Cloning and characterization of the LvCTL genes encoding C-type lectin from white-leg shrimp ( Litopenaeus vannamei)

Background Lectins are carbohydrate-binding protein domains. The C-type lectin designates a requirement for calcium for binding. Proteins contain C-type lectin domains that have a diverse range of functions, including cell-cell adhesion, immune response to pathogens, and apoptosis. This study aimed to investigate the characters of LvCTL-encoding genes from white-leg shrimp ( Litopenaeus vannamei) in Central Vietnam. Methods Two PCR products (LvCTL3 and LvCTL4) were cloned and sequenced. The structure and characterization of LvCTL proteins were predicted using bioinformatics tools. Results The results showed that the LvCTL3 gene was 444 nucleotides in length and 98.87% similar to the published LvCTL3 gene (accession number: KF156943). The polypeptide sequence had 147 amino acids, which were 97.28% identical to the reference sequence (AGV68681) and the LvCTL4 gene had a length of 417 nucleotides and homology of 99.52% compared to the published gene (KM387560). The deduced polypeptide sequence had 138 amino acids, and was 100% similar to the reference sequence (AKA64754). The LvCTL3 had a molecular weight of 16.91 kDa and an isoelectric point (pI) of 4.66, while LvCTL4 had 15.75 and 4.58 kDa, respectively. The structure prediction results showed that LvCTL3 and LvCTL4 had one domain (CTLD), LvCTL3 had two α helices and nine β sheets, and LvCTL4 had two α helices and eight β sheets. Conclusions Our results provide essential information for the heterologous expression and biosynthesis production of C-type lectins.

number: KF156943).The polypeptide sequence had 147 amino acids, which were 97.28% identical to the reference sequence (AGV68681) and the LvCTL4 gene had a length of 417 nucleotides and homology of 99.52% compared to the published gene (KM387560).The deduced polypeptide sequence had 138 amino acids, and was 100% similar to the reference sequence (AKA64754).The LvCTL3 had a molecular weight of 16.91 kDa and an isoelectric point (pI) of 4.66, while LvCTL4 had 15.75 and 4.58 kDa, respectively.The structure prediction results showed that LvCTL3 and LvCTL4 had one domain (CTLD), LvCTL3 had two α helices and nine β sheets, and LvCTL4 had two α helices and eight β sheets.
Introduction C-type lectins (CTLs) are proteins/glycoproteins capable of reversibly binding to carbohydrates by non-covalent bonds without altering their structure (Zhang et al., 2009).Lectins can bind to cells with glycoproteins or microbial surface glycolipids and are considered pattern recognition receptors (PRR) recognizing and releasing invading microorganisms in the system.The shrimp lectin protein family is very rich and diverse; members of the eight PRR families have been identified in several shrimp species, based on structure and specificity to different sugars of the carbohydrate recognition domain (CRD).These families are lipopolysaccharide and b-1,3-glucan binding proteins (LGBPs), CTLs, galectin, thioester-containing proteins (TEP), fibrinogen-related proteins (FREP), scavenger receptors (SR), down syndrome cell adhesion molecules (DSCAM) and Toll-like receptors (TLR) (Wang and Wang, 2013).According to form and function, lectins are classified as Calnexin, C-, L-, P-, I-, R-, and S-type lens (Janeway and Medzhitov, 2002;Zhang et al., 2009).Among the lectins, CTL is the most diverse and well-studied (Zhang et al., 2009).
CTLs are a group of proteins that play an essential role in many animal biological processes, including cell signaling and pathogen recognition.CTLs are a superfamily of more than 1,000 proteins identified by having one or more wellcharacterized C-type lectin-like domains (CTLDs).They are divided into 17 subgroups based on their species and domain organization (Weis et al., 1998;Zelensky and Gready, 2005).CTL also plays an essential role in mammalian innate immunity.The mannose-binding lectin (MBL) is primarily expressed in the liver and released into the plasma, where it recognizes potentially pathogenic bacteria and binds to microbial surfaces.Animals can also activate the complement pathway (Thiel and Gadjeva, 2009).Similarly, CTLs are also important immune recognition receptors in invertebrates.CTLs in shrimp can currently be divided into three subgroups based on domain composition and organization: those containing only one C-type lectin domain (CTLD), those having two CTLDs, and those containing one CTLD and another part (Wang and Wang, 2013).Among them, the group containing only one CTLD was more common than the few CTLs in shrimp containing two CTLDs.The presence of two or more binding sites for each lectin molecule allows it to bind to many different cell types.The prevalence of CTLD in invertebrates is much higher than in vertebrates.Typically, CTLD is the seventh most common domain in the Caenorhabditis elegans genome, but only the 43 rd most common domain in the human genome (Zelensky and Gready, 2005).Shrimp CTLD can bind multiple ligands and constructs inducible expression of CTLs.Recent studies have revealed multiple functions and mechanisms of shrimp CTLs in antimicrobial and antiviral immunity.These protective functions of CTLs in the shrimp species Fenneropenaeus chinensis are based on their ability to recognize and bind CTLDs (Sun et al., 2008).In the framework of this study, we present the results of cloning and characterization of the LvCTL genes isolated from white-leg shrimp (Litopenaeus vannamei) in Thua Thien Hue as a prerequisite for the production of recombinant LvCTL proteins for further research.

