Plant endophytic fungus is an important biological resource and has shown significant applications in medicine and in the biological control of agricultural pests and diseases. In order to study the diversity of endophytic fungi of potato (Solanum tuberosum), samples from three regions of Yunnan Province, namely, Dehong Mangshi, Dali Xizhou, and Lincang Shuangjiang. The endophytic fungi in potato roots, stems and tubers were isolated and cultured using the method of tissue block isolation. Well-grown colonies were purified using the method of tip mycelium selection. The endophytic fungi were identified by morphological methods and ITS sequence analysis. Then, the colonization rate, isolation rate and diversity index of the endophytic fungi were calculated and analyzed. The results were as follows: (1) A total of 98 endophytic fungi were isolated, including 40 strains samples from Dehong Mangshi, 27 strains from Dali Xizhou and 31 strains from Lincang Shuangjiang. (2) The endophytic fungi isolated from potato were identified to include 10 orders, 10 families and 13 genera, mostly of the Ascomycota and Basidiomycota, with Fusarium and Penicillium as the dominant fungi. Five species of fungi, Emericella rugulosa, Fusarium sambucinum, Stereum hirsutum, Psathyrella sulcatotuberculosa and Epicoccum catenisporum were first reported to be isolated from potato plants. (3) Potato tuber had the highest colonization rate of endophytic fungi and root had the lowest; while the isolation rate of endophytic fungi was highest in potato root and lowest in stem; trend of diversity index of endophytic fungi in different tissues were H′root > H′tuber > H′stem. In conclusion, the endophytic fungi in Yunnan potato plants are highly diverse, with different endophytic fungal dominants in potato samples among the three localities, potato root has the richest endophytic fungal populations and the highest isolation rate, and are therefore the most suitable material for endophytic fungal isolation. The above results provide a reference for later investigation of the antagonistic effect of endophytic fungi on pathogens in potato.

采用基因组挖掘的方法从球孢白僵菌基因组中获得了一条杂合PKS/NRPS基因(命名为Bbpks1),通过生物信息学分析预测了该基因的功能,检测了该基因在不同碳源、氮源培养基上的表达情况.结果显示:Bbpks1基因长度为11838 bp,编码3945个氨基酸,其结构域顺序为KS-AT-DH-MT-KR-ACP-C-A-PP-SDR;BbPKS1蛋白与布氏虫草、半翅轮枝菌、塔宾曲霉等真菌中的杂合PKS/NRPS蛋白序列(GenBank登录号分别为OAA40809.1、CEJ94083.1、OJI88893.1)聚为一个分支,且与参与细胞松弛素和伊快霉素合成的蛋白的亲缘关系较近,可推测该基因在球孢白僵菌中参与2,4-吡咯酮型化合物的合成;Bbpks1基因在含有乳糖、肌醇碳源的培养基上表达量较高,在以麦芽糖作为碳源的培养基上不表达,在含不同氮源的培养基上均大量表达.

通过对地衣型真菌皮革肾岛衣转录组数据的分析,挖掘出1条非还原型聚酮合酶基因,通过RT-PCR首次克隆得到该基因的cDNA全长(NpPKS2),并通过生物信息学手段分析其基因和蛋白氨基酸序列,采用荧光定量PCR技术分析该基因在不同培养基上的表达情况.结果显示:NpPKS2基因全长6249 bp,编码2082个氨基酸;生物信息学分析显示该基因编码1种非还原型聚酮合酶,结构域顺序为SAT-KS-AT-PT-ACP-TE,根据聚类分析和结构域分析,推断NpPKS2为苔色酸合成酶;荧光定量分析显示地衣型真菌皮革肾岛衣中的NpPKS2基因用BMG培养基培养时表达量最高.

地衣中含有种类丰富并具有生物活性的聚酮类化合物,目前对地衣聚酮类化合物的生物合成尚不清楚.本研究通过对皮革肾岛衣地衣型真菌转录组数据的分析,获得一个新的含有甲基转移酶结构域的非还原型聚酮合酶(NR-PKS)基因(NpPKS1),利用RT-PCR技术首次从皮革肾岛衣地衣型真菌中克隆得到该基因全长cDNA,并检测不同培养基对NpPKS1基因表达的影响.结果 显示:NpPKS1基因cDNA全长7 857 bp,编码2 618个氨基酸;该蛋白不存在信号肽,在细胞质基质中发挥作用;与含有甲基转移酶结构域的构巢曲霉(Aspergillus nidulans,XP_664052)、壳球孢菌(Macrophomina phaseolina,EKG19938)聚为一支;表达分析显示该基因在不同培养基上的表达量差异极其显著,在BMG培养基上的表达量最高,其相对表达量可达39.15.本研究为下一步异源表达皮革肾岛衣地衣型真菌的聚酮合酶、探讨皮革肾岛衣聚酮化合物的生物合成及基因资源的有效利用提供必要的研究材料.

