热带作物学报

引物名称Primer name	上游引物（5'–3'）Forward primer (5'–3')	下游引物（5'–3'）Reverse primer (5'–3')
GAPDH	ATCAACGGTTTCGGAAGGATCGG	TTCTGACCGAAGAGGAGGGTCTTG
GID1	ACCAAGAGCCTCATTATCGTGTCG	CCTTCTCACGGTACACGAGCTT
PIE1	CCTGAAAAGAGTGCGTTTGTCGTCTG	TCACTTATAAGCTCAGGTGAGGGTGC
FT1	ATCCCACATGAACGCG	GTAGCAAATCACCTCG
GA20ox1	TGACAAGGTGGTCAAC	CGACCT
GA20ox2	CACGGCATATGCCAGAGAACCCACATCACG	TCTCCTTCCACGGCAGCTT
	CGAACGGGTCCGGGATCTTT

引物名称Primer name	上游引物（5'–3'）Forward primer (5'–3')	下游引物（5'–3'）Reverse primer (5'–3')
GAPDH	ATCAACGGTTTCGGAAGGATCGG	TTCTGACCGAAGAGGAGGGTCTTG
GID1	ACCAAGAGCCTCATTATCGTGTCG	CCTTCTCACGGTACACGAGCTT
PIE1	CCTGAAAAGAGTGCGTTTGTCGTCTG	TCACTTATAAGCTCAGGTGAGGGTGC
FT1	ATCCCACATGAACGCG	GTAGCAAATCACCTCG
GA20ox1	TGACAAGGTGGTCAAC	CGACCT
GA20ox2	CACGGCATATGCCAGAGAACCCACATCACG	TCTCCTTCCACGGCAGCTT
	CGAACGGGTCCGGGATCTTT

通路Pathway	代谢通路ID Pathway ID	差异基因数目Counts of DEGs	P-value
Biosynthesis of amino acids	sbi01230	226	4.45E-07
Purine metabolism	sbi00230	103	2.67E-06
Carbon metabolism	sbi01200	247	0.0004462
Phagosome	sbi04145	79	0.0005084
Glycerophospholipid metabolism	sbi00564	113	0.0007
Inositol phosphate metabolism	sbi00562	75	0.0008856
Pentose phosphate pathway	sbi00030	63	0.0011618
Phosphatidylinositol signaling system	sbi04070	73	0.0011657
Ribosome	sbi03010	249	0.0035088
Endocytosis	sbi04144	144	0.0037752
Glyoxylate and dicarboxylate metabolism	sbi00630	72	0.0044091
Oxidative phosphorylation	sbi00190	101	0.0065322
Spliceosome	sbi03040	180	0.0065768
Pyrimidine metabolism	sbi00240	67	0.0081457
Arginine biosynthesis	sbi00220	38	0.0100436
Autophagy-other	sbi04136	45	0.0145776
Glycolysis/Gluconeogenesis	sbi00010	133	0.0183614
Pyruvate metabolism	sbi00620	75	0.0198884
Ribosome biogenesis in eukaryotes	sbi03008	103	0.0221835
Citrate cycle (TCA cycle)	sbi00020	50	0.0232448
Ether lipid metabolism	sbi00565	32	0.0244963
2-Oxocarboxylic acid metabolism	sbi01210	49	0.0262444
Nicotinate and nicotinamide metabolism	sbi00760	21	0.0269251
Phenylalanine, tyrosine and tryptophan biosynthesis	sbi00400	40	0.0283316
Valine, leucine and isoleucine degradation	sbi00280	39	0.0322748
Proteasome	sbi03050	62	0.0351436
Sulfur metabolism	sbi00920	38	0.0367322
Selenocompound metabolism	sbi00450	18	0.0451696
Peroxisome	sbi04146	101	0.0468568
RNA degradation	sbi03018	94	0.0478577

通路Pathway	代谢通路ID Pathway ID	差异基因数目Counts of DEGs	P-value
Biosynthesis of amino acids	sbi01230	226	4.45E-07
Purine metabolism	sbi00230	103	2.67E-06
Carbon metabolism	sbi01200	247	0.0004462
Phagosome	sbi04145	79	0.0005084
Glycerophospholipid metabolism	sbi00564	113	0.0007
Inositol phosphate metabolism	sbi00562	75	0.0008856
Pentose phosphate pathway	sbi00030	63	0.0011618
Phosphatidylinositol signaling system	sbi04070	73	0.0011657
Ribosome	sbi03010	249	0.0035088
Endocytosis	sbi04144	144	0.0037752
Glyoxylate and dicarboxylate metabolism	sbi00630	72	0.0044091
Oxidative phosphorylation	sbi00190	101	0.0065322
Spliceosome	sbi03040	180	0.0065768
Pyrimidine metabolism	sbi00240	67	0.0081457
Arginine biosynthesis	sbi00220	38	0.0100436
Autophagy-other	sbi04136	45	0.0145776
Glycolysis/Gluconeogenesis	sbi00010	133	0.0183614
Pyruvate metabolism	sbi00620	75	0.0198884
Ribosome biogenesis in eukaryotes	sbi03008	103	0.0221835
Citrate cycle (TCA cycle)	sbi00020	50	0.0232448
Ether lipid metabolism	sbi00565	32	0.0244963
2-Oxocarboxylic acid metabolism	sbi01210	49	0.0262444
Nicotinate and nicotinamide metabolism	sbi00760	21	0.0269251
Phenylalanine, tyrosine and tryptophan biosynthesis	sbi00400	40	0.0283316
Valine, leucine and isoleucine degradation	sbi00280	39	0.0322748
Proteasome	sbi03050	62	0.0351436
Sulfur metabolism	sbi00920	38	0.0367322
Selenocompound metabolism	sbi00450	18	0.0451696
Peroxisome	sbi04146	101	0.0468568
RNA degradation	sbi03018	94	0.0478577