Ethical approval
Animal use in this study was allowed by Hue University of Animal Ethics Committee with certificate reference number: HUVN0018, April 10 th , 2022.Principal Researcher: Nguyen Quang Linh.All efforts were undertaken to minimize the suffering of animals.

Animals
White-leg shrimp (L.vannamei) with a weight of ~20 g per head, which were collected from shrimp ponds; the samples were aseptically dissected to collect the hepatopancreas shrimp samples and stored in liquid nitrogen, and used directly for RNA extraction.

Isolation and cloning of LvCTL gene
Total RNA from hepatopancreas samples was extracted using the Gene JET RNA Purification Kit (Thermo Scientific, USA) according to the manufacturer's instructions.Electrophoresis was conducted to check the total RNA obtained on 1% agarose gel with TAE 1X at 70V for 30 min by using Power Pac 3000 (Bio-Rad).The cDNA biosynthesis was performed using Revert Aid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, USA), and the obtained product was used directly for PCR amplification.

REVISED Amendments from Version 2
We would like to change in first author and add more one institution, where PhD thesis has been done by the author.Other minor changes were implemented throughout.
The sequences of LvCTL encoding genes were obtained from GenBank to design a specific primer (Table 1).PCR was performed using the following conditions: 95°C for 2 min, and then 40 cycles of 95°C for 1 min, 55°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 7 min.PCR products were confirmed by electrophoresis at 70V for 35 min on 1% agarose gel with TAE 1X stained SafeViewTM Classic (abm, Canada).PCR products were recovered from agarose gels and purified using the GeneJET Gel Extraction kit (Thermo Scientific, USA).Purified PCR products were cloned into pGEM T-easy vector with T4 DNA Ligase for 1 hour at room temperature (Promega, USA), then, the recombinant vectors were transferred into Escherichia coli TOP10 bacteria using the heat shock method.The recombinant vectors were selected on LB medium (Luria Bertani Agar, Merck, Germany) supplemented with 50 μg/mL Amp, X-Gal/IPTG.E. coli cells carrying the recombinant vector (white colonies) were then inoculated with 5 mL of liquid LB medium supplemented with 50 μg/mL Amp, and DNA plasmids were isolated using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, USA).The isolated DNA plasmid was examined by 1.0% agarose gel electrophoresis at 70V for 35 min and used for LvCTL sequencing (Ha et al., 2018).The sequence of the LvCTL genes was analyzed using the Sanger method (First base Company, Malaysia).The results were checked using BioEdit software and then compared with those published in GenBank using the BLAST tool.A phylogenetic tree of CTL encoding genes was built using MEGA 11 software with the Neighbor-Joining algorithm (Tamura et al., 2021).
Characterization and molecular structure of the CTL encoding genes Characterization of LvCTL proteins was determined using bioinformatics tools, including amino acid sequence translation with Expasy, protein domain using SMART (Letunic et al., 2021), spatial structure model using Phyre2 (Kelley et al., 2015), isoelectric point using IPC2 (Li et al., 2014).

