Objective:The objective of this study was to analyse fungal composition and exploit application potential in the Bantou(BT)agarwood-forming trunk of Aquilaria sinensis.Methods:BT agarwood is a naturally formed agarwoo...Objective:The objective of this study was to analyse fungal composition and exploit application potential in the Bantou(BT)agarwood-forming trunk of Aquilaria sinensis.Methods:BT agarwood is a naturally formed agarwood that was collected after cutting.Total genomic DNA of the fungi in BT agarwood was extracted by the hexadecyltrimethy ammonium bromide(CTAB)method,followed by PCR amplification and library construction.The effective tags were obtained by the HiSeq2500 platform,and the data were subjected to bioinformatics and statistical analyses.Results:A total of 7850040 effective tags were obtained,Ascomycota was the most abundant fungus at the phylum level,with a relative abundance of 56.36%–61.44%,followed by Basidiomycota,with a relative abundance of 10.49%–20.39%.Dothideomycetes,Agaricomycetes and Sordariomycetes were dominant at the class level,accounting for 26.21%–33.88%,8.40%–17.66%,and 18.41%–24.11%,respectively.Lignosphaeria,Phaeoacremonium and Hermatomyces were dominant at the genus level,with relative abundances of 6.25%–7.64%,1.95%–9.05%and 1.5%–5.4%,respectively.Diversity and richness analysis showed that the fungal composition in the agarwood formation sites(agarwood layer,upper agarwood layer and lower agarwood layer)were significantly lower than those in the decomposing layer and the healthy layer.That is,the fungal diversity and richness were significantly reduced during agarwood formation by the action of open wounds.The fungal community structure in the decomposing layer and agarwood formation sites obviously differed from that in the healthy layer.The number of Aspergillus taxa in agarwood formation sites decreased significantly(healthy layer is 0.5%,decomposing layer is 0.022%,upper agarwood layer is 0.012%,agarwood layer is 0.01%,and lower agarwood layer is 0.013%),indicating that agarwood may contain potential substances to inhibit the growth of Aspergillus.Conclusion:Agarwood from agarwood formation sites contains potential substances that inhibit Aspergillus,which provides valuable information for the control of the genus of Aspergillus.展开更多
Notoginseng Radix et Rhizoma(Sanqi in Chinese)is a precious traditional Chinese herbal medicine.It has the effect of dispersing blood stasis and stopping bleeding,reducing swelling and fixing pain.However,it tends to ...Notoginseng Radix et Rhizoma(Sanqi in Chinese)is a precious traditional Chinese herbal medicine.It has the effect of dispersing blood stasis and stopping bleeding,reducing swelling and fixing pain.However,it tends to contaminate with harmful fungi during storage,which may make it much less effective.In order to understand the fungal contamination of Notoginseng Radix et Rhizoma and master its composition of the exogenous fungi.The surface fungi of Notoginseng Radix et Rhizoma samples collected from six Chinese provinces and districts were investigated by using dilution plate method.Detection of aflatoxins by UPLC-MS/MS.The results showed that Penicillium citrinum was dominantly isolated from Notoginseng Radix et Rhizoma samples from No.1 to No.4.Aspergillus flavus,which produces aflatoxin,was dominantly isolated from Notoginseng Radix et Rhizoma samples from No.5 and No.6.In addition,kinds of mycotoxin were assayed which were produced by three of those identified A.flavus.All three fungi strains produced aflatoxin B1(AFB1)and one strain HBSQ1-5 additionally produced other three kinds of mycotoxin,AFB2,AFG1 and AFG2.It is the results implied that it will be very important to take serious cautions when using Notoginseng Radix et Rhizoma.As well as,understanding the composition of the exogenous fungi of Notoginseng Radix et Rhizoma and the strains of toxin-producing fungi,which can play an important role in guiding the storage of Notoginseng Radix et Rhizoma.展开更多
