海甘蓝发育种子中FAE1的表达模式与芥酸积累

doi:10.7668/hbnxb.20194753

摘要/Abstract

摘要：

为了探究海甘蓝控制超长链脂肪酸合成的关键基因FAE1的表达模式及其对发育种子中芥酸合成的影响,针对已克隆的5个FAE1同源基因(CaFAE1-1~CaFAE1-5),以苗期根、茎、叶及不同发育时期(花后5~25 d)的种子为材料,采用实时荧光定量PCR(qRT-PCR)技术分析其表达模式;并采用气相质谱技术(GC-MS)检测不同发育时期种子中的脂肪酸相对含量,重点探讨不同CaFAE1基因表达量与种子芥酸含量的相关性。结果表明,相对种子而言,5个CaFAE1基因在根、茎、叶组织中的表达水平极低;而在花后5~25 d种子中,CaFAE1-3的表达量均为最高,且随种子发育进程呈逐渐升高趋势;其他4个CaFAE1基因的表达量均呈先升后降趋势,均于花后20 d达到最高值,随后CaFAE1-5和CaFAE1-4表达量迅速下降。GC-MS分析结果表明,花后5~15 d种子中,长链饱和脂肪酸C16∶0和C18∶0及单不饱和脂肪酸C18∶1相对含量均显著高于超长链脂肪酸含量,随后其含量快速下降,表明CaFAE1以C18∶0和C18∶1为底物的脂肪酸碳链延长主要发生在花后15 d及以后,且终产物主要为C22∶0和C22∶1。CaFAE1基因表达量与脂肪酸组分含量相关性分析结果表明,在花后5~25 d种子中,5个CaFAE1的表达量均与超长链脂肪酸含量呈正相关,且CaFAE1-1、CaFAE1-2和CaFAE1-3与芥酸相对含量呈显著或极显著正相关。由此推测,多拷贝、高活性FAE1的高水平表达可能是海甘蓝种子中芥酸含量高的主要成因。

关键词: 海甘蓝, 发育种子, 芥酸, FAE1, 表达模式, 实时荧光定量PCR

Abstract:

To explore the expression patterns of the FAE1s controlling the biosynthesis of very-long-chain fatty acids and their effects on the synthesis of erucic acid in the developing seeds of Crambe abyssinica,the expression levels of the five cloned FAE1 homologous genes(CaFAE1-1—CaFAE1-5)were analyzed in the roots,stems,and leaves of seedlings and in the developing seeds(5—25 d after flowering)by using Real-time Quantitative PCR(qRT-PCR)technology,and the fatty acid components in seeds at different developmental stages were detected by gas chromatography-mass spectrometry(GC-MS)method,and finally the correlation between the expression level of individual CaFAE1 and the erucic acid content in seeds was emphasized.The results showed that,compared to seeds,all five CaFAE1s expressed at an extremely low level in root,stem,and leaf tissues.In the 5—25 d after flowering seeds,CaFAE1-3 always exhibited the highest expression level,and it increased gradually with the seed development process;while the expression levels of the other four CaFAE1s showed a trend of first increasing and then decreasing,all reached their highest values in 20 d after flowering seeds,and then CaFAE1-5 and CaFAE1-4 decreased rapidly.The GC-MS analysis results showed that the relative contents of long-chain saturated fatty acids C16:0 and C18∶0,as well as monounsaturated fatty acid C18∶1,were significantly higher than those of very-long-chain fatty acids in 5—15 d after flowering seeds,afterward their contents rapidly decreased,indicating that the carbon chain elongation of fatty acids by CaFAE1 using C18∶0 and C18∶1 as substrates mainly occurred after 15 d after flowering,and the end-products were dominantly C22∶0 and C22∶1.The correlation analysis between the expression levels of CaFAE1s and the content of fatty acid components demonstrated that in 5—25 d after flowering seeds,the expression levels of the five CaFAE1s showed highly significant or significant positive correlation with the content of very-long-chain fatty acids,and CaFAE1-1,CaFAE1-2,and CaFAE1-3 showed highly significant or significant positive correlation with the content of erucic acid.Collectively,it is speculated that the convergence of multiple functional copies of CaFAE1 accompanied by high-level expression of high-active CaFAE1 may be the main cause of high erucic acid content in Crambe abyssinica seeds.

