Cloning and Functional Analysis of Lateral Branching Regulatory Gene BoBRC1 in Broccoli

doi:10.16420/j.issn.0513-353x.2024-0057

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (9): 1997-2007.doi: 10.16420/j.issn.0513-353x.2024-0057

• Genetic & Breeding · Germplasm Resources · Molecular Biology • Previous Articles Next Articles

Cloning and Functional Analysis of Lateral Branching Regulatory Gene BoBRC1 in Broccoli

LIU Yuxiang¹^,², HAN Fengqing², ZHAO Xinyu², LIU Yumei², LI Zhansheng²^,^**(), $\boxed{\hbox{FANG Zhiyuan}}$¹^,²^,^**()

¹ College of Horticulture，Hunan Agricultural University，Changsha 410125，China
² State Key Laboratory of Vegetable Biobreeding，Institute of Vegetables and Flowers，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2024-03-01 Revised:2024-08-12 Online:2024-09-25 Published:2024-09-19
Contact: LI Zhansheng, $\boxed{\hbox{FANG Zhiyuan}}$

Abstract

Abstract:

In this study，the BoBRC1 gene was cloned from the elite broccoli inbred line L461. Expression pattern analysis showed that BoBRC1 had the highest expression level in axillary bud tissues，and changed dynamically with axillary bud growth. According to the sequence of BoBRC1，two targets were designed on the first exon，and a CRISPR/Cas9 gene editing vector was constructed. The construct was introduced into L461 by Agrobacterium-mediated genetic transformation，and a total of 18 plants were confirmed as positive transgenic. The results showed that three plants harbored mutations in at least one of the targeted regions corresponding to an editing efficiency of 16%. Among them L461-3 was edited in both Target 1 and Target 2 while L461-1 and L461-11 were edited in Target 2. Compared with the wild type，the homologous editing mutants showed a significant increase in the number and length of lateral branches. In the bobrc1 mutant，qRT-PCR analysis of key genes downstream of lateral branch gene BoBRC1 showed a significant decrease in the expression of the BoHB21，BoHB53 and BoNCED3 genes while the expression of the BoHB40 significantly increased. This study verified the function of BoBRC1 gene in the regulation of lateral branch development in broccoli，and knocking out can promote the growth of broccoli lateral branches.

Key words: broccoli, CRISPR/Cas9, gene cloning, functional analysis, BoBRC1, lateral branch regulation

LIU Yuxiang, HAN Fengqing, ZHAO Xinyu, LIU Yumei, LI Zhansheng, $\boxed{\hbox{FANG Zhiyuan}}$. Cloning and Functional Analysis of Lateral Branching Regulatory Gene BoBRC1 in Broccoli[J]. Acta Horticulturae Sinica, 2024, 51(9): 1997-2007.

Figures/Tables 8

References 30

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用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
基因全长扩增 Full-length gene amplification	Bol3-F	TGATTGTCTTCGTCAGTG
基因全长扩增 Full-length gene amplification	Bol3-R	ATGTCCAGGAGTCATAAA
靶点测序 Target sequencing	BRC1-1122F	CTTCCCTTGAACAACCCT
靶点测序 Target sequencing	BRC1-1122R	ATTGCTGCCTCTGTCCTC
定量引物 Primers for qRT-PCR	BOL3-1014F	CAACTTCTCAAGACCCAA
定量引物 Primers for qRT-PCR	BOL3-1014R	TGTAAGCCGAACAACTCT
编辑载体构建引物 Primers for CRISPR/Cas9 vector construction	16069-F	CAGTGGTCTCATGCAGCAAGATCAAAACGGCTAAA
编辑载体构建引物 Primers for CRISPR/Cas9 vector construction	16069-R	CAGTGGTCTCAAAACTCGTGTACCGGCGTTGTTCG
阳性苗鉴定 Identification of transgenic seedling	BarH-F	ATGAGCCCAGAACGACGCCCG
阳性苗鉴定 Identification of transgenic seedling	BarH-R	TCAAATCTCGGTGACGGGCAGG
菌落PCR PCR of colonies	M13-F	GTAAAACGACGGCCAGT
菌落PCR PCR of colonies	M13-R	CAGGAAACAGCTATGAC

