转录组和蛋白组联合筛选饲用燕麦株高性状候选基因

doi:10.11686/cyxb2024418

摘要/Abstract

摘要：

饲用燕麦是一种高产、优质且抗逆性强的饲草，在我国饲草产业的发展中占据重要地位。本研究分别对饲用燕麦高株高（编号972）与低株高（编号1289）的极端材料取穗下茎节和节间组织进行高通量转录组测序（RNA-Seq）和蛋白组定量分析，筛选差异表达基因（differentially expressed genes， DEGs）和差异表达蛋白（differentially expressed proteins， DEPs）。转录组分析筛选到22762个差异表达基因；蛋白组分析获得3934个差异表达蛋白；进一步的联合分析发现1147个差异表达基因（蛋白）重叠出现于转录组及蛋白组分析中。通过对重叠基因（蛋白）进行GO功能富集分析和KEGG信号通路分析，发现很多基因被显著富集到与细胞生长、代谢和细胞壁形成的通路上。进一步结合转录因子分析，筛选到10个饲用燕麦株高性状相关的候选基因。对候选基因进行qRT-PCR验证，得到了测序结果的可靠性。此外，对10个候选基因的组织表达特异性分析发现候选基因在燕麦茎、茎节中均有较高表达，而在其他组织中表达量较低，表明以上候选基因可能参与饲用燕麦的株高发育过程。综上，通过转录组与蛋白组联合分析，结合差异基因功能注释及转录因子分析，筛选到10个饲用燕麦株高性状相关的候选基因。以上候选基因主要通过调控细胞生长、代谢和细胞壁发育参与饲用燕麦株高性状的形成。本研究为进一步探究饲用燕麦株高性状形成的分子机制奠定了基础，并为今后饲用燕麦株高性状的生物育种提供了关键候选基因。

关键词: 饲用燕麦, 株高, 转录组, 蛋白组, 联合分析

Abstract:

Forage oat （Avena sativa） is a high-yielding， high-quality， and stress-resistant forage that plays a significant role in China’s forage industry. In this study， high-throughput transcriptome sequencing （RNA-Seq） and quantitative proteomic analysis were performed on stem nodes and internode tissues from high-stalk （No.972） and low-stalk （No.1289） forage oat varieties. The aim was to identify differentially expressed genes （DEGs） and differentially expressed proteins （DEPs） between these two varieties to discover plant height-related genes in A. sativa. We identified 22762 DEGs and 3934 DEPs between the two germplasm lines. Further integrated analyses revealed 1147 overlapping DEGs/DEPs in the transcriptome and proteome datasets. Gene Ontology （GO） enrichment and KEGG pathway analysis indicated that these 1147 overlapping genes/proteins were significantly enriched in pathways associated with cell growth， metabolism， and cell wall formation. Among these DEGs/DEPs， 10 candidate transcription factors were identified through transcription factor analysis， and their transcription profiles were validated using quantitative real-time polymerase chain reaction （qRT-PCR） analyses. The transcription patterns of the ten candidate genes were highly consistent with those predicted from the transcriptomic and proteomic data， confirming the reliability of the sequencing results. Tissue-specific analyses of their transcriptional profiles showed that these ten candidate genes exhibited higher transcript levels in stems and nodes but lower levels in other tissues， suggesting that they play roles in regulating plant height in forage oat. In summary， through integrated transcriptomic and proteomic analyses， along with differential gene function annotation and transcription factor analyses， we identified ten candidate genes related to plant height in forage oat. These candidate genes primarily regulate processes such as cell growth， metabolism， and cell wall development， contributing to plant height formation in forage oat. These findings provide a foundation for further exploration of the molecular mechanisms underlying plant height and offer key candidate genes for forage oat breeding programs.

Key words: forage oat, plant height, transcriptome, proteome, combined analysis

张志鹏, 蒋庆雪, 周昕越, 苗童, 唐俊, 仪登霞, 王学敏, 马琳. 转录组和蛋白组联合筛选饲用燕麦株高性状候选基因[J]. 草业学报, 2025, 34(9): 147-161.

Zhi-peng ZHANG, Qing-xue JIANG, Xin-yue ZHOU, Tong MIAO, Jun TANG, Deng-xia YI, Xue-min WANG, Lin MA. Screening of candidate genes for plant height in forage oat （Avena sativa） through combined transcriptome and proteome analysis[J]. Acta Prataculturae Sinica, 2025, 34(9): 147-161.