Objective In plant, squalene epoxidase （SE） catalyzes the first oxygenation step in the biosynthetic pathway of triterpenoid and phytosterol, representing one of the rate-limiting enzymes in this pathway. Bupleurum ...Objective In plant, squalene epoxidase （SE） catalyzes the first oxygenation step in the biosynthetic pathway of triterpenoid and phytosterol, representing one of the rate-limiting enzymes in this pathway. Bupleurum chinense is an important medicinal herb with its major active constituents such as triterpenoid saponins and saikosaponins. In order to obtain the series of enzymatic genes involved in saikosaponin biosynthesis, a cDNA of SE, designated BcSEI, was cloned from B. chinense. Methods The BcSEI gene was cloned by homology-based PCR and 5＇/3＇ RACE methods from the adventitious roots of B. chinense. The physical and chemical parameters of BcSE1 protein were predicted by protparam. In order to discover hints in amino acid sequences on the dominant functions in the biosynthesis of saponin or phytosterol, sequences of SE from other plants were downloaded from NCBI for sequences alignment and phylogenetic analysis. BcSEI was cloned into a yeast mutant KLNI （MATa, ergl.＇：URA3, leu2, ura3, and trpl） to verify the enzyme activity of BcSE1. Additionally, the tissue-specific expression and methyl jasmonate （MeJA） inducibility of BcSEI were investigated using quantitative real-time PCR. Results The predicted protein of BcSE1 is highly similar to SEs from other plants sharing amino acid sequence identities of up to 88%. The BcSEI can functionally complement with yeast SE gene （ERGI） when expressed in the KLNI mutant （MATa, ergl：：URA3, leu2, ura3, and trpl）. Using as controls with ^-amyrin synthase （G-AS） which is presumed to catalyze the first committed step in saikosaponin biosynthesis and a cycloartenol synthase （CAS） relating to the phytosterol biosynthesis, the transcript of BcSE1 was significantly elevated by MeJA in adventitious roots of B. chinenseand the transcript of BcSElwas most abundant in the fruits and flowers of plants, followed by that in the leaves and roots, and least in stems. Conclusion It is the first time to illustrate the molecular information of SE in B. chinense and to clone the full-length SEgene in plants of genus Bupleurum L.展开更多
The CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPRassociated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene ...The CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPRassociated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene editing technology in 2013. Over the subsequent years, it has received extensive attention owing to its easy manipulation, high efficiency, and wide application in gene mutation and transcriptional regulation in mammals and plants. The process of CRISPR/Cas is optimized constantly and its application has also expanded dramatically. Therefore, CRISPR/Cas is considered a revolutionary technology in plant biology. Here, we introduce the mechanism of the type II CRISPR/Cas called CRISPR/Cas9, update its recent advances in various applications in plants, and discuss its future prospects to provide an argument for its use in the study of medicinal plants.展开更多
Dracaena,a remarkably long-lived and slowly maturing species of plant,is world famous for its ability to produce dragon’s blood,a precious traditional medicine used by different cultures since ancient times.However,t...Dracaena,a remarkably long-lived and slowly maturing species of plant,is world famous for its ability to produce dragon’s blood,a precious traditional medicine used by different cultures since ancient times.However,there is no detailed and high-quality genome available for this species at present;thus,the molecular mechanisms that underlie its important traits are largely unknown.These factors seriously limit the protection and regeneration of this rare and endangered plant resource.Here,we sequenced and assembled the genome of Dracaena cochinchinensis at the chromosome level.The D.cochinchinensis genome covers 1.21 Gb with a scaffold N50 of 50.06 Mb and encodes 31619 predicted protein-coding genes.Analysis showed that D.cochinchinensis has undergone two whole-genome duplications and two bursts of long