Fengdan α-linolenic acid synthase encoding gene and its application

Abstract

The present invention belongs to the field of agricultural biotechnology, and discloses three key enzyme-encoding genes for α-linolenic acid synthesis in Paeonia ostii and their applications. Among them, the gene encoding stearoyl-ACP desaturase (PoSAD) in Paeonia ostii regulates the conversion of stearic acid into oleic acid, the gene encoding fatty acid desaturase 2 (PoFAD2) in Paeonia ostii regulates the conversion of oleic acid into linoleic acid, and the gene encoding fatty acid desaturase 3 (PoFAD3) in Paeonia ostii regulates the conversion of linoleic acid into α-linolenic acid. The present invention also relates to the applications of the proteins encoded by the above three genes. By using the expression of these genes in plant seeds, wild Paeonia ostii and its similar oil-using plant materials that synthesize α-linolenic acid are selected and bred to evaluate their high-quality and high-yield varieties; by using these genes and their parents for biological breeding, hybridization or other breeding methods, the ratios and / or contents of oleic acid, linoleic acid or α-linolenic acid synthesized by oil-using Paeonia ostii and its similar plants are directionally improved and enhanced, and oil varieties rich in specific unsaturated fatty acids, especially rich in α-linolenic acid, are cultivated.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and more particularly to the Fengdan α-linolenic acid synthase genes PoSAD (SEQ ID No.1), PoFAD2 (SEQ ID No.3) and PoFAD3 (SEQ ID No.5), and the proteins PoSAD (SEQ ID No.2), PoFAD2 (SEQ ID No.4) and PoFAD3 (SEQ ID No.6) encoded thereby. The present invention also relates to the cloning and application methods of the PoSAD, PoFAD2 and PoFAD3 genes. The fatty acid dehydrogenases and their encoding genes of the present invention are of great significance for screening and improving oil-using Fengdan and its similar plant varieties containing the above genes and the characteristic unsaturated fatty acid content composition regulated thereby. Background Art

[0002] Peonies (Paeonia spp.) have been widely used as ornamental flowers and Chinese medicinal materials Cortex Moutan for more than 1,600 years (Li J J, Zhang X F, Zhao X Q, et al. Chinese Peonies [M]. China Encyclopedia Press, 2011; Liu T F, Li C H, Wan T T. Chronology of Ancient Gardens. Chinese Landscape Architecture, 2005, 21(5): 76-78.). Fengdan (Paeonia ostiivar. lishizhenii) has always been used as a rootstock to breed ornamental peonies and as a medicinal material Cortex Moutan for planting. It is a plant of the leathery flower disc subgroup of the Paeonia section of the Paeoniaceae family and is propagated by seeds. Fengdan has strong stress resistance and wide adaptability, and has extremely strong resistance to various abiotic stresses such as drought, cold, and mild salinity, and can adapt to a variety of environments and climates (Wang Y, Dong C, Xue Z, et al. De novo, transcriptome sequencing and discovery of genes related to copper tolerance in Paeonia ostii [J]. Gene, 2016, 576(1 Pt 1): 126-135.). It has been widely introduced in 28 provinces, municipalities and autonomous regions in China (Hong Kong, Macao and Taiwan have not been counted), and is an important stress-resistant greening pioneer tree species of the Paeonia section plants that can be planted in arid and difficult areas.

