Construction and Practice of Foundation Experiment Teaching System in National Excellent Microbiology Course / 微生物学通报

Microbiology experiment existing independently from microbiology theoretical curriculum is an indispensable compulsory course in contemporary life science. This article presents the principle applied by the National Excellent Microbiology Course teaching group in Nankai University, which is to strengthen the undergraduates’ basic skills of conducting microbiology experiments. With an aim to enhance the core competitiveness of the undergraduates, we have established the three-level experimental contents. A new multilevel teaching pattern focusing on basic skill training as the cornerstone has been applied to enhance the overall competences of the students and to stimulate their innovation abilities. Students’ experimental accomplishment will also be taken into consideration when their experiment results are evaluated, which helps to standardizing their research ethics.

Protoplast transformation of Mortierella isabellina with hygromycin B resistance plasmid PD4 / 生物工程学报

A strain Mortierella isabellina M6-22-4, which was sensitive to hygromycin B, was selected by treating parental spores with N-methyl-N' -Nitro-N-nitrosoguanidine (MNNG). Protoplasts of the strain Mortierella isabellina M6-22-4 were transformed successfully to hygromycin B resistance using the PD4 plasmid, which contains the Escherichia coli hph gene under the control of Mortierella alpina his H4.1 promoter. The PD4 plasmid was introduced by PEG/CaCl2 treatment. Transformation frequencies of 1.6 - 2.8 transformants/microg of DNA were achieved. Then they were successively incubated to non-selected PDA plates for 10 generations. About 31.6% transformants only from digested plasmid were mitotically stable and showed different hygromycin B resistance when they were incubated back to selection plates. The results of PCR and Southern analysis in three transformants indicated that the plasmid PD4 had been integrated into the fungal genome with 1 - 2 copies. This is the first report of Mortierella isabellina transformation system and supplies an important tool for further research into genetic manipulation of this filamentous fungus.

Type III secretion study of popN- mutant of Pseudomonas aeruginosa and proteases degradation / 生物工程学报

Pseudomonas aeruginosa is an important opportunistic human pathogen. It encodes many virulence factors and one of them is type III secretion system (TTSS). Effectors proteins can be delivered into host cells directly by this system, causing necrosis or apoptosis. popN gene is the first gene in the popN operon of TTSS gene cluster. To investigate its function, popN gene deletion mutant was generated in this study, and we found this mutant can secrete effectors proteins constitutively under non-inducting condition in DMEM medium containing serum. The results indicated that PopN is a negative regulator of the TTSS expression. However, no secreted effector proteins were detectable when the popN- mutant was grown in LB medium under non-inducting condition. To investigate the possible reasons, effects of growth status and protease (s) inhibitors on the TTSS were investigated. We present evidences that indicate protease mediated degradation of secreted effector proteins played a key role in the phenotypic inconsistency of popN- mutant.

A method using long primers for cloning the upstream sequence of delta-6 fatty acid desaturases gene of Thamnidium elegans by nested inverse PCR / 生物工程学报

Thamnidium elegans is a kind of phycomycete that produces essential unsaturated fatty acids, particularly y-linolenic acid. In this process, delta6-Fatty acid desaturase (D6D) plays a key role due to its enzymatic properties that catalyze the delta6 site dehydrogenation of precursor linoleic acid (18:2delta(9, 12) n-6) and a-linolenic acid (18:3delta(9, 12, 15) n-3). This reaction is the first and rate-limiting step of highly unsaturated fatty acids (HUFA) synthesis pathways. After we have isolated and cloned the gene coding delta6-fatty acid desaturase from Thamnidium elegans As3.2806 (GenBank accession number DQ099380), our interest focuses on the promotion and regulation of the gene transcription. To achieve this aim, we designed long primers and used nested inverse PCR to amplify DNA flanking sequences. First, genome of Thamnidium elegans was extracted and digested with restriction enzymes EcoR I and Kpn I , respectively. Then we ligated the digested DNA with T4 ligase at low concentration which is propitious for linear DNA to joint intromolecule. According to the sequence of delta6-fatty acid desaturase gene of Thamnidium elegans, we designed a couple of 35nt long inverse primers and two couples of shorter inverse primers for inverse PCR. Three rounds of PCR reactions were performed. In the primary reaction, the ligated DNA was used as a template, and the product was used as the template of the secondary reaction, the tertiary reaction was achieved in the same way. After all the three rounds of reactions, we got a nice product about 4 kb from the EcoR I digested sample, in which a 1.3kb 5' upstream sequence (GenBank accession number DQ309425) of delta6-fatty acid desaturase gene containing several putative regulatory elements including TATA. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans detla6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3 kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans delta6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome.

A Modified Method for Isolating DNA from Fungus / 微生物学通报

Genomic DNA of two fungi Thamnidium elegans and Umbelopsis isabellina were extracted with an amended Cetyltrimethyl Ammonium Bromide (CTAB) method. This modified method uses repeated freezing in liquid nitrogen and thawing with combination of shocking with glass beads to replace of the tra- ditional method. Quality and concentration of DNA extracted by the modified methodwere tested. Compared with the traditional method, higher yield and purity of genomic DNA were obtained with less amount ofmy- celium. The result indicted that this is a simple and highly efficient method, which is suitable to treat many samples at one time and for basic molecular experiments, such as restriction endonuclease reaction and PCR.