百合叶烧病发病过程叶片细胞和差异表达基因分析

葛金涛; 王江英; 汤雪燕; 孙明伟; 腾年军; 朱朋波; 赵统利; 吴秋月; 邵小斌

doi:10.19303/j.issn.1008-0384.2022.002.012

百合叶烧病发病过程叶片细胞和差异表达基因分析

doi: 10.19303/j.issn.1008-0384.2022.002.012

1.
连云港市农业科学院，江苏　连云港　222000
2.
南京农业大学园艺学院，江苏　南京　210095
3.
连云港市海州区农业农村局，江苏　连云港　222006

基金项目: 连云港市农业科学院青年基金项目（QNJJ2004）；江苏省政策引导类计划项目(苏北科技专项)（SZ-LYG202039）；江苏省农业科技自主创新资金[CX(20)3025]

详细信息

作者简介:
葛金涛（1987−），男，硕士，助理研究员，主要从事园艺作物育种研究（E-mail：672643419@qq.com）

通讯作者:
邵小斌（1972−），男，学士，研究员，主要从事园艺作物育种与栽培技术研究（E-mail：382748523@qq.com）

中图分类号: S 682.29
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出版历程
- 收稿日期: 2021-11-19
- 录用日期: 2021-11-19
- 修回日期: 2021-12-30
- 刊出日期: 2022-02-25

Changes in Cell Structure and Gene Expression of Lily Leaves with Necrosis

1.
Lianyungang Academy of Agricultural Sciences, Lianyungang, Jiangsu　222000, China
2.
College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu　210095, China
3.
Bureau of Agricultural and Rural Affairs of Haizhou District, Lianyungang, Jiangsu　222006, China

摘要

摘要: 目的明确百合叶烧病发病过程中百合叶片细胞结构的变化，探究百合叶烧病发病的分子调控机理。方法通过扫描电镜和透射电镜观察东方百合Tarrango正常叶片、轻度叶烧叶片和重度叶烧叶片超微结构。并通过比较东方百合Tarrango正常叶片、叶烧叶片、正常叶片喷钙和叶烧叶片喷钙4种处理的转录组测序数据，对差异表达基因进行序列对比和分类分析。结果百合叶片近轴面表皮细胞大小随叶烧程度的加深而减小，而液泡失水是导致表皮细胞体积缩小的原因。转录组测序共获得349 537条unigenes，平均长度为513.25 bp。124 405条unigenes获得注释，占unigenes总数的35.59%。共发现7 185个差异表达基因，包括5 860个特异差异表达基因和1 325个共同差异表达基因。KEGG富集分析显示，“代谢途径”“丙酮酸代谢”“次生代谢产物的生物合成”和“光合作用生物的碳固定作用”的基因在4组试验处理都有富集。百合叶烧病发病过程中显著下调基因有FOLK、PLD1_2、ATPeF1B 和KCS、CALM、ENO和pel等；而在叶烧叶片喷钙后，表达量上调的基因有CALM、CPK、EIN2、AUX1、PLD1_2和SORD等。结论缺钙是导致百合叶烧病的重要因素，由缺钙引起的液泡失水和细胞皱缩等病害表征可能受脱落酸、乙烯和生长素等激素调控。
- 百合 /
- 叶烧病 /
- 电镜 /
- 转录组 /
- 差异表达基因
Abstract: Objective Changes in the cell structure and gene expression of leaf caused by the pathogenesis of lily upper leaf necrosis (ULN) were studied to understand the molecular mechanism of the disease on Oriental Lily Tarrango (Lilium tarrango) . Methods Ultrastructure of the leaves from normal as well as mildly and severely ULN-infected lily plants was examined under a scanning electron microscope (SEM) and a projection electron microscope (TEM). Transcriptome sequences of the leaf specimens with or without calcium spraying were compared to identify the differentially expressed genes. Result The size of epidermal cells on the adaxial leaf surface shrank as the ULN worsened with vacuolar water loss. A total of 7 185 differentially expressed genes were identified that included 5 860 specific and 1 325 common differentially expressed genes. The KEGG enrichment analysis showed that the genes were enriched in the metabolic pathways, pyruvate metabolism, biosynthesis of secondary metabolites, and carbon fixation of photosynthetic organisms in all 4 groups of specimens with or without calcium spraying. FOLK, PLD1_2, ATPeF1B, KCS, CALM, ENO, and pel were significantly downregulated during the progress of ULN on the leaves. After the calcium spraying, CALM, CPK, EIN2, AUX1, PLD1_2, and SORD were upregulated. Conclusion Calcium deficiency was deemed to be the key factor that led to ULN on the lily plants. The deficiency produced the symptoms, such as vacuolar moisture loss and cellular shrinkage, might be regulated by the hormone metabolisms related to abscisic acid, ethylene, and auxin.
- Oriental Lily Tarrango /
- lily upper leaf necrosis /
- electron microscopy /
- transcriptome /
- differentially expressed gene

