RU2795627C1 - Set of synthetic oligonucleotides for determining the level of expression of the dhn1, dhn7, sgs3, cpk, cyp450, erd3, ccoaomt1, lea, and act genes in scotch pine (pinus sylvestris l.) by real-time pcr - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the sets of synthetic oligonucleotides and can be used to determine the level of expression of Scotch pine (Pinus sylvestris L.) genes by real-time PCR.

EFFECT: increasing the reliability of assessing the level of expression of Scots pine genes (DHN1, DHN7, SGS3, CPK, CYP450, ERD3, CCOAOMT1, LEA and ACT) through the use of fluorescently labeled probes complementary to the inner part of the amplified DNA which leads to an increase in the total length of synthetic nucleotides and reduction of the risk of non-specific amplification.

1 cl, 2 dwg, 1 ex

Description

The invention relates to the field of molecular biology and forest biotechnology, in particular to sets of synthetic oligonucleotides, and can be used to determine the level of gene expression of Scotch pine ( Pinus sylvestris L. ) by real-time PCR.

A significant disadvantage of traditional methods for obtaining varieties and forms of tree species, such as hybridization and mutagenesis, is the length of the process, since it is necessary to conduct a long evaluation of the resulting hybrid or mutant progeny in specially designed test cultures. At the same time, the object of research is only the signs and properties formed on the basis of the created genotype. The development of molecular genetic research methods can significantly reduce the duration of the breeding process and increase its efficiency through direct study of the expression of genes associated with the formation of economically important traits and properties of trees.

At present, the solution of fundamental and applied scientific problems in the field of molecular biology and forest biotechnology requires studying the level of gene expression. One of the approaches to studying the level of gene expression is to determine the amount of products of the work of genes - messenger RNA (mRNA) by reverse transcription polymerase chain reaction (RT-PCR). In this case, the fixation of the results can be performed by endpoint using electrophoresis after using classical PCR or using fluorescent detection when using real-time PCR (RT-PCR). Methods for fluorescent detection of RT-PCR results are based on the use of dye-labeled probes or on the use of intercalators (SYBR Green I/II and SYBR Gold). A significant limitation of the use of the above approach is the lack of highly specific oligonucleotides for certain genes, which allow determining the presence or absence of target gene expression, as well as quantifying the expression level by real-time PCR in relation to specific plant species.

The sequences of primers used to determine the level of expression of the following genes of Scotch pine ( Pinus sylvestris ) with the detection of PCR results by endpoint by electrophoresis are known from the literature:

Expression of retrotransposon-like sequences in Scots pine (Pinus sylvestris) in response to heat stress. Environmental and Experimental Biology. 2011. 9: 121-127];1. Primers for LEA genes (late embryogenesis abundant protein) F- TCCGCAGAGGTTACAGACATCG, R- 5'CTATTTGCGC TCAGGAGTCGAA-3; P5CS2 (delta-1-pyrroline-5-carboxylate synthase F-GATCCCAAGAGGTCAGCA, R-GAATCCTGCTTGTGCTTATTCC; AbaH (abscisic acid and water-stress induced protein) F -AGGACAACGT TAATTCTGGCTC, R-AATCGGCCTTATAACCAGTGTCG; GapC1 (glyceraldehyde-3-phosphate dehydrogenase) F -ACGGTTTTGGTCGAATTG, R-CCCACGAGCTCGATATCAT [Voronova A., Jansons A.,

Expression of retrotransposon-like sequences in Scots pine (Pinus sylvestris) in response to heat stress. Environmental and Experimental Biology. 2011. 9: 121-127];

2. Primers to the Peroxidase F gene - GCTCTAGCGGCTAAAAGAGT and R - GAGGTCTGTGACGTTGAGA [Kovaleva VA, Hrunyk NI, Yusypovych Yu.M., Gout RT Expression of the peroxidase dene from Pinus Sylvestris L. in seedlings under abiotic stress. Scientific Bulletin of NLTU of Ukraine. 2016. 26(8): 87-95];

3. Primers for family genesDifensin PsDef1F-GGGATGATGCAGGTTCAAGT, R-ACATTTTCTGCCAGCCACAT;PsDef2F-TCCACTCAGTGCCCCTTTTTC, R-ACCAGCCGAAAGTGCTACTG;PsDef3F-AACCATTGGGATGATGGC, R-GCACTTTCGGCTGGTGAC;PsDef4F-TGTGCTGCTCGTGTTAG, R-CGTTGGAAACCCTTCAGTA;PsDef5.1. F- TCTTGCTACTTGCAATACGT, R-GATACATGGTTTCTCGCAGA and gene60S ribosomal proteinF-CAAAGCTTGCAAAAAGCACA, R-TTCCCTTCCCCTTCTTGTCT [Shalovilo Yu. I., Yusipovich Yu. M., Kovalova V. A., Gut R. T. Influx of phytohormones on gene expression of defensins in scotch pine. Biological Studios. 2015. 9 (1): 15-24 (Shalovylo Y.I., Yusypovych Y.M., Kovaleva V.A., R.T. Gout Shalovylo Y.I. The effect of phytohormones on expression of defensin gene in Scots pine. Studia Biologica. 2015. 9 (1): 15-24 )];