图/表 15

表1 材料具体信息

Table 1 Materials details

编号

Code

名称

Name

材料来源

Source of material

972

Dookie 10

澳大利亚，维多利亚州Victoria， Australia

1289

X61-7

美国，佛罗里达州Florida， United States

表2 实时荧光定量 PCR引物设计序列

Table 2 Primer sequences of real-time fluorescence quantitative PCR

基因ID Gene ID	正向引物Forward primer （5′-3′）	反向引物Reverse primer （5′-3′）
AVESA.00010b.r1.2CG3384710	CATCACCGCCAACATCACC	GCAGCCTCCACGATCTCC
AVESA.00010b.r1.3CG0669440	AAGAAGTTGGTGGTTATTGG	CTGCTTCCTTACCTCTCC
AVESA.00010b.r1.4AG2537620	CGGACCTACAACCAGAACC	CAGCCCAAAGTGCCTCTC
AVESA.00010b.r1.5DG0437330	CGGTCTTCTTGCGAATGG	CGATGTACTCCACGAAACC
AVESA.00010b.r1.3CG2847600	ATGCTCTTCACCGTCTCC	CTGGTTGATGTAGTCCTTGG
AVESA.00010b.r1.4AG1741330	GCATAGCGAGCAGTGAAGAG	ACACGGAGATCAGCAGAGC
AVESA.00010b.r1.2CG1087750	CTCCTCCTCCTGCTCGTC	GTGCCCTTGAACCTGTGG
AVESA.00010b.r1.2DG0384520	TGTGCTACGGGAGAAACG	AACTTGATGCCGCTATTCG
AVESA.00010b.r1.2CG0426050	ACCTTCACGCTCAACTTCTCC	GCTCTCCACGCAGTTCTCC
AVESA.00010b.r1.4AG0885780	AACTTCCCGTGCTCTGATCC	GTCGTCTCGTCGGCTTCC

图1 株高极端燕麦材料的表型分析972：高株高种质High plant height germplasm； 1289：低株高种质Low plant height germplasm. A：成熟期的植株（标尺=10 cm）Plants at maturity （scale bar=10 cm）； C：节数、各茎节长度和穗长表型（标尺=5 cm）Number of nodes， length of each stem node and spike length phenotype （scale bar=5 cm）. **： P<0.01.

Fig.1 Phenotypic observations of extreme plant height materials

表3 转录组测序数据质控分析

Table 3 Quality test of transcriptome sequencing data

样品名称 Sample	过滤后数据 Clean reads （M）	过滤后序列 Clean bases （G）	碱基质量>30的占比Q₃₀ （%）	GC含量 GC content （%）	唯一比对（对比率） Uniquely mapped （mapping ratio， %）	多方比对（对比率） Multiple mapped （mapping ratio， %）
H-1	43.09	6.11	94.99	53.21	83.39	14.16
H-2	43.05	6.13	95.07	52.98	83.80	14.22
H-3	43.08	6.13	94.84	53.08	83.92	14.09
L-1	43.13	6.33	94.06	51.53	85.83	11.67
L-2	44.62	6.57	94.58	51.38	85.79	11.62
L-3	42.36	6.23	93.99	51.16	85.83	11.60

图2 蛋白质鉴定基本信息A：肽段长度范围分布Distribution of peptide length ranges； B：蛋白质分子量的分布Distribution of protein molecular weights.

Fig.2 Basic information for protein identification

图3 极端材料差异表达基因和差异表达蛋白上调和下调基因数目

Fig.3 Number of differentially expressed genes and differentially expressed proteins up- and down-regulated genes in extreme materials

图4 转录组和蛋白组关联分析A：差异表达转录组与蛋白组Venn图Venn diagram of differentially expressed transcriptome and proteome； B：转录组与蛋白组表达相关性分析Transcriptome and proteome expression correlation analysis； C：差异表达转录组与蛋白组数目统计Statistics of the number of differentially expressed transcriptome and proteome.

Fig.4 Transcriptome and proteome association analysis

图5 相同变化趋势相关因子的GO富集分析A：转录本与蛋白共同上调的GO富集分析GO enrichment analysis of transcripts and proteins jointly up-regulated； B：转录本与蛋白共同下调的GO富集分析GO enrichment analysis of transcripts and proteins jointly down-regulated.