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图 1 不同叶烧程度百合叶片

注：A，百合Tarrango正常叶片；B，百合Tarrango轻度叶烧叶片；C，百合Tarrango重度叶烧叶片。图2同。

Figure 1. Leaves from different degrees of ULN-infection

Note: A: Leaf from normal lily plant; B: Leaf from lily plant mildly infected by ULN; C: Leaf from lily plant severely infected by ULN. Same for Fig. 2.

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图 2 百合叶片近轴面超微结构

Figure 2. Ultrastructure of lily leaves in adaxial view

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图 3 百合叶片远轴面超微结构

注：A，百合Tarrango重度叶烧叶片；B，百合Tarrango轻度叶烧叶片；C，百合Tarrango正常叶片。

Figure 3. Ultrastructure of lily leaves in abaxial view

Note: A, Leaf from lily plant severely infected by ULN; B, Leaf from lily plant mildly infected by ULN; C, Leaf from normal lily plant.

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图 4 百合叶片透射电镜下超微结构

注：A和B：百合Tarrango正常叶片；C和D：百合Tarrango轻度叶烧叶片；E和F：百合Tarrango重度叶烧叶片。

Figure 4. Ultrastructure of lily leaves shown by TEM

Note: A and B: normal leaf of lily Tarrango; C and D: mildly ULN leaf of Lily Tarrango; E and F: severely Lily ULN leaf of Lily Tarrango.

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图 5 差异表达基因的表达谱分析

注：A，差异表达基因的聚类分析；B，差异表达基因的数量；C，差异表达基因的主成分分析；D，差异基因维恩图分析。

Figure 5. Expression profiling of differentially expressed genes

Note: A: Cluster analysis on differentially expressed genes; B: Number of differentially expressed genes; C: Principal component analysis on differentially expressed genes; D: Venn diagram of differential genes.

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图 6 差异表达基因的GO富集分析

注：1，催化活性；2，结合；3，转运活性；4，结构分子活性；5，电子载体；6，核酸结合转录因子活性；7，酶调节活性；8，抗氧化活性；9，分子传感器活性；10，细胞组分；11，细胞器；12，膜部分；13，细胞器部分；14，细胞膜；15，高分子复合物；16，胞外区；17，类核；18，细胞连接；19，代谢过程；20，细胞过程；21，单一生物过程；22，生物调节；23，应激反应；24，定位；25，发育过程；26，多生物过程；27，免疫系统过程；28，组织细胞组成或生物起源；29，生殖过程；30，多细胞生物过程；31，繁殖；32，细胞外区域部分；33，细胞外基质；34，细胞外基质成分；35，运动；36，生长；37，突触部分。

Figure 6. GO enrichment analysis on differentially expressed genes

Note: 1: catalytic activity; 2: binding; 3: transporter activity; 4: structural molecule activity; 5: electron carrier activity; 6: nucleic acid binding transcription factor activity; 7: enzyme regulator activity; 8: antioxidant activity; 9: molecular transducer activity; 10: cell part; 11: organelle; 12: membrane part;13: organelle part; 14: membrane; 15: macromolecular complex; 16: extracellular region; 17: nucleoid; 18: cell junction; 19: metabolic process; 20: cellular process; 21: single-organism process; 22: biological regulation; 23: response to stimulus; 24: localization; 25: developmental process; 26: multi-organism process; 27: immune system process; 28: cellular component organization or biogenesis; 29: reproductive process; 30: multicellular organismal process; 31: reproduction; 32: extracellular region part; 33: extracellular matrix; 34: extracellular matrix component; 35: locomotion; 36: growth; 37: synapse part.

