Biomics

Нокаутное CRISPR/Cas редактирование геномов растений может применяться как при проведении модельных экспериментов, направленных на разработку этой технологии, так и для внесения целевых мутаций, приводящих к созданию форм растений с новыми полезными признаками. Важным аспектом, ускоряющим получение желаемых результатов, является быстрая валидация факта проведенного редактирования. Опосредованно таковой может служить органолептический контроль произошедших изменений в геноме путем визуальной оценки или с помощью обоняния. Соответственно в качестве репортерных генов для этих целей могут быть ген фитоендесатуразы, нарушение которого вызывает побеление растений, либо ген бетаин альдегид дегидрогеназы 2 (BADH2), нарушение которого ведет к изменению метаболизма γ‑аминомасляного альдегида и появлению 2‑ацетил-1-пирролина, обладающего сильным ароматом, напоминающим запах попкорна. История изучения гена BADH2 берет начало с душистого риса Oryza sativa разновидности basmati, несущего природную мутацию в данном гене и за счет этого накапливающего 2‑ацетил-1-пирролин. В настоящее время ген BADH2, характеризующийся высоким эволюционным консерватизмом, и помимо риса секвенирован у нескольких видов растений из разных семейств. Путем нокаутирования гена BADH2 создан ряд сельскохозяйственных культур, среди которых сорго, арахис, рапс, рис, соя. При этом CRISPR/Cas нокаутное редактирование в отдельных случаях может проводиться без встраивания в геном чужеродной ДНК, имитируя тем самым природные мутационные процессы, что согласно законодательствам некоторых стран позволяет не считать их ГМО.

CRISPR/Cas редактирование, рис, растения, бетаин альдегид дегидрогеназа 2, BADH2, запах, аромат, 2 ацетил-1-пирролин

1. Геращенков Г.А, Рожнова Н.А, Кулуев Б.Р. и др. Дизайн РНК-гидов для CRISPR/CAS редактирования геномов растений. Молекулярная биология. 2020. 54(1). 29-50. doi: 10.31857/S0026898420010061
2. Кулуев Б.Р., Гумерова Г.Р., Михайлова Е.В. и др. Доставка CRISPR/CAS-компонентов в клетки высших растений для редактирования их геномов. Физиология растений. 2019. 66(5). 339-353. doi: 10.1134/S0015330319050117
3. Михайлова Е.В., Хуснутдинов Э.А., Чемерис А.В. и др. Доступный арсенал для CRISPR/Cas геномного редактирования растений. Физиология растений. 2022. 69(1). 38-53.doi: 10.31857/S0015330322010134
4. Bhatt V, Ramkumar TR, Barvkar V et al. Targeted disruption of OsBADH2 induces basmati aroma in the popular indica rice variety IR-64 using CRISPR/Cas9. Plant Physiology Reports. doi: 10.1007/s40502-025-00901-z
5. Bigyan KC, Pandit R, Regmi R et al. Genetic basis of rice aroma gene and its application in rice genetics and breeding: A review. Russian Journal of Agricultural and Socio-Economic Sciences 111(3). 170-175. doi: 10.18551/rjoas.2021-03.20
6. Bradbury LM, Fitzgerald TL, Henry RJ et al. The gene for fragrance in rice. Plant Biotechnol J. 2005. 3(3). 363-370. doi: 10.1111/j.1467-7652.2005.00131.x
7. Brocker C, Vasiliou M, Carpenter S et al. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics. Planta. 2013. 237(1). 189-210. doi: 10.1007/s00425-012-1749-0
8. Bulle M, Venkatapuram AK, Abbagani S, Kirti PB. CRISPR/Cas9 based genome editing of Phytoene desaturase (PDS) gene in chilli pepper (Capsicum annuum). J Genet Eng Biotechnol. 2024. 22(2). 100380. doi: 10.1016/j.jgeb.2024.100380
9. Buttery RG, Ling LC, Juliano BO. 2-Acetyl-1-Pyrroline: An Important Aroma Component of Cooked Rice. Chemistry and Industry (London). (23). 958-959.
