谷子苗期氮高效转录组分析与基因挖掘

doi:10.13304/j.nykjdb.2024.0288

中国农业科技导报 ›› 2024, Vol. 26 ›› Issue (10): 41-57.DOI: 10.13304/j.nykjdb.2024.0288

谷子苗期氮高效转录组分析与基因挖掘

夏雪岩(), 崔纪菡(), 黄玫红, 郭帅, 刘猛, 赵宇, 鲁一薇, 赵文庆, 王京新, 李顺国()

河北省农林科学院谷子研究所，国家谷子改良中心，农业农村部特色杂粮遗传改良与利用重点实验室（部省共建），河北省杂粮重点实验室，石家庄 050035

收稿日期:2024-04-11 接受日期:2024-05-29 出版日期:2024-10-15 发布日期:2024-10-18
通讯作者: 夏雪岩,李顺国
作者简介:夏雪岩 E-mail：xyxia7808@126.com
崔纪菡，E-mail：cuijihancau@126.com。第一联系人：夏雪岩和崔纪菡为共同第一作者。
基金资助:
国家谷子高粱产业技术体系建设项目(CARS-06-14.5-A23);河北省农林科学院基本科研业务费包干制项目(HBNKY-BGZ-02);河北省现代农业产业技术体系杂粮杂豆产业创新团队建设(HBCT2023050101)

Analysis of High-efficiency Transcriptome of Nitrogen in Millet Seedlings and Gene Mining

Xueyan XIA(), Jihan CUI(), Meihong HUANG, Shuai GUO, Meng LIU, Yu ZHAO, Yiwei LU, Wenqin ZHAO, Jingxin WANG, Shunguo LI()

Key Laboratory for Genetic Improvement and Utilization of Special Grains of the Ministry of Agriculture and Rural Affairs （jointly established by the Ministry and the Province），National Millet Improvement Center，Key Laboratory of Miscellaneous Grains in Hebei Province，Millet Research Institute，Hebei Academy of Agricultural and Forestry Sciences，Shijiazhuang 050035，China

Received:2024-04-11 Accepted:2024-05-29 Online:2024-10-15 Published:2024-10-18
Contact: Xueyan XIA,Shunguo LI

摘要/Abstract

摘要：

氮利用效率是影响谷子产量和品质的重要因素。筛选氮高效谷子品种、挖掘控制氮利用效率的重要基因及解析其作用机制对于提高谷子氮利用效率、减少肥料使用、保护环境和保障粮食安全具有重要意义。通过比较氮高效和氮低效谷子品种在不同氮水平条件下表型性状和氮吸收能力发现，氮高效品种比低效品种具有较高的铵态氮和硝态氮吸收能力。设置3个氮素水平（0、3和6 mmol·L^-1），在谷苗两叶一心期处理7 d后，运用转录组测序（RNA-seq）技术比较不同品种在各个处理之间的差异表达基因（differentially expressed genes，DEGs）。结果表明，以0 mmol·L^-1氮处理为对照，共获得16 000个DEGs。通过GO和KEGG富集分析发现，氨基酸合成与代谢、防御相关代谢、乙醛酸和二羧酸代谢、光合生物的碳固定、丙酮酸代谢、卟啉与叶绿素代谢、MAPK信号通路-植物、氨基糖和核苷酸糖代谢在调控氮代谢生物反应过程中发挥重要作用。并预测到3 348个转录因子，分属46个家族，其中预测转录因子数量最多的是MYB-related家族（189个），其次为bHLH家族（182个）、WRKY家族（158个）、MYB家族（156个）和HB-other家族（153个）。通过WGCNA分析筛选出K_ME值排名前30位的基因，其中有7个基因被注释到基因信息。RT-qPCR结果显示，Seita.9G488700、Seita.2G368800和Seita.5G063300表达趋势与RNA-seq结果一致，主要调控糖转运蛋白和碳水化合物运输与代谢过程。说明氨基酸合成与代谢、光合生物的碳固定、乙醛酸和二羧酸代谢途径等信号通路在谷子氮高效利用方面发挥重要作用。研究结果筛选到3个与氮高效利用相关的基因，完善了谷子氮高效基因组注释信息，为氮高效基因资源的挖掘利用提供理论指导。

关键词: 谷子, 氮高效, 转录组学, 氮高效基因

Abstract:

