Detection and quantification of splicing variants of Hd3a gene in oil palm

https://doi.org/10.22146/ijbiotech.88327

Aqwin Polosoro(1), Wening Enggarini(2), Kusumawaty Kusumanegara(3), Toto Hadiarto(4), Miftahudin Miftahudin(5), Ence Darmo Jaya Supena(6*)

(1) Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Bogor 16680, Indonesia; Research Center for Genetic Engineering, National Research and Innovation Agency, Bogor 16911, Indonesia
(2) Research Center for Genetic Engineering, National Research and Innovation Agency, Bogor 16911, Indonesia
(3) Research Center for Horticulture, National Research and Innovation Agency, Bogor 16911, Indonesia
(4) Research Center for Genetic Engineering, National Research and Innovation Agency, Bogor 16911, Indonesia
(5) Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Bogor 16680, Indonesia
(6) Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Bogor 16680, Indonesia
(*) Corresponding Author

Abstract


Alternative splicing is a complex process that contributes to the generation of diverse mRNA and protein isoforms, including in oil palm (Elaeis guineensis). Despite their importance, many functions of alternative splicing genes remain poorly characterized. This study aims to investigate splicing variants of gene encoding Heading date 3a in E. guineensis (EgHd3a) using the GenBank database and ClustalW algorithm. To ensure the data accuracy and reliability of design isoform‐ specific primers, special emphasis is given to primer design techniques and validation using polymerase chain reaction (PCR) and quantitative real‐time (qRT)‐PCR analysis. The designed primers demonstrated high specificity and discrimination between mRNA specimens. Nucleotide variations at the 3’‐end influenced the specificity of primers with the addition of GC composition. Furthermore, qRT‐PCR analysis revealed a strong correlation between Ct values and gene concentration for the isoforms which indicates a reliable amplification of EgHd3a. Although two isoforms, Hd3a‐X2 and Hd3a‐X3, showed slightly higher than acceptable PCR efficiency values, caution is advised to prevent non‐specific amplification. Despite the challenge posed by the limitation of primer positioning due to alternative splicing, the chosen primer proved optimal for analysis. This study highlights the importance of considering alternative splicing in gene quantification experiments and provides insights into the critical steps, methods, and quality control measures necessary for accurately detecting alternative splicing events, contributing to understanding this complex biological process.


Keywords


Alternative splicing; Hd3a; Primer; mRNA; qRT‐PCR



References

Adams G. 2020. A beginner’s guide to RT-PCR, qPCR and RT-qPCR. Biochem. (Lond). 42(3):48–53. doi:10.1042/bio20200034.

Bak A, Emerson JB. 2019. Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection. BMC Biotechnol. 19(1):1–12. doi:10.1186/s12896-019-0571-1.

Baralle FE, Giudice J. 2017. Alternative splicing as a regulator of development and tissue identity. Nat. Rev. Mol. Cell Biol. 18(7):437–451. doi:10.1038/nrm.2017.27.

Busk PK. 2014. A tool for design of primers for microRNA-specific quantitative RT-qPCR. BMC Bioinformatics 15(1):29. doi:10.1186/1471-2105-15- 29.

Bustin S, Huggett J. 2017. qPCR primer design revisited. Biomol. Detect. Quantif. 14:19–28. doi:10.1016/j.bdq.2017.11.001.

Fu XD, Ares M. 2014. Context-dependent control of alternative splicing by RNA-binding proteins. Nat. Rev. Genet. 15(10):689–701. doi:10.1038/nrg3778.

Harvey SE, Cheng C. 2016. Methods for characterization of alternative RNA splicing. Methods Mol. Biol. 1402:229–241. doi:10.1007/978-1-4939-3378-5_18.

Hassankhah A, Rahemi M, Ramshini H, Sarikhani S, Vahdati K. 2020. Flowering in Persian walnut: Patterns of gene expression during flower development. BMC Plant Biol. 20(1):1–10. doi:10.1186/s12870- 020-02372-w.

Jadav KK, Rajput N, Singh AP, Sarkhel BC, Shrivastav AB. 2016. A species-specific ARMS PCR test for detection of Indian wild pig DNA. Indian J. Anim. Sci. 86(6):67–70. URL https://www.researchgate.net /publication/323355376.

Kalendar R, Shustov AV, Akhmetollayev I, Kairov U. 2022. Designing allele-specific competitiveextension PCR-based assays for high-throughput genotyping and gene characterization. Front. Mol. Biosci. 9:1–13. doi:10.3389/fmolb.2022.773956.