[0003] In recent years, it has been found that the seeds of the Robinia pseudoacacia are rich in oil. The oil content of the seeds is higher than 20%, and the oil content of the shelled seeds is as high as 30% (Xiu Yu. Basic research on the mechanism of FpDREB2A gene regulating the drought resistance of Robinia pseudoacacia taproot growth and the selection and improvement of drought-resistant high-quality materials [D]. Beijing Forestry University, 2016.). Fengdan has single petals, normal pistil and stamen development, and large seed yield. It is the only oil-bearing peony variety with estimated seed yield, which reaches 2,250-6,000 kg / ha (Jiang Peijun. 1,500 mu of oil-bearing peonies in Heze enter the "harvest period" with an acreage yield of about 200 kg [Z]. 201382.), which is similar to the yields of soybean and rapeseed (Wang X, Jiang GL, Green M, et al. Identification and validation of quantitative trait loci for seed yield, oil and protein contents in two recombinant inbred line populations of soybean [J]. Molecular Genetics & Genomics, 2014, 289 (5): 935-949; Kelly AA, Shaw E, Powers SJ, et al. Suppression of the SUGAR-DEPENDENT1, triacylglycerol lipase family during seed development enhances oil yield in oilseed rape (Brassica napus L.)[J].Plant Biotechnology Journal,2013,11(3):355-361;Shi T,Li R,Zhao Z,et al.QTL for yield traits and their associationwith functional genes in response to phosphorus deficiency in Brassica napus[J].Plos One,2012,8(1):1970.). According to the oil content of 20%, the oil yield of Fengdan seed can reach 450-1,200kg / ha. The content of unsaturated fatty acids in Fengdan seed kernel is 89-92%, among which α-linolenic acid (C18:3Δ 9c,12c,15c, a content of omega-3 unsaturated fatty acids) higher than 40% (Xiuyu. Basic research on the mechanism of FpDREB2A gene regulating the taproot growth and drought resistance of Robinia pseudoacacia and the selection and improvement of drought-resistant and high-quality materials [D]. Beijing Forestry University, 2016.). Fengdan has been expanded from traditional ornamental and medicinal plants to an important woody oil resource and new resource food (Announcement No. 9 of the Ministry of Health of the People's Republic of China in 2011 [EB / OL]. www.moh.gov.cn 2011-03-29).

[0004] Fengdan seed oil mainly exists in the endosperm and its composition is mainly unsaturated fatty acids. Aflatoxin B1, lead, arsenic, mercury, chromium, and peroxide value all meet the national food hygiene standards (Xiu Yu. Basic research on the mechanism of FpDREB2A gene regulating the growth of Robinia pseudoacacia taproot and drought resistance and breeding and improvement of drought-resistant and high-quality materials [D]. Beijing Forestry University, 2016.). Its α-linolenic acid content is much higher than that of the other four main woody vegetable oils (olive oil, 0 - 1.5%; palm oil, 0.3%; coconut oil, 0 - 0.2%; camellia oil: 0.5 - 0.9%) (Bezard J, Bugaut M, Clement G. Triglyceride composition of coconut oil [J]. Journal of Oil&Fat Industries, 1971, 48(3):134 - 139; Sabelli P A, Dante R A, Nguyen H N, et al. Expression, regulation and activity of a B2-type cyclin inmitotic and endoreduplicating maize endosperm [J]. Frontiers in Plant Science, 2013, 5(5):561; López-Villalobos A, Rol, Hornung. Changes in fatty acidcomposition during development of tissues of coconut (Cocos nucifera L.) embryos in the intact nut and in vitro [J]. Journal of Experimental Botany, 2001, 52(358):933 - 942; Nevin K G, Rajamohan T. Virgin coconut oil supplementeddiet increases the antioxidant status in rats [J]. Food Chemistry, 2006, 99(2):260 - 266; Terés S, Barcelócoblijn G, Benet M, et al. Oleic acid content isresponsible for the reduction in blood pressure induced by olive oil [J].Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(37): 13811 - 13816; Torstensen B E,. Lie, L.Lipidmetabolism and tissue composition in Atlantic salmon(Salmo salar L.)-Effectsof capelin oil,palm oil,and oleic acid-enriched sunflower oil as dietary lipid sources[J].Lipids,2012,35(6):653-664;Wang Y,Tan XF,Xie P,et al.Thephysical and chemical properties of the seed oil and its fatty acidcompositions of Camellia oleifera superior clones species[J].Journal ofCentral South University of Forestry&Technology,2011,31(6):70-74.), helps to reduce the risk of diseases such as diabetes, asthma, prostate cancer and breast cancer (Leitzmann MF,Stampfer MJ,Michaud DS,et al.Dietary intake of n-3and n-6fatty acids and the risk ofprostate cancer[J].American Journal of Clinical Nutrition, 2004, 80 (1): 204-216; Myles IA, Pincus NB, Fontecilla NM, et al. Effects of parental omega-3fattyacid intake on offspring microbiome and immunity [J]. Plos One, 2014, 9 (1): e87181; Thorsdottir I, Hill J, Ramel A. Omega-3 fatty acid supply from milk associates with Lower type 2diabetes in men and coronary heart disease in women[J].Preventive Medicine, 2004, 39(3): 630 - 634; Wu M, Harvey K A, Ruzmetov N, et al. Omega-3 polyunsaturated fatty acids attenuate breast cancer growth through activation of a neutral sphingomyelinase-mediated pathway[J]. International Journal of Cancer Journal International Du Cancer, 2005, 117(3): 340 - 348.). And the content ratios of oleic acid (C18:1Δ 9c ), linoleic acid (C18:2Δ 9c,12c ), and α-linolenic acid are 23%: 25%: 44% respectively, approximately 1: 1: 2, and it is also an important health food for human brain health (Deng R X, Liu Z, Qin L L, et al. Optimization of supercritical CO2 extraction and analysis of chemical composition of peony seed oil[J]. Food Science, 2010, 10: 142 - 145; Li S S, Yuan R Y, Chen L G, et al. Systematic qualitative and quantitative assessment of fatty acids in the seeds of 60 tree peony (Paeonia section Moutan DC.) cultivars by GC-MS[J]. Food Chemistry, 2015, 173: 133 - 140. McNamara R K, Carlson S E. Role of omega-3 fatty acids in brain development and function: potential implications for the pathogenesis and prevention of psychopathology[J]. Prostaglandins, Leukotrienes & Essential Fatty Acids, 2006, 75(4 - 5): 329 - 349.).

