Cloning of phytase coding gene

At present, there is little research on phytase gene of Trichoderma. Up to now, only AJ543399(2003) and patent US 75 1083 1 (2009) have been published in GenBank. Luo et al. (20 1 1) amplified the genomic DNA of Trichoderma T2-1 by PCR, and obtained a specific DNA fragment with a length of about 1.7 kb. The sequencing results showed that the DNA fragment contained the complete sequence of phytase coding gene and three intron sequences, in which the phytase gene was 1443 bp, encoding 480 amino acids, a signal peptide sequence at the 5' end encoded 23 amino acids, and the remaining 457 amino acid residues were the amino acid sequences of mature phytase. The tertiary structure of the amino acid sequence encoded by this gene was predicted, and it was found to be phosphomonoesterase.

Juck Zhang et al. (20 12) identified the phytase-producing ability of Trichoderma strain 148 on agar medium containing calcium phytate, and all the tested Trichoderma strains had phytase activity, which confirmed that phytase coding genes existed widely in Trichoderma population. 2 1 strain was screened from 14 Trichoderma species, and degenerate primer P8205 was designed according to phytase conserved sequence. P500-2 was used to amplify the phytase gene fragment of Trichoderma 1 1 strain from 17 strain, and the sequence was determined. The sequencing results showed that its size was about 1 100 bp. Through sequence comparison, it was found that there was a conserved sequence of about 500 bp near the C-terminal motif except the active site, but there were obvious differences in other regions, which indicated that the phytase gene sequence of Trichoderma showed the characteristics of diversity.

Trichoderma phytase genes showed diversity characteristics. The similarity of phytase gene among Trichoderma species is high, but there are obvious differences among Trichoderma species, and the phylogenetic relationship is basically consistent with its gene sequence. Comparing the obtained phytase gene sequence with the patent sequence of Trichoderma reesei phytase gene (patent US 75 1083 1), it was found that the homology between them was very low, and no histidine phytase active site was found in this gene. Therefore, it is speculated that the phytases produced by different Trichoderma reesei may not all belong to the same phytase, and only 14 Trichoderma strains were obtained in this experiment. At the same time, in view of the potential of Trichoderma phytase gene diversity analysis as a phylogenetic marker (Juck Zhang et al., 20 12) (Figure 10. 13).

Table 10. 13 phytase gene amplification of Trichoderma strain