Abstract
Sesamum indicum is widely cultivated in India for its superior agronomic characters like adaptation to the warm climate of India and resistance to drought and pathogen attack. However, the yield of sesame per unit area of land is quite low. In this context, the genes involved with reproductive tissue development is very important since they are directly involved with crop productivity. Therefore, identification and isolation of anther-specific genes through PCR-Select cDNA subtraction between cDNAs of anthers and leaves was carried out. Sequencing of ~ 700 clones from the cDNA subtraction library followed by BLASTX analysis using sesame database resulted in the identification of 163 annotated unique transcripts, of which 18 transcripts showed homology with the genes of exclusive anther-/pollen-specific functions. Furthermore, anther-specific expression of some of the sesame transcripts as well as already identified anther-specific genes of Arabidopsis and rice have been monitored during development of sesame anthers. Subsequently, full-length cDNA sequence and the upstream sequence of an anther-specific β-1 3-glucanase gene was isolated. Transgenic tobacco plants were developed with GUS gene under the control of a 437-bp upstream sequence along with the first exon and first intron of the gene. Monitoring of GUS gene expression pattern indicated exclusive expression of the promoter in the tapetal cell layer before tetrad formation in the anther. Interestingly, upon treatment with ABA as well as drought stress, GUS was found to express in the vegetative parts of the plant such as leaves and roots as well.
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Abbreviations
- ABA :
-
Abscisic acid
- bp:
-
Base pair
- PMC:
-
Pollen mother cells
- CDS`:
-
Coding DNA sequence
- cDNA:
-
Complementary deoxyribonucleic acid
- kb:
-
Kilobase
- MS:
-
Murashige and Skoog
- PDB:
-
Protein data bank
- PCR:
-
Polymerase chain reaction
- RNase A:
-
Ribonuclease A
- RT-PCR:
-
Reverse transcription polymerase chain reaction
- T1:
-
First-generation transgenic line
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Acknowledgements
The authors are indebted to the Director, Bose Institute, Kolkata, for providing the necessary facilities for carrying out our research. We are grateful to University Grants Commission for providing fellowship to the author. We are also indebted to Department of Biotechnology, Government of India, for funding in the form of project.
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The study was conceived, planned, and executed by SP, MM, and DB. All experimental works were carried out at different stages of the study by SP, SM, AB, BM, MM, US, AM, and AD. Time to time technical guidance, whenever necessary, was offered by DB, MM, and SP. The manuscript was edited and prepared by SP, MM, and DB. All authors read and approved the final manuscript prepared for submission by SP, MM, and DB.
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Electronic Supplementary Material
Supplementary Figure S1
Agarose gel profile of the subtracted cDNA population and its validation: (A) The cDNA obtained from the anthers of sesame buds was subtracted from that of the leaves following the protocol provided with the PCR-Select cDNA Subtraction Kit from Clontech. Lane 1 shows the 500 bp ladder, Lane 2 shows the test sample (subtracted cDNAs, lane 3 shows the unsubtracted control cDNA, and lane 4 shows the 100 bp ladder. (B) The subtracted cDNA population was then purification using QIAGEN purification kit. Further, the purified cDNA was alcohol precipitated and then run on a 1% Agarose gel to check the quality. Lane 1 shows the 100 bp ladder; Lane 2 shows the purified subtracted cDNA population. (C) The subtracted and unsubtracted libraries were checked for the expression of the GAPDH gene. Reduction in the expression of GAPDH in the subtracted population is an evidence of successful subtraction. Lanes 1&5 depict 18 cycles of PCR for the subtracted and unsubtracted libraries respectively. Similarly, lanes 2&6, 3&7 and 4&8 depict 23, 28 and 33 cycles of either libraries as shown. For the unsubtracted cDNA library, the expression of GADPH is strong between 18 and 33 PCR cycles. However, the expression of the same is markedly low in the subtracted cDNA population even after 33 cycles, whereas no expression is seen in from 18 to 28 PCR cycles. Thus, it is apparent that proper and efficient subtraction has taken place. C.No. Stands for a number of cycles. (PDF 629 kb)
Supplementary Figure S2
Expression pattern of the 17 selected transcripts in the different tissues of sesame: RT-PCR data showing the amplification of 12 out of 17 transcripts in anther tissue, whereas no comparable amplification of the same was detected in other tissues of the plant in the gel. GAPDH is taken as internal control. (JPEG 1249 kb)
Supplementary Figure S3