Results
Isolating and cloning of the LvCTL genes After PCR with specific primers, the results showed that two DNA fragments were amplified and expressed approximately at 450 and 420 bp in size, respectively.According to the theoretical length of the cDNA fragment of the LvCTL3 and LvCTL4 genes.PCR reaction using specific primers with pGEM vector as template was performed, electrophoresis images showed very specific PCR products (Figure 1), which proved that LvCTL genes was successfully attached to pGEM vector T-easy (referred to as the recombinant vector pGEM/LvCTL3 and pGEM/LvCTL4).Therefore, the recombinant vectors were used to analyze the nucleotide sequences.The previous reports indicate several CTLs in some shrimp species, such as: Litopenaeus vannamei, L. schmitti, L. setiferus, Fenneropenaeus chinensis, F. merguiensis, Penaeus monodon, Marsupenaeus japonicus and Macrobrachium rosenbergii (Wang and Wang, 2013).We constructed a phylogenetic tree from the LvCTL3 and LvCTL4 deduced amino acid sequences that isolated from genes compared      with the published reference sequences of other shrimp species.The results show that LvCTL3 and LvCTL4 deduced amino acid sequence belong to two groups in the dendrogram and our arrangements were similar to the published of white-leg shrimp (L.vannamei) but different from other shrimp or crustacean species (Figure 6).

Characteristic determination and molecular structure of the gene encoding CTLs
In our study, the nucleotide sequence of LvCTL3 PCR product was 444 bp in length, encoding a polypeptide sequence of 147 amino acids.The nucleotide sequence of LvCTL3 PCR product was 417 bp in length, encoding a polypeptide sequence of 138 amino acids.These were fragment of genes containing the open reading frame (ORF) region cut off the peptide signal, intended to be used to produce recombinant LvCTL protein for other studies related to recombinant protein production.For the conserved domain determined using the SMART program, the ORF sequence of LvCTL3 had one domain (CTLD) from amino acid position 14 to 146.LvCTL3 had a molecular weight of 16.91 kDa, and the isoelectric point (pI) was 4.66.The results of spatial structure prediction showed that the LvCTL3 molecule had two alpha helices (16%) and nine beta strands (28%) (Figure 7).Meanwhile, the ORF sequence of LvCTL4 had one domain (amino acid 1 to 137), a molecular weight of 15.75 kDa and a pI was 4.58.LvCTL4 structure had two alpha helices (17%) and eight beta strands (32%) (Figure 8).

