热带作物学报

植株类型Plant type	组织Tissue	编号No.	过滤前的reads数Total raw reads/Mb	过滤后的reads数Total clean reads/Mb	过滤后的碱基总数Total clean bases/Gb	碱基识别准确率Clean reads Q20/%	碱基识别准确率Clean Reads Q30/%	过滤后的reads比例Clean reads ratio/%	比对上参考基因组的clean reads比例Total mapping ratio/%	唯一比对上参考基因组某一位置的clean reads比例Uniquely mapping ratio/%
Mu	茎	M7d_2_1	45.44	44.59	6.69	98.41	94.65	98.13	93.58	73.61
	茎	M7d_2_2	43.69	43.00	6.45	98.28	94.31	98.43	94.17	73.63
	茎	M7d_2_3	45.48	44.17	6.63	98.53	95.10	97.12	94.55	74.53
	叶	M7d_3_1	45.44	44.63	6.69	98.35	94.37	98.21	94.13	73.35
	叶	M7d_3_2	41.89	41.14	6.17	98.15	93.91	98.21	94.12	72.67
	叶	M7d_3_3	43.69	43.00	6.45	98.37	94.43	98.42	94.79	74.05
ME34	茎	S7d_2_1	47.36	44.68	6.70	98.14	93.91	94.32	91.64	71.61
	茎	S7d_2_2	43.69	42.69	6.40	97.88	92.97	97.71	91.21	70.77
	茎	S7d_2_3	43.69	42.63	6.39	97.69	92.39	97.57	92.47	70.63
	叶	S7d_3_1	43.32	42.35	6.35	97.87	93.00	97.76	93.17	71.16
	叶	S7d_3_2	43.69	42.78	6.42	98.00	93.31	97.91	93.23	71.91
	叶	S7d_3_3	45.45	44.33	6.65	98.23	94.21	97.54	93.41	71.96

植株类型Plant type	组织Tissue	编号No.	过滤前的reads数Total raw reads/Mb	过滤后的reads数Total clean reads/Mb	过滤后的碱基总数Total clean bases/Gb	碱基识别准确率Clean reads Q20/%	碱基识别准确率Clean Reads Q30/%	过滤后的reads比例Clean reads ratio/%	比对上参考基因组的clean reads比例Total mapping ratio/%	唯一比对上参考基因组某一位置的clean reads比例Uniquely mapping ratio/%
Mu	茎	M7d_2_1	45.44	44.59	6.69	98.41	94.65	98.13	93.58	73.61
	茎	M7d_2_2	43.69	43.00	6.45	98.28	94.31	98.43	94.17	73.63
	茎	M7d_2_3	45.48	44.17	6.63	98.53	95.10	97.12	94.55	74.53
	叶	M7d_3_1	45.44	44.63	6.69	98.35	94.37	98.21	94.13	73.35
	叶	M7d_3_2	41.89	41.14	6.17	98.15	93.91	98.21	94.12	72.67
	叶	M7d_3_3	43.69	43.00	6.45	98.37	94.43	98.42	94.79	74.05
ME34	茎	S7d_2_1	47.36	44.68	6.70	98.14	93.91	94.32	91.64	71.61
	茎	S7d_2_2	43.69	42.69	6.40	97.88	92.97	97.71	91.21	70.77
	茎	S7d_2_3	43.69	42.63	6.39	97.69	92.39	97.57	92.47	70.63
	叶	S7d_3_1	43.32	42.35	6.35	97.87	93.00	97.76	93.17	71.16
	叶	S7d_3_2	43.69	42.78	6.42	98.00	93.31	97.91	93.23	71.91
	叶	S7d_3_3	45.45	44.33	6.65	98.23	94.21	97.54	93.41	71.96

序号No.	GO分类GO classification	基因数目Number of genes
1	nitrogen compound metabolic process	414
2	organonitrogen compound metabolic process	287
3	cellular nitrogen compound metabolic process	242
4	regulation of nitrogen compound metabolic process	163
5	organonitrogen compound biosynthetic process	118
6	cellular nitrogen compound biosynthetic process	109
7	nitrogen compound transport	61
8	organonitrogen compound catabolic process	52
9	positive regulation of nitrogen compound metabolic process	48
10	cellular nitrogen compound catabolic process	19
11	negative regulation of nitrogen compound metabolic process	19
12	response to nitrogen compound	13
13	response to organonitrogen compound	9
14	reactive nitrogen species metabolic process	3
15	nitrogen cycle metabolic process	3
16	cellular response to nitrogen compound	3
17	cellular response to nitrogen levels	3
18	cellular response to reactive nitrogen species	2

序号No.	GO分类GO classification	基因数目Number of genes
1	nitrogen compound metabolic process	414
2	organonitrogen compound metabolic process	287
3	cellular nitrogen compound metabolic process	242
4	regulation of nitrogen compound metabolic process	163
5	organonitrogen compound biosynthetic process	118
6	cellular nitrogen compound biosynthetic process	109
7	nitrogen compound transport	61
8	organonitrogen compound catabolic process	52
9	positive regulation of nitrogen compound metabolic process	48
10	cellular nitrogen compound catabolic process	19
11	negative regulation of nitrogen compound metabolic process	19
12	response to nitrogen compound	13
13	response to organonitrogen compound	9
14	reactive nitrogen species metabolic process	3
15	nitrogen cycle metabolic process	3
16	cellular response to nitrogen compound	3
17	cellular response to nitrogen levels	3
18	cellular response to reactive nitrogen species	2

序号No.	GO分类GO classification	基因数目Number of genes
1	nitrogen compound metabolic process	716
2	cellular nitrogen compound metabolic process	497
3	organonitrogen compound metabolic process	393
4	regulation of nitrogen compound metabolic process	262
5	cellular nitrogen compound biosynthetic process	203
6	organonitrogen compound biosynthetic process	169
7	nitrogen compound transport	135
8	organonitrogen compound catabolic process	77
9	positive regulation of nitrogen compound metabolic process	74
10	cellular nitrogen compound catabolic process	41
11	negative regulation of nitrogen compound metabolic process	37
12	response to nitrogen compound	16
13	response to organonitrogen compound	10
14	cellular response to nitrogen compound	5
15	cellular response to nitrogen levels	5
16	cellular response to organonitrogen compound	3
17	cellular response to nitrogen starvation	3
18	cellular response to reactive nitrogen species	2
19	reactive nitrogen species metabolic process	2
20	nitrogen utilization	2
21	nitrogen cycle metabolic process	2

