生物技术通报 ›› 2022, Vol. 38 ›› Issue (11): 210-219.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0173
赵燚1(), 雷建峰2, 刘敏1, 胡子曜1, 代培红1, 刘超1, 李月1, 刘晓东1()
收稿日期:
2022-02-15
出版日期:
2022-11-26
发布日期:
2022-12-01
作者简介:
赵燚,男,硕士研究生,研究方向:作物逆境分子生物学;E-mail:基金资助:
ZHAO Yi1(), LEI Jian-feng2, LIU Min1, HU Zi-yao1, DAI Pei-hong1, LIU Chao1, LI Yue1, LIU Xiao-dong1()
Received:
2022-02-15
Published:
2022-11-26
Online:
2022-12-01
摘要:
探索CLCrV介导的VIGE体系的承载力。通过CLCrV介导的sgRNA投送系统将基因编辑载体注射过表达Cas9的棉花,提取棉花基因组DNA,利用PCR/RE的方法筛选有效的sgRNA。然后利用同样的方法来检测CLCrV介导的VIGE体系的承载力。基于棉花GhBsr-k1基因成功构建了6个基因编辑载体,其中2个基因编辑载体实现了对棉花GhBsr-k1基因的靶向编辑;将完整的基因编辑载体元件构建到CLCrV病毒载体上并没有检测到棉花细胞中发生基因编辑。针对棉花GhBsr-k1基因,筛选到两个有效的sgRNA。但承载整个CRISPR/Cas9系统的CLCrV载体难以在棉花叶片中实现有效的基因编辑。
赵燚, 雷建峰, 刘敏, 胡子曜, 代培红, 刘超, 李月, 刘晓东. CLCrV介导的VIGE体系承载力的研究[J]. 生物技术通报, 2022, 38(11): 210-219.
ZHAO Yi, LEI Jian-feng, LIU Min, HU Zi-yao, DAI Pei-hong, LIU Chao, LI Yue, LIU Xiao-dong. Research on the Carrying Capacity of CLCrV-mediated VIGE System[J]. Biotechnology Bulletin, 2022, 38(11): 210-219.
引物Primer | 序列Sequence(5'-3') | 用途Application |
---|---|---|
GhBsr-k1-sgRNA1F GhBsr-k1-sgRNA1R | GATTGCAGCGTTTCTACCATGGAT AAACATCCATGGTAGAAACGCTGC | 构建GhBsr-k1-sgRNA1 Construction of GhBsr-k1-sgRNA1 |
GhBsr-k1-sgRNA2F GhBsr-k1-sgRNA2R | GATTGGTGTACTTGGTAAGATCTT AAACAAGATCTTACCAAGTACACC | 构建GhBsr-k1-sgRNA2 Construction of GhBsr-k1-sgRNA2 |
GhBsr-k1-sgRNA3F GhBsr-k1-sgRNA3R | GATTGCGCTGGCATTACCATGGGC AAACGCCCATGGTAATGCCAGCGC | 构建GhBsr-k1-sgRNA3 Construction of GhBsr-k1-sgRNA3 |
GhBsr-k1-sgRNA4F GhBsr-k1-sgRNA4R | GATTGTTAATCAGGGGACTGCAGT AAACACTGCAGTCCCCTGATTAAC | 构建GhBsr-k1-sgRNA4 Construction of GhBsr-k1-sgRNA4 |
GhBsr-k1-sgRNA5F GhBsr-k1-sgRNA5R | GATTGCTGGACCGGATTTAGCAAT AAACATTGCTAAATCCGGTCCAGC | 构建GhBsr-k1-sgRNA5 Construction of GhBsr-k1-sgRNA5 |
GhBsr-k1-sgRNA6F GhBsr-k1-sgRNA6R | GATTGGTCGTGCCTACGCTGCAGA AAACTCTGCAGCGTAGGCACGACC | 构建GhBsr-k1-sgRNA6 Construction of GhBsr-k1-sgRNA6 |
M-GhBsr-k1-F1 M-GhBsr-k1-R1 | CCCTTCAGTCTTTTAATGGC ACTCCATTCTGAAACCCAAG | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
M-GhBsr-k1-F2 M-GhBsr-k1-R2 | GTCACAGGATGCACGTAAAG AAAGCCACCCAGAACTCAGA | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
M-GhBsr-k1-F3 M-GhBsr-k1-R3 | GAAGTGTGTGCTTAAACTTGTC ACTGCTCTGGTAGGATATGG | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
M-GhBsr-k1-F4 M-GhBsr-k1-R4 | GTTTAGAATACAATGCAGAAACTTTC GACAAGTTTAAGCACACACTTC | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
M-GhBsr-k1-F5 M-GhBsr-k1-R5 | GGATATCTGCAGCTGTGTCT ACTCCATTCTGAAACCCAAG | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
M-GhBsr-k1-F6 M-GhBsr-k1-R6 | ACCATAATGCCCGATACTTGC ATAGAAGTGACACCTGAATCATC | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
CLCrVA-F CLCrVA-R | GCTGCTTGTATGTTTGGGTG CAAGGGAGAGTAGTTGGCA | CLCrV-A病毒检测 CLCrV-A virus detection |
表1 引物及用途
Table 1 Primer and application
引物Primer | 序列Sequence(5'-3') | 用途Application |
---|---|---|
