Molecular cloning and characterization of FLOWERING LOCUS T (FT)gene from kinnow mandarin (Citrus reticulata)

DIVYA  RANA; POOJA  MANCHANDA; HARLEEN  KAUR; GURUPKAR SINGH  SIDHU; PARVEEN  CHHUNEJA

doi:10.56093/ijas.v94i7.147433

Authors

DIVYA RANA Punjab Agricultural University, Ludhiana, Punjab 141 004, India
POOJA MANCHANDA Punjab Agricultural University, Ludhiana, Punjab 141 004, India
HARLEEN KAUR Punjab Agricultural University, Ludhiana, Punjab 141 004, India
GURUPKAR SINGH SIDHU Punjab Agricultural University, Ludhiana, Punjab 141 004, India
PARVEEN CHHUNEJA Punjab Agricultural University, Ludhiana, Punjab 141 004, India

https://doi.org/10.56093/ijas.v94i7.147433

Keywords:

CrFT, Early flowering, Florigen, Kinnow mandarin, PEBPs

Abstract

FLOWERING LOCUS T (FT) is considered as a crucial regulator involved in early flowering induction which functions by switching the vegetative phase into reproductive phase. The role of early flowering has been elucidated by many workers but till date, there has been no report on cloning of FT gene from Kinnow mandarin (Citrus reticulata Blanco). An experiment was conducted during 2022–23 at Punjab Agricultural University, Ludhiana, Punjab for molecular cloning and characterization of FT gene from Kinnow mandarin. During the study, FT gene was cloned into pGEM-T Easy vector and characterized using in silico analysis. Sequence analysis revealed that the FT gene is comprised of 822 nucleotides, including a 5’- untranslated region (UTR) of 137 bp, a coding sequence (CDS) of 534 bp, and a 3’-UTR region of 151 bp. The BLASTn results of CrFT CDS showed high similarity to FT-like genes of other citrus species including C. unshiu, C. sinensis, and C. clementina which were found homologous to Phosphatidylethanolamine binding protein (PEBP). The predicted molecular weight of protein was approximately 20 kDa and isoelectric point (pI) was 6.73 indicating slightly acidic nature of the protein. The instability, aliphatic and GRAVY (Grand Average of Hydropathy) indices of the protein were determined as 40.97, 73.16 and -0.491, respectively. This study would help to elucidate the molecular mechanism behind flowering in Kinnow mandarin and facilitate the molecular breeding studies targeted to obtain early flowering phenotypes.

Downloads

Download data is not yet available.

References

Abe M, Kobayashi Y, Yamamoto S, DaimonY, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi M, Goto K and Araki T. 2005. FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309: 1052–56. https://doi.org/10.1126/science.1115983

Ashkenazy H, Abadi S, Martz E, Chay O, Mayrose T, Pupko T and Ben-Tal N. 2016. ConSurf 2016: An improved methodology to estimate and visualize evolutionary conservation in macro- molecules. Nucleic Acids Research 8(44): 344–50. https://doi. org/10.1093/nar/gkw408

Chomczynski P and Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162(1): 156–59. https://doi.org/10.1006/abio.1987.9999

Cohen S N, Chang A C and Hsu L. 1972. Non-chromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Proceedings of the National Academy of Sciences 69(8): 2110–14. https://doi.org/10.1073/pnas.69.8.2110

Endo T, Shimada T, Fujii H, Kobayashi Y, Araki T and Omura M. 2005. Ectopic expression of an FT homolog from Citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.). Transgenic Research 14: 703–12. https://doi.org/10.1007/s11248-005-6632-3

FAO. 2021. Agricultural Data. Food and Agricultural Organization (FAO), Rome, Italy.

Henderson I R, Liu F, Drea S, Simpson G G and Dean C. 2005. An allelic series reveals essential roles for LFY in plant development in addition to flowering-time control. Development 132(16): 3597–3607. https://doi.org/10.1242/dev.01924

Jang S, Choi S C, Li H Y, An G and Schmelzer E. 2015. Functional characterization of Phalaenopsis aphrodite flowering genes PaFT1 and PaFD. Plos One 10(8): e0134987. https://doi.org/10.1371/journal.pone.0134987

Kaur H, Manchanda P, Sidhu G S and Chhuneja P. 2024. Genome- wide identification and characterization of flowering genes in Citrus sinensis (L.) Osbeck: A comparison among C. medica L., C. reticulata Blanco, C. grandis (L.) Osbeck and C. clementina. BMC Genomic Data 25: 20. https://doi.org/10.1186/s12863-024-01201-5

Kiyak A and Mutlu A G. 2023. Molecular cloning, characterization and expression profile of FLOWERING LOCUS T (FT) gene from Prunus armeniaca (L.) South African Journal of Botany 155: 330–39. https://doi.org/10.1016/j.sajb.2023.02.026

Lee H, Suh S S, Park E, Cho E, Ahn J H, Kim S G, Lee J S, Kwon Y M and Lee I. 2000. The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes and Development 14: 2366–76. www.genesdev.org/cgi/

https://doi.org/10.1101/gad.813600

Manchanda P, Kaur H, Kaur R K, Kaur A, Kaur J, Kaur S and Sidhu G S. 2022a. Optimization of extraction of bioactive phenolics and their antioxidant potential from callus and leaf extracts of Citrus sinensis (L.) Osbeck, C. reticulata Blanco and C. maxima (Burm.) Merr. Journal of Food Measurement and Characterization 17: 1343–53. https://doi.org/10.1007/s11694-022-01695-6