序号No.	GO分类GO classification	基因数目Number of genes
1	nitrogen compound metabolic process	716
2	cellular nitrogen compound metabolic process	497
3	organonitrogen compound metabolic process	393
4	regulation of nitrogen compound metabolic process	262
5	cellular nitrogen compound biosynthetic process	203
6	organonitrogen compound biosynthetic process	169
7	nitrogen compound transport	135
8	organonitrogen compound catabolic process	77
9	positive regulation of nitrogen compound metabolic process	74
10	cellular nitrogen compound catabolic process	41
11	negative regulation of nitrogen compound metabolic process	37
12	response to nitrogen compound	16
13	response to organonitrogen compound	10
14	cellular response to nitrogen compound	5
15	cellular response to nitrogen levels	5
16	cellular response to organonitrogen compound	3
17	cellular response to nitrogen starvation	3
18	cellular response to reactive nitrogen species	2
19	reactive nitrogen species metabolic process	2
20	nitrogen utilization	2
21	nitrogen cycle metabolic process	2

基因登录号Gene accession No.	叶片转录组差异基因倍数log₂ Ratio	茎转录组差异基因倍数log₂ Ratio	基因注释Gene description
LOC117860115	3.21	2.23	Partner protein of NRT2, Activator for NRT2, High-affinity nitrate transport
LOC117858072	1.73	1.16	co-factor for nitrate, reductase and xanthine dehydrogenase 7
LOC117860105	5.32	1.91	Partner protein of NRT2, Activator for NRT2, High-affinity nitrate transport
LOC117846891	1.41	1.21	Low-affinity nitrate transporter, Root-to-shoot nitrate transport, Vascular bundle development
LOC117853975	1.08	1.30	Similar to Nitrate-induced NOI protein-like protein
LOC117861149	1.07	1.09	Similar to carbonic anhydrase
BGI_novel_G000242	1.14	1.28	5'-AMP-activated protein kinase, regulatory beta subunit
BGI_novel_G000243	–1.42	–2.55	5'-AMP-activated protein kinase, regulatory beta subunit
LOC117860265	–1.11	–1.36	Determination of the grain yield, Modulation of nitrogen utilization
LOC117850052	–1.56	–2.68	Serine/threonine protein kinase-related domain containing protein

基因登录号Gene accession No.	叶片转录组差异基因倍数log₂ Ratio	茎转录组差异基因倍数log₂ Ratio	基因注释Gene description
LOC117860115	3.21	2.23	Partner protein of NRT2, Activator for NRT2, High-affinity nitrate transport
LOC117858072	1.73	1.16	co-factor for nitrate, reductase and xanthine dehydrogenase 7
LOC117860105	5.32	1.91	Partner protein of NRT2, Activator for NRT2, High-affinity nitrate transport
LOC117846891	1.41	1.21	Low-affinity nitrate transporter, Root-to-shoot nitrate transport, Vascular bundle development
LOC117853975	1.08	1.30	Similar to Nitrate-induced NOI protein-like protein
LOC117861149	1.07	1.09	Similar to carbonic anhydrase
BGI_novel_G000242	1.14	1.28	5'-AMP-activated protein kinase, regulatory beta subunit
BGI_novel_G000243	–1.42	–2.55	5'-AMP-activated protein kinase, regulatory beta subunit
LOC117860265	–1.11	–1.36	Determination of the grain yield, Modulation of nitrogen utilization
LOC117850052	–1.56	–2.68	Serine/threonine protein kinase-related domain containing protein

狗尾草生物量增大突变体的转录组分析及氮素代谢相关基因挖掘

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张丽丽 ¹^,² , 赵海旭 ¹^,² , 胡帅 ¹^,² , 霍姗姗 ¹^,² , 夏启玉 ¹^,² , 郭安平 ¹^,² , 赵辉 ¹^,²^,^*

热带作物学报 | 组学与生物技术 2025,46(4): 853-865

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热带作物学报 |组学与生物技术 2025 , 46 (4) : 853 -865

狗尾草生物量增大突变体的转录组分析及氮素代谢相关基因挖掘

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张丽丽¹^,², 赵海旭¹^,², 胡帅¹^,², 霍姗姗¹^,², 夏启玉¹^,², 郭安平¹^,², 赵辉¹^,²^,^*

作者信息

^1.中国热带农业科学院三亚研究院/海南省南繁生物安全与分子育种重点实验室，海南三亚　572024

^2.中国热带农业科学院热带生物技术研究所，海南海口　571101

通讯作者:

^* 赵辉（ZHAO Hui），E-mail：zhaohui@itbb.org.cn。

Transcriptome Analysis and Nitrogen Metabolism Related Gene Mining of a Setaria viridis Mutant with Increased Biomass

Lili ZHANG¹^,², Haixu ZHAO¹^,², Shuai HU¹^,², Shanshan HUO¹^,², Qiyu XIA¹^,², Anping GUO¹^,², Hui ZHAO¹^,²^,^*

Affiliations

^1.Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences / Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-season Reproduction Regions, Sanya, Hainan 572024, China

