酸性蛋白酶基因Asp在红曲霉中的同源表达及序列分析

从红曲霉基因组中扩增出酸性蛋白酶基因Asp的编码区,构建其同源表达载体p BC-Hygro-Asp并导入到农杆菌GV3101备用。利用根癌农杆菌介导法将重组质粒导入红曲霉,并筛选获得Asp基因同源重组转化子,实现了酸性蛋白酶基因在红曲霉中的同源表达。转化子中酸性蛋白酶基因表达量是野生型菌株的3.30倍,能达到高产酸性蛋白酶的目的。对红曲霉酸性蛋白酶基因序列进行分析,结果显示该基因产物有两个天冬氨酸蛋白酶活性位点,为亲水性分泌蛋白,不参与信号转导,且与赤曲霉酸性蛋白酶有相同进化速率。     

根癌农杆菌介导载体pBI121转化少根根霉条件的研究

考察了脂肪酶生产菌株少根根霉N1023 G418的敏感性,结果表明,G418对少根根霉最小抑制浓度为500μg/mL。研究了不同条件对载体pBI121转化少根根霉的影响,结果表明,以根癌农杆菌LBA4404为介导菌,少根根霉N1023为受体菌株,G418为筛选标记的最佳转化条件为:乙酰丁香酮(AS)300μmol/L、根癌农杆菌的初始浓度OD600为0.6、共培养时间2 d。在此条件下pBI121对少根根霉的转化率达到112 cfu/106 cfu孢子。转化菌株经过5次连续培养后仍然稳定。通过上述研究,建立了根癌农杆菌介导载体pBI121转化少根根霉的方法。这是首次成功建立少根根霉的转化方法,为进一步的研究奠定了基础。     

平菇疏水蛋白Po.hyd基因RNAi载体构建及转化

为研究平菇疏水蛋白基因Po.hyd的功能,首次构建了Po.hyd的RNAi载体,并通过根癌农杆菌介导获得了平菇转化子。通过聚合酶链式反应（polymerase chain reaction,PCR）扩增Po.hyd序列,利用Golden Gate克隆法构建了干扰载体p1302-GG-ihyd,将其导入根癌农杆菌GV3101中,并在乙酰丁香酮的诱导下完成根癌农杆菌对平菇幼嫩菌丝的介导转化。转化子经PCR检测、荧光显微镜观察、实时定量荧光PCR技术（real-time PCR）以及Southern杂交等技术进行验证。结果显示,本研究最终获得1 株可稳定遗传的平菇转化子,该转化子具有潮霉素抗性,且在荧光显微镜下可检测到绿色荧光信号,Po.hyd基因的表达量为野生型的43%,干扰载体T-DNA片段以单拷贝的形式整合在转化子基因组内。     

葡萄糖氧化酶基因在黑曲霉中的分泌表达

通过PCR扩增从一株葡萄糖氧化酶生产菌黑曲霉中克隆得到1818bp葡萄糖氧化酶基因GOD,将GOD基因与糖化酶基因的5’同源臂、3’同源臂进行重叠延伸,构建葡萄糖氧化酶基因的表达框。将此表达框插入载体pSZH中,构建黑曲霉同源重组表达载体pSZH-GOD。将载体pSZH-GOD通过冻融法转化农杆菌AGL1,进而通过农杆菌介导法转化高产糖化酶的黑曲霉。经潮霉素筛选和PCR鉴定获得8株重组菌株。对8株重组菌株的发酵液上清液进行酶活测定,结果表明葡萄糖氧化酶基因在高产糖化酶的黑曲霉中得到了分泌表达,静置培养6d后其分泌表达的葡萄糖氧化酶酶活最高可达1.166U/mL,而在出发菌株中检测不到葡萄糖氧化酶的分泌表达。同时,重组菌株胞内表达的葡萄糖氧化酶最高酶活可达11.45U/mL,是出发菌株的266倍。此研究结果为简化葡萄糖氧化酶发酵生产工艺、提高酶产量提供了新的途径。     

