农杆菌介导海洋草酸青霉转化体系及聚酮合酶Pks生物学功能*

为研究南海柳珊瑚共附生草酸青霉SCSGAF0023的聚酮合酶(PKS)生物学功能,采用农杆菌介导法构建草酸青霉SCSGAF0023的Pks敲除株ΔPks,比较野生菌株及ΔPks的生长发育及环境适应性差异。以草酸青霉SCSGAF0023分生孢子为受体,p0380-hygB为双元载体,成功实现草酸青霉SCSGAF0023的遗传转化。结果表明:农杆菌浓度为OD₆₀₀=0.5,在200μmol/L 乙酰丁香酮(AS)诱导下与10⁷个/ml草酸青霉SCSGAF0023孢子于25℃共孵育时转化效率最高。基于上述转化体系,成功获得Pks敲除株ΔPks,并首次证实Pks正向调控草酸青霉SCSGAF0023产孢,但不影响其对环境的适应性。这为进一步系统研究真菌PKSs及聚酮化合物对真菌生长发育与环境适应性的影响提供素材。     

Cmku70基因敲除对蛹虫草生长发育的影响

ku70和ku80是非同源末端连接修复通路的关键基因,在一些丝状真菌中其基因敲除株可作为底盘菌株,提高同源重组效率和基因敲除效率。本研究从蛹虫草基因组中鉴定得到Cmku70及Cmku80基因,分别编码分子量为71.50kDa和80.96kDa的蛋白,均含有Ku core结构域,预测均定位于细胞核。系统进化分析表明Ku70和Ku80蛋白在真菌中广泛存在,且具有保守性。通过农杆菌介导的同源重组法敲除Cmku70,发现不影响蛹虫草菌丝生长、见光转色、分生孢子形成及形态等无性生长过程,但敲除后不能形成子实体,因此Cmku70敲除株不宜用作蛹虫草生长发育相关基因高效敲除的底盘菌株。     

金针菇G蛋白偶联受体基因的CRISPR/Cas9基因组编辑载体构建及转化研究

通过氨基酸同源比对（Blast P）以及金针菇冷诱导前后菌丝阶段和原基阶段的转录组数据分析,获得了金针菇中的两个假定G蛋白偶联受体基因Fvgpcr1、Fvgpcr2。对获得的金针菇假定G蛋白偶联受体基因Fvgpcr1、Fvgpcr2构建了基因组编辑（CRISPR/Cas9）的pCAMBIA0390-hph-Fvcas9-Fvgpcr1- sgRNA1/sgRNA2、pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1/sgRNA2等4个表达载体。通过农杆菌介导（ATMT）将表达载体pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA转化金针菇菌丝体,采用潮霉素和头孢毒素低浓度初筛和高浓度复筛,经两段筛选获得金针菇拟转化子。经对拟转化子进行PCR鉴定、RT-qPCR检测和Western杂交验证,结果显示表达载体pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA成功整合进金针菇基因组中,FvCas9蛋白正常表达,但未得到Fvgpcr基因敲除突变体。本研究利用农杆菌介导转化法在金针菇中构建了CRISPR/Cas9敲除体系,对后续目标基因的敲除有着重要意义。     

农杆菌介导的马尔尼菲青霉基因转化技术的建立及优化

目的建立农杆菌介导的马尔尼菲青霉(PM)基因转化技术,并对该技术条件进行优化。方法以二元质粒p DHt/SK为载体,通过农杆菌介导将pyr G基因插入马尔尼菲青霉尿嘧啶缺陷株SPM4(pyr G-,nia D-)中,在不含尿嘧啶的培养基中筛选阳性转化子。运用PCR验证重组子。进一步对影响转化效率的农杆菌类型、共培养浓度、转化媒介、共培养温度、共培养时间、乙酰丁香酮(AS)等六个条件进行优化。结果 PCR验证pyr G基因成功的插入SPM4中,所得到转化子可稳定传代,通过条件优化,得到转化子约300个/106个细胞。选用AGL-1,以农杆菌共培养浓度为OD600=0.8,AS浓度为200μmol/L,无膜IM固体共培养基为介质,25℃共培养48 h为最适转化条件。结论成功建立了农杆菌介导PM基因转化技术,简化并优化了转化条件,该方法可用于PM基因功能研究。     