^2.Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China

出版时间: 2025-04-25 doi: 10.3969/j.issn.1000-2561.2025.04.008

文章导航

摘要

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生物量大小是植物进化适应和产量的关键因素，获得高产作物品种一直是作物育种的基本要求。本研究在对狗尾草ME34的组织培养过程中，发现1株生物量变大的突变体植株Mu，其性状可以稳定遗传给下一代。通过对狗尾草表型观察和幼苗、成苗的农艺性状统计分析，发现狗尾草突变体幼苗的生物量（鲜重和干重）及成苗的株高均显著高于狗尾草野生型植株，狗尾草突变体成苗的穗长和种子长度也显著长于狗尾草野生型植株，且均达极显著水平。同时，对ME34和Mu的叶和茎进行转录组测序，并对部分基因进行荧光定量PCR验证。差异表达基因分析发现，氮素代谢和调控相关的差异表达基因占差异基因总数的20%左右。GO功能和KEGG富集分析同样发现，生物量大小与氮素的代谢、调控有着密切的关系。利用DEGseq方法分析差异基因的变化，获得了参与氮素吸收、运输、同化和再利用的相关基因，如硝酸盐转运蛋白NRT、铵转运蛋白AMT、硝酸还原酶NR、亚硝酸还原酶NiR、谷氨酸合成酶GOGAT、NLP家族转录因子以及丝氨酸苏氨酸蛋白激酶等。本研究为谷子等作物的育种工作提供重要分子依据和基础。

关键词

狗尾草 / 生物量 / 转录组测序 / 氮素利用效率

Abstract

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Biomass is a key factor in plant evolutionary adaptation and yield, and obtaining high-yield crop varieties is always a basic requirement for crop breeding. In this study, a mutant Mu with increased biomass was discovered during the tissue culture of Setaria viridis ME34, and the trait can be stably inherited to the next generation. Through the observation of the phenotype of S. viridis and statistical analysis of the agronomic traits of seedlings and adults, it was found that the biomass (fresh and dry weight) of the mutant seedlings and the adult plant height of S. viridis mutant was significantly higher than that of the wild-type plants, and the spike length and seed length were also significantly longer than those of the S. viridis wild-type plants, reaching a highly significant level. Transcriptome sequencing was performed on the leaves and stems of ME34 and Mu, and some genes were validated by RT-PCR. Differential expression gene analysis revealed that the genes related to nitrogen metabolism and regulation accounted for about 20% of the total differential genes. GO functional and KEGG enrichment analysis also revealed a close relationship between biomass and nitrogen metabolism and regulation. The DEGseq method was used to analyze the changes in the differentially expressed genes, and some related genes involved in nitrogen absorption, transport, assimilation, and reuse were obtained, such as nitrate transporter NRT, ammonium transporter AMT, nitrate reductase NR, nitrite reductase NiR, glutamate synthase GOGAT, NLP family transcription factors, and serine threonine protein kinase. Through the above research, important molecular basis and foundation are provided for the breeding work of crops such as foxtail millet.

Key words

Setaria viridis / biomass / transcriptome sequencing / nitrogen utilization efficiency

引用本文

张丽丽, 赵海旭, 胡帅, 霍姗姗, 夏启玉, 郭安平, 赵辉. 狗尾草生物量增大突变体的转录组分析及氮素代谢相关基因挖掘. 热带作物学报, 2025 , 46 (4) : 853 -865 . DOI: 10.3969/j.issn.1000-2561.2025.04.008

Lili ZHANG, Haixu ZHAO, Shuai HU, Shanshan HUO, Qiyu XIA, Anping GUO, Hui ZHAO. Transcriptome Analysis and Nitrogen Metabolism Related Gene Mining of a Setaria viridis Mutant with Increased Biomass[J]. Chinese Journal of Tropical Crops, 2025 , 46 (4) : 853 -865 . DOI: 10.3969/j.issn.1000-2561.2025.04.008

正文

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狗尾草（Setaria viridis）属单子叶植物纲禾本科黍亚科，是重要粮食作物谷子（S. italica）的野生近缘种。谷子是单子叶植物禾本科狗尾草属植物，二者核型基本相同，带型相近，基因组序列几乎一致^[1]，因此狗尾草也是研究谷子基因的重要模型。与谷子相比，狗尾草具有生长周期短（1.5~2个月）、植株矮小、易于种植（尤其是室内培养箱大量种植）、能产生大量自交系种子等优点。因此，研究狗尾草对C4植物如谷子、玉米、高粱、甘蔗等，尤其是对同属的谷子具有重要的借鉴作用，为快速挖掘谷子优良基因奠定基础。

氮素是植物生长和发育的重要营养元素，是限制作物产量和品质的关键因素^[2]。随着全球人口对粮食日益增长的需求，氮肥在农业生产中的投入也在逐年增加，然而，投入的氮肥只有不到一半被作物吸收，剩余的则以一氧化二氮的形式排放到大气中或以硝酸盐的形式排到地下，造成一系列严重的环境问题，因此，提高农作物自身的氮素利用效率（nitrogen use efficiency，NUE）显得更为重要^[3]。提高NUE可有效提高作物产量、减少氮肥需求、减轻环境污染、实现粮食安全和确保农业可持续发展。

本课题组在对狗尾草ME34组织培养过程中发现了1株生物量变大的突变体Mu，其株高、穗长及籽粒大小均显著增加，并且该性状能够稳定遗传。生物量对于植物的生长发育和最终产量都具有重要影响。通常植物的生物量越大，意味着其进行光合作用的能力越强，生长更为旺盛，能量转化效率也相应提高，从而有可能获得更高的产量。因此，在农作物生产过程中，农民经常关注农作物的生物量，通过采取合理的栽培管理措施来增加农作物的生物量，以期达到提高产量的目的。其中，籽粒大小是植物进化适应和种子产量的关键因素^[4-7]，大种子的幼苗被认为在胁迫条件下具有更强的生存能力，而小种子的植物物种则被认为具有更好的繁殖后代的能力^[8]。本研究利用转录组测序，对狗尾草野生型ME34和狗尾草生物量变大的突变体材料Mu的叶和茎分别进行转录组测序，分析突变体材料与野生型材料相比所引起的基因变化，挖掘与植物生物量大小相关的基因资源，尤其是与氮素代谢和调控相关的差异基因，从而为下一步谷子的育种工作提供一定的分子线索和基础。

1　材料与方法

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1.1　材料

1.1.1　植物材料

以狗尾草野生型ME34品系（本实验室保存）和狗尾草野生型ME34的突变体Mu（中国热带农业科学院热带生物技术研究所提供）为植物材料，筛选与狗尾草生物量变大的性状相关基因。