采用农杆菌介导转化法的泡盛曲霉表达载体的构建

为在泡盛曲霉中表达外源基因、克服泡盛曲霉常规转化法效率低下的瓶颈,本研究拟构建采用农杆菌介导转化法的泡盛曲霉表达载体。通过PCR,从糖化酶基因(glaA)高效表达的泡盛曲霉菌株SG1 基因组DNA 扩增获得glaA 2.1 kb 启动子片段(包含信号肽编码序列)及1.1kb 终止子片段,构建了外源基因表达盒。以根瘤农杆菌双元载体pCAMBIA1302 为基础,删除非必需区段及多余限制酶位点,插入上述外源基因表达盒及来自丝状真菌标记载体pAN7-1 的潮霉素抗性标记,构建采用农杆菌介导转化法的泡盛曲霉分泌整合型表达载体pSUAA52am。转化结果表明：采用原生质体法的转化效率为21 个转化子/106 分生孢子,而农杆菌介导法可达1106 个转化子/106 分生孢子,是原生质体法的53 倍。构建载体成功利用glaA 表达盒与农杆菌介导转化法的高效特性,可为利用泡盛曲霉高效表达外源基因奠定基础。     

Bar基因双元载体的快速构建及其在蛹虫草中的功能验证

为获得草铵膦完全抑制蛹虫草生长的最小浓度和一种快速构建Bar基因双元载体的方法,本文首先采用浓度梯度法研究草铵膦对蛹虫草的抑制效果,其次采用双接头PCR(DJ-PCR)构建Bar基因表达盒,然后分别采用T4 DNA连接酶法和同源重组法把Bar基因表达盒插入双元载体pAg1-H3的T-DNA区域,以构建Bar基因双元载体pAg-Bar,并比较T4DNA连接酶法和同源重组法的连接效果,最后通过农杆菌介导转化法来验证载体pAg-Bar的有效性及Bar基因在蛹虫草中的功能。结果表明:草铵膦完全抑制蛹虫草分生孢子生长的最小质量浓度为400μg/mL;采用DJ-PCR方法可快速构建Bar基因表达盒;采用T4 DNA连接酶法和同源重组法均可实现双元载体pAg-Bar的构建,而同源重组法更快速、高效;采用农杆菌介导转化法可把双元载体pAg-Bar中的Bar基因表达盒整合入蛹虫草的基因组,使蛹虫草转化子具有草铵膦抗性。本研究建立的载体构建方法具有快速、高效的特点,适用于抗性基因载体、敲除载体、超表达载体等载体的构建,为蛹虫草基因功能的鉴定提供技术支撑。     

基于CRISPR/Cas9系统的金针菇基因组编辑载体构建

采用高效基因编辑系统CRISPR/Cas9,构建金针菇基因Mads-8敲除载体及转化体系。以转录组数据为依据,通过分析基因Mads-8序列信息,选择高效的靶位点序列,同时加入筛选标记基因hph构建过渡载体sgRNA-T,通过菌落PCR鉴定和测序来验证靶位点序列是否正确插入。以表达载体pg Fvs-cas9为框架,酶切连接后构建重组敲除载体pg Fvs-Cas9-gRNA,PCR和酶切鉴定敲除载体。再以PEG介导的金针菇原生质体转化表达载体,在潮霉素抗性平板上筛选拟转化子,以拟转化子基因组DNA为模板扩增Cas9基因。结果显示,靶位点序列成功插入敲除载体且序列正确,重组质粒成功转化进金针菇的基因组。对金针菇基因组敲除载体的构建,为后续金针菇相关基因的功能验证及育种研究提供载体材料及理论依据。     

阿魏酸酯酶在黑曲霉中的同源表达

利用食品级黑曲霉表达系统同源表达阿魏酸酯酶是提高阿魏酸酯酶产量、降低生产成本的有效途径。利用PCR技术扩增黑曲霉阿魏酸酯酶A基因(faeA)的编码区,构建黑曲霉表达载体pSZHG-faeA,并通过农杆菌介导法转化黑曲霉。经筛选获得将faeA基因整合到糖化酶基因位点的同源重组转化子4株。在酶摇瓶发酵条件下,重组菌株培养液上清中的阿魏酸酯酶活性最高达18.6mU/mL。SDS-PAGE分析显示,4株重组菌株都有约36ku目的蛋白条带,表达量为188~262μg/mL。结果表明,实现了阿魏酸酯酶在黑曲霉中的同源表达,为食品级阿魏酸酯酶的大规模工业化生产奠定了基础。     

影响根癌农杆菌的电击转化条件

采用电击转化方法将双元载体质粒pCAM3300导入根癌农杆菌LBA4404,研究了不同实验条件对转化率的影响.结果表明:当电场强度为11 kV/cm时,转化率最高,每微克DNA得到9.8×103CFU;在细胞OD600为1.0时进行电转化,转化率最高,每微克DNA得到9.54×103CFU;在一定的细胞浓度范围内,电击转化率与细菌浓度密切相关,转化率随着细菌浓度的上升而上升;质粒大小与电击转化率之间没有明确的关系.将含pCAMBIA3300的根癌农杆菌与粗糙脉胞菌共培养转化,在含有膦化麦黄桐(PPT)(405 mg/L)的平板上筛选到转化子.PCR扩增转化子的染色体,初步证明Bar基因整合进粗糙脉胞菌的染色体中.     