4. Primers for the genes Lipid-transfer protein F-ATGGCTGTGAAGAAGATG, R-TCAGTGAACCTTGGAACAG; 60S ribosomal protein F-CAAAGCTTGCAAAAAGCACA, R-TTCCCTTCCCCTTCTTGTCT [Grunik N. I., Kovalova V. A., Gut R. T. Peculiarities of expression of the gene of lipid transfer protein in the organs of pine ( Pinus sylvestris L.). Biological Studios. 2012. 6(2): 151-160 (Hrunyk NI, Kovaleva VA, Gout RT Patterns of expression of lipid-transfer protein gene in organs of scots pine ( Pinus sylvestris L.). Studia Biologica. 2012. 6(2): 151-160)];

5. Primers to PsWUS/WOX5 genes (wuschel homeobox protein WOX) F-TCACAGCTCAAACGATACGG, R- CAGGGGCGTCCACTTCTAT; Actin F- CCAACAGGGAGAAAATGACG, R- CTGACGCCACATTTACCAAA [Nardmann J., Reisewitz P., Werr W. et al. Discrete Shoot and Root Stem Cell-Promoting WUS/WOX5 Functions Are an Evolutionary Innovation of Angiosperms. Mol. Biol. Evol. 2009. 26(8):1745-1755].

Also known from the literature are the primer sequences used to determine the level of expression by real-time PCR using the SYBR Green intercalating dye of the following Scots pine ( Pinus sylvestris ) genes:

1. Primers for ADC genes (arginine decarboxylase) F-AGTCCGTGTGGCCTGTAATC, R-TGCACAGACACAACGTCAAA; SPDS (spermidine synthase) F-CCAACGTCCCATTAACCCTA, R-TGGCAAACAAAATGATGCTG; ACL5 (tSpm synthase) F-ACTGCTCACATTCCGTCCT, R-TTCGCCTTTGATTCTCTTGCT; DAO (diamine oxidase) F-AATGGGGAAGTTGGGAGTTC, R-CCCTCCTCAGTTTTCCAGTG; CAT (catalase) F-GGGAGGCAAACCTATGTGAA, R-TTGGTTGCATGACTGTGGTT; RAD51 (DNA repair gene) F-TATGGGGAATTTCGAACAGG, R-GTTCCCTCGGCATCAATAAA; KU80 (KU80-like protein) F-GCGGCTATGAGAATGTGGTT, R-AAAAATCACCTGGAGCCATTC; ATG5 (autophagy-related gene) F-GGGTGAAGACAGCGTGAAAT, R-GCCATAGCTCTATTTGATCTG; ATG8 (autophagy-related gene) F-CCTGCTGATCTGACAGTTGGT, R-AGCAGCAGTTGGAGGTAGGT [Salo HM, Sarjala T., Jokela A. et al. Moderate stress responses and specific changes in polyamine metabolism characterize Scots pine somatic embryogenesis. tree physiology. 2016. 36: 392-402];

2. Primers for CAT genes (catalase) F-GGGAGGCAAACCTATGTGAA, R-TTGGTTGCATGACTGTGGTT; βG (β-glucosidase) F-CAAATCTGTTTGTGCCGTTG, R-CTGACGAGCAATTCCCTGTT; RBR (retinoblastoma-related protein) F-ACAGGAAGCAACCTAGTGC; R-TCCACTGTCTCATGCCCCTAA (Vuosku J., Sutela S., Kestilä J. et al. Expression of catalase and retinoblastoma-related protein genes associates with cell death processes in Scots pine zygotic embryogenesis. BMC Plant Biology. 2015. 15:88];

3. Primers for ADC genes (arginine decarboxylase) F-AGTCCGTGTGGCCTGTAATC, R-TGCACAGACACAACGTCAAA; SPDS (spermidine synthase) F-CCAACGTCCCATTAACCCTA, R-TGGCAAACAAAATGATGCTG; ACL5 (tSpm synthase) F-ACTGCTCACATTCCGTCCT, R-TTCGCCTTTGATTCTCTTGCT; DAO (diamine oxidase) F-AATGGGGAAGTTGGGAGTTC, R-CCCTCCTCAGTTTTCCAGTG; PAO (flavoprotein-containing polyamine) F-CGAAATTGCAGAACCTCCAC, R-CGGCCACGAACTACTCATCT; SAMDC (S-adenosyl methionine decarboxylase) F-GCTTCGGCGAGGAAATATCTTA, R-TGTTTGCGGTCCAGTTG; SAMS (s-adenosyl methionine synthetase) F-ACTGCAAAGTGCTGGTT, R-ATGGGTCAGTGGCATAAG; LEA (late embryogenesis abundant protein) F-ACCCTCGCAGAGGTTACAGACA, R-TTGGCCTTCACTGACCCAGGA; CAT (catalase) F-GGGAGGCAAACCTATGTGAA, R-TTGGTTGCATGACTGTGGTT; ICE1 F-TTAGCTTGCTCTGCCCGAAA, R-TCACTTCCCAGTCCCAATGC; R2R3-MYB8 (R2R3-MYB transcription factor MYB8) F-CTTCCTGGAAAGATTTAATAGTGT, R-GGAGCCTGCAATACCCATA; PAL (phenylalanine ammonia-lyase) F-GAGGGAATTTCCAGGGCACA, R-GATCTCGGCCCCTTTCAGTC; psSTS (pinosylvin-forming stilbene synthase) F-ATGTTTCCGTACTCGCTCATAAC, R-ACAAGTTCAAGCGAATATGTGAA; UBQ (putative ubiquitin) F-GAAGGAGCAGTGGAGTCCTG, R-CAATTTCAGGGACGAGGA; TUBA (alpha tubulin) F-TGGCCGCATCTTCCTTGCCG, R-GATGGCCAGTGCCCAGCGA; [Muilu-Mäkelä R., Vuosku J., Saarinen M. et al. Coping with spring frost-effects on polyamine metabolism of Scots pine seedlings. iForest. 2017. 10: 227-236];

