Analysis of α-Linolenic Acid Content and Related Genes in Seeds of Sea Buckthorn Cultivars

ZHANG Tong; LYU Zhong-rui; WEI Ji-hua; ZHANG Guo-yun; LUO Hong-mei; HE Cai-yun

doi:10.13275/j.cnki.lykxyj.2020.04.001

Volume 33 Issue 4

Aug. 2020

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Analysis of α-Linolenic Acid Content and Related Genes in Seeds of Sea Buckthorn Cultivars

1.
Key Laboratory of Tree Breeding and Cultivation, National Forestry and Grassland Administration, Research Institute of Forestry, Chinese academy of forestry, Beijing　100091, China
2.
Experimental Center of Desert Forestry, Chinese Academy of Forestry, Dengkou　015200, Inner Mongolia, China

Corresponding author: HE Cai-yun, hecy@caf.ac.cn
Received Date: 2020-02-27
Accepted Date: 2020-05-22

Abstract

Objective To study the differences of gene regulation in seeds of two sea buckthorn (Hippophae rhamnoides) cultivars with different α-linolenic acid content and to find the method of further increase the content of α-linolenic acid in H. rhamnoides seed. Method The difference in α-linolenic acid content and the expressed genes in the seeds of two H. rhamnoides cultivars were analyzed and determined by gas chromatography-mass spectrometry and transcriptomics. Result The proportion of α-linolenic acid in H. rhamnoides 'Mongolia' (XY) and H. rhamnoides ‘Sinensis’ (FN) seeds was different. The fatty acid with the highest content of XY was α-linolenic acid, followed by linoleic acid. The content of α-linolenic acid in FN was lower than that of linoleic acid. In semi-mature stage (T2), the expression levels of gene FAD2 and gene FAD3 in XY were 3.83 times and 13.63 times that in FN respectively. The expression of gene FAD7 in XY was 2.09 times and 1.72 times that in FN in immature stage (T1) and T2respectively. It was consistent with the trend of linoleic acid and α-linolenic acid content. On the contrary, the expressions of gene LOX3.1 and gene LOX3.2 in FN were 8.02 times and 7.12 times that of XY in T2 respectively. Conclusion The high accumulation of α-linolenic acid in H. rhamnoides seeds is due to the synergistic effect of multiple genes. High expression of gene FAD2, FAD3, FAD7 and low expression of gene LOX3.1, LOX3.2 contribute to the accumulation of α-linolenic acid.
- Hippophae rhamnoides,
- α-linolenic acid,
- cultivars,
- coordinated regulation of multi genes

References

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沈阳化工大学材料科学与工程学院沈阳 110142

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沙棘(Hippophae rhamnoides L.)是胡颓子科沙棘属的一种雌雄异株的灌木或小乔木，耐旱性和耐寒性良好。沙棘果实具有独特的药用和营养价值，在中国和俄罗斯有上百年的药用历史^[1]。沙棘果实富含维生素、有机酸、氨基酸、脂肪酸、抗氧化剂和类黄酮^[2-4]，在炎症性疾病、肝脏性疾病、动脉粥样硬化等疾病的治疗以及保护心脏等方面具有显著功效^[5]。在沙棘种子含有的众多脂肪酸中，α-亚麻酸（α-Linolenic acid，C18:3N3）的含量十分丰富，且α-亚麻酸对大脑发育、心血管健康、炎症治疗等都具有重要作用^[6-9]，但α-亚麻酸是人体必需脂肪酸，不能在人体内合成，只能通过食物摄取。豆油、花生油等常见植物食用油中的主要脂肪酸为油酸、亚油酸、棕榈酸和硬脂酸^[10-11]，α-亚麻酸含量极低，而沙棘中高积累20.3%~36.3%的α-亚麻酸^[12]，并且沙棘作为耐旱木本植物可以在沙地、荒山、盐碱地等边际土地生长，可避免与大田油料作物争夺土地，是理想的α-亚麻酸供给对象。

目前，油料作物脂肪酸合成途径及调控基因的研究较多，在沙棘的相关研究中多围绕脂肪酸组分及脂肪酸测定方法展开，少有针对沙棘中α-亚麻酸合成机制的研究。本研究选取α-亚麻酸含量存在差异的2个沙棘品种种子，对其含量差异及相关基因进行分析，在基因表达水平上研究沙棘中α-亚麻酸合成及代谢规律，为培育高α-亚麻酸含量的沙棘良种奠定理论基础。