GhBsr-k1-sgRNA1F GhBsr-k1-sgRNA1R | GATTGCAGCGTTTCTACCATGGAT AAACATCCATGGTAGAAACGCTGC | 构建GhBsr-k1-sgRNA1 Construction of GhBsr-k1-sgRNA1 |
GhBsr-k1-sgRNA2F GhBsr-k1-sgRNA2R | GATTGGTGTACTTGGTAAGATCTT AAACAAGATCTTACCAAGTACACC | 构建GhBsr-k1-sgRNA2 Construction of GhBsr-k1-sgRNA2 |
GhBsr-k1-sgRNA3F GhBsr-k1-sgRNA3R | GATTGCGCTGGCATTACCATGGGC AAACGCCCATGGTAATGCCAGCGC | 构建GhBsr-k1-sgRNA3 Construction of GhBsr-k1-sgRNA3 |
GhBsr-k1-sgRNA4F GhBsr-k1-sgRNA4R | GATTGTTAATCAGGGGACTGCAGT AAACACTGCAGTCCCCTGATTAAC | 构建GhBsr-k1-sgRNA4 Construction of GhBsr-k1-sgRNA4 |
GhBsr-k1-sgRNA5F GhBsr-k1-sgRNA5R | GATTGCTGGACCGGATTTAGCAAT AAACATTGCTAAATCCGGTCCAGC | 构建GhBsr-k1-sgRNA5 Construction of GhBsr-k1-sgRNA5 |
GhBsr-k1-sgRNA6F GhBsr-k1-sgRNA6R | GATTGGTCGTGCCTACGCTGCAGA AAACTCTGCAGCGTAGGCACGACC | 构建GhBsr-k1-sgRNA6 Construction of GhBsr-k1-sgRNA6 |
M-GhBsr-k1-F1 M-GhBsr-k1-R1 | CCCTTCAGTCTTTTAATGGC ACTCCATTCTGAAACCCAAG | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
M-GhBsr-k1-F2 M-GhBsr-k1-R2 | GTCACAGGATGCACGTAAAG AAAGCCACCCAGAACTCAGA | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
M-GhBsr-k1-F3 M-GhBsr-k1-R3 | GAAGTGTGTGCTTAAACTTGTC ACTGCTCTGGTAGGATATGG | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
M-GhBsr-k1-F4 M-GhBsr-k1-R4 | GTTTAGAATACAATGCAGAAACTTTC GACAAGTTTAAGCACACACTTC | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
M-GhBsr-k1-F5 M-GhBsr-k1-R5 | GGATATCTGCAGCTGTGTCT ACTCCATTCTGAAACCCAAG | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
M-GhBsr-k1-F6 M-GhBsr-k1-R6 | ACCATAATGCCCGATACTTGC ATAGAAGTGACACCTGAATCATC | PCR扩增涵盖GhBsr-k1基因A/D亚组靶位点区域 PCR amplification covers the target site region of GhBsr-k1 gene A/D subgroup |
CLCrVA-F CLCrVA-R | GCTGCTTGTATGTTTGGGTG CAAGGGAGAGTAGTTGGCA | CLCrV-A病毒检测 CLCrV-A virus detection |
图1 VIGE编辑载体构建 A:1-6为B-Zero-AtU6-26∷GhBsr-k1-sgRNA(1-6)克隆载体酶切结果;B:1-6为VIGE编辑载体CLCrV-AtU6-26∷GhBsr-k1-sgRNA(1-6)酶切结果。M:Trans2K Plus II DNA marker
Fig. 1 Construction of VIGE editing gene vector A:1-6 are B-Zero-AtU6-26∷GhBsr-k1-sgRNA(1-6)cloning vector digestion. B:1-6 are VIGE editing vector CLCrV-AtU6-26∷GhBsr-k1-sgRNA(1-6)digestion. M:Trans2K Plus II DNA marker
图2 基因编辑载体筛选和突变检测 A:WT为对照载体基因组PCR扩增产物Nco I酶切消化后的结果,泳道1-5分别瞬时表达CLCrV-AtU6-26∷GhBsr-k1-sgRNA1基因编辑载体的10个单株混样基因组PCR产物Nco I酶切消化后的结果;B:瞬时表达CLCrV-AtU6-26∷GhBsr-k1-sgRNA1基因编辑载体未消化的PCR产物缺乏Nco I酶切位点(存在突变),克隆和测序分析后得到的突变类型;C:CLCrV-AtU6-26∷GhBsr-k1-sgRNA1的突变类型测序峰图;D:WT为对照载体基因组PCR产物Pst I酶切消化后的结果,泳道1为瞬时表达CLCrV-AtU6-26∷GhBsr-k1-sgRNA6基因编辑载体的10个单株混样基因组PCR产物Pst I酶切消化后的结果;E:瞬时表达CLCrV-AtU6-26∷GhBsr-k1-sgRNA6基因编辑载体未消化的PCR扩增产物缺乏Pst I酶切位点(存在突变),克隆和测序分析后得到的突变类型;F:CLCrV-AtU6-26∷GhBsr-k1-sgRNA6的突变类型峰图。红色下划线表示靶序列上对应的酶切位点;“M”表示突变类型;“+”碱基插入用蓝色大写字母表示;“-”碱基缺失用红色破折号表示;黄色突出表示GhBsr-k1基因A/D亚组差异碱基。M:Trans2K Plus II DNA marker