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表 1 12个cDNA文库的过滤数据

Table 1. Clean data of 12 cDNA library

样品 Sample	平均长度 Average length/bp	总序列数 The total number of sequences	总碱基数 Total base number /bp	Q20含量 Q20 content /%	Q30含量 Q30 content /%	GC含量 GC /%
TarCK1	148.70	46691744	6943031355	98.11	94.32	50.55
TarCK2	148.77	49717384	7396527911	98.26	94.67	49.80
TarCK3	148.63	40433794	6009572727	98.15	94.35	52.27
TarCa1	148.74	47758784	7103493445	98.15	94.44	51.41
TarCa2	148.87	40229314	5988834979	98.07	94.30	52.04
TarCa3	149.00	46500744	6928790175	98.07	94.28	51.59
TNCK1	148.98	43959714	6549244364	98.15	94.42	50.78
TNCK2	148.79	40479744	6022881697	98.23	94.59	49.17
TNCK3	148.87	45992514	6846847661	98.08	94.21	49.91
TNCa1	149.00	44762836	6669452846	98.10	94.41	52.32
TNCa2	148.96	46342564	6903233715	98.04	94.15	51.03
TNCa3	148.89	48524526	7224858395	98.09	94.31	52.03
合计 Total		541393662	80686769270

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表 2 转录组序列组装分析

Table 2. Summary of transcriptome assembly

序列类型 Sequence type	重叠群 Contig	序列 Unigene
最短序列长度 Minimum sequence Length/bp	25	201
最长序列长度 Maximum sequence length/bp	15 734	11 377
序列平均长度 Mean sequence length/bp	53.76	513.25
N50长度 N50 length/bp	48	686
(A+T)/%	51.94	55.72
(C+G)/%	48.06	44.28
序列总数 The total number of sequences	19 585 575	349 537
总碱基数量 Total base number/bp	1 052 959 813	179 400 360

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表 3 Unigene 的长度及数量统计

Table 3. Unigene length and quantity statistics

长度 Length/bp	数量 Number	比例 Percentage/%
<200	0	0.00
200～500	265 367	75.92
500～1 000	44113	12.62
1 000～1 500	17 752	5.08
1 500～2 000	10 495	3.00
≥2 000	11 809	3.38
总数 Total	349 537	100

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表 4 显著差异表达基因

Table 4. Significantly differentially expressed genes

对照组 Group	基因编号 Gene ID	差异倍数 Log2 fold change	基因名称 KO_name	基因定义 KO_definition
TarCK/TNCK	DN74111_c0_g1_i2	−6.76	FOLK	法呢醇醇激酶 Farnesol kinase
TarCK/TNCK	DN42826_c0_g2_i2	−6.56	PLD1_2	磷脂酶D1/2 Phospholipase D1/2
TarCK/TNCK	DN73032_c0_g2_i1	−3.14	ATPeF1B	膜上ATP合酶 F-type H+-transporting ATPase subunit beta
TarCK/TNCK	DN59908_c0_g1_i2	−1.88	KCS	3-酮脂酰辅酶A合成酶基因 3-ketoacyl-CoA synthase
TarCK/TNCK	DN88233_c0_g1_i2	−1.08	ENO	烯醇酶 Enolase
TarCK/TNCK	DN45172_c0_g1_i1	−1.00	CALM	钙调蛋白 Calmodulin
TarCK/TNCK	DN75575_c0_g1_i1	6.00	AAO3	脱落醛氧化酶 Abscisic-aldehyde oxidase
TNCK/TNCa	DN75425_c0_g1_i6	−4.83	ABF	ABA响应元件结合因子 ABA responsive element binding factor
TNCK/TNCa	DN89025_c0_g1_i3	−2.34	MFP2	烯酰辅酶A水合酶/3-羟酰辅酶A脱氢酶 Enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase
TNCK/TNCa	DN10149_c0_g1_i1	1.09	AUX1	生长素流入载体蛋白 Auxin influx carrier
TNCK/TNCa	DN36010_c0_g1_i1	1.20	CALM	钙调蛋白 Calmodulin
TNCK/TNCa	DN81084_c0_g4_i2	1.29	CPK	钙依赖性蛋白激酶 Calcium-dependent protein kinase
TNCK/TNCa	DN132576_c0_g1_i1	1.64	CML	钙结合蛋白CML Calcium-binding protein CML
TNCK/TNCa	DN55671_c1_g1_i8	2.01	PLD1_2	磷脂酶D1/2 Phospholipase D1/2
TNCK/TNCa	DN1778_c0_g3_i1	2.45	SORD	L-艾杜糖醇-2-脱氢酶 L-iditol 2-dehydrogenase
TNCK/TNCa	DN48191_c0_g1_i1	2.61	EIN2	乙烯不敏感蛋白2 Ethylene-insensitive protein 2

