球毛壳菌降解天然木质纤维素能力差异及酶系基因分析简

目的：以不同植物中分离到的4株内生球毛壳菌NK102、NK103、NK104和NK105为对象,研究不同生态来源球毛壳菌降解木质素和纤维素的能力。方法：首先采用羧甲基纤维素和纤维素刚果红平板检测各菌株的纤维素降解能力,并利用Bavendamm平板反应检测各菌株的木质素降解能力;将4株菌分别培养在以微晶纤维素、杨树叶和木屑为惟一碳源的液体培养基中,通过检测培养液中纤维素酶和漆酶的酶活力,比较各菌株分解利用天然木质纤维素材料的能力,连续培养12d后检测培养液中次级代谢产物的合成情况;利用已测序的球毛壳菌CBS148．51的基因组信息,寻找编码木质纤维素降解酶类的基因,为球毛壳菌分解利用木质纤维素提供分子生物学依据。结果：NK102、NK103、NK104和NK105在羧甲基纤维素培养基和纤维素刚果红培养基上都能够生长并形成水解圈;Bavendamm平板反应显示4株菌降解木质素的能力由强到弱依次是NK103、NK102、NK105和NK104。4株菌都能分解利用微晶纤维素、杨树叶和木屑,分泌纤维素酶和漆酶,其中NK102在以木屑为碳源的培养基上纤维素酶活力最强,达到0．76U/mL发酵液,NK103在以杨树叶为碳源的培养基上漆酶活力最强。与此同时,4株菌在发酵培养过程中都能够稳定地合成球毛壳甲素（ChA）,ChA产量受到碳源影响,在以杨树叶为碳源的培养基上,NK104的ChA产量最高,可达到14．88mg/L发酵液。利用已测序的球毛壳菌CBS148．51的基因组信息,寻找到119个编码纤维素半纤维素酶的基因、8个编码漆酶的基因和2个编码锰过氧化物酶的基因,球毛壳菌具有完整的降解纤维素半纤维素的酶体系,在木质纤维素降解真菌的开发过程中具有重要的研究价值。结论：本研究为球毛壳菌木质纤维素降解过程的研究及该菌种的开发利用奠定了基础。     

球毛壳菌对植物病原真菌和根结线虫的生物防治潜力

球毛壳菌(Chaetomium globosum)隶属于子囊菌门、核菌纲、粪壳菌目、毛壳菌科、毛壳菌属真菌,广泛分布于空气、土壤等多种自然环境中,也是植物最常见的内生真菌之一。球毛壳菌能产生种类繁多的次级代谢产物,且其次级代谢产物具有抗真菌和杀线虫等多种生物活性,因而被制作成杀菌剂和杀线虫剂等生物农药,对植物病原真菌和根结线虫(Meloidogyne spp.)有良好的生物防治潜力。本文从球毛壳菌及其次级代谢产物抗植物病原真原菌和杀根结线虫两个方面进行综述,并探讨球毛壳菌对植物病原真菌的生防机制,为植物病原真菌及根结线虫的综合治理和新型生物农药的开发提供参考。     

丝状真菌次级代谢产物生物合成的表观遗传调控

丝状真菌产生的次级代谢产物是新药的重要来源之一,其生物合成过程受到众多因素的调控。最近的研究表明,表观遗传对多种丝状真菌次级代谢产物的生物合成具有调控作用。DNA和组蛋白的甲基化与乙酰化修饰是目前所知的丝状真菌主要的表观遗传调控形式。通过过表达或缺失相关表观修饰基因和利用小分子表观遗传试剂改变丝状真菌染色体的修饰形式,不仅可以提高多种已知次级代谢产物产量,而且可以通过激活沉默的生物合成基因簇诱导丝状真菌产生新的未知代谢产物。丝状真菌表观遗传学正逐渐成为真菌菌株改良的新策略以及挖掘真菌次级代谢产物合成潜力的强有力手段。     

