Screening and Verification of Minor Genes Related to Fatty Acid Metabolism in High Oleic Acid Rapeseed (Brassica napus)
WANG Xiao-Dan, XIAO Gang, ZHANG Zhen-Qian*, GUAN Chun-Yun
Southen Regional Collaborative Innovation Center for Grain and Oil Crops/Hunan Provincial Key Laboratory of Rice and Rapeseed Breeding for Disease Resistance, Hunan Agricultural University, Changsha 410128, China
Abstract The genetic mechanism of oleic acid in rapeseed (Brassica napus) is complex. The main gene FAD2 (fatty acid desaturase 2) is studied mostly, but little about minor genes now. In order to discover new related genes and promote the research of fatty acid metabolism in high oleic acid rapeseed, 20 high oleic acid rapeseed strains were used as materials in this study. Not only were their fatty acid composition tested by gas chromatography (GC), but also the expressions of OPR (1,2-oxo-phytodienoic acid reductase) and AT(acetyltransferase) gene, which screened from microRNA sequencing and related to fatty acid metabolism, were determined by qRT-PCR. Meanwhile, the relationship between the expression of the 2 genes and the fatty acid composition were analyzed. The results showed that the content of oleic acid was negatively correlated with linoleic acid and α-linolenic acid. Linoleic acid, α-linolenic acid and palmitic acid were positively correlated with each other. The expression patterns of OPR and AT genes were similar during the whole growth period. Their expression increased gradually at seedling stage, the highest expression was in blooming flowers at flowering stage, and decreased gradually at pod stage, while the expression level of OPR gene was much lower than that of internal reference gene, and it was opposite with AT. The analysis between expression of 2 genes and fatty acid composition was found that AT gene was significantly or extremely significantly correlated with oleic acid, linoleic acid and α-linolenic acid in leaves of 5~6 leaf stage, budding stage and selfed seeds of 35 d; OPR was positively correlated with oleic acid and negatively correlated with linoleic acid and linolenic acid during the whole pod stage. The 2 genes might be related with oleic acid synthesis as the minor genes. Besides, AT could be used for early screening of high oleic acid materials. The results of this study could promote the research of molecular mechanism in high oleic acid rapeseed.
WANG Xiao-Dan,XIAO Gang,ZHANG Zhen-Qian, et al. Screening and Verification of Minor Genes Related to Fatty Acid Metabolism in High Oleic Acid Rapeseed (Brassica napus)[J]. 农业生物技术学报, 2019, 27(7): 1171-1178.
[1] 谷晓娜, 刘振库, 王丕武, 等. 2015. hrpZPsta转基因大豆中定量表达与疫霉根腐病和灰斑病抗性相关研究[J]. 中国油料作物学报, 37(1): 35-40. (Gu X N, Liu Z K, Wang P W, et al.Gene expression and disease resistance of transformed hrpZPsta gene in transgenic soybean[J]. Chinese Journal of Oil Crop Sciences, 37(1): 35-40.) [2] 何路军. 2003. N-乙酰基转移酶的研究进展[J]. 国外医学. 输血及血液学分册, (06): 529-531. (He L J.2003. Research of N-acetyltransferase[J]. Foreign Medical Sciences (Section of Blood Transfusion and Heanatology), (06): 529-531.) [3] 江南, 谭晓风, 张琳, 等. 2014. 基于RNA-Seq的油茶种子α-亚麻酸代谢途径及相关基因分析[J]. 林业科学, 50(08): 68-75. (Jiang N, Tan X F, Zhang L, et al.2014. Gene analysis of α-linolenic acid metabolism of Camellia oleifera seeds based on RNA-Seq[J]. Scientia Silvae Sinicae, 50(08): 68-75.) [4] 刘芳, 刘睿洋, 彭烨, 等. 2015. 甘蓝型油菜BnFAD2-C1基因全长序列的克隆、表达及转录调控元件分析[J]. 