亚麻荠CsFAD基因家族鉴定及α-亚麻酸合成关键基因CsFAD3.3的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-1058

摘要/Abstract

摘要：

目的亚麻荠（Camelina sativa (L.) Crantz）是一种具有开发潜力的十字花科油料作物，其种子油富含不饱和脂肪酸。脂肪酸脱氢酶（fatty acid desaturase, FAD）是催化不饱和脂肪酸合成的关键酶，鉴定亚麻荠CsFAD基因家族并解析关键基因CsFAD3.3在α-亚麻酸（α-linolenic acid, ALA）合成中的功能，可为亚麻荠种子油脂品质改良提供理论支撑。方法利用全基因组数据结合生物信息学方法，对亚麻荠CsFAD基因进行系统鉴定，分析其染色体分布、系统发育关系、基因结构和保守基序；结合转录组和RT-qPCR数据分析基因的组织表达模式；采用亚细胞定位验证CsFAD3.3的细胞定位；通过转基因亚麻荠和烟草进行功能验证。结果共鉴定54个CsFAD基因，不均匀地分布在20条染色体上。系统发育树分析将其划分为6个亚家族，与基因结构和保守基序相一致。表达谱分析显示，CsFAD基因在不同的亚组中表达模式不相同，但在同一亚家族的表达模式一致，其中CsFAD3亚家族成员（尤其CsFAD3.3）主要在种子中表达。亚细胞定位证实CsFAD3.3定位于内质网。功能验证显示，过表达CsFAD3.3显著提高亚麻荠种子中ALA含量。此外，CsFAD3.3在本氏烟草（Nicotiana benthamiana）中的瞬时表达与在普通烟草（N. tabacum）中的稳定遗传转化，分别显著提升了叶片及转基因种子中的ALA含量。结论 CsFAD3.3定位于内质网，是亚麻荠ALA合成的关键限速酶。过表达CsFAD3.3基因显著提高了亚麻荠种子、烟草叶片及烟草种子中的ALA含量，证实了其功能的保守性。

关键词: 亚麻荠, FAD基因家族, CsFAD3.3基因, 表达模式分析, 功能验证, α-亚麻酸

Abstract:

Objective Camelina sativa (L.) Crantz is a cruciferous oilseed crop with great development potential and its seed oil is rich in unsaturated fatty acids. Fatty acid desaturases (FADs) are key enzymes that catalyze the biosynthesis of unsaturated fatty acids. The identification of the CsFAD gene family in camelina and the functional characterization of the key gene CsFAD3.3 involved in α-linolenic acid (ALA) biosynthesis may provide theoretical support for improving the oil quality of camelina seeds. Method Genome-wide data combined with bioinformatics approaches were used to identify the CsFAD gene family in camelina and to analyze chromosomal distribution, phylogenetic relationships, gene structures, and conserved motifs. Transcriptome data and RT-qPCR were employed to examine tissue-specific expression patterns. Subcellular localization assays were conducted to determine the localization of CsFAD3.3, and transgenic camelina and tobacco were generated for functional validation. Result A total of 54 CsFAD genes were identified, unevenly distributed across 20 chromosomes. Phylogenetic analysis classified them into six subfamilies, consistent with conserved structures and motif features. Expression profiling showed that CsFAD genes exhibited diverse expression patterns across different subgroups but similar patterns within the same subfamily. Notably, members of the CsFAD3 subfamily, especially CsFAD3.3, were predominantly expressed in seeds. Subcellular localization confirmed that CsFAD3.3 was located in the endoplasmic reticulum. Functional assays demonstrated that overexpression of CsFAD3.3 significantly increased ALA content in camelina seeds. Moreover, both the transient expression in N. benthamiana leaves and the stable transformation in N. tabacum seeds demonstrated that CsFAD3.3 could significantly enhance ALA accumulation in recipient leaves and transgenic seeds, respectively. Conclusion CsFAD3.3 is localized in the endoplasmic reticulum and functions as a key rate-limiting enzyme for ALA biosynthesis in camelina. Overexpression of CsFAD3.3 significantly increased ALA content in camelina seeds, tobacco leaves, and tobacco seeds, confirming its functional conservation.

