Cloning and Functional Analysis of the Soybean GmRIQ2 Promoter

Round 1

Reviewer 1 Report

The revised and resubmitted article entitled "Cloning and Functional Analysis of the Soybean GmRIQ2 Promoter" has improved significantly in the revised version. But still, I could see many changes needed and go through a major revision before it is acceptable for publication. The authors need to modify the manuscript after considering the following comments carefully. 

Line 38: ........applications in plant genetic engineering due to their and highly efficient expression throughout.... 
Change to ........applications in plant genetic engineering due to their highly efficient expression throughout ...
Line 90: Table 1 doesn't have PGmRIQ2-F and PGmRIQ2-R primers, please check.
Line 96: Is it XbarI or XbaI restriction enzyme used? In Figure 1, it is shown as Xba1. Please clarify. If it's Xba1, change it throughout in manuscript.
Line 137: ........primer pairs were PCR-amplified as shown in Table 1 to detect positive roots. Need to specify which primer pairs from Table 1 are used for detecting positive roots?
Line 154: After soaking, the unopened flower buds were soaked.............. Please check the sentence
The DNA ladder marking in the gel pictures is not proper and needs to modify with appropriate arrows for the marker size. Figure 7B gel pictures are not good quality and publishable, need to change.
Figure 7 legend need to edit according to the gel pictures provided. When multiple gel pictures are provided, please give numbers to all and accordingly explain in the figure legend. It is not clear why the authors performed gradient PCR from 52 to 58? Need explanation. Also, M: DNA Marker (DL2000) repeated.  

The discussion needs to improve further. 
 

Author Response

The revised and resubmitted article entitled "Cloning and Functional Analysis of the Soybean GmRIQ2 Promoter" has improved significantly in the revised version. But still, I could see many changes needed and go through a major revision before it is acceptable for publication. The authors need to modify the manuscript after considering the following comments carefully. 

Line 38: ........applications in plant genetic engineering due to their and highly efficient expression throughout.... 
Change to ........applications in plant genetic engineering due to their highly efficient expression throughout ...

Thanks. We have done.

Line 90: Table 1 doesn't have PGmRIQ2-F and PGmRIQ2-R primers, please check.

Thanks. We have done. Should be PGmRIQ2-1661-F and PGmRIQ2-1661-R

Line 96: Is it XbarI or XbaI restriction enzyme used? In Figure 1, it is shown as Xba1. Please clarify. If it's Xba1, change it throughout in manuscript.

It is Xbar I in Figure 1. We have checked.

Line 137: ........primer pairs were PCR-amplified as shown in Table 1 to detect positive roots. Need to specify which primer pairs from Table 1 are used for detecting positive roots?

PGmRIQ2-1661-F,PGmRIQ2-1661-R

Line 154: After soaking, the unopened flower buds were soaked.............. Please check the sentence

Thanks. We have done.

Line 327-333：The DNA ladder marking in the gel pictures is not proper and needs to modify with appropriate arrows for the marker size. Figure 7B gel pictures are not good quality and publishable, need to change. Figure 7 legend need to edit according to the gel pictures provided. When multiple gel pictures are provided, please give numbers to all and accordingly explain in the figure legend. It is not clear why the authors performed gradient PCR from 52 to 58? Need explanation. Also, M: DNA Marker (DL2000) repeated.

We have added the DNA ladder marking and arrows. The legend of Fig. 7 and the number both have been explained. The PCR were performed at 56°C. 

Line 404：The discussion needs to improve further. 

We have revised discussion part, mainly added the content of “GmRIQ2 gene promoter expression of vascular tissue specificity”

Reviewer 2 Report

Comments to the Author

Overall, the research was done properly and the paper is well written. Below are a few suggestions that the authors should consider as they revise the manuscript.

Introduction

Line 64 – 71. There is no clear objective(s). First the author mentioned investigation, how they determined functions of promoter and etc. At the end of the paragraph authors even discussed results.

Material and Methods

Line 74. Add more information/details regarding the soybean cultivars, such as MG growth habit.

Line 79. Describe the soil in details.

Line 81. Add more information about the Murashige-Skoog media.

Line 82. Seedlings were grown in each type of soil. More details.

Line 94. Please, be more specific about the software and add more information, such as Version and manufacture.

Table 1. Align primers to the left, instead of center.

Line 114 to 155. Most add more information on how bioinformatic analysis was executed.

