米曲霉尿嘧啶营养缺陷型突变体筛选及转化体系建立

米曲霉(Aspergillus oryzae)是一种十分重要的工业微生物，但由于其菌丝体和孢子均含有多个细胞核，并且对多种抗性药物具有抗性，导致其遗传转化和分子生物学研究较其他模式丝状真菌困难。目前米曲霉遗传转化主要采用营养缺陷型作为筛选标记。尿嘧啶营养缺陷型是米曲霉转化中最常用的一种营养缺陷型标记，其筛选标记基因pyrG编码乳清酸核苷-5′-磷酸脱羧酶为尿嘧啶的前体尿苷合成所必需。本研究以米曲霉3.042为背景，利用紫外线诱变和5-氟乳清酸(5-FOA)胁迫筛选获得5株尿嘧啶营养缺陷型菌株，经DNA测序5株菌株均为pyrG基因不同位点突变，确定为尿嘧啶营养缺陷型。以筛选到的尿嘧啶营养缺陷型菌株为背景，利用农杆菌介导的米曲霉转化系统，成功构建了米曲霉尿嘧啶营养缺陷型为筛选标记的农杆菌转化体系。     

蛹虫草磷酸甲羟戊酸激酶和焦磷酸甲羟戊酸脱羧酶基因的功能分析

【目的】确定蛹虫草甲羟戊酸途径中的2个关键酶——磷酸甲羟戊酸激酶(CmErg8)和焦磷酸甲羟戊酸脱羧酶(CmErg19)的功能及其对麦角甾醇和虫草素含量的影响。【方法】通过生物信息学分析鉴定蛹虫草中CmErg8和CmErg19,并采用酵母互补确定其功能是否保守;以蛹虫草尿嘧啶营养缺陷型CmΔpyrG为背景菌株,利用农杆菌介导的转化方法对CmErg8和CmErg19进行过表达,观察其对麦角甾醇和虫草素含量的影响。【结果】CmErg8和CmErg19不能互补酵母erg8和erg19突变体的温度敏感表型;CmErg8和CmErg19过表达菌株中麦角甾醇和虫草素含量均有所增加,特别是CmErg19基因过表达可以使虫草素含量提升5倍左右。【结论】本研究揭示了蛹虫草CmErg8和CmErg19的功能,并且发现蛹虫草麦角甾醇合成通路基因可能会影响虫草素含量。     

灵芝尿嘧啶营养缺陷型菌株的筛选和分子鉴定

【背景】营养缺陷型是一种应用广泛的分子标记,但是目前在灵芝中还未有研究和应用报道。【目的】为灵芝遗传转化研究、杂交育种和菌种鉴别提供亲本材料和技术支持。【方法】采用紫外光诱变、单单杂交、孢子单核化的方法从灵芝单核体菌株出发得到尿嘧啶营养缺陷型双核体菌株。【结果】获得8株稳定的尿嘧啶营养缺陷型单核体突变菌株和7株尿嘧啶营养缺陷型双核体菌株。【结论】灵芝尿嘧啶营养缺陷型菌株在添加外源营养物的基础上可恢复正常生长,可以为灵芝遗传转化体系的构建和灵芝育种提供材料。     

基于CRISPR/Cas9的蛹虫草无抗性标记转化技术的构建

传统的真菌遗传改造方法需要抗性标记,但目前可使用的抗性标记基因非常有限,导致蛹虫草遗传改造面临着抗性基因数量不足的问题,且尚未能实现多个目的基因的连续敲入或敲除,因此在蛹虫草中建立高效的无抗性标记转化技术显得尤为重要。本研究利用CRISPR/Cas9技术对蛹虫草的Cmura5基因进行编辑,通过内源5S-1、5S-2和U6启动子对gRNA进行转录,结果表明使用U6启动子对Cmura5基因的编辑效率达到了100%。在尿嘧啶缺陷型菌株Cmura5^-中,回补野生型Cmura5基因可实现正向选择,即野生型菌株可以在基础培养基上生长。利用设计的同源臂对Cmura5基因进行回收,可以实现反向选择,即野生型在含有5-氟乳清酸培养基中生长受到抑制。以尿嘧啶缺陷型Cmura5^-为出发菌株,利用无抗性标记转化技术,导入一个重组质粒效率为75%;连续导入2个重组质粒效率为80%;连续导入3个重组质粒效率为100%;连续导入4个重组质粒效率为50%,平均转化效率为75.7%,每一轮的标记回收率均在100%,实现了4个外源基因在蛹虫草中同时表达。     