Fig. 2 Gene editing vector screening and mutation detection A:WT:PCR/RE assay of negative control genome before and after the Nco I enzyme digestion,1-5:PCR/RE assay of CLCrV-AtU6-26∷GhBsr-k1-sgRNA1 of 10 single plant sample after Nco I enzyme digestion. B:CLCrV-AtU6-26∷GhBsr-k1-sgRNA1 editing vector of the undigested PCR products lacked Nco I sites(due to the presence of a mutation)that were subsequently purified,cloned,and analyzed by sequencing. C:CLCrV-AtU6-26∷GhBsr-k1-sgRNA1 mutation type sequencing peak map. D:WT:PCR/RE assay of negative control genome before and after the Pst I enzyme digestion. 1:PCR/RE assay of CLCrV-AtU6-26∷GhBsr-k1-sgRNA6 of 10 single plant sample after Pst I enzyme digestion. E:CLCrV-AtU6-26∷GhBsr-k1-sgRNA6 editing vector of the undigested PCR products lacked Pst I sites(due to the presence of a mutation)that were subsequently purified,cloned,and analyzed by sequencing. F:CLCrV-AtU6-26∷GhBsr-k1-sgRNA6 mutation type sequencing peak map. “M” indicates the mutation sequence;“+” are denoted with red capital letters;“-” are shown as red dashes;yellow highlighted the differential bases in A/D subgroup of GhBsr-k1 gene. M:Trans2K Plus II DNA marker
图3 sgRNA的二级结构预测 A:sgRNA1的二级结构;B:sgRNA2的二级结构;C:sgRNA3的二级结构;D:sgRNA4的二级结构;E:sgRNA5的二级结构;F:sgRNA6的二级结构
Fig. 3 Prediction of secondary structure of sgRNA A:The secondary structure of sgRNA1. B:The secondary structure of sgRNA2. C:The secondary structure of sgRNA3. D:The secondary structure of sgRNA4. E:The secondary structure of sgRNA5. F:The secondary structure of sgRNA6
图4 CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1基因编辑载体构建和突变检测 A:CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1基因编辑载体示意图;B:泳道1为CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1基因编辑载体三酶切结果;C:新陆早26和YZ-1瞬时表达CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1单株样品基因组PCR扩增产物Nco I酶切消化后的结果;WT为对照载体基因组PCR扩增产物Nco I酶切消化后的结果;D:新陆早26和YZ-1瞬时表达CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1单株混合样品基因组PCR扩增产物Nco I酶切消化后的结果;WT为对照载体基因组PCR扩增产物Nco I酶切消化后的结果;E:新陆早26和YZ-1单株样品CLCrV-A病毒检测结果,GhBsr-k1为内参基因;N为阴性对照;P为阳性对照。M:Trans2K Plus II DNA marker
Fig. 4 CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1 editing vector construction and mutation detection A:CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1 gene editing vector diagram. B:lane 1:CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1 gene editing vector was digested with three enzymes. C:PCR/RE assay of CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1 of Xinluzao 26 and YZ-1 single plant sample after Nco I enzyme digestion. WT:PCR/RE assay of negative control genome before and after the Nco I enzyme digestion. D:PCR/RE assay of CLCrV-35S∷Cas9-AtU6-26∷GhBsr-k1-sgRNA1 of Xinluzao26 and YZ-1 mix single plant sample after Nco I enzyme digestion. WT:PCR/RE assay of negative control genome before and after the Nco I enzyme digestion.E:Detection of CLCrV-A virus in individual samples of Xinluzao 26 and YZ-1,GhBsr-k1 is internal reference gene. N indicates negative control,and P indicates positive control. M:Trans2K Plus II DNA marker
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