CRISPR/Cas9编辑MeHNL基因创制低生氰糖苷木薯

doi:10.13560/j.cnki.biotech.bull.1985.2024-0520

生物技术通报 ›› 2024, Vol. 40 ›› Issue (9): 11-19.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0520

• 薯类作物生物技术专题（专题主编：徐建飞，尚轶） • 上一篇下一篇

CRISPR/Cas9编辑MeHNL基因创制低生氰糖苷木薯

童玮婧¹^,²(), 罗数¹, 陆新露¹, 沈建福³, 陆柏益³, 李开绵⁴, 马秋香¹(), 张鹏¹^,²()

1.中国科学院分子植物科学卓越创新中心，上海 200032
2.中国科学院大学，北京 100049
3.浙江大学生物系统工程与食品科学学院，杭州 310058
4.中国热带农业科学院热带作物品种资源研究所，海口 571101

收稿日期:2024-05-30 出版日期:2024-09-26 发布日期:2024-10-12
通讯作者: 马秋香，女，副研究员，研究方向：薯类分子育种；E-mail: qxma@cemps.ac.cn；
张鹏，男，研究员，研究方向：薯类分子育种；E-mail: zhangpeng@cemps.ac.cn
作者简介:童玮婧，女，博士研究生，研究方向：薯类分子育种；E-mail: tongweijing@cemps.ac.cn
基金资助:
国家重点研发计划(2023YFD1600605-4);热带作物生物育种全国重点实验室2023年度开放课题项目(1630052024001);农业农村部国家现代农业产业技术体系项目(CARS-11)

CRISPR/Cas9 Editing MeHNL Gene to Generate Cassava Plants with Low Cyanogenic Glycoside

TONG Wei-jing¹^,²(), LUO Shu¹, LU Xin-lu¹, SHEN Jian-fu³, LU Bai-yi³, LI Kai-mian⁴, MA Qiu-xiang¹(), ZHANG Peng¹^,²()

1. CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Science, Shanghai 200032
2. University of Chinese Academy of Sciences, Beijing 100049
3. College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058
4. Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101

Received:2024-05-30 Published:2024-09-26 Online:2024-10-12

摘要/Abstract

摘要：

【目的】木薯（Manihot esculenta Crantz）中含有潜在毒性的生氰糖苷，其食用安全性受到影响且导致加工成本增加。因此，利用生物技术开展低生氰糖苷木薯的培育具有重要意义。【方法】利用CRISPR/Cas9技术对木薯醇氰酶基因MeHNL进行了编辑。该基因编码催化生氰糖苷分解的α-羟基腈裂解酶，编辑靶点位于第1个外显子上，通过农杆菌介导的稳定转化获得了27株阳性植株。【结果】测序分析显示，其中26个株系被编辑，编辑效率高达96.3%。编辑类型主要包括碱基的插入和缺失，少数为碱基替换和大片段缺失。氰化物检测试剂盒染色和HPLC测定分析表明，编辑株系中的氢氰酸和生氰糖苷含量均显著降低。与非编辑植株相比，编辑植株的叶片细长，暗示了MeHNL可能对木薯的生长发育产生影响。【结论】利用CRISPR/Cas9技术获得了低氰化物的木薯种质，为开展生氰糖苷代谢影响木薯生长发育的研究提供了材料。

关键词: 木薯, 基因编辑, 醇氰酶, 低氰化物, 生氰糖苷

Abstract:

【Objective】 Because cassava（Manihot esculenta Crantz）contains potentially toxic cyanogenic glucosides, its food safety is affected, which leads to the increase of processing costs. It is of great significance to cultivate cassava with low cyanogenic glycosides by biotechnology.【Method】 CRISPR/Cas9 technology was used to edit the α-hydroxynitrile lyase gene MeHNL. This gene encodes α-hydroxynitrile lyase that catalyzes the decomposition of cyanogenic glycosides. The editing target is located on its first exon, and 27 positive plants were obtained through Agrobacterium-mediated stable transformation.【Result】Sequencing analysis indicated successful editing in 26 out of 27 transgenic plants, and a high editing efficiency was achieved by 96.3%. The editing types predominantly comprised base insertions and deletions, alongside minor base substitutions and large fragment deletions. HCN colorimetric kit and HPLC analysis confirmed a significant reduction in hydrocyanic acid and cyanogenic glycosides in the mutant lines. Additionally, transgenic plants demonstrated slender leaves compared to non-transgenic counterparts, implicating MeHNL's impact on plant growth and development.【Conclusion】The cassava germplasm with low cyanide is obtained by CRISPR/Cas9 technology, which provides materials for exploring the study of cyanogenic glycoside metabolism on cassava growth and development.

