Antrodia cinnamomea, an edible and medicinal fungus with significant economic value and application prospects, is rich in terpenoids, benzenoids, lignans, polysaccharides, and benzoquinone, succinic and maleic derivatives. In this study, the transcriptome of A. cinnamomea cultured on the wood substrates of Cinnamomum glanduliferum (YZM), C. camphora (XZM), and C. kanehirae (NZM) was sequenced using the high-throughput sequencing technology Illumina HiSeq 2000, and the data were assembled by de novo strategy to obtain 78,729 Unigenes with an N50 of 4,463 bp. Compared with public databases, about 11,435, 6,947, and 5,994 Unigenes were annotated to the Non-Redundant (NR), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genome (KEGG), respectively. The comprehensive analysis of the mycelium terpene biosynthesis-related genes in A. cinnamomea revealed that the expression of acetyl-CoA acetyltransferase (AACT), acyl-CoA dehydrogenase (MCAD), 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA), mevalonate pyrophosphate decarboxylase (MVD), and isopentenyl diphosphate isomerase (IDI) was significantly higher on NZM compared to the other two wood substrates. Similarly, the expression of geranylgeranyltransferase (GGT) was significantly higher on YZM compared to NZM and XZM, and the expression of farnesyl transferase (FTase) was significantly higher on XZM. Furthermore, the expressions of 2,3-oxidized squalene cyclase (OCS), squalene synthase (SQS), and squalene epoxidase (SE) were significantly higher on NZM. Overall, this study provides a potential approach to explore the molecular regulation mechanism of terpenoid biosynthesis in A. cinnamomea.

MYB转录因子在真菌的生长发育和胁迫响应中发挥多种作用.为了解牛樟芝MYB的功能,本研究利用本地BLAST对牛樟芝基因组进行比对分析,获得了 9个牛樟芝MYB基因序列,并对牛樟芝MYB转录因子进行功能预测分析,利用转录组测序分析MYB基因在不同段木培养条件下子实体和菌丝体中的表达情况.蛋白理化性质分析发现,9个牛樟芝MYB转录因子多为不稳定蛋白,理论等电点介于4.92～8.51;亚细胞定位预测结果显示有1个转录因子(ACMYB1)定位在细胞质中,推测其可能参与细胞质基因的转录调控;有8个定位在细胞核中,占总蛋白的88.9％.二级结构预测发现以无规则卷曲和a-螺旋为主要结构,β-转角为次要结构.将9个牛樟芝MYB转录因子与5个已知功能的MYB蛋白共同构建系统进化树,14个MYB转录因子分为Ⅰ、Ⅱ两个分支,其中ACMYB1、ACMYB5与冬虫夏草中参与菌丝生长的MYB-6聚为一支,ACMYB9与冬虫夏草中参与子实体生长的MYB-3聚为一支,ACMYB6与顶头孢霉AcMYBA聚为一支.分析牛樟芝MYB转录因子在不同段木菌丝和子实体中的相对表达量,发现ACMYB1、ACMYB5、ACMYB6均在香樟段木菌丝体阶段高表达,推测其参与了牛樟芝菌丝体的生长,ACMYB9基因在香樟段木子实体阶段表达量上调,说明ACMYB9可能参与调控牛樟芝子实体的发育.本研究结果可为进一步挖掘研究牛樟芝MYB转录因子的功能奠定基础.

为深入了解牛樟芝Zn（Ⅱ）2Cys6转录因子的功能,利用本地Blast对牛樟芝全基因组的蛋白序列进行全局比对,鉴定出20个牛樟芝Zn（Ⅱ）2Cys6转录因子蛋白序列。通过生物信息学方法对Zn（Ⅱ）2Cys6转录因子进行结构和功能预测,并利用转录组测序分析Zn（Ⅱ）2Cys6转录因子在不同段木培养条件下菌丝体和子实体的表达情况。结果表明:20个Zn（Ⅱ）2Cys6转录因子均具有Cy6型锌指基序,属于GAL4型锌簇蛋白转录因子,其氨基酸数量为225～1 384个;理论等电点平均pI值为6.91,均为不稳定蛋白;其中,AcCys6-17存在跨膜结构;AcCys6-9、AcCys6-14定位在细胞质中;平均每个转录因子含9个外显子,AcCys6-6有25个外显子,二级结构以无规卷曲为主要结构。Zn（Ⅱ）2Cys6转录因子基因表达在不同段木的菌丝体和子实体存在差异,有11个转录因子在香樟段木菌丝体表达量高,AcCys6-16在香樟段木子实体表达量高; AcCys6-11在牛樟段木菌丝体表达量高; AcCys6-4在云南樟段木菌丝体表达量最高。