根癌农杆菌介导的日本曲霉转化体系的建立

【目的】通过根癌农杆菌介导的方法构建日本曲霉转化子库,从而筛选出高产甘没氧化酶的日本曲霉突变菌株。【方法】本文通过三亲杂交的方法将双元载体pBI-hphII转移至根癌农杆菌EHA105中并作为侵染菌株,以日本曲霉As5999为受体菌株,建立了农杆菌介导的日本曲霉转化体系,构建了突变体库,并对影响转化效率的根癌农杆菌浓度,乙酰丁香酮(As)加入与否,共培养时间,共培养温度等因素进行了分析。【结果】对转化子的PCR检测和Southern杂交分析表明,T-DNA已整合进日本曲霉基因组中,随机挑选的9个转化子连续转接10代后均能稳定遗传。【结论】该转化体系的建立为筛选出高产甘油氧化酶的日本曲霉突变菌株奠定了基础。     

农杆菌介导的斑玉蕈遗传转化

【目的】将农杆菌介导的转化应用于重要的工厂化栽培食用菌斑玉蕈中,建立稳定的农杆菌介导的斑玉蕈遗传转化技术。【方法】将构建的双元载体pYN6982转入农杆菌LBA4404菌株中,以斑玉蕈SIEF3133菌株打碎的双核菌丝为受体材料,利用根癌农杆菌介导的转化方法进行斑玉蕈转化试验。【结果】经潮霉素抗性筛选、PCR鉴定以及有丝分裂稳定性试验验证,表明潮霉素磷酸转移酶基因(hph)已经整合到斑玉蕈的基因组中;转基因斑玉蕈菌丝在荧光显微镜下可以观测到绿色荧光,表明增强型绿色荧光蛋白基因(egfp)已经在转基因斑玉蕈菌株中获得了表达;通过PCR检测,随机挑选的8个转基因斑玉蕈菌株中有2个可以扩增出载体转移DNA(T-DNA)边界重复序列外的卡那霉素基因(kan)序列。【结论】获得了稳定遗传和表达的斑玉蕈转基因菌株,建立了农杆菌介导的斑玉蕈遗传转化方法。农杆菌介导的斑玉蕈遗传转化中,存在载体T-DNA边界重复序列之外的DNA序列转移到转基因斑玉蕈中的现象,有待进一步研究。     

卷枝毛霉pyrG基因缺陷突变株的诱变筛选与鉴定

为制备新的遗传筛选标记用于构建高产番茄红素的工程菌株,实验运用化学诱变的手段,以N-甲基-N'-硝基-N-亚硝基胍为诱变剂,以番茄红素生产菌株卷枝毛霉MU616为出发菌株,诱变获得5株尿嘧啶缺陷型突变株,突变株在基本培养基中培养5天后仍不能生长。通过同源转化带有pyrG基因(编码乳清酸核苷-磷酸盐脱羧酶)的质粒pEPM1确定突变株Mt1、Mt4和Mt5为pyrG基因缺陷突变株。随之对pyrG突变株进行生长特性的研究和产番茄红素性能的检测,结果表明,其中突变株Mt4的生物量为(9.0±0.6)g/L,番茄红素产量在黑暗和光照条件下分别为(1 648±185)μg/g和(3 234±281)μg/g,均与出发菌株相似,适宜作为进行卷枝毛霉转化的带有遗传标记的受体菌。pyrG基因缺陷突变菌株的获得对构建高产番茄红素的卷枝毛霉工程菌具有重要的意义。     