[0005] In summary, Paeonia ostii has strong stress resistance and wide adaptability; its seed yield is high and the oil content is rich; a large amount of unsaturated fatty acids are contained in its seed oil, especially rich in omega-3 unsaturated fatty acids which are beneficial to human health. In 2011, the National Development and Reform Commission of the People's Republic of China has approved Paeonia ostii as a new resource food (Su J, Ma C, Liu C, et al. Hypolipidemic activity of peony seed oil rich in α-linolenic, is mediated through inhibition of lipogenesis and upregulation of fatty acid β-oxidation[J]. Journal of Food Science, 2016, 81(4): H1001-H1009.), making it an important woody healthy oil variety that can be popularized and applied in the vast arid and semi-arid regions of our country.

[0006] The spatio-temporal expression of genes related to fatty acid synthesis is highly correlated with the fatty acid composition in plant seeds (Gu K, Yi C, Tian D, et al. Expression of fatty acid and lipid biosynthetic genes in developing endosperm of Jatropha curcas [J]. Biotechnology for Biofuels, 2012, 5(1):47.). In endosperm tissue, malonyl-CoA undergoes 7 rounds of reactions to generate C16:0-ACP and is extended to generate C18:0-ACP; C18:0-ACP is dehydrogenated by stearoyl-ACP desaturase (SAD) to produce C18:1-ACP and hydrolyzed to generate oleic acid; oleic acid is sequentially dehydrogenated by fatty acid desaturase 2 (FAD2) and fatty acid desaturase 3 (FAD3) to generate linoleic acid and α-linolenic acid (Bates P D, Stymne S, Ohlrogge J, et al. Biochemical pathways in seed oil synthesis [J]. Current Opinion in Plant Biology, 2013, 16(3):358-364; Huang J, Tong Z, Zhang Q, et al. The mechanism of high contents of oil and oleic acid revealed by transcriptomic and lipidomic analysis during embryogenesis in Carya cathayensis, Sarg [J]. Bmc Genomics, 2016, 17(1):1-18.).At present, the SAD genes have been cloned from plants such as Arabidopsis thaliana, Camellia oleifera, and Jatropha curcas (Zhang Dangquan, Tan Xiaofeng, Chen Hongpeng, et al. Cloning and bioinformatics analysis of the full-length cDNA of the SAD gene from Camellia oleifera [J]. Scientia Silvae Sinicae, 2008, 44(02): 155-159; Luo Tong. Study on cold resistance, cloning and expression of the SAD gene in Jatropha curcas [D]. Sichuan University, 2006.); the FAD2 genes have been cloned from plants such as Brassica napus, Arachis hypogaea, and Zea mays (Tan Xiaofeng, Chen Hongpeng, Zhang Dangquan, et al. Cloning and sequence analysis of the full-length cDNA of the FAD2 gene from Camellia oleifera [J]. Scientia Silvae Sinicae, 2008, 44(03): 70-75; Huang Bingyan, Zhang Xinyou, Miao Lijuan, et al. Analysis of fatty acids in transgenic seeds of Arachis hypogaea by RNAi vector transformation of the FAD2 gene [J]. Chinese Journal of Oil Crop Sciences, 2008, 30(03): 290-293; Tao Fang, Zhu Suwen, Fan Jun, et al. Cloning and sequence analysis of the FAD2 gene from Zea mays [J]. Acta Phytophysiol Sin, 2006, 32(6): 649-656.), and the FAD3 genes have been obtained from plants such as Perilla frutescens, Linum usitatissimum, and Jatropha curcas (Fu Yuhua, Chen Xin, Wang Wenquan. Cloning and subcellular localization of the FAD3 gene from Jatropha curcas [J]. Chinese Journal of Tropical Crops, 2012, 33(11): 2030-2034; Bai Ruiying, Lu Junxing, Huang Xinglin, et al. Cloning and expression analysis of the FAD3 gene from Perilla frutescens [J]. Molecular Plant Breeding, 2015(12); Chen Fang. Cloning, vector construction and genetic transformation of the FAD3 gene from Linum usitatissimum [D]. Gansu Agricultural University, 2014.).