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参考文献(24)

[1]	CHANG Y C, ALBANO J P, MILLER W B. William B Miller. Oriental hybrid lily cultivars vary in susceptibility to upper leaf necrosis [J]. Acta Horticulturae, 2008, 766(766): 433−440.
[2]	CHANG Y C, MILLER W B. The relationship between leaf enclosure, transpiration, and upper leaf necrosis on Lilium 'Star gazer' [J]. Journal of the American Society for Horticultural Science, 2004, 129(1): 128−133. doi: 10.21273/JASHS.129.1.0128
[3]	CHANG Y C, GRACE-MARTIN K, MILLER W B. Efficacy of exogenous calcium applications for reducing upper leaf necrosis in lilium `Star Gazer' [J]. Hortscience A Publication of the American Society for Horticultural Science, 2004, 39(2): 272−275.
[4]	TSAI Y H, SUSILO H, CHANG Y. Effects of temperature and defoliation on upper leaf necrosis in lilium ‘star gazer’ [J]. Acta Horticulturae, 2011, 886(886): 289−297.
[5]	杨爽. 设施百合品种筛选与“叶烧病”发生机理的研究[D]. 北京: 北京林业大学, 2012. YANG S. Studies on screening and mechanism of the upper leaf necroses of facility lily cultivars[D]. Beijing: Beijing Forestry University, 2012. (in Chinese)
[6]	MORTAZAVI S N, KARIMI V, AZIMI M H. Pre-harvest foliar application of humic acid, salicylic acid and calcium chloride to increase quantitative and qualitative traits of Lilium longiflorum cut flowers [J]. Journal of Science & Technology of Greenhouse Culture, 2015, 6(3): 37−46.
[7]	MIRABBASI N, NIKBAKHT A, ETEMADI N, et al. Effect of different concentrations of potassium silicate, nano-silicon and calcium chloride on concentration of potassium, calcium and magnesium, chlorophyll content and number of florets of Asiatic lily cv. 'Brunello' [J]. Journal of Science & Technology of Greenhouse Culture, 2013, 4(14): 41−50.
[8]	FITZPATRICK A H, BHANDARI J, CROWELL D N. Farnesol kinase is involved in farnesol metabolism, ABA signaling and flower development in Arabidopsis [J]. The Plant Journal, 2011, 66(6): 1078−1088. doi: 10.1111/j.1365-313X.2011.04572.x
[9]	LI S, HUANG M, DI Q, et al. The functions of a cucumber phospholipase D alpha gene (CsPLDα) in growth and tolerance to hyperosmotic stress [J]. Plant Physiology and Biochemistry, 2015, 97: 175−186. doi: 10.1016/j.plaphy.2015.10.006
[10]	TODD J, POST-BEITTENMILLER D, JAWORSKI J G. KCS₁ encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana [J]. The Plant Journal, 1999, 17(2): 119−130. doi: 10.1046/j.1365-313X.1999.00352.x
[11]	SNEDDEN W A, FROMM H. Calmodulin as a versatile calcium signal transducer in plants [J]. The New Phytologist, 2001, 151(1): 35−66. doi: 10.1046/j.1469-8137.2001.00154.x
[12]	VOLL L M, HAJIREZAEI M R, CZOGALLA-PETER C, et al. Antisense inhibition of enolase strongly limits the metabolism of aromatic amino acids, but has only minor effects on respiration in leaves of transgenic tobacco plants [J]. The New Phytologist, 2009, 184(3): 607−618. doi: 10.1111/j.1469-8137.2009.02998.x
