组蛋白去乙酰化酶抑制剂（HDACi）对小麦基因编辑效率的影响及转录组学分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0645

摘要/Abstract

摘要：

主要研究组蛋白去乙酰化酶抑制剂（HDACi）与染色质状态以及CRISPR/Cas9的编辑效率之间的关系。利用不同浓度的尼克酰胺（0,2.5和5 mmol/L）和丁酸钠（0,5和10 mmol/L）对小麦幼苗处理7 d和14 d,结果显示丁酸钠处理会抑制幼苗的生长,而尼克酰胺对幼苗影响较小。对尼克酰胺处理的小麦幼苗进行转录组测序,发现了一些有利于促进染色质状态开放的基因：6个甲基转移酶合成通路基因。此外对未发生编辑的TaAGO4a基因编辑转基因小麦材料的T₂代进行尼克酰胺处理,结果显示5 mmol/L处理14 d时检测到1株3A和3B基因组均杂合编辑的植株,编辑效率从0提高到8.3%,其它处理组和对照组均没有检测到编辑。本研究证明尼克酰胺确实可以提高小麦基因编辑效率,为提高小麦基因编辑效率提供了一种新策略。

关键词: 小麦, 组蛋白去乙酰化酶抑制剂, 染色质状态, CRISPR/cas9, 转录组

Abstract:

This study focuses on the relationship between histone deacetylase inhibitors（HDACi）and chromatin status and CRISPR/Cas9 editing efficiency. Different concentrations of nicotinamide（0,2.5,and 5 mmol/L）and sodium butyrate（0,5,and 10 mmol/L）were used to treat wheat seedlings for 7 d and 14 d,and the results showed that sodium butyrate seriously inhibited wheat growth,however,nicotinamide had little effect on it. Moreover,transcriptome sequencing of wheat seedlings treated with nicotinamide revealed some genes which were conducive to promoting the opening of the chromatin state,including 6 methyltransferase synthesis pathway genes. In addition,the TaAGO4a transgenic wheat materials in T₂ generation that had not been edited were treated with nicotinamide,and results showed that a heterozygous mutant plant with mutations in the homologous alleles on 3A and 3B chromosomes was detected in the 5 mmol/L treatment for 14 d,and the editing efficiency increased from 0 to 8.3%;there was no mutant found in other treatments or control group. This study proves that nicotinamide can indeed improve the editing efficiency of wheat genes as a new strategy.

Key words: wheat, histone deacetylation inhibitor, chromatin state, CRISPR/Cas9, RNA-seq

代文双, 刘会云, 杜庆国, 邹枨, 王轲. 组蛋白去乙酰化酶抑制剂（HDACi）对小麦基因编辑效率的影响及转录组学分析[J]. 生物技术通报, 2021, 37(1): 2-14.

DAI Wen-shuang, LIU Hui-yun, DU Qing-guo, ZOU Cheng, WANG Ke. Effect of Histone Deacetylase Inhibitor（HDACi）on CRISPR Editing Efficiency of Wheat and Transcriptomics Analysis[J]. Biotechnology Bulletin, 2021, 37(1): 2-14.

图/表 12

表1 PCR引物序列表

引物名称	引物序列（5'-3'）
AGO4a486F	AGACTTTGCCTATGATGGTGA
AGO4a1404R	AAATCTTACCTTAGCCCAATCA
Ago4aA1629R	ACTTACATACAACAAATGGCGATATA
Ago4aB749F	GAAACGACAGTCCAGGAAATG
Ago4aD698F	GTTGCTCATGTAACTCTGTTGTTG

图1 不同的组蛋白乙酰化抑制剂对小麦幼苗生长的影响 A：丁酸钠处理14 d小麦幼苗生长状况;B：尼克酰胺处理7 d小麦幼苗生长状况;C：尼克酰胺处理14 d小麦幼苗生长状况;D：尼克酰胺不同时间处理下幼苗株高统计

表2 测序数据质控与比对情况统计

样本	非冗余序列数	Q20/%	Q30/%	GC/%	比对率/%
ctrl7d_1	59882027	97.08	93.21	56.72	91.21
ctrl7d_2	46792049	97.12	93.43	56.84	91.71
t7d-2.5_1	51954951	96.74	92.69	57.80	90.85
t7d-2.5_2	46624524	96.50	92.18	57.68	90.57
t7d-5_1	43606982	96.87	92.91	57.30	91.48
t7d-5_2	44386513	97.08	93.08	57.24	91.89
ctrl14d _1	47377531	97.23	93.32	57.09	91.90
ctrl14d _2	62520474	97.15	93.33	58.19	92.52
t14d-2.5_1	44413018	96.83	92.58	57.21	91.76
t14d-2.5_2	65941651	96.97	92.94	57.65	91.87
t14d-5_1	48123569	96.92	93.16	58.24	90.87
t14d-5_2	50461326	97.23	93.39	58.17	92.49

图2 主成分分析图

图3 差异表达基因分析 A：样本差异表达基因（黑色）、上调表达基因（白色）和下调表达基因（灰色）直方图;B：S2（t7d-2.5）,S3（t7d-5）,S5（t14d-2.5）,S6（t14d-5）异表达基因韦恩图

图4 差异表达基因（DEGs）GO富集分析 A：7 d DEGs GO分析;B：14 d DEGs GO分析。其中regulated分组中“▲”表示表达上调,“●”表示表达下调

表3 主要GO trems统计表

处理时间/d	GO terms	基因数目	类别	调控方式
7	nicotianamine synthase activity	15	MF	down
7	nicotianamine biosynthetic process	15	BP	down
7	O-methyltransferase activity	6	MF	down
7	transaminase activity	6	MF	up
7	cell division	4	BP	up

图5 主要基因的Pathway分布图

图6 T1代转基因植株bar基因及突变类型检测情况 A：T1代转基因植株bar基因PCR检测。M：2 kb DNA marker;1：阳性对照（质粒）;2：阴性对照（水）;3-8：QA147-1;9-14：QA167-2;15-19：QA167-7。B：利用通用引物对T1代转基因植株中TaAGO4a基因的PCR-RE检测,M：2 kb DNA marker;1-4：QA147-1;5-8：QA167-2;9-12：QA167-7;13：受体对照Fielder;14：未酶切的PCR产物

图7 14 d T2代转基因植株bar基因及突变类型检测情况 A：T2代转基因植株bar基因PCR检测。M：2 kb DNA marker;1：阳性对照（质粒）;2：阴性对照（水）;3-8：QA147-1;9-15：QA167-2;16-22：QA167-7。B：利用通用引物对T2代转基因植株TaAGO4a基因的PCR-RE检测。M：2 kb DNA marker;1-6：14-CK;7-13：14-2.5;14-19：14-5;20：受体对照Fielder;21：未酶切的PCR产物

表4 各处理编辑情况统计

样本	尼克酰胺浓度/（mmol·L^-1）	处理数	检测植株数	Cas9阳性	编辑情况	编辑效率
7-CK	0	14	19	12	0	0
7-2.5	2.5	14	32	18	0	0
7-5	5	14	18	12	0	0
14-CK	0	14	19	12	0	0
14-2.5	2.5	14	32	18	0	0
14-5	5	14	18	12	1He	8.3%

图8 突变体材料编辑类型检测 A：利用A、B、D引物对T2代转基因植株TaAGO4a基因的PCR-RE检测。1-4：14-CK;5-8：14-2.5;9-13：14-5;14：受体对照Fielder;15：未酶切的PCR产物。M：2 kb DNA marker。B：编辑植株的测序结果

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