[0007] Transcriptome analysis of Paeonia ostii showed that stearoyl-ACP desaturase gene (PoSAD), fatty acid desaturase 2 gene (PoFAD2), and fatty acid desaturase 3 gene (PoFAD3) were highly expressed during endosperm development (Xiu Yu. Basic research on the mechanism of regulating the taproot growth and drought resistance of Robinia pseudoacacia by FpDREB2A gene and breeding improved drought-resistant and high-quality materials [D]. Beijing Forestry University, 2016; Song Shuxiang, Guo Xianfeng, Ma Yan, et al. Cloning and expression analysis of fatty acid desaturase gene PoFAD_2 from Paeonia ostii [J]. Acta Horticulturae Sinica, 2016, 43(02): 347-355.). It was confirmed that PoSAD, PoFAD2, and PoFAD3 genes in Paeonia ostii co-regulated the synthesis and accumulation of α-linolenic acid in Paeonia ostii seeds. Their division of labor was clear and the process was continuous. They were key genes inseparable from the synthesis of α-linolenic acid. The PoSAD, PoFAD2, and PoFAD3 genes described in the present invention had relatively high similarities with the fatty acid desaturases of the herbaceous plant Paeonia lactiflora in the same genus, which were 94%, 97%, and 97% respectively, indicating that the same genus was not the same; the PoFAD2 gene described in the present invention had the highest similarity with the FAD2 gene of Paeonia ostii reported in the literature (Song Shuxiang, Guo Xianfeng, Ma Yan, et al. Cloning and expression analysis of fatty acid desaturase gene PoFAD_2 from Paeonia ostii [J]. Acta Horticulturae Sinica, 2016, 43(02): 347-355.) but had two obvious characteristics: First, there were 37 more nucleotides in the 3' non-coding region; Second, there were 2 different amino acid residues in the encoded protein. PoFAD2 of the present invention and FAD2 of Paeonia ostii in the literature (Song Shuxiang et al., 2016) might be homologous genes but not the same gene, and their differences might be closely related to post-transcriptional regulation of genes and the activity of encoded proteins (Xiu Y, Iqbal A, Zhu C, et al. Improvement and transcriptome analysis of root architecture by overexpression of Fraxinus pennsylvanica DREB2A transcription factor in Robinia pseudoacacia L.'Idaho'. [J]. Plant Biotechnology Journal, 2016.). The above-mentioned PoSAD, PoFAD2, and PoFAD3 genes in Paeonia ostii had important theoretical significance for improving and enhancing the edible value and quality of Paeonia suffruticosa Andr. seed oil; they had important application value for screening wild varieties with high yields of characteristic unsaturated fatty acids, directionally improving oil-using varieties containing characteristic unsaturated fatty acids, and evaluating high-quality and high-yield varieties with the content and proportion of characteristic unsaturated fatty acids meeting people's needs. Summary of the Invention

[0008] The first key point of the invention: The present invention provides the key enzyme genes PoSAD (SEQ ID No.1), PoFAD2 (SEQ ID No.3), PoFAD3 (SEQ ID No.5) for α-linolenic acid synthesis in Paeonia ostii, and the proteins (SEQ ID No.2, SEQ ID No.4, SEQ ID No.6) encoded by the key enzyme genes for α-linolenic acid synthesis in Paeonia ostii.

