Identification and Functions of JHE 6 Specifically Expressed in Bombyx mori Silk Gland
and Chun Liu 1,2,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsJHE is a key catabolic enzyme of JH that is involved in the growth, development, and metamorphosis of insects The author identified the JHE genes in the silkworm and found that BmJHE6 was specifically expressed in the silk gland. The author regulated down the expression of BmJHE6 through RNAi and found that the development of the silk gland was changed and also the signal of JH pathway genes. It was interesting that Bmjh6 was secreted into the silk gland lumen and cocoon silk, maybe BmJHE6 have some new function. This work offered some information of silk gland development and silk protein synthesis by JH in silkworms. At the same time, it also provides a reference for the study of other insect JHE. Before publishing on insects, there are some questions need to answer and revision.
1. Fig. 2, Fig. 3
Because of the Bmjhe may have multiple family members, the specificity of the antibody is crucial. The author should provide the evidence of Bmjhe6 antibody specificity and the antibody preparation process.
2. Table S2
Bioinformatics analysis and molecular experimental validated that the specific expression of Bmjhe6 in silk glands. However, why the other members of the Bmjhe family appear in the data of cocoon silk proteomics?
3. Fig.5
Does the interference experiment affect the expression of other key enzymes in the JH synthesis of silkworm, such as jheh?
4. Can the solubility Bmjhe6 recombinant protein decompose JHI and JHIII？
5. Does the author consider further exploring its function by overexpressing Bmjhe6 in the posterior silk gland?
6. After downregulated the expression of Bmjhe6, the weight of larvae and silk glands increased. Does it mean the increase of silk protein? Has the author investigated the changes of cocoon layer rate?
7. As a catabolic enzyme of JH, it is difficult to understand why it is secreted into the cocoon silk. How did the author think about this issue?
Comments on the Quality of English Languagesame as above
Author Response
Dear reviewer：
Thank you for acknowledging our work and for providing us with valuable feedback that enabled us to enhance the quality of our manuscript. We sincerely appreciate your time and effort in reviewing our work. We have carefully reviewed your comments and made point-to-point revisions to address the issues you raised, we believe that these modifications have substantially strengthened the overall quality of our work, please see the responses below. All modifications have been marked in yellow. Please see the PDF file.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsAuthors study Bmjhe6 in Bombyx mori silk gland using numerous methods (computational, qRT-PCR, western bolt, etc), individuating among other things the Bmjhe6 specific expression in the middle silk gland (MSG) with high level in its anterior region. Authors have done consistent experimental work. The Discussion chapter is reasonable. The text and figure drafting instead must be improved.
Authors’ affiliation is missing for Keli Wu, Hongguo Yang, Tingcai Cheng.
Affiliation 2 is Jinfeng Laboratory. Chongqing 401329, China. Is it independent non-governmental Laboratory or it belongs to some University?
Methods, pg3, line 139. Primary antibody against Bmjhe6: is it monoclonal or polyclonal antibody? Where this antibody was purchased? The incubation for 2 hrs at 37C is not conventional incubation conditions, please check if 37C is right.
Methods, pg3, line 149. The antibody anti-Bmjhe6 mentioned here, is it the same as in line 139?
Figure 1. The characters in the Panel D is not readable. Please, increase the figure size. The characters in the Panels A, C, D are in the limit to be readable in A4 print version of the paper. Please, increase the character size.
Figure 2, panel E and Figure S2, panel B: the point symbols used in the graph are too similar and small, thus it’s difficult to distinguish between the lines. Change the symbols to larger or open/close symbols or use the colours for different lines.
Figure 2 legend: 5L1D – 5L7D abbreviations are not defined in the figure legend.
Figure 3, panel A. The characters are in the limit to be readable in A4 print version of the paper. Please, increase the character size.
Figure 3 legend. The figure title “Effects of JHA and 20E on Bmjhe6 expression of silk gland” describes only the panel C and F. Please, use adequate more generic Figure title.
Figure 4 IS COMPLETELY MISSING (probably, it’s erroneously substituted with Figure 3, which is doubled in the text)
Figure 5 legend. Confusion in the panel description. In the figure Ser2 and Ser3 are in panel A and B respectively, but in the legend they are both indicated in the panel A. Please, control and fix the whole figure legend.
Figure S1, panel A, red characters are too small.
Discussion, line 335. The affirmation “..as demonstrated by many genes’ RNAi experiments such is silk gene” needs the citation.
References: strange symbol [J] is present on all citations in Reference list.
Author Response
Dear reviewer：
Thank you for acknowledging our work and for providing us with valuable feedback that enabled us to enhance the quality of our manuscript. We sincerely appreciate your time and effort in reviewing our work. We have carefully reviewed your comments and made point-to-point revisions to address the issues you raised, we believe that these modifications have substantially strengthened the overall quality of our work, please see the responses below. All modifications have been marked in yellow. Please see the PDF file.
Author Response File: Author Response.pdf