Fig.5 GO enrichment analysis of correlators with the same trend change

图6 相同变化趋势相关因子的KEGG通路分析A：转录本与蛋白共同上调的KEGG通路分析Analysis of KEGG pathway jointly up-regulated by transcripts and proteins； B：转录本与蛋白共同下调的KEGG通路分析Analysis of KEGG pathway jointly down-regulated by transcripts and proteins.

Fig.6 KEGG pathway analysis of correlators with the same change trend

图7 不同变化趋势相关因子的GO富集分析A：转录本下调、蛋白上调的GO富集分析GO enrichment analysis of transcript down-regulation and protein up-regulation； B：转录本上调、蛋白下调的GO富集分析GO enrichment analysis of transcript up-regulation and protein down-regulation.

Fig.7 GO enrichment analysis of correlators with different trends

图8 不同变化趋势相关因子的KEGG通路分析A：转录本下调、蛋白上调的KEGG通路分析Analysis of KEGG pathway with transcript down-regulation and protein up-regulation； B：转录本上调、蛋白下调的KEGG通路分析Analysis of KEGG pathway with transcript up-regulation and protein down-regulation.

Fig.8 KEGG pathway analysis of different trend correlators

表4 转录因子分类

Table 4 Classification of transcription factors

变化趋势 Change trend	转录因子家族 Transcription factor family	数量 Number
转录本与蛋白共同上调Transcripts and proteins jointly up-regulated	Nin-like	1
转录本与蛋白共同下调Transcripts and proteins jointly down-regulated	Trihelix	3
	FAR1	1
	ERF	1
转录本下调、蛋白上调Transcript down-regulation and protein up-regulation	TCP	1
	bHLH	1
	NF	1
转录本上调、蛋白下调Transcript up-regulation and protein down-regulation	Whirly	1
	NAC	1
	B3	1

表5 候选基因

Table 5 The candidate genes

基因ID Gene ID	注释 Exegesis	基因差异倍数Gene fold change	趋势 Tendencies	蛋白差异倍数Protein fold change	趋势 Tendencies
AVESA.00010b.r1.2CG3384710	BHLH结构域蛋白质BHLH domain-containing protein	2.27	下调Down	1.92	下调Down
AVESA.00010b.r1.3CG0669440	GIDA葡萄糖抑制分裂蛋白GIDA glucose inhibited division protein	4.41	上调Up	1.31	上调Up
AVESA.00010b.r1.4AG2537620	糖苷水解酶家族17 Glycosyl hydrolases family 17	4.14	上调Up	2.18	上调Up
AVESA.00010b.r1.5DG0437330	未定性蛋白Uncharacterized protein	4.85	下调Down	1.20	上调Up
AVESA.00010b.r1.3CG2847600	色氨酸氨基转移酶相关蛋白Tryptophan aminotransferase related protein	5.65	下调Down	3.59	下调Down
AVESA.00010b.r1.4AG1741330	过氧化物酶Peroxidase	4.69	上调Up	2.50	上调Up
AVESA.00010b.r1.2CG1087750	γ-硫堇家族γ-thionin family	3.70	下调Down	1.28	上调Up
AVESA.00010b.r1.2DG0384520	糖基水解酶家族17 Glycosyl hydrolases family 17	4.70	上调Up	1.85	上调Up
AVESA.00010b.r1.2CG0426050	谷氨酸脱羧酶Glutamate decarboxylase	3.02	下调Down	1.25	上调Up
AVESA.00010b.r1.4AG0885780	乙烯反应性转录因子ERF109 Ethylene-responsive transcription factor ERF109	9.04	下调Down	1.89	上调Up

图9 转录组数据的qRT-PCR验证

Fig.9 qRT-PCR validation of transcriptome data

图10 候选基因的组织特异性表达A：苗期根Seedling stage root； B：苗期地上Seedling stage aboveground； C：拔节期根Shooting stage root； D：拔节期茎Shooting stage stem； E：拔节期叶Shooting stage leaf； F：拔节期茎节Shooting stage stem nodes； G：孕穗期根Booting stage root； H：孕穗期茎Booting stage stem； I：孕穗期叶Booting stage leaf； J：孕穗期茎节Booting stage stem nodes； K：孕穗期穗Booting stage spike； L：抽穗期根 Heading stage root； M：抽穗期茎Heading stage stem； N：抽穗期叶Heading stage leaf； O：抽穗期茎节Heading stage stem nodes； P：抽穗期穗Heading stage spike.