1.1.2　试剂耗材

使用Agilent 2100 Bioanalyzer（Agilent RNA 6000 Nano Kit）检测total RNA的浓度、RIN值、28S/18S和片段大小；使用紫外分光光度计NanoDrop^TM检测植物样本的纯度；转录组测序仪器采用DNBSEQ-T7。

1.2　方法

1.2.1　狗尾草的培养

选取经休眠处理的狗尾草ME34和突变体Mu，干燥其成熟种子，用无菌水清洗多次后播种于放有3层滤纸并用无菌水完全浸湿的培养皿中，每皿培养基约放置20粒种子，于24 ℃光/暗培养（12 000 lx，16 h/8 h，50%~60%湿度），7 d后分别对狗尾草ME34和Mu的叶和茎冻样0.5 g，以供提取RNA。同时，对在培养皿里生长7 d的ME34和Mu幼苗（完整植株）直接称量，测量鲜质量，然后将狗尾草幼苗置于60 ℃烘箱，24 h后称量干质量。

将生长7 d的幼苗转移至盆中，于30 ℃光培养/22 ℃暗培养（12 000 lx，16 h/8 h，50%~60%湿度）45 d后，测量成苗的株高和穗长，60 d后测量收获的种子长度。

1.2.2　文库构建及转录组分析

狗尾草ME34、Mu叶和茎的RNA提取，转录组的文库构建及基础的生物信息学分析均由深圳华大基因股份有限公司完成。本项目使用DNBSEQ平台完成转录组的测序。转录组差异基因的筛选标准为Fold Change≥2和Q<0.001。

1.2.3　转录组及农艺性状数据统计分析

分别取30株ME34和Mu植株幼苗进行总生物量统计（鲜质量和干质量）。对ME34和Mu的成苗进行株高、穗长和种子长度测量，采用Excel 2016软件进行数据分析。利用DPS数据处理系统对试验数据进行统计、差异显著性检验和相关性分析。

1.2.4　转录组荧光定量验证

将转录组测序时提取的狗尾草ME34、Mu的叶和茎的总RNA通过反转录试剂盒合成cDNA。以反转录产物为模板，以狗尾草ME34看家基因actin（登录号：Sevir.9 G114100，）为内参，对转录组部分基因进行荧光定量PCR。荧光定量PCR程序为：95 ℃预变性3 min；95 ℃变性15 s，60 ℃退火30 s，72 ℃延伸35 s，共40个循环。引物序列为Actin-F：CTTCCAGCCATCTTTCATT；Actin-R：CCAGA CTCGTCGTACTCAG。

2　结果与分析

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2.1　表型观察及农艺性状的统计分析

通过对狗尾草野生型ME34和突变体Mu成苗的表型观察及农艺性状进行统计分析发现，相较于野生型，狗尾草突变体Mu的株高更高，生物量更大，突变体株高增加了21.36%，并且达到极显著差异（P=0.003<0.01）；相较于野生型，狗尾草突变体的穗长更长，增加了41.79%，并且达到极显著差异（P=0<0.01）；突变体M₀代植株的种子与野生型相比更大更长，突变体籽粒长度增加了36.50%，并且达到极显著差异（P=0.0007<0.01）；突变体M₁代植株的种子与野生型相比延续了上一代的性状，种子依然更大更长（图1，图2）。

对生长7 d的狗尾草野生型ME34和突变体Mu（共30株）的生物量进行统计分析发现，相较于野生型，突变体鲜重增加51.53%，并且达到极显著差异（P=0.0001<0.01）；突变体干重增加57.00%，并且达到极显著差异（P=0<0.01）（图2）。

2.2　狗尾草样本转录组测序数据质量分析

本研究共检测了12个样品，样品比对基因组的平均比对率为93.37%，比对基因集的平均比对率为83.80%（表1）。预测的新基因为923个，共检测到表达的基因数为26 041，其中已知的基因为25 131个，预测的新基因为910个。测序的原始数据经过过滤及测序质量控制，12个样本中的Q20（表示碱基识别错误的概率为1%）所占比例均大于97%，Q30（表示碱基识别错误的概率为0.1%）所占比例均大于92%，以上结果表明测序数据质量可靠，可用于后续分析。

2.3　转录组基因差异表达分析

在狗尾草野生型ME34和狗尾草突变体Mu的叶转录组差异分析中，共发现3167个差异基因，其中上调基因1893个，下调基因1274个；在狗尾草野生型ME34和狗尾草突变体Mu茎的转录组差异分析中，共发现4667个差异基因，其中上调基因2157个，下调基因2510个（图3）。在狗尾草野生型ME34和狗尾草突变体Mu的叶和茎的交叉转录组差异分析中发现1737个共同差异表达的基因。

2.4　转录组差异表达基因GO（gene gntology）功能富集分析

GO富集分析通常依据细胞组分、分子功能和生物过程这3个方向进行分类，图4为狗尾草野生型叶片和突变体叶片（ME34-leaf vs Mu-leaf）转录组差异基因的GO功能富集分析的前十名。在细胞组成的分类中，差异基因主要集中在膜的固有成分、膜的组成部分等部位；在分子功能分类中，差异基因主要集中在ADP结合、腺苷核糖核苷酸结合等功能；在生物过程分类中，差异基因主要集中在吲哚烷基胺生物合成过程、色氨酸生物合成过程、β-葡聚糖生物合成过程、植物型原代细胞壁生物合成以及纤维素生物合成过程等生物过程。在狗尾草野生型茎和突变体茎（ME34-stem vs Mu-stem）的转录组差异基因的GO功能富集分析如图5所示，在细胞组成分类中，差异基因主要集中在非膜结合细胞器、细胞内非膜结合细胞器等部位；在分子功能分类中，差异基因主要集中在ADP结合、腺苷核糖核苷酸结合等功能；在生物过程分类中，差异基因主要集中在防御反应、对压力的反应等生物过程。