1. 实验材料

沙棘种子采自中国林业科学研究院沙漠林业实验中心（内蒙古磴口县），2个沙棘品种——蒙古大果沙棘向阳（Hippophae rhamnoides ‘Mongolia’）（XY）和中国沙棘丰宁（H. rhamnoides ‘Sinensis’）（FN）的种子分别采自3个不同的发育时期，依次为未成熟期（T1）、半成熟期(T2)和成熟期(T3)，在这3个时期沙棘种子的种皮颜色依次呈现为白色、黑白相间斑点色、黑色。因沙棘种皮与果皮的转色期基本一致，为保证时期界定更加精准，同时利用色差计测量沙棘果皮的红绿色指标 a 值和黄蓝指标 b 值，计算色泽比（h=a/b），种皮呈白色且果皮h值介于−0.18～−0.10的界定为T1时期，种皮呈黑白相间斑点色且果皮h值介于0.20～0.28的界定为T2时期，种皮呈黑色且果皮h值介于0.52～0.60的界定为T3时期。沙棘果实采集后快速将种子从果实中剥离，放入液氮中进行速冻，然后转移至−80℃冰箱长久保存，用于下一步分析。

2. 实验方法

2.1. 脂肪酸提取

将样品冰上解冻，取50 mg样品于2 mL离心管中。加入1 mL的氯仿甲醇(2:1 v/v)溶液，超声30 min。取上清液，加入1%硫酸甲醇溶液2 mL，80℃水浴，甲酯化30 min，随后加入1 mL的正己烷萃取，5 mL的纯水洗涤。吸取上清液500 μL，加入25 μL的水杨酸甲酯（500 mg·L⁻¹）作为内标，混匀加入进样瓶，进样量1 μL，分流比 10∶1，分流进样，进行GC-MS 检测。

2.2. 色谱-质谱分析

样品采用 AgilentDB-WAX 毛细管柱(30 m×0.25 mmID×0.25 μm)气相色谱系统进行分离。程序升温：初始温度为 50℃，保持 3 min，随后以10℃·min⁻¹的速度升温至220℃并维持 20 min。载气为氦气，载气流速1.0 mL·min⁻¹。样本队列中每间隔6个实验样本设置1个QC样本，用于检测和评价系统的稳定性及重复性。采用 Agilent7890A/5975C 气-质联用仪进行质谱分析。进样口温度 280℃，离子源温度230℃，传输线温度250℃。电子轰击电离（EI）源，SIM 扫描方式，电子能量70 eV。采用 MSDChemStation 软件提取色谱峰面积及保留时间。绘制标准曲线，计算样品中中长链脂肪酸的含量。

2.3. 转录组测序分析

按照Qingen（Qingen，DE）试剂盒^[13]操作说明对样品中的总RNA进行提取，并去除总RNA中DNA片段的残留。分别使用NanoPhotometer 分光光度仪、Qbuit 2.0 荧光计和Qubit RNA 检测试剂盒以及Agilent 2100和RNA Nano 6000试剂盒检测RNA纯度、RNA浓度以及RNA完整性。RNA检测合格后进行文库制备。使用Illumina HiSeq 2000 对RNA文库进行测序，获得原始测序数据。为了保证测序数据质量，对原始数据进行过滤，从而获得高质量数据（Clean data），用于后续生物信息分析。首先使用DESeq R（1.18.0）^[14]进行基因表达量分析，筛选差异表达基因，然后通过GOseq R数据包^[15]和KOBAS（2.0）^[16]分别对差异表达基因进行GO功能注释和KEGG通路分析。

4. 讨论

沙棘种子中富含不饱和脂肪酸，其中，α-亚麻酸含量一般较高。α-亚麻酸作为人体必需脂肪酸，具有多种有益的生理功能，因此，为了进一步提高沙棘种子中α-亚麻酸含量，培育沙棘新品种和良种，在分子水平上研究沙棘中α-亚麻酸的合成代谢规律至关重要。亚油酸是α-亚麻酸合成的底物，脱饱和生成亚麻酸^[17-18]。本研究选取的2个沙棘品种中，α-亚麻酸/亚麻酸的比值存在显著差异，XY种子中α-亚麻酸是含量最高的脂肪酸，亚油酸含量次之，而在FN中正相反，含量最高的脂肪酸是亚油酸，α-亚麻酸含量次之。因此，基于这2个品种存在的差异挖掘沙棘种子中调控亚油酸向α-亚麻酸转化的关键基因以及抑制α-亚麻酸分解的关键基因，探究α-亚麻酸合成代谢规律。