Discussion
The results of sequence analysis showed a change in the isolated LvCTL genes compared with the published genes.The isolated LvCTL3 fragment was 444 bp in size (including the stop codon), 98.87% homologous to the published gene (439/444 nucleotides compared with KF156943) (Figure 2).The change of the LvCTL3 gene resulted in a difference in the sequence of the deduced amino acid sequence, the highest similarity obtained was 97.28% (143/147 amino acids) compared with the reference sequence AGV68681.Meanwhile, the LvCTL4 gene fragment was 417 bp, 99.52% homologous to the gene of KM387560 (415/417 nucleotides) (Figure 3).However, the change of the LvCTL4 gene resulted in no change in the sequence of the deduced amino acid sequence (100% similarity) compared with the reference sequence (AKA64754).
According to Li et al. (2014), the LvCTL3 gene from shrimp (L.vannamei) has a length of 579 bp, in which the coding segment is 492 bp (from nucleotide positions 25 to 516), encoding a polypeptide chain with a length of 163 amino acids, and has an estimated molecular weight of 17.9 kDa, an estimated pI of 4.80 (Li et al., 2014).The LvCTL4 gene, also from L. vannamei shrimp, has a length of 563 bp, in which the coding segment is 471 bp (from nucleotide positions 27 to 497).
The ORF encoded a protein of 156 amino acids consisting of a single CTLD (residues 19-155) and a putative signal peptide (residues 1-18).Tissue expression analysis showed LvCTL4 was distributed with high levels in the gills, intestine, epithelium and hepatopancreas (Li et al., 2015).Meanwhile, LvTRAF3 was also cloned and characterized from the shrimp L.vannamei; it had a transcript of 3,865 bp, with an ORF of 1,002 bp, and encoded a polypeptide of 333 amino acids, with a calculated molecular weight of 38.6 kDa (Li et al., 2020).TRAF3 functions as a regulator of innate immune response that involves many cellular processes.
When comparing to the whiteleg shrimp (L.vannamei) deduced amino acid sequence from LvCTL3 genes, the resulted in 97.28% similarity to the reference sequence of AGV68681 or 96.60% to XP_027212325, while LvCTL4 deduced amino acid sequence was 100% similar to the Genbank reference sequence of AKA64754 and XP_027228060.The LvCTL3 and LvCTL4 deduced amino acid sequence were 60.69% (XP_042883674) and 80.43% (QEX50549) homologous to kuruma prawn (Penaeus japonicus), respectively.Compared with black tiger shrimp (Penaeus monodon), the LvCTL3 deduced amino acid sequence was 59.18% similar to the reference sequence by XP_037787306 while LvCTL4 was 88.21% similar to that of XP_037800826.Compared with other crustacean species, the difference in LvCTL3 and LvCTL4 sequences was higher.The similarity of the LvCTL3 deduced amino acid sequence was only 45.58% compare to the AYD41573 reference sequence from red swamp crayfish (Procambarus clarkii) or 40.25 % to the XP_042233235 reference sequence from the American lobster (Homarus americanus).Meanwhile, LvCTL4 amino acid sequence was only 59.12% similar to the XP_045611199 reference sequence from red swamp crayfish, 60.14% to XP_042237721 from the American lobster, or 57.258% to the XP_045118055 sequence from gazami crab (Portunus trituberculatus).
The amino acid sequence alignments of two proteins (Figure 4 & Figure 5) that may cause protein secondary structure shifts (Li et al., 2015).The form of a protein is related to its function.Knowledge of protein's 3D structure is a huge hint for understanding how protein's work.Although other refinement tools such as homology-modeling tools based on high sequence similiritues provide higher quality structure.However, our 3D structures have been modelled with 99.9% and 99.8% confidence by the single highest scoring template with the 3D structure of LvCTL3 and LvCTL4, respectively.This showed that the function of these proteins is not changed.Protein folding is determined by the physicochemical properties that are encoded in the amino acids.Although most of the predictions were highly accurate, the system that we used is not perfect.Programs such as Alpha Fold will exponentially increase our general understanding of different biological processes.So the 3D structure of these proteins should be further study by another program to provide higherquality structures.And the simulation of these proteins at pH of shrimp stomach (5.7) should be considered for further experiments.

Conclusions
In this study, two LvCTL3 and LvCTL4 genes from white-leg shrimp were successfully isolated, cloned, and sequenced.The ORF of LvCTL deduced amino acid sequences from gene had homology levels of 98.87% (LvCTL3, KF156943) and 99.52% (LvCTL4, KM387560 respectively) compared to the published reference sequence.The predicted LvCTL proteins have one conserved domain and are defined as C-type lectins.They can be used for heterologous expression and scale-up production of recombinant C-type lectin in the future to add to the aquatic feed to enhance immunity and prevent disease in shrimp.