大豆荚壳杀根结线虫成分的分离及其对抗氧化酶活性的影响

采用液-液分配萃取方法,从大豆荚壳甲醇提取物中分离获得石油醚、氯仿、乙酸乙酯、正丁醇和水的萃取物,测定了其对南方根结线虫J2的毒杀活性,结果表明正丁醇萃取物比其它4种萃取物有更高的杀虫活性,24和48 h对线虫校正死亡率分别为57.76%和67.72%.将正丁醇萃取物通过硅胶柱层析和薄层层析,得到8个组分,通过杀虫活性试验,得到组分2和7为主要的杀虫活性组分,24和48 h对线虫校正死亡率分别为65.93%、66.65%和69.25%、68.51%.进一步就组分2和7对J2抗氧化酶活性的影响进行了研究,结果表明两种组分对谷胱甘肽转硫酶和过氧化氢酶活性都有一定的抑制作用,其中两种组分对过氧化氢酶活性的抑制效果差异不显著,组分7对谷胱甘肽转硫酶活性具有显著的抑制效果,24和48 h的抑制率分别可达72.36%和50.21%.说明大豆荚壳的杀线虫活性成分可能通过影响虫体内的氧化代谢,使线虫体内氧化和抗氧化作用失衡,最终导致线虫死亡.     

多粘类芽胞杆菌KM2501-1杀南方根结线虫活性产物研究

【目的】南方根结线虫(Meloidogyne incognita)是一种危害严重的土传性植物病原线虫,给农业生产造成了巨大的经济损失,前期研究发现多粘类芽胞杆菌(Panebacillus polymyxa) KM2501-1具有很好的温室防治南方根结线虫效果,且可产生多种挥发性杀线虫活性物质,但对其非挥发性产物是否有杀线虫活性没有研究。本研究拟进一步分离鉴定其产生的杀线虫活性代谢产物,发掘新的杀线虫药物。【方法】对菌株KM2501-1进行液体发酵并离心收集发酵上清液,通过硅胶柱层析、高效液相色谱分离等方法得到高纯度的杀线虫活性物质,并通过液相色谱质谱联用分析、核磁共振等技术鉴定杀线虫活性物质的结构。【结果】生物活性检测显示,多粘类芽胞杆菌KM2501-1发酵上清液具有较强的南方根结线虫触杀活性,并能有效抑制南方根结线虫卵孵化,体外杀线虫效率高达87.66%,抑制卵孵化效率达92.26%。结构鉴定结果显示多粘类芽胞杆菌产生的杀线虫活性物质为环二肽类物质cyclo (Pro-Phe),800 mg/L的cyclo(Pro-Phe)杀线虫效率达84.75%。进一步的显微观测结果表明,与对照组相比,活性物质cyclo(Pro-Phe)处理后的根结线虫肠道组织紊乱、结构发生破坏。【结论】多粘类芽胞杆菌KM2501-1产生的cyclo (Pro-Phe)是一个具有杀线虫新功能的活性物质,其可能通过破坏线虫肠道杀死线虫。     

表观遗传调控植物叶片衰老的研究进展

植物叶片衰老是一个非常重要的发育过程,涉及大分子的有序分解从而将营养物质从叶片转移到其他器官,对植物的生存和适应至关重要。叶片衰老主要受植物的发育调控,但同时也受内部和外部环境因素的影响,涉及高度复杂的基因调控网络和多层级的调控。近年来的研究表明表观遗传是调控植物叶片衰老的一种重要调控方式。该研究综述了植物叶片衰老过程中的表观遗传调控机制,包括组蛋白修饰、DNA甲基化、ATP依赖的染色质重塑和非编码RNA介导的调控,并对该领域今后的发展方向进行了展望。     

多倍体植物的表观遗传现象

表观遗传现象是指基因表达发生改变但不涉及DNA序列的变化, 它存在于许多植物的多倍体化过程中,而且能够在代与代之间传递。表观遗传变异包括基因沉默、DNA甲基化、核仁显性、休眠转座子激活和基因组印记等方面。这种现象可能是由于基因组间的相互作用直接诱发基因沉默或基因表达改变所致;也可能由DNA甲基化之外的组蛋白编码的改变引起;或者与甲基化不足、染色质重组或转座子激活等有关。表观遗传变异在提高基因表达的多样性,引起遗传学和细胞学上的二倍化,以及促进基因组间的相互协调等方面起着重要作用。文章综述了植物多倍体化过程中的表观遗传现象及其在多倍体植物基因组进化中的作用,并在此基础上提出了今后在这方面的研究途径。     