作物学报, 41(11): 1663-1670. (Liu F, Liu R Y, Peng Y, et al.2015. Cloning and expression of BnFAD2-C1 gene involved in Brassica napus and analysis of transcription regulation elements[J]. Acta Agronomica Sinica, 41(11): 1663-1670.) [5] 刘列钊, 王欣娜, 阎星颖, 等. 2012. 航天诱变高油酸甘蓝型油菜突变体分子标记的筛选[J]. 中国农业科学, 45(23): 4931-4938. (Liu L Z, Wang X N, Yan X Y, et al.2012. Molecular maker screen for high oleic acidin space flight mutant Brassica napus[J]. Scientia Agricultura Sinica, 45(23): 4931-4938.) [6] 王晓丹, 陈浩, 张振乾, 等. 2019. 2个抗病相关基因与高油酸油菜病害关系分析[J]. 核农学报, 33(05): 894-901. (Wang X D, Chen H, Zhang Z Q, et al.2019. Relationship between two resistance gene and diseases in high oleic acid rapeseed[J]. Journal of Nuclear Agricultural Sciences, 33(05): 894-901.) [7] 夏德安, 刘春娟, 吕世博, 等. 2015. 植物组蛋白乙酰基转移酶的研究进展[J]. 生物技术通报, 31(07): 18-25. (Xia D A, Liu C J, Lv S B, et al.2015. Research progress of plant histone acetyltransferases[J]. Biotechnology Bulletin, 31(07): 18-25.) [8] 肖旭峰, 张袆, 杨寅桂. 2017. 菜心组蛋白乙酰转移酶基因BrcuHAC1克隆与表达分析[J]. 核农学报, (12): 41-49. (Xiao X F, Zhang H, Yang Y G.2017. Cloning and expression analysis of histone acetyltransferase gene (BrcuHAC1) in flowering Chinese cabbage[J]. Journal of Nuclear Agricultural Sciences, (12): 41-49.) [9] 徐明月, 肖庆生, 张学昆, 等. 2013. 油菜干旱相关基因的表达及其与耐旱生理指标的相关性[J]. 中国油料作物学报, 35(05): 557-563. (Xu M Y, Xiao Q S, Zhang X K, et al.2013. Correlation between drought-related genes expression and physiological indicators in rapeseed[J]. Chinese Journal of Oil Crop Sciences, 35(05): 557-563.) [10] 杨柳, 李东欣, 谭太龙. 2018. 不同油酸含量油菜脂肪酸含量分析[J]. 作物研究, 32(05): 390-394, 402.(Yang L, Li D X, Tan T L. 2018. Analysis of fatty acid content in rapeseed with different oleic acid contents[J]. Crop Research, 32(05): 390-394, 402.) [11] 张振乾, 胡庆一, 官春云. 2016. 高油酸油菜研究现状, 存在的问题及发展建议[J]. 作物研究, 30(4): 462-474. (Zhang Z Q, Hu Q Y, Guan C Y.2016. Present research situation,questions and developmental advises of high oleic acid rapeseed[J]. Crop Research, 30(4): 462-474.) [12] Barkley N A, Chamberlin K D C, Wang M L, et al.2010. Development of a Real-time PCR genotyping assay to identify high oleic acid peanuts (Arachis hypogaea L.)[J]. Molecular Breeding, 25(3): 541-548. [13] Barkley N A, Wang M L, Pittman R N.2011. A Real-time PCR genotyping assay to detect FAD2A SNPs in peanuts (Arachis hypogaea L.)[J]. Electronic Journal of Biotechnology, 14(1): 9-10. [14] Chen L, Chen L, Zhang X, et al.2018. Identification of miRNAs that regulate silique development in Brassica napus[J]. Plant Science, 269: 106-117. [15] de Azevedo Souza C, Kim S S, Koch S, et al.2009. A novel fatty Acyl-CoA synthetase is required for pollen development and sporopollenin biosynthesis in Arabidopsis[J]. Plant Cell, 21(2): 507-525. [16] de Winter J C, Gosling S D, Potter J.2016. Comparing the pearson and spearman correlation coefficients across distributions and sample sizes: A tutorial using simulations and empirical data[J]. Psychological Methods, 21(3): 273-90. [17] Deng W, Liu C, Pei Y, et al.2007. Involvement of the histone acetyltransferase AtHAC1 in the regulation of flowering time via repression of FLOWERING LOCUS C in Arabidopsis[J]. Plant Physiology, 143(4): 1660-1668. [18] Gillingham L G, Gustafson J A, Han S Y, et al.2011. High-oleic rapeseed (Canola) and flaxseed modulate serum lipids and inflammatory biomarkers in hypercholesterolaemic subjects[J]. The British Journal of Nutrition, 105(3): 417-427. [19] Han R, Jian C, Lv J, et al.2014. Identification and characterization of microRNAs in the flag leaf and developing seed of wheat (Triticum aestivum L.)