Parin Chaivisuthangkura
Srinakharinwirot University, Bangkok, Bangkok, Thailand This manuscript described the isolation of CTL genes from L. vannamei with the intention to express the recombinant protein in the future.The isolated PCR products, LvCTL3 and LvCTL4 were sequenced, analyzed and the structure was predicted.
The designed primers must indicate the nucleotide positions of PCR products to provide the intention of amplification.In Table 1, there were no explanation for each primer pair.Why did you amplify LvCTLD, LvAV,LvdlrCTL and what were those regions?In addition, the successful of PCR amplifications was only occurs with CTL3 and CTL4.What about the other 3 products in Figure 1?The discussion must be added. 1.
How many clones of recombinant plasmids were sequenced since there were some discrepancies of nucleotide sequence compared with the GenBank reports?Only one clone is not enough to obtain the correct sequence.

2.
Did you use the proofreading enzyme to do the PCR amplification? 3.
The first paragraph of results must be rewritten since there were plenty of errors that cause confusion to the audience.

4.
The gel picture of Figure 1 must be redone.The marker was not clear and the size of PCR products did not seem to be different.How many percent gel did you use?

5.
The first paragraph of discussion must be moved to result section.The explanation of Figure 4 and 5 must be added in the results.

6.
Why did you discuss about LvTRAF3?This should be deleted.7.
The authors should consider adding a Table for the percent similarity of amino acid sequences among CTLs from various shrimp species.

Minor points Introduction, L6: use "beta instead of b"
L9: Did you mean lectins instead of lens Methods Animals: Did you mean 20 g per shrimp?
Rewrite the whole section since there are plenty of grammatical errors.Figure 6: Please choose between Litopenaeus vannamei and Penaeus vannamei.Only one term must be used.The legend should be "The phylogenetic tree of LvCTL3……..amino acid sequences.Figure 7 legend should be "The predicted LvCTL3 structure" Figure 8 legend should be "The predicted LvCTL4 structure" Discussion: What did you mean by "the system that we used is not perfect"?The underlying data: Do you have any Western blot image in this project?Did you mean DNA gel pictures?

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: innate immunity in shrimp.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Reviewer Report 15 July 2024 https://doi.org/10.5256/f1000research.156535.r295548 © 2024 Lin T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ta-Hui Lin
Department of Basic Medical Science, Xiamen Medical College, Xiamen, Fujian, China I confirm that I have read this submitted material and believe that I have the appropriate professional knowledge to verify that it meets acceptable scientific standards.But as mentioned above, I have significant reservations.Some vocabulary and grammar errors in the article need to be corrected.The description of the results in the abstract should be written in this way to be correct."The molecular weight of LvCTL3 is 16.91 kDa and its isoelectric point (pI) is 4.66, while the molecular weight and pI of LvCTL4 are 15.75 kDa and 4.58, respectively."At the same time, new articles can be used as a reference for structural and functional analysis.The authors have satisfactorily responded to all my questions and made the necessary changes to the manuscript.Therefore, the manuscript can be accepted for indexing in its current form.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.Does the deletion in the DNA sequence of LvCTL3 and LvCTL4 in your study might affect its functions?

4.
According to your discussion, what is the purpose of building a 3D model of these two proteins?

5.
Authors should include amino acid sequence alignments of two proteins to highlight the differences.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Not applicable Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Biotechnology, molecular modelling, immunology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
proteins work.Although other refinement tools, such as homology-modelling tools based on high sequence similarities, provide higher-quality structure.However, our 3D structures have been modelled with 99.9% and 99.8% confidence by the single highest-scoring template with the 3D structure of LvCTL3 and LvCTL4, respectively.This showed that the function of these proteins is not changed.Protein folding is determined by the physicochemical properties that are encoded in the amino acids.Although most of the predictions were highly accurate, our system is imperfect.Programs such as Alpha Fold will exponentially increase our general understanding of different biological processes.So the 3D structure of these proteins should be further studied by another program to provide higher-quality structures.And the simulation of these proteins at the pH of the shrimp stomach (5.7) should be considered for further experiments.The authors didn't discuss any information about protein structural characteristics.According to a previous study, shrimp stomach pH is approximately 5.7, so maybe authors should consider simulation the protein at that pH for further experiments 4 Does the deletion in the DNA sequence of LvCTL3 and LvCTL4 in your study might affect its functions?5 According to your discussion, what is the purpose of building a 3D model of these two proteins?6 Authors should include amino acid sequence alignments of two proteins to highlight the differences.