Manchanda P, Kaur M, Sharma S and Sidhu G S. 2022b. Biotechnological interventions for reducing juvenility in perennials. Horticulturae 91(1): 33. https://doi.org/10.3390/ horticulturae9010033

Manchanda P, Kaur H, Kaur R K, Kaur J, Kaur M and Sidhu G S. 2023a. Effect of solvent ratio, temperature and time on extraction of bioactive compounds and their antioxidant potential from callus, leaf and peel extracts of Citrus pseudolimon Taraka. Journal of Food Measurement and Characterization 17: 6180–90. https://doi.org/10.1007/s11694-023-02111-3

Manchanda P, Kaur H, Khan F, Sidhu G S, Hunjan M S, Chhuneja P and Bains N S. 2023b. Agroinfiltration-based transient genome editing for targeting phytoene desaturase gene in kinnow mandarin (Citrus reticulata Blanco). Molecular Biotechnology 2003: 1–12 https://doi.org/10.1007/s12033-023-00980-z

Matsuda N, Ikeda K, Kurosaka M, Takashina T, Isuzugawa K, Endo T and Omura M. 2009. Early flowering phenotype in transgenic pears (Pyrus communis L.) expressing the CiFT gene. Journal of the Japanese Society for Horticultural Science 78(4): 410–16.

Nishikawa F, Endo T, Shimada T, Fujii H, Shimizu T and Omura M. 2008. Isolation and characterization of a Citrus FT/TFL1 homologue (CuMFT1), which shows quantitatively preferential expression in Citrus seeds. Journal of the Japanese Society for Horticultural Science 77(1): 38–46.

Notaguchi M, Abe M, Kimura T, Daimon Y, Kobayashi T, Yamaguchi A, Tomita Y, Dohi K, Mori M and Araki T. 2008. Long-distance, graft-transmissible action of Arabidopsis FLOWERING LOCUS T protein to promote flowering. Plant and Cell Physiology 49(11): 1645–58. https://doi.org/10.1093/pcp/pcn154

Pena L, Martın-Trillo M, Juarez J, Pina J A, Navarro L and Martinez-Zapater J M. 2001. Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time. Nature biotechnology 19(3): 263–67. https://doi.org/10.1038/85719

Petri C, Alburquerque N, Faize M, Scorza R and Dardick C. 2018. Current achievements and future directions in genetic engineering of European plum (Prunus domestica L.). Transgenic Research 27: 225–40. https://doi.org/10.1007/s11248-018-0072-3

Searle I, He Y H, Turck F, Vincent C, Fornara F, Krober S, Amasino R A and Coupland G. 2006. The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. Genes and Development 20: 898–912. https://doi.org/10.1101/gad.373506

Soares J M, Weber K C, Qiu W, Stanton D, Mahmoud L M, Wu H, Huyck P, Zale J, Al Jasim K, Grosser J W and Dutt M. 2020. The vascular targeted citrus FLOWERING LOCUS T3 gene promotes non-inductive early flowering in transgenic Carrizo rootstocks and grafted juvenile scions. Scientific Reports 1: 21404. https://doi.org/10.1038/s41598-020-78417-9

Tamura K, Stecher G and Kumar S. 2021. MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution 38(7): 3022–27. https://doi.org/10.1093/molbev/msab120

Taoka K, Ohki I, Tsuji H, Furuita K, Hayashi K, Yanase T, Yamaguchi M, Nakashima C, Purwestri Y A, Tamaki S, Ogaki Y, Shimada C, Nakagawa A, Kojima C and Shimamoto K. 2011. 14-3-3 proteins act as intracellular receptors for rice Hd3a florigen. Nature 31: 332–35. https://doi.org/10.1038/nature10272

Velazquez K, Aguero J, Vives M C, Aleza P, Pina J A, Moreno P, Navarro L and Guerri J. 2016. Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding. Plant Biotechnology Journal 14(10): 1976–85. https://doi.org/10.1111/pbi.12555

Wickland D P and Hanzawa Y. 2015. The FLOWERING LOCUS T/TERMINAL FLOWER 1 gene family: Functional evolution and molecular mechanisms. Molecular Plant 8(7): 983–97. https://doi.org/10.1016/j.molp.2015.01.007

Wilkie J D, Sedgley M and Olesen T. 2008. Regulation of floral initiation in horticultural trees. Journal of Experimental Botany 59: 3215–28. https://doi.org/10.1093/jxb/ern188

Yang J and Zhang Y. 2015. I-TASSER server: New development for protein structure and function predictions. Nucleic Acids Research 1(43): 174–81. https://doi.org/10.1093/nar/gkv342

Yoo S K, Chung K S, Kim J, Lee J H, Hong S M, Yoo S J, Yoo S Y, Lee J S and Ahn J H. 2005. Constans activates suppressor of overexpression of constans 1 through FLOWERING LOCUS T to promote flowering in Arabidopsis. Plant Physiology 139: 770–78.

Zhang L, Hao Y, Lin S and Gao Y. 2016 Molecular Characterization of FT and FD homologs from Eriobotrya deflexa Nakai forma koshunensis. Frontiers in Plant Science 7: 8. https://doi. org/10.3389/fpls.2016.00008