4. Primers for ADC genes (arginine decarboxylase) F-AGTCCGTGTGGCCTGTAATC, R-TGCACAGACACAACGTCAAA; AIH (agmatine iminohydrolase) F-TACCACATGCCTGCTGAATG, R-TCAGCAAAGACCGTTGACC; CPA (N-carbamoylputrescine amidohydrolase) F-TTCAGTCCAGGTGACACAGG, R-CTCTTGCTGCCTCTGGAAAC; SPDS (spermidine synthase) F-CCAACGTCCCATTAACCCTA, R-TGGCAAACAAAATGATGCTG; ACL5 (tSpm synthase) F-ACTGCTCACATTCCGTCCT, R-TTCGCCTTTGATTCTCTTGCT; SAMDC (S-adenosyl methionine decarboxylase) F-AAGGGGCAGCATTTCCACT, R-CTCCACCAGTGCTTCAAGGT; DAO (diamine oxidase) F-AATGGGGAAGTTGGGAGTTC, R-CCCTCCTCAGTTTTCCAGTG; PAO (flavoprotein-containing polyamine) F-CGAAATTGCAGAACCTCCAC, R-CGGCCACGAACTACTCATCT; CAT (catalase) F-GGGAGGCAAACCTATGTGAA, R-TTGGTTGCATGACTGTGGTT; MCA (metacaspase) F-CGGGAGAGGACATGCTAAAA, R-CTGGCCTAATTTTCCCAACA; TAT-D (TAT-D nuclease) F-TGGATGTTCCTTAAAGACAGTGG, R-TCTCACAGTATGGAGCGTCTG; E2F (E2F transcription factor) F-TGTGCTTGAGGGGATAGGAC, R-TTCAACCTCTCCAGGTCTCG; RBR (retinoblastoma-related protein) F-GGAATTGCAAAGATCCAACAA, R-ATCGCCTCGATTCAACAAAG [Vuosku J., Suorsa M., Ruottinen M. et al. Polyamine metabolism during exponential growth transition in Scots pine embryogenic cell culture. tree physiology. 2012. 32: 1274-1287];

5. Primers for the genes Dirigent protein F- GTTAAGCACAAAAGTGAATCCAAG, R- TTCTACTTTTATGACATGAAGAACACC; Kunitz-type protease inhibitor F- AATGAATGAGAGACGCACCA, R- TCACAGCAACACAGTCGTTTT; Laccase F- CTGCTCAAAACATAAGCACAGC, R-GAACTTTCACTCTCGTGTCTTTCA; Transcription factor (NAC family) F-TGGAAAGCTACTGGGACAGAC, R-AGAGCCTTTTTCACCCCAAC; Cinnamoyl-CoA reductase F- GCTCACATTTTAGTTTATGAGACACG, R- CGGTGGAGGCTACATTCTG; Terpene synthase F- GCTCTCAGAATGTCGAAAGCA, R- GAAACCAGACACCAATGTTCC; Chalcone synthase F- CCGAATCCAAACAGAACTCC, R- CGCAGAATGGATGTTCGAC; β-glucosidase F- TTTCCGGCTGTGCAATTT, R-CTACGATGTGGGGATTCTCG; Serine-type protease inhibitor F- AGCAATTGCGAACACACACT, R- TGCACAAGGAATTGGAGAGA; Pectin methylesterase F-ATGGCTACGGCTTGAGAATG, R-GTCAATGGATTCGCTGCAA; Phospholipase A F- AGAGGCCCCAGTTCTAAGGT, R- CACAGGGAGCTTCGTTATGG; OPDA reductase F- TCCCGTCATAGACCACAATG, R-GTTCAGGACACTGATTAGGTGGT; Nitrate transporter F- AGTTGGAATTTCAGCGCAAC, R- ATCCCCATCACAGAATCCTCTT; Transcription factor F- CCTGAGAGCCTCTACGTCAATAG, R- TGCTGTAAGTTCACGCCAAA; Oxidoreductase F- ACATCCATGGCCAATTTTTC, R- GTTGGCTAAACGAGGAGCAC; Invertase F- ACAGCCATCCGCATCATAA; R-GCACATGAAAACCTCCATCC; Hydrolase F- GAGATCGGGGATGAGGAGAT, R- AGGCTGGGATGACCAAATC; Cation/proton exchanger F- AGTTGGGTCCAATTGGGAAT, R- CAATACTGATGCGCTGATGG; TUBA F-GATCTGGTTCAGCCTGATGG, R-CCAGTCCGTACCTCGTCAAT; EF1-a F-CACCTTGGGAGTGAAGCAAATG, R-GGGAGTAGTGGCATCCATCTTG (Kovalchuk A., Raffaello T., Jaber E. et al. Activation of defense pathways in Scots pine barkafter feeding by pine weevil (Hylobius abietis). BMC Genomics. 2015. 16:352 ];