[0009] The second key point of the invention: The present invention provides a preparation method for the fatty acid dehydrogenase genes PoSAD, PoFAD2 and PoFAD3. Extract RNA from the developing endosperm tissue of Paeonia ostii. Then, construct a transcriptome library for high-throughput transcriptome sequencing. After sequence splicing alignment and data bioinformatics analysis, predict the key enzyme gene sequences for α-linolenic acid synthesis during the development of Paeonia ostii endosperm. Then, use the above key gene sequences to compare with other species through the BLAST online program on GenBank to obtain the conserved sequences of Paeonia ostii fatty acid dehydrogenase genes. Design primers to amplify the gene conserved sequences by RT-PCR. Use the conserved sequences to design primers to obtain the 3'-end and 5'-end sequences of the genes by the RACE method. Use the DNAMAN software to splice the obtained gene fragments to respectively obtain the full-length cDNA sequences of the Paeonia ostii PoSAD, PoFAD2 and PoFAD3 genes. Finally, design primers and use RT-PCR to amplify the full-length ORF sequences of the genes.

[0010] The third key point of the invention: The present invention provides the applications of the PoSAD, PoFAD2 and PoFAD3 genes in screening and directionally improving plant varieties containing specific unsaturated fatty acid compositions. According to the molecular assisted breeding method provided by the present invention, design primers using the conserved sequences of the PoSAD, PoFAD2 and PoFAD3 genes, extract Paeonia ostii endosperm RNA, and detect the gene expression levels in Paeonia ostii endosperm by real-time fluorescence quantitative PCR (RT-qPCR) to evaluate and screen high α-linolenic acid oil-use Paeonia ostii varieties. The present invention respectively constructs recombinant expression vectors for the PoSAD, PoFAD2 and PoFAD3 genes, and uses Agrobacterium tumefaciens transformed with the recombinant expression vectors to infect plant materials to obtain transgenic plant seeds, which changes the contents of characteristic unsaturated fatty acids in the oils of the transgenic plant seeds. Description of the Drawings

[0011] From the following detailed description in conjunction with the drawings, the above features and advantages of the present invention will become more obvious, wherein:

[0012] Figure 1 . The electrophoresis diagram of Paeonia ostii endosperm RNA extracted by the CTAB-LiCl method at three developmental stages. A: CTAB-LiCl method; B: SDS method; C: Trizol method; D: guanidine isothiocyanate method.

[0013] Figure 2 . Full-length electrophoresis patterns of the PoSAD, PoFAD2, and PoFAD3 genes in the present invention. M: DNA Marker; 1: PoSAD; 2: PoFAD2; 3: PoFAD3.

[0014] Figure 3 . ORF sequence and protein prediction sequence of the PoSAD gene.

[0015] Figure 4 . ORF sequence and protein prediction sequence of the PoFAD2 gene.

[0016] Figure 5 . ORF sequence and protein prediction sequence of the PoFAD3 gene.

[0017] Figure 6 . Expression characteristics of the PoSAD, PoFAD2, and PoFAD3 genes in Paeonia ostii endosperm.

[0018] Figure 7 . Schematic diagram of the construction strategy of the expression vectors of the PoSAD, PoFAD2, and PoFAD3 genes.

[0019] Figure 8 . Changes in the contents of characteristic fatty acids in the seeds of transgenic plants (Arabidopsis thaliana). Detailed implementation manners

[0020] The present invention will be further illustrated by the following examples. However, the examples are for illustrative purposes only and are not intended to limit the scope and spirit of the present invention.

[0021] Example 1

[0022] Extract RNA from Paeonia ostii endosperm tissue

[0023] Select the endosperm tissue of Paeonia ostii seeds and extract RNA according to the CTAB-LiCl method. The specific extraction method is as follows: Preheat the CTAB buffer (2% CTAB; 2% PVP; 0.1 mol / L Tris-HCl; 25 mmol / L EDTA; 2.0 mol / L NaCl; 0.5 g / L spermidine; pH = 8.0) for RNA extraction at 65 °C. Weigh about 100 mg of Paeonia ostii endosperm tissue and grind it into powder in liquid nitrogen, quickly transfer it to a centrifuge tube containing 800 μL of CTAB extraction buffer, add 16 μL of β-mercaptoethanol, and invert to mix evenly. Incubate at 65 °C for 5 min and then transfer to ice for cooling. Add an equal volume of chloroform / isoamyl alcohol (v:v = 24:1), invert to mix evenly, centrifuge at 10,000 rpm at 4 °C for 10 min. Carefully aspirate the supernatant into a new centrifuge tube. Repeat this step once. Carefully aspirate the supernatant, add 1 / 4 volume of 10 mol / L LiCl (stored at -20 °C), mix well and precipitate overnight in an ice bath. Centrifuge at 10,000 rpm at 4 °C for 20 min. Discard the supernatant, and dissolve the precipitate in 600 μL of SDS solution (0.5%, w / v). Add an equal volume of chloroform / isoamyl alcohol, invert to mix evenly. Centrifuge at 10,000 rpm at 4 °C for 10 min. Carefully aspirate the supernatant into a new centrifuge tube. Repeat this step once. Aspirate about 400 μL of the supernatant, add 2.5 volumes of absolute ethanol (-20 °C) and 1 / 10 volume of 3 mol / L NaAc solution, and precipitate at -20 °C for 2 h. Centrifuge at 12,000 rpm at 4 °C for 15 min and discard the supernatant. Add 1 mL of 70% ethanol to wash the precipitate, centrifuge at 12,000 rpm at 4 °C for 5 min and discard the supernatant. Add 30 μL of DEPC water to dissolve the precipitate.

