Bioinformatics analysis of BnFAD2 gene in Brassica napus L.

doi:10.14088/j.cnki.issn0439-8114.2021.11.029

Abstract

Abstract: The gene AtFAD2 was taken as reference sequence, homologous sequence comparison was conducted by BLAST to obtain the homologous copies of FAD2 gene in the genomes of Brassica oleracea, Brassica rapa and Brassica napus. Bioinformatics methods were used to analyze the components of the amino acid sequence, the basic physical and chemical properties of the protein, subcellular localization, hydrophilicity and hydrophobicity, the secondary and tertiary structure of the protein, and the conserved domain of the FAD2 gene editing targets and sgRNA design. Two FAD2 homologus protein sequence were obtained from B. rapa, B. deracea, B.napus. Both subcells were localized in the endoplasmic reticulum, and the peptide chain showed hydrophobness and stability. The predicted secondary and tertiary structure of the protein showed that it was mainly composed of -helix and irregular curl. The conserved structural domain of the protein showed that FAD2 gene was highly sequence conserved, belonging to the Membrane-FADs-like superfamily, and contained PLN02505 conserved structural domain. Phylogenetic tree analysis showed that Brassica napus L was closely related to B. oleracea, B. rapa and B. juncea. The CRISPR-Cas9 gene-editing target analysis revealed that there were 10 highly conserved target sites in the FAD2 gene of Brassica napus L., and the corresponding sgRNA was designed.

Key words: Brassica napus L., FAD2, bioinformatics, sgRNA, oleic acid

CLC Number:

S565.4
Q789

CHEN Zhen, HE Xiao-ying, WANG Jing-qiao, LI Gen-ze, SHU Zheng-qi, ZHAO Kai-qin, CHENG Xiao-mao, ZU Feng. Bioinformatics analysis of BnFAD2 gene in Brassica napus L.[J]. HUBEI AGRICULTURAL SCIENCES, 2021, 60(11): 134-140.