terminal repeat insertions.The expansion of two gene classes,cis-zeatin O-glucosyltransferase and small auxin upregulated RNA,were found to account for its longevity and slow growth.Two transcription factors(bHLH and MYB)were found to be core regulators of the flavonoid biosynthesis pathway,and reactive oxygen species were identified as the specific signaling molecules responsible for the injuryinduced formation of dragon’s blood.Our study provides high-quality genomic information relating to D.cochinchinensis and significant insight into the molecular mechanisms responsible for its longevity and formation of dragon’s blood.These findings will facilitate resource protection and sustainable utilization of Dracaena.展开更多
Objective To identify the genes of WRKY transcription factors（TFs） from roots of Bupleurum chinense and genes that potentially regulate saikosaponin（SS） biosynthesis.Methods Firstly,the subfamily cluster analysis ...Objective To identify the genes of WRKY transcription factors（TFs） from roots of Bupleurum chinense and genes that potentially regulate saikosaponin（SS） biosynthesis.Methods Firstly,the subfamily cluster analysis was mainly based on Arabidopsis thaliana WRKYs for 27 putative WRKY TFs selected from previous transcriptome sequencing data.Secondly,qPCR was used to screen such genes of WRKY TFs that could be induced by NaCI and PEG6000 in adventitious roots of B.chinense.Meanwhile,saikosaponins（SSs） in treated adventitious roots were determined by HPLC.The roots were collected at 0,2,4,8,12,24,48,and 72 h after treatments,and 120 h only for PEG.Finally,the tissue-specific expression was analyzed on screened genes by qPCR.Results The 27 genes were grouped into three categories：There were nine in Group Ⅰ,15 in Group Ⅱ,and two in Group Ⅲ.Four genes of WRKYTFs,BCWRKY6,BCWRKY16,BCWRKY32,and BCWRKY35 were obviously induced by NaCI in adventitious roots of B.chinense,while only BCWRKY32 was induced by PEG.The content of SSs increased at different levels in NaCI and PEG6000 treatment.Three genes including BCWRKY6,BCWRKY32,and BCWRKY35,expressed most in roots,were similar to the accumulation pattern of SS.Conclusion The three WRKY genes,BCWRKY6,BCWRKY32,and BCWRKY35,may be involved in the biosynthesis of SS.展开更多
Objective: We are trying to verify how a transcription factor BcbZIP134 regulates the synthesis of saikosaponin using specific antibody. However, it is hard to obtain this soluble protein expressed in vitro, a prerequ...Objective: We are trying to verify how a transcription factor BcbZIP134 regulates the synthesis of saikosaponin using specific antibody. However, it is hard to obtain this soluble protein expressed in vitro, a prerequisite for antibody preparation. So we explored a condition in which the soluble protein can efficiently express followed by preparation of the specific polyclonal antibody.Methods: Firstly, the cDNA of BcbZIP134 from Bupleurum chinense was expressed in Transetta(DE3) E.coli. Different concentrations of IPTG(0.05 and 0.5 mmol/L) and different culture temperatures(16 and37 °C) were explored for efficient expression of target protein. Then, the expressed protein with His Tag was purified using Ni Sepharose 6 Fast Flow. Different concentrations of imidazole elution(15, 60, and300 mmol/L) were used to elute the target protein. The purified protein of BcbZIP134 was used to immunize rabbits. Using the purified polyclonal antibody, the expression of BcbZIP134 in transgenic B. chinense hairy root lines was analyzed by Western blot assays.Results: Under the conditions, IPTG 0.5 mmol/L, 16 °C, and overnight, recombinant protein of BcbZIP134 was obtained in high content using pEASY?-Blunt E1 vector and Transetta(DE3) E. coli. Anti-serum against BcbZIP134 was obtained with a high titer of 1: 51 200 after immunization in rabbits. The polyclonal antibody could react with BcbZIP134 overexpressed in transgenic B. chinense hairy root lines.Conclusion: An efficient protein expression system in E. coli was constructed and the soluble recombinant protein for BcbZIP134 was obtained. By using this protein, the specific and high-performance polyclonal antibody was prepared. Furthermore, by using BcbZIP134 overexpressed hairy roots, the availability of the polyclonal antibody was verified by Western Blotting.展开更多
