Cloning and expression analyses of a cellobiohydrolase gene from Auricularia heimuer

Figures & data

Table 1. Primers used in this research.

Name	Sequence (5′-3′)
A-cbhF	5′-CGCGGATCCATGTTCCAAGCCGCAGCTCT     BamHI
A-cbhR	5′-CCCAAGCTTTTACAGGCACTGGGAGTAATAGTCG  HindIII
RV-M	5′-GAGCGGATAACAATTTCACACAGG-3′
M13-47	5′-CGCCAGGGTTTTCCCAGTCACGAC-3′
pET1	5′-TAATACGACTCACTATAGGG-3′
pET2	5′-GCTAGTTATTGCTCAGCGG-3′
A-cbh-qF	5^′-GAGCAGTGGAACTCTGTCGGAA-3′
A-cbh-qR	5^′-GGCATCGGTAGGGTAGTTGGAG-3′
Actin-F	5^′-GCCCTCGTTCCTTGGTCTTG-3′
Actin-R	5^′-GTGCTCAACGACGCCAGGAT-3′

The underlined portions are restriction sites.

Figure 1. Fruiting bodies stage of A. heimuer, including primordia (a), ear buds (b), immature period (c), and maturity period (d).

Figure 2. Nucleotide sequence of A-cbh and deduced amino-acid sequence of A-cbh from A. heimuer.

Figure 3. Amino acid composition of A-cbh.

Figure 4. Putative conserved domains of the A-cbh protease (NCBI Conserved Domains search result).

Figure 5. Transmembrane regions prediction of the A-cbh protein.

Figure 6. Tertiary structure prediction of the A-cbh protein.

Figure 7. Phylogenetic analysis of A-cbh of A. heimuer.

Table 2. Homologous sequences of A-cbh protein.

Species	Accession ID
Exidia glandulosa	KZV94040.1
Exidia glandulosa	KZV94032.1
Exidia glandulosa	KZV87868.1
Auricularia subglabra	EJD53243.1
Auricularia subglabra	EJD55681.1
Flammulina velutipes	BAJ07535.1
Pleurotus ostreatus	KDQ29416.1
Clohesyomyces aquaticus	ORY11399.1
Leucoagaricus gongylophorus	CDJ79819.1
Agaricus bisporus var. bisporus	XP_006463825.1
Volvariella volvacea	AAT64006.1
Volvariella volvacea	AAT64007.1
Hypsizygus marmoreus	RDB20585.1
Hypsizygus marmoreus	RDB20782.1
Hypsizygus marmoreus	RDB20742.1
Hypsizygus marmoreus	RDB24361.1
Paenibacillus terrae	AET59390.1

Figure 8. Heterologous protein expression in E. coli after IPTG (1 mmol/L) induction. M: protein molecular weight marker (Tiangen, Beijing, China); Lane 1: E. coli transformed with the empty vector; Lane 2: E. coli transformed with pET32a-A-cbh induced with IPTG at 0 h; Lane 3: E. coli transformed with pET32a-A-cbh induced with IPTG at 2 h; Lane 4: E. coli transformed with pET32a-A-cbh induced with IPTG at 4 h; Lane 5: E. coli transformed with pET32a-A-cbh induced with IPTG at 6 h; Lane 6: E. coli transformed with pET32a-A-cbh induced with IPTG at 8 h; Lane 7: E. coli transformed with pET32a-A-cbh induced with IPTG at 10 h.

Figure 9. Transcription levels of A-cbh at different stages of A. heimuer.