[0024] The obtained RNA was detected by 1% agarose gel electrophoresis and Figure 1 the results shown that the 18S and 28S bands of RNA extracted by the CTAB-LiCl method were complete and clear.

[0025] Example 2

[0026] Clone the PoSAD, PoFAD2, and PoFAD3 genes

[0027] Transcriptome sequencing and analysis: Use the method described in Example 1 to extract the total RNA of Paeonia ostii endosperm tissue, enrich mRNA with Oligo(dT) and construct a cDNA library, Illumina HiSeq TMTranscriptome sequencing was performed in 2000. After sequencing, adapter sequences and low-quality sequences were removed, and de novo assembly was carried out using Velvet software to obtain transcripts (Zerbino D R, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs [J]. Genome Research, 2008, 18(5): 821-829.). The above transcripts were respectively aligned to the GO (http: / / www.geneontology.org / ), COG (http: / / www.ncbi.nlm.nih.gov / COG / ), and KEGG (http: / / www.genome.jp / kegg / ) databases to obtain the biological function annotations of the transcripts. According to the gene function annotations and gene expression levels of the transcriptome, the splicing sequences of Paeonia ostii PoSAD, PoFAD2, and PoFAD3 genes were obtained respectively.

[0028] Paeonia ostii seeds were collected. After extracting RNA by the method of Example 1, genomic DNA contamination was removed using RNase Free-DNase (Promega), and then cDNA sequences were obtained using a reverse transcription kit (Promega).

[0029] Cloning of the conserved sequences of PoSAD, PoFAD2, and PoFAD3 genes. According to the transcriptome splicing sequences of Paeonia ostii PoSAD, PoFAD2, and PoFAD3 genes, the primer sequences are shown in Table 1 below. The RT-PCR reaction system includes: 0.5 μl of LA Taq (5 U / μl, TaKaRa), 5 μl of Taq Buffer II (10×, TaKaRa), 8 μl of dNTP Mixture (2.5 mM, TaKaRa), 2 μl of cDNA template (100 ng / μl), 1 μl of each of the upstream and downstream primers (10 μM), and 32.5 μl of Nuclease-free Water. The reaction procedure: 94 °C, 10 min (1 cycle); 95 °C, 30 s, 52 °C, 30 s, 72 °C, 1 min (30 cycles); 72 °C, 10 min (1 cycle). The products obtained by RT-PCR were ligated to the pGEM-T easy vector (Promega) using T4 DNA ligase (Promega), and DH5α (Tiangen) was transformed by the heat shock method. The conserved sequences of PoSAD, PoFAD2, and PoFAD3 genes were obtained by sequencing.

[0030] Table 1. Primers for amplifying conserved sequences

[0031]

[0032] The full-length sequences of PoSAD, PoFAD2, and PoFAD3 genes were obtained by 3’RACE and 5’RACE. According to the conserved sequences of Paeonia ostii PoSAD, PoFAD2, and PoFAD3 genes, the primer sequences are shown in Table 2 below. The 3’-end sequences of PoSAD, PoFAD2, and PoFAD3 genes were cloned by 3’RACE method (TaKaRa) respectively.

[0033] Table 2. Primers for amplifying 3’-end sequences

[0034]

[0035] According to the conserved sequences of Paeonia ostii PoSAD, PoFAD2, and PoFAD3 genes, the primer sequences are shown in Table 3 below. The 5’-end sequences of PoSAD, PoFAD2, and PoFAD3 genes were cloned by 5’RACE method (TaKaRa) respectively.