Key words: Crambe abyssinica, Developing seeds, Erucic acid, Fatty acid elongase 1, Expression pattern, qRT-PCR

胡馨月, 王津, 聂婉虹, 龚超, 许本波, 张学昆, 赵福永. 海甘蓝发育种子中FAE1的表达模式与芥酸积累[J]. 华北农学报, 2024, 39(3): 35-42. doi: 10.7668/hbnxb.20194753.

HU Xinyue, WANG Jin, NIE Wanhong, GONG Chao, XU Benbo, ZHANG Xuekun, ZHAO Fuyong. Expression Pattern of FAE1s and Erucic Acid Accumulation in Developing Seeds of Crambe abyssinica[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 35-42. doi: 10.7668/hbnxb.20194753.

http://www.hbnxb.net/CN/Y2024/V39/I3/35

图/表 7

表1 海甘蓝CaFAE1及内参基因CaActin2 qRT-PCR分析引物

Tab.1 Information of primers of CaFAE1 and CaActin2 for qRT-PCR

引物名称 Primer name	引物序列(5'—3') Sequence (5'—3')	扩增子/bp Amplicon	扩增效率/% Amplification efficiency
CaFAE1-1-Fq	AACGCATACCGGAGCTGAC	156	96.60
CaFAE1-1-Rq	GGAAGAATCAACGGACCCAAC
CaFAE1-2-Fq	TTCACACGGTCCGAACACAT	119	98.80
CaFAE1-2-Rq	GGCAACATCGGTTATGTCCTTAG
CaFAE1-3-Fq	TGAGTTGGCATACATAGAGGCA	110	95.30
CaFAE1-3-Rq	TTAAAGCTACCCAAACCGCACT
CaFAE1-4-Fq	AACGCATACCGGAGCTGAC	118	88.20
CaFAE1-4-Rq	AACCGTTCGACCAGCAACC
CaFAE1-5-Fq	GGTGCGCTGGAAAATCTATTCA	137	93.50
CaFAE1-5-Rq	TTGCTTCGGAGCTTGAAAGTATTG
CaActin2-Fq	CAGTGTCTGGATCGGTGGTT	91	96.30
CaActin2-Rq	TGGACCTGCCTCGTCATACT

图1 十字花科不同属间β-Actin2基因DNA序列比对结果(部分) 图中所示十字花科β-Actin2基因DNA序列均下载自NCBI网站(https://www.ncbi.nlm.nih.gov/)。箭头所示序列位置为海甘蓝Actin2基因qPCR扩增引物设计区。

Fig.1 DNA sequence alignment results of β-Actin2 among different genera in the Cruciferae family (partial) All the DNA sequences of β-Actin2 in the Cruciferae family displayed in the figure were downloaded from the NCBI website (https://www.ncbi.nlm.nih.gov/).The sequence regions indicated by the arrows were the designed qPCR primers for CaActin2.

图2 海甘蓝根、茎和叶组织中CaFAE1的表达水平不同小写字母表示不同基因间在P<0.05水平上的差异显著。图3同。

Fig.2 The expression levels of CaFAE1s in the root, stem, and leaf tissues of Crambe abyssinica Different lowercase letters indicate significant differences between different genes at the P<0.05 level. The same as Fig.3.