基金funded by the National Key Research and Development(No.2022YFC3501504)the Key Project of Research and Development Project of Hainan Province(No.ZDYF2022SHFZ035)the CAMS Innovation Fund for Medical Sciences(CIFMS)(No.2021-I2M-1-032)。
文摘Objective:The objective of this study was to analyse fungal composition and exploit application potential in the Bantou(BT)agarwood-forming trunk of Aquilaria sinensis.Methods:BT agarwood is a naturally formed agarwood that was collected after cutting.Total genomic DNA of the fungi in BT agarwood was extracted by the hexadecyltrimethy ammonium bromide(CTAB)method,followed by PCR amplification and library construction.The effective tags were obtained by the HiSeq2500 platform,and the data were subjected to bioinformatics and statistical analyses.Results:A total of 7850040 effective tags were obtained,Ascomycota was the most abundant fungus at the phylum level,with a relative abundance of 56.36%–61.44%,followed by Basidiomycota,with a relative abundance of 10.49%–20.39%.Dothideomycetes,Agaricomycetes and Sordariomycetes were dominant at the class level,accounting for 26.21%–33.88%,8.40%–17.66%,and 18.41%–24.11%,respectively.Lignosphaeria,Phaeoacremonium and Hermatomyces were dominant at the genus level,with relative abundances of 6.25%–7.64%,1.95%–9.05%and 1.5%–5.4%,respectively.Diversity and richness analysis showed that the fungal composition in the agarwood formation sites(agarwood layer,upper agarwood layer and lower agarwood layer)were significantly lower than those in the decomposing layer and the healthy layer.That is,the fungal diversity and richness were significantly reduced during agarwood formation by the action of open wounds.The fungal community structure in the decomposing layer and agarwood formation sites obviously differed from that in the healthy layer.The number of Aspergillus taxa in agarwood formation sites decreased significantly(healthy layer is 0.5%,decomposing layer is 0.022%,upper agarwood layer is 0.012%,agarwood layer is 0.01%,and lower agarwood layer is 0.013%),indicating that agarwood may contain potential substances to inhibit the growth of Aspergillus.Conclusion:Agarwood from agarwood formation sites contains potential substances that inhibit Aspergillus,which provides valuable information for the control of the genus of Aspergillus.
基金the CAMS Innovation Fund for Medical Sciences(CIFMS)(2021-I2M-1-032 and 2017-I2M-1-013)the National Key Research and Development Project(2022YFC3501504).
文摘Notoginseng Radix et Rhizoma(Sanqi in Chinese)is a precious traditional Chinese herbal medicine.It has the effect of dispersing blood stasis and stopping bleeding,reducing swelling and fixing pain.However,it tends to contaminate with harmful fungi during storage,which may make it much less effective.In order to understand the fungal contamination of Notoginseng Radix et Rhizoma and master its composition of the exogenous fungi.The surface fungi of Notoginseng Radix et Rhizoma samples collected from six Chinese provinces and districts were investigated by using dilution plate method.Detection of aflatoxins by UPLC-MS/MS.The results showed that Penicillium citrinum was dominantly isolated from Notoginseng Radix et Rhizoma samples from No.1 to No.4.Aspergillus flavus,which produces aflatoxin,was dominantly isolated from Notoginseng Radix et Rhizoma samples from No.5 and No.6.In addition,kinds of mycotoxin were assayed which were produced by three of those identified A.flavus.All three fungi strains produced aflatoxin B1(AFB1)and one strain HBSQ1-5 additionally produced other three kinds of mycotoxin,AFB2,AFG1 and AFG2.It is the results implied that it will be very important to take serious cautions when using Notoginseng Radix et Rhizoma.As well as,understanding the composition of the exogenous fungi of Notoginseng Radix et Rhizoma and the strains of toxin-producing fungi,which can play an important role in guiding the storage of Notoginseng Radix et Rhizoma.