2.5　转录组差异表达基因KEGG（kyoto encyclopedia of genes and genomes）通路分析

通过KEGG富集分析发现，在ME34-leaf vs Mu-leaf差异表达基因显著富集的前20个通路中，包含参与细胞过程、环境信息过程、遗传信息过程和新陈代谢过程，如ABC转运蛋白和植物激素信号转导，黄曲霉毒素生物合成，苯丙氨酸、酪氨酸和色氨酸的生物合成以及油菜素内酯生物合成等（图6）。在ME34-stem vs Mu-stem差异表达基因显著富集的前20个通路中，包括ABC转运蛋白，磷脂酰肌醇信号系统，植物激素信号转导，MAPK信号通路——植物、苯丙氨酸、酪氨酸和色氨酸的生物合成，肌醇磷酸盐代谢，烟酸和烟酰胺代谢，不饱和脂肪酸的生物合成及β-丙氨酸代谢等（图7）。

2.6　与氮素代谢和调控相关差异表达基因的统计、热图分析及荧光定量RCR验证

与氮素代谢和调控相关差异表达基因的统计结果显示，ME34-leaf vs Mu-leaf转录组中共发现18个与氮素代谢和调控相关的GO分类，去除重复的基因后，共富集到621个差异基因，占叶片转录组中差异基因总数的19.61%，其中上调基因396个，下调基因225个（表2）。ME34-stem vs Mu-stem转录组中共发现21个与氮素代谢和调控相关的GO分类，去除重复的基因后，共富集到1048个差异基因，占茎转录组中差异基因总数的22.46%，其中上调基因430个，下调基因618个。叶片和茎的转录组中与氮素代谢和调控相关的共同差异表达基因331个，占2个转录组中共同差异表达基因总数的19.06%（表3）。

通过基因注释，GO功能富集和KEGG通路分析得到的与氮素代谢、调控最为直接相关的差异基因制成热图（图8）。ME34-leaf vs Mu-leaf转录组中共筛选到28个差异基因，其中上调基因24个，下调基因4个。ME34-stem vs Mu-stem转录组中共筛选到32个差异基因，其中上调基因16个，下调基因16个。在2个组织中共筛选出10个与氮素代谢、调控共同直接相关的差异基因，其中上调基因7个，下调基因3个（表4），且这10个基因在2个组织中表达模式相似。

从上述制成热图的2个转录组基因中各选6个基因进行荧光定量PCR验证（图9），以检验转录组数据的可靠性。对比图8和图9中的基因表达情况可知，基因的表达趋势一致。说明转录组数据真实可靠。

3　讨论

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本课题组在狗尾草野生型ME34的组织培养中发现了1株株高、穗长和种子长度均显著高于野生型的突变体Mu材料，生长1周的突变体狗尾草的生物量显著高于野生型狗尾草。为了挖掘和生物量变大性状相关的目标基因，分别对野生型狗尾草ME34和突变体狗尾草Mu叶和茎的转录组进行分析，发现无论是叶片的转录组还是茎的转录组，与氮素代谢、调控相关的差异表达基因占总的差异基因总数的20%左右，推测与氮素的代谢、调控相关基因对狗尾草生物量的大小至关重要。

通过转录组学的叶片GO功能富集分析中的生物过程分类发现，有一半以上的生物过程，如吲哚烷基胺生物合成过程、色氨酸生物合成过程、β-葡聚糖生物合成过程、植物型原代细胞壁生物合成以及纤维素生物合成过程都直接或者间接与氮素的代谢有密切关系。叶片和茎的KEGG富集分析中，苯丙氨酸、酪氨酸和色氨酸的生物合成，烟酸和烟酰胺代谢，黄曲霉毒素生物合成，油菜素内酯生物合成以及β-丙氨酸代谢等新陈代谢过程均与氮素的代谢有着密不可分的关系。通过转录组中与氮素代谢、调控相关的GO分类及基因注释，结合GO功能富集和KEGG通路分析，得到与氮素代谢、调控最为直接相关的差异基因。

NUE是一个涉及遗传和环境因素的复杂性状，且主要受到氮素吸收、运输、同化和再利用效率这几个因素的影响^[9]。植物对氮素的吸收转运根据土壤中氮源的不同，主要分为以下3种形式：酰胺态氮、硝态氮和铵态氮的吸收转运。硝酸盐的吸收和同化已成为影响作物氮利用率的重要因素^[10]。通过分析转录组的差异基因发现，在叶片转录组中，硝酸盐转运蛋白（nitrate transporter，NRT）基因，比如LOC117860105、LOC117860115、LOC117866632、LOC117842703、LOC117846891、LOC117845475、LOC117857667和LOC117866639全部上调表达。在茎转录组中，硝酸盐转运蛋白基因，如LOC117860105、LOC117860115和LOC117846891上调表达，LOC117836569、LOC117866281、LOC117852271、和LOC117866651下调表达。硝态氮是植物从土壤中吸收和利用氮源的主要形式，其吸收和转运主要由NPF（nitrate transport l/peptide transport family）基因家族成员完成。谷子SiNPF4.12基因协同调控了谷子氮素吸收利用和根系与籽粒的生长发育，有塑应用于作物氮高效高产育种^[11]。近年来，硝酸盐转运蛋白基因作为一个重要的家族已被用于作物育种，以提高氮素利用率^[12-18]，OsNRT1.1A（OsNPF6.3）是硝酸盐转运蛋白家族的成员，参与调节氮的利用和开花，为同时实现高产和早熟提供了目标，OsNRT.1A不仅可以上调硝酸盐和铵等氮利用相关基因的表达，还可以调控开花相关基因。与野生型相比，osnrt1.1a突变体表现出氮利用率降低和开花延迟，相比之下，osnrt1.1a在水稻中的过表达大大提高了氮利用效率（nitrogen use efficiency，NUE）和粮食产量，成熟时间也显著缩短^[19]。在玉米中对氮供应响应最强的基因是ZmNRT2.1和ZmNRT2.2，氮饥饿处理后，其在根部的表达增加了10倍以上。然而，在恢复氮供应24 h后，其表达恢复到对照水平^[20]。高粱的水培试验表明^[21]，在氮饥饿条件下，NRT2.2、NRT2.3、NRT3.1、NRT4.3、NRT4.4和NRT6.3在氮耐受和氮敏感基因型中的表达均上调。并且，与氮敏感基因型相比，氮耐受基因型中硝酸盐转运蛋白基因NRT2.4、NRT3.1和NRT4.5的转录本更丰富。由此推测这3个硝酸盐转运蛋白基因的过表达可能会提高氮敏感基因型对氮饥饿的耐受性，从而提高高粱氮素利用效率。在番茄中，LeNRT2.3介导低浓度硝酸盐的转运，LeNRT2.3的过表达可以增加番茄根系对