途径Pathway	基因名称Gene name	基因编号Gene number	表达变化倍数log₂ (Fold Change)	功能注释Function annotation
开花整合子Floral integrations	FT1	Sh03_g001310	7.958152092	Protein RICE FLOWERING LOCUS T 1
	MADS-15	Sh02_g000890	13.13985093	MADS-box transcription factor 15
	MADS-16	Sh10_g017730	5.717251945	MADS-box transcription factor 16
花分生组织决定基因Floral meristem identity genes	RAP2-11	Sh02_g018350	–7.810617389	Ethylene-responsive transcription factor RAP2-11
RAP2-3	Sh02_g030860	–1.628805404	Ethylene-responsive transcription factor RAP2-3
RAP2-7	Sh09_g002170	1.242907698	Ethylene-responsive transcription factor RAP2-7
RAP2-4	Sh02_g011650	–1.191152161	Ethylene-responsive transcription factor RAP2-4
PFT1	Sh02_g010230	1.684690279	Phytochrome and flowering time 1 protein
PIF3	Sh02_g002020	1.145448931	Transcription factor PIF3
CIB5	Sh06_g005950	–1.991067928	Protein CRYPTOCHROME INTERACTING BASIC-HELIX-LOOP-HELIX 5
CRY2	Sh06_g005950	-1.568184262	Cryptochrome 2
CRY-DASH	Sh10_g015300	-1.668928566	Cryptochrome DASH, chloroplastic/mitochondrial
CIB1	Sh04_g019390	1.27080514	Protein CRYPTOCHROME INTERACTING BASIC-HELIX-LOOP-HELIX 1
COL4	Sh07_g007600	–3.004809988	Zinc finger protein CONSTANS-LIKE 4
光周期Photoperiod	COL5	Sh04_g004530	–2.138158501	Zinc finger protein CONSTANS-LIKE 5
COL9	Sh10_g010100	2.716167887	Zinc finger protein CONSTANS-LIKE 9
COL16	Sh01_g009230	–1.814080263	Zinc finger protein CONSTANS-LIKE 16
COL16	Sh_239A02_contig-2_g000020	–1.742909918	Zinc finger protein CONSTANS-LIKE 16
COL5	Sh06_g008770	–1.263104823	Zinc finger protein CONSTANS-LIKE 5
PIE1	Sh02_g011510	–2.374910569	Protein PHOTOPERIOD-INDEPENDENT EARLY-FLOWERING 1
SEF	Sh_231L07_g000030	2.932200999	Protein SERRATED LEAVES AND EARLY FLOWERING
RUP1	Sh04_g000580	–1.698504889	WD repeat-containing protein RUP1
GI	Sh01_g036290	–2.982276809	Protein GIGANTEA
春化作用Vernalization	VRN2	Sh03_g010670	3.001678826	Polycomb group protein VERNALIZATION 2
	GID1	Sh09_g010540	–3.198956386	Similar to GID1-like gibberellin receptor
赤霉素途径Gibberellin pathway	GID1L2	Sh02_g002840	4.206398003	Gibberellin receptor GID1L2
GID1	Sh10_g016190	1.585422559	Gibberellin receptor GID1
GID1L2	Sh10_g007510	1.362567614	Gibberellin receptor GID1L2
GA20ox1	Sh01_g000490	1.60517892	Gibberellin 20 oxidase 1
GA20ox2	Sh06_g017130	1.486081164	Gibberellin 20 oxidase 2

途径Pathway	基因名称Gene name	基因编号Gene number	表达变化倍数log₂ (Fold Change)	功能注释Function annotation
开花整合子Floral integrations	FT1	Sh03_g001310	7.958152092	Protein RICE FLOWERING LOCUS T 1
	MADS-15	Sh02_g000890	13.13985093	MADS-box transcription factor 15
	MADS-16	Sh10_g017730	5.717251945	MADS-box transcription factor 16
花分生组织决定基因Floral meristem identity genes	RAP2-11	Sh02_g018350	–7.810617389	Ethylene-responsive transcription factor RAP2-11
RAP2-3	Sh02_g030860	–1.628805404	Ethylene-responsive transcription factor RAP2-3
RAP2-7	Sh09_g002170	1.242907698	Ethylene-responsive transcription factor RAP2-7
RAP2-4	Sh02_g011650	–1.191152161	Ethylene-responsive transcription factor RAP2-4
PFT1	Sh02_g010230	1.684690279	Phytochrome and flowering time 1 protein
PIF3	Sh02_g002020	1.145448931	Transcription factor PIF3
CIB5	Sh06_g005950	–1.991067928	Protein CRYPTOCHROME INTERACTING BASIC-HELIX-LOOP-HELIX 5
CRY2	Sh06_g005950	-1.568184262	Cryptochrome 2
CRY-DASH	Sh10_g015300	-1.668928566	Cryptochrome DASH, chloroplastic/mitochondrial
CIB1	Sh04_g019390	1.27080514	Protein CRYPTOCHROME INTERACTING BASIC-HELIX-LOOP-HELIX 1
COL4	Sh07_g007600	–3.004809988	Zinc finger protein CONSTANS-LIKE 4
光周期Photoperiod	COL5	Sh04_g004530	–2.138158501	Zinc finger protein CONSTANS-LIKE 5
COL9	Sh10_g010100	2.716167887	Zinc finger protein CONSTANS-LIKE 9
COL16	Sh01_g009230	–1.814080263	Zinc finger protein CONSTANS-LIKE 16
COL16	Sh_239A02_contig-2_g000020	–1.742909918	Zinc finger protein CONSTANS-LIKE 16
COL5	Sh06_g008770	–1.263104823	Zinc finger protein CONSTANS-LIKE 5
PIE1	Sh02_g011510	–2.374910569	Protein PHOTOPERIOD-INDEPENDENT EARLY-FLOWERING 1
SEF	Sh_231L07_g000030	2.932200999	Protein SERRATED LEAVES AND EARLY FLOWERING
RUP1	Sh04_g000580	–1.698504889	WD repeat-containing protein RUP1
GI	Sh01_g036290	–2.982276809	Protein GIGANTEA
春化作用Vernalization	VRN2	Sh03_g010670	3.001678826	Polycomb group protein VERNALIZATION 2
	GID1	Sh09_g010540	–3.198956386	Similar to GID1-like gibberellin receptor
赤霉素途径Gibberellin pathway	GID1L2	Sh02_g002840	4.206398003	Gibberellin receptor GID1L2
GID1	Sh10_g016190	1.585422559	Gibberellin receptor GID1
GID1L2	Sh10_g007510	1.362567614	Gibberellin receptor GID1L2
GA20ox1	Sh01_g000490	1.60517892	Gibberellin 20 oxidase 1
GA20ox2	Sh06_g017130	1.486081164	Gibberellin 20 oxidase 2