基金Open Research Fund of State Key Laboratory Breeding Base of Systematic Research,Development and Utilization of Chinese Medicine Resources 2014KFJJ05
文摘Objective In plant, squalene epoxidase （SE） catalyzes the first oxygenation step in the biosynthetic pathway of triterpenoid and phytosterol, representing one of the rate-limiting enzymes in this pathway. Bupleurum chinense is an important medicinal herb with its major active constituents such as triterpenoid saponins and saikosaponins. In order to obtain the series of enzymatic genes involved in saikosaponin biosynthesis, a cDNA of SE, designated BcSEI, was cloned from B. chinense. Methods The BcSEI gene was cloned by homology-based PCR and 5＇/3＇ RACE methods from the adventitious roots of B. chinense. The physical and chemical parameters of BcSE1 protein were predicted by protparam. In order to discover hints in amino acid sequences on the dominant functions in the biosynthesis of saponin or phytosterol, sequences of SE from other plants were downloaded from NCBI for sequences alignment and phylogenetic analysis. BcSEI was cloned into a yeast mutant KLNI （MATa, ergl.＇：URA3, leu2, ura3, and trpl） to verify the enzyme activity of BcSE1. Additionally, the tissue-specific expression and methyl jasmonate （MeJA） inducibility of BcSEI were investigated using quantitative real-time PCR. Results The predicted protein of BcSE1 is highly similar to SEs from other plants sharing amino acid sequence identities of up to 88%. The BcSEI can functionally complement with yeast SE gene （ERGI） when expressed in the KLNI mutant （MATa, ergl：：URA3, leu2, ura3, and trpl）. Using as controls with ^-amyrin synthase （G-AS） which is presumed to catalyze the first committed step in saikosaponin biosynthesis and a cycloartenol synthase （CAS） relating to the phytosterol biosynthesis, the transcript of BcSE1 was significantly elevated by MeJA in adventitious roots of B. chinenseand the transcript of BcSElwas most abundant in the fruits and flowers of plants, followed by that in the leaves and roots, and least in stems. Conclusion It is the first time to illustrate the molecular information of SE in B. chinense and to clone the full-length SEgene in plants of genus Bupleurum L.
文摘The CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPRassociated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene editing technology in 2013. Over the subsequent years, it has received extensive attention owing to its easy manipulation, high efficiency, and wide application in gene mutation and transcriptional regulation in mammals and plants. The process of CRISPR/Cas is optimized constantly and its application has also expanded dramatically. Therefore, CRISPR/Cas is considered a revolutionary technology in plant biology. Here, we introduce the mechanism of the type II CRISPR/Cas called CRISPR/Cas9, update its recent advances in various applications in plants, and discuss its future prospects to provide an argument for its use in the study of medicinal plants.
基金supported by the program of the CAMS Initiative for Innovative Medicine(2021-1-I2M-032)the National Natural Science Foundation of China(82173925 and 81573525)the National Key Research and Development Program of China(2018YFC1706401).
文摘Dracaena,a remarkably long-lived and slowly maturing species of plant,is world famous for its ability to produce dragon’s blood,a precious traditional medicine used by different cultures since ancient times.However,there is no detailed and high-quality genome available for this species at present;thus,the molecular mechanisms that underlie its important traits are largely unknown.These factors seriously limit the protection and regeneration of this rare and endangered plant resource.Here,we sequenced and assembled the genome of Dracaena cochinchinensis at the chromosome level.The D.cochinchinensis genome covers 1.21 Gb with a scaffold N50 of 50.06 Mb and encodes 31619 predicted protein-coding genes.Analysis showed that D.cochinchinensis has undergone two whole-genome duplications and two bursts of long terminal repeat insertions.The expansion of two gene classes,cis-zeatin O-glucosyltransferase and small auxin upregulated RNA,were found to account for its longevity and slow growth.Two transcription factors(bHLH and MYB)were found to be core regulators of the flavonoid biosynthesis pathway,and reactive oxygen species were identified as the specific signaling molecules responsible for the injuryinduced formation of dragon’s blood.Our study provides high-quality genomic information relating to D.cochinchinensis and significant insight into the molecular mechanisms responsible for its longevity and formation of dragon’s blood.These findings will facilitate resource protection and sustainable utilization of Dracaena.