的吸收以及

从根到茎的运输，从而增加生物量和果实重量^[22]。TANG等^[23]通过全基因组关联分析，鉴定出硝酸盐转运蛋白OsNPF6.1HapB的优势单倍型，氮转运蛋白OsNPF6.1HapB由转录因子OsNAC42反式调节，通过激活水稻对硝酸盐的吸收来提高NUE。

铵态氮在土壤中主要以NH₄⁺的形式存在，铵转运蛋白（AMT）在铵（NH₄⁺）的吸收和转运中起着至关重要的作用^[24]。在叶片转录组中，铵转运蛋白（ammonium transporter，AMT），如LOC117846954、LOC117840316和LOC117862543全部上调表达。铵可以被铵转运蛋白（AMT）吸收^[25-27]，OsAMT1;1在低和高NH₄⁺条件下会显著促进对NH₄⁺的吸收，在次优和最佳氮条件下提高了种子产量^[28-29]。木薯MeAMT1基因转化拟南芥后在缺铵反应中比野生型植物生长更好，表明MeAMT1在低铵反应中起着重要作用，该研究为在其他植物中高效利用氮提供了基础^[30]。木薯MeAMT2（MeAMT2.3、MeAMT2.5和MeATM2.6）在缺铵条件下表达上调。互补实验表明，用MeAMT2.3、MeAMT2.5或MeATM2.6转化的酵母突变株TM31019b在缺铵条件下比未转化的酵母细胞生长更好，这表明MeAMT2.3、MeAMT2.5和MeATM2.2可能是木薯缺铵反应的主要因素，该研究为进一步研究木薯氮素高效利用提供了依据^[31]。甘蔗ScAMT1.1与水稻AMT1.1具有91.57%的同源性，甘蔗ScAMT1.1在水稻中稳定过表达后，在低氮处理下，ScAMT1.1过表达转基因水稻的株高和鲜重分别比野生型高36.48%和51.55%。转基因植物中铵同化关键酶GS和GDH的活性以及铵同化关键基因（包括GS1.1、GS1.2、GDH、Fd-GOGAT和NADH-GOGAT2）的表达水平均显著高于野生型。在盆栽试验中，转基因水稻的粒数和单株产量分别比野生型高6.44%和9.52%。甘蔗ScAMT1.1在低氮肥条件下具有提高铵同化能力和转基因水稻产量的潜力，该研究为改良氮利用率高的甘蔗品种提供了重要的功能基因^[32]。增强铵的吸收和再活化将为未来提高作物氮利用率提供一种有前景的策略。

氮素在进行同化时，硝酸根离子在胞质中通过硝酸还原酶（nitrate reductase，NR）还原成亚硝酸根离子，然后再通过亚硝酸还原酶（nitrite reductase，NiR）还原成铵根离子，最后经过GS/GOGAT（谷氨酰胺合成酶/谷氨酸合成酶，glutamine synthetase / glutamate synthase）循环合成谷氨酸后被植物生长所利用。在叶片转录组中，NADPH依赖性硝酸还原酶（NR）LOC117860413下调表达。在茎转录组中亚硝酸还原酶LOC117866314上调表达，在叶片转录组中，还有参与氮素同化过程的基因LOC117847605和LOC117847606全部上调表达。在茎转录组中，谷氨酸合成酶基因LOC117857834和LOC117848374下调表达。籼稻和粳稻亚种在硝酸盐同化能力和NUE方面存在差异，GAO等^[33]发现这种差异的主要成分是由编码NADH/NADPH依赖性硝酸还原酶（NR）的基因OsNR2的等位基因变异引起的，籼稻OsNR2表现出更大的NR活性，籼稻OsNR2还可以通过与编码硝酸盐转运蛋白的基因OsNRT1.1B相互作用促进硝酸盐的摄取，这些特性使籼稻OsNR2能够提高有效分蘖数、籽粒产量和NUE。在籼稻品种中，过表达OsNADH-GOGAT基因可使籽粒重量增加^[34]。OsNADH-GOGAT2的突变会导致了水稻小穗数和生产力的显著降低，证明了OsNADH-GOGAT2在叶片向种子再动员中的协调作用^[35]。在茎的转录组中发现2个NLP（NIN-LIKE PROTEIN）家族转录因子LOC117836353和LOC117844922，全部下调表达。YU等^[36]通过GWAS的方法在水稻中鉴定了一种与NUE相关的NLP4蛋白，发现OsNLP4可以反式高度激活编码亚硝酸还原酶的关键氮同化基因OsNiR，OsNLP4-OsNiR通过增强氮同化和NUE，来最终增加水稻的有效分蘖数和产量。

氮再活化利用是氮利用效率的关键组成部分。硝酸盐转运蛋白NRT1.7负责将源叶中储存的过量硝酸盐运输到韧皮部，并促进硝酸盐分配到库叶，在氮饥饿条件下，nrt1.7突变体表现出生长迟缓，表明nrt1.7介导的储存硝酸盐的源库再动员对于维持植物的生长非常重要^[37]。增强源库硝酸盐再活化是提高氮素利用率和作物产量的新策略。

