstly, which was extremely sensitive to wounding and responded to the early damage.