Kaneko-Suzuki M, Kurihara-Ishikawa R, OkushitaTerakawa C, Kojima C, Nagano-Fujiwara M, Ohki I, Tsuji H, Shimamoto K, Taoka KI. 2018. TFL1-like proteins in rice antagonize rice FT-like protein in inflorescence development by competition for complex formation with 14-3-3 and FD. Plant Cell Physiol. 59(3):458–468. doi:10.1093/pcp/pcy021.

Klepikova AV, Kasianov AS, Gerasimov ES, Logacheva MD, Penin AA. 2016. A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling. Plant J. 88(6):1058– 1070. doi:10.1111/tpj.13312.

Li C, Zhang Y, Zhang K, Guo D, Cui B, Wang X, Huang X. 2015. Promoting flowering, lateral shoot outgrowth, leaf development, and flower abscission in tobacco plants overexpressing cotton FLOWERING LOCUS T (FT)-like gene GhFT1. Front. Plant Sci. 6(June):1– 14. doi:10.3389/fpls.2015.00454.

Li D, Zhang J, Li J. 2020. Primer design for quantitative real-time PCR for the emerging Coronavirus SARS-CoV-2. Theranostics 10(16):7150– 7162. doi:10.7150/thno.47649.

Li KC, Ding ST, Lin EC, Wang L, Lu YW. 2014. Melting analysis on microbeads in rapid temperaturegradient inside microchannels for single nucleotide polymorphisms detection. Biomicrofluidics 8(6):1– 15. doi:10.1063/1.4902907.

Liu YY, Yang KZ, Wei XX, Wang XQ. 2016. Revisiting the phosphatidylethanolamine-binding protein (PEBP) gene family reveals cryptic FLOWERING LOCUS T gene homologs in gymnosperms and sheds new light on functional evolution. New Phytol. 212(3):730–744. doi:10.1111/nph.14066.

Mao Y, Sun J, Cao P, Zhang R, Fu Q, Chen S, Chen F, Jiang J. 2016. Functional analysis of alternative splicing of the FLOWERING LOCUS T orthologous gene in Chrysanthemum morifolium. Hortic. Res. 3:16058. doi:10.1038/hortres.2016.58.

Mardalisa, Suhandono S, Yanti N, Rozi F, Nova F, Primawati. 2021. Bioinformatic analysis in designing mega-primer in overlap extension PCR cloning (OEPC) technique. Int. J. Informatics Vis. 5(2):139– 143. doi:10.30630/joiv.5.2.459.

Pal A. 2022. Rapid amplification of cDNA ends (RACE). Protoc. Adv. Genomics Allied Tech. p. 505–535. doi:10.1007/978-1-0716-1818-9_21.

Polosoro A, Enggarini W, Hadiarto T, Miftahudin, Supena EDJ. 2024. Optimizing RNA extraction and cloning techniques: a case study of cloning the Heading date 3a gene in oil palm [manuscript submitted for publication]. Ph.D. thesis, Departement of Plant Biology, IPB University, Bogor.

Polosoro A, Enggarini W, Hadiarto T, Supena ED, Suharsono. 2021. In silico screening of oil palm early and continuously flowering gene candidates for faster breeding program. In: IOP Conf. Ser. Earth Environ. Sci., volume 762. p. 012063. doi:10.1088/1755- 1315/762/1/012063.

Purwestri YA, Ogaki Y, Tsuji H, Shimamoto K. 2012. Functional analysis of OsKANADI1, a florigen Hd3a interacting protein in rice (Oryza sativa L.). Indones. J. Biotechnol. 17(2):169–176. doi:10.22146/ijbiotech.7860.

Shang X, Cao Y, Ma L. 2017. Alternative splicing in plant genes: A means of regulating the environmental fitness of plants. Int. J. Mol. Sci. 18(2):1–18. doi:10.3390/ijms18020432.

Shao C, Cai F, Zhang Y, Bao Z, Shi G, Bao M, Zhang J. 2022. Regulation of alternative splicing of PaFT and PaFDL1, the FT and FD homologs in Platanus acerifolia. Gene 830:146506. doi:10.1016/j.gene.2022.146506.

Svec D, Tichopad A, Novosadova V, Pfaffl MW, Kubista M. 2015. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments. Biomol. Detect. Quantif. 3:9–16. doi:10.1016/j.bdq.2015.01.005.

Szakonyi D, Duque P. 2018. Alternative splicing as a regulator of early plant development. Front. Plant Sci. 9:1–9. doi:10.3389/fpls.2018.01174.