疏棉状嗜热丝孢菌耐热脂肪酶在无孢黑曲霉中的高效表达

综合考虑密码子偏爱性、RNA稳定性以及自由能等因素,对疏棉状嗜热丝孢菌(Thermomyces lanuginosus)耐热脂肪酶(tll)基因序列进行密码子优化并全基因合成。将合成的tll基因连接到克隆载体,然后亚克隆到表达载体pHGWPT-tll,采用根癌农杆菌介导转化无孢黑曲霉SH-2。经过三丁酸甘油酯平板、SDS-PAGE以及Western Blotting鉴定,成功表达了tll基因。发酵72h,转化子脂肪酶酶活最高达36U/mL。转化子经等离子诱变得到的突变株的脂肪酶比活力比出发菌株提高约37%。     

Construction of an autonomously replicating plasmid in n-alkane-assimilating yeast, Candida tropicalis

A transformation system using the autonomously replicating plasmid in the n-alkane-assimilating and asporogenic diploid yeast, Candida tropicalis, was developed. For the cloning of a DNA fragment containing a potential autonomously replicating sequence (ARS) from the genomic DNA of C. tropicalis, the ura3 mutant obtained using ethylmethane sulfonate as the host and the URA3 gene amplified by PCR using the C. tropicalis genomic DNA as a selectable marker were prepared. Comparison of ARSs among yeasts revealed that the consensus sequence found in S. cerevisiae was also present in C. tropicalis. The autonomously replicating plasmid containing the putative ARS as the shuttle vector, capable of replicating in both E. coli and C. tropicalis, was first constructed. The transformation system using this plasmid, in addition to the integrative transformation system, will be applicable to genetic studies of C. tropicalis.     

Protoplast Transformation of Alkalophilic Bacillus sp. by Plasmid DNA

A method for transformation of alkalophilic Bacillus sp. A-59 (ATCC21591, alkaline amylase producing bacterium) with plasmid DNA was developed. Plasmid pHWl containing a chloramphenicol acetyltransferase gene was used as a vector, because chloramphenicol was the only antibiotic found to be stable at alkaline pH condition. Transformation was carried out by a protoplast method and addition of polyethylene glycol (PEG; final concentration 22.5%) was essential for transformation. Optimum pH values of the media and buffers were slightly higher than those used in transformation of Bacillus subtilis protoplasts. Through optimization of transformation conditions for alkalophilic Bacillus sp., the transformation efficiency was increased to approximately 10⁶ transformants per μg plasmid DNA.     

Effect of Non-Ionic Surfactants and Beta-Ionone on the Morphology of Blakeslea trispora and Carotenoids Production from Cheese Whey in Submerged Aerobic Growth: A Statistical Approach

The effect of non-ionic surfactants and β-ionone on morphology of Blakeslea trispora and carotenoids production from deproteinized hydrolyzed whey in submerged aerobic growth was investigated. Also, a central composite design was employed to determine the maximum carotenoids concentration at optimum values for the process variables (Tween 80, Span 80, β-ionone). The fit of the model was found to be good. Tween 80 and Span 80 had a strong linear effect on carotenoids production. The concentration of carotenoids was significantly affected by Tween 80 – Span 80 and Span 80 – β-ionone interactions as well as by the negative quadratic effect of β-ionone. The optimum medium composition for the maximum carotenoids production (100.0 ± 5.0 mg/g biomass dry weight) was found in deproteinized hydrolyzed whey supplemented with Tween 80 (33.6 g/L), Span 80 (68.7 g/L), and β-ionone (2.6 g/L). This result indicated that the optimization strategy led to an increase in carotenoids production by 33-fold. The carotenoids content in B. trispora were β-carotene, γ-carotene, and lycopene. The composition of carotenoids depends of the amount of nonionic surfactants and β-ionone added to the cheese whey. The medium composition influenced the morphogenesis of B. trispora and product formation. The addition of surfactants into the medium changed the morphology of the microorganism from solid aggregates to loose aggregates and resulted in a substantial increase in pigment production. B. trispora growing in submerged aerobic growth is able to develop complex morphologies which have been classified into three major groups: freely dispersed hyphae, clumps, and pellets. These parameters are responsible for the production of carotenoids.     