Key words: cassava, gene editing, HNL, low cyanide, cyanogenic glycosides

童玮婧, 罗数, 陆新露, 沈建福, 陆柏益, 李开绵, 马秋香, 张鹏. CRISPR/Cas9编辑MeHNL基因创制低生氰糖苷木薯[J]. 生物技术通报, 2024, 40(9): 11-19.

TONG Wei-jing, LUO Shu, LU Xin-lu, SHEN Jian-fu, LU Bai-yi, LI Kai-mian, MA Qiu-xiang, ZHANG Peng. CRISPR/Cas9 Editing MeHNL Gene to Generate Cassava Plants with Low Cyanogenic Glycoside[J]. Biotechnology Bulletin, 2024, 40(9): 11-19.

图/表 7

参考文献 35

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引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Use
target-Fp	GATTGCTTGGAGAAACTCCCTCAAG	Target gene
target-Rp	AAACCTTGAGGGAGTTTCTCCAAGC	Target gene
M13-Fp	TGTAAAACGACGGCCAGT	Identification of positive clones
Hpt II-Fp	TTCTACACAGCCATCGGTCC	Identification of positive transgenic plants
Hpt II-Rp	CCCATGTGTATCACTGGCAA	Identification of positive transgenic plants
HNLgRNA-Fp	GCACCACTCACAGAAAAATCCAAAG	PCR amplification of target region
HNLgRNA-Rp	ATGTGGCTTAATTAGAATACCGTCT	PCR amplification of target region

引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Use
target-Fp	GATTGCTTGGAGAAACTCCCTCAAG	Target gene
target-Rp	AAACCTTGAGGGAGTTTCTCCAAGC	Target gene
M13-Fp	TGTAAAACGACGGCCAGT	Identification of positive clones
Hpt II-Fp	TTCTACACAGCCATCGGTCC	Identification of positive transgenic plants
Hpt II-Rp	CCCATGTGTATCACTGGCAA	Identification of positive transgenic plants
HNLgRNA-Fp	GCACCACTCACAGAAAAATCCAAAG	PCR amplification of target region
HNLgRNA-Rp	ATGTGGCTTAATTAGAATACCGTCT	PCR amplification of target region

Type	MeHNL-sgRNA target sites	Indel	Lines
WT	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA		WT
1	CTTGG - - - - - - - - - - - - - - - -- - - GGAAAAGGTCATCA CTTGG - - - - - - - - - - - - - - - -- - - GGAAAAGGTCATCA	-16	L1
2	CTTGGAGAAACTCCCTCAAGGGGAAAAAGGTCATCA CTTGGAGAAACTCCC - -AAGGAGAAGAGGTCATCA	+1, -2, 2	L2, L7
3	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCAAAAAGGGGAAAAGGTCATCA	+1, +3	L3, L11
4	CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA	+1	L4
5	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACT- - - - - - - - - - - - - - - - - - - - GTCATCA	+1, -16	L5
6	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCAAAAAGGGGAAAAGGTCATCA	+3	L6, L9, L16
7	CTTGGAGAAACTCCCTCGAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCC - - - AGGGGAAAAGGTCATCA	+1, -3	L10
8	CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCCAAAGGGGAAAAGGTCATCA	+1, +2	L12, L18, L31
9	CTTGGAGAAACTCCCTC - - - - - - - - - - - - - - - - CATCA CTTGGAGAAACTCCCGTCAAGGGGAAAAGGTCATCA	-13, +1	L13
10	CTTGGAGAAACTCC- - - - - - -GGCAAAAGGTCATCA CTTGGAGAAACTAGCTCCAAGGGGAAAAGGTCATCA	-7, +1, +1, 2	L14
11	CTTGGAGAAACTCCCTC- AGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTC- AGGGGAAAAGGTCATCA	-1	L15
12	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCC- -AATAAGGGGAAAAGGTCATCA	-2, +3	L17
13	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCA CTTGGAGAAACTCCC- -AAAAGGGGAAAAGGTCATCA	+1, -2, +2	L19, L32
14	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCT- -TCA AAGGGGAAAAGGTCATCA	+1, -2, +3	L21
15	CTTGGAGAAACTCCCTCAAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCTGGAAGGGGAAAAGGTCATCA	+1, +3	L24
16	CTCTGAACCCTTATTGACTTTCTTGGAGAAACTCCCTCAAGGGG CTCTGAA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGGGG	-31	L25
17	CTTGGAGAAA - - - - - - - - AGGGGAAAAGGTCATCA CTTGGAGAAA - - - - - - - - AGGGGAAAAGGTCATCA	-8	L26
18	CTTGGAGAAACTCCCTCAAG - - - - - - - - - - - - - - - - - - TCATCA CTTGGAGAAA - - - - - - GGGGAAATGAAGGGGAAAAGGTCATCA	-9, -7, +9	L29