为了解牛樟芝中聚酮化合物的生物合成机理及聚酮合酶基因功能,从牛樟芝基因组挖掘并克隆得到一个部分还原型PKS(PR-PKS)基因(AcPKS3),并对其进行生物信息学分析及表达谱分析.结果显示,AcPKS3(GenBank登录号:MG988206)DNA全长8286 bp,有22个内含子,其外显子共编码2285个氨基酸;结构域依次为KS-AT-KR-ACP-SDR,各结构域的活性保守位点为β-酮基合成酶(DTACSS)、酰基转移酶(GHSAGETA)、酮基还原酶(YLLVGGIG)、酰基转移酶(YGLDSITSA)、短链醇脱氢酶与NAD(P)H结合的N端保守序列(IT-GTTGSFG)及活性保守位点(YTESK);AcPKS3与6-甲基水杨酸合成酶的亲缘关系较近;不同碳源中葡萄糖,不同氮源中牛肉浸粉、酪蛋白胨、土豆蛋白胨可促进AcPKS3基因表达.本研究为牛樟芝聚酮合酶功能研究及牛樟芝基因资源利用提供参考.

以蒜头果(Malania oleifera)为实验材料,基于转录组数据分析,采用RT-PCR方法获得蒜头果3-酮酯酰-CoA合酶(KCS)基因的cDNA序列,命名为MoKCS1,GenBank登录号为MK210592.序列分析显示MoKCS1基因cDNA全长为1 539 bp,编码512个氨基酸,属于KCS家族.序列比对分析显示MoKCS1拥有KCS家族特有的3个功能保守结构域,与榴莲(Durio zibethinus)KCS的蛋白序列同源性为80.66％;与已知超长链KCS蛋白进行系统进化分析显示,MoKCS1独立形成一个分支.荧光定量PCR分析表明,MoKCS1在蒜头果果实膨大期的表达量最高,而在叶中几乎不表达.

以蒜头果(Malania oleifera)为实验材料,基于转录组数据分析,采用RT-PCR方法克隆获得蒜头果3-酮酯酰-CoA还原基因,命名为MoKCR1,GenBank登录号为:KX421278.序列分析显示MoKCR1基因cDNA开放阅读框全长为963 bp,编码320个氨基酸,属于KCR家族.序列比对分析显示MoKCR1具有KCR蛋白所具有的NADH结合结构域[G(X)3GXG(X)3A(X)3A(X)2G]和裂解有关的关键结构域[Y(X)3K],蒜头果KCR与木薯(Manihot esculenta) KCR的蛋白序列同源性为82.81％.与己知超长链KCR蛋白进行系统进化分析显示MoKCR1与巨尾桉(Eucalyptus grandis)关系较近.荧光定量PCR分析表明MoKCR1在蒜头果果实膨大期的表达量最高.本研究为最终揭示蒜头果神经酸生物合成提供了研究基础.

以蛹虫草的菌丝体为材料,利用基因组挖掘的方式从蛹虫草基因组中获得其聚酮合酶(Polyketide syn-thase,PKS)基因,对这些PKS基因进行生物信息学分析以推断它们的功能,并检测它们在不同培养基上的表达情况.结果表明:蛹虫草中含有13个PKS基因(CmPKS 1-13),包括2个非还原型聚酮合酶(Non-reducing PKS,NR-PKS),5个部分还原的聚酮合酶(Partial-reducing PKS,PR-PKS),6个高度还原型聚酮合酶(Highly reducing PKS,HR-PKS);聚类分析显示CmPKS 1、CmPKS 3可能参与链格孢吡喃酮(alternapyrone),CmPKS 4可能参与黄曲霉素,CmPKS 5可能参与美伐他汀,CmPKS 6可能参与分生孢子色素,CmPKS 8可能参与伊快霉素的生物合成,其余PKS与生物合成未知化合物的PKS聚为一支;半定量PCR显示CmPKS 7和CmPKS 9在4种培养基上均强烈表达;CmPKS 3、CmPKS 6在4种培养基上微弱表达;CmPKS 10、CmPKS 11和CmPKS 12只在GL培养基上强烈表达,在其余3种培养基上微弱表达;CmPKS 1和CmPKS 4只在GL培养上微弱表达,CmPKS 8仅在GS培养基上微弱表达;CmPKS 2、CmPKS 5和CmPKS 13在检测的培养基中都不表达.本研究为进一步异源表达蛹虫草中的PKS基因及其功能鉴定、 具新颖结构聚酮化合物的发掘奠定基础.