6. Primers for the PsFTL2 genes (flowering locus T) F-AGCAGGCAGGTTAACAATGG, R-TTGGATCGCTTGGACTGGGA; GI (gigantea) F-GCAGATGGACTATGGAACC, R-GCTTTATGCCAATCTTGAAC; PRR1 (pseudo-response regulator) F-ACTCCAATACCAACAGTACCAA, R-ATATGTGAGGAAAGCTGATGC; ZTL (zeitlupe) F-ATGCAGCCTATGAATGACAC, R-CCAAATCCAGAATGAAAACATCG; Actin F-TGACATGGAGAAGATTTGGC, R-CATACATAGCAGGCACATTG; GAPDH (glyceraldehyde-3-phosphate dehydrogenase) F-CTGGTGTCTTCACCGACAAA, R-GGTGCTCATTAACCCCAACA [Avia K., Karkkainen K., Lagercrantz U. and Savolainen O. Association of FLOWERING LOCUS T/TERMINAL FLOWER1-like gene FTL2 expression with growth rhythm in Scots pine Pinus sylvestris). New Phytologist. 2014. 204: 159-170.];

7. Primers to the PsDef1 gene (defensin 1) F-CAACGGGAAGTTGCGATT, R-TCAAGGGCAGGGTTTGTAAC (Jaber E., Xiao Ch., Asiegbu FO Comparative pathobiology of Heterobasidion annosum during challenge on Pinus sylvestris and Arabidopsis roots: an analysis of defensin gene expression in two pathosystems Planta 2014 239:717-733];

8. Primers for PsHAP3A genes (LEC-type HAP3 gene) F-AGCTCGATCTTCGGTCTTCA, R-TGCCAACTTCGGACATCATA; PsVP1 (viviparous1) F-GTGCCCAATTTGGTTAATGG, R-GTGCCATCAAAAGACAGCAGA [Uddenberg D., Valladares S., Abrahamsson M. et al. Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor. planta. 2011. 234:527-539.]

9. Primers for the Ef1-a (Elongation factor 1-α) genes F- ACTCTGGCAAGTCCACCACT, R- TCCTTCTCAAAACGCTCGAT; GH3 (GH3 homologue) F-ACGCTATCAGCTCACTGGTC, R-TTGGCTATGCGAAGAATCTC; iaa88 (transcription factor) F-GCAAATTCAAAGCCTAACGA, R-GCTGAGGAAAGCTCATGGTA; MtN21-like-a (noduline like) F- CTTTAAGCGGAGGGATGAAT, R-TTCCACGAAGAAAGCAAAAG; MtN21-like-b (noduline like) F- TTGGTCTCCTCGTCATCAAT, R- TCAGAAGAGAGCCGACAATC; 5NG4 (noduline like) F- CGCTGGATTCCATATCGTTT, R- AAGGGCCAATCAACATCAAG; Clv1-like (clavata like) F- TAATAATCTGAGCGGCAGCA, R- TTCGAGGAAACATCAAGCTG (Heller G., Lundén K., Finlay RD et al. Expression analysis of Clavata1-like and Nodulin21-likegenes from Pinus sylvestris during ectomycorrhiza formation. Mycorrhiza (2012) 22 :271-277];

10. Primers for PsTPS1 genes (Terpene synthases 1) F-TACGGAATGCTAGACGAACTG, R-GCGGCCAGTTCAGACGAACTG; PsTPS2 (Terpene synthases 2) F-GGATCAGATTCTACCAGACG, R-GTTATGTCGTCCTTGACTCG; PsTPS3 (Terpene synthases 3) F-AGGGTATATGCAAGAGTCGC, R-GTATACACATTCCAAGAGCTGC; Ubi (ubiquitin) F-GTTGATTTTTGCTGGCAA, R-CACCTCTCAGACGAAGTA [Köpke D., Schröder R., Fischer HM et al. Does egg deposition by herbivorous pine sawflies affect transcription of sesquiterpene synthases in pine? planta. 2008. 228:427-438];

11. Primers for PER genes (peroxidase) F-GCAGGTACCTTTCCATGATCG, R-ATTCTCGCCATTGCTGCT; AMP (antimicrobial peptide) F-TGCCCACGCAGTGAAATA, R-TGTTTGTGTTGGTAAGCTTGTTTT; Thau (thaumatin) F-TTGTTGCCACCATTCAGAGT, R-TGCTCAATTGCCAAGGTTC; metallothionein like protein F-GCACTTGCAGTCATTTTCCA, R-GGGATTCCAGATGGATGGTA; PR-10 (pathogenesis-related 10) F-TTTATTTTGCACGGTGAACG, R-TCATGTTGAGGAAAGTGGAGAA; MAP kinase F-GCCCTGCTCAAAATCAAAAT, R-TAGCAAGTCTGCATGGCATC [Sun H., Paulin L., Alatalo E., Asiegbu FO et al. Response of living tissues of Pinus sylvestris to the saprotrophic biocontrol fungus Phlebiopsis gigantea. tree physiology. 2011. 31: 438-451];