Essential involvement of the Bacillus subtilis ABC transporter, EcsB, in genetic transformation of purified DNA but not native DNA from protoplast lysates

Involvement of the Bacillus subtilis ABC transporter EcsB in genetic transformation with native DNA from protoplast lysate (LP transformation) was investigated using an ecsB deletion mutant constructed by fusion polymerase chain reaction. In these experiments, the non-transformability phenotype of the ecsB mutant was reversed and high numbers of transformants generated (1.5 × 10⁵/μg DNA). The relative efficiency of transformation (RET) of ecsB to wild type (1.2 × 10^{− 2}) was a thousand times higher using native chromosomal DNA than the RET obtained from purified DNA (< 8.6 × 10^{− 6}). Similar transformation efficiencies were observed using native plasmid DNA. These results rule out a primary role for EcsB as a competence gene regulator. DNA-binding proteins attached to native DNA are not present in purified DNA preparations, and it is possible that such proteins could account for the transformability of the ecsB mutant. Because EcsB may play a role in protein(s) export, we tested exogenous proteins to identify functional replacements. We found that bovine serum albumin (fraction V) partially suppressed the phenotype of the ecsB mutation, leading to transformability with purified DNA. Linkage analysis of the ecsB mutant by LP co-transformation produced a higher co-transformation ratio (42% and 20%) at a distance of 34 kb and 121 kb in the ecsB mutant, compared to the wild-type strain, AYG2 (30.5% and 12.3%). The stimulatory linkage effect observed could be derived from a regulating gene involved in homologous recombination.     

Role of ComFA in controlling the DNA uptake rate during transformation of competent Bacillus subtilis

The roles of ComFA and ComEC in DNA uptake by competent Bacillus subtilis were analyzed by transformation with DNA in protoplast lysates (LP transformation). Deletion mutants of comFA and comEC and putative Walker A mutants (K152N, K152Q, K152E) of comFA were constructed by fusion polymerase chain reaction. Transformants of comEC mutant with purified DNA and DNA in protoplast lysate were not obtained, which shows a lack of transformation ability and backwards recombination of the mutant. Transformants of the comFA mutant were obtained by LP transformation (1.8 × 10(4) transformants/μg DNA). Low relative efficiency of transformation (RET) of comFA compared to wild type (4.3 × 10(-4)) showed an important role for comFA in DNA uptake. Walker A mutants showed 1.8-19 × 10(-4) RET, suggesting a dependence on ATPase activity for transformation. Co-transformation between short linkages was only detected in comFA mutants. The results demonstrated that ComFA controlled the DNA uptake rate. The interpretation was further supported by analyzing the plasmid used in LP transformation of the comFA mutant. The RET of comFA compared to the wild type was 2.7 × 10(-2), 60-fold higher than that with chromosomal DNA (4.3 × 10(-4)). Following addition of DNA into comFA culture, transformants were obtained after 15 min, with the number of transformants increasing over time. The kinetics strongly suggested that in comFA mutants, formation of another DNA uptake complex without ComFA would be a lengthy process.     

Study on Flax Genetic Transformation Mediated by Agrobacterium tumefaciens

Abstract

Flax is an important natural material for the linen spinning industry and as an oil crop in China. Flax products worth US $100 million are exported every year. Recently, with the development of a market economy and China's entry into the WTO, farmers and flax factories have shown a need for new varieties that have a short vegetation period, are resistant to herbicides and lodging, and have a high fiber content. In order to resolve these problems quickly, we have studied flax genetic transformation since 1998.

The paper is about flax genetic transformation by Agrobacterium tumefaciens strain EHA105. The explants were hypocotyl segments from 5-7 day old seedlings. The segments cannot form a callus when the concentration of kanamycin is 30 mg/L and 50 mg/L. Therefore, 50 mg/L of kanamycin was selected as an efficient concentration to select the transgenic plants. The segments of hypocotyl were treated with EHA105 and then were inoculated in different media (Ms, B5, N6). The experiment showed that Ms is a preference medium for flax genetic transformation. Agrobacteriumtieated segments of hypocotyl were cultured on the surface of modified Ms medium (Ms agar medium supplemented with 3.5 mg/L IAA, 2 mg/L KT, 150 mg/L LH). Callus was formed by 71.4%-81.6% of the hypocotyls. Callus inoculated in a regenerative medium of Ms with little BA and NAA could regenerate at the rate of 31.8%-40.2%, and 87.4% of regenerative plants could root in the medium of 1/2 Ms supplemented with 0.001 mg/L NAA. This transformation system has been tested by the GUS-INT gene. After co-cultivation of the segments of hypocotyl and GUS-INT, the segments of hypocotyl were moved to callus medium. After 3 days, the transformation rate of new callus was 72.0%; after 4 weeks the transformation rate of callus was 24.0%; the transformation rate of regenerative plants was 7.5%. The experiment allowed for the establishing of a flax genetic transformation system mediated by Agrobacterium tumefaciens.     