图3 不同发育时期海甘蓝种子中CaFAE1相对表达量

Fig.3 Relative expression levels of CaFAE1s in Crambe abyssinica seeds at different developmental stages

图4 CaFAE1基因在种子发育进程中的表达水平动态变化

Fig.4 Dynamic changes in the expression level of CaFAE1s during seed development process

图5 海甘蓝不同发育时期种子中脂肪酸组分及相对含量

Fig.5 Fatty acid components and relative abundance in seeds of Crambe abyssinica at different developmental stages

图6 海甘蓝发育种子中CaFAE1基因表达量与脂肪酸组分相对含量的相关性

Fig.6 The correlation between the expression levels of CaFAE1 genes and the relative content of fatty acids in the developing seeds of Crambe abyssinica ^*.P<0.05;^**.P<0.01;^***.P<0.001。

参考文献 32

[1]	Leonard E C. High-erucic vegetable oils[J]. Industrial Crops and Products, 1992, 1(2/3/4):119-123.doi:10.1016/0926-6690(92)90009.
[2]	吴关庭, 郎春秀, 陈锦清. 芥酸的生产及其衍生产品开发[J]. 中国油脂, 2007, 32(6):27-31.doi:10.3321/j.issn:1003-7969.2007.06.007.
	Wu G T, Lang C X, Chen J Q. Production of erucic acid and development of its derivative products[J]. China Oils and Fats, 2007, 32(6):27-31.
[3]	Wang P D, Xiong X J, Zhang X B, Wu G, Liu F. A review of erucic acid production in Brassicaceae oilseeds:progress and prospects for the genetic engineering of high and low-erucic acid rapeseeds(Brassica napus)[J]. Frontiers in Plant Science, 2022, 13:899076.doi:10.3389/fpls.2022.899076.
[4]	Mietkiewska E, Giblin E M, Wang S, Barton D L, Dirpaul J, Brost J M, Katavic V, Taylor D C. Seed-specific heterologous expression of a Nasturtium FAE gene in Arabidopsis results in a dramatic increase in the proportion of erucic acid[J]. Plant Physiology, 2004, 136(1):2665-2675.doi:10.1104/pp.104.046839. pmid: 15333757
[5]	Guan R, Lager I, Li X Y, Stymne S, Zhu L H. Bottlenecks in erucic acid accumulation in genetically engineered ultrahigh erucic acid Crambe abyssinica[J]. Plant Biotechnology Journal, 2014, 12(2):193-203.doi:10.1111/pbi.12128. pmid: 24119222
[6]	Pushkarova N, Yemets A. Biotechnological approach for improvement of Crambe species as valuable oilseed plants for industrial purposes[J]. RSC Advances, 2022, 12(12):7168-7178.doi:10.1039/d2ra00422d. pmid: 35424652
[7]	王幼平, 罗鹏, 李旭峰. 海甘蓝的初步研究[J]. 云南植物研究, 1995(2):169-174.
	Wang Y P, Luo P, Li X F. Preliminery study on Crambe abyssinica[J]. Acta Botanica Yunnanica, 1995(2):169-174.
[8]	Qi W C, Tinnenbroek-Capel I E M, Salentijn E M J, Zhang Z, Huang B Q, Cheng J H, Shao H B, Visser R G F, Krens F A, van Loo E N. Genetically engineering Crambe abyssinica—a potentially high-value oil crop for salt land improvement[J]. Land Degradation & Development, 2018, 29:1096-1106.doi:10.1002/ldr.2847.
[9]	Samarappuli D, Zanetti F, Berzuini S, Berti M T. Crambe(Crambe abyssinica hochst):a non-food oilseed crop with great potential:a review[J]. Agronomy, 2020, 10(9):1380.doi:10.3390/agronomy10091380.