[13]	SUN H, HAO P, GU L, et al. Pectate lyase-like Gene GhPEL76 regulates organ elongation in Arabidopsis and fiber elongation in cotton [J]. Plant Science, 2020, 293: 110395. doi: 10.1016/j.plantsci.2019.110395
[14]	KHAN M, IMRAN Q M, SHAHID M, et al. Nitric oxide- induced AtAO3 differentially regulates plant defense and drought tolerance in Arabidopsis thaliana [J]. BMC Plant Biology, 2019, 19(1): 602. doi: 10.1186/s12870-019-2210-3
[15]	时欢, 李蓉, 高玉莹, 等. 文心兰乙烯不敏感基因EIN2的克隆及表达分析 [J]. 西北植物学报, 2018, 38(9):1613−1619. SHI H, LI R, GAO Y Y, et al. Cloning and expression of ethylene insensitivity gene EIN2 in oncidesa [J]. Acta Botanica Boreali-Occidentalia Sinica, 2018, 38(9): 1613−1619.(in Chinese)
[16]	VANDENBUSSCHE F, PETRÁSEK J, ZÁDNÍKOVÁ P, et al. The auxin influx carriers AUX1 and LAX3 are involved in auxin-ethylene interactions during apical hook development in Arabidopsis thaliana seedlings [J]. Development (Cambridge, England), 2010, 137(4): 597−606. doi: 10.1242/dev.040790
[17]	王东, 牛蓓, 宋君, 等. 基于银耳转录组测序的多糖代谢途径分析 [J]. 西南农业学报, 2019, 32(6):1347−1352. WANG D, NIU B, SONG J, et al. Study on fructose and mannose metabolism pathway of Tremella fuciformis based on transcriptome [J]. Southwest China Journal of Agricultural Sciences, 2019, 32(6): 1347−1352.(in Chinese)
[18]	FINKELSTEIN R, GAMPALA S S L, LYNCH T J, et al. Redundant and distinct functions of the ABA response loci ABA-INSENSITIVE(ABI)₅ and ABRE-BINDING FACTOR (ABF)₃ [J]. Plant Molecular Biology, 2005, 59(2): 253−267. doi: 10.1007/s11103-005-8767-2
[19]	ARENT S, CHRISTENSEN C E, PYE V E, et al. The multifunctional protein in peroxisomal beta-oxidation: Structure and substrate specificity of the Arabidopsis thaliana protein MFP₂ [J]. The Journal of Biological Chemistry, 2010, 285(31): 24066−24077. doi: 10.1074/jbc.M110.106005
[20]	王五宏, 钟新民, 李必元, 等. 盐钙胁迫下大白菜干烧心病的发生及矿质营养分配 [J]. 核农学报, 2012, 26(8):1204−1208. WANG W H, ZHONG X M, LI B Y, et al. Effects of calcium and salt stress on tipburn and nutrition distribution of Chinese cabbage [J]. Acta Agriculturae Nucleatae Sinica, 2012, 26(8): 1204−1208.(in Chinese)
[21]	程涣, 苏同兵, 于拴仓, 等. 大白菜钙运输基因ECA和钙响应基因CAS在缺钙胁迫下的表达分析 [J]. 植物生理学报, 2015, 51(4):566−572. CHENG H, SU T B, YU S C, et al. Expression analysis of Ca²⁺ transport and response genes, ECA and CAS, in cabbage under calcium deficiency condition [J]. Plant Physiology Journal, 2015, 51(4): 566−572.(in Chinese)
[22]	王娟, 王倩, 陈清. 结球莴苣“烧边”成因及其调控措施的研究进展 [J]. 中国蔬菜, 2005, 25(B10):32−35. WANG J, WANG Q, CHEN Q. Research progress on causes and control measures of "edge burning" in lettuce [J]. China Vegetables, 2005, 25(B10): 32−35.(in Chinese)
[23]	WELTI R, LI W, LI M, et al. Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis [J]. The Journal of Biological Chemistry, 2002, 277(35): 31994−32002. doi: 10.1074/jbc.M205375200
[24]	ZHANG Y, ZHU H, ZHANG Q, et al. Phospholipase Dα1 and phosphatidic acid regulate NADPH oxidase activity and production of reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis [J]. The Plant Cell, 2009, 21(8): 2357−2377. doi: 10.1105/tpc.108.062992