The tissue culture for Dendrocalams fugongensis was conducted using its seeds as explants. The results showed that disinfection for 60s with 75% alcohol and disinfection for 30 min with 0.1% HgCl2were most effective approach. The optimal multiple shoots inducing medium was MS+6-BA 2mg/L +IBA 0.5mg/L +KT 0.2mg/L. The optimal shoots multiplication culture medium was MS+6-BA 3mg/L +NAA 0.2mg/L. The optimal rooting cul-ture medium was MS+IBA 1mg/L +NAA 0.5mg/L. After 90 days transplanted into greenhouse, the survival rate of young plants were over 81%.

The separation and purification of endophytic fungi from the stalks and leaves of Aquilaria sinensis (Lour.) Gilg.were studied by tissue block separation.The antimicrobial activities of the crude extract of endophytic fungi were studied by filter paper method,and the minimum inhibitory concentration (MIC) was determined.The results showed that 10 endophytic fungi were obtained from Aquilaria sinensis (Lour.) Gilg..The fungi names are as follows:Colletotrichum gloeosporioides,Arthrinium rasikravindrii,Xylaria adscendens,Cladosporium perangustum,Fusarium tricinctum.,Cladosporium sphaerospermum,Diaporthe sp.,Phomopsis amygdali,Phomopsis sp.,Periconia macrospinosa.A.rasikravindrii,Diaporthe sp.,P.amygdali,Phomopsis sp.,P.macrospinosa were first seperated from Aquilaria sinensis (Lour.) Gilg..Among them,the crude extracts of C.gloeosporioides,C.perangustum,Fusarium sp.,Diaporthe sp.and P.macrospinosa have antibacterial activitis toward 13 species of test bacteria after the liquid culture.The inhibition zone of Diaporthe sp.toward vibrio parahaemolyticus can reach (17.1 ± 0.04) mm.The minimum inhibitory concentration of crude extract of Fusarium tricinctum.toward Bacillus subtilis,Streptococcus agalactiae,Bacillus subtilis,Bacillus subtilis,Staphylococcus aureus,Micrococcus luteus and Vibrio parahaemolyticus was 3.125 mg · mL-1.This study laid the foundation for the development and utilization of antimicrobial compounds in endophytic fungi of Aquilaria sinensis (Lour.) Gilg..

Metarhizium spp.are highly pathogenic insect fungi, which widely parasitize in a variety of insects. The insect pathogenic fungi from Cordyceps sinensis and Cordyceps militaris have a good medical and health func-tions, which can produce variety of active metabolites.In this study, four species of Metarhizium were cultured in liquid culture.The culture medium was extracted with ethyl acetate, which finally obtain the primary extract of Metarhizium spp..The antimicrobial activities of the four ethyl acetate extracts of Metarhizium spp.to 13 patho-gens were evaluated using the inhibition zone method, and the minimum inhibitory concentration(MIC)of the cor-responding pathogens was determined.The results showed that the ethyl acetate extracts produced by the four spe-cies of Metarhizium had a certain antibacterial activity.Four extracts of Metarhizium spp.had inhibitory effects on Bacillus cereus and Bacillus lentus.The minimum inhibitory concentration of the extract of Metarhizium guizhouense on Bacillus cereus was 6.45 mg/mL, and the MIC for Straphylococcus haemolyticus was 3.23 mg/mL.The extracts of Metarhizium brunneum had inhibitory effect on the Bacillus cereus, Bacillus lentus, Vibrio parahaemolyticus, Straphylococcus haemolyticus and Micrococcus luteus.The minimum inhibitory concentration of Metarhizium brunne-um extract on Micrococcus luteus was 13.9 mg/mL.This study laid the foundation for the further study, and for development and utilization of antimicrobial compounds of Metarhizium spp.