Line 129. Describe how soybean seeds were sterilized.

Line 137. CTBA reference paper.

Line 152 – 153. Add more information about mixed nutrient soil and describe conditions in the incubator.

Line 200. Again, add information about mixed nutrient soil and describe conditions in the greenhouse.

Results

Line 224. BDGP online tool, add reference.

Table 4. Justify Function to the left instead of center.

Table 5. Justify Agrobacterium to the left instead of center.

Author Response

Overall, the research was done properly and the paper is well written. Below are a few suggestions that the authors should consider as they revise the manuscript.

Introduction

Line 64 – 71. There is no clear objective(s). First the author mentioned investigation, how they determined functions of promoter and etc. At the end of the paragraph authors even discussed results.

We have rewritten this paragraph.

Material and Methods

Line 74. Add more information/details regarding the soybean cultivars, such as MG growth habit.

We have done. KF16 is a main cultivar in Heilongjiang province. The transformation efficiency of DN50 was higher than other cultivars.

Line 79. Describe the soil in details.

Line 79-82 The soybean plants were grown in black soil:vermiculite (1:1). They both were purchased locally.

Line 81. Add more information about the Murashige-Skoog media.

Murashige-Skoog Basal Salt Mixture medium supplemented with 4.43 g/L MS (Phyto Technology Laboratory, USA), 30 g/L sucrose and 8 g/L agar, adjusted to pH 5.8.

Line 82. Seedlings were grown in each type of soil. More details.

Seedlings of Arabidopsis thaliana were grown in black soil:vermiculite (1:1).

Line 94. Please, be more specific about the software and add more information, such as Version and manufacture.

Thanks. We have done.

Table 1. Align primers to the left, instead of center.

Thanks. We have done.

Line 114 to 155. Most add more information on how bioinformatic analysis was executed.

We have done.

Line 129. Describe how soybean seeds were sterilized.

The completely and healthy soybean seeds (DN50) were selected and disinfected with 10% sodium hypochlorite (NaClO) solution for 15 h, and then rinsed with water 3 times.

Line 137. CTBA reference paper.

Thanks. We have done

Line 152 – 153. Add more information about mixed nutrient soil and describe conditions in the incubator.

We check the content and rewrite.

Line 200. Again, add information about mixed nutrient soil and describe conditions in the greenhouse.

We have rewritten.

Results

Line 224. BDGP online tool, add reference.

Thanks. We have done.

Table 4. Justify Function to the left instead of center.

Thanks. We have done.

Table 5. Justify Agrobacterium to the left instead of center.

Thanks. We have done.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have significantly improved the manuscript by adding relevant pieces of information and missing data points. The manuscript would have improved further if the authors have given a little more effort and concentrated on minor points.

For example, figure 6 and 7C have a-k and a-l, respectively but in the manuscript, it has mentioned just 6C and 7C only. It is worth incorporating the relevant sub numbering. for example lines, 320 to 329 GUS expression conveys critical pieces of information of the promoter truncation without mentioning the relevant sub numbering (like 6C-a; 6C-e, and so on). Like that, I could see multiple places the readers need to check the relevant result figures. I would suggest incorporating this for better clarity. 

Gel pictures of Figures 5 and 6 the ladder size mark with arrows.

When reviewers pointed out general comments as mentioned above, it is not particular for one figure. It is the author's responsibility to make it uniform and attend the full manuscript. 

 

Author Response

The authors have significantly improved the manuscript by adding relevant pieces of information and missing data points. The manuscript would have improved further if the authors have given a little more effort and concentrated on minor points.

For example, figure 6 and 7C have a-k and a-l, respectively but in the manuscript, it has mentioned just 6C and 7C only. It is worth incorporating the relevant sub numbering. For example lines, 320 to 329 GUS expression conveys critical pieces of information of the promoter truncation without mentioning the relevant sub numbering (like 6C-a; 6C-e, and so on). Like that, I could see multiple places the readers need to check the relevant result figures. I would suggest incorporating this for better clarity. 

Thanks for this question. We thought the sub numbering in figure 6 and 7 is necessary, which can explain the differences of different classification. So we added the sub numbering in manuscript.

Gel pictures of Figures 5 and 6 the ladder size mark with arrows.

OK, we have done.

When reviewers pointed out general comments as mentioned above, it is not particular for one figure. It is the author's responsibility to make it uniform and attend the full manuscript. 