植物叶片衰老的表观遗传调控

叶片是植物重要的光合器官, 它的衰老由外界环境刺激和内源发育信号所启动, 复杂的基因调控网络参与衰老过程的精确调控。最新研究表明, 植物通过对基因表达的重编程, 在表观遗传水平上调节着叶片衰老过程。该文简要介绍了表观遗传的分子机制, 在此基础上重点综述了组蛋白修饰、染色质重塑、DNA甲基化及小RNAs途径对叶片衰老调控的最新研究进展, 同时讨论了该领域存在的问题和未来研究方向。     

放线菌Snea253代谢产物及寡糖对南方根结线虫活性的影响

本文针对前期筛选获得的高效放线菌Snea253菌株代谢产物中杀线虫活性成分进行试管法和薄层层析法预试,结果显示活性物质含糖和氨基酸。贝氏皿浸没法研究了Snea253代谢产物中杀线虫活性物质和已知寡糖对南方根结线虫卵孵化和二龄幼虫J2的影响,结果表明:Snea253活性物质对南方根结线虫卵孵化的相对抑制率为84.95%,供试寡糖中除麦芽糖和蔗糖有不显著的抑制作用外,半乳糖、果糖和木糖显著促进了南方根结线虫卵孵化;乳糖、葡萄糖和山梨糖均具有促进作用,但与对照相比不显著。Snea253活性物质对J2的校正死亡率为98.21%,与各寡糖处理和对照相比差异显著;葡萄糖、乳糖和蔗糖处理J2无死亡现象,反而利于南方根结线虫J2的存活。研究结果显示普通寡糖对南方根结线虫基本无毒性,而放线菌Snea253代谢产物中氨基糖类物质则对线虫有毒性。     

纤维素降解球毛壳菌NK-102的高效遗传转化方法

摘要:【目的】在球毛壳菌(Chaetomium globosum)NK-102 中,建立菌株特异性转化体系。【方法】构建新的抗性标记pUCATPH-Pgap,转化效率优于pUCATPH 和pCM768。建立了PEG-原生质体和根癌农杆菌(Agrobacterium tumefaciens)EHA105 介导的两种转化方法。【结果】原生质体转化效率为30－50 个转化子/10 μg DNA,抗性标记pUCATPH-Pgap 效率最高。EHA105 介导转化率达到3.2×102 转化子/107 孢子。Sout     

Chaetomium globosum for reducing primary inoculum of Diaporthe phaseolorum f. sp. meridionalis in soil-surface soybean stubble in field conditions

The potential of an antibiotic-producing isolate of Chaetomium globosum (CgA-1) to suppress Diaporthe phaseolorum f. sp. meridionalis (Dpm) in soybean stubble was studied in field microplots of no-tillage, minimum-tillage, and shallow plowing. Mature soybean stems colonized in vitro with Dpm were spread on the soil surface and C. globosum ascospore suspension, without nutrient supply, was sprayed over the entire plot prior to any tillage operation. Perithecial formation and survival of Dpm in soybean stems, concomitantly with colonization by C. globosum, were monitored for a 180-day period (mid-autumn through winter and mid-spring), which is the normal interval between soybean harvest and sowing. The proportion of soybean stem segments occupied by Dpm and number of perithecia formed decreased linearly with time and showed a strong negative correlation with increase in the occupation by C. globosum. At the end of the study, which coincided with the soybean sowing season, the soybean stubble was free from viable Dpm and was colonized by C. globosum. The effectiveness of C. globosum in eliminating the pathogen from surface-borne residue or harrowed-in residue was similar but much slower than in the shallow-plowed microplots. C. globosum successfully competed with major interfering fungi such as, Trichoderma, Nigrospora, and Fusarium in colonizing the soybean stems above and under the soil surface. The data provide strong evidence for use of the antibiotic-producing isolate of C. globosum to control soybean stem canker disease.     