12. Primers for the RAD51 genes F-TATGGGGAATTTCGAACAGG, R-GTTCCCTCGGCATCAATAAA; KU80 F-GAATGGCTCCAGGTGATTTT, R-AGGCGTTTATTTCCCTTGCT; LIG (DNA ligase) F-GCATTAGCCCTGTTCATCGT, R-CGCTTGGTCTGGATTCTTGT; MCA (metacaspase) F-CGGGAGAGGACATGCTAAAA, R-CTGGCCTAATTTTCCCAACA; TAT-D (Tat-D nuclease) (F-TGGATGTTCCTTAAAGACAGTGG, R-TCTCACAGTATGGAGCGTCTG; ACT (actin) F-GGACAGGTCATTACCGTTGG, R-GATACCCGCTGCTTCCATT; UBI (ubiquitin) F-GAAGGAGCAGTGGAGTCCTG, R-CAATTTCAGGGACGAGAGGA; GAPDH (glyceraldehyde- 3-phosphate dehydrogenase ) F- CTGGTGTCTTCACCGACAAA, R- GGTGCTCATTAACCCCAACA (Vuosku J., Sarjala T., Jokela et al. A. One tissue, two fates: different roles of megagametophyte cells during Scots pine embryogenesis. Journal of Experimental Botany. 2009. 60(4) : 375-1386];

13. Primers for membrane intrinsic protein/Porine MIP1 genes F-GGCCAGAAAAGTTGTCCTTCGC, R-ATGAAGCCCTTCACAACACC; putative auxin induced F-TGGCTCCTAAGCCCACTAAT, R-CTCCACCCACTATGAACACG; anthocyanidine synthase F-GATGCGATAAAGGGAGTTGC, R-GGAGCACTTTTCTGCTTTT; disease resistance protein F-ACAATTGCCCGAAATTGAAG, R-ATTGCAACCCACCATTCTCT; unknown protein 2 F-AAAGGCATCCCCTGTTTCTT, R-GGACTAGCCCTGGGACTACC; unknown protein 3 F-ATTCATTGCGCTTTTCTGCT, R-CGCTTTGTGTTTAGCAGGTG; unknown protein 4 F-CGAAGCCATTGCTTTTCTTC, R-TCCTCATGGATCGAATCTCC; aldehyde dehydrogenase homolog F-CGTGGGGAATTCTCGTTTTA, R-TTGAGTGCAAGCCTAGCTGA; endoglucanase 1 F-AAGTCCGGATTCTTCAGTGC, R-AGAGTGCTGCAATGACTCCA; PR10/BetV1 F-AGCGAGCTGGACCTGTAACT, R-GGGCAAGGCTATCTTCCTTT; Clavata1 F-TAATAATCTGAGCGGCAGCA, R-TTCGAGGAAACATCAAGCTG; tau class glutathione S-transferase F-AATACATCGAGGAGGCATGG, R-TATGGGTCTTCGGGCATAAG; subtilisine like protease precursor F-CATGAACTATCCGACCATCG, R-GAGGCGGATCCTATGTTTGT; kanamycin F-GGACGGCGGCTTTGTTG, R-CTGCGTTGTCGGGAAGATG [Heller G., Adomas A., Li G. et al. Transcriptional analysis of Pinus sylvestris roots challenged with the ectomycorrhizal fungus Laccaria bicolor. BMC Plant Biology. 2008. 8:19];

14. Primers to ADC genes (arginine decarboxylase) F- AGAAATTGGGGATGCTGGAT, R- GCCATCACGATTGTATTCACC; ODC (ornithine decarboxylase) F- AAGCGGTGAAGCCATTAAAA, R- TTGCGTTGCAGACGTATTTC; Act (Actin) F- GCTTGCTTATGTAGCCCTTGA, R- GGTCTTGGCAATCCACATCT [Vuosku J., Jokela A., Läärä E.et al. Consistency of Polyamine Profiles and Expression of Arginine Decarboxylase in Mitosis during Zygotic Embryogenesis of Scots Pine. plant physiology. 2006. 142: 1027-1038].

The listed oligonucleotides are strictly specific to these genes and cannot be used to study the expression of the DHN1 (dehydrin 1), DHN7 (dehydrin 7), SGS3 (suppressor of gene silencing), CPK (calcium-dependent protein kinase), CYP450 (cytochrome P450) genes. ), ERD3 (early responsive to dehydration 3), CCOAOMT1 (caffeoyl-CoA-O-methyltransferase 1) , except for the LEA (late embryogenesis abundant protein) and ACT (actin) genes. For the LEA and ACT genes, there are no oligonucleotide sequences in the literature for studying expression using fluorescently labeled probes. However, the use of gene-specific fluorescently labeled probes together with specific primers can significantly reduce the likelihood of non-specific DNA amplification by increasing the total length of synthetic nucleotides for PCR, since when using an intercalating dye, only forward and reverse primers are used, and complementary to the internal part is not used. amplified DNA probe.

Based on the analysis of the prior art, a technical task was set to develop a set of synthetic oligonucleotides, consisting of forward primers, reverse primers and fluorescently labeled probes specific to the DHN1, DHN7, SGS3, CPK, CYP450, ERD3, CCOAOMT1, LEA and ACT genes of Scots pine.

The technical result consists in increasing the reliability of assessing the level of expression of Scots pine genes ( DHN1, DHN7, SGS3, CPK, CYP450, ERD3, CCOAOMT1, LEA and ACT ), due to the use of fluorescently labeled probes complementary to the inner part of the amplified DNA, which leads to an increase in the total length synthetic nucleotides and reduce the risk of non-specific amplification.