Another revision
Added 3rd affiliation of the first author: Tran Vinh Phuong 1, 3 1.
Updated another affiliation (Ex: Faculty of Fisheries, University of Agriculture and Forestry, Hue University, Thua Thien Hue, 49000, Vietnam) 2.

Underlying data 3.
Updated 2 new links, Genbank, to replace old ones.https://www.ncbi.nlm.nih.gov/nuccore/OP584994.1/and https://www.ncbi.nlm.nih.gov/nuccore/OP584995.1/The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com

Figure 2 .
Figure 2. The similarity of the nucleotide sequences of the isolated LvCTL3 gene and the published LvCTL3 gene (KF156943).

Figure 3 .
Figure 3.The similarity of the nucleotide sequences of the isolated LvCTL4 gene and the published LvCTL4 gene (KM387560).

Figure 4 .
Figure 4.The similarity of the deduced amino acid of the isolated from LvCTL3 gene and the published LvCTL3 sequence (AGV68681).

Figure 5 .Figure 6 .
Figure 5.The similarity of the deduced amino acid of the isolated from LvCTL4 gene and the published LvCTL4 sequence (AKA64754).

References 1 .
Xue Q, Yang B, Luo K, Luan S, et al.: Molecular Characterization and Expression Analysis of the C-Type Lectin Domain Family 4 Member F in Litopenaeus vannamei against White Spot Syndrome Virus.Animals (Basel).2024; 14 (8).PubMed Abstract | Publisher Full Text Is the work clearly and accurately presented and does it cite the current literature?Yes Is the study design appropriate and is the work technically sound?Yes Are sufficient details of methods and analysis provided to allow replication by others?Yes If applicable, is the statistical analysis and its interpretation appropriate?Not applicable Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Yes Competing Interests: No competing interests were disclosed.Reviewer Expertise: Biochemistry, Molecular Biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.Reviewer Report 11 July 2024 https://doi.org/10.5256/f1000research.156535.r295546© 2024 Wang M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Mengqiang WangOcean University of China, Qingdao, China F1KR00CDE F1R-VER156535-R C-type lectins are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity.The manuscript, Cloning and characterization of the LvCTL genes encoding C-type lectin from white-leg shrimp (Litopenaeus vannamei), cloned and characterized two C-type lectin genes (LvCTL3 and LvCTL4) from white-leg shrimp, which provided essential information for the heterologous expression and biosynthesis production of C-type lectins.The provided detailed procedure of their experiments in Methods section, the laboratory experiments were well designed and executed.The overall experimental design is sound and solid, the results are convincing in general.The current study provides heterologous expression and scale-up production of recombinant C-type lectin in the future to add to the aquatic feed to enhance immunity and prevent disease in shrimp.The manuscript qualifies for indexing.Is the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Yes Competing Interests: No competing interests were disclosed.Reviewer Expertise: Innate immunity in aquaculture.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.assistance, so version 3 would be nice for publication in the last version.Thank you so much; see you soon in the next manuscript./doi.org/10.5256/f1000research.147015.r205043© 2023 Tran-Van H.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Hieu Tran-Van Laboratory of Biosensors, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh, Vietnam

Fig. 4 .
The amino acid similarity of the isolated LvCTL3 gene and the published LvCTL3 gene (AGV68681) and Fig. 5.The similarity of the amino acid of the isolated LvCTL4 gene and the published LvCTL4 gene (AKA64754) and discussed in the text (purple colour) 2 Should model reference proteins of Li et al. publication to give readers a better visualization of amino acid differences that may cause protein secondary structure shifts. 3

Table 2 .
Characteristic of the gene encoding LvCTLs.