Fig.10 Tissue-specific expression of candidate genes

参考文献 31

[1]	Liu W H， Jia Z F， Liang G L. The current situation， problems and suggestions for the development of China’s oat feed industry. Qinghai Science and Technology， 2020， 27（3）： 82-85.
	刘文辉，贾志锋，梁国玲. 我国饲用燕麦产业发展现状及存在的问题和建议. 青海科技， 2020， 27（3）： 82-85.
[2]	Ye X L， Gan Z， Wan Y， et al. Advances and perspectives in forage oat breeding. Acta Prataculturae Sinica， 2023， 32（2）： 160-177.
	叶雪玲，甘圳，万燕，等. 饲用燕麦育种研究进展与展望. 草业学报， 2023， 32（2）： 160-177.
[3]	Chen X， Wu B， Zhang Z W. Evaluation of adaptability and stability for important agronomic traits of oat （Avena spp.） germplasm resources. Journal of Plant Genetic Resources， 2016， 17（4）： 577-585.
	陈新，吴斌，张宗文. 燕麦种质资源重要农艺性状适应性和稳定性评价. 植物遗传资源学报， 2016， 17（4）： 577-585.
[4]	Milach S， Rines H W， Phillips R L， et al. Inheritance of a new dwarfing gene in oat. Crop Science， 1998， 38（2）： 356-360.
[5]	Brown P D， Mckenzie R， Mikaelsen K. Agronomic， genetic， and cytologic evaluation of a vigorous new semidwarf oat. Crop Science， 1980， 20（3）： 303-306.
[6]	Xu Y H. Fine mapping of oat dwarfing gene Dw6. Chengdu： Sichuan Agricultural University， 2023.
	徐颖红. 燕麦矮秆基因Dw6的精细定位. 成都：四川农业大学， 2023.
[7]	Milach S C K， Rines H W， Phillips R L. Molecular genetic mapping of dwarfing genes in oat. Theoretical & Applied Genetics， 1997， 95（5/6）： 783-790.
[8]	Morikawa T. Genetic analysis on dwarfness of wild oat， Avena fatua. The Janpanese Journal of Genetics， 1989， 64： 363-371.
[9]	Zhang X B. Map-based cloning and functional analysis of leaf shape gene CFL2 and plant height gene SD38 in rice. Chongqing： Southwest University， 2021.
	张孝波. 水稻叶形基因CFL2和株高基因SD38的图位克隆与功能分析. 重庆：西南大学， 2021.
[10]	Yamamuro C， Ihara Y， Wu X， et al. Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint. The Plant Cell， 2000， 12（9）： 1591-1605.
[11]	Zhang J， Liu X， Li S， et al. The rice semi-dwarf mutant sd37， caused by a mutation in CYP96B4， plays an important role in the fine-tuning of plant growth. PLoS One， 2014， 9（2）： e88068.
[12]	Schefe J H， Lehmann K E， Buschmann I R， et al. Quantitative real-time RT-PCR data analysis： Current concepts and the novel “gene expression’s CT difference” formula. Journal of Molecular Medicine， 2006， 84（11）： 901-910.
[13]	Yang F， Ye R， Ma C， et al. Toxicity evaluation， toxin screening and its intervention of the jellyfish Phacellophora camtschatica based on a combined transcriptome-proteome analysis. Ecotoxicology and Environmental Safety， 2022， 46（6）： 1-12.
[14]	Luo J. Brassica napus L. dwarf traits proteome and transcriptome joint analysis. Guiyang： Guizhou Normal University， 2021.
	罗京. 甘蓝型油菜矮化性状的蛋白质组和转录组联合分析. 贵阳：贵州师范大学， 2021.
[15]	Li H， He X W， Gao Y F， et al. Integrative analysis of transcriptome， proteome， and phosphoproteome reveals potential roles of photosynthesis antenna proteins in response to brassinosteroids signaling in maize. Plants， 2023， 12（6）： 1290-1307.
[16]	Lin J， Zheng X， Xia J， et al. Integrative analysis of the transcriptome and proteome reveals the molecular responses of tobacco to boron deficiency. BMC Plant Biology， 2024， 24（1）： 689-707.
