Cordyceps militaris and culture conditions
Cordyceps militaris 01(CGMCC 3.14242) was obtained from the Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences. And the C. militaris 01 was maintained on potato dextrose agar slants and cultured at 25 °C for 7 days [14]. The C. militaris wild-type (WT) strains for transformation experiments was preserved by our group and cultured on sabouraud medium. A 0.5-cm2 mycelial block of C. militaris was transferred to shake flasks (250 mL) containing 50 mL of submerge fermentation medium (SFM: 2%glucose, 0.70% (NH4)2SO4, 0.05% K2HPO4·3H2O, 0.05% KH2PO4, 0.05% MgSO4·7H2O, 0.10% L-Glycine). Inoculated submerge fermentation flasks were shaken at 180 rpm at 25 °C for 9 days.
Recombinant Vector Construction
Escherichia coli DH5α with plasmid pCAMBIA-PgpdA-Tcbh1-hph-PtrpC (11852 base pair) was used in this work [34]. PCAMBIA-PgpdA-Tcbh1-hph-PtrpC was used as a T-DNA donor for maintenance of constructs and for C. militaris WT transformation. The plasmid also had hph (hygromycin B phosphotransferase) gene for transformants selection with Trpc promoter and cbh1 terminator (Fig. 5). The C. militaris 01 gk, pgm, ugp, and ugdh gene were amplified by using primers (F1 ~ 4, R1 ~ 4) respectively (Table 3). Plasmid pCAMBIA-PgpdA-Tcbh1-hph-PtrpC was linearized and amplified using primers (F5, R5) (Table 3). Seamless cloning enzyme pEASY-Uni Seamless Cloning and Assembly Kit used throughout this study was purchased from TransGen Biotech (Beijing City, China).
Table 3
Primers used in this experiment.
primers | Sequences (5’-3’) | notes |
gk-F1 | GCAGACATCACAATGGGCCTGCAAGAAGAAACCAAAA | Cloning |
gk-R1 | TTTCGCCACGGAGCCTAATGACGCCTCCAGTACCACAAC | Cloning |
pgm-F2 | GCAGACATCACAATGGACGTCAAGACTGTTGAGTTTA | Cloning |
pgm-R2 | TTTCGCCACGGAGCTCAAGTGCGGACATTAGGCTCGTCG | Cloning |
ugp-F3 | GCAGACATCACAATGGTTGTGGCAGGGGGGAGGAGGG | Cloning |
ugp-R3 | TTTCGCCACGGAGCTTAATGCTCAAGCAGGCGTAGCGAG | Cloning |
ugdh-F4 | GCAGACATCACAATGTCTTCATCCATCGTCGACACCG | Cloning |
ugdh-R4 | TTTCGCCACGGAGCTTAGAAGCGGTGTTGACGACCAACG | Cloning |
Plasmid-F5 | AGCTCCGTGGCGAAAGCCTGACGCA | Cloning |
Plasmid-R5 | CATTGTGATGTCTGCTCAAGCGGGGT | Cloning |
F6 | CCCCGCTTGAGCAGACATCACAATG | Identification |
R6 | TGCGTCAGGCTTTCGCCACGGAGCT | Identification |
F7 | CTATTCCTTTGCCCTCGGACGA | Identification |
R7 | ATGCCTGAACTCACCGCGACGT | Identification |
F8 | GTATATTCATCTTCCCATCCAAGAACCT | Sequencing |
R8 | ATATTTGAAAAGGGTCAGAAGTAGATAC | Sequencing |
F9 | ACCTATTCCTTTGCCCTCGGACGA | Cloning |
R9 | CAAGTTGGTCTCCAACAGTGCTTT | Cloning |
F10 | TTGGAGACCAACTTGGCTTGTATCTCTACACACAGG | Cloning |
R10 | GGCAAAGGAATAGGTAAGTTGGTCTCCAACAGTGCTT | Cloning |
The Ti mini-recombinant vector with the gene was transformed into E. coli DH5α, which were grown on Luria–Bertani (LB) agar plates containing 100 µg/mL kanamycin. Then it was identified by PCR and further confirmed by DNA sequencing (TsingKe, Beijing). E. coli DH5α transformants are stored in -80 °C and ready for use.