此外，在叶片和茎的转录组中，都有一个丝氨酸苏氨酸蛋白激酶LOC117850052下调表达。在植物中，氮素是影响植物生长和发育的关键营养元素之一，而丝氨酸/苏氨酸蛋白激酶在植物对氮素的响应和利用中发挥着重要作用。熊延教授团队的研究揭示了TOR（target of rapamycin）蛋白激酶在植物氮素营养中的作用机制，TOR是一种高度保守的丝氨酸/苏氨酸蛋白激酶，在植物中，无机氮（如硝酸根与铵根）和有机氮（如谷氨酰胺等氨基酸）以彼此相互独立的信号方式激活ROP2-TOR信号通路，从而调控茎尖生长和发育的分子机制，这一发现为理解植物如何感知和响应氮素营养提供了新的视角，同时也揭示了丝氨酸/苏氨酸蛋白激酶在氮素利用率中的关键作用^[38]。

综上所述，获得高产、高氮利用效率的作物品种一直是作物育种的一项艰巨任务。提高氮利用效率的过程包括氮吸收、氮从根到茎的运输、氮同化和氮再分配，每一步都是提高氮利用率不可或缺的。本研究通过一个狗尾草生物量变大突变体的转录组分析，挖掘了与狗尾草氮素代谢和调控等可能与生物量大小相关的基因，如硝酸盐转运蛋白NRT、铵转运蛋白AMT、硝酸还原酶NR、亚硝酸还原酶NiR、谷氨酸合成酶GOGAT、NLP家族转录因子以及丝氨酸苏氨酸蛋白激酶等与NUE密切相关的基因，为下一步谷子及同属的作物育种提供理论基础与科学依据。

基金

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海南省院士创新平台科研专项(YSPTZX202102)
三亚崖州湾科技城科技专项(SKJC-2022-PTDX-030)
海南省自然科学基金项目(323MS086)

参考文献

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2025年第46卷第4期

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doi: 10.3969/j.issn.1000-2561.2025.04.008

接收时间：2024-09-24
首发时间：2026-06-24
出版时间：2025-04-25

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出版历史

收稿日期：2024-09-24
录用日期：2024-11-19

基金

海南省院士创新平台科研专项(YSPTZX202102)

三亚崖州湾科技城科技专项(SKJC-2022-PTDX-030)

海南省自然科学基金项目(323MS086)

作者信息

^1.中国热带农业科学院三亚研究院/海南省南繁生物安全与分子育种重点实验室，海南三亚　572024

^2.中国热带农业科学院热带生物技术研究所，海南海口　571101

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^* 赵辉（ZHAO Hui），E-mail：zhaohui@itbb.org.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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植株类型Plant type	组织Tissue	编号No.	过滤前的reads数Total raw reads/Mb	过滤后的reads数Total clean reads/Mb	过滤后的碱基总数Total clean bases/Gb	碱基识别准确率Clean reads Q20/%	碱基识别准确率Clean Reads Q30/%	过滤后的reads比例Clean reads ratio/%	比对上参考基因组的clean reads比例Total mapping ratio/%	唯一比对上参考基因组某一位置的clean reads比例Uniquely mapping ratio/%
Mu	茎	M7d_2_1	45.44	44.59	6.69	98.41	94.65	98.13	93.58	73.61
	茎	M7d_2_2	43.69	43.00	6.45	98.28	94.31	98.43	94.17	73.63
	茎	M7d_2_3	45.48	44.17	6.63	98.53	95.10	97.12	94.55	74.53
	叶	M7d_3_1	45.44	44.63	6.69	98.35	94.37	98.21	94.13	73.35
	叶	M7d_3_2	41.89	41.14	6.17	98.15	93.91	98.21	94.12	72.67
	叶	M7d_3_3	43.69	43.00	6.45	98.37	94.43	98.42	94.79	74.05
ME34	茎	S7d_2_1	47.36	44.68	6.70	98.14	93.91	94.32	91.64	71.61
	茎	S7d_2_2	43.69	42.69	6.40	97.88	92.97	97.71	91.21	70.77
	茎	S7d_2_3	43.69	42.63	6.39	97.69	92.39	97.57	92.47	70.63
	叶	S7d_3_1	43.32	42.35	6.35	97.87	93.00	97.76	93.17	71.16
	叶	S7d_3_2	43.69	42.78	6.42	98.00	93.31	97.91	93.23	71.91
	叶	S7d_3_3	45.45	44.33	6.65	98.23	94.21	97.54	93.41	71.96

植株类型Plant type

组织Tissue

编号No.

