Changes in Cell Structure and Gene Expression of Lily Leaves with Necrosis
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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。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.
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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 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.
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 /bpQ20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_definitionTarCK/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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