Type	MeHNL-sgRNA target sites	Indel	Lines
WT	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA		WT
1	CTTGG - - - - - - - - - - - - - - - -- - - GGAAAAGGTCATCA CTTGG - - - - - - - - - - - - - - - -- - - GGAAAAGGTCATCA	-16	L1
2	CTTGGAGAAACTCCCTCAAGGGGAAAAAGGTCATCA CTTGGAGAAACTCCC - -AAGGAGAAGAGGTCATCA	+1, -2, 2	L2, L7
3	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCAAAAAGGGGAAAAGGTCATCA	+1, +3	L3, L11
4	CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA	+1	L4
5	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACT- - - - - - - - - - - - - - - - - - - - GTCATCA	+1, -16	L5
6	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCAAAAAGGGGAAAAGGTCATCA	+3	L6, L9, L16
7	CTTGGAGAAACTCCCTCGAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCC - - - AGGGGAAAAGGTCATCA	+1, -3	L10
8	CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCCAAAGGGGAAAAGGTCATCA	+1, +2	L12, L18, L31
9	CTTGGAGAAACTCCCTC - - - - - - - - - - - - - - - - CATCA CTTGGAGAAACTCCCGTCAAGGGGAAAAGGTCATCA	-13, +1	L13
10	CTTGGAGAAACTCC- - - - - - -GGCAAAAGGTCATCA CTTGGAGAAACTAGCTCCAAGGGGAAAAGGTCATCA	-7, +1, +1, 2	L14
11	CTTGGAGAAACTCCCTC- AGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTC- AGGGGAAAAGGTCATCA	-1	L15
12	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCC- -AATAAGGGGAAAAGGTCATCA	-2, +3	L17
13	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCA CTTGGAGAAACTCCC- -AAAAGGGGAAAAGGTCATCA	+1, -2, +2	L19, L32
14	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCT- -TCA AAGGGGAAAAGGTCATCA	+1, -2, +3	L21
15	CTTGGAGAAACTCCCTCAAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCTGGAAGGGGAAAAGGTCATCA	+1, +3	L24
16	CTCTGAACCCTTATTGACTTTCTTGGAGAAACTCCCTCAAGGGG CTCTGAA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGGGG	-31	L25
17	CTTGGAGAAA - - - - - - - - AGGGGAAAAGGTCATCA CTTGGAGAAA - - - - - - - - AGGGGAAAAGGTCATCA	-8	L26
18	CTTGGAGAAACTCCCTCAAG - - - - - - - - - - - - - - - - - - TCATCA CTTGGAGAAA - - - - - - GGGGAAATGAAGGGGAAAAGGTCATCA	-9, -7, +9	L29

CRISPR/Cas9编辑MeHNL基因创制低生氰糖苷木薯

CRISPR/Cas9 Editing MeHNL Gene to Generate Cassava Plants with Low Cyanogenic Glycoside

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