Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of Sphenophorus levis
Abstract
:1. Introduction
2. Results
2.1. Sphenophorus levis Midgut Transcriptome and Identification of Putative Pseudo-Cysteine Proteases
2.2. Identification of Putative Proteases
2.3. Relative Expression of Sl-CathL-CS in Different Parts of the Larvae
2.4. Heterologous Expression of Sl-CathL-CS and Sl-CathL-mutSC
2.5. Enzymatic Characterization of Sl-CathL-CS and Sl-CathL-mutSC
2.5.1. Sl-CathL-CS and Sl-CathL-mutSC Protease Activity
2.5.2. Competition Assay
2.6. Pull-Down
2.7. Static Protein Ligand Docking
3. Discussion
4. Materials and Methods
4.1. RNA Extraction
4.2. Library Preparation and Illumina Sequencing
4.3. Quality Control, Preprocessing and de Novo Assembly
4.4. Identification of Putative Pseudo-Cysteine Proteases
4.5. Relative Gene Expression of Sl-CathL-CS in Different Developmental Stages and Parts of the Larvae
4.6. Heterologous Expression of Sl-CathL-CS and Sl-CathLCS-mutSC
4.7. Enzymatic Characterization of Sl-CathL-CS and Sl-CathL-mutSC
4.7.1. Sl-CathL-CS and Sl-CathL-mutSC Protease Activity
4.7.2. Competition Assay
4.8. Pull-Down
4.9. Static Protein-Ligand Docking
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Number of raw reads | 70,720,844 |
Number of readings after filtering | 146,145 |
Average length of contigs (bp) | 1131.23 |
N50 length (bp) | 2382 |
GC Percent | 37.20 |
Primer | Sequence (5′–3′) | Purpose | Amplicon (bp) |
---|---|---|---|
Sl-CathL-CS_fw: | CCGAATTCAGTTCGGAGCTGAACATGG | ORF cloning | 984 |
Sl-CathL-CS_rv | ATTCTTATGCGGCCGCATCGATTTCGACGTAGGCAGC | ||
Sl-CathL-mutSCforw | ACCAAGGAGAATGGGATACATGTTGGGCTTTCTCCACTATTGC | Site-directed mutagenesis | |
Sl-CathL-mutSC_rev | GCAATAGTGGAGAAAGCCCAACATGTATCCCATTCTCCTTGGT | ||
Sl_GADPH_qF | CAACTGGCGTTTTTACCACA | Real Time PCR | 104 |
Sl_GADPH_qR | AACATACATTGGGGCGTCA | ||
Sl_CathL-CS_qF: | ATACGACTGGAGGGAGCAGA | Real Time PCR | 108 |
Sl_CathL-CS_qR | ATGGCGTAGGCACTTTCAAC |
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Shibao, P.Y.T.; Ferro, M.; de Paula, F.F.P.; Lima, B.S.; Henrique-Silva, F. Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of Sphenophorus levis. Int. J. Mol. Sci. 2021, 22, 11476. https://doi.org/10.3390/ijms222111476
Shibao PYT, Ferro M, de Paula FFP, Lima BS, Henrique-Silva F. Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of Sphenophorus levis. International Journal of Molecular Sciences. 2021; 22(21):11476. https://doi.org/10.3390/ijms222111476
Chicago/Turabian StyleShibao, Priscila Yumi Tanaka, Milene Ferro, Fernando Fonseca Pereira de Paula, Bruno Salata Lima, and Flávio Henrique-Silva. 2021. "Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of Sphenophorus levis" International Journal of Molecular Sciences 22, no. 21: 11476. https://doi.org/10.3390/ijms222111476