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
基因全长扩增 Full-length gene amplification	Bol3-F	TGATTGTCTTCGTCAGTG
基因全长扩增 Full-length gene amplification	Bol3-R	ATGTCCAGGAGTCATAAA
靶点测序 Target sequencing	BRC1-1122F	CTTCCCTTGAACAACCCT
靶点测序 Target sequencing	BRC1-1122R	ATTGCTGCCTCTGTCCTC
定量引物 Primers for qRT-PCR	BOL3-1014F	CAACTTCTCAAGACCCAA
定量引物 Primers for qRT-PCR	BOL3-1014R	TGTAAGCCGAACAACTCT
编辑载体构建引物 Primers for CRISPR/Cas9 vector construction	16069-F	CAGTGGTCTCATGCAGCAAGATCAAAACGGCTAAA
编辑载体构建引物 Primers for CRISPR/Cas9 vector construction	16069-R	CAGTGGTCTCAAAACTCGTGTACCGGCGTTGTTCG
阳性苗鉴定 Identification of transgenic seedling	BarH-F	ATGAGCCCAGAACGACGCCCG
阳性苗鉴定 Identification of transgenic seedling	BarH-R	TCAAATCTCGGTGACGGGCAGG
菌落PCR PCR of colonies	M13-F	GTAAAACGACGGCCAGT
菌落PCR PCR of colonies	M13-R	CAGGAAACAGCTATGAC

基因名称	拟南芥同源基因	青花菜同源基因	引物名称	引物序列（5′-3′）
Gene name	Arabidopsis homologous gene	Broccoli homologous gene	Primer name	Primer sequence
BoHB21	At2g18550	BolC9t54306H	HB21-2F	TCATTTCTCAGATGTACCCTAG
BoHB21	At2g18550	BolC9t54306H	HB21-2R	CATTGCCTCCTTCTCCAC
BoHB40	At4g36740	BolC1t00227H	HB40-2F	AAAGATTGGCAGAAAGAG
BoHB40	At4g36740	BolC1t00227H	HB40-2R	AGGTTACCGTAGTCGTCA
BoHB53	At5g66700	BolC9t54057H	HB53-2F	GAAGCCAACTACGCACCC
BoHB53	At5g66700	BolC9t54057H	HB53-2R	AGCCATCCCAATACTCCA
BoNCED3	At3g14440	BolC1t04961H	NCED3-2F	CATCCAGGAGCGTCAGTT
BoNCED3	At3g14440	BolC1t04961H	NCED3-2R	GCTCTGGCGTAGAATAGCA

基因名称	拟南芥同源基因	青花菜同源基因	引物名称	引物序列（5′-3′）
Gene name	Arabidopsis homologous gene	Broccoli homologous gene	Primer name	Primer sequence
BoHB21	At2g18550	BolC9t54306H	HB21-2F	TCATTTCTCAGATGTACCCTAG
BoHB21	At2g18550	BolC9t54306H	HB21-2R	CATTGCCTCCTTCTCCAC
BoHB40	At4g36740	BolC1t00227H	HB40-2F	AAAGATTGGCAGAAAGAG
BoHB40	At4g36740	BolC1t00227H	HB40-2R	AGGTTACCGTAGTCGTCA
BoHB53	At5g66700	BolC9t54057H	HB53-2F	GAAGCCAACTACGCACCC
BoHB53	At5g66700	BolC9t54057H	HB53-2R	AGCCATCCCAATACTCCA
BoNCED3	At3g14440	BolC1t04961H	NCED3-2F	CATCCAGGAGCGTCAGTT
BoNCED3	At3g14440	BolC1t04961H	NCED3-2R	GCTCTGGCGTAGAATAGCA

Cloning and Functional Analysis of Lateral Branching Regulatory Gene BoBRC1 in Broccoli

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