FAD2催化油酸转化为亚油酸^[19-21]，FAD2基因表达量下降使亚油酸合成受阻，进而影响α-亚麻酸的生成，而FAD2基因的高表达可以为α-亚麻酸的合成提供充足的底物。2个沙棘品种种子中的亚油酸含量在T2时期快速积累，与T2时期基因FAD2的表达水平急剧上升相一致。在XY和FN种子中，T1和T2时期之间的亚油酸含量差异显著。T3时期基因FAD2的表达量降低，亚油酸含量积累速度明显下降。

α-亚麻酸在FAD3和FAD7的催化下由亚油酸脱饱和产生^[17-18]。FAD3与FAD7同属于ω-6 型脂肪酸去饱和酶，已有的研究表明，基因FAD3在植物种子α-亚麻酸的合成过程中起到关键作用^[22-24]。实验表明，拟南芥的FAD3基因在大豆中异位表达可使大豆中α-亚麻酸含量增加，大豆的FAD3C基因在芝麻中特异性表达同样也可使芝麻中α-亚麻酸的含量显著提高^[25]。此外，Wu等将麻风树的JcFAD3基因在拟南芥的种子中特异性表达使拟南芥种子中α-亚麻酸的含量提高了20.50%~24.94%；同时亚油酸的含量相比野生型降低了11.4%~23.5%^[26]。在烟草中过表达拟南芥FAD7基因，同样使烟草中α-亚麻酸的含量升高且亚油酸含量下降^[23]。

在T1和T2时期，XY中基因FAD3和基因FAD7的表达量远高于FN，尤其在T2时期，基因FAD3在XY和FN中的表达差异显著，在XY中的表达量是FN中的13.63倍，调控大量亚油酸脱饱和生成α-亚麻酸，因此，XY种子中α-亚麻酸的含量大于亚油酸的含量，而FN中亚油酸含量比α-亚麻酸高。这解释了为何基因FAD2在XY种子中的表达量高于FN，但亚油酸含量却是FN种子大于XY种子。

基因LOX是α-亚麻酸代谢过程中的关键基因，是调控α-亚麻酸转化为其他物质的第一步，调控α-亚麻酸氧化，生成脂肪酸氢过氧化物^[27-29]。基因LOX3.1和基因LOX3.2在XY和FN发育的T1时期表达量处于3个时期的最大值，抑制α-亚麻酸的积累，协同基因FAD2、FAD3、FAD7在T1时期的低表达水平，导致T1时期α-亚麻酸含量处于低水平，T2时期基因LOX3.1和LOX3.2表达量下调，基因FAD2、FAD3、FAD7表达量急剧上升，因而α-亚麻酸含量在T2时期显著增长。同时，基因LOX3.1和LOX3.2在FN种子中的表达量高于XY种子，抑制FN种子中α-亚麻酸的积累，使FN种子中α-亚麻酸积累量低于XY。

沙棘种子中α-亚麻酸的高积累源于多个基因的协同调控作用，基因FAD2、FAD3、FAD7的高表达协同基因LOX3.1和LOX3.2的低表达使α-亚麻酸的高积累得以实现。

本研究在基因和代谢水平上研究沙棘中α-亚麻酸合成代谢规律，对进一步提高沙棘种子中α-亚麻酸含量、培育沙棘良种具有重要意义。

5. 结论

通过对2个不同沙棘品种种子中中长链脂肪酸和转录组数据联合分析得出：沙棘种子中α-亚麻酸的高积累源于多个基因的协同调控作用。基因FAD2调控油酸去饱和生成亚油酸，其高表达为α-亚麻酸的合成提供了充足的底物。α-亚麻酸在基因FAD3和基因FAD7的调控下由亚油酸去饱和产生，基因FAD3和基因FAD7的高表达促进亚油酸向α-亚麻酸转化，使α-亚麻酸积累，同时使亚油酸含量下降。沙棘种子中基因LOX3.1和LOX3.2是α-亚麻酸代谢过程中的关键基因，其表达水平下降有利于种子中α-亚麻酸的积累。

Reference (29)