Inactivation of OGG1 increases the incidence of G?·?C→T?·?A transversions in Saccharomyces cerevisiae : evidence for endogenous oxidative damage to DNA in eukaryotic cells

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. To investigate the biological role of the OGG1 gene, mutants were constructed by partial deletion of the coding sequence and insertion of marker genes, yielding ogg1::TRP1 and ogg1::URA3 mutant strains. The disruption of the OGG1 gene does not compromise the viability of haploid cells, therefore it is not an essential gene. The capacity to repair 8-OxoG has been measured in cell-free extracts of wild-type and ogg1 strains using a 34mer DNA fragment containing a single 8-OxoG residue paired with a cytosine (8-OxoG/C) as a substrate. Cell-free extracts of the wild-type strain efficiently cleave the 8-OxoG-containing strand of the 8-OxoG/C duplex. In contrast, cell-free extracts of the Ogg1-deficient strain have no detectable activity that can cleave the 8-OxoG/C duplex. The biological properties of the ogg1 mutant have also been investigated. The results show that the ogg1 disruptant is not hypersensitive to DNA-damaging agents such as ultraviolet light at 254 nm, hydrogen peroxide or methyl methanesulfonate. However, the ogg1 mutant exhibits a mutator phenotype. When compared to those of a wild-type strain, the frequencies of mutation to canavanine resistance (Can^R) and reversion to Lys⁺ are sevenfold and tenfold higher for the ogg1 mutant strain, respectively. Moreover, using a specific tester system, we show that the Ogg1-deficient strain displays a 50-fold increase in spontaneously occurring G · C→T · A transversions compared to the wild-type strain. The five other base substitution events are not affected by the disruption of the OGG1 gene. These results strongly suggest that endogeneous reactive oxygen species cause DNA damage and that the excision of 8-OxoG catalyzed by the Ogg1 protein contributes to the maintenance of genetic stability in S. cerevisiae. Received: 6 September 1996 / Accepted: 22 October 1996     

The Full-Length, Cytoplasmic C-Terminus of the β2-Adrenergic Receptor Expressed in E. coli Acts as a Substrate for Phosphorylation by Protein Kinase A, Insulin Receptor Tyrosine Kinase, GRK2, but Not Protein Kinase C and Suppresses Desensitization when Expressed in Vivo

The ability of the cytoplasmic, full-length C-terminus of the β2-adrenergic receptor (BAC1) expressed in Escherichia coli to act as a functional domain and substrate for protein phosphorylation was tested. BAC1 was expressed at high-levels, purified, and examined in solution as a substrate for protein phosphorylation. The mobility of BAC1 on SDS–PAGE mimics that of the native receptor itself, displaying decreased mobility upon chemical reduction of disulfide bonds. Importantly, the C-terminal, cytoplasmic domain of the receptor expressed in E. coli was determined to be a substrate for phosphorylation by several candidate protein kinases known to regulate G-protein-linked receptors. Mapping was performed by proteolytic degradation and matrix-assisted laser desorption ionization, time-of-flight mass spectrometry. Purified BAC1 is phosphorylated readily by protein kinase A, the phosphorylation occurring within the predicted motif RRSSSK. The kinetic properties of the phosphorylation by protein kinase A displayed cooperative character. The activated insulin receptor tyrosine kinase, which phosphorylates the beta-adrenergic receptor in vivo, phosphorylates BAC1. The Y364 residue of BAC1 was predominantly phosphorylated by the insulin receptor kinase. GRK2 catalyzed modest phosphorylation of BAC1. Phosphorylation of the human analog of BAC1 in which Cys341 and Cys378 were mutated to minimize disulfide bonding constraints, displayed robust phosphorylation following thermal activation, suggesting under standard conditions that the population of BAC1 molecules capable of assuming the “activated” conformer required by GRKs is low. BAC1 was not a substrate for protein kinase C, suggesting that the canonical site in the second cytoplasmic loop of the intact receptor is preferred. The functional nature of BAC1 was tested additionally by expression of BAC1 protein in human epidermoid carcinoma A431 cells. BAC1 was found to act as a dominant-negative, blocking agonist-induced desensitization of the beta-adrenergic receptor when expressed in mammalian cells. Thus, the C-terminal, cytoplasmic tail of this G-protein-linked receptor expressed in E. coli acts as a functional domain, displaying fidelity with regard to protein kinase action in vivo and acting as a dominant-negative with respect to agonist-induced desensitization.