Isolation of full-length β-1,3-glucanase (BISI27f) gene by 5′ RACE and its upstream sequence: (A) Full-length β-1,3-glucanase (SiBG) gene was isolated through 5’RACE. An 813 bp long EST sequence was obtained after the cDNA subtraction which had significant homology with the Nicotiana tabecum β-1,3-glucanase gene. (B) The isolated fragment was incorporated into a TA clone and sequenced. Primers were designed from it’s 5′ region, and then 5’RACE genome walking procedure was performed to isolate the upstream sequence of the SiBG gene. A 512 nt sequence of the upstream element was isolated from the genome walking. (C) Further 5′ walking was performed in a similar way which resulted in a total of 712 nt long upstream sequences. (PDF 458 kb)
Supplementary Figure S4
(a) Schematic representation of the isolation of upstream and 1st intron nucleotide sequence of β-1,3-glucanase (BISI27f) gene through cDNA subtraction followed by 5′ RACE and PCR mediated genome walking. (b) Schematic representation of the construct containing the 437 bp upstream sequence, first exon, first intron and the fusion sequence of the sesame β 1,3-glucanase gene with the GUS reporter gene. (PPTX 51 kb)
Supplementary Figure S5
Restriction analysis of the SiBGpro-GUS cloned in the plant expression vector called pPZPY112. A. Lanes 1 & 3 show digestion of clones with the enzymes HindIII and EcoRI which gives a pop-out at the size of around 2.8 kb which corresponded with the size of the desired cassette. Lanes 2 & 4 show digestion of clones with the enzymes HindIII and BamHI which gives a pop-out of around 670 kb which corresponds to the size of the SiBGpro which was cloned between these given sites. Lane 5 is the 1 kb plus ladder. The clones were also checked for the presence of the SiBGpro sequence and that of the kanamycin resistance gene by using the specific primers in a PCR reaction. B. Lanes 1–4 show the amplicon of the promoter sequence (437 bp) in the clones and lanes 5–8 show the presence of the kanamycin resistance gene (~500 bp). (PDF 340 kb)
Supplementary Figure S6
Induction pattern of SiBG gene upon ABA and drought treatment in sesame leaves: 4 weeks old sesame plants were treated with 20 μM ABA for 6 h and 20% PEG for 48 h each. The control plants were treated only with water. In control samples (Lane 1), there was no amplification of the β-1,3-glucanase (SiBG), whereas the ABA treated (Lane 2) and the PEG treated (Lane 3) plants showed a distinct induction of the same. GAPDH was taken as the internal control in both the cases. (JPEG 148 kb)
Supplementary Figure S7
Splicing of the first intron of the gene in anthers and upon ABA and drought treatment of leaves. (a) Amplification of the SiBG gene sequence along with the first intron through RT-PCR using GUS gene specific reverse primer and SiBG gene specific 5’end forward primer (NBG-f) from the total cDNA of transgenic Nicotiana tabecum plants, treated with ABA/dehydration stress. Lane 1 shows amplification using genomic DNA which results in a band of a larger size (100 bp larger). Lanes 2 and 3 show amplification in anther cDNAs of stages II and III. Lanes 4, 5 and 6 show amplification from the cDNAs of leaves treated with ABA at 10 μM, 20 μM and 30 μM concentrations. Lane 7 showed control without stress. Lanes 8, 9 and 10 show amplification from cDNAs of leaves exposed to drought stress (days 3, 5 and 7 respectively). (b) In sesame also we have tested the expression of β-1,3-glucanase (SiBG) on external application of ABA at a concentration of 10 μM, 20 μM and 30 μM as well as exposing sesame plants under drought for 7 days. Lane 1 shows amplification using genomic DNA which results in a band of a larger size (100 bp larger). Lanes 2 and 3 show amplification in anther cDNAs of stages II and III. Lanes 4, 5 and 6 show amplification from the cDNAs of leaves treated with ABA at 10 μM, 20 μM and 30 μM concentrations. Lane 7 showed control without stress. Lanes 8, 9 and 10 show amplification from cDNAs of leaves exposed to drought stress (days 3, 5 and 7 respectively). In both the cases, RT-PCR analysis of the ABA/drought treated sesame cDNA using the primers NBG-f (+1 bp to +21 bp) and NBG-r (+132 bp to +150 bp) designed from 5’region of the gene showed induction of the gene. NBG-f – AGCTGTCGACAAGCTTCGACGGCCCGGGCTGGTAAC. NBG-r- AGCTGGATCCAGCACCTGCCAGTGAAAACC. GUS r- CAATTGCCCGGCTTTCTTGTAAC (PDF 629 kb) (PNG 81 kb)
Supplementary Table S1
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Supplementary Table S2
(DOCX 15 kb)
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Parveen, S., Mazumder, M., Bhattacharya, A. et al. Identification of Anther-Specific Genes from Sesame and Functional Assessment of the Upstream Region of a Tapetum-Specific β-1,3-glucanase Gene. Plant Mol Biol Rep 36, 149–161 (2018). https://doi.org/10.1007/s11105-017-1054-y
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DOI: https://doi.org/10.1007/s11105-017-1054-y