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     

根癌农杆菌介导的Pr1基因在贵阳腐霉的组成性表达

【目的】构建组成性表达贵阳腐霉Pr1基因的载体pCambia-hph-Pr1,转化贵阳腐霉,以获得高毒力的基因工程转化菌株。【方法】以双元载体pCambia-Pnos-PNN-hph为基本骨架,将Pr1基因置于组成型启动子PgpdA和终止子TtrpC之间,获得含Pr1基因的表达元件。以贵阳腐霉菌丝体为受体材料,利用根癌农杆菌介导的遗传转化法转化贵阳腐霉,观察转化株的生物学特征和灭蚊毒力。【结果】转化株的菌丝生长速度和游动孢子产量与野生株无显著性差异。生物测定表明转化株对蚊幼虫平均致死率达到32.9%,比野生株提高了约一倍,并且使受试蚊幼虫生长速度明显减缓。【结论】利用根癌农杆菌介导的遗传转化获得了遗传稳定的高毒力菌株。     

类鼻疽伯克霍尔德菌bopA基因敲除株的构建及生物学特征

【【背景】类鼻疽杆菌是一种能够引起人类疾病甚至死亡的胞内寄生菌,Ⅲ型分泌系统在该菌入侵上皮细胞、逃避宿主免疫以及毒力因子的分泌过程中发挥重要作用,其中bopA基因为TTSS-3基因编码的重要效应蛋白,在类鼻疽杆菌的免疫逃逸中发挥重要作用。【目的】构建类鼻疽杆菌bopA基因敲除菌株,并对其生物学特征进行初步研究。【方法】构建pK18mobSacB-ΔbopA自杀质粒,通过大肠杆菌S17-1λpair以接合的方式转入类鼻疽杆菌,利用同源重组敲除了bopA基因,并用蔗糖平板筛选出菌株,最后在细胞和动物水平检测敲除菌株的表型变化。【结果】构建了bopA敲除的类鼻疽菌株,并通过细胞和动物实验证实敲除bopA基因后,细菌的细胞侵袭和胞内存活以及体内定殖能力都显著降低。【结论】利用同源重组成功构建类鼻疽bopA基因敲除株,为深入研究该基因的作用靶点奠定了实验基础。     

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.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响

【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77 d,但差异不显著;除1龄幼虫体重增加(0.0773 mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

Evaluation of Beauveria bassiana and B. brongniartii strains and four wild-type fungal species against adults of Bactrocera oleae and Ceratitis capitata

The virulence of two isolates of the hyphomycete fungi, Beauveria bassianaand B. brongniartii, and additional fungal species isolated from diseased Bactrocera oleae pupae and Sesamia nonagrioideslarvae were assessed against adults of the olive fruit fly B. oleae and the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae). Contact and oral bioassays revealed that moderate to high mortality rates for the olive fruit fly occurred when the adults were exposed to conidia of Mucor hiemalis, Penicillium aurantiogriseum, P. chrysogenum and B. bassianaisolates. A strain of M. hiemalis isolated from S. nonagrioides larvae was the most toxic resulting in 85.2% mortality to the olive fruit fly adults. B. brongniartiiand B. bassiana were the most pathogenic to the C. capitataadults causing 97.4 and 85.6% mortality. Metabolites collected from the M. hiemalis and P. chrysogenum isolates were toxic to adults of both species.     

The phylogenetic placement of the Leucogastrales, including Mycolevis siccigleba (Cribbeaceae), in the Albatrellaceae using morphological and molecular data

The morphology of many hypogeous fungi converges on a homogeneous reduced form, suggesting that disparate lineages are subject to a uniform selection pressure. The primary goal of this study was to evaluate the morphology and infer the phylogeny of the Leucogastrales with Mycolevis siccigleba using a Bayesian methodology. A comprehensive morphological assessment was used for an a priori phylogenetic inference to guide the sequencing effort. All structures except spore ornamentation pointed to the Albatrellaceae as the most likely sister taxon. Polyporoletus sublividus, a close relative of Albatrellus, produces ornamented basidiospores with a similar structure to M. siccigleba basidiospores. The ITS from 30 taxa was used for the molecular phylogenetic analysis. P. sublividus was found sister to Mycolevis. Leucophleps spinispora and L. magnata formed a group sister to the Polyporoletus/Mycolevis group, whereas Leucogaster was polyphyletic with respect to the core of the Leucogastrales and sister to A. caeruleoporus. This relationship was expected as previously undescribed chlamydospores produced by members of Albatrellus had a similar morphology to the basidiospores of L. rubescens.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.