基于RNA-Seq的甘蔗响应鞭黑粉菌侵染的早花相关基因挖掘

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王致远 ¹^,² , 梁鸿翠 ² , 蔡健和 ³ , 陈保善 ¹^,²^,⁴^,^* , 许雄彪 ¹^,²^,⁴^,^*

热带作物学报 | 组学与生物技术 2024,45(6): 1102-1109

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热带作物学报 |组学与生物技术 2024 , 45 (6) : 1102 -1109

基于RNA-Seq的甘蔗响应鞭黑粉菌侵染的早花相关基因挖掘

全屏

王致远¹^,², 梁鸿翠², 蔡健和³, 陈保善¹^,²^,⁴^,^*, 许雄彪¹^,²^,⁴^,^*

作者信息

^1.广西甘蔗生物学重点实验室，广西南宁　530004

^2.广西大学农学院，广西南宁　530004

^3.广西农业科学研究院植物保护研究所，广西南宁　530007

^4.亚热带农业生物资源保护与利用国家重点实验室，广西南宁　530004

通讯作者:

^* 陈保善（CHEN Baoshan），E-mail：chenyaoj@gxu.edu.cn

许雄彪（XU Xiongbiao），E-mail：xiongbiaox@gxu.edu.cn。

Identification of Early Flowering Related Genes of Sugarcane in Response to Sporisorium scitamineum Infection Based on RNA-Seq

Zhiyuan WANG¹^,², Hongcui LIANG², Jianhe CAI³, Baoshan CHEN¹^,²^,⁴^,^*, Xiongbiao XU¹^,²^,⁴^,^*

Affiliations

^1.Guangxi Key Laboratory of Sugarcane Biology, Nanning, Guangxi 530004, China

^2.College of Agriculture, Guangxi University, Nanning, Guangxi 530004, China

^3.Plant Protection Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi 530007, China

^4.State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Nanning, Guangxi 530004, China

出版时间: 2024-06-25 doi: 10.3969/j.issn.1000-2561.2024.06.002

文章导航

摘要

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甘蔗鞭黑粉菌（Sporisorium scitamineum）是引起甘蔗鞭黑穗病（sugarcane smut）的重要病原菌，侵染甘蔗后通常诱导顶端分生组织产生灰黑色“黑鞭”，在部分甘蔗品种（系）中能诱导植株提前开花，但其作用机制仍不明确。为了探究甘蔗响应鞭黑粉菌侵染的开花相关基因及其表达模式，本研究以同期健康和感染鞭黑粉菌的桂糖42甘蔗品种为实验材料进行转录组测序分析，共筛选出了3276个显著差异表达基因，其中1677个基因上调表达（P<0.05），1599个基因下调表达（P<0.05）。GO富集分析发现差异表达基因主要集中在生物合成过程、核糖体、核糖核蛋白复合体、核糖体结构成分等小类中。KEGG富集结果显示差异表达基因主要集中在氨基酸的生物合成、嘌呤代谢、碳代谢、吞噬作用等代谢通路。筛选到30个与开花途径相关的差异表达基因，并对其中的FT1、PIE1、GID1、GA20ox-1、GA20ox-2等基因进行了qRT-PCR验证，qRT-PCR结果与转录组测序结果基本一致，验证了转录组测序结果的可靠性。本研究结果为解析甘蔗鞭黑粉菌诱导甘蔗提前开花表型的分子机理提供了重要的候选基因，对甘蔗的育种也有重要的参考价值。

关键词

甘蔗 / 甘蔗鞭黑粉菌 / 转录组 / 开花相关基因

Abstract

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Sporisorium scitamineum, the pathogen of destructive disease smut, usually causes abnormal growth of meristematic tissue and forms a “black whip” in meristem tissues. In some cases, it induces formation of flowering structures in some sugarcane cultivars (genotypes), but the exact mechanism remains unclear. In order to explore flowering related genes and the expression patterns in sugarcane in response to infection by S. scitamineum, the healthy and S. scitamineum infected ‘guitang42’ sugarcane cultivar samples were analyzed by transcriptome sequencing. A total of 3276 differentially expressed genes (DEGs) were identified, of which 1677 genes were up-regulated (P<0.05) and 1599 genes down-regulated (P<0.05). The DEGs were mostly enriched in the biosynthesis process, ribosome, ribonucleoprotein complex, ribosome structural components, etc. according to GO enrichment analysis. The KEGG analysis showed that the DEGs were mostly enriched in amino acid biosynthesis, purine metabolism, carbon metabolism, phagocytosis, and other metabolic pathways. Also, out of 30 DEGs were identified to be associated with flowering, and the representative genes such as FT1, PIE1, GID1, GA20ox-1, GA20ox-2 were validated by qRT-PCR. The qRT-PCR results were consistent with the transcriptomic data, confirming the dependability of the transcriptome sequencing results. The findings would present essential candidate genes for revealing the mechanism of early flowering of sugarcane plants caused by S. scitamineum, and lay the foundation for disease resistance breeding.

