青花菜侧枝调控基因BoBRC1的克隆及功能分析

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

园艺学报 ›› 2024, Vol. 51 ›› Issue (9): 1997-2007.doi: 10.16420/j.issn.0513-353x.2024-0057

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

青花菜侧枝调控基因BoBRC1的克隆及功能分析

刘宇香¹^,²^,^*, 韩风庆²^,^*, 赵鑫雨², 刘玉梅², 李占省²^,^**(), $\boxed{\hbox{方智远}}$¹^,²^,^**()

¹ 湖南农业大学园艺学院，长沙 410125
² 中国农业科学院蔬菜花卉研究所，蔬菜生物育种全国重点实验室，北京 100081

收稿日期:2024-03-01 修回日期:2024-08-12 出版日期:2024-09-25 发布日期:2024-09-19
通讯作者:
^** E-mail: lizhansheng@caas.cn;
fangzhiyuan@caas.cn
作者简介:
*共同第一作者
基金资助:
国家重点研发计划项目(2022YFF1003000); 国家自然科学基金项目(32302544); 国家自然科学基金项目(32172580); 中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS); 现代农业产业技术体系建设专项资助(CARS-23); 内蒙古自治区科技重大专项(2021ZD0001); 乌兰察布市揭榜挂帅项目(2022JB006)

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 Published:2024-09-25 Online:2024-09-19

摘要/Abstract

摘要：

从青花菜优良自交系L461中克隆了BoBRC1，表达模式分析表明其在腋芽组织中表达量最高，并随腋芽生长动态变化。根据其基因序列，在第1个外显子上设计2个靶标，构建CRISPR-Cas9基因编辑载体，通过农杆菌介导的遗传转化法导入自交系L461中，获得18个阳性株系。测序结果显示，有3个株系在至少1个靶标处发生了基因突变，编辑效率为16%。其中L461-3株系在靶标1和靶标2均发生编辑事件，L461-1和L461-11株系仅在靶标2处发生编辑事件。与野生型相比，T₁代纯合突变体侧枝数明显增加，侧枝长度变长。对bobrc1纯合突变体中侧枝发育基因BoBRC1下游关键基因进行qRT-PCR分析发现，BoHB21、BoHB53、BoNCED3基因表达量显著降低，BoHB40基因表达量显著增高。以上研究结果证实了BoBRC1基因参与青花菜侧枝发育调控，敲除该基因可促进青花菜侧枝生长。

关键词: 青花菜, CRISPR/Cas9, 基因克隆, 功能分析, BoBRC1, 侧枝调控

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

刘宇香, 韩风庆, 赵鑫雨, 刘玉梅, 李占省, $\boxed{\hbox{方智远}}$. 青花菜侧枝调控基因BoBRC1的克隆及功能分析[J]. 园艺学报, 2024, 51(9): 1997-2007.

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.

图/表 8

表1 本试验中所用的引物信息

Table 1 Information of primers used in this study

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

图1 青花菜BoBRC1系统发育进化树

Fig. 1 Phylogenetic tree of BoBRC1 and its orthologous in multiple species

图2 BoBRC1基因在不同长度腋芽和不同组织中的表达模式不同小写字母表示P < 0.05时差异显著。

Fig. 2 Expression patterns of BoBRC1 gene in axillary buds of different lengths and in different tissues Different lowercase letters indicate statistical differences at P < 0.05.

图3 CRISPR/Cas9表达载体结构图（A）和农杆菌介导的转基因流程（B）

Fig. 3 Structure of CRISPR/Cas9 expression vectors（A）and Agrobacterium-mediated transformation processes（B）

图4 青花菜野生型（WT）和T0代基因编辑株系（L461-1，L461-3，L461-11）BoBRC1靶位点突变蓝色为PAM序列，绿色为靶标，红色为插入和缺失。

Fig. 4 BoBRC1 target site mutations in broccoli wild-type（WT）and T0 generation gene-edited lines（L461-1，L461-3，L461-11） PAM sequences are marked in blue，targets are marked in green，insertions and deletions are marked in red.

图5 野生型（WT）与T1代BoBRC1编辑植株（L461-1-T1、L461-3 -T1）的表型及其侧枝数和长度

Fig. 5 Phenotype，number and length of lateral branches of wild-type（WT）and T1 generation BoBRC1-edited plants（L461-1-T1，L461-3-T1） * P < 0.05，** P < 0.01，*** P < 0.001.

表2 青花菜BoHB21、BoHB40、BoHB53和BoNCED3相关引物信息

Table 2 Information of primers for BoHB21，BoHB40，BoHB53 and BoNCED3 in broccoli

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

图6 青花菜bobrc1突变体（L461-1）与野生型（WT）中BoHB21、BoHB40、BoHB53和BoNCED3的表达量星号表示统计学上的显著差异，*** P < 0.001。

Fig. 6 Analysis of BoHB21，BoHB40，BoHB53 and BoNCED3 gene expression in bobrc1 mutant（L461-1）and wild type（WT） The asterisk denotes statistically significant differences，*** P < 0.001.

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青花菜侧枝调控基因BoBRC1的克隆及功能分析

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

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青花菜侧枝调控基因BoBRC1的克隆及功能分析

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

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