10. Buttery RG, Stern DJ, Ling LC. Studies on Flavor Volatiles of Some Sweet Corn Products. Agric. Food Chem. 1994. 42(3). 791–795. doi: 10.1021/jf00039a038
11. Buttery RG, Turnbaugh JG, Ling LC. Contribution of volatiles to rice aroma. Agric. Food Chem. 1988. 36(5). 1006–1009. doi: 10.1021/jf00083a025
12. Chandanshive S, Mathure S, Nadaf A. Multiple mutations in BADH2 gene reveal the novel fragrance allele in indica rice (Oryza sativa). J Genet. 2024. 103. 13. doi: 10.1007/s12041-024-01464-y
13. Chen M, Wei X, Shao G et al. Fragrance of the rice grain achieved via artificial microRNA induced down regulation of OsBADH2. Plant Breeding. 2012. 131(5). 584-590. doi: 10.1111/j.1439-0523.2012.01989.x
14. Cordeiro GM, Christopher MJ, Henry RJ et al. Identification of microsatellite markers for fragrance in rice by analysis of the rice genome sequence. Molecular Breeding. 2002. 9. 245–250. doi.org/10.1023/A:1020350725667
15. Fitzgerald TL, Waters DL, Henry RJ. Betaine aldehyde dehydrogenase in plants. Plant Biol (Stuttg). 2009. 11(2). 119-130. doi: 10.1111/j.1438-8677.2008.00161.x
16. Goff SA, Ricke D, Lan TH et al. A draft sequence of the rice genome (Oryza sativa ssp. japonica). Science. 2002. 296(5565). 92-100. doi: 10.1126/science.1068275
17. He D, Zhou R, Huang C et al. Improvement of Flowering Stage in Japonica Rice Variety Jiahe212 by Using CRISPR/Cas9 System. Plants (Basel). 13(15). 2166. doi: 10.3390/plants13152166
18. Henderson SW, Henderson ST, Goetz M et al. Efficient CRISPR/Cas9-Mediated Knockout of an Endogenous Phytoene Desaturase Gene in T1 Progeny of Apomictic Hieracium Enables New Strategies for Apomixis Gene Identification. Genes (Basel). 11(9). 1064. doi: 10.3390/genes11091064
19. Huang Y, Huang L, Cheng M et al. , Li C, Zhou X, Ullah A, Sarfraz S, Khatab A, Xie G. Progresses in biosynthesis pathway, regulation mechanism and potential application of 2-acetyl-1-pyrroline in fragrant rice. Plant Physiol Biochem. 215.109047. doi: 10.1016/j.plaphy.2024.109047
20. Hui S, Li H, Mawia AM et al. Production of aromatic three-line hybrid rice using novel alleles of BADH2. Plant Biotechnol J. 2022. 20(1). 59-74. doi: 10.1111/pbi.13695
21. Imran M, Shafiq S, Ashraf U et al. Biosynthesis of 2-Acetyl-1-pyrroline in Fragrant Rice: Recent Insights into Agro-management, Environmental Factors, and Functional Genomics. J Agric Food Chem. 71(10). 4201-4215. doi: 10.1021/acs.jafc.2c07934
22. Jin Q, Waters D, Cordeiro GM et al. A single nucleotide polymorphism (SNP) marker linked to the fragrance gene in rice (Oryza sativa). Plant Science. 2003. 165(2). 359-364. doi: 10.1016/S0168-9452(03)00195-X
23. Juwattanasomran R, Somta P, Chankaew S et al. A SNP in GmBADH2 gene associates with fragrance in vegetable soybean variety "Kaori" and SNAP marker development for the fragrance. Theor Appl Genet. 122(3). 533-541. doi: 10.1007/s00122-010-1467-6
24. Khandagale KS, Chavhan R, Nadaf AB. RNAi-mediated down regulation of BADH2 gene for expression of 2-acetyl-1-pyrroline in non-scented indica rice IR-64 (Oryza sativa). 3 Biotech. 2020. 10(4). 145. doi: 10.1007/s13205-020-2131-8
25. Liao Y, Li M, Wu H et al. Generation of aroma in three-line hybrid rice through CRISPR/Cas9 editing of betaine aldehyde dehydrogenase2 (OsBADH2). Physiol Plant. 2024. 176(1). e doi: 10.1111/ppl.14206
26. Niu X, Tang W, Huang W et al. RNAi-directed downregulation of OsBADH2 results in aroma (2-acetyl-1-pyrroline) production in rice (Oryza sativa). BMC Plant Biol. 2008. 8. 100. doi: 10.1186/1471-2229-8-100
27. Okpala NE, Mo Z, Duan M et al. The genetics and biosynthesis of 2-acetyl-1-pyrroline in fragrant rice. Plant Physiol Biochem. 2019. 135. 272-276. doi: 10.1016/j.plaphy.2018.12.012
28. Pavese V, Moglia A, Corredoira E et al. First Report of CRISPR/Cas9 Gene Editing in Castanea sativa Mill. Front Plant Sci. 25(12). 728516. doi: 10.3389/fpls.2021.728516
29. Qian L, Jin H, Yang Q et al. A Sequence Variation in GmBADH2 Enhances Soybean Aroma and Is a Functional Marker for Improving Soybean Flavor. Int J Mol Sci. 23(8). 4116. doi: 10.3390/ijms23084116
30. Senthil K, Rathinam M, Parashar M et al. Establishing a CRISPR/Cas9 genome editing framework in pigeonpea (Cajanus cajan L.) by targeting phytoene desaturase (PDS) gene disruption. J Genet Eng Biotechnol. 23(1). 100465. doi: 10.1016/j.jgeb.2025.100465