Development of a green fluorescent tagged strain of Aspergillus carbonarius to monitor fungal colonization in grapes

An enhanced green fluorescent protein has been used to tag an OTA-producing strain of Aspergillus carbonarius (W04-40) isolated from naturally infected grape berries. Transformation of the fungus was mediated by Agrobacterium tumefaciens. The most efficient transformation occurred when the co-cultivation was done with 10⁴ conidia due to higher frequency of resistance colonies (894 per 10⁴ conidia) and lower background obtained. To confirm the presence of the hph gene in hygromycin resistant colonies, 20 putative transformants were screened by PCR analysis. The hph gene was identified in all the transformants. Variation on the expression levels of the eGFP was detected among the transformants and 50% of them appeared bright green fluorescent under the microscope. Microscopic analysis of all the bright fluorescent transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the conidia. eGFP expression in A. carbonarius was shown to be stable in all transformants. Confocal Laser scanning microscopy images of grape berries infected with the eGFP transformant demonstrated fungal penetration into the berry tissues. OTA production was importantly increased in the eGFP transformant in comparison with the wild type strain and pathogenicity on grape berries was slightly decreased after four days of inoculation. However, no differences in virulence were found after seven days of inoculation, thus allowing utilization of this eGFP mutant for in situ analysis of A. carbonarius infection of grape berries. To our knowledge, this is the first report describing the construction of a GFP-tagged strain belonging to Aspergillus section Nigri for monitoring Aspergillus rot on grape berries.     

Contamination with storage fungi of human food from Cameroon

In a mycological study, a total of 95 human food samples were investigated to evaluate the incidence of fungal contamination in Cameroon by conventional identification method and partly confirmed by DNA sequencing. The isolated fungal spp. were further studied to determine their toxigenic potentials. The investigation revealed the predominance of Aspergillus and Penicillium with 96% of samples contaminated with at least one species of these fungi, whereas the incidence of co-contamination of samples was 85%. Aspergillus flavus and Aspergillus parasiticus (Flavi section) were the most predominant species contaminating mainly maize and peanuts. In addition, P. crustosum and P. polonicum were the most common contaminants belonging to the genus Penicillium. On the other hand, A. ochraceus (Circumdati section) registered a low incidence rate of 5%, including other members of the Aspergillus group. Other members of the genera Rhizopus and Alternaria spp. were also registered in the study. A majority of fungal strains of A. ochraceus, A. parasiticus, P. crustosum and P. polonicum isolated were toxigenic, producing the mycotoxins tested for, while none was detected in cultures of A. fumigatus. The high incidence rate of fungi contamination coupled with their potentials in producing mycotoxins gives a strong indication that the samples tested may likely be contaminated with various mycotoxins. There is need for further study to assess the incidence of mycotoxins contamination in similar food samples.     

蜂粮中Lactobacillus kunkeei的益生特性研究

蜂粮是植物花粉、蜂蜜和蜜蜂唾液的发酵混合物,是营养丰富的天然食材。作者从蜂粮中分离到61株乳酸杆菌,经16S rRNA基因测序,鉴定为Lactobacillus kunkeei。首先,用邻苯二甲醛法对61株L.kunkeei进行胆固醇去除能力检测,其中5株菌胆固醇去除能力均大于15%,菌株B35对胆固醇的去除率达到(29.07±1.30)%。随后,对这5株L.kunkeei进行酸耐受性、胆盐耐受性、抗生素耐药性及抑菌等益生特性进行研究。耐酸实验和耐胆盐实验结果表明,5株L.kunkeei均具有良好的耐酸性和胆盐耐受性。药敏试验结果表明,5株L.kunkeei对4种临床常用抗生素(红霉素、克林霉素、庆大霉素、万古霉素)均敏感。通过琼脂扩散法抑菌实验,发现5株L.kunkeei对大肠埃希氏菌、金黄色葡萄球菌均有一定的抑菌作用。B35菌株的细胞黏附性能良好,对Caco-2细胞的黏附率达到3.32%。从蜂粮中分离到5株具有良好益生特性的菌株,其中菌株B35的益生特性最佳,为益生菌的开发与利用提供了新的菌种资源。