[17]	Wang Y J， Lu W J， Zhao J， et al. Transcriptome dynamics of dominant maize dwarf Dwarf11 （D11） revealed by RNA-seq and co-expression analysis. Plant Molecular Biology Reporter， 2017， 35（3）： 355-365.
[18]	Sui J M. Fine mapping of one semidwarf gene sdt3 and candidate-gene screening and functional analysis of the other semidwarf gene sdg in rice （Oryza sativa L.） . Yangzhou： Yangzhou University， 2006.
	隋炯明. 水稻半矮秆基因sdt3的精细定位和sdg的克隆与功能分析. 扬州：扬州大学， 2006.
[19]	Gong Y S， Wei S H， Peng Z S， et al. Genetic study on plant height and its components， partial yield traits in durum wheat ‘ANW16F’. Southwest China Journal of Agricultural Sciences， 2021， 34（2）： 229-235.
	龚胤书，魏淑红，彭正松，等. 硬粒小麦ANW16F株高及构成因子与部分产量性状遗传研究. 西南农业学报， 2021， 34（2）： 229-235.
[20]	Wu M Y. Genetic analysis and gene mapping of three （semi-） dwarf genes in rice. Beijing： Chinese Academy of Agricultural Sciences， 2020.
	吴明月. 三个（半）矮秆水稻基因的遗传分析和基因定位. 北京：中国农业科学院， 2020.
[21]	Lv H K， Zhang J， Wang T Y， et al. The maize d2003， a novel allele of VP8， is required for maize internode elongation. Plant Molecular Biology， 2014， 84（3）： 243-257.
[22]	Peng Z， Li X， Yang Z， et al. A new reduced height gene found in the tetraploid semi-dwarf wheat landrace Aiganfanmai. Genetics and Molecular Research， 2011， 10（4）： 2349-2357.
[23]	Song J， Li L， Liu B Y， et al. Fine mapping of reduced height locus RHT26 in common wheat. Theoretical and Applied Genetics， 2023， 136（3）： 62-72.
[24]	Chen L， Yang Y， Mishina K， et al. RNA-seq analysis of the peduncle development of Rht12 dwarf plants and primary mapping of Rht12 in common wheat. Cereal Research Communications， 2020， 48（2）： 1-9.
[25]	Shan Q Q. Analysis of allelic variaions of wheat plant height regulation genes Rht-1 and GID1 and their interaction mechansim. Zhengzhou： Henan Agricultural University， 2018.
	单强强. 小麦株高调控基因Rht-1和GID1的等位变异分析及其编码蛋白的互作机制. 郑州：河南农业大学， 2018.
[26]	Wang H M. Characterization of the expression patterns of tomato SlGH3.2 and its potential functions in rice plants. Nanjing： Nanjing Agricultural University， 2015.
	王慧敏. 番茄SlGH3.2的表达特征及在水稻中的功能研究分析. 南京：南京农业大学， 2015.
[27]	Ai G. Functional dissection of SlGH3-15 in tomato. Wuhan： Huazhong Agricultural University， 2017.
	艾国. 番茄SlGH3-15基因的功能解析. 武汉：华中农业大学， 2017.
[28]	Kazuhito A， Fumio T. C-terminal extension of rice glutamate decarboxylase （OsGAD2） functions as an autoinhibitory domain and overexpression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells. Journal of Experimental Botany， 2007， 58（10）： 2699-2707.
[29]	Jia Y T. Function analysis of transcription factor bHLH146 in Arabidopsis thaliana. Changchun： Jilin University， 2022.
	贾雨彤. 拟南芥转录因子bHLH146的功能研究. 长春：吉林大学， 2022.
[30]	Qi W W， Sun F， Wang Q J， et al. Rice ethylene-response AP2/ERF factor OsEATB restricts internode elongation by down-regulating a gibberellin biosynthetic gene. Plant Physiology， 2011， 157（1）： 216-228.
[31]	Ma Z M， Wu T， Huang K， et al. A novel AP2/ERF transcription factor， OsRPH1， negatively regulates plant height in rice. Frontiers in Plant Science， 2020， 11（13）： 709-724.