Agrobacterium transformation
Agrobacterium tumefaciens strain AGL-1 (provided by Dr. Linian Cai of Zhejiang University, China) was cultured on LB medium containing 25 µg/mL rifampin (28 ℃). The recombinant vector carrying the target gene were extracted from E. coli DH5α and transformed into A. tumefaciens [35]. The transformed clones are then screened from LB agar plates containing 25 µg/mL rifampicin and 50 µg/mL kanamycin. A. tumefaciens clones was transferred in induction liquid medium (AIM: 0.145% KH2PO4, 0.205% K2HPO4, 0.06% MgSO4·7H2O, 0.03% NaCl, 0.01‰ CaCl2, 0.001‰ FeSO4, 0.05% NH4NO3, 5 mL/L glycerol, 0.2% glucose, 5 mL/L trace element stock solution, 40 mL/L MES buffer (1 mol/L, pH = 5.5)) containing 200 µM acetyl syringone (AS) and shaken at 180 rpm for activation (28 °C). Induction solid medium (CO-AIM: add 1.5% agar to IM medium and reduces glucose by half) was prepared for cocultivation of pre-cultured A. tumefaciens clones and C. militaris WT. Selective CO-IM (CO-AIM without AS) containing 200 µg/mL hygromycin B, 200 µg/mL ceftriaxone sodium, and 200 µg/mL kanamycin was used for inhibiting A. tumefaciens growth and screening for C. militaris transformants.
Fungal transformation and identification
A. tumefaciens, carrying the binary vector pCAMBIA-PgpdA-Tcbh1-hph-PtrpC with hph gene under the Aspergillus nidulans Trpc promoter, was used to introduce C. militaris 01 gene (gk, pgm, ugp, ugdh) to C. militaris WT with the already described method. In order to test the inhibition ability of hygromycin B to the growth of C. militaris WT, different concentrations of hygromycin B (0, 50, 100, 150, 200 and 250 µg/mL) were added to the sabouraud medium plates, and then conidiospores were inoculated at a same concentration. The hygromycin B concentration at which C. militaris WT cannot grow was selected as the hygromycin B screening concentration for C. militaris transformantion.
A. tumefaciens clones was grown for 36 h on LB with kanamycin (50 µg/mL), rifampin (25 µg/mL) in a shaker (180 rpm, 28 °C). Then 1% (v/v) A. tumefaciens were transferred to shake flasks (250 mL) which containing 50 mL AIM, and re-incubated at 28 °C until the OD600 was 0.80. C. militaris WT conidia were obtained by cultivating the fungi on PDA plates for 7 day and the plates was washed gently with sterile water. Hemocytometer was used to calculated the conidiospores density of conidial suspension that has been filtered by four layers of sterile gauze to remove large particles. The conidial suspension was diluted to 106/mL with sterile water and mixed with the equal volume (150 µL) of A. tumefaciens clones that have been induced to obtain a mixed solution.
The mixed solution was inoculated onto a sterile cellophane sheet (diameter 90 mm), and placed on CO-AIM medium (24 °C, 48 h) with three replicates. After co-cultivation, the cellophane was transferred to the selective CO-IM while being covered by another layer of selective CO-IM. C. militaris transformants were picked by sterile toothpick and transferred to sabouraud medium containing 200 µg/mL hygromycin B. To determine the genetic stability of the transformants, all C. militaris transformants were cultured on sabouraud medium (25 °C, 4 d) containing 200 µg/mL hygromycin B. At the end of each generation of genetic stability cultivation, a spot of mycelia at the edge of each transformant was picked with a sterile toothpick and transferred to a new sabouraud medium plate (hygromycin B 200 µg/mL), which was repeated additional four times.
Potential transformants resistant to hygromycin B were verified by PCR, and confirmed by DNA sequencing. C. militaris transformants mycelia were harvested, frozen in liquid nitrogen, and ground to powder. The genomic DNA was extracted using the urea method. Standard PCR and primers (F6, R6; F7, R7) were used to detect whether Target-gene and hph were successfully recombined into the genomic DNA of potential transformants.
Sampling, analysis of residual sugar in medium and EPS production
A 0.5-cm2 mycelial block of C. militaris WT and all transformants were inoculated to shake flasks (250 mL) containing 50 mL PDB (PDA without ager, 200 µg/mL ceftriaxone sodium), and the flasks were shaken at 180 rpm at 25 °C for 3 days to obtain a seed liquid. A 1% (v/w) seed liquid was transferred to 50 mL of SFM (200 µg/mL ceftriaxone sodium) in a 250 mL flask and cultivated for 9 days. The sample was vacuum filtered to obtain mycelium, washed three times with deionized water and dried to constant weight at 50 ℃.
The fermentation sample was centrifuged at 10,000 × g (model: 5810R, Eppendorf, Hamburg Germany) for 10 minutes at 4 °C, and the supernatant was taken to measure the glucose and EPS concentrations, respectively. As the carbon source in the medium is derived from glucose, the residual amount of glucose in the fermentation supernatant was determined by the 3,5-dinitrosalicylic acid method described by Miller [36]. The crude EPS was precipitated by addition of four volumes of absolute ethanol and stored at 4 °C overnight. After centrifugation at 13000 g for 15 min, the precipitate was suspended in 1 M NaOH at 60 °C for 1 h. Phenol-sulfuric acid method was used for the determination of EPS in the supernatant [37].
Statistics Processing
All dates are the average of three independent sample measurements and expressed as the mean ± standard error. The error bars indicate the standard deviations from the means of triplicates. Statistical analyses were performed with SPSS 16.0 (SPSS Inc., Chicago, USA).