Key words

sugarcane / Sporisorium scitamineum / transcriptome / flowering-related genes

引用本文

王致远, 梁鸿翠, 蔡健和, 陈保善, 许雄彪. 基于RNA-Seq的甘蔗响应鞭黑粉菌侵染的早花相关基因挖掘. 热带作物学报, 2024 , 45 (6) : 1102 -1109 . DOI: 10.3969/j.issn.1000-2561.2024.06.002

Zhiyuan WANG, Hongcui LIANG, Jianhe CAI, Baoshan CHEN, Xiongbiao XU. Identification of Early Flowering Related Genes of Sugarcane in Response to Sporisorium scitamineum Infection Based on RNA-Seq[J]. Chinese Journal of Tropical Crops, 2024 , 45 (6) : 1102 -1109 . DOI: 10.3969/j.issn.1000-2561.2024.06.002

正文

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甘蔗（Saccharum spp.）是世界上最大的商业作物，在全球超120个国家和地区广泛种植。甘蔗是主要的制糖原料，世界上大约80%的糖来源于甘蔗^[1]，我国90%的食糖也来源于蔗糖^[2]。甘蔗黑穗病（sugarcane smut）是世界范围内为害甘蔗最为严重的病害之一，可造成甘蔗产量和品质的严重损失^[3-4]。其病原菌为甘蔗鞭黑粉菌（Sporisorium scitamineum），通过生长初期的蔗株幼芽顶端实现侵染^[5]。蔗株被侵染后表现出分蘖增多、蔗叶淡绿细长、顶叶尖挺、蔗茎细小和节疏等症状^[6-7]。随着鞭黑粉菌的不断蔓延，在梢头逐渐形成由植物组织和真菌组成的不分支、方向朝下向内卷曲的鞭状物，俗称“黑鞭”^[7]。除上述常见症状外，甘蔗鞭黑粉菌侵染与甘蔗开花密切相关，颜梅新等^[8]通过孢子悬浮液接种试验，发现甘蔗鞭黑粉菌能诱导桂柳05-136甘蔗品种提前开花。

开花是被子植物生活史中的重要组成部分，影响植物对不同环境的适应、营养发育、生物量积累和谷物产量等^[9-10]。开花是一个由5种基因通路组成的复杂网络协调调控的生物学过程，包括光周期、春化、自主、赤霉素和年龄^[9，11-14]。此外，土壤营养、供水及温度等因素也可以抑制、促进或破坏开花过程^[15-17]。开花整合基因FT（FLOWERING LOCUS T）和SOC1（SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1），将上述这些复杂的调节因子整合后进一步传递到下游的花分生组织基因（floral meristem identity gene）中，从而启动开花^[12]。甘蔗的开花同样受到许多因素，如昼长、昼夜温度、湿度、甘蔗的生理成熟度，养分有效性，以及纬度等的影响^[18]。目前，关于甘蔗开花相关基因的研究相对较少，对于甘蔗鞭黑粉诱导甘蔗提前开花的研究更是未见报道。

甘蔗开花与产量是一个矛盾的过程，一方面生殖生长导致糖分向花序运输，降低了蔗茎中蔗糖的积累；另一方面，甘蔗亲本诱导开花难且花期不遇成为影响甘蔗杂交育种和新亲本创制的重要“瓶颈”。如何精准调控甘蔗的开花过程是平衡甘蔗产量和育种的重点和难点，具有重要的生产实际意义。本研究基于RNA-Seq测序技术，对甘蔗鞭黑粉菌侵染前、后2个时期的甘蔗叶片进行测序分析，挖掘并筛选与甘蔗开花相关的差异表达基因，为甘蔗开花调控和育种提供理论基础。

1　材料与方法

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1.1　材料

本研究所用甘蔗品种桂糖42号取自广西亚热带农科新城及广西大学甘蔗试验基地，分别选取被甘蔗鞭黑粉菌侵染但“黑鞭”尚未抽出的甘蔗+1叶为试验材料，以同龄期健康甘蔗植株+1叶为对照，各采集3株叶片样本均匀混合，置于–80 ℃超低温冰箱保存。

1.2　方法

1.2.1　RNA提取及质量检测

对照组和试验组甘蔗叶片组织总RNA由北京诺禾致源科技股份有限公司提取，以Nanodrop分光光度计检测RNA纯度，采用Agilent 2100核酸分析仪对提取的总RNA进行质量检测。

1.2.2　文库构建及测序

对质量检测合格的甘蔗叶片总RNA，利用Oligo（dT）磁珠富集含poly（A）尾的mRNA，然后加入NEB Fragmentation Buffer将mRNA打断成短片段，以mRNA为模板合成cDNA第一链，然后加入缓冲液、dNTPs和DNA Polymerase Ⅰ合成cDNA第二链，随后对双链cDNA进行纯化、末端修复、加尾并连接测序接头，最后进行片段大小选择和PCR富集，获得cDNA文库。文库检测合格后进行Illumina NovaSeq 6000平台测序。

1.2.3　测序数据处理与分析

测序得到的原始图像数据文件经CASAVA碱基识别（base calling）分析转化为原始测序序列（original sequence）。对原始数据（raw reads）进行过滤，去除带接头和N比率大于10%及低质量的reads，得到高质量序列（clean reads）。将clean reads与甘蔗参考基因组数据库（https://sugarcane-genome.cirad.fr/）进行比对和分类注释。

1.2.4　差异表达基因筛选

对测序得到的原始reads进行评估，经TMM标准化处理后，获得对照组和试验组的基因表达量。应用edgeR软件进行样本间差异分析，得到P值后进行多重假设检验校正，通过控制PDR值，并以|log₂（Fold Change）|≥1和P≤0.05为标准对差异表达基因进行筛选。

1.2.5　差异表达基因本位数据库（GO）和Pathway显著性富集（KEGG）分析

基于Gene Ontology（GO），分别从分子功能（molecular function，MF）、生物过程（biological process，BP）和细胞组分（cellular component，CC）3个方面对差异表达基因进行GO注释。基于KEGG数据库，应用超几何检验，找出与整个基因组背景相比，在差异表达基因中显著性富集的Pathway，采用Fisher进行精确检验，通过Bonferroni校正法进行校正，得到差异基因显著富集的GO功能和代谢通路，从中筛选显著差异表达基因进行重点分析。