Tellinghuisen J, Spiess AN. 2015. Bias and imprecision in analysis of real-time quantitative polymerase chain reaction data. Anal. Chem. 87(17):8925–8931. doi:10.1021/acs.analchem.5b02057.

Tsuji H, Tachibana C, Tamaki S, Taoka KI, Kyozuka J, Shimamoto K. 2015. Hd3a promotes lateral branching in rice. Plant J. 82(2):256–266. doi:10.1111/tpj.12811.

Whankaew S, Ruttajorn K, Madsen CK, Asp T, Xu L, Nakkaew A, Phongdara A. 2019. An EgHd3a-like and its alternatively spliced transcripts in the oil palm (Elaeis guineensis). Songklanakarin J. Sci. Technol. 41(2):332–340. doi:10.14456/sjst-psu.2019.42.

Wilkinson ME, Charenton C, Nagai K. 2020. RNA Splicing by the Spliceosome. Annu. Rev. Biochem. 89:359–388. doi:10.1146/annurev-biochem-091719- 064225.

Wu Y, Wei J, Choi SC, Fei Y, Xiong F. 2019. Rice florigen gene Hd3a has conserved functions in callus development. Acta Physiol. Plant. 41(7):125. doi:10.1007/s11738-019-2914-x.

Xia W, Liu R, Zhang J, Mason AS, Li Z, Gong S, Zhong Y, Dou Y, Sun X, Fan H, Xiao Y. 2020. Alternative splicing of flowering time gene FT is associated with halving of time to flowering in coconut. Sci. Rep. 10(1):1–11. doi:10.1038/s41598-020-68431-2.

Yang S, Tang F, Zhu H. 2014. Alternative splicing in plant immunity. Int. J. Mol. Sci. 15(6):10424–10445. doi:10.3390/ijms150610424.

Yun HR, Chen C, Kim JH, Kim HE, Karthik S, Kim HJ, Chung YS, Baek HS, Sung S, Kim HU, Heo JB. 2023. Genome-edited HEADING DATE 3a knockout enhances leaf production in Perilla frutescens. Front. Plant Sci. 14:1–12. doi:10.3389/fpls.2023.1133518.

Zhang A, Jin L, Yarra R, Cao H, Chen P, John Martin JJ. 2022. Transcriptome analysis reveals key developmental and metabolic regulatory aspects of oil palm (Elaeis guineensis Jacq.) during zygotic embryo development. BMC Plant Biol. 22(1):1–13. doi:10.1186/s12870-022-03459-2.

Zuhar LM. 2019. Determination of reference genes for normalisation of gene expression study of Ganoderma-infected oil palms. J. Oil Palm Res. 31(4):550– 560. doi:10.21894/jopr.2019.0051.

Adams G. 2020. A beginner’s guide to RT-PCR, qPCR and RT-qPCR. Biochem. (Lond). 42(3):48–53. doi:10.1042/bio20200034.

Bak A, Emerson JB. 2019. Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection. BMC Biotechnol. 19(1):1–12. doi:10.1186/s12896- 019-0571-1.

Baralle FE, Giudice J. 2017. Alternative splicingas a regulator of development and tissue identity. Nat. Rev. Mol. Cell Biol. 18(7):437–451.doi:10.1038/nrm.2017.27.Busk PK. 2014. A tool for design of primers formicroRNA-specific quantitative RT-qPCR. BMCBioinformatics 15(1):29. doi:10.1186/1471-2105-15-29.Bustin S, Huggett J. 2017. qPCR primer designrevisited. Biomol. Detect. Quantif. 14:19–28.doi:10.1016/j.bdq.2017.11.001.

Fu XD, Ares M. 2014. Context-dependent control of alternative splicing by RNA-binding proteins. Nat. Rev. Genet. 15(10):689–701. doi:10.1038/nrg3778.

Harvey SE, Cheng C. 2016. Methods for characterization of alternative RNA splicing. Methods Mol. Biol. 1402:229–241. doi:10.1007/978-1-4939-3378-5_18.

Hassankhah A, Rahemi M, Ramshini H, Sarikhani S, Vahdati K. 2020. Flowering in Persian walnut: Patterns of gene expression during flower development. BMC Plant Biol. 20(1):1–10. doi:10.1186/s12870- 020-02372-w.

Jadav KK, Rajput N, Singh AP, Sarkhel BC, Shrivastav AB. 2016. A species-specific ARMS PCR test for detection of Indian wild pig DNA. Indian J. Anim. Sci. 86(6):67–70. URL https://www.researchgate.net /publication/323355376.

