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作物学报 ›› 2022, Vol. 48 ›› Issue (11): 2765-2773.doi: 10.3724/SP.J.1006.2022.14143

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ChIP-seq分析花生中AhGLK1与AhHDA1调控的下游靶基因网络

刘星1(), 苏良辰1, 李丽梅2, 李玲2,*()   

  1. 1遵义医科大学珠海校区生物工程系, 广东珠海 519041
    2华南师范大学生命科学学院 / 广东省植物发育生物工程重点实验室, 广东广州510631
  • 收稿日期:2021-08-10 接受日期:2021-11-29 出版日期:2022-11-12 网络出版日期:2021-12-17
  • 通讯作者: 李玲
  • 作者简介:第一作者联系方式: E-mail: liuxingas@126.com
  • 基金资助:
    本研究由贵州省科学技术厅基金项目(ZK2021-127);国家自然科学基金项目(31671600)

Downstream target gene network regulated by AhGLK1 and AhHDA1 using ChIP-seq in peanut

LIU Xing1(), SU Liang-Chen1, LI Li-Mei2, LI Ling2,*()   

  1. 1Department of Bioengineening, Zhuhai Campus, Zunyi Medical University, Zhuhai 519041, Guangdong, China
    2Guangdong Provincial Key Laboratory of Biotechnology for Plant Development / School of Life Sciences, South China Normal University, Guangzhou 510631, Guangdong, China
  • Received:2021-08-10 Accepted:2021-11-29 Published:2022-11-12 Published online:2021-12-17
  • Contact: LI Ling
  • Supported by:
    The Department of Science and Technology of Guizhou Province(ZK2021-127);The National Natural Science Foundation of China(31671600)

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摘要：

花生(Arachis hypogaea L.)是重要的经济油料作物, 其生长发育、产量与品质受干旱影响。为深入了解花生的抗旱机理, 本研究通过ChIP-seq对组蛋白去乙酰化酶AhHDA1和转录因子AhGLK1富集的DNA序列进行分析, 揭示两者调控的下游靶基因网络。通过比对分析, GLK-IP获得6571万clean beads, HDA-IP获得6390万clean beads, Input获得7006万clean beads, 唯一比对率分别为74.97%、76.81%和76.75%。GLK-IP获得714个peak, HDA-IP获得543个peak。Peak在基因的外显子、内含子、上游、下游和基因间等功能元件均有分布。GO富集结果显示, AhGLK1-IP和AhHDA1-IP的peak相关基因在分子功能中的富集分别为35.1%和32.8%, 在生物学过程中的富集分别为39.3%和44.2%, 在细胞组分中的富集分别为25.5%和22.8%。KEGG信号通路富集结果显示, AhGLK1-IP相关基因显著富集在“代谢途径(metabolic pathways)”、“抗生素生物合成(biosynthesis of antibiotics)”、“二羧酸代谢(glyoxylate and dicarboxylate metabolism)”、“不同环境中微生物代谢(microbial metabolism in diverse environments)”、“碳代谢(carbon metabolism)”、“次生代谢生物合成(biosynthesis of secondary metabolites)”和“氨基酸生物合成(biosynthesis of amino acids)。而AhHDA1-IP相关基因在“N聚糖生物合成(N-glycan biosynthesis)”、“精氨酸和脯氨酸代谢(arginine and proline metabolism)”和“苯丙氨酸代谢(phenylalanine metabolism)”通路显著富集。AhGLK1-IP和AhHDA1-IP共同富集的peak有4个, 在AhGLK1-IP和AhHDA1-IP特异富集的基序(motif)中存在共同的保守序列AGAA/T。研究结果为深入认识AhGLK1和AhHDA1基因的功能和了解花生响应干旱胁迫和旱后恢复生长中的调控机制具有参考价值。

关键词: 花生, ChIP-seq, AhGLK1, AhHDA1, 靶基因

Abstract:

Peanut (Arachis hypogaea L.) is an important economic and edible oil crop whose growth, yield and quality are seriously affected by drought stress. In order to further investigate the mechanism of drought tolerance in peanut, chromatin immunoprecipitation sequence (ChIP-seq) was conducted to analyze immunoaffinity separation of specific binding DNA fragments with AhHDA1 (Arachis hypogaea L. histone deacetylase 1) and transcription factor AhGLK1 (Arachis hypogaea L. Golden2-like 1) for sequence identification, revealing the downstream target gene networks regulated by AhHDA1 and AhGLK1. Through alignment and analysis, 65.71, 63.90, and 70.06 million clean beads were obtained for GLKHDA and input IP from the sequencing run, respectively. The unique alignment of reads to the reference genome sequence were 74.97%, 76.81%, and 76.75%, respectively. GLK-IP obtained 714 peaks and HDA-IP obtained 543 peaks. Peaks were distributed into exons, introns, upstream, downstream, intergenic and other functional elements. GO enrichment revealed that 35.1% and 32.8% of the peak-related genes of AhGLK1-IP and AhHDA1-IP were enriched in “molecular function”. 39.3% and 44.2% were enriched in “biological process” and 25.5% and 22.8% were enriched in “cellular components”, respectively. KEGG pathway enrichment showed that AhGLK1-IP related genes were significantly enriched in metabolic, biosynthesis of antibiotics, glyoxylate and dicarboxylate metabolism, microbial metabolism in diverse environments, carbon metabolism, biosynthesis of secondary metabolites and biosynthesis of amino acids pathways. Whereas, AhHDA1-IP related genes were significantly enriched in N-glycan biosynthesis, arginine, proline and phenylalanine metabolism pathways. Moreover, there were four common peaks enriched both in AhGLK1-IP and AhHDA1-IP. A common conserved motif sequences (AGAA/T) was presented in the motif sequences specifically enriched by AhGLK1-IP and AhHDA1-IP. These results of the study have some reference value for further understanding the functions of AhGLK1 and AhHDA1 genes and the regulatory mechanism of peanuts in response to drought stress and post-drought recovery growth.

