生物技术通报 ›› 2021, Vol. 37 ›› Issue (1): 2-14.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0645
收稿日期:
2020-05-26
出版日期:
2021-01-26
发布日期:
2021-01-15
作者简介:
代文双,女,硕士研究生,研究方向:生物信息的整合与模拟;E-mail: 基金资助:
DAI Wen-shuang(), LIU Hui-yun, DU Qing-guo, ZOU Cheng(), WANG Ke()
Received:
2020-05-26
Published:
2021-01-26
Online:
2021-01-15
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。
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.
引物名称 | 引物序列(5'-3') |
---|---|
AGO4a486F | AGACTTTGCCTATGATGGTGA |
AGO4a1404R | AAATCTTACCTTAGCCCAATCA |
Ago4aA1629R | ACTTACATACAACAAATGGCGATATA |
Ago4aB749F | GAAACGACAGTCCAGGAAATG |
Ago4aD698F | GTTGCTCATGTAACTCTGTTGTTG |
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 |
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 |
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 |
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 |
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
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% |
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% |
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