[0036] Table 3. Primers for amplifying 5’-end sequences

[0037]

[0038] The full-length sequences of PoSAD (SEQ ID No.1), PoFAD2 (SEQ ID No.3), and PoFAD3 (SEQ ID No.5) genes were obtained by splicing with DNAMAN software.

[0039] For the cloning of the ORF sequences of PoSAD, PoFAD2, and PoFAD3 genes, the primer sequences are shown in Table 4 below for RT-PCR. The RT-PCR reaction system includes: 25 μl of PrimeSTAR Max Premix (2×, TaKaRa), 2 μl of cDNA template (100 ng / μl), 1.5 μl of each upstream and downstream primer (10 μM), and 20 μl of Nuclease-free Water. The reaction program is: 94 °C, 3 min (1 cycle); 94 °C, 15 s, 52 °C, 15 s, 72 °C, 30 s (35 cycles); 72 °C, 10 min (1 cycle).

[0040] Table 4. Primers for amplifying gene ORF sequences

[0041]

[0042] By performing RT-PCR on the RNA extracted in Example 1, the product was detected by 1% agarose gel electrophoresis to obtain as Figure 2The results are shown as follows. In the figure, RT-PCR in lanes 2, 3, and 4 respectively yielded a band with a size of approximately 1479 bp, 1333 bp, and 1523 bp.

[0043] After recovering the above 3 bands (UPTECH), add A tails (TaKaRa), ligate with pGEM-T easy vector (Promega), and then transform competent Escherichia coli DH5α cells (Tiangen) by heat shock method. After PCR detection, gene sequencing was performed (BGI). The sequencing results confirmed that: in the experiment, the so-called 1479-bp sequence was the PoSAD gene, and the sequence was included in SEQ ID No.1; the so-called 1333-bp sequence was the PoFAD2 gene, and the sequence was included in SEQ ID No.3; the so-called 1523-bp sequence was the PoFAD3 gene, and the sequence was included in SEQ ID No.5.

[0044] Analysis of the protein sequences encoded by PoSAD, PoFAD2, and PoFAD3 genes. The ORF of the genes was predicted using the DNAMAN software with the universal codon. The SEQ ID No.1 sequence translated into 396 amino acid residues (SEQ ID No.2) ( Figure 3 ), and the predicted protein size was 45.13 KDa, and the isoelectric point was 5.86. Using the BLAST alignment tool for online alignment, it can be seen that the predicted amino acid sequence had more than 80% similarity with the SAD genes of other plants (Paeonia lactiflora, Citrus reticulata, Vitis vinifera, Theobroma cacao), verifying that this sequence was the SAD gene sequence regulating oleic acid synthesis. The SEQ ID No.3 sequence translated into 383 amino acid residues (SEQ ID No.4) ( Figure 4 ), and the predicted protein size was 44.43 KDa, and the isoelectric point was 7.10. The predicted amino acid sequence had more than 80% similarity with the FAD2 genes of other plants (Paeonia lactiflora, Davidia involucrata, Sesamum indicum, Coffea arabica), verifying that this sequence was the FAD2 gene sequence regulating linoleic acid synthesis. The SEQ ID No.5 sequence translated into 435 amino acid residues (SEQ ID No.6) ( Figure 5 ), and the predicted protein size was 49.93 KDa, and the isoelectric point was 7.42. The predicted amino acid sequence had more than 70% similarity with the FAD3 genes of other plants (Paeonia lactiflora, Vitis vinifera, Nelumbo nucifera, Eucalyptus globulus), verifying that this sequence was the FAD3 gene sequence regulating α-linolenic acid synthesis.

[0045] Example 3

[0046] Expression characteristics of PoSAD, PoFAD2, and PoFAD3 genes

[0047] Detection of the expression levels of PoSAD, PoFAD2, and PoFAD3 genes. Seeds of Paeonia ostii at six different developmental stages (S1 - S6) were collected. After extracting RNA using the method of Example 1 and removing genomic DNA contamination with DNase, cDNA sequences were obtained by reverse transcription. RT-qPCR primers were designed according to the gene sequences obtained in Example 2. The fluorescence quantitative PCR method was used to detect the expression levels of PoSAD, PoFAD2, and PoFAD3 genes in Paeonia ostii seeds at different developmental stages. The reaction system included: 10 μl RT-qPCR Mix (Promega), 10 ng cDNA template, 0.3 μl of each upstream and downstream primer (10 μM), and 8.4 μl ddH2O. The PCR program was: 95°C, 10 min (1 cycle); 95°C, 15 s, 52°C, 20 s, 72°C, 30 s (40 cycles); 72°C, 10 min (1 cycle). Using the Ubiquitin gene as an internal reference and taking the stage with the lowest expression level of the target gene as the standard, the relative expression levels of the target genes were calculated using the 2 -ΔΔCt method. The results were obtained as shown in Figure 6 . The PoSAD, PoFAD2, and PoFAD3 genes were all expressed during the development of Paeonia ostii seeds. Among them, the PoSAD gene started to be highly expressed from the S2 stage, and the PoFAD2 and PoFAD3 genes started to be highly expressed from the S4 stage.