[10]	Mietkiewska E, Brost J M, Giblin E M, Barton D L, Taylor D C. Cloning and functional characterization of the fatty acid elongase 1(FAE1)gene from high erucic Crambe abyssinica cv.Prophet[J]. Plant Biotechnology Journal, 2007, 5(5):636-645.doi:10.1111/j.1467-7652.2007.00268.x. pmid: 17565584
[11]	Li X Y, van Loo E N, Gruber J, Fan J, Guan R, Frentzen M, Stymne S, Zhu L H. Development of ultra-high erucic acid oil in the industrial oil crop Crambe abyssinica[J]. Plant Biotechnology Journal, 2012, 10(7):862-870.doi:10.1111/j.1467-7652.2012.00709.x.
[12]	Guo Y M, Mietkiewska E, Francis T, Katavic V, Brost J M, Giblin M, Barton D L, Taylor D C. Increase in nervonic acid content in transformed yeast and transgenic plants by introduction of a Lunaria annua L.3-ketoacyl-CoA synthase(KCS)gene[J]. Plant Molecular Biology, 2009, 69(5):565-575.doi:10.1007/s11103-008-9439-9.
[13]	Cheng J H, Salentijn E M J, Huang B Q, Krens F A, Dechesne A C, Visser R G F, van Loo E N. Isolation and characterization of the omega-6 fatty acid desaturase(FAD2)gene family in the allohexaploid oil seed crop Crambe abyssinica Hochst[J]. Molecular Breeding, 2013, 32(3):517-531.doi:10.1007/s11032-013-9886-0.
[14]	王幼平, 罗鹏. 海甘蓝的农学性状观察及评价[J]. 中国油料, 1995(1):16-18.
	Wang Y P, Luo P. Observation of agronomic characteristics and evaluation of Crambe abyssinica[J]. Chinese Journal of Oil Crop Sciences, 1995(1):16-18.
[15]	王幼平, 罗鹏, 高宏波. 高芥酸油料植物海甘蓝的引种和利用[J]. 植物学通报, 1998(3):23-28.
	Wang Y P, Luo P, Gao H B. Introduction and utilization of high erucic acid oil plant Crambe abyssinica[J]. Chinese Bulletin of Botany, 1998(3):23-28.
[16]	王幼平, 蓝乐夫, 贾勇炯, 罗鹏. 高芥酸油料作物海甘蓝的引种栽培研究[J]. 作物学报, 1999, 25(1):130-133.doi:10.3321/j.issn:0496-3490.1999.01.022.
	Wang Y P, Lan L F, Jia Y J, Luo P. Studies on introduction and cultivation of Crambe abyssinica with high erucic acid content[J]. Acta Agronomica Sinica, 1999, 25(1):130-133.
[17]	韩凤英, 王津, 胡馨月, 徐劲松, 许本波, 张学昆, 赵福永. 不同超长链脂肪酸延长酶基因FAE1芥酸合成功能的比较[J]. 植物遗传资源学报, 2023, 24(2):569-583.doi:10.13430/j.cnki.jpgr.20220913001.
	Han F Y, Wang J, Hu X Y, Xu J S, Xu B B, Zhang X K, Zhao F Y. Comparison of erucic acid biosynthesis of the FAE1 genes encoding the very-long-chain fatty acid elongase from different plant species[J]. Journal of Plant Genetic Resources, 2023, 24(2):569-583.
[18]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4):402-408.doi:10.1006/meth.2001.1262. pmid: 11846609
[19]	James D W Jr, Lim E, Keller J, Plooy I, Ralston E, Dooner H K. Directed tagging of the Arabidopsis FATTY ACID ELONGATION1(FAE1)gene with the maize transposon activator[J]. The Plant Cell, 1995, 7(3):309-319.doi:10.1105/tpc.7.3.309.
[20]	Han J X, Lühs W, Sonntag K, Zähringer U, Borchardt D S, Wolter F P, Heinz E, Frentzen M. Functional characterization of β-ketoacyl-CoA synthase genes from Brassica napus L.[J]. Plant Molecular Biology, 2001, 46(2):229-239.doi:10.1023/A:1010665121980. pmid: 11442062