The technical result is achieved by the fact that a set of synthetic oligonucleotides for determining the level of expression of the DHN1 , DHN7 , SGS3 , CPK , CYP450 , ERD3 , CCOAOMT1 , LEA and ACT genes of Scotch pine ( Pinus sylvestris L.) by real-time PCR, including the following sequences:

DHN1_F: GGAAGTGGAGAAGAAAGTAGGTT

DHN1_R: CCGGGGAGTTTCTCTTTGATTTTA

DHN1_Z: FAM-TAACAAGAAGGAAGGGGAAGGGAAAGAAGAAGAAG-BHQ1

DHN7_F: GGCAAAAAGAAGGAAGATGAGGG

DHN7_R: GGCAGCTTCTGTTTTGATTTTATCAAC

DHN7_Z: FAM-TTCTTCCCCTCTTCACATCTACCCACCCTCAGT-BHQ1

SGS3_F: ATCGGAGCTTGGAAGGGGA

SGS3_R: CTTGGTTTCCATTCCTCTGTTG

SGS3_Z: FAM-TAAGGAACGGGGATGGAAGGAGTCAGG-BHQ1

CPK_F: CTACAAAGCAGCATCAGCAGTG

CPK_R: CGATGGTCTTGCACGCGAAC

CPK_Z: FAM-TCGGGCGTGGGCAGTTCGGCGTA-BHQ1

CYP450_F: ATTTGGATTGGCTCGATT

CYP450_R: GATGGTTTCCCGTGTAAT

CYP450_Z: FAM-TGCGGAGAAAATAATAGACGACCA-BHQ1

ERD3_F: GTTGATGGTCTTTTTCAGTGCAG

ERD3_R: CTGCGGCTAGATTCTTGACA

ERD3_Z: FAM-TGTGTTGTTGGAAATGGACCGTATATTAAGACCTG-BHQ1

CCOAOMT1_F: TGCAGTATATATTGGAAACGAGC

CCOAOMT1_R: TTCTTGGCGTTAATGAGCTTC

CCOAOMT1_Z: FAM-TACTTCTGCCGATGAGGGTCAATTTCTGGG-BHQ1

LEA_F: TATGATCATGACCTACCAATTGGG

LEA_R: CGTAAGGCACTGTCACTGGAA

LEA_Z: FAM-TCTGGAACAATTGTAGATCCTGGGTCAGTGAAGG-BHQ1

ACT_F: TGAGCTTCGAGTTGCTCCAG

ACT_R: CCAGTTGTACGACCACTTGC

ACT_Z: FAM-TCCCAAGGCAAACAGAGAGAAGATGACTCAG-BHQ1

The invention is illustrated by graphic materials.

Figure 1. Real-time PCR results using specific primers and probes for the CPK gene.

Figure 2. Relative transcription levels of CPK, CYP450, CCOAOMT1, SGS3, ERD3, DHN1, DHN7, and LEA genes in Scotch pine seedling needles under no stress (2 hours after watering) and under severe stress (10 days after watering). Gene expression values were calculated relative to the expression level of the Actin gene ( ACT ).

The essence of the invention.

The technical result is the creation of a set of synthetic oligonucleotides, which makes it possible to determine the level of expression of the DHN1, DHN7, SGS3, CPK, CYP450, ERD3, CCOAOMT1, LEA and ACT genes of Scotch pine with high efficiency and specificity by real-time PCR. The kit includes forward primers (F), reverse primers (R) and probes (Z) for each gene having the following nucleotide sequences:

1) DHN1(dehydrin 1) gene:

DHN1_F: GGAAGTGGAGAAGAAAGTAGGTT

DHN1_R: CCGGGGAGTTTCTCTTTGATTTTA

DHN1_Z: FAM-TAACAAGAAGGAAGGGGAAGGGAAAGAAGAAGAAG-BHQ1

2) DHN7 gene (dehydrin 7):

DHN7_F: GGCAAAAAGAAGGAAGATGAGGG

DHN7_R: GGCAGCTTCTGTTTTGATTTTATCAAC

DHN7_Z: FAM-TTCTTCCCCTCTTCACATCTACCCACCCTCAGT-BHQ1

3) SGS3 (suppressor of gene silencing):

SGS3_F :ATCGGAGCTTGGAAGGGGA

SGS3_R : CTTGGTTTCCATTCCTCTGTTG

SGS3_Z : FAM-TAAGGAACGGGGATGGAAGGAGTCAGG-BHQ1

4) CPK (calcium-dependent protein kinase):

CPK_F: CTACAAAGCAGCATCAGCAGTG

CPK_R: CGATGGTCTTGCACGCGAAC

CPK_Z: FAM-TCGGGCGTGGGCAGTTCGGCGTA-BHQ1

5) CYP450 (cytochrome P450):

CYP450_F: ATTTGGATTGGCTCGATT

CYP450_R: GATGGTTTCCCGTGTAAT

CYP450_Z: FAM-TGCGGAGAAAATAATAGACGACCA-BHQ1

6) ERD3 (early responsive to dehydration 3):

ERD3_F: GTTGATGGTCTTTTTCAGTGCAG

ERD3_R: CTGCGGCTAGATTCTTGACA

ERD3_Z: FAM-TGTGTTGTTGGAAATGGACCGTATATTAAGACCTG-BHQ1

7) CCOAOMT1 (caffeoyl-CoA-O-methyltransferase 1):

