CLCrV介导的VIGE体系承载力的研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0173

摘要/Abstract

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。

关键词: 棉花叶皱缩病毒, VIGE, 棉花, GhBsr-k1

Abstract:

This work aims to explore the carrying capacity of CLCrV-mediated VIGE system. Through CLCrV-mediated sgRNA delivery system，the gene editing vector was injected into cotton overexpressing Cas9，and the genomic DNA of cotton was extracted. The effective sgRNA was screened by PCR/RE method. Then the same method was used to detect the carrying capacity of the CLCrV-mediated VIGE system. Six gene-editing vectors were successfully constructed based on the cotton GhBsr-k1 gene，and two of them achieved targeted editing of cotton GhBsr-k1 gene. The complete gene editing vector components were constructed on the CLCrV vector and no gene editing was detected in cotton cells. Two effective sgRNAs were screened for the GhBsr-k1 gene in cotton. However，CLCrV vectors carrying the entire CRISPR/Cas9 system are difficult to achieve efficient gene editing in cotton leaves.

Key words: cotton leaf crumple virus, VIGE, cotton, GhBsr-k1

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.

图/表 5

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

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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