生物技术通报 ›› 2022, Vol. 38 ›› Issue (4): 97-105.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1344
• 作物品质遗传与改良专题(专题主编: 刘巧泉 教授) • 上一篇 下一篇
Olalekan Amoo(), 胡利民, 翟云孤, 范楚川(), 周永明
收稿日期:
2021-10-25
出版日期:
2022-04-26
发布日期:
2022-05-06
通讯作者:
范楚川,男,博士,教授,研究方向:油菜产量性状遗传解析和基因编辑技术在油菜中的应用;E-mail: fanchuchuan@mail.hzau.edu.cn作者简介:
Olalekan Amoo,男,硕士研究生,研究方向:油菜遗传育种;E-mail: olalekan@webmail.hzau.edu.cn基金资助:
Olalekan Amoo(), HU Li-min, ZHAI Yun-gu, FAN Chu-chuan(), ZHOU Yong-ming
Received:
2021-10-25
Published:
2022-04-26
Online:
2022-05-06
摘要:
植物的株型决定于其分枝方式,而改良分枝方式是提高作物产量的有效方式之一。目前关于油菜分枝调控的分子机理研究较少,缺乏用于株型改良的种质资源。本研究以油菜BRANCHED1(BRC1)为目的基因,利用CRISPR/Cas9基因编辑技术靶向突变油菜BRC1基因,通过农杆菌介导的遗传转化获得再生苗,PCR技术筛选出转基因阳性单株,再利用Hi-TOM方法进行靶点突变基因型的测序分析,证实基因编辑产生的定点突变可稳定遗传。最后对获得的突变体进行表型观察发现,BRC1的BnaC01g34090D和BnaA01g26700D的双拷贝纯合突变体表现出明显的多分枝表型,研究结果表明BnaBRC1参与油菜中的分枝和株型调控,为后续研究油菜的株型调控分子机理提供了重要研究材料。
Olalekan Amoo, 胡利民, 翟云孤, 范楚川, 周永明. 利用基因编辑技术研究BRANCHED1参与油菜分枝过程的调控[J]. 生物技术通报, 2022, 38(4): 97-105.
Olalekan Amoo, HU Li-min, ZHAI Yun-gu, FAN Chu-chuan, ZHOU Yong-ming. Regulation of Shoot Branching by BRANCHED1 in Brassica napus Based on Gene Editing Technology[J]. Biotechnology Bulletin, 2022, 38(4): 97-105.
引物 Primer | 序列 Sequence(5'-3') | 用途 Purpose |
---|---|---|
CBnaC01-F1 | GAGACCATAACCACACACAAC | Cloning BnaC01g34090D |
CBnaC01-F2 | TGTGGACAGTCGAGGATAGA | |
CBnaC01-R | TCCATGTAGACAGAGACGATTT | |
CBnaA01-F1 | GAGACCATAACCACACACAAC | Cloning BnaA01g26700D |
CBnaA01-F2 | TGTGGACAGTCGAGGATAGA | |
CBnaA01-R | GACGATTGCCCACAAAGTAAA | |
CBnaC05-F | CAGTACTACCACCACCATCAAT | Cloning BnaC05g51920D |
CBnaC05-R | TCGGGATCTGAGATAGCCTATAA | |
CBnaCnn-F | ACAACAACAACAGAAGATCTCTCAG | Cloning BnaCnng23770D |
CBnaCnn-R | CATGAGGTCTCTTGGTCTCTCC | |
CBnaA03-F1 | ACAACAGGTCTTTCAGTACTACC | Cloning BnaA03g34820D |
CBnaA03-F2 | CAAGGACCGGCGTTAGG | |
CBnaA03-R | CATGAGGTCTCTTGGTCTCTC | |
BnBRC1T1-F | gtcAAGCATCATGCAGGAACATG | BnaBRC1 target construction |
BnBRC1T1-R | aaacCATGTTCCTGCATGATGCT | |
BnBRC1T2-F | attGCAAAGGGTCAGGTCCTGAA | |
BnBRC1T2-R | aaacTTCAGGACCTGACCCTTTG | |
BnBRC1T3-F | gtcACGGAGAGATTCTCACTCCCA | |
BnBRC1T3-R | aaacTGGGAGTGAGAATCTCTCCG | |
BnBRC1T4-F | gtcATGGATATCTCCGTAACTCTC | |
BnBRC1T4-R | aaacGAGAGTTACGGAGATATCCA | |
BnBRC1T5-F | attGTCGCCATGTCGAGGACCTCT | |
BnBRC1T5-R | aaacAGAGGTCCTCGACATGGCGA | |
BnBRC1T6-F | attGAGGAGATGGTCCATGTCAG | |
BnBRC1T6-R | aaacCTGACATGGACCATCTCCT | |
PB-L | GCGCGCgGTctcGCTCGACTAGTATGG | Transgenic positive detection |
PB-R | GCGCGCggtctcTACCGACGCGTATCC | |
PSH 20A-1F | ggagtgagtacggtgtgcGAGACCATAACCACACACAAC | BnaBRC1 editing check |
PSH 20A-1R | gagttggatgctggatggCTCATTCTACGATCTCTTGTCCC | |
PSH 20A-2F | ggagtgagtacggtgtgcGGGACAAGAGATCGTAGAATGAG | |
PSH 20A-2R | gagttggatgctggatggATGTATACGTACGGGTTTGGG | |
PSH 20A-3F | ggagtgagtacggtgtgcACACACATAGAAGATTCCCAGAG | |
