利用CRISPR/Cas9编辑草莓FