薰衣草叶片对低温胁迫的生理与分子响应机制

doi:10.11983/CBB22046

摘要/Abstract

摘要： 薰衣草(Lavandula angustifolia)作为名贵的芳香植物, 其生长、繁育、品质和产量均受低温影响。前期研究已获得1个耐低温薰衣草品种。该研究将对其处理的温度从20°C降至0°C, 揭示薰衣草响应冷胁迫的生理及分子调控机制, 同时结合薰衣草的细胞质膜透性、可溶性糖和蛋白质含量及抗氧化酶活性等生理变化。采用转录组学和生物信息学方法挖掘分析相关耐寒基因, 并探讨外施水杨酸缓解-10°C冻胁迫的可行性。研究发现7个编码脂肪酸去饱和酶和转移酶的基因(LaFADs)、3个参与合成可溶性糖的基因(LaBAM1和LaSS2)、19个编码胚胎晚期丰富蛋白的基因(LaLEAs)及7个编码过氧化物酶的基因(LaPODs), 这些基因在低温胁迫下均上调表达, 指导薰衣草合成并积累保护物质, 维持膜稳定性以应对胁迫。此外, 150 mg·L^-1水杨酸预处理能有效缓解植株冻害, 可作为低温保护剂。该研究丰富了薰衣草重要抗逆基因家族的遗传背景, 为后续分子遗传学功能分析和定向品种改良奠定基础。

关键词: 薰衣草, 冷害, 冻害, 转录组学分析, 胚胎发育晚期丰富蛋白

Abstract: The growth and reproduction as well as quality and yield of Lavandula angustifolia, a valuable aromatic plant, are severely affected by low temperatures. In our previous study, a low-temperature tolerant lavender variety was obtained. This study revealed the physiological and molecular regulatory mechanisms of L. angustifolia in response to cold stress. The relevant cold tolerance genes were mined and analyzed using transcriptomic and bioinformatics approaches. The feasibility of external application of salicylic acid to alleviate -10°C freezing stress was also explored. The results showed that seven genes encoding fatty acid desaturases and transferases (LaFADs), three genes involved in the synthesis of soluble sugars (LaBAM1, LaSS2), 19 genes encoding late embryonic abundant proteins (LaLEAs), and seven genes encoding peroxidases (LaPODs) were found to be up-regulated at low temperatures, directing L. angustifolia to synthesize and accumulate protective substances which maintained membrane stability in response to stress. In addition, salicylic acid pretreatment at 150 mg·L^-1was effective in alleviating freezing damage to plants and could be used as a low-temperature protectant. This study provides a basis for further understanding molecular mechanism of L. angustifolia responding to low-temperature stress.

Key words: Lavandula angustifolia, chilling injury, freezing injury, transcriptomic analysis, late embriogenesis abundant protein (LEA)

蔺海娇, 曲嘉琪, 刘祎男, 苑泽宁. 薰衣草叶片对低温胁迫的生理与分子响应机制. 植物学报, 2022, 57(5): 611-622.

Lin Haijiao, Qu Jiaqi, Liu Yinan, Yuan Zening. Physiological and Molecular Response Mechanisms Under Low-temperature Stress in Lavandula angustifolia Leaves. Chinese Bulletin of Botany, 2022, 57(5): 611-622.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22046

https://www.chinbullbotany.com/CN/Y2022/V57/I5/611

图/表 13

表1 qRT-PCR反应所用引物

Table 1 Primers used for qRT-PCR

Gene ID	Gene name	Primer sequence (5′-3′)
DN12834_c2_g2	LaLEA1	F: CCAAGGCAATCTCTGCTCTCA	R: CTTTGGATCCGGAGCCTTTCT
DN11187_c0_g4	LaLEA2	F: TCTACGTCTTCATCTTCGCCG	R: GCGTTGTAGTTTGCCAGAGTG
DN10094_c0_g1	LaERD10	F: ATATGACGACGCACCGACTG	R: TCCGGTGCGTAATCCGAATC
DN14123_c1_g1	LaFAB2	F: CGACTTTTGTCTCCCACGGA	R: TGTCCCATCTGGGTCGATCT
DN16955_c0_g1	LaPOD2	F: CATGGACTATGTCACGCCGA	R: GCTCGAAGAAAGCAATGGCA
DN18853_c0_g2	LaBAM1	F: CCCACGGCGAGAGAATTGTA	R: TTTGAGCGATGGGGACGTAG
	GADPH	F: TAGGAGGTGGCAGGACATCA	R: CCCTTTACCCGTCACGTTGT

表2 植物冷害的症状分级

Table 2 Symptoms grading of plant chilling injury

Levels of chilling injury	Standard of judgment
1	Leaf present green and there are no symptoms of chilling injury
2	Only the edge of the leaf is yellow
3	Leaf withered and yellow part of < 50%, the green part is more
4	50%-100% of the leaf is dry and yellow, with less green
5	The whole plant is wilting
6	The aboveground parts dried up and died

表3 基因功能注释结果统计

Table 3 The statistical result of unigene functional annotation

Database	Number of annotated unigene	Percentage of annotated unigene (%)
NT	32797	42.73
NR	48554	63.26
Uniprot (BLASTX)	37801	49.25
Uniprot (BLASTP)	25631	33.39
PFAM	22340	29.11
eggNOG	28488	37.12
GO	24430	31.83
KEGG	15496	20.19
KOG	28787	37.51

图1 低温胁迫下薰衣草叶片的DEGs火山图(0°C vs 20°C)

Figure 1 DEGs volcano map of Lavandula angustifolia lea- ves under low-temperature stress (0°C vs 20°C)

图2 低温胁迫下薰衣草叶片DEGs的GO与KEGG富集情况(0°C vs 20°C) BP: 生物学过程; MF: 分子功能; CC: 细胞组分

Figure 2 GO and KEGG enrichment of DEGs in Lavandula angustifolia leaves under low-temperature stress (0°C vs 20°C) BP: Biological process; MF: Molecular function; CC: Cellular component

图3 薰衣草膜稳定性相关耐寒基因表达的变化及编码蛋白的互作关系 (A) 基因表达的变化(0°C vs 20°C); (B) 蛋白网络互作。FPKM: 每千个碱基的转录每百万映射读取的片段

Figure 3 Changes in expression of cold tolerance genes related to membrane stability of Lavandula angustifolia and the interaction network of these encoding proteins (A) Gene expression changes (0°C vs 20°C); (B) Protein interaction network. FPKM: Fragments per kilobase per million mapped fragments

图4 薰衣草与近缘物种LEA的进化树和保守基序分布(A)及seq LOGO (B)

Figure 4 The phylogenetic tree, conservative motif distribution (A) and seq LOGO (B) of LEA proteins in Lavandula angustifolia and related species

表4 薰衣草LaLEAs家族的理化信息

Table 4 Physical and chemical information of LaLEAs of Lavandula angustifolia

Gene ID	Gene name	Number of amino acids (aa)	Molecular weight (kDa)	Theoretical pI	Grand average of hydropathicity (GRAVY)	Aliphatic index	Instability index	Subcellular localization
DN12834_c2_g2	LaLEA1	100	10.90	10.32	-0.484	73.40	52.96	-
DN11187_c0_g4	LaLEA2	128	14.16	10.09	-0.088	74.77	22.26	Chloroplasts
DN13720_c1_g4	LaLEA3	264	28.68	5.12	-0.933	59.73	14.27	Chloroplasts
DN9448_c0_g7	LaLEA4	193	21.28	10.13	0.123	92.49	43.01	Chloroplasts
DN10042_c1_g5	LaLEA5	176	19.78	10.52	-0.463	83.01	34.04	Chloroplasts
DN20068_c2_g4	LaLEA6	122	13.28	10.07	-0.204	71.15	35.17	-
DN13618_c0_g1	LaLEA7	120	13.63	9.68	-0.299	61.08	16.35	Nucleus
DN7134_c0_g1	LaLEA8	208	22.93	9.97	-0.062	92.74	28.05	-
DN16104_c0_g1	LaLEA9	219	24.60	9.97	-0.357	72.19	41.89	-
DN9448_c0_g2	LaLEA10	132	14.84	10.06	-0.475	67.42	24.52	-
DN19299_c2_g2	LaLEA11	320	35.11	9.63	-0.674	55.44	89.14	-
DN11058_c1_g6	LaLEA12	124	13.00	8.83	-1.085	41.94	29.73	Mitochondrial
DN17935_c0_g1	LaLEA13	228	25.24	10.37	-0.224	89.74	42.60	-
DN5364_c0_g1	LaLEA14	229	25.76	9.85	-0.035	90.66	40.29	-
DN13618_c0_g3	LaLEA15	134	14.75	10.01	-0.440	64.03	24.39	-
DN24875_c0_g1	LaLEA16	121	12.51	8.78	-1.135	31.98	32.24	Nucleus
DN9962_c0_g1	LaLEA17	225	24.75	9.69	-0.036	98.67	35.53	Cytoplasm
DN10042_c1_g1	LaLEA18	203	22.61	10.07	-0.226	95.02	30.10	-
DN11740_c4_g1	LaLEA19	133	13.80	4.64	-0.514	72.56	19.99	Chloroplasts
DN11740_c4_g1	LaLEA20	356	37.92	5.88	-1.015	52.16	17.85	Chloroplasts
DN4252_c0_g1	LaLEA21	259	26.30	4.89	-0.167	82.43	30.36	Cytoplasm
DN6726_c0_g1	LaLEA22	204	22.22	10.42	-0.135	88.97	40.07	-
DN10094_c0_g1	LaERD10	155	17.51	5.79	-1.632	45.94	48.98	-

图5 20°C与0°C下薰衣草中编码可溶性糖和蛋白合成基因的聚类图

Figure 5 Cluster diagram of genes encoding soluble sugar and protein synthesis in Lavandula angustifolia under 20°C and 0°C

图6 薰衣草与其近缘物种的进化树及其LaPODs在低温胁迫下的表达变化(0°C vs 20°C)

Figure 6 The evolutionary tree of Lavandula angustifolia and it’s relative species, and LaPODs expression changes under low-temperature stress (0°C vs 20°C)

图7 薰衣草耐寒基因在低温胁迫下的表达水平验证(0°C vs 20°C)

Figure 7 Expression of cold tolerance genes in Lavandula angustifolia under low-temperature stress (0°C vs 20°C)

图8 在冷害与冻害条件下薰衣草叶片的生理变化(A-E)及水杨酸(SA)对冻害的缓解效果(F) MDA: 丙二醛; SS: 可溶性糖; SP: 可溶性蛋白; POD: 过氧化物酶。不同小写字母表示不同处理间差异显著(P<0.05)。

Figure 8 Physiological changes of Lavandula angustifolia leaves under chilling and freezing conditions (A-E) and alleviation of freezing injury by salicylic acid (SA) (F) MDA: Malondialdehyde; SS: Soluble sugars; SP: Soluble proteins; POD: Peroxidase. Different lowercase letters indicate significant differences among different treatments (P<0.05).

图9 薰衣草叶片响应低温并提高耐寒性途径示意图

Figure 9 Schematic diagram of the way that Lavandula angustifolia leaves respond to low temperature and improve cold tolerance

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