PSH 20A-3R | gagttggatgctggatggAGGCTGTTCGCGATCTTTAT | |
PSH 20A-4F | ggagtgagtacggtgtgcCCTTTTTCTCACTTCGAATCCG | |
PSH 20A-4R | gagttggatgctggatggTTGGTTTCTGAGGGCTCAAT | |
PSH 20B-1F | ggagtgagtacggtgtgcTCTTACAAGCACCTTCTTCTTTTTC | |
PSH 20B-1R | gagttggatgctggatggCAAGGGCTCAACGAGAGATT | |
PSH 20B-2F | ggagtgagtacggtgtgcTTGACCACCACCATCATCAG | |
PSH 20B-2R | gagttggatgctggatggGGGCTCAATGAGGTGAGATT | |
PSH 20B-CF | ggagtgagtacggtgtgcCGGCACAGCAAGATCAAAAC | |
PSH 20B-CR | gagttggatgctggatggGCTTGTGTGAGCAACCATTC |
表1 本研究中使用的引物
Table 1 Primers used in this study
引物 Primer | 序列 Sequence(5'-3') | 用途 Purpose |
---|---|---|
CBnaC01-F1 | GAGACCATAACCACACACAAC | Cloning BnaC01g34090D |
CBnaC01-F2 | TGTGGACAGTCGAGGATAGA | |
CBnaC01-R | TCCATGTAGACAGAGACGATTT | |
CBnaA01-F1 | GAGACCATAACCACACACAAC | Cloning BnaA01g26700D |
CBnaA01-F2 | TGTGGACAGTCGAGGATAGA | |
CBnaA01-R | GACGATTGCCCACAAAGTAAA | |
CBnaC05-F | CAGTACTACCACCACCATCAAT | Cloning BnaC05g51920D |
CBnaC05-R | TCGGGATCTGAGATAGCCTATAA | |
CBnaCnn-F | ACAACAACAACAGAAGATCTCTCAG | Cloning BnaCnng23770D |
CBnaCnn-R | CATGAGGTCTCTTGGTCTCTCC | |
CBnaA03-F1 | ACAACAGGTCTTTCAGTACTACC | Cloning BnaA03g34820D |
CBnaA03-F2 | CAAGGACCGGCGTTAGG | |
CBnaA03-R | CATGAGGTCTCTTGGTCTCTC | |
BnBRC1T1-F | gtcAAGCATCATGCAGGAACATG | BnaBRC1 target construction |
BnBRC1T1-R | aaacCATGTTCCTGCATGATGCT | |
BnBRC1T2-F | attGCAAAGGGTCAGGTCCTGAA | |
BnBRC1T2-R | aaacTTCAGGACCTGACCCTTTG | |
BnBRC1T3-F | gtcACGGAGAGATTCTCACTCCCA | |
BnBRC1T3-R | aaacTGGGAGTGAGAATCTCTCCG | |
BnBRC1T4-F | gtcATGGATATCTCCGTAACTCTC | |
BnBRC1T4-R | aaacGAGAGTTACGGAGATATCCA | |
BnBRC1T5-F | attGTCGCCATGTCGAGGACCTCT | |
BnBRC1T5-R | aaacAGAGGTCCTCGACATGGCGA | |
BnBRC1T6-F | attGAGGAGATGGTCCATGTCAG | |
BnBRC1T6-R | aaacCTGACATGGACCATCTCCT | |
PB-L | GCGCGCgGTctcGCTCGACTAGTATGG | Transgenic positive detection |
PB-R | GCGCGCggtctcTACCGACGCGTATCC | |
PSH 20A-1F | ggagtgagtacggtgtgcGAGACCATAACCACACACAAC | BnaBRC1 editing check |
PSH 20A-1R | gagttggatgctggatggCTCATTCTACGATCTCTTGTCCC | |
PSH 20A-2F | ggagtgagtacggtgtgcGGGACAAGAGATCGTAGAATGAG | |
PSH 20A-2R | gagttggatgctggatggATGTATACGTACGGGTTTGGG | |
PSH 20A-3F | ggagtgagtacggtgtgcACACACATAGAAGATTCCCAGAG | |
PSH 20A-3R | gagttggatgctggatggAGGCTGTTCGCGATCTTTAT | |
PSH 20A-4F | ggagtgagtacggtgtgcCCTTTTTCTCACTTCGAATCCG | |
PSH 20A-4R | gagttggatgctggatggTTGGTTTCTGAGGGCTCAAT | |
PSH 20B-1F | ggagtgagtacggtgtgcTCTTACAAGCACCTTCTTCTTTTTC | |
PSH 20B-1R | gagttggatgctggatggCAAGGGCTCAACGAGAGATT | |
PSH 20B-2F | ggagtgagtacggtgtgcTTGACCACCACCATCATCAG | |
PSH 20B-2R | gagttggatgctggatggGGGCTCAATGAGGTGAGATT | |
PSH 20B-CF | ggagtgagtacggtgtgcCGGCACAGCAAGATCAAAAC | |
PSH 20B-CR | gagttggatgctggatggGCTTGTGTGAGCAACCATTC |
图2 BnaBRC1基因的Motif分析及保守结构域分析 A:BRC1同源基因在甘蓝型油菜和拟南芥中的Motif分析;B:油菜和拟南芥中BRC1同源拷贝中保守结构域的预测