CCOAOMT1_F: TGCAGTATATATTGGAAACGAGC

CCOAOMT1_R: TTCTTGGCGTTAATGAGCTTC

CCOAOMT1_Z: FAM-TACTTCTGCCGATGAGGGTCAATTTCTGGG-BHQ1

8) LEA (late embryogenesis abundant protein):

LEA_F: TATGATCATGACCTACCAATTGGG

LEA_R: CGTAAGGCACTGTCACTGGAA

LEA_Z: FAM-TCTGGAACAATTGTAGATCCTGGGTCAGTGAAGG-BHQ1

9) ACT (actin) :

ACT_F: TGAGCTTCGAGTTGCTCCAG

ACT_R: CCAGTTGTACGACCACTTGC

ACT_Z: FAM-TCCCAAGGCAAACAGAGAGAAGATGACTCAG-BHQ1

Determination of the level of expression is carried out in a known manner through the use of a fluorescently labeled probe, which allows monitoring the dynamics of accumulation of the polymerase chain reaction product in real time. A fluorescently labeled real-time PCR probe is a synthetic oligonucleotide to which a fluorophore molecule (eg, carboxyfluorescein (FAM) molecule) and a fluorescence quencher molecule (eg, Black Hole Quenchers (BHQ1) molecule) are attached.

The created set of synthetic oligonucleotides makes it possible to effectively and with high specificity evaluate the level of expression of the DHN1, DHN7, SGS3, CPK, CYP450, ERD3, CCOAOMT1, LEA, and ACT genes by real-time PCR. The high specificity of amplification of the target DNA region is ensured by an increase in the total length of synthetic oligonucleotides complementary to the target region to 61-82 base pairs, compared with the detection of PCR results based on the SYBR Green intercalating dye, when two primers are used, with a total length of about 40-50 base pairs.

Method for carrying out the invention.

The created set of synthetic olgonucleotides is used as follows.

1. Collection of tissue samples from Scots pine. Needles, buds, and roots can serve as initial material for studying gene expression.

2. Isolation of total RNA from plant tissue using one of the known methods. Classical liquid-phase methods can be used to isolate RNA, including extraction of nucleic acids with a lysing CTAB buffer and purification with chloroform. Also, commercial kits of reagents and columns can be used to obtain RNA preparations.

3. Obtaining complementary DNA (cDNA) using a reverse transcription reaction using a universal oligo(dT) primer using standard protocols. The reverse transcription reaction is carried out using commercial kits containing a reverse transcriptase enzyme preparation (eg, murine leukemia virus reverse transcriptase, MMLV reversetase).

4. Real-time PCR using the obtained cDNA preparations and the created set of synthetic oligonucleotides. Real-time PCR is carried out using specialized equipment (real-time PCR thermal cycler) in accordance with the following temperature regime:

1) for oligonucleotides specific to SGS3, CPK, CYP450, CCOAOMT1 and ACT genes: preliminary denaturation at 95°C - 10 min; 45 cycles: denaturation 95°C - 15 sec, annealing 57°C - 30 sec, elongation 72°C - 30 sec+fluorescence signal detection;

2) for oligonucleotides specific to DHN1, DHN7 and ERD3 genes: preliminary denaturation at 95°C - 10 min; 45 cycles: denaturation 95°С - 15 sec, annealing 61(- 30 sec, elongation 72°С - 30 sec + fluorescence signal detection.

5. Analysis of the results of real-time PCR and assessment of changes in the expression level of the DHN1, DHN7, SGS3, CPK, CYP450, ERD3, CCOAOMT1 and ACT genes compared with the AST gene, related to the so-called housekeeping genes ) whose expression level is relatively constant. To quantify the content of the matrix in the sample during PCR-RT, the Ct indicator is used - the polymerase chain reaction cycle at which the fluorescence level exceeded the threshold value.

An example of the use of the invention.

The invention was used to study the variability in the expression level of the DHN1 , DHN7 , SGS3 , CPK , CYP450 , ERD3, CCOAOMT1 and LEA genes in Scots pine seedlings under water stress. The Actin gene ( AST ) was used as a reference gene for calculating relative expression. Seedlings of Scotch pine with a closed root system were exposed to drought for a long time, and needle samples were taken at certain time intervals to isolate total RNA. Total RNA was isolated from needles according to the protocol proposed by M. Su et al. [Su M., Zang W., Yao N., Huang M. Isolation of high quality RNA from various Populus tissues. Physiology of plants. 2009. V.56. No. 5. S.791-795.].

For the synthesis of single-stranded cDNA, a commercial kit "MMLV RT kit" (Evrogen, Russia) and the manufacturer's protocol were used:

1) for each RNA sample, mixture No. 1 was prepared in PCR tubes, total volume 9 µl:

2 µl - RNA solution (1-2 µg RNA)

1 µl - oligo(dT) primer

6 µl - deionized water

The tubes were incubated for 2 min at +70°C;

2) prepared mixture No. 2 (the volumes are indicated for 1 RNA sample, the total volume is 11 µl):

4 µl - 5X buffer for the synthesis of the first strand cDNA (first-strand buffer)

2 µl - DTT (20 mM)

2 µl - dNTP mix (10 mM)

1 µl - MMLV revertase

2 µl - deionized water

3) mix #2 was added to mix #1, mixed and incubated for 60 min at 42°C.

