Modified cellulase gene and expression vector and application thereof

[0036] Example 1

[0037] In this example, Aspergillus niger and Trichoderma kansei were used as the laboratory preservation strains, E. coli DH5α and the cloning plasmid pMD19-T were purchased from TAKARA. PCR amplification primer design, the complete gene sequence of Aspergillus niger and Trichoderma konsi were found from NCBI, and primers were designed downstream of the signal peptide sequence of the coding region of CBHⅠ gene, and XbaⅠ restriction site was added to the forward primer. The reverse primer is added to the Pst I restriction site, and the protective bases GC and CCG are added to the 5'end of the reverse primer (this primer is synthesized by Shanghai Shenggong Biological Engineering Co., Ltd.).

[0038] Trichoderma Koningii primers:

[0039] The forward primer K1 is shown in SEQ NO.5: 5'-GC T CTA GA A TGT ATC GGA AGT TGG CCG TC Xba Ⅰ

[0040] The reverse primer K2 is shown in SEQ NO.6: 5'-CCG CTC GAG TTA CAG GCA CTG AGA GTA GTA Pst Ⅰ

[0041] Aspergillus niger primers:

[0042] Forward primer H1 is shown in SEQ NO.7: 5'-GC T CTA GA A TGC ATC AAC GTG CCC TTC TCT TTT Xba Ⅰ

[0043] The reverse primer H2 is shown in SEQ NO. 8: 5'-CCG CTC GAG TTA TGC GGA AGC GCT GAA GGT CGA GT Pst Ⅰ

[0044] Example 2 PCR amplification of cellulase gene

[0045] Inoculate Trichoderma konsi and Aspergillus niger respectively in PDA medium, culture for 48h on a shaker at 120 rpm at 30℃, filter the bacteria with sterile filter paper and absorb water, use RNA extraction kit to extract Trichoderma konsi and Aspergillus niger Of total RNA.

[0046] Among them, the PDA medium is prepared, and MgSO is weighed 4 0.15g, soluble starch 3g, glucose 10g, KH 2 PO 4 1g, yeast extract 1g, tryptone 2.5g, dissolve in 400mL of distilled water, fully stir to dissolve, dilute to 500mL with distilled water. 121°C, 1.05×10 5 Sterilize with Pa autoclave for 20min, for use.

[0047] Establish a cDNA library of cellulase gene, treat total RNA with DNase I, react at 37°C for 20-30 minutes, and then inactivate it in a PCR machine at 80°C for 10 minutes; configure the following system in the microtube according to Table 1.

[0048] Table 1 Reverse transcription reaction system (μL)

[0049]

[0050] (3) After being kept at 70°C for 10 minutes, quickly freeze on ice for more than 2 minutes;

[0051] (4) After centrifugation for a few seconds, configure the following solutions in the above microtube according to Table 2;

[0052] Table 2 Reverse transcription reaction system (μL)

[0053]

[0054] (5) Keep 42℃ for 1h;

[0055] (6) After incubating at 70°C for 15 minutes, cooling on ice, the obtained cDNA is used for PCR amplification.

[0056] Using the above cDNA as a template, K1, K2/H1, and H2 as primers, respectively, RNase free water and 2×EsTaq MasterMix were added, mixed with a vortex shaker, and PCR amplification was performed. The reaction system is 50 μL, and the composition is shown in Table 3.

[0057] Table 3 shows two cellulase gene PCR reaction systems (μL)

[0058]

[0059] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30s, 60-63°C (different depending on the cellulase gene) 30s, extension at 72°C for 80s, 35 cycles; extension at 72°C for 10 minutes. After the reaction, use 1% agarose gel electrophoresis for detection, such as figure 1 As shown, the cellulase bands of Trichoderma konsei and Aspergillus niger obtained by PCR amplification are clear, and the position and size are consistent with the corresponding cellulase genes. The PCR product band size is selected to meet the expectations for the next step.

[0060] Example 3 Connection and transformation of cellulase gene

[0061] To connect the cellulase gene to the pMD19-vector, configure the cellulase gene PCR product connection system according to Table 4, and connect overnight at 4°C.

[0062] Table 4 Reaction system of PCR product and pMD19-T vector ligation (μL)

[0063]

[0064] Place the well-grown E. coli plate in a refrigerator at 4°C and develop color for several hours. Such as figure 2 As shown, E. coli without recombinant plasmid has β-galactosidase activity, the center of the colony is light blue, and the periphery is dark blue; while the E. coli containing recombinant plasmid has no β-galactosidase activity, the colony It is milky white for preliminary screening.

[0065] Gene preparation, PCR amplification of the cellulase gene that has been sequenced, and the use of an agarose purification and recovery kit to recover the PCR product to obtain the cellulase gene.