References

[1] 刘成,冯中朝,肖唐华,等.我国油菜产业发展现状、潜力及对策[J].中国油料作物学报,2019,41(4):485-489.
[2] HU Q, HUA W, YIN Y, et al.Rapeseed research and production in China[J]. The crop journal, 2017(5):127-135.
[3] SHI J, LANG C, WANG F, et al.Depressed expression of FAE1 and FAD2 genes modifies fatty acid profiles and storage compounds accumulation in Brassica napus seeds[J]. Plant Sci,2017,263:177-182.
[4] ABBADI A,LECKBAND G.Rapeseed breeding for oil content, quality and sustainability[J]. Eur J Lipid Sci Technol,2011,113:1198-1206.
[5] ROMAN O, HEYD B, BROYART B, et al.Oxidative reactivity of unsaturated fatty acids from sunflower, high oleic sunflower and rapeseed oils subjected to heat treatment, under controlled conditions[J]. LWT Food Sci Technol,2013,52:49-59.
[6] YAMAKI T, NAGAMINE I, FUKUMOTO K, et al.High oleic peanut oil modulates promotion stage in lung tumorigenesis of mice treated with methyl nitrosourea[J]. Food Sci Technol Res,2005,11:231-235.
[7] BURNS M J, BARNES S R, BOWMAN J G, et al.QTL analysis of an intervarietal set of substitution lines in Brassica napus:(i) Seed oil content and fatty acid composition[J]. Heredity,2003,90:39-48.
[8] HUANG J Q, ZHANG T, ZHANG Q X, et al.The mechanism of high contents of oil and oleic acid revealed by transcriptomic and lipidomic analysis during embryogenesis in Caryacathayensis Sarg[J]. BMC Genomics, 2016, 17(1):113.
[9] CHAPMAN K D, OHLROGGE J B.Compartmentation of triacylglycerol accumulation in plants[J]. The journal of biological chemistry, 2012, 287(4): 2288-2294.
[10] OKULEY J, LIGHTNER J E, FELDMANN K A, et al.Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis[J]. The plant cell, 1994, 6(1): 147-158.
[11] PANDEY M K, WANG M L, QIAO L, et al.Identification of QTLs associated with oil content and mapping FAD2 genes and their relative contribution to oil quality in peanut (Arachis hypogaea L.)[J]. BMC genet, 2014,15:133.
[12] LI L Y, WANG X L, GAI J Y, et al.Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean[J]. Journal of plant physiology, 2007, 164(11):1516-1526.
[13] PIRJO T, VILKKI J,VIHINEN M.Mapping and cloning of FAD2 gene to developallele-specific PCR for oleic acid in spring turnip rape (Brassica rapa ssp. oleifera)[J]. Molecular breeding, 1998, 4(6):543-550.
[14] MIKKILINENI V, ROCHEFORD T R.Sequence variation and genomic organization of fatty acid desaturase-2 (fad2) and fatty acid desaturase-6 (fad6) cDNAs in maize[J]. Theor Appl genet,2003,106:1326-1332.
[15] BELÓ A, ZHENG P, LUCK S, et al.Whole genome scan detects an allelic variant of fad2 associated with increased oleic acid levels in maize[J]. Mol genet genomics, 2008,279:1-10.
[16] REN J, GUO R, CHEN L, et al.Single nucleotide polymorphisms of fad2 gene from tung tree, Verniciafordii, a potential biodiesel plant[J]. Euphytica ,2013,194:93-107.
[17] HERNÁNDEZ M L,MANCHA M, MARTÍNEZ-RIVAS J M. Molecular cloning and characterization of genes encoding two microsomal oleate desaturases (FAD2) from olive[J]. Phytochemistry, 2005, 66(12):1417-1426.
[18] HU X, SULLIVAN-GILBERT M, GUPTA M, et al.Mapping of the loci controlling oleic and linolenic acid contents and development of fad2 and fad3 allele-specific markers in canola (Brassica napus L.)[J]. Theor Appl genet,2006,113(3):497-507.
[19] YANG Q, FAN C, GUO Z, et al.Identification of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents[J]. Theor Appl genet,2012,125(4):715-729.
[20] WELLS R, TRICK M, SOUMPOUROU E, et al.The control of seed oil polyunsaturate content in the polyploid crop species Brassica napus[J]. Mol breed,2014,33:349-362.
[21] LIU F, WANG G, LIU R, et al.The promoter of fatty acid desaturase on chromosome C5 in Brassica napus drives high-level expression in seeds[J]. Plant biotechnol rep ,2016,10:369-381.
[22] ZHAO Q, WU J, CAI G, et al.A novel quantitative trait locus on chromosome A9 controlling oleic acid content in Brassica napus[J]. Plant Biotechnol J,2019,17(12):2313-2324.
[23] YANG J,XING G,NIU L,et al.Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B[J]. Transgenic Res,2018,27:155-166.
[24] LEE K-R, KIM E-H, ROH K H, et al.High-oleic oilseed rapes developed with seed-specific suppression of FAD2 gene expression[J]. Applied biological chemistry,2016,59(4):669-676.
[25] GASTEIGER E,HOOGLAND C,GATTIKER A,et al.Protein identification and analysis tools on the ExPASy server[M]. Totowa,NJ:Humana Press,2005.571-607.
[26] LETUNIC I,DOERKS T,BORK P.SMART:Recent updates,new developments and status in 2015[J]. Nucleic Acids Res,2015,43(D1):D257-D260.
[27] LARKIN M A, BLACKSHIELDS G, BROWN N P, et al.Clustal W and Clustal X version 2.0[J]. Bioinformatics,2007,23(21):2947-2948.
[28] BAILEY T L, ELKAN C.Fitting a mixture model by expectation maximization to discover motifs in biopolymers[J]. Proc Int Conf Intell Syst Mol Biol,1994,2:28-36.
[29] KUMAR S, STECHER G, LI M, et al.MEGA-X:Molecular evolutionary genetics analysis across computingplatforms[J]. Molecular biology & evolution, 2018.
[30] SHAFIEI-KOIJ F, RAVICHANDRAN S, BARTHET V J, et al.Evolution of carthamus species revealed through sequence analyses of the fad2 gene family[J]. Physiol Mol Biol plants,2020,26(3):419-432.
[31] ZHAO M, WANG W, WEI L, et al.The evolution and biocatalysis of FAD2 indicate its correlation to the content of seed oil in plants[J]. Int J Mol Sci,2019,20(4):849.
[32] YUAN M, ZHU J, GONG L, et al.Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing[J]. BMC biotechnol,2019,19(1):24.
[33] AL AMIN N, AHMAD N, WU N, et al.CRISPR-Cas9 mediated targeted disruption of FAD2-2 microsomal omega-6 desaturase in soybean (Glycine max. L)[J]. BMC biotechnol,2019,19(1):9.
[34] JIANG W Z, HENRY I M, LYNAGH P G, et al.Significant enhancement of fatty acid composition in seeds of the allohexaploid,Camelina sativa, using CRISPR/Cas9 gene editing[J]. Plant biotech J,2017,15:648-657.
[35] TIAN Y, CHEN K, LI X, et al.Design of high-oleic tobacco (Nicotiana tabacum L.) seed oil by CRISPR-Cas9-mediated knockout of NtFAD2-2[J]. BMC plant Biol,2020,20:233.
[36] LAWRENSON T, SHORINOLA O, STACEY N, et al.Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease[J]. Genome Biol,2015,16:258.