4) to stop the reaction, the mixture was heated for 10 min at 70°C, then placed on ice.

For all cDNA samples, RT-PCR was sequentially performed in triplicate with specific oligonucleotides under the following temperature conditions:

1) for oligonucleotides specific to SGS3, CPK, CYP450, CCOAOMT1 and ACT genes: preliminary denaturation at 95°C - 10 min; 45 cycles: denaturation 95°C - 15 sec, annealing 57°C - 30 sec, elongation 72°C - 30 sec+fluorescence signal detection;

2) for oligonucleotides specific to DHN1, DHN7, ERD3 and LEA genes: preliminary denaturation at 95°C - 10 min; 45 cycles: denaturation 95°С - 15 sec, annealing 61(- 30 sec, elongation 72°С - 30 sec + fluorescence signal detection.

For real-time PCR, ready-made mixture for PCR "qPCRmix-HS" (Evrogen, Russia) and CFX96 Touch Thermal Cycler (Bio-Rad, USA) were used. The reaction mixture for real-time PCR, with a total volume of 20 µl, contained: qPCRmix-HS - 4 µl, cDNA - 2.0 µl, forward and reverse primers - 0.3 µl each, fluorescent probe (FAM dye) - 0.3 µl , deionized water - 13.1 µl.

After the completion of the amplification reaction, the data are stored in the form of graphs illustrating the rate of accumulation of amplification products (figure 1) and Ct values (threshold cycle) determined in the CFX Manager program. The Ct values were transferred to Microsoft Excel and mathematical data processing was performed. The relative level of gene transcription (R) was calculated by formula (1):

где

Where

where ΔCt is the difference between the values of the threshold cycles for the reference and target genes.

The level of gene transcription was expressed in relative units (relative units) (figure 2).

Results. As a result of studying the variability of gene expression in the needles of Scotch pine seedlings, a 4.6-9.1-fold increase in the level of transcription of the DHN1 , DHN7 and LEA genes under the influence of water stress was revealed.

The positive effect of the proposed invention is the creation of a unique set of synthetic oligonucleotides for studying the level of gene expression (DHN1, DHN7, SGS3, CPK, CYP450, ERD3, CCOAOMT1, LEA and ACT), providing high reliability and reducing the risk of non-specific DNA amplification during polymerase chain reaction for due to the use of fluorescently labeled probes, which leads to an increase in the total length of synthetic nucleotides.

The work was carried out using the resources of the Center for Collective Use "Ecology, biotechnologies and processes for obtaining environmentally friendly energy carriers" of the Volga State Technological University, Yoshkar-Ola, with the financial support of the Ministry of Science and Higher Education of the Russian Federation (agreement No. 075-15-2021-674 dated 28.07.21 ).

Claims (28)

A set of synthetic oligonucleotides for determining the level of expression of the DHN1 , DHN7 , SGS3 , CPK , CYP450 , ERD3 , CCOAOMT1 , LEA and ACT genes of Scotch pine ( Pinus sylvestris L.) by real-time PCR, including the following sequences: DHN1_F: GGAAGTGGAGAAGAAAGTAGGTT DHN1_R: CCGGGGAGTTTCTCTTTGATTTTA DHN1_Z: FAM-TAACAAGAAGGAAGGGGAAGGGAAAGAAGAAGAAG-BHQ1 DHN7_F: GGCAAAAAGAAGGAAGATGAGGG DHN7_R: GGCAGCTTCTGTTTTGATTTTATCAAC DHN7_Z: FAM-TTCTTCCCCTCTTCACATCTACCCACCCTCAGT-BHQ1 SGS3_F: ATCGGAGCTTGGAAGGGGA SGS3_R: CTTGGTTTCCATTCCTCTGTTG SGS3_Z: FAM-TAAGGAACGGGGATGGAAGGAGTCAGG-BHQ1 CPK_F: CTACAAAGCAGCATCAGCAGTG CPK_R: CGATGGTCTTGCACGCGAAC CPK_Z: FAM-TCGGGCGTGGGCAGTTCGGCGTA-BHQ1 CYP450_F: ATTTGGATTGGCTCGATT CYP450_R: GATGGTTTCCCGTGTAAT CYP450_Z: FAM-TGCGGAGAAAATAATAGACGACCA-BHQ1 ERD3_F: GTTGATGGTCTTTTTCAGTGCAG ERD3_R: CTGCGGCTAGATTCTTGACA ERD3_Z: FAM-TGTGTTGTTGGAAATGGACCGTATATTAAGACCTG-BHQ1 CCOAOMT1_F: TGCAGTATATATTGGAAACGAGC CCOAOMT1_R: TTCTTGGCGTTAATGAGCTTC CCOAOMT1_Z: FAM-TACTTCTGCCGATGAGGGTCAATTTCTGGG-BHQ1 LEA_F: TATGATCATGACCTACCAATTGGG LEA_R: CGTAAGGCACTGTCACTGGAA LEA_Z: FAM-TCTGGAACAATTGTAGATCCTGGGTCAGTGAAGG-BHQ1 ACT_F: TGAGCTTCGAGTTGCTCCAG ACT_R: CCAGTTGTACGACCACTTGC ACT_Z: FAM-TCCCAAGGCAAACAGAGAGAAGATGACTCAG-BHQ1

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