1.2.6　qRT-PCR验证

为验证转录组数据的可靠性，利用qRT-PCR对部分差异表达基因进行验证。利用HiScript III RT SuperMix for qPCR（+gDNA wiper）试剂盒（Vazyme，Cat. R323-01）合成cDNA第一链，采用ChamQ Universal SYBR qPCR Master Mix试剂盒（Vazyme，Cat. Q711-02）检测基因的表达量，以GAPDH基因作为内参，用SnapGene软件设计qRT-PCR引物（表1）。反应体系为：cDNA模板（cDNA原液稀释10倍）2 μL，10 μmol/L正、反向引物各0.4 μL，2×ChamQ Universal SYBR qPCR Master Mix 10 μL，加入Nuclease-free water至总体积为20 μL，在Light-Cycler 96实时荧光定量PCR仪（Roche）上进行qRT-PCR反应。荧光定量PCR反应程序为：95 ℃预变性30 s；95 ℃变性10 s，60 ℃退火延伸30 s，循环40次。所有试验均设置3次生物学重复和3次实验重复。运用2^–ΔΔC_T法进行数据处理。

2　结果与分析

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2.1　测序数据质控结果分析

试验组（Smut +1leaf）和对照组（Control + 1leaf）的原始数据raw reads数分别为45 626 696条和46 125 906条，经过滤得到clean reads数分别为42 937 438条和44 276 602条，测序cDNA读取量分别为6.44 Gb和6.64 Gb，Q₂₀分别为98.05%和96.7%，Q₃₀分别为94.17%和91.19%，GC含量分别为50.48%和52.72%。说明测序质量较高，满足后续生物信息学分析要求。

2.2　差异表达基因筛选

将对照组与试验组的测序数据进行比对分析，结果如图1所示，共有3276个基因差异表达，其中1677个基因显著上调表达（P<0.05），1599个基因显著下调表达（P<0.05）。

2.3　差异表达基因的GO功能注释和KEGG分析

GO功能分析表明，差异表达基因被富集到生物过程、细胞组分和分子功能等3大类和785小类中，其中生物过程390个，细胞组分107个，分子功能288个。生物过程中有28个小类显著富集，细胞组分中有7个小类显著富集，分子功能中有16个小类显著富集。选取3个大类中富集程度高和差异基因占比大的小类作图（图2），其中生物过程主要富集在酰胺生物合成过程（amide biosynthetic process）、翻译（translation）、肽生物合成过程（peptide biosynthetic process）、细胞酰胺代谢过程（cellular amide metabolic process）、肽代谢过程（peptide metabolic process）；细胞组份主要富集在核糖体（ribosome）、核糖核蛋白复合体（ribonucleoprotein complex）、非膜结合细胞器（non-membrane-bounded organelle）、胞内非膜结合细胞器（intracellular non-membrane-bounded organelle）、细胞质部分（cytoplasmic part）；分子功能主要富集在核糖体的结构成分（structural constituent of ribosome）、结构分子活性（structural molecule activity）及rRNA结合（rRNA binding）等。

利用KEGG数据库对筛选到的差异表达基因进行功能分类和pathway注释，结果显示（表2）：甘蔗鞭黑粉菌侵染前后的差异表达基因定位到了118个代谢通路中，显著富集到了30个代谢通路中，如氨基酸的生物合成（biosynthesis of amino acids）、嘌呤代谢（purine metabolism）、碳代谢（carbon metabolism）、吞噬作用（phagosome）、甘油磷脂代谢（glycerophospholipid metabolism）等。

2.4　开花相关基因的差异表达分析

从转录组数据中筛选到30个与开花途径相关的差异表达基因，其中包括开花整合子（floral integrations）基因1个；花分生组织决定基因（floral meristem identity genes）6个；光周期（photoperiod）途径相关基因16个；春化途径相关基因1个；赤霉素途径相关基因6个（表3）。

2.5　开花相关基因的qRT-PCR验证

选取GID1、FT1、PIE1、GA20ox-1、GA20ox-2等5个与植物开花途径相关基因进行qRT-PCR验证（图3），结果显示，与健康对照相比，甘蔗鞭黑粉菌侵染的甘蔗+1叶片中FT1、GA20ox-1、GA20ox-2基因显著上调表达，而GID1和PIE1基因显著下调表达。虽然这3个基因的转录组测序结果和qRT-PCR结果在基因差异表达倍数并非完全一致，但是二者之间反映出的差异表达变化趋势是一致的，说明转录组数据的可靠性。

3　讨论

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开花是植物由营养生长向生殖生长转变的重要阶段，在植物生活史中扮演着非常重要的作用。甘蔗开花的精准高效调控对于蔗茎产量和遗传育种具有重要意义。本研究通过转录组测序分析，筛选出3276个差异表达基因，其中1677个基因上调表达，1599个基因下调表达。结合植物开花调控途径对差异表达基因进一步分析，筛选出30个与开花途径相关的差异表达基因。FLOWERING LOCUS T（FT）成花基因作为下游开花整合基因，可以整合多个调控途径中的花发育信号分子，促进开花相关基因的表达，促进植物开花^[19]。然而，过表达甘蔗ScFT1基因可抑制拟南芥开花^[20]。本研究转录组测序分析发现在甘蔗鞭粉菌侵染后ScFT1基因呈显著上调表达，同时qRT-PCR也验证了这一结果。推测甘蔗鞭粉菌侵染后诱导甘蔗提前开花的现象，可能是通过影响上游的开花途径导致的。