组蛋白甲基化与去乙酰化对蛇足石杉内生真菌盘长孢状刺盘孢合成石杉碱甲的影响

以蛇足石杉Huperzia serrata内生真菌盘长孢状刺盘孢Cg01菌株为研究对象,利用PEG介导的同源重组转化体系,对Cg01组蛋白甲基化酶基因（histone methyltransferases,HMT）CgClr4和组蛋白去乙酰化酶基因（histone deacetylase,HDAC）CgClr3、CgSir2进行基因敲除与回补,并通过实时荧光定量PCR（RT-qPCR）检测了回补株中对应基因表达量以及高效液相色谱HPLC检测突变体菌株中石杉碱甲huperzine A（HupA）产量。结果显示3个基因敲除突变体菌株ΔCgClr4、ΔCgClr3、ΔCgSir2的HupA产量分别为255μg/L、270μg/L、244μg/L,与野生型菌株相比分别下降了21.3%、16.6%、24.7%。在基因回补突变体菌株ΔCgClr4/CgClr4、ΔCgClr3/CgClr3、ΔCgSir2/CgSir2中,相应回补基因表达均与野生型无显著性差异,其HupA产量分别为351.9μg/L、334.7μg/L、331μg/L,回补菌株的HupA产量回复到野生型水平。结果表明这3个基因均具有调控内生真菌盘长孢状刺盘孢Cg01合成HupA的作用,为研究蛇足石杉内生真菌中石杉碱甲的合成调控机制提供了理论基础和新的思路。     

两种不同来源的绿色木霉降解木质纤维素研究

利用纤维素酶高产菌绿色木霉Trichoderma viride降解木质纤维素是实现废料资源化的重要手段。本研究选取来自不同生境的两株T. viride,分别以玉米秸秆和甘草药渣为基质,测定两者滤纸纤维素酶（filter paper cellulase,FPase）活性和还原糖产量。从时间、温度、水分、pH 4个方面比较两株T. viride的环境适应性和不同基质的差异性。结果表明,以玉米秸秆为基质,T. viride XJ最适初始料液比为1:4-1:5.5,T. viride AG最适初始料液比为1:5-1:5.5。初始料液比1:5.5时,T. viride AG产FPase活性显著高于T. viride XJ。两株T. viride最适发酵温度均为28℃,各温度处理下不同菌株间无显著差异。两株T. viride均表现为还原糖消耗。以甘草药渣为基质,T. viride XJ最适初始料液比为1:2-1:2.5,T. viride AG最适初始料液比为1:3-1:3.5。料液比高于1:3,T. viride AG产FPase活性显著高于T. viride XJ。T. viride AG最适发酵温度为28℃,T. viride XJ最适发酵温度为23-28℃。温度低于28℃,T. viride XJ产FPase活性显著高于T. viride AG。两株T. viride均表现为还原糖积累。两株T. viride最适初始pH均为6-7,最适发酵时间均为3d。最优发酵条件下FPase活性：T. viride AG>T. viride XJ。对T. viride产FPase诱导能力：甘草药渣>玉米秸秆。变差分解表明两株T. viride产FPase活性差异主要源于菌株对生境的生态适应。比较分析菌种来源、基质类型、环境条件对T. viride发酵效果的影响,将有助于该菌大规模应用性研究。     

利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*

目的: 利用CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) 系统构建玉米中心蛋白(Centrin)的表达载体,经转化后分析其对玉米生长发育的影响。方法: 针对ZmCen基因的第一个外显子设计sgRNA,将其连入pOMS01-Cas9-ZmCen-sgRNA表达载体,转化农杆菌GV3101后,侵染玉米自交系材料B104的愈伤组织,经继代、诱导、分化成苗,筛选出转基因后代。对T₀代和T₁代基因组DNA进行PCR验证、测序及表型分析。结果: 成功构建ZmCen的表达载体。侵染农杆菌后,PCR测序显示,T₀ 代和T₁ 代突变率分别为 20.13% 和 64.52%,其中T₁ 代的纯合缺失突变率为5%。序列分析表明,ZmCen基因的编辑靶点附近发生了碱基的替换、插入或缺失。经与野生型表型比对发现,ZmCen 突变体T₁代植株出现发育缓慢且雄花序不完全发育表型,纯合突变体植株雄花序则完全不发育。结论: 通过 CRISPR/Cas9技术成功地对玉米ZmCen基因进行了编辑,ZmCen突变体的获得为玉米雄性器官发育相关基因的研究奠定了基础。     

安徽黄山红豆杉内生真菌多样性与生态分布

为了了解红豆杉Taxus chinensis内生真菌的种类及分布特点,通过采样,分离,培养与鉴定,对安徽黄山红豆杉内生真菌的群落结构及生态分布进行了研究.结果共分离出14属107株内生真菌,其中青霉属Penicillium,曲霉属Aspergillus,交链孢属Alternaria和拟青霉属Paecilomyces为优势种群.同时内生真菌的多样性具有组织专一性,以根和皮中分布较多,分别是分离菌株总数的37.38%和26.17%.     