Key words: Camelina sativa (L.) Crantz, FAD gene family, CsFAD3.3 gene, expression pattern analysis, functional validation, α-linolenic acid

高慧玲, 贾峥嵘, 薛金爱, 宋亚楠, 贾小云, 李润植. 亚麻荠CsFAD基因家族鉴定及α-亚麻酸合成关键基因CsFAD3.3的功能分析[J]. 生物技术通报, 2026, 42(10): 122-133.

GAO Hui-ling, JIA Zheng-rong, XUE Jin-ai, SONG Ya-nan, JIA Xiao-yun, LI Run-zhi. Genome-wide Identification of the CsFAD Gene Family in Camelina sativa and Functional Characterization of the Key Gene CsFAD3.3 Involved in α-linolenic Acid Biosynthesis[J]. Biotechnology Bulletin, 2026, 42(10): 122-133.

图/表 10

图1 亚麻荠CsFAD基因家族的系统发育树、基序、保守结构域和基因结构

Fig. 1 Phylogenetic tree, motif, conserved domain and gene structure of CsFAD gene family in camelina

图2 亚麻荠CsFAD基因家族的进化树

Fig. 2 Phylogenetic tree of CsFAD gene family in camelina

图3 亚麻荠CsFAD基因家族染色体定位

Fig. 3 Chromosome mapping of CsFAD gene family in camelina

图4 亚麻荠CsFAD基因的种内共线性分析

Fig. 4 Intraspecific synteny analysis of CsFAD genes in camelina

图5 亚麻荠与拟南芥（A）、白菜（B）FAD基因的共线性分析

Fig. 5 Synteny analysis of FAD genes between Camelina sativa, Arabidopsis thaliana (A), and Brassica rapa (B)

图6 亚麻荠CsFAD启动子序列顺式作用元件

Fig. 6 Cis-element analysis in camelina CsFAD promoter sequences

图7 亚麻荠CsFAD基因家族成员在不同组织及种子发育阶段的表达热图

Fig. 7 Expression heatmap of CsFAD family genes across various tissues and seed developmental stages in camelina

图8 CsFAD3基因在不同组织及种子发育阶段的表达谱（A）以及亚麻荠种子发育过程中油含量的动态变化（B）采用RT-qPCR分析CsFAD3基因在根、茎、叶、花和开花后10、20、30和40 d种子的表达模式。以CsActin基因为内参基因，采用2-ΔΔCt方法计算相对表达量。数据来源于3次生物学重复，以均值±标准差表示（n=3）。采用单因素方差分析（ANOVA）后进行Tukey事后检验，不同小写字母表示组间差异显著（P<0.05），下同

Fig. 8 Expression profiles of CsFAD3 gene in different tissues and seed developmental stages (A) as well as dynamic changes in oil content during seed development of camelina (B)The expression patterns of CsFAD3 genes in roots, stems, leaves, petals, and seeds at 10, 20, 30, and 40 days after flowering (DAF) were analyzed by RT-qPCR. CsActin was used as the internal reference gene, and the relative expression levels were calculated using the 2-ΔΔCt method. Data represent the mean ± standard deviation (SD) from three biological replicates (n=3). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed to evaluate significant differences among groups, and different lowercase letters indicate statistically significant differences (P<0.05), the same below

图9 CsFAD3.3蛋白的亚细胞定位

Fig. 9 Subcellular localization of the CsFAD3.3 protein

图10 过表达CsFAD3.3基因在亚麻荠种子中总脂含量（A）及脂肪酸成分（B）分析

Fig. 10 Analysis of total lipid content (A) and fatty acid composition (B) in camelina seeds overexpressing CsFAD3.3

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