[J]. BMC Genomics, 15: 289. [20] He Y, Zhang H, Sun Z, et al.2017. Jasmonic acid-mediated defense suppresses brassinosteroid-mediated susceptibility to Rice black streaked dwarf virus infection in rice[J]. New Phytologist, 214(1): 388-399. [21] Li N, You X, Chen T, et al.2013. Global profiling of miRNAs and the hairpin precursors: Insights into miRNA processing and novel miRNA discovery[J]. Nucleic Acids Research, 41(6): 3619-34. [22] Liu R Y, Liu F, Guan C Y.2016. Cloning and expression analyses for BnFAD2 genes in Brassica napus[J]. Acta Agronomica Sinica, 42(7): 1000-1008. [23] Long W, Hu M, Gao J, et al.2018. Identification and functional analysis of two new mutant BnFAD2 alleles that confer elevated oleic acid content in rapeseed[J]. Frontiers in Genetics. 9: 399-419. [24] Meng Y, Shao C, Ma X, et al.2012. Expression-based functional investigation of the organ-specific microRNAs in Arabidopsis[J]. PLoS One, 7(11): e50870. [25] Pfaffl M W.2001. A new mathematical model for relative quantification in Real-time RT-PCR[J]. Nucleic Acids Research, 29(9): 45. [26] Peng Q, Hu Y, Wei R, et al.2010. Simultaneous silencing of FAD2 and FAE1 genes affects both oleic acid and erucic acid contents in Brassica napus seeds[J]. Plant Cell Reports, 29(4): 317-325. [27] Schaller F, Biesgen C, Mussig C, et al.2000. 12-Oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis[J]. Planta, 210: 979. [28] Schierholt?A,?Becker?H C,?Ecke?W.?2000. Mappinga?high?oleic?acid?mutation?in?winteroilseed?rape?(Brassica?napus?L.)[J]. Theoretical?and?Applied?Genetics, 101(5-6):?897-901. [29] Sivaraman I, Arumugam N, Sodhi Y S, et al.2004. Development of high oleic and low linoleic acid transgenics in a zero erucic acid Brassica juncea, L. (Indian mustard) line by antisense suppression of the fad2 gene[J]. Molecular Breeding, 13(4): 365-375. [30] Song J B, Shu X X, Shen Q, et al.2015. Altered fruit and seed development of transgenic rapeseed (Brassica napus) over-expressing microRNA394[J]. PLoS One, 10(5): e0125427. [31] Stintzi A, Weber H, Reymond P, et al.2001. Plant defense in the absence of jasmonic acid: The role of cyclopentenones[J]. Plant Biology, 98: 12837. [32] Strassner J, Schaller F, Frick U B, et al.2002. Characterization and cDNA-microarray expression analysis of 12-oxophytodienoate reductases reveals differential roles for octadecanoid biosynthesis in the local versus the systemic wound response[J]. Plant Journal, 32: 585-601. [33] Wang Z, Qiao Y, Zhang J, et al.2017. Genome wide identification of microRNAs involved in fatty acid and lipid metabolism of Brassica napus by small RNA and degradome sequencing[J]. Gene, 619: 61-70. [34] Wu L, Zhang Q, Zhou H, et al.2009. Rice microRNA effector complexes and targets[J]. Plant Cell, 21(11): 3421-3435. [35] Zhang H, Zhang Z, Xiong T, et al.2018. The CCCH-type transcription factor BnZFP1 is a positive regulator to control oleic acid levels through the expression of diacylglycerol O-acyltransferase 1 gene in Brassica napus[J]. Plant Physiology and Biochemistry. 132: 633-640. [36] Zhao Y T, Wang M, Fu S X, et al.2012. Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production-and development-correlated expression and new small RNA classes[J]. Plant physiology, 158(2): 813-823.