深圳大鹏湾六种珊瑚共附生真菌多样性研究

本研究使用6种不同培养基对深圳大鹏湾6种珊瑚上的共附生真菌进行平板涂布法分离培养,结合ITS-rDNA基因序列特征和形态学特征进行鉴定来研究深圳大鹏湾海域珊瑚共附生真菌的多样性。共分离培养出457株真菌菌株,分属于7个属,分别为红酵母属Rhodotorula（180株）、曲霉属Aspergillus（170株）、木霉属Trichoderma（50株）、青霉属Penicillium（34株）、枝孢属Cladosporium（21株）、透孢黑团壳属Massarina（1株）和弯颈霉属Tolypocladium（1株）,其中优势属为红酵母属Rhodotorula和曲霉属Aspergillus,占菌株分离总数的76.59%。单独鹿角珊瑚上分离到的共附生真菌数量和种类最多,为160株9种;在该种珊瑚上还分离出弯颈霉属Tolypocladium sp.,该属真菌此前从未在珊瑚共附生真菌的研究中分离出来。本研究还发现SDA培养基分离出的共附生真菌数量最多,CDA培养基的真菌数量最少,表明不同培养基分离的共附生真菌多样性存在差异。     

利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*

目的: 利用CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) 系统构建玉米中心蛋白(Centrin)的表达载体,经转化后分析其对玉米生长发育的影响。方法: 针对ZmCen基因的第一个外显子设计sgRNA,将其连入pOMS01-Cas9-ZmCen-sgRNA表达载体,转化农杆菌GV3101后,侵染玉米自交系材料B104的愈伤组织,经继代、诱导、分化成苗,筛选出转基因后代。对T₀代和T₁代基因组DNA进行PCR验证、测序及表型分析。结果: 成功构建ZmCen的表达载体。侵染农杆菌后,PCR测序显示,T₀ 代和T₁ 代突变率分别为 20.13% 和 64.52%,其中T₁ 代的纯合缺失突变率为5%。序列分析表明,ZmCen基因的编辑靶点附近发生了碱基的替换、插入或缺失。经与野生型表型比对发现,ZmCen 突变体T₁代植株出现发育缓慢且雄花序不完全发育表型,纯合突变体植株雄花序则完全不发育。结论: 通过 CRISPR/Cas9技术成功地对玉米ZmCen基因进行了编辑,ZmCen突变体的获得为玉米雄性器官发育相关基因的研究奠定了基础。     

Coccystes undoxylophus

Ohne Zusammenfassung     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Aquila pennata undminuta

Ohne Zusammenfassung     

UeberAquila pennata undminuta

Ohne Zusammenfassung     

Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.     

Chemical composition, in situ ruminal degradability and post-ruminal disappearance of dry matter and crude protein from the halophytic plants Kochia scoparia, Atriplex dimorphostegia, Suaeda arcuata and Gamanthus gamacarpus

Samples of Kochia (K. scoparia), Atriplex (A. dimorphostegia), Suaeda (S. arcuata) and Gamanthus (G. gamacarpus) were collected and analyzed for chemical composition including crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDFom), acid detergent fiber (ADFom), non-protein N (NPN), Ca, P, Na, K, Cl, Mg, Fe, Cu and Se. In addition, in situ ruminal degradability and post-ruminal disappearance of dry matter (DM) and CP of the samples using a mobile bag technique were determined. Results indicate that the chemical composition of Kochia and Atriplex was notably different from those of Suaeda and Gamanthus. All of these halophytic plants had high concentrations of Na, K, Cl, Cu and Se, and low levels of Ca, P and Mg. The rapidly degradable fractions of DM and CP (g/g) of Kochia (0.31 and 0.35, respectively) and Atriplex (0.39 and 0.50, respectively) were lower than for Suaeda (0.53 and 0.55, respectively) and Gamanthus (0.56 and 0.66, respectively). Ruminal DM and CP disappearance of Kochia (444 and 517 g/kg, respectively) and Atriplex (472 and 529 g/kg, respectively) were lower (P<0.05) than those of Suaeda (553 and 577 g/kg, respectively) and Gamanthus (663 and 677 g/kg, respectively) (P<0.05) using the mobile bag technique. Suaeda had the lowest (P<0.05) NDFom and ADFom disappearance (214 and 232 g/kg, respectively) in the rumen. Kochia scoparia and Atriplex dimorphostegia have more beneficial chemical nutritive components and digestible values versus Suaeda arcuata and Gamanthus gamacarpus.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Flavonoids of Astilbe and Rodgersia compared to Aruncus

The flavonoid profiles of Astilbe (four taxa studied) and Rodgersia (two taxa studied) are based on simple flavonol glycosides. Astilbe has 3-O-mono-, 3-O-di-, and 3-O-triglycosides of kaempferol, quercetin, and myricetin, while Rodgersia has only mono- and diglycosides of kaempferol and quercetin. Astilbe×arendsii was also shown to accumulate dihydrochalcone glycosides. The flavonoid profile of Rodgersia is the simplest recorded so far in the herbaceous Saxifragaceae. The flavonoids of two species of Aruncus were shown to be based upon kaempferol and quercetin 3-O-mono- and 3-O-diglycosides. One of the species also exhibited an eriodictyol glycoside. The triglycoside differences were not considered important, but the differences in myricetin occurrences were taken as evidence against derivation of Saxifragaceae from an Aruncus-like ancestor. Should such an event be proposed, however, serious consideration would have to be given to the current pattern of myricetin occurrence in the two families.