Molecular Cloning and Expression Analysis of the Endogenous Cellulase Gene MaCel1 in Monochamus alternatus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Test Materials
2.2. RNA Isolation and cDNA Synthesis
2.3. Amplification of the Intermediate Fragment of MaCel1
2.4. Full-Length RACE of MaCel1
2.5. Prokaryotic Expression and Purification of MaCel1
2.6. Determination of Enzyme Activity of MaCel1
2.7. Bioinformatics Analysis
2.8. Quantitative Expression of MaCel1
2.9. Prediction of Endogenous Cellulase in M. alternatus
3. Results
3.1. Prediction of Endogenous Cellulase in M. alternatus
3.2. Cloning and Sequence Analysis of MaCel1
3.3. Similarity Analysis of MaCel1
3.4. Prokaryotic Expression of MaCel1 Gene and Determination of Enzyme Activity of MaCel1
3.5. Expression Analysis of MaCel1
4. Discussion
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
Appendix A
Family | Enzyme Activity or Function | Gene ID | EC# (Putative) |
---|---|---|---|
GH1 | Beta-glucosidase | c89763 | EC 3.2.1.21 |
GH3 | Beta-glucosidase | c31589 | EC 3.2.1.21 |
GH3 | Beta-glucosidase | c124724 | EC 3.2.1.21 |
GH3 | Beta-glucosidase | c114480 | EC 3.2.1.21 |
GH5 | Endo-β-1,4-glucanase | c39850 | EC 3.2.1.4 |
GH5 | Endo-β-1,4-glucanase | c77902 | EC 3.2.1.4 |
GH9 | Endo-β-1,4-glucanase | c81097 | EC 3.2.1.4 |
GH9 | Endo-β-1,4-glucanase | c94844 | EC 3.2.1.4 |
GH45 | Endo-β-1,4-glucanase | c71020 | EC 3.2.1.4 |
GH45 | Endo-β-1,4-glucanase | c79607 | EC 3.2.1.4 |
GH45 | Endo-β-1,4-glucanase | c86570 | EC 3.2.1.4 |
GH48 | Exo-β-1,4-glucanase | c34374 | EC 3.2.1.91 |
GH48 | Exo-β-1,4-glucanase | c83558 | EC 3.2.1.91 |
GH48 | Exo-β-1,4-glucanase | c95129 | EC 3.2.1.91 |
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Primer Name | Primer Sequence | Primer Use |
---|---|---|
A408F1 | GTACTAGACTATGTTTGGG | Sequence verification of transcriptome |
A408R1 | CTATAGATCTCCTTTTAACC | |
A408-1(GSP1) | GGTAGACACCCCGAAA | 5′ RACE |
A408-2(GSP2) | CCTTCTGGGAACTGGAGC | |
A408-3(GSP3) | ATCCTCAGCGATGACACTT | |
3′977-1 | GGCTACAGTGAAAGATTCGGATTGTACC | 3′ RACE |
3′977-2 | AGAAATCGGTGCAGTGGTACAAACA | |
P1944F | GATCCGAATTGATGTTTGGGTACGCTGGCCTGGC | Coding sequences (CDS) amplification of MaCel1 |
P1944R | CCGCTCGAGTAGATCTCCTTTTAACCTGCTTGAA | |
GAPDH F | AGAAAGTTATTATCTCCGCTCCA | RT-qPCR internal reference gene |
GADPH R | CCATACCAGTTAGTTTGCCATT | |
Cel-HX-1F | AGGTCAGGAAGGCAATGGATA | RT-qPCR target gene |
Cel-HX-1R | AAGGTAGTTCGCCGTGGTG |
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Li, Y.; Chen, H.; Chu, X.; Ma, Q.; Liang, G.; Wu, S.; Wang, R.; Tigabu, M.; Zhang, F.; Hu, X. Molecular Cloning and Expression Analysis of the Endogenous Cellulase Gene MaCel1 in Monochamus alternatus. Forests 2020, 11, 1372. https://doi.org/10.3390/f11121372
Li Y, Chen H, Chu X, Ma Q, Liang G, Wu S, Wang R, Tigabu M, Zhang F, Hu X. Molecular Cloning and Expression Analysis of the Endogenous Cellulase Gene MaCel1 in Monochamus alternatus. Forests. 2020; 11(12):1372. https://doi.org/10.3390/f11121372
Chicago/Turabian StyleLi, Yachao, Hao Chen, Xu Chu, Qiuyu Ma, Guanghong Liang, Songqing Wu, Rong Wang, Mulualem Tigabu, Feiping Zhang, and Xia Hu. 2020. "Molecular Cloning and Expression Analysis of the Endogenous Cellulase Gene MaCel1 in Monochamus alternatus" Forests 11, no. 12: 1372. https://doi.org/10.3390/f11121372
APA StyleLi, Y., Chen, H., Chu, X., Ma, Q., Liang, G., Wu, S., Wang, R., Tigabu, M., Zhang, F., & Hu, X. (2020). Molecular Cloning and Expression Analysis of the Endogenous Cellulase Gene MaCel1 in Monochamus alternatus. Forests, 11(12), 1372. https://doi.org/10.3390/f11121372