[0048] Table 6. Primers for RT-qPCR detection

[0049]

[0050]

[0051] Example 4

[0052] Functional identification of PoSAD, PoFAD2, and PoFAD3 genes

[0053] Construction of plant expression vectors. Construction of PoSAD, PoFAD2, and PoFAD3 gene expression vectors, and the construction strategy was as shown in Figure 7As shown in the figure. Primers were designed according to the following sequence to add double restriction enzyme cleavage sites of SpeI and SalI. The above two restriction enzyme cleavage sites were introduced at both ends of the gene by PCR method (SpeI at the upstream and SalI at the downstream), and then ligated to the T vector and transformed into Escherichia coli DH5α. The plasmid extracted was double digested to obtain a gene fragment with sticky ends; the pBin438 vector (see patent number: ZL00103561.4; Li Taiyuan, Tian Yingchuan, Qin Xiaofeng, etc. Research on highly insect-resistant transgenic tobacco [J]. Science in China, 1994, 24(3): 276-282.) was double digested and the large fragment was recovered. The target gene was inserted into the pBin438 plant expression vector using T4 DNA ligase to obtain three expression vectors pBin438-P 35S-35S -PoSAD, pBin438-P 35S-35S -PoFAD2 and pBin438-P 35S-35S -PoFAD3. After sequencing verification, it was transformed into Agrobacterium tumefaciens strain GV3101.

[0054] Table 5. Primers for constructing plant expression vectors

[0055]

[0056] The characteristic unsaturated fatty acid content in the seeds of transgenic Arabidopsis thaliana was measured. The pBin438 vector was used as a negative control, and pBin438-P 35S-35S -PoSAD, pBin438-P 35S-35S -PoFAD2 and pBin438-P 35S-35S -PoFAD3 expression vectors were respectively transformed into Arabidopsis thaliana (by Agrobacterium-mediated pollen tube pathway method). After harvesting the seeds (T1 generation), transgenic Arabidopsis thaliana was screened on MS medium containing 100 mg / L kanamycin to obtain transgenic Arabidopsis thaliana seeds (T2 generation).

[0057] The fatty acids of transgenic Arabidopsis thaliana seeds were extracted, methylated, and their fatty acid composition was analyzed by high performance gas chromatography (Liu Y F, Li Q T, Lu X, et al. Soybean GmMYB73, promotes lipid accumulation in transgenic plants [J]. Bmc Plant Biology, 2013, 14(1): 1-16.). As Figure 8As shown, the main fatty acid content in transgenic Arabidopsis thaliana seeds changed significantly, and the corresponding unsaturated fatty acid content increased. Among them, the contents of C18:1 and C20:1 in transgenic Arabidopsis thaliana seeds overexpressing the PoSAD gene increased significantly, the content of C18:2 in transgenic Arabidopsis thaliana seeds overexpressing the PoFAD2 gene increased significantly, and the content of C18:3 in transgenic Arabidopsis thaliana seeds overexpressing the PoFAD3 gene increased significantly.

Claims

1. Use of the fatty acid desaturase gene PoFAD3 in the directed improvement of plant varieties with a specific unsaturated fatty acid composition, wherein a recombinant expression vector of the PoFAD3 gene is constructed, and transgenic plant seeds are obtained by infecting plant materials with Agrobacterium tumefaciens transformed with the recombinant expression vector, and the content of specific unsaturated fatty acids in the oil of the transgenic plant seeds is changed, wherein the amino acid sequence encoded by the fatty acid desaturase gene PoFAD3 is as shown in SEQ ID No.6, and the specific unsaturated fatty acid is α-linolenic acid.

2. The use according to claim 1, wherein the nucleotide sequence of the fatty acid desaturase gene PoFAD3 is as shown in SEQ ID No.

5.

3. The use according to claim 1 or 2, wherein the transgenic plant is Arabidopsis thaliana.

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