聚酮和非核糖体多肽的复合化合物具有独特的生理活性，它们由聚酮合酶/非核糖体肽合成酶（PKS/NRPS）催化合成。目前球孢白僵菌Beauveria bassiana中含SDR结构域的PKS/NRPS酶的生物合成机制尚不清楚，采用基因挖掘技术从球孢白僵菌基因组中分离得到1个PKS/NRPS基因（命名Bbpks2），利用生物信息学分析对其功能进行预测并检测该基因在以6.0 g·L-1麦芽提取物和3.0 g·L-1酵母提取物为基本氮源培养基，7种碳源添加物和以1.8 g·L-1麦芽糖和6.0 g·L-1葡萄糖为基本碳源培养基，4种氮源添加物培养基上的具体表达情况，其中每种添加物含量为4.0 g·L-1。结果显示：Bbpks2基因长度为12 051 bp，编码4 016个氨基酸；其结构域组织顺序为KS-AT-DH-MT-KR-ACP-C-A-PP-SDR，是一种含有SDR结构域的PKS/NRPS；系统进化分析发现，BbPKS2与球孢白僵菌JEF007菌株（PMB64475.1）、头状虫草Tolypocladium capitatum（PNY25600.1）等的PKS/NRPS蛋白聚在同个分支中，可能参与一种聚酮/非核糖体多肽类化合物的生物合成；比较不同氮源、碳源添加物对Bbpks2基因表达的影响，发现该基因在添加了乳糖的培养基上的表达量是其他碳源添加物的3.4倍以上，添加了牛肉浸粉的培养基上表达量是其他氮源添加物的1.3倍以上。该研究为下一步通过异源表达鉴定Bbpks2基因的具体功能，及其调控机理研究和基因资源利用奠定基础。

α-蒎烯合成酶(α-pinene synthase,APS)是α-蒎烯生物合成的关键酶.本研究利用同源克隆及基因组步移法从思茅松中克隆得到一个α-蒎烯合成酶基因,命名为PkA PS (GenBank登录号为KX394684),基因全长3 523 bp,含有10个内含子,其cDNA全长1956 bp,编码651个氨基酸残基.生物信息学分析显示PkAPS属于萜烯合成酶家族蛋白;系统进化树分析显示PkAPS与马尾松α-蒎烯合成酶亲缘关系最近.基因表达分析显示:PkA PS在高产脂思茅松个体各组织的表达量均高于低产产脂思茅松个体;而在高产脂思茅松不同组织中的表达比较中,PkA PS在小枝中的表达量最高.本研究将有利于进一步研究思茅松α-蒎烯合成酶调控机理及培育高产α-蒎烯思茅松新品种的研究.

从开黄花的滇牡丹转录组中分离了3个CHS基因,利用生物信息学预测其基因功能,并比较不同花发育时期CHS基因的表达量.结果显示:3个CHS基因的cDNA全长分别为1173、1185、1128 bp,依次命名为PdCHS1(GenBank登录号MK516264)、PdCHS2(GenBank登录号MK516265)和PdCHS3(GenBank登录号MK516266),分别编码390、394和375个氨基酸;这3个CHS基因编码的蛋白均为无信号肽的非分泌蛋白,均含查尔酮合成酶活性位点基序(G/A)FGPG.聚类结果显示,滇牡丹中的3个CHS蛋白归属于不同分支.基因表达结果显示,PdCHS1基因在花蕾期和花蕊中表达量较高,PdCHS2基因在末花期和初花期表达量较高,PdCHS3基因在末花期和花蕾期表达量较高.这3个CHS基因分别参与不同次生代谢产物的合成,推测PdCHS1参与柚皮素查尔酮,PdCHS2参与芪类化合物,PdCHS3参与聚酮类化合物的生物合成.

