利用基因编辑技术研究BRANCHED1参与油菜分枝过程的调控

doi:10.13560/j.cnki.biotech.bull.1985.2021-1344

摘要/Abstract

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。

关键词: CRISPR/Cas9, BRC1, 分枝, 株型, 油菜

Abstract:

Plant architecture depends on shoot branching patterns,thus the improvement of shoot branching pattern is an efficient way of increasing crop yield. However,the molecular mechanism of shoot branching in rapeseed（Brassica napus L.）is not properly understood yet. Also,genetic resources for the improvement of plant architecture in agricultural crops are lacking. In the present study,we conducted the successful application of CRISPR/Cas9 genome editing technology for the targeted mutation of BRANCHED1（BRC1）in rapeseed. The transformed seedlings were acquired using the Agrobacterium-mediated transformation and the transgenic positive plants were selected using PCR. Hi-TOM sequencing analysis was performed to detect the sequences site-targeted mutated genes. The results demonstrated that the induced mutations were heritably transmitted to successive generations. Phenotypic observation of the obtained mutants showed that the double-copy homologous mutants from the BnaC01g34090 and BnaA01g26700D of BRC1 gene presented significant much shoot branching. Collectively,our results revealed that BnaBRC1 is involved in the shoot branching and in the regulation of plant architecture,thus providing the critical study materials to understant the mechanisms involved in the regulation of plant architecture in B. napus.

Key words: CRISPR/Cas9, BRC1, shoot branching, plant architecture, Brassica napus

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.

图/表 8

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	Cloning BnaC05g51920D
CBnaCnn-F	ACAACAACAACAGAAGATCTCTCAG	Cloning BnaCnng23770D
CBnaCnn-R	CATGAGGTCTCTTGGTCTCTCC	Cloning BnaCnng23770D
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	Transgenic positive detection
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	Cloning BnaC05g51920D
CBnaCnn-F	ACAACAACAACAGAAGATCTCTCAG	Cloning BnaCnng23770D
CBnaCnn-R	CATGAGGTCTCTTGGTCTCTCC	Cloning BnaCnng23770D
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	Transgenic positive detection
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1 BnaBRC1蛋白系统进化分析

Fig.1 Phylogenetic analysis of BnaBRC1 protein

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3 BnaBRC1基因5个同源拷贝在油菜的不同组织中的转录丰度

Fig.3 Transcript abundances of five BnaBRC1 copies in different tissues of B. napus

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

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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