赤霉素（gibberellins，GAs）是一类重要的植物激素，在调节植物生长和抵抗生物和非生物胁迫中具有重要作用^[21-22]。在赤霉素信号通路中，GA受体GIBBERELLIN INSENSITIVE DWARF1（GID1）感知GA信号后，与下游DELLA蛋白N-端DELLA/TVHYNP结构域互作，促进DELLA经SCF^GID2/SLY1泛素/26S蛋白酶体途径降解^[23-24]，正向调控植株茎伸长、花和果实发育、叶片扩张及种子萌发等生物学过程^[25-27]。Gibberellin 20 oxidase（GA20ox）是赤霉素合成过程中的一类重要的限速酶，GA20ox及Gibberellin 3 oxidase（GA3ox）通过一系列催化反应，将赤霉素前期GA12转化为具有生物活性的GA1和GA4^[28-30]。GA20ox-2被认为是一个“绿色革命”基因，其突变导致水稻的矮杆化，从而大大提高了产量^[31]。本研究发现与对照相比，甘蔗鞭黑粉菌侵染的甘蔗+1叶中ScGA20ox-1、ScGA20ox-2基因均呈现显著上调表达，而ScGID1基因呈显著下调表达，这与GID1正向调控开花的结果不一致^[25-26]。此外，酵母双杂交（yeast two hybrid，Y2H）和双分子荧光互补实验（BiFC）研究发现，ScGID1能与ScGA20ox2在体外和体内发生互作（数据待发表）。推测甘蔗鞭黑粉菌侵染一方面下调ScGID1表达，另一方面促进上游ScGA20ox等赤霉素合成相关基因的表达，干扰了ScGID1与ScGA20ox的相互作用，从而诱导甘蔗早花的表型。

不依赖光周期的早花基因PHOTOPERIOD INDEPENDENT EARLY FLOWERING1（PIE1）能负调控拟南芥开花过程，PIE1突变能抑制FLOWERING LOCUS C（FLC）介导的迟花表型，并诱导非FLC依赖的早花表型^[32]。本研究发现在甘蔗鞭黑粉菌侵染的甘蔗+1叶中ScPIE1基因转录水平极显著下调表达，推测甘蔗鞭黑粉菌诱导甘蔗早花现象可能与ScPIE1基因的表达下调有关。

在拟南芥中CONSTANS-Like（COL）基因是光周期诱导开花途径中的关键转录因子^[33]，研究发现AtCOL4是长日照和短日照的开花抑制因子，COL4过表达导致开花延迟^[34]；而过表达AtCOL5可诱导短日照拟南芥开花^[35]；COL9通过抑制Ehd1（early heading date 1）基因延迟水稻的开花时间^[36]；COL16基因在水稻中通过上调开花阻碍基因Ghd7（grain number，plant height and heading date 7）来抑制开花^[37]。本研究发现ScCOL4和ScCOL16显著下调表达，而COL4和COL16在其他作物中存在抑制开花的现象，推测甘蔗鞭黑粉菌可能通过下调这2个基因的表达从而诱导甘蔗早花。而COL5和COL9在甘蔗中的表达模式和功能可能与其他作物存在一定的差异。

乙烯响应因子（ethylene-responsive transcription factor RAP）RAP2-3、RAP2-4、RAP2-7等属于AP2/ERF类转录因子，参与植物花发育、果实成熟、种子发育和萌发以及响应逆境胁迫的过程^[38]。APETALA2（AP2）基因在拟南芥中直接或间接调控花发育过程中其他基因的表达，如FT^[39]和SOC1基因^[40]，进而抑制植物开花。本研究发现RAP2-11、RAP2-3、RAP2-4基因表达下调，推测这3个基因可能响应了甘蔗鞭黑粉菌的侵染，进而诱导甘蔗早花现象。

CRY2（cryptochrome 2）、CRY-DASH（cryptochrome-DASH）都属于隐花色素基因家族，而CIB1（cryptochrome-interacting basic-helix-loop-helix 1）、CIB5（cryptochrome-interacting basic-helix-loop-helix 5）是一类重要的转录因子，可以在蓝光的作用下与CRY2互作，进而促进拟南芥开花^[41]。本研究发现CIB1基因在甘蔗鞭黑粉菌侵染后显著上调表达，推测甘蔗鞭黑粉菌可能通过上调CIB1表达，进而促进甘蔗早花。

关于甘蔗鞭黑粉菌诱导甘蔗早花现象的研究鲜见报道，本研究基于RNA-Seq技术初步筛选了甘蔗响应鞭黑粉菌侵染的与开花途径相关的差异表达基因，并对部分基因的表达模式进行了验证分析，为甘蔗开花调控和育种提供了理论基础。这些基因是如何响应甘蔗鞭黑粉菌侵染，具体互作因子以及诱导提前开花的分子机理，还有待进一步深入研究。

基金

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广西自然科学基金项目(2023GXNSFBA026058)
广西大学甘蔗专项科研项目(2022GZB011)

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2024年第45卷第6期

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doi: 10.3969/j.issn.1000-2561.2024.06.002

接收时间：2023-09-23
首发时间：2026-06-24
出版时间：2024-06-25

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出版历史

收稿日期：2023-09-23
修回日期：2023-10-22

基金

广西自然科学基金项目(2023GXNSFBA026058)

广西大学甘蔗专项科研项目(2022GZB011)

作者信息

^1.广西甘蔗生物学重点实验室，广西南宁　530004

^2.广西大学农学院，广西南宁　530004

^3.广西农业科学研究院植物保护研究所，广西南宁　530007

^4.亚热带农业生物资源保护与利用国家重点实验室，广西南宁　530004

通讯作者:

^* 陈保善（CHEN Baoshan），E-mail：chenyaoj@gxu.edu.cn

许雄彪（XU Xiongbiao），E-mail：xiongbiaox@gxu.edu.cn。

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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引物名称Primer name	上游引物（5'–3'）Forward primer (5'–3')	下游引物（5'–3'）Reverse primer (5'–3')
GAPDH	ATCAACGGTTTCGGAAGGATCGG	TTCTGACCGAAGAGGAGGGTCTTG
GID1	ACCAAGAGCCTCATTATCGTGTCG	CCTTCTCACGGTACACGAGCTT
PIE1	CCTGAAAAGAGTGCGTTTGTCGTCTG	TCACTTATAAGCTCAGGTGAGGGTGC
FT1	ATCCCACATGAACGCG	GTAGCAAATCACCTCG
GA20ox1	TGACAAGGTGGTCAAC	CGACCT
GA20ox2	CACGGCATATGCCAGAGAACCCACATCACG	TCTCCTTCCACGGCAGCTT
	CGAACGGGTCCGGGATCTTT