Kalendar R, Shustov AV, Akhmetollayev I, Kairov

U. 2022. Designing allele-specific competitiveextension PCR-based assays for high-throughput

genotyping and gene characterization. Front. Mol.

Biosci. 9:1–13. doi:10.3389/fmolb.2022.773956.

Kaneko-Suzuki M, Kurihara-Ishikawa R, OkushitaTerakawa C, Kojima C, Nagano-Fujiwara M, Ohki I, Tsuji H, Shimamoto K, Taoka KI. 2018. TFL1-like proteins in rice antagonize rice FT-like protein in inflorescence development by competition for complex formation with 14-3-3 and FD. Plant Cell Physiol. 59(3):458–468. doi:10.1093/pcp/pcy021.

Klepikova AV, Kasianov AS, Gerasimov ES, Logacheva MD, Penin AA. 2016. A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling. Plant J. 88(6):1058– 1070. doi:10.1111/tpj.13312.

Li C, Zhang Y, Zhang K, Guo D, Cui B, Wang X, Huang X. 2015. Promoting flowering, lateral shoot outgrowth, leaf development, and flower abscission in tobacco

plants overexpressing cotton FLOWERING LOCUS T (FT)-like gene GhFT1. Front. Plant Sci. 6(June):1– 14. doi:10.3389/fpls.2015.00454.

Li D, Zhang J, Li J. 2020. Primer design for quantitative real-time PCR for the emerging Coronavirus SARS-CoV-2. Theranostics 10(16):7150– 7162. doi:10.7150/thno.47649.

Li KC, Ding ST, Lin EC, Wang L, Lu YW. 2014. Melting analysis on microbeads in rapid temperaturegradient inside microchannels for single nucleotide polymorphisms detection. Biomicrofluidics 8(6):1– 15. doi:10.1063/1.4902907.

Liu YY, Yang KZ, Wei XX, Wang XQ. 2016. Revisiting the phosphatidylethanolamine-binding protein (PEBP) gene family reveals cryptic FLOWERING LOCUS T gene homologs in gymnosperms and sheds new light on functional evolution. New Phytol. 212(3):730–744. doi:10.1111/nph.14066.

Mao Y, Sun J, Cao P, Zhang R, Fu Q, Chen S, Chen F, Jiang J. 2016. Functional analysis of alternative splicing of the FLOWERING LOCUS T orthologous gene in Chrysanthemum morifolium. Hortic. Res. 3:16058. doi:10.1038/hortres.2016.58.

Mardalisa, Suhandono S, Yanti N, Rozi F, Nova F, Primawati. 2021. Bioinformatic analysis in designing mega-primer in overlap extension PCR cloning (OEPC) technique. Int. J. Informatics Vis. 5(2):139– 143. doi:10.30630/joiv.5.2.459.

Pal A. 2022. Rapid amplification of cDNA ends (RACE). Protoc. Adv. Genomics Allied Tech. p. 505–535. doi:10.1007/978-1-0716-1818-9_21.

Polosoro A, Enggarini W, Hadiarto T, Miftahudin, Supena EDJ. 2024. Optimizing RNA extraction and cloning techniques: a case study of cloning the Heading date 3a gene in oil palm [manuscript submitted for publication]. Ph.D. thesis, Departement of Plant Biology, IPB University, Bogor.

Polosoro A, Enggarini W, Hadiarto T, Supena ED, Suharsono. 2021. In silico screening of oil palm early and continuously flowering gene candidates for faster breeding program. In: IOP Conf. Ser. Earth Environ. Sci., volume 762. p. 012063. doi:10.1088/1755- 1315/762/1/012063.

Purwestri YA, Ogaki Y, Tsuji H, Shimamoto K. 2012. Functional analysis of OsKANADI1, a florigen Hd3a interacting protein in rice (Oryza sativa

L.). Indones. J. Biotechnol. 17(2):169–176.

doi:10.22146/ijbiotech.7860.

Shang X, Cao Y, Ma L. 2017. Alternative splicing in plant genes: A means of regulating the environmental fitness of plants. Int. J. Mol. Sci. 18(2):1–18. doi:10.3390/ijms18020432.

Shao C, Cai F, Zhang Y, Bao Z, Shi G, Bao M,

Zhang J. 2022. Regulation of alternative splicing of PaFT and PaFDL1, the FT and FD homologs in Platanus acerifolia. Gene 830:146506. doi:10.1016/j.gene.2022.146506.

Svec D, Tichopad A, Novosadova V, Pfaffl MW, Kubista



DOI: https://doi.org/10.22146/ijbiotech.88327

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