本研究通过对皮革肾岛衣(Nephrmopsis pallescens)地衣型真菌转录组数据的分析,首次克隆得到一种高度还原型聚酮合酶(Highly reducing PKS)基因全长,并对该基因进行生物信息学分析,并检测该基因在不同培养基上的表达量.该基因cDNA全长7491 bp(命名为NpPKS1),可编码2496个氨基酸,是一种稳定存在于细胞质基质中的非分泌蛋白;结构域与其他真菌的洛伐他汀九酮合成酶(LNKS)结构域极为相似,且聚类分析与其他真菌的洛伐他汀九酮合成酶(LNKS)聚为一类;在以葡萄糖和麦芽糖作为碳源的情况下,番茄浸粉、牛肉浸粉、酪蛋白胨等作为氮源可强烈刺激NpPKS1基因的表达,而胰蛋白胨对该基因的表达存在一定程度的抑制效应.本研究为皮革肾岛衣地衣型真菌中的基因资源利用、聚酮化合物异源表达和其合成机制的研究奠定了基础.

目的 获得牛樟芝聚酮合酶基因(AcPKS1)全长,对其进行生物学分析并分析该基因在不同培养基上的表达差异.方法 通过对牛樟芝基因组分析获得牛樟芝聚酮合酶基因,通过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为模板克隆得到AcPKS1基因全长,并对该基因进行生物信息学分析及在不同培养基上的表达谱分析.结果 AcPKS1全长6 348 bp,含有6个内含子和7个外显子,外显子编码2 115个氨基酸;通过生物信息学分析,推测该基因为真菌Ⅰ型非还原型PKS,结构域为SAT-KS-PT-ACP-ACP-TE,系统树分析显示AcPKS1与其他未知功能的聚酮合酶(PKS)聚为一支,说明AcPKS1可能是一种新的聚酮化合物环化方式;表达谱分析表明,葡萄糖为AcPKS1基因表达的必要条件,且葡萄糖含量与AcPKS基因表达量呈正相关.结论 本研究为AcPKS1功能鉴定以及牛樟芝基因资源利用奠定基础.

Usnea longissima has a long history of use as a traditional medicine. Several bioactive compounds, primarily belonging to the polyketide family, have been isolated from U. longissima. However, the genes for the biosynthesis of these compounds are yet to be identified. In the present study, three different types of non-reducing polyketide synthases (UlPKS2, UlPKS4, and UlPKS6) were identified from a cultured lichen-forming fungus of U. longissima. Phylogenetic analysis of product template domains showed that UlPKS2 and UlPKS4 belong to group IV, which includes the non-reducing polyketide synthases with an methyltransferase (MeT) domain that are involved in methylorcinol-based compound synthesis; UlPKS6 was found to belong to group I, which includes the non-reducing polyketide synthases that synthesize single aromatic ring polyketides, such as orsellinic acid. Reverse transcriptase-PCR analysis demonstrated that UlPKS2 and UlPKS4 were upregulated by sucrose; UlPKS6 was downregulated by asparagine, glycine, and alanine.

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.

This study was aimed at evaluating the growth promoting effect of symbiotic algal polyol (ribitol) and plant hormones on the lichen-forming fungi (LFF), Ramalina farinacea (CH050010 and 40403) and Ramalina fastigiata. The addition of ribitol to basal (malt-yeast extract) medium enhanced the relative growth rates of all three LFF. R. farinacea (CH050010), R. farinacea (40403) and R. fastigiata (H06127) showed 35.3%, 29.0% and 29.3% higher growth rates, respectively, compared to the control. IBA (indole-3-butyric acid) and TIBA (2,3,5-tridobenzoic acid) also increased growth rates of the LFF by 34 to 64% and 7 to 28%, respectively, compared to the control. The combination of ribitol with IBA or TIBA synergistically increased the growth of all LFF. For example, ribitol and IBA treatments increased growth rates of R. farinacea (CH050010), R. farinacea (40403) and R. fastigiata (H06127) by 79.4%, 40.3% and 72.8% in, respectively, compared to those grown on the basal medium. The stimulating effect of ribitol and IBA on the LFF growth induced vertical development of the fungal mass in culture. We suggest that lichen-forming fungal growth of Ramalina lichens can be stimulated aposymbiotically by supplementing polyols and plant hormones to the basal medium in the mass production of lichen secondary metabolites under large scale culture conditions.