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作物学报

作物学报 ›› 2024, Vol. 50 ›› Issue (7): 1658-1668.doi: 10.3724/SP.J.1006.2024.34196

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

通过CRISPR/Cas9建立豌豆基因组大片段敲除体系

黄淑贤1(), 刘荣1, 李冠2, 疏琴1, 徐斐1, 宗绪晓1,*(), 杨涛1,*()   

  1. 1中国农业科学院作物科学研究所, 北京 100081
    2山东省农业科学院农作物种质资源研究所, 山东济南 250100
  • 收稿日期:2023-11-19 接受日期:2024-01-31 出版日期:2024-07-12 网络出版日期:2024-06-15
  • 通讯作者: *杨涛, E-mail: yangtao02@caas.cn;宗绪晓, E-mail: zongxuxiao@caas.cn
  • 作者简介:E-mail: xianshuhuang@163.com
  • 基金资助:
    国家自然科学基金项目(32241042);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-08-G11);作物基因资源与育种全国重点实验项目

Establishment of large fragment knockout in pea genome by CRISPR/Cas9 technology

HUANG Shu-Xian1(), LIU Rong1, LI Guan2, SHU Qin1, XU Fei1, ZONG Xu-Xiao1,*(), YANG Tao1,*()   

  1. 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    2Institute of Crop Germplasm Resources, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China
  • Received:2023-11-19 Accepted:2024-01-31 Published:2024-07-12 Published online:2024-06-15
  • Contact: *E-mail: yangtao02@caas.cn; E-mail: zongxuxiao@caas.cn
  • Supported by:
    National Natural Science Foundation of China(32241042);China Agriculture Research System of MOF and MARA(CARS-08-G11);State Key Laboratory of Crop Gene Resources and Breeding

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摘要：

豌豆是世界上主要食用豆类作物之一, 因其丰富的营养价值和良好的生态价值而受到越来越多的重视。CRISPR/Cas9作为一种生物育种的新型技术手段, 已广泛应用于各种作物的品种改良中, 但在豌豆中的应用报道非常少见。本研究以“中豌6号”品种为试验材料, 采用CRISPR/Cas9技术手段成功地实现了豌豆基因组中Psat01G0240600-T1、Psat03G0303300-T1和Psat03G0304700-T1基因的大片段缺失, 其大片段敲除效率分别是24.1%、13.0%和3.6%。进一步对结果分析发现, 不同靶点的编辑效率相差较大, 并且大片段缺失的效率取决于2个靶点中较低编辑效率的靶点。本研究利用发状根体系探索了CRISPR/Cas9在豌豆中的应用, 首次在豌豆中实现了大片段敲除, 对于豌豆的研究具有重要意义。

关键词: 豌豆, CRISPR/Cas9, 发状根, 大片段敲除

Abstract:

Pea is one of the main food legumes in the world and has been paid more and more attention due to its rich nutritional value and good ecological value. CRISPR/Cas9 as a new biology breeding tool has been widely used in many crops, however, its usage in pea is very limited. In this study, we successfully achieved large fragment deletion of the Psat01G0240600-T1, Psat03G0303300-T1, and Psat03G0304700-T1 genes in “Zhongwan 6” variety by CRISPR/Cas9 technology. The large fragment knockout ratio was 24.1%, 13.0%, and 3.6%, respectively. Further analysis revealed that there were significant differences in editing efficiency among different target sites and large fragment deletion depend on the specific target site with the lower editing efficiency between the two target sites. In this study, we explored the application of CRISPR/Cas9 by using the hairy root system in pea and achieved large fragment knockout for the first time in pea, which is of great significant for the research of peas.

Key words: pea, CRISPR/Cas9, hairy root, large fragment knockout

表1

检验基因序列引物"

引物名称Primer name 引物序列Primer sequences (5′-3′)
Psat01G0240600-T1-FP ATGTCAAGTGGTAGTAGAGACCCTC
Psat01G0240600-T1-RP TCATCGGCATAACCTTCTTCCACC
Psat03G0303300-T1-1-FP ATGCCTAGGAATATGGTCGATCCTC
Psat03G0303300-T1-1-RP GAGAGTGCCTATCTTGACAC
Psat03G0303300-T1-2-FP CGGTGAAAACACTCACGTGTATGTG
Psat03G0303300-T1-2-RP TTAGCATCTCCTTCCACCGCAGCCG
Psat03G0304700-T1-FP ATGGCAGGTAGTAGCAGGAATCCTC
Psat03G0304700-T1-RP TTATCTAAATGTTCTTCCACCAGAGCC

表2

载体构建引物"