为了研究牛樟芝中PKS基因与化合物之间的关系,该研究通过对牛樟芝基因组分析获得牛樟芝聚酮合酶基因,以此序列为模板设计含有起始密码子和终止密码子的特异引物并以牛樟芝cDNA为模板克隆获得一个高度还原型PKS(HR-PKS)基因全长,命名为AcPKS2;对AcPKS2基因进行生物信息学分析,并比较该基因在不同培养基上的表达量.结果表明:AcPKS2全长7842 bp,有24个内含子,其外显子共编码2613个氨基酸,该蛋白的相对分子质量为293.5 kDa,理论等电点pI为5.78.用CDD分析其结构域显示,该基因属于HR-PKS,其结构域组织排列为KS-AT-DH-MT-ER-KR-ACP-TE,8个结构域其活性位点分别为 β-酮基合成酶(DTACSSSL)、酰基转移酶(GHSIGETA)、脱水酶(RNDGSTSPL)、甲基转移酶(SFDIITAFDV)、烯酰还原酶(HAGVSSPAA)、酮基还原酶(GSPGQANYTAA)、酰基转移酶(YGLDSLTSVRL)、硫酯酶(KQPNGPY).系统发育树显示AcPKS2与其他化合物未知的HR-PKS蛋白聚为一支,结构域和系统进化树分析显示该基因可能编码一种新的含TE结构域高度还原型聚酮合酶;表达分析结果显示葡萄糖和果糖能够诱导该基因的表达.

In order to searching the NRPS gene, the putative compound or protein of Ramalina intermedia lichen forming fungi, we obtained a novel NRPS gene(RiNRPS)by analysed the transcriptom of R.intermedia lichen forming fungi.We checked the RiNRPS gene using the BLAST, phylogenetic tree analysis and antiSMASH, NRPS predictor, NaPDoS, Norine bioinformatics, the results showed that: the relative molecular weight of the RiNRPS gene is 702 699.9, pI is 5.83, and unstable protein; the full length of RiNRPS open reading framer takes 19 098bp, and codes 6 362 amino acids, non-exist signal peptide, is an unsecreted protein, locates in the cytoplasmic matrix; it might code HC-toxin synthetase.The HC-toxin synthetase could catalyze Orn, Gln, Gln and Cys into cyclic tetrapeptides, the compound may be an polypeptides having the bioactivity of toxin, and the consti-tutional formula is C21H32N4O6.The study should apply some basis of gene mining of NRPS gene from Ramalina intermedia and the biosynthesis of nonribosomal peptides.

Malania oleifera has a wild plant resource with huge prospective development, as their fruits abound in nervonic acids, and it is the special monotypic plant genera in China. To acquire the variety and distribution of endophytic fungi in Malania oleifera tree, we took the sample from two habitat of bare limestone mountain and limestone holt, respectively, isolated and purified the endophytic fungi from the roots, barks and leaves of Malania oleifera using the traditional separation methods, identificated these fungus according to their character of phenotype and r DNA-ITS. The research showed that there were 112 endophytic fungus from all the samples, belongs to 10 genus, and Purpureocillium, Acaromyces, Trichoderma is the domaint fungi genus, the preponderant strain is Purpureocillium lilacinum. The difference of separation rate of endophytic fungi in the different tissue of Malania oleifera is very obviously, the endophytic fungi in the root distributes the most widespread, the separation rate in the roots and leaves are both 100％ in two habitat, and the rate of barks is 56.7％, 63.3％, respectively. The effect of different habitats on the endophytic fungi of Malania oleifera was apparently, the diversity index of roots, barks and leaves in the exposed limestone mountain was severally 0.756, 1.518, 0.662, and it was 1.412, 1.439 and0.974, respectively. The fungal diversity of barks were analogous in both habitats, and the diversity of roots and leaves in the limestone forest land both manifested higher than the bare limestone mountain.