引物名称Primer name

上游引物（5'–3'）Forward primer (5'–3')

下游引物（5'–3'）Reverse primer (5'–3')

GAPDH

ATCAACGGTTTCGGAAGGATCGG

TTCTGACCGAAGAGGAGGGTCTTG

GID1

ACCAAGAGCCTCATTATCGTGTCG

CCTTCTCACGGTACACGAGCTT

PIE1

CCTGAAAAGAGTGCGTTTGTCGTCTG

TCACTTATAAGCTCAGGTGAGGGTGC

FT1

ATCCCACATGAACGCG

GTAGCAAATCACCTCG

GA20ox1

TGACAAGGTGGTCAAC

CGACCT

GA20ox2

CACGGCATATGCCAGAGAACCCACATCACG

TCTCCTTCCACGGCAGCTT

CGAACGGGTCCGGGATCTTT

通路Pathway	代谢通路ID Pathway ID	差异基因数目Counts of DEGs	P-value
Biosynthesis of amino acids	sbi01230	226	4.45E-07
Purine metabolism	sbi00230	103	2.67E-06
Carbon metabolism	sbi01200	247	0.0004462
Phagosome	sbi04145	79	0.0005084
Glycerophospholipid metabolism	sbi00564	113	0.0007
Inositol phosphate metabolism	sbi00562	75	0.0008856
Pentose phosphate pathway	sbi00030	63	0.0011618
Phosphatidylinositol signaling system	sbi04070	73	0.0011657
Ribosome	sbi03010	249	0.0035088
Endocytosis	sbi04144	144	0.0037752
Glyoxylate and dicarboxylate metabolism	sbi00630	72	0.0044091
Oxidative phosphorylation	sbi00190	101	0.0065322
Spliceosome	sbi03040	180	0.0065768
Pyrimidine metabolism	sbi00240	67	0.0081457
Arginine biosynthesis	sbi00220	38	0.0100436
Autophagy-other	sbi04136	45	0.0145776
Glycolysis/Gluconeogenesis	sbi00010	133	0.0183614
Pyruvate metabolism	sbi00620	75	0.0198884
Ribosome biogenesis in eukaryotes	sbi03008	103	0.0221835
Citrate cycle (TCA cycle)	sbi00020	50	0.0232448
Ether lipid metabolism	sbi00565	32	0.0244963
2-Oxocarboxylic acid metabolism	sbi01210	49	0.0262444
Nicotinate and nicotinamide metabolism	sbi00760	21	0.0269251
Phenylalanine, tyrosine and tryptophan biosynthesis	sbi00400	40	0.0283316
Valine, leucine and isoleucine degradation	sbi00280	39	0.0322748
Proteasome	sbi03050	62	0.0351436
Sulfur metabolism	sbi00920	38	0.0367322
Selenocompound metabolism	sbi00450	18	0.0451696
Peroxisome	sbi04146	101	0.0468568
RNA degradation	sbi03018	94	0.0478577

通路Pathway

代谢通路ID Pathway ID

差异基因数目Counts of DEGs

P-value

Biosynthesis of amino acids

sbi01230

226

4.45E-07

Purine metabolism

sbi00230

103

2.67E-06

Carbon metabolism

sbi01200

247

0.0004462

Phagosome

sbi04145

0.0005084

Glycerophospholipid metabolism

sbi00564

113

0.0007

Inositol phosphate metabolism

sbi00562

0.0008856

Pentose phosphate pathway

sbi00030

0.0011618

Phosphatidylinositol signaling system

sbi04070

0.0011657

Ribosome

sbi03010

249

0.0035088

Endocytosis

sbi04144

144

0.0037752

Glyoxylate and dicarboxylate metabolism

sbi00630

0.0044091

Oxidative phosphorylation

sbi00190

101

0.0065322

Spliceosome

sbi03040

180

0.0065768

Pyrimidine metabolism

sbi00240

0.0081457

Arginine biosynthesis

sbi00220

0.0100436

Autophagy-other

sbi04136

0.0145776

Glycolysis/Gluconeogenesis

sbi00010

133

0.0183614

Pyruvate metabolism

sbi00620

0.0198884

Ribosome biogenesis in eukaryotes

sbi03008

103

0.0221835

Citrate cycle (TCA cycle)