Yes. We know. Thanks for reviewer’s question and suggestion.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The authors present research in which they analyze the soybean GmRIQ2 promoter.  The cloned promoter region was fused to GUS and analyzed for GUS expression and activity.  Promoter deletion mutants were also investigated.  Functionality of promoter was characterized.  Discrepancies between the cloned promoter and previous in plant analysis was observed for drought and high-salt.  These results should be of interest to the plant community.

           

Suggested changes:

            Review for English grammar

            All table and figure legends need to be expanded to provide a detailed description of the information being presented.

            The authors need to be careful how they use the term PCR.  They should specify each time if they are evaluating genomic DNA or cDNA.

            Figures 8, 9 and 10: define CK

Line 58: specify the year RIQ was discovered

Lines 62-63: remove “upstream” from “upstream promoter”

Line 84: provide a citation for the bioinformatic analysis, for BDGP

Lines 95-96: remove “(represented by underlined letters)”, add to Table 1 legend

Figure 1. is missing GUS in blue box

Line 124: how was the correct sequence determined?

Figure 2: promoter regions are not proportional to bp length, this would help significantly

            Line 127: specify what is in the parenthesis

Line 211: was DNase used during cDNA synthesis? What cDNA kit was used, provide company name.

Line 244: add bp numbers on right side, underline primer sequences, describe colors used in the figure.

Lines 335-336: why was time 0 not included?

Line 340: specify the optimal response times and refer to figure 8 analysis.

Lines 403-406: expand discussion

Lines 421-425: expand discussion

 

Reviewer 2 Report

The article entitled "Cloning and Functional Analysis of the Soybean GmRIQ2 Promoter" is interesting. The authors cloned the GmRIQ2 promoter and identified multiple cis-elements with the help of bioinformatics. The authors also validated the full-length promoter using GUS in different crops to study the spatiotemporal expression. Through 5' deletion constructs attached to GUS gene, the authors developed transgenics in Arabidopsis thaliana and validated the presence of cis-elements by challenging using various hormones and stress conditions.

The full-length promoter cloned is 1661bp, but the sequence provided looks bigger, need clarification on this. There are three 5' deletion constructs developed which I am not getting any clue from the sequence provided from which part of the promoter sequence these are developed. Is the sequence provided is positive or negative strand? The authors need to clearly mention all the details like the transcription start site, highlight the primer sequences used in the promoter for creating 5' deletion constructs, highlight the core promoter regions identified. Since the entire manuscript depends on the promoter data this information is very critical. In Table 4 the positions of the cis-motifs need to be cross-checked. I believe this does not fall in line with the positions mentioned in the text for the ABA, IAA, and MeJA cis-elements.

I strongly feel the manuscript needs a thorough language check, in many places I find it hard to follow the real meaning and it can derail the readers. I suggest the manuscript for a major revision and address all the comments and suggestions provided before considering it further. 

Specific comments

1. Line 39: ........ which leads to balance and the occurrence of gene silencing. What do you mean by balance? Please check the statement.
2. Line 47-48: .... in the food and industrial materials industries. Please reframe this.
3. Line 89-90: Mention what sort of polymerase is used for PCR amplification of PGmRIQ2. 
4. Line 95: .... the XbarI.... Please check.
5. Figure 1. Schematic diagram of the pCAMBIA3301PGmRIQ2::GUS construct. Please mention the GUS in the diagram.
6. Line 138: Mention which primer pair was used for PCR amplification from Table 1.
7. Table 2: Can you explain what is the function of DTT in the SCCM? Is this media composition is a published one or standardized by the authors? If it's published it is better to provide the reference for the readers.
8. Write Arabidopsis thaliana in full form at the start and then follow a common pattern of writing (A. thaliana or full form).
9. Figure 4: Is the sequence provided is positive or negative strand? Please mark the direction of the promoter sequence, predicted transcription initiation sites, and the primer positions in the sequence for generating different deletions constructs. It will be easy for the readers.
10. Line 226-227: ......four core promoter regions in the PGmRIQ2 sequence. Need to highlight these regions in Figure 4.
11. Line 252-253: Need to explain what is 403 bp amplification. Is it CaMV35S or GUS? Also, provide the primer sequence information in one of the Tables.
12. I believe the three constructs developed are from the top region of the promoter sequence and I could see more cis-elements at the bottom of the provided sequence, for eg. CGTCAC for MeJA and ACGTG ABRE. Why these elements not considered for the deletion fragments?