Key words: Arachis hypogaea L., chromatin immunoprecipitation sequence, AhGLK1, AhHDA1, target gene

表1

ChIP-seq数据产出统计"

样品名
Sample name		 序列总数
No. of total sequences		 碱基总数
No. of total bases		 GC
(%)		 Q20
(%)		 Q30
(%)
GLK-IP 65,712,234 9,856,835,100 41.50 97.60 93.96
HDA-IP 63,903,912 9,85,586,800 40.73 97.61 93.96
Input 70,062,804 10,509,420,600 40.49 97.43 93.58

表2

数据比对结果统计"

样品名
Sample		 总序列数
No. of total
sequences		 比对序列数
No. of aligned
sequences		 比对率
Aligned rate
(%)		 唯一比对序列数
No. of unique aligned
sequences		 唯一比对率
Unique aligned rate
(%)
GLK-IP 65,133,710 60,248,717 92.50 48,828,873 74.97
HDA-IP 63,362,432 59,643,659 94.13 48,666,448 76.81
Input 69,408,066 68,607,870 98.85 53,268,396 76.75

表3

Peak信息统计"

样品名
Sample name		 Peak数
Peak number		 Peak总长度
Peak total
length		 Peak平均长度
Peak average length		 Peak总序列深度
Peak total sequence depth		 Peak平均序列度
Peak average sequence degree		 基因组比例
Genome rate
(%)
GLK-IP 714 416,446 583 11,224 15 0.02
HDA-IP 543 298,118 549 7764 14 0.01

图1

AhGLK1和AhHDA1富集基因的peak分布"

图2

AhGLK1和AhHDA1富集基因GO分类分析"

图3

AhGLK1和AhHDA1富集基因KEGG pathway分析 A: AhGLK1-IP 相关基因; B: AhHDA1-IP相关基因。"

表4

AhGLK1和AhHDA1-IP相关基因在不同KEGG通路中的分布"

样品
Sample name		 KEGG通路
KEGG pathway		 基因个数
Gene number		 基因ID
Gene ID
AhGLK-IP 代谢途径
Metabolic pathways		 20 Arahy.14YEDZ, Arahy.46RTPJ, Arahy.46W7S4, Arahy.6Q1KS5, Arahy.8T1UF6, Arahy.C0Q7J8, Arahy.CQFZ3U, Arahy.D1ZPVC, Arahy.IC3HGI, Arahy.LT225G, Arahy.MY1TBE, Arahy.NDL7Z2, Arahy.S45BII, Arahy.S8DJY1, Arahy.SKV00U, Arahy.SP7CPF, Arahy.U4VZKL, Arahy.VD5UEC, Arahy.YS6PIM, Arahy.ZZNJ3G
抗生素生物合成
Biosynthesis of antibiotics		 8 Arahy.46W7S4, Arahy.8T1UF6, Arahy.C0Q7J8, Arahy.CQFZ3U, Arahy.NDL7Z2, Arahy.S45BII, Arahy.SP7CPF, Arahy.U4VZKL
二羧酸代谢
Glyoxylate and dicarboxylate metabolism		 3 Arahy.NDL7Z2, Arahy.S45BII, Arahy.VD5UEC
不同环境中微生物代谢
Microbial metabolism in diverse environments		 6 Arahy.CQFZ3U, Arahy.NDL7Z2, Arahy.S45BII, Arahy.SP7CPF, Arahy.U4VZKL, Arahy.VD5UEC
碳代谢
Carbon metabolism		 5 Arahy.CQFZ3U, Arahy.NDL7Z2, Arahy.S45BII, Arahy.U4VZKL, Arahy.VD5UEC
次生代谢生物合成
Biosynthesis of secondary metabolites		 12 Arahy.46W7S4, Arahy.6Q1KS5, Arahy.8T1UF6, Arahy.C0Q7J8, Arahy.CQFZ3U, Arahy.IC3HGI, Arahy.NDL7Z2, Arahy.S45BII, Arahy.SP7CPF, Arahy.U4VZKL, Arahy.YS6PIM, Arahy.ZZNJ3G
AhHDA-IP N聚糖生物合成
N-Glycan biosynthesis		 3 Arahy.6B4W14,Arahy.7LT28F, Arahy.YXCZ83
精氨酸和脯氨酸代谢
Arginine and proline metabolism		 3 Arahy.EJ6Q9X, Arahy.GRS0A1, Arahy.UIG78U
苯丙氨酸代谢
Phenylalanine metabolism		 2 Arahy.21PEZK, Arahy.SGZ2CH

图4

AhGLK1和AhHDA1共同富集peak分布"

图5

AhGLK1和AhHDA1富集基序序列"

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