31. Schieberle P. Quantitation of Important Roast-Smelling Odorants in Popcorn by Stable Isotope Dilution Assays and Model Studies on Flavor Formation during Popping. Agric. Food Chem. 1995. 43(9). 2442–2448. doi: 10.1021/jf00057a024
32. Shan Q, Zhang Y, Chen K et al. Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology. Plant Biotechnol J. 13(6). 791-800. doi: 10.1111/pbi.12312
33. Shi Y, Zhao G, Xu X et al. Discovery of a new fragrance allele and development of functional markers for identifying diverse fragrant genotypes in rice. Mol Breeding. 2014. 33. 701–708. doi.org/10.1007/s11032-013-9986-x
34. Tian Y, Zhou Y, Gao G et al. Creation of Two-Line Fragrant Glutinous Hybrid Rice by Editing the Wx and OsBADH2 Genes via the CRISPR/Cas9 System. Int J Mol Sci. 24(1). 849. doi: 10.3390/ijms24010849
35. Thimmaraju R, Bhagyalakshmi N, Narayan Ms et al. In vitro culture of Pandanus amaryllifolius and enhancement of 2‐acetyl‐1‐pyrroline, the major flavouring compound of aromatic rice, by precursor feeding of L‐proline. J Science Food Agriculture. 2005. 85(15). 2527-2534. DOI: 10.1002/jsfa.2286
36. Usman B, Nawaz G, Zhao N et al. Generation of High Yielding and Fragrant Rice (Oryza sativa) Lines by CRISPR/Cas9 Targeted Mutagenesis of Three Homoeologs of Cytochrome P450 Gene Family and OsBADH2 and Transcriptome and Proteome Profiling of Revealed Changes Triggered by Mutations. Plants (Basel). 2020. 9(6). 788. doi: 10.3390/plants9060788
37. Vaia G, Pavese V, Moglia A et al. Knockout of phytoene desaturase gene using CRISPR/Cas9 in highbush blueberry. Front Plant Sci. 13. 1074541. doi: 10.3389/fpls.2022.1074541
38. Wakte K, Zanan R, Hinge V et al. Thirty-three years of 2-acetyl-1-pyrroline, a principal basmati aroma compound in scented rice (Oryza sativa): a status review. J Sci Food Agric. 2017. 97(2). 384-395. doi: 10.1002/jsfa.7875
39. Wang Y, Liu X, Zheng X et al. Creation of aromatic maize by CRISPR/Cas. J Integr Plant Biol. 63(9). 1664-1670. doi: 10.1111/jipb.13105
40. Wei X, Handoko DD, Pather L et al. Evaluation of 2-acetyl-1-pyrroline in foods, with an emphasis on rice flavour. Food Chem. 531-544. doi: 10.1016/j.foodchem.2017.04.005
41. Wu J, Xu J, He A et al. Generating fragrant oilseed rape using CRISPR/Cas9-mediated gene editing. Plant Physiol. 197(1) kiae660. doi: 10.1093/plphys/kiae660
42. Wu W, Miao R, Li Z et al. CRISPR/Cas9-mediated editing of BADH2 and Wx genes for the development of novel aromatic and soft-textured black and red rice. Physiol Plant. 177(2). e70194. doi: 10.1111/ppl.70194
43. Xie H, Song M, Cao X et al. Breeding exceptionally fragrant soybeans for soy milk with strong aroma. J Integr Plant Biol. 66(4). 642-644. doi: 10.1111/jipb.13631
44. Xue L, Qu P, Zhao H et al. Creation of fragrant peanut using CRISPR/Cas9. J Integr Plant Biol. 67(6). 1438-1440. doi: 10.1111/jipb.13864
45. Yu J, Hu S, Wang J. et al. A draft sequence of the rice genome (Oryza sativa ssp. indica). Science. 2002. 296(5565). 79-92. doi: 10.1126/science.1068037
46. Yundaeng C, Somta P, Tangphatsornruang S et al. A single base substitution in BADH/AMADH is responsible for fragrance in cucumber (Cucumis sativus), and development of SNAP markers for the fragrance. Theor Appl Genet. 128(9). 1881-1892. doi: 10.1007/s00122-015-2554-5
47. Yundaeng C, Somta P, Tangphatsornruang S et al. Gene discovery and functional marker development for fragrance in sorghum (Sorghum bicolor (L.) Moench). Theor Appl Genet. 126(11). 2897-2906. doi: 10.1007/s00122-013-2180-z
48. Zhang C, Yun P, Xia J et al. CRISPR/Cas9-mediated editing of Wx and BADH2 genes created glutinous and aromatic two-line hybrid rice. Mol Breed. 43(4). 24. doi: 10.1007/s11032-023-01368-2
49. Zhang D, Tang S, Xie P et al. Creation of fragrant sorghum by CRISPR/Cas9. J Integr Plant Biol. 64(5). 961-964. doi: 10.1111/jipb.13232
50. Zhang Y, He Q, Zhang S et al. De novo creation of popcorn-like fragrant foxtail millet. J Integr Plant Biol. 65(11). 2412-2415. doi: 10.1111/jipb.13556
51. Zhao G, Deng L, Liu X et al. A mutation in CmoBADH results in the production of 2-acetyl-1-pyrroline, conferring a 'taro-like' aroma in Cucurbita moschata. Theor Appl Genet. 138(11). 265. doi: 10.1007/s00122-025-05050-3