[21]	Rossak M, Smith M, Kunst L. Expression of the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana[J]. Plant Molecular Biology, 2001, 46(6):717-725.doi:10.1023/a:1011603923889. pmid: 11575726
[22]	淮东欣. 调控超长链脂肪酸合成关键基因对植物种子中脂肪酸组成的影响[D]. 武汉: 华中农业大学, 2015.doi:10.7666/d.Y2803593.
	Huai D X. Effects of key genes regulating the synthesis of super-long chain fatty acids on fatty acid composition in plant seeds[D]. Wuhan: Huazhong Agricultural University, 2015.
[23]	Cheng J H, Salentijn E M J, Huang B Q, Denneboom C, Qi W C, Dechesne A C, Krens F A, Visser R G F, van Loo E N. Detection of induced mutations in CaFAD2 genes by next-generation sequencing leading to the production of improved oil composition in Crambe abyssinica[J]. Plant Biotechnology Journal, 2015, 13(4):471-481.doi:10.1111/pbi.12269.
[24]	韩凤英. 芥酸高合成能力关键基因FAE1的鉴定及其油菜遗传转化[D]. 荆州: 长江大学, 2023.
	Han F Y. Identification of FAE1,a key gene with high erucic acid synthesis ability,and its genetic transformation in rapeseed[D]. Jingzhou: Yangtze University, 2023.
[25]	Siebel J, Pauls K P. Inheritance patterns of erucic acid content in populations of Brassica napus microspore-derived spontaneous diploids[J]. Theoretical and Applied Genetics, 1989, 77(4):489-494.doi:10.1007/BF00274268. pmid: 24232714
[26]	吴江生. 甘蓝型油菜芥酸含量的遗传研究[J]. 湖北农业科学, 1989(7):16-17,33.
	Wu J S. Genetic study on erucic acid content in Brassica napus[J]. Hubei Agricultural Sciences, 1989(7):16-17.33.
[27]	赵福永, 高震, 严寒, 田志宏. 野芥芥酸合成酶基因fae1的克隆与序列分析[J]. 华北农学报, 2009, 24(5):40-44. doi:10.7668/hbnxb.2009.05.009.
	Zhao F Y, Gao Z, Yan H, Tian Z H. Gene cloning and sequence analysis of fatty acid elongase 1(fae1) in Sinapis arvensis L.[J]. Acta Agriculturae Boreali-Sinica, 2009, 24(5):40-44.
[28]	Guan R, Li X Y, Hofvander P, Zhou X R, Wang D N, Stymne S, Zhu L H. RNAi targeting putative genes in phosphatidylcholine turnover results in significant change in fatty acid composition in Crambe abyssinica seed oil[J]. Lipids, 2015, 50(4):407-416.doi:10.1007/s11745-015-4004-1. pmid: 25753896
[29]	Li X Y, Mei D S, Liu Q, Fan J, Singh S, Green A, Zhou X R, Zhu L H. Down-regulation of crambe fatty acid desaturase and elongase in Arabidopsis and Crambe resulted in significantly increased oleic acid content in seed oil[J]. Plant Biotechnology Journal, 2016, 14(1):323-331.doi:10.1111/pbi.12386.
[30]	Sagun J V, Yadav U P, Alonso A P. Progress in understanding and improving oil content and quality in seeds[J]. Frontiers in Plant Science, 2023, 14:1116894.doi:10.3389/fpls.2023.1116894.
[31]	Liu Y H, Du Z L, Lin S L, Li H M, Lu S P, Guo L, Tang S. CRISPR/Cas9-targeted mutagenesis of BnaFAE1 genes confers low-erucic acid in Brassica napus[J]. Frontiers in Plant Science, 2022, 13:848723.doi:10.3389/fpls.2022.848723.
[32]	赵福永, 王津. 一种海甘蓝突变基因CaFAE1-3及其在合成芥酸中的应用:CN116555299A[P]. 2023-08-08
	Zhao F Y, Wang J. A mutated gene of CaFAE1-3 in Crambe abyssinica and its application in the synthesis of erucic acid[P]. 2023-08-08

选择文件类型/文献管理软件名称

选择包含的内容