The colors of the leaves in Paeonia delavayi may be pure green, green-red, or dark red according to their habitat. The goal of this study was to compare the type and the total concentrations of anthocyanins and flavonols in red and green P. delavayi. The constituents and concentration of anthocyanins and flavonols were identified and determined by high-performance liquid chromatography with diode array detection and mass spectrometry. Cyanidin-3, 5-di-O-glucoside, cyanidin-3-O-glucoside, and peonidin-3-O-glucoside were first determined in red P. delavayi. There were no anthocyanins in green P. delavayi. Seven flavonols were identified in both red and green P. delavayi with differences in their concentrations. The concentrations of total anthocyanins and flavonoids were determined at 525 and 360nm using the external standard method. The total anthocyanins in red P. delavayi were 152.24mg/100g in leaves and 78.92mg/100g in stems. The total flavonoids were 805.4mg/100g in leaves and 438.3mg/100g in stems, which were much higher than in green P. delavayi for both leaves and stems. This study reports composition and concentration differences in red and green P. delavayi based on ecological habitat.

以野生滇牡丹为试验材料,以其转录组数据为基础,采用反转录PCR技术克隆得到一个具完整开放阅读框的类黄酮7-O-葡萄糖基转移酶基因(Pd7GT),并通过生物信息学分析手段、 基因表达对该基因进行分析.结果显示:该基因全长1446bp(GenBank登录号为KX394687),可编码481个蛋白氨基酸;生物信息学分析发现Pd7GT蛋白C末端含有典型糖基转移酶识别区(WAPQV)和UDP-葡萄糖基配体绑定位点(HCGWNS)的PSPG盒子,其蛋白序列GWAPQVMILEHEAVGGFVTHCGWNSTLEGISAGLPLVTWPIFAEQFYNEK,与可可(XP 007042481)、Herra-nnia umbratica(XP 021298085)、 毛果杨(XP 006379195)、 巨桉(XP 010066837)等以葡萄糖为糖基配体的类黄酮7-O-糖基转移酶聚为一类;Pd7GT基因在组织茎中表达量最高、 不同花发育时期的花谢期表达量最高、 不同颜色花瓣中黄花花瓣表达量最高.本研究为滇牡丹糖基转移酶异源表达、 分子育种等研究提供一定的基础,为未来通过基因工程培育具新颖花色和抗性的滇牡丹新品种提供必要材料.

In angiosperms, the 2'-O-glucosyltransferases (2'GT) can affect the biosynthesis of color pigment molecules, and adding glucosyl ligand to flavonoids is the key enzyme to form xanthophyl. The present study cloned a flavonoid 2'-O-glycosyltransferase (Pd2'GT) gene with complete ORF from Paeonia delavayi for the first time with the method of RT-PCR, and its bioinformatics and transcriptional pattern analysis were carried out. The results showed that the full length of Pd2'GT gene was 1 428 bp, encoding a protein of 475 amino acids. The protein had no signal peptide and transmembrane structure, which was an unstable protein functioning in cytoplasmic matrix.The identity between the nucleotide sequence of Pd2'GT and the glycosyltransferase gene of Vitis vinifera was78％, indicating that the Pd2'GT gene might encode a new protein. Its protein sequence contained glycosyltransferase superfamily domain PSPG, and its amino acid residue sequence was WAPQVAILSHRATGGFVSHCGWNSILESL WFG VPIAALPMYAEQQ, including the typical identifiable sites of glycosyltransferase and the binding sites ofUDP sugar moiety. This protein clustered together with the 2'GT of Malus domestica (NP_001315903), Pyrus communis (D3 UAG1), Cyclamen persicum (BAF75895), and Catharanthus roseus (BAF75901), and the sugar acceptor of this group was chalcone. The transcriptional pattern analysis clarified that the highest expression of Pd2'GT gene was in the yellow petals of P. delavyi, and the highest accumulation level of this gene was in the budding phase. In this study, the Pd2'GT gene was isolated and cloned through the analysis of the transcriptional data of Paeonia delavayi, which could provide research reference for the breeding of new ornamental peony by genetic engineering.