基金CAMS Innovation Fund for Medical Sciences(CIFMS)(2016-I2M-2-003)
文摘Objective To identify the genes of WRKY transcription factors（TFs） from roots of Bupleurum chinense and genes that potentially regulate saikosaponin（SS） biosynthesis.Methods Firstly,the subfamily cluster analysis was mainly based on Arabidopsis thaliana WRKYs for 27 putative WRKY TFs selected from previous transcriptome sequencing data.Secondly,qPCR was used to screen such genes of WRKY TFs that could be induced by NaCI and PEG6000 in adventitious roots of B.chinense.Meanwhile,saikosaponins（SSs） in treated adventitious roots were determined by HPLC.The roots were collected at 0,2,4,8,12,24,48,and 72 h after treatments,and 120 h only for PEG.Finally,the tissue-specific expression was analyzed on screened genes by qPCR.Results The 27 genes were grouped into three categories：There were nine in Group Ⅰ,15 in Group Ⅱ,and two in Group Ⅲ.Four genes of WRKYTFs,BCWRKY6,BCWRKY16,BCWRKY32,and BCWRKY35 were obviously induced by NaCI in adventitious roots of B.chinense,while only BCWRKY32 was induced by PEG.The content of SSs increased at different levels in NaCI and PEG6000 treatment.Three genes including BCWRKY6,BCWRKY32,and BCWRKY35,expressed most in roots,were similar to the accumulation pattern of SS.Conclusion The three WRKY genes,BCWRKY6,BCWRKY32,and BCWRKY35,may be involved in the biosynthesis of SS.
基金supported by the CAMS Innovation Fund for Medical Sciences(CIFMS)(2016-I2M-2-003)National Standardization Project of Traditional Chinese Medicine(ZYBZH-Y-JIN-34)
文摘Objective: We are trying to verify how a transcription factor BcbZIP134 regulates the synthesis of saikosaponin using specific antibody. However, it is hard to obtain this soluble protein expressed in vitro, a prerequisite for antibody preparation. So we explored a condition in which the soluble protein can efficiently express followed by preparation of the specific polyclonal antibody.Methods: Firstly, the cDNA of BcbZIP134 from Bupleurum chinense was expressed in Transetta(DE3) E.coli. Different concentrations of IPTG(0.05 and 0.5 mmol/L) and different culture temperatures(16 and37 °C) were explored for efficient expression of target protein. Then, the expressed protein with His Tag was purified using Ni Sepharose 6 Fast Flow. Different concentrations of imidazole elution(15, 60, and300 mmol/L) were used to elute the target protein. The purified protein of BcbZIP134 was used to immunize rabbits. Using the purified polyclonal antibody, the expression of BcbZIP134 in transgenic B. chinense hairy root lines was analyzed by Western blot assays.Results: Under the conditions, IPTG 0.5 mmol/L, 16 °C, and overnight, recombinant protein of BcbZIP134 was obtained in high content using pEASY?-Blunt E1 vector and Transetta(DE3) E. coli. Anti-serum against BcbZIP134 was obtained with a high titer of 1: 51 200 after immunization in rabbits. The polyclonal antibody could react with BcbZIP134 overexpressed in transgenic B. chinense hairy root lines.Conclusion: An efficient protein expression system in E. coli was constructed and the soluble recombinant protein for BcbZIP134 was obtained. By using this protein, the specific and high-performance polyclonal antibody was prepared. Furthermore, by using BcbZIP134 overexpressed hairy roots, the availability of the polyclonal antibody was verified by Western Blotting.