Fig.2 Motif analysis and conserved domain structures of BnaBRC1 gene A:Analysis of conserved motifs in the protein sequence of homologous copies of the BRC1 gene in B. napus,and A. thaliana. B:Prediction of the conserved domains in the homologous copies of BRC1 in B. napus and A. thaliana
图4 BnaBRC1基因结构与靶点序列及双元质粒载体示意图 a:BnaBRC1基因包括4个外显子,靶点(S1-S6)位置用黑色的竖线标注,方向用带箭头的水平黑线标注。靶点序列中PAM碱基都用红色的字体和下划线强调;b:双元载体SBRC1a和SBRC1b的载体示意图
Fig.4 BnaBRC1 gene structure with target sequences and schematics of binary plasmid vectors a:The BnaBRC1 gene structure includes 4 exons separated by three introns. The vertical line in the gene structure indicates the target site,and the arrow indicates the sgRNA direction. The target sequences are shown with the PAM highlighted in red and underlined. b:Schematic presentation of binary vector SBRC1a and SBRC1b.
图5 BnaBRC1突变体株系在T0代和T1代中S2和S3靶位点突变基因型 CRISPR/Cas9诱导的插入和缺失都用红色的字体或实线表示;PAM序列用红色的字体和下划线强调。“A”,“C”为野生型基因型,“a”,“c”为突变基因型;“-”和“+”分别表示缺失或插入的碱基
Fig.5 Mutated alleles at the S2 and S3 target sites of BnaBRC1 mutants from T0 to T1 generation CRISPR/Cas9-induced insertions and deletions are indicated by red font and red hyphens respectively,while the PAM is underlined and highlighted in red. “A” and “C” are the wild type alleles of the target gene on A and C chromosome copy,and “a”and “c” are the mutated alleles of the target gene on A and C chromosome copy. “_” and “ +” indicate the deletion and insertion of bases,respectively
图6 BnaBRC1突变体的表型观察 A,B:分别为BnaBRC1突变体的叶片数目和株高的统计;C,D:分别为苗期和成熟期野生型材料WT(AA1CC1CC2)和突变体材料(aa1cc1CC2)的株型。其中数据表示平均值+ SD,其中n ≥ 10;标尺=3 cm
Fig.6 Phenotypes of BnaBRC1 mutants A and B:Statistical analysis of leaf numbers and plant height(cm)of BnaBRC1 mutants,respectively. C and D:Phenotypes at the vegetative and flowering stage,respectively,WT refers to wild type(AA1CC1CC2),aacc1CC2 refers to double copy of homozygous BnaBRC1 mutant. The data and error bars represent the mean ± SD,n ≥ 10,and bar = 3 cm
靶点 On-target site | 基因 Gene | 测序植株数目 Number of sequenced plants | 脱靶位点数 Putative off-target site | 脱靶编辑 Off-target editing |
---|---|---|---|---|
S1 | BnaBRC1 | 72 | 9 | No |
S2 | BnaBRC1 | 72 | 14 | No |
S3 | BnaBRC1 | 72 | 11 | No |
S4 | BnaBRC1 | 54 | 18 | No |
S5 | BnaBRC1 | 54 | 18 | No |
S6 | BnaBRC1 | 54 | 16 | No |
表2 在T0代突变体中检测每个sgRNA的脱靶活性
Table 2 Detection of potential off-target effects for each sgRNA target site in T0 mutated plants
靶点 On-target site | 基因 Gene | 测序植株数目 Number of sequenced plants | 脱靶位点数 Putative off-target site | 脱靶编辑 Off-target editing |
---|---|---|---|---|
S1 | BnaBRC1 | 72 | 9 | No |
S2 | BnaBRC1 | 72 | 14 | No |
S3 | BnaBRC1 | 72 | 11 | No |
S4 | BnaBRC1 | 54 | 18 | No |
S5 | BnaBRC1 | 54 | 18 | No |
S6 | BnaBRC1 | 54 | 16 | No |
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