引物名称Primer name 引物序列Primer sequence (5′-3′)
CmYLCV FP TGCTCTTCGCGCTGGCAGACATACTGTCCCAC
Psat01G0240600-T1-gRNA1 RP TCGTCTCCAACCCTTGGTTGCTGCCTATACGGCAGTGAACCTG
Psat01G0240600-T1-gRNA1 FP TCGTCTCAGGTTATTGTTGGGTTTTAGAGCTAGAAATAGC
Psat01G0240600-T1-RNA2 RP TCGTCTCACAGGAATATCAGCTGCCTATACGGCAGTGAAC
Psat01G0240600-T1-RNA2 FP TCGTCTCACCTGCAACTACTGTTTTAGAGCTAGAAATAGC
oCsy-E RP TGCTCTTCTGACCTGCCTATACGGCAGTGAAC
Psat03G0303300-T1-gRNA1 RP TCGTCTCCAACCAACTGAGACTGCCTATACGGCAGTGAACCTG
Psat03G0303300-T1-gRNA1 FP TCGTCTCAGGTTAACCGTCCGTTTTAGAGCTAGAAATAGC
Psat03G0303300-T1-gRNA2 RP TCGTCTCATTGACAGTTGAACTGCCTATACGGCAGTGAAC
Psat03G0303300-T1-gRNA2 FP TCGTCTCATCAAAGAGAGCGGTTTTAGAGCTAGAAATAGC
Psat03G0304700-T1-gRNA1 RP TCGTCTCAGGTGACTCGGAGCTGCCTATACGGCAGTGAAC
Psat03G0304700-T1-gRNA1 FP TCGTCTCACACCTATGGTAGGTTTTAGAGCTAGAAATAGC
Psat03G0304700-T1-gRNA2 RP TCGTCTCAGTCGTGGCCTTTCTGCCTATACGGCAGTGAAC
Psat03G0304700-T1-gRNA2 FP TCGTCTCACGACCCACCTCAGTTTTAGAGCTAGAAATAGC
Check FP CTAGAAGTAGTCAAGGCGGC
MBF GTAAAACGACGGCCAGT

表3

Hi-TOM测序引物"

引物名称Primer name 引物序列Primer sequence (5′-3′)
Psat01G0240600-T1-1-Hi-FP GGAGTGAGTACGGTGTGCGGCTGTGAATTCAAACCTTCT
Psat01G0240600-T1-1-Hi-RP GAGTTGGATGCTGGATGGGCATCTGGATCCACCATGATC
Psat01G0240600-T1-2-Hi-FP GGAGTGAGTACGGTGTGCGAAAATGCTATGACTATG
Psat01G0240600-T1-2-Hi-RP GAGTTGGATGCTGGATGGCAACATCATCATCACAAGC
Psat03G0303300-T1-1-Hi-FP GGAGTGAGTACGGTGTGCCTGTATCTTTGAGTGTTG
Psat03G0303300-T1-1-Hi-RP GAGTTGGATGCTGGATGGCGTTACTAGGGCTAGGTG
Psat03G0303300-T1-2-Hi-FP GGAGTGAGTACGGTGTGCGGGAAAGAGGCAGTGTTT
Psat03G0303300-T1-2-Hi-RP GAGTTGGATGCTGGATGGTTAGCATCTCCTTCCACC
Psat03G0304700-T1-1-Hi-FP GGAGTGAGTACGGTGTGCAGGAATCCTCTCGCTGTTGG
Psat03G0304700-T1-1-Hi-RP GAGTTGGATGCTGGATGGGATCGTTTCCACCAACATTC
Psat03G0304700-T1-2-Hi-FP GGAGTGAGTACGGTGTGCGGTGACTGATATTCCAGC
Psat03G0304700-T1-2-Hi-RP GAGTTGGATGCTGGATGGTTCTGTCGCCATCCTGGA

表4

大片段缺失检测引物"

引物名称Primer name 引物序列Primer sequence (5′-3′)
Psat01G0240600-T1dpdFP TGGTCCAACTTACTTGCCTTGA
Psat01G0240600-T1dpdRP GCAGTGATTCCCAATTGAGTGTA
Psat03G0303300-T1dpdFP AGACCAAACGAGGTACGGTT
Psat03G0303300-T1dpdRP TGCAATGAACTAACCCCCGC
Psat03G0304700-T1dpdFP GTCACGGACGTGAGCAAAACGACATGG
Psat03G0304700-T1dpdRP ACGACACACACATGGATAAACACTGC

图1

豌豆FT基因家族蛋白保守基序"

图2

靶点的选择 绿色方框: 外显子; 黑色实线: 内含子; 蓝色字母: 靶点; 红色字母: PAM。"

图3

编辑载体的示意图 绿色方框: CmYLCV启动子; 蓝色方框: 特异性核糖核酸内切酶Csy4; 灰色方框: gRNA; 橙色方框: 35S终止子。"

图4

3个基因单个靶点的编辑效率"

图5

靶点各种突变情况分析 A: 6个靶点的突变情况分析, 灰色柱子为缺失突变类型, 白色柱子为其他突变类型; B: 6个靶点缺失类型及其效率; C: 6个靶点缺失类型的平均效率。"

表5

3个基因大片段缺失效率"

基因 ID
Gene ID		 鉴定的样品数量
Number of samples
identified		 大片段缺失的数量
Number of large
fragment deletion		 突变频率
Mutation frequency (%)		 片段缺失的大小
Size of the missing
fragments (bp)
Psat01G0240600-T1 87 21 24.1 161-967
Psat03G0303300-T1 77 10 13.0 1642-1673
Psat03G0304700-T1 84 3 3.6 228-809

附图1

编辑基因大片段缺失电泳图 Psat01G0240600-T1、Psat03G0303300-T1和Psat03G0304700-T1的琼脂糖凝胶电泳示意图, 蓝色箭头指向的条带表示测序出现大片段的样品。"

附图2

Psat01G0240600-T1、Psat03G0303300-T1和Psat03G0304700-T1的大片段缺失样品PCR测序结果 蓝色突出显示代表靶点序列, 红色突出显示代表PAM位点, 橙色突出显示代表插入序列, 绿色突出显示代表碱基突变, “—//—”代表碱基省略, “------//------”代表片段缺失。"

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