In order to establish the peony petals chromatographic fingerprints, the fresh Peony petals collected from different introduction regionalization were detected by high performance liquid chromatography (HPLC). The related chromatographic conditions were as follows: Eclipse plus C18 column (4. 6×150mm, 5μm), with aceto-nitrile and 1％ acetic acid as mobile phase in gradient mode. The detective wavelength was 360nm, the flow rate was 0. 8mL/ min and the column temperature was 35℃. Chromatographic fingerprints of peony petals from different introduction region were established based on the analysis of similarities of composition. The results showed that:The method had a good repeatability and reproducibility and a total of 6 main common peaks was found in the fin-gerprints of peony petals in 9 different samples, which could be used as the main basis for the identification of peo-ny petals products and provide a quality control method for peony petals products.

为研究牛樟芝(Antrodia camphorata)萜类生物合成MVA途径中关键酶3-羟-3-甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutary CoA reductase,HMGR)的调控机制,从牛樟芝基因组中分离并克隆出AcHMGR基因,对其进行生物信息学分析、不同碳氮源添加物对该基因的诱导表达情况进行分析.分析显示:AcHMGR基因含7个外显子、6个内含子;开放阅读框为3 402 bp,编码1 133个氨基酸残基组成蛋白质序列;AcHMGR包含HMGR典型的多肽位点,即2个HMG-CoA结合基序:PLG (I/V) AGPLK (I/V) DG和AEGTLVASTSRG,2个NADP结合基序:TGDAMGMNMI和IEVGT (I/V) GGGT;分子系统进化分析显示,AcHMGR蛋白序列与其他多孔菌科真菌的HMGR聚为一支;表达谱分析显示:碳源中的果糖、氮源中的酪蛋白胨诱导表达AcHMGR基因的能力最强.本研究为探讨牛樟芝萜类,尤其是三萜类化合物的生物合成及调控机理提供了帮助.

为明确滇西德宏州是否适合进行蒜头果产业化种植,利用M axent模型对蒜头果的适生区进行预测,并在德宏进行了小规模的试种试验.结果显示:在气候条件上,德宏州处于蒜头果的低适生到中适生区,蒜头果可能有一定的存活生长量,但规模化种植风险大.适生区分析与试种试验均表明德宏州不适合蒜头果的规模化种植.

Malania oleifera,an endemic plant species,is naturally distributed in karst region of southeast Yunnan and west Guangxi,China. This plant is a potential species with high economic value as its seed oil contains rich nervonic acid,and is also an excellent tree for the restoration and reconstruction of vegetation in karst region.Much attention has been paid to exploitation and utilization of this species in recent years. In order to understand fruiting characters of wild trees,we analyzed the fruit yield of wild trees,fruit and pit characteristics from six sites. The results were as follows:(1)Fruit yield was significantly different among wild trees. The coefficient of variation(CV)was 136.38%. There was positive correlation between fruit yield and tree crown.(2)The mean size of fruit was 37.10-40.36 mm in longitudinal diameter,and 41.15-45.03 mm in transverse diameter of CV ranged from 7.28% to 9.31%. The mean fruit weight was 35.77-47.29 g with a range in CV from 18.99% to 21.44%.(3)The mean size of pit was 27.50-31.69 mm and 30.94-34.16 mm respectively in longitudinal and transverse diameter.CV was from 6.47%to 10.99%.The mean pit weight was 14.03-18.77 g,and CV was from 17.37% to 22.68%.(4)The mean longitudinal thickness of pericarp was 4.33-4.80 mm,and transverse thickness was 5.10-5.44 mm. Their CV was 12.92%-26.91%. The mean weight of pericarp was 21.62-28.51 g,and CV was 20.01%-24.12%. These results indicate that the yield, size and weight of fruits of wild Malania oleifera have abundant phenotypic diversity, which can provide various source of excellent character selection for oriented cultivation and exploitation.