过滤前的reads数Total raw reads/Mb

过滤后的reads数Total clean reads/Mb

过滤后的碱基总数Total clean bases/Gb

碱基识别准确率Clean reads Q20/%

碱基识别准确率Clean Reads Q30/%

过滤后的reads比例Clean reads ratio/%

比对上参考基因组的clean reads比例Total mapping ratio/%

唯一比对上参考基因组某一位置的clean reads比例Uniquely mapping ratio/%

茎

M7d_2_1

45.44

44.59

6.69

98.41

94.65

98.13

93.58

73.61

茎

M7d_2_2

43.69

43.00

6.45

98.28

94.31

98.43

94.17

73.63

茎

M7d_2_3

45.48

44.17

6.63

98.53

95.10

97.12

94.55

74.53

叶

M7d_3_1

45.44

44.63

6.69

98.35

94.37

98.21

94.13

73.35

叶

M7d_3_2

41.89

41.14

6.17

98.15

93.91

98.21

94.12

72.67

叶

M7d_3_3

43.69

43.00

6.45

98.37

94.43

98.42

94.79

74.05

ME34

茎

S7d_2_1

47.36

44.68

6.70

98.14

93.91

94.32

91.64

71.61

茎

S7d_2_2

43.69

42.69

6.40

97.88

92.97

97.71

91.21

70.77

茎

S7d_2_3

43.69

42.63

6.39

97.69

92.39

97.57

92.47

70.63

叶

S7d_3_1

43.32

42.35

6.35

97.87

93.00

97.76

93.17

71.16

叶

S7d_3_2

43.69

42.78

6.42

98.00

93.31

97.91

93.23

71.91

叶

S7d_3_3

45.45

44.33

6.65

98.23

94.21

97.54

93.41

71.96

序号No.	GO分类GO classification	基因数目Number of genes
1	nitrogen compound metabolic process	414
2	organonitrogen compound metabolic process	287
3	cellular nitrogen compound metabolic process	242
4	regulation of nitrogen compound metabolic process	163
5	organonitrogen compound biosynthetic process	118
6	cellular nitrogen compound biosynthetic process	109
7	nitrogen compound transport	61
8	organonitrogen compound catabolic process	52
9	positive regulation of nitrogen compound metabolic process	48
10	cellular nitrogen compound catabolic process	19
11	negative regulation of nitrogen compound metabolic process	19
12	response to nitrogen compound	13
13	response to organonitrogen compound	9
14	reactive nitrogen species metabolic process	3
15	nitrogen cycle metabolic process	3
16	cellular response to nitrogen compound	3
17	cellular response to nitrogen levels	3
18	cellular response to reactive nitrogen species	2

序号No.

GO分类GO classification

基因数目Number of genes

nitrogen compound metabolic process

414

organonitrogen compound metabolic process

287

cellular nitrogen compound metabolic process

242

regulation of nitrogen compound metabolic process

163

organonitrogen compound biosynthetic process

118

cellular nitrogen compound biosynthetic process

109

nitrogen compound transport

organonitrogen compound catabolic process

positive regulation of nitrogen compound metabolic process

cellular nitrogen compound catabolic process

negative regulation of nitrogen compound metabolic process

response to nitrogen compound

response to organonitrogen compound

reactive nitrogen species metabolic process

nitrogen cycle metabolic process

cellular response to nitrogen compound

cellular response to nitrogen levels

cellular response to reactive nitrogen species

序号No.	GO分类GO classification	基因数目Number of genes
1	nitrogen compound metabolic process	716
2	cellular nitrogen compound metabolic process	497
3	organonitrogen compound metabolic process	393
4	regulation of nitrogen compound metabolic process	262
5	cellular nitrogen compound biosynthetic process	203
6	organonitrogen compound biosynthetic process	169
7	nitrogen compound transport	135
8	organonitrogen compound catabolic process	77
9	positive regulation of nitrogen compound metabolic process	74
10	cellular nitrogen compound catabolic process	41
11	negative regulation of nitrogen compound metabolic process	37
12	response to nitrogen compound	16
13	response to organonitrogen compound	10
14	cellular response to nitrogen compound	5
15	cellular response to nitrogen levels	5
16	cellular response to organonitrogen compound	3
17	cellular response to nitrogen starvation	3
18	cellular response to reactive nitrogen species	2
19	reactive nitrogen species metabolic process	2
20	nitrogen utilization	2
21	nitrogen cycle metabolic process	2

序号No.

GO分类GO classification

基因数目Number of genes

nitrogen compound metabolic process

716

cellular nitrogen compound metabolic process

497

organonitrogen compound metabolic process

393

regulation of nitrogen compound metabolic process

262

cellular nitrogen compound biosynthetic process

203

organonitrogen compound biosynthetic process

169

nitrogen compound transport

135

organonitrogen compound catabolic process

positive regulation of nitrogen compound metabolic process

cellular nitrogen compound catabolic process

negative regulation of nitrogen compound metabolic process

response to nitrogen compound

response to organonitrogen compound

cellular response to nitrogen compound

cellular response to nitrogen levels

cellular response to organonitrogen compound

cellular response to nitrogen starvation

cellular response to reactive nitrogen species

reactive nitrogen species metabolic process

nitrogen utilization

nitrogen cycle metabolic process

基因登录号Gene accession No.	叶片转录组差异基因倍数log₂ Ratio	茎转录组差异基因倍数log₂ Ratio	基因注释Gene description
LOC117860115	3.21	2.23	Partner protein of NRT2, Activator for NRT2, High-affinity nitrate transport
LOC117858072	1.73	1.16	co-factor for nitrate, reductase and xanthine dehydrogenase 7
LOC117860105	5.32	1.91	Partner protein of NRT2, Activator for NRT2, High-affinity nitrate transport
LOC117846891	1.41	1.21	Low-affinity nitrate transporter, Root-to-shoot nitrate transport, Vascular bundle development
LOC117853975	1.08	1.30	Similar to Nitrate-induced NOI protein-like protein
LOC117861149	1.07	1.09	Similar to carbonic anhydrase
BGI_novel_G000242	1.14	1.28	5'-AMP-activated protein kinase, regulatory beta subunit
BGI_novel_G000243	–1.42	–2.55	5'-AMP-activated protein kinase, regulatory beta subunit
LOC117860265	–1.11	–1.36	Determination of the grain yield, Modulation of nitrogen utilization
LOC117850052	–1.56	–2.68	Serine/threonine protein kinase-related domain containing protein

基因登录号Gene accession No.

叶片转录组差异基因倍数log₂ Ratio

茎转录组差异基因倍数log₂ Ratio

基因注释Gene description

LOC117860115

3.21

2.23

Partner protein of NRT2, Activator for NRT2, High-affinity nitrate transport

LOC117858072

1.73

1.16

co-factor for nitrate, reductase and xanthine dehydrogenase 7

LOC117860105

5.32

1.91

Partner protein of NRT2, Activator for NRT2, High-affinity nitrate transport

LOC117846891

1.41

1.21

Low-affinity nitrate transporter, Root-to-shoot nitrate transport, Vascular bundle development

LOC117853975

1.08

1.30

Similar to Nitrate-induced NOI protein-like protein

LOC117861149

1.07

1.09

Similar to carbonic anhydrase

BGI_novel_G000242

1.14

1.28

5'-AMP-activated protein kinase, regulatory beta subunit

BGI_novel_G000243

–1.42

–2.55

5'-AMP-activated protein kinase, regulatory beta subunit

LOC117860265

–1.11

–1.36

Determination of the grain yield, Modulation of nitrogen utilization

LOC117850052

–1.56

–2.68

Serine/threonine protein kinase-related domain containing protein