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组分 Compositions	XY			FN
组分 Compositions	T1	T2	T3	T1	T2	T3
C10:0	1.04±0.09 b	1.75±0.13 ab	2.43±0.69 a	1.04±0.07 b	1.72±0.20 ab	2.24±0.20 a
C12:0	1.11±0.27 a	2.06±0.78 a	5.62±0.32 a	0.34±0.40 a	1.34±0.17 a	2.09±1.25 a
C13:0	0.08±0.05 b	0.30±0.08 ab	0.35±0.188 a	0.08±0.02 b	0.31±0.10 ab	0.48±0.10 a
C14:0	20.70±2.32 c	40.23±2.638 a	53.98±18.17 a	17.66±1.34 c	40.85±7.66 a	58.91±7.54 a
C14:1	136.34±14.20 a	145.86±15.94 a	122.48±14.74 a	121.50±9.22 a	121.08±9.35 a	117.06±7.36 a
C15:0	6.20±1.43 c	19.42±1.16 b	28.45±10.91 ab	6.84±0.91 c	24.40±5.20 b	37.00±5.97 a
C15:1	16.90±2.09 a	14.37±2.21 ab	8.59±0.85 c	14.65±0.78 ab	12.06±4.19 abc	10.52±1.83 bc
C16:0	1 219.70±135.84 b	2 337.41±132.23 a	2 873.02±915.18 a	1 141.08±69.79 b	2 482.63±282.95 a	3 165.25±346.54 a
C16:1	38.04±6.73 c	70.55±12.28 bc	112.18±36.82 ab	28.40±4.84 c	86.75±22.13 ab	126.20±25.12 a
C17:0	7.89±1.39 b	16.93±1.34 a	19.21±6.58 a	9.02±0.85 b	15.92±1.62 a	21.51±2.86 a
C17:1	66.01±7.57 a	51.99±6.67 ab	47.64±2.13 ab	56.48±12.46 ab	44.60±3.13 b	51.85±7.10 ab
C18:0	695.59±62.14 c	1 330.90±97.82 ab	1 656.18±553.21 a	634.31±23.71 c	1 035.51±73.38 bc	1 280.61±137.68 ab
C18:1	78.99±20.21 d	1 824.10±207.78 c	3 447.79±1 190.81 a	101.66±20.47 d	2 197.36±133.31 bc	3 029.64±464.58 ab
C18:2	311.23±62.81 d	4 231.52±246.51 c	6 879.55±2 327.19 ab	265.09±48.17 d	5 028.36±863.33 bc	7 443.04±795.32 a
C18:3N3	298.54±76.45 c	4 980.34±261.85 ab	7 463.53±2 636.16 a	223.36±46.56 c	4 307.42±690.29 b	6 526.07±969.49 ab
C20:0	19.50±3.83 c	102.61±13.30 b	200.82±74.68 a	16.14±2.06 c	94.96±7.90 b	139.33±19.39 b
C20:1	7.42±1.60 c	50.74±4.18 b	90.17±30.25 a	5.87±0.58 c	53.50±4.71 b	83.41±16.89 a
C20:2	0.93±0.22 c	8.69±0.52 b	14.62±5.51 ab	0.81±0.13 c	9.72±3.29 b	16.89±3.08 a
C21:0	3.85±0.72 b	8.44±0.77 ab	11.93±3.97 a	3.76±0.51 b	8.15±1.48 ab	11.94±2.08 a
C20:3N3	1.90±0.21 bc	3.02±0.23 ab	3.86±0.93 a	1.53±0.28 c	2.51±0.55 bc	3.94±0.72 a
C20:5N3	0 a	0.15±0.08 a	1.04±0.79 a	0 a	0.33±0.03 a	0.87±0.61 a
C22:0	12.20±1.62 c	36.59±4.23 b	57.26±20.45 ab	14.29±2.13 c	44.80±9.54 b	68.61±10.83 a
C22:1N9	24.60±3.00 ab	29.29±6.16 a	26.30±5.35 ab	16.34±0.58 b	21.45±3.73 ab	20.85±4.84 ab
C23:0	2.00±0.44 c	10.73±1.28 b	18.25±7.01 a	2.41±0.44 c	9.76±2.18 b	15.84±3.27 ab
C24:0	4.74±0.60 c	17.42±2.00 b	29.66±10.75 a	5.35±0.46 c	18.09±3.08 b	26.69±4.54 ab
C24:1	4.05±3.29 a	11.48±0.91 a	6.68±3.95 a	4.38±3.87 a	3.94±3.01 a	4.47±1.55 a
C22:6N3	2.62±0.11 c	3.43±0.05 a	3.25±0.25 ab	2.55±0.08 c	3.07±0.09 b	3.26±0.18.00 ab
注：同行不同字母代表显著差异（P< 0.05）。　　Note: Different letters in the same line represent significant differences（P< 0.05）

品种 Cultivar	C18:3N3/C18:2
品种 Cultivar	T2	T2-1	T2-2	T2-3
XY	1.182a	1.21	1.17	1.16
FN	0.862b	0.92	0.75	0.92
注：不同字母代表显著差异（P<0.05），T2-1、T2-2、T2-3代表T2时期的3个生物学重复样本。　　Notes: Different letters in the same column represent significant differences（P<0.05）.T2-1、T2-2 and T2-3represent three biological replicates of the T2 period.

Analysis of α-Linolenic Acid Content and Related Genes in Seeds of Sea Buckthorn Cultivars