Nitrogen use efficiency is an important factor affecting the yield and quality of millet. It is of great significance to screen millet varieties with high nitrogen efficiency and explore the important genes controlling nitrogen use efficiency and their mechanism for improving nitrogen use efficiency of millet， reducing fertilizer use， protecting the environment and ensuring food security. By comparing phenotypic traits， nitrogen absorption capacity， it showed that nitrogen-efficient varieties had high ammonium nitrogen and nitrate nitrogen absorption capacity than nitrogen inefficient varieties. 3 nitrogen level treatments were set， 0， 3 and 6 mmol·L^-1 and after the rice seedlings at two-leaf one-stage were treated for 7 d. Taking 0 mmol·L^-1 nitrogen treatment as control， a total of 16 000 differential expression genes （DEGs） were obtained by RNA-seq. Through GO and KEGG enrichment analysis， it was found that amino acid synthesis and metabolism， defense-related metabolism， acetaldehyde acid and dicarboxylic acid metabolism， and carbon fixation of photosynthetic organisms， pyruvate metabolism， porphyrin and chlorophyll metabolism， MAPK signaling pathway-plant， amino sugar and nucleotide sugar metabolism played important roles in regulating the biological reaction of nitrogen metabolism. 3 348 transcription factors were predicted， which belonged to 46 families， among which the largest number of predicted transcription factors is the MYB-related family （189）， followed by the bHLH family （182）， the WRKY family （158）， the MYB family （156） and HB- Other families （153）. Through WGCNA analysis， the top 30 genes with K_ME values were screened， and 7 of them were annotated to genetic information. RT-qPCR results showed that the expression trends of Seita.9G488700， Seita.2G368800 and Seita.5G063300 were basically same as RNA-seq results， which mainly regulated the transportation and metabolism of glucose transporters and carbohydrates. .

Key words: millet, nitrogen efficiency, transcriptomics, nitrogen efficient genes

中图分类号:

S515

夏雪岩, 崔纪菡, 黄玫红, 郭帅, 刘猛, 赵宇, 鲁一薇, 赵文庆, 王京新, 李顺国. 谷子苗期氮高效转录组分析与基因挖掘[J]. 中国农业科技导报, 2024, 26(10): 41-57.

Xueyan XIA, Jihan CUI, Meihong HUANG, Shuai GUO, Meng LIU, Yu ZHAO, Yiwei LU, Wenqin ZHAO, Jingxin WANG, Shunguo LI. Analysis of High-efficiency Transcriptome of Nitrogen in Millet Seedlings and Gene Mining[J]. Journal of Agricultural Science and Technology, 2024, 26(10): 41-57.

图/表 6

表1 不同处理营养液配方 (mL)

Table 1 Nutrient solution formula of differnet treatment

成分 Component	N₀	N₃	N₆
1.0 mol·L^-1 Ca（NO₃）₂·4H₂O	0	1.5	3
0.5 mol·L^-1 K₂SO₄	5	5	5
1.0 mol·L^-1 CaCl₂	5	3.5	2
1.0 mol·L^-1 MgSO4·7H₂O	2	2	2
1.0 mol·L^-1 KH₂PO₄	1	1	1
0.5% Fe-EDTA	1	1	1
微量元素母液 Trace element mother liquor	1	1	1
ddH₂O	985	985	985
总体积Total volume	1 000	1 000	1 000

表2 实时荧光定量PCR的引物序列

Table 2 Primer sequence of real-time fluorescence quantitative PCR.

基因名称 Gene name	引物名称 Primer name	引物序列 Primer sequence（5’-3’）
Seita.9G488700	GS-488700-F1	AAGAAGCACGAGGACAAGGA
	GS-488700-R1	GTGGTGTAGGAGGCGAAGAA
Seita.2G368800	GS-368800-F2	CAAGGAACGAAACGGTGAGA
	GS-368800-R2	CAAGCGGGCTGTAGAGGAC
Seita.5G063300	GS-63300-F2	GGACCAGAAGACCGTCAACTC
	GS-63300-R2	TCGGGAACGCCGTGAGGAAC
SiActin	SiActin-F	GGCAAACAGGGAGAAGATGA
	SiActin-R	GAGGTTGTCGGTAAGGTCACG

图1 氮素处理下4个谷子品种的表型差异注：*和**分别表示P<0.05和P<0.01水平差异显著；不同大小写字母表示P<0.01和P<0.05水平差异显著。

Fig. 1 Phenotypic differences of four millet varieties under nitrogen treatmentNote： * and ** indicate significant different at P<0.05 and P<0.01 levels， respectively； different captial and small letters indicate significant differences at P<0.01 and P<0.05 levels， respectively.

图2 4个谷子品种在不同氮素处理下的差异表达基因韦恩分析

Fig.2 Venn analysis of differentially expressed genes in 4 millet varieties under different nitrogen treatments

图5 不同氮素处理下WGCNA分析A：基因和模块的树状图；B：MEbrown模块中连接程度（KME）排名前30基因的共表达网络；C：模块-性状关联分析，括号内数值为模块与表型的相关性P值

Fig. 5 Analysis of WGCNA under different nitrogen treatmentsA： Dendrogram of genes and modules； B： Co-expression network of top 30 genes highly connected （KME） in the MEbrown with high； C： Module-trait association， the values in bracket represent the P value of the correlation between the module and phenotype

图6 RT-qPCR验证结果

Fig. 6 RT-qPCR validation result

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谷子苗期氮高效转录组分析与基因挖掘

Analysis of High-efficiency Transcriptome of Nitrogen in Millet Seedlings and Gene Mining

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