sbi00020

0.0232448

Ether lipid metabolism

sbi00565

0.0244963

2-Oxocarboxylic acid metabolism

sbi01210

0.0262444

Nicotinate and nicotinamide metabolism

sbi00760

0.0269251

Phenylalanine, tyrosine and tryptophan biosynthesis

sbi00400

0.0283316

Valine, leucine and isoleucine degradation

sbi00280

0.0322748

Proteasome

sbi03050

0.0351436

Sulfur metabolism

sbi00920

0.0367322

Selenocompound metabolism

sbi00450

0.0451696

Peroxisome

sbi04146

101

0.0468568

RNA degradation

sbi03018

0.0478577

途径Pathway	基因名称Gene name	基因编号Gene number	表达变化倍数log₂ (Fold Change)	功能注释Function annotation
开花整合子Floral integrations	FT1	Sh03_g001310	7.958152092	Protein RICE FLOWERING LOCUS T 1
	MADS-15	Sh02_g000890	13.13985093	MADS-box transcription factor 15
	MADS-16	Sh10_g017730	5.717251945	MADS-box transcription factor 16
花分生组织决定基因Floral meristem identity genes	RAP2-11	Sh02_g018350	–7.810617389	Ethylene-responsive transcription factor RAP2-11
RAP2-3	Sh02_g030860	–1.628805404	Ethylene-responsive transcription factor RAP2-3
RAP2-7	Sh09_g002170	1.242907698	Ethylene-responsive transcription factor RAP2-7
RAP2-4	Sh02_g011650	–1.191152161	Ethylene-responsive transcription factor RAP2-4
PFT1	Sh02_g010230	1.684690279	Phytochrome and flowering time 1 protein
PIF3	Sh02_g002020	1.145448931	Transcription factor PIF3
CIB5	Sh06_g005950	–1.991067928	Protein CRYPTOCHROME INTERACTING BASIC-HELIX-LOOP-HELIX 5
CRY2	Sh06_g005950	-1.568184262	Cryptochrome 2
CRY-DASH	Sh10_g015300	-1.668928566	Cryptochrome DASH, chloroplastic/mitochondrial
CIB1	Sh04_g019390	1.27080514	Protein CRYPTOCHROME INTERACTING BASIC-HELIX-LOOP-HELIX 1
COL4	Sh07_g007600	–3.004809988	Zinc finger protein CONSTANS-LIKE 4
光周期Photoperiod	COL5	Sh04_g004530	–2.138158501	Zinc finger protein CONSTANS-LIKE 5
COL9	Sh10_g010100	2.716167887	Zinc finger protein CONSTANS-LIKE 9
COL16	Sh01_g009230	–1.814080263	Zinc finger protein CONSTANS-LIKE 16
COL16	Sh_239A02_contig-2_g000020	–1.742909918	Zinc finger protein CONSTANS-LIKE 16
COL5	Sh06_g008770	–1.263104823	Zinc finger protein CONSTANS-LIKE 5
PIE1	Sh02_g011510	–2.374910569	Protein PHOTOPERIOD-INDEPENDENT EARLY-FLOWERING 1
SEF	Sh_231L07_g000030	2.932200999	Protein SERRATED LEAVES AND EARLY FLOWERING
RUP1	Sh04_g000580	–1.698504889	WD repeat-containing protein RUP1
GI	Sh01_g036290	–2.982276809	Protein GIGANTEA
春化作用Vernalization	VRN2	Sh03_g010670	3.001678826	Polycomb group protein VERNALIZATION 2
	GID1	Sh09_g010540	–3.198956386	Similar to GID1-like gibberellin receptor
赤霉素途径Gibberellin pathway	GID1L2	Sh02_g002840	4.206398003	Gibberellin receptor GID1L2
GID1	Sh10_g016190	1.585422559	Gibberellin receptor GID1
GID1L2	Sh10_g007510	1.362567614	Gibberellin receptor GID1L2
GA20ox1	Sh01_g000490	1.60517892	Gibberellin 20 oxidase 1
GA20ox2	Sh06_g017130	1.486081164	Gibberellin 20 oxidase 2

途径Pathway

基因名称Gene name

基因编号Gene number

表达变化倍数log₂ (Fold Change)

功能注释Function annotation

开花整合子Floral integrations

FT1

Sh03_g001310

7.958152092

Protein RICE FLOWERING LOCUS T 1

MADS-15

Sh02_g000890

13.13985093

MADS-box transcription factor 15

MADS-16

Sh10_g017730

5.717251945

MADS-box transcription factor 16

花分生组织决定基因Floral meristem identity genes

RAP2-11

Sh02_g018350

–7.810617389

Ethylene-responsive transcription factor RAP2-11

RAP2-3

Sh02_g030860

–1.628805404

Ethylene-responsive transcription factor RAP2-3

RAP2-7

Sh09_g002170

1.242907698

Ethylene-responsive transcription factor RAP2-7

RAP2-4

Sh02_g011650

–1.191152161

Ethylene-responsive transcription factor RAP2-4

PFT1

Sh02_g010230

1.684690279

Phytochrome and flowering time 1 protein

PIF3

Sh02_g002020

1.145448931

Transcription factor PIF3

CIB5

Sh06_g005950

–1.991067928

Protein CRYPTOCHROME INTERACTING BASIC-HELIX-LOOP-HELIX 5

CRY2

Sh06_g005950

-1.568184262

Cryptochrome 2

CRY-DASH

Sh10_g015300

-1.668928566

Cryptochrome DASH, chloroplastic/mitochondrial

CIB1

Sh04_g019390

1.27080514

Protein CRYPTOCHROME INTERACTING BASIC-HELIX-LOOP-HELIX 1

COL4

Sh07_g007600

–3.004809988

Zinc finger protein CONSTANS-LIKE 4

光周期Photoperiod

COL5

Sh04_g004530

–2.138158501

Zinc finger protein CONSTANS-LIKE 5

COL9

Sh10_g010100

2.716167887

Zinc finger protein CONSTANS-LIKE 9

COL16

Sh01_g009230

–1.814080263

Zinc finger protein CONSTANS-LIKE 16

COL16

Sh_239A02_contig-2_g000020

–1.742909918

Zinc finger protein CONSTANS-LIKE 16

COL5

Sh06_g008770

–1.263104823

Zinc finger protein CONSTANS-LIKE 5

PIE1

Sh02_g011510

–2.374910569

Protein PHOTOPERIOD-INDEPENDENT EARLY-FLOWERING 1

SEF

Sh_231L07_g000030

2.932200999

Protein SERRATED LEAVES AND EARLY FLOWERING

RUP1

Sh04_g000580

–1.698504889

WD repeat-containing protein RUP1

Sh01_g036290

–2.982276809

Protein GIGANTEA

春化作用Vernalization

VRN2

Sh03_g010670

3.001678826

Polycomb group protein VERNALIZATION 2

GID1

Sh09_g010540

–3.198956386

Similar to GID1-like gibberellin receptor

赤霉素途径Gibberellin pathway

GID1L2

Sh02_g002840

4.206398003

Gibberellin receptor GID1L2

GID1

Sh10_g016190

1.585422559

Gibberellin receptor GID1

GID1L2

Sh10_g007510

1.362567614

Gibberellin receptor GID1L2

GA20ox1

Sh01_g000490

1.60517892

Gibberellin 20 oxidase 1

GA20ox2

Sh06_g017130

1.486081164

Gibberellin 20 oxidase 2