In order to explore the potential antibacterial medicinal value of insect fungi and find new com-pounds with novel structures and bioactivity. In this study, the single-cell multi-product strategy (OSMAC) acti-vates were used in liquid culture of Metarhizium guizhouense. The effects of different carbon sources, nitrogen sources,inoculation amount and inoculation time on antibacterial activity of liquid culture extracts of Metarhizium guizhouense were detected. The results showed that the extracts of Metarhizium guizhouense could inhibit the growth of Bacillus cereus,Bacillus lentus and Straphylococcus haemolyticus of 13 common pathogenic bacteria. The minimum inhibitory concentrations of the extracts to 3 pathogenic bacteria were 6.25 mg/mL, 1.56 mg/mL, 6.25 mg/mL, respectively. The effects of different carbon and nitrogen sources on the antibacterial activity of liquid culture ex-tracts of Metarhizium guizhouense was significant, and the extracts of Metarhizium guizhouense grown in starch and potato culture medium showed strongest antibacterial activity. Different inoculation amount had little effects on the antibacterial of extracts of Metarhizium guizhouense. The effects of different culture time on the antibacterial effect of mycelium extract was significant. The antibacterial activity of extract of Metarhizium guizhouense increased with the increase of culture time.

The way of supercritical CO2 fluid extraction was used in oil peony, and the effects of different con-ditions of different kinds of cosolvents and dosage on the yield of extraction of oil peony were studied. GC-MS tech-nology was used for analysis of fatty acids composition in the oil peony. The result shows that at the extraction condi-tion:35 MPa for pressure, 25 g/min for CO2 flow rate, 2 h for extraction time and 40℃ for temperature, the oil yield with shell has been increased 16. 82% after using ethyl acetate as cosolvent. As well the oil yield with shell has been increased 6. 55% after using petroleum ether as cosolvent. And the oil yield with shell has been increased 12. 89% after using n-hexane as cosolvent. As can be seen from the results the oil yields with shell all have im-proved after using of cosolvents, and ethyl acetate has shown the optimum efficiency in oil yields in supercritical CO2 fluid extraction. Comprehensively studied the dosages of 3 cosolvents, the optimal dosage of cosolvents was 10% of CO2 flow rate of ethyl acetate. Peony oil fatty acid composition analysis showed that, oil peony riched in un-saturated fatty acid, especially α-linolenic acid, which take up 78. 95% content of the total fatty acid.

In Simao pine(Pinus kesiya var. langbianensis)breeding programs,lack of co-dominant genetic markers constrains the development of molecular marker assisted breeding. The aim of this study is to develop SSR molecular markers using transcriptome sequencing data. Searching the SSR loci from 59636 unigenes of P. kesiya var. langbianensis with MISA software,total 3745 SSRs were obtained, accounting for 6.28% of the total unigenes,averagely one SSR per 11.36 kb. Trinucleotide and dinucleotide repeats were dominant types among SSRs with the ratio of 49% and 24%,and others were only 27%. AGC/CTG was the most trinucleotide motif and AT/TA was the most dinucleotide motif. The primers were designed and synthetized based on randomly selected 224 SSR loci,verification by agarose gel electrophoresis revealed that 29 loci showed clear polymorphism. While only 12 of the 29 loci satisfied the multiple fragments of SSR repeat motifs in capillary electrophoresis and their amplification efficiencies were higher than 85%. Genetic diversity of 42 Simao pine samples from Jingdong and Puwen populations was investigated with these 12 fluorescently labeled primers. A total of 35 alleles were detected,the polymorphism information content(PIC)of all loci ranged from 0.0932-0.4809(the mean was 0.3069). Among these,4 loci were classified as lowly polymorphic ones(0 < PIC <0.25)and 8 loci as moderately polymorphic ones(0.25 < PIC <0.5). Conclusively,these 12 SSRs may be used in future studies on genetic diversity,linkage mapping,gene location and cloning,providing the technical support for molecular marker assisted breeding and mutation in P. kesiya var. langbianensis.

为研究滇重楼内生真菌的抑菌活性,先从滇重楼块茎中分离得到98株内生真菌,对峙试验结果表明,其中有8株内生真菌对供试植物病原菌立枯丝核菌、尖孢镰刀菌和烟草黑胫病菌有一定的抑制作用;内生真菌发酵液初提物抑菌试验结果表明,3株内生真菌发酵液的提取物对供试植物病原菌有一定的抑制作用,分别为PPC-25、PPC-43、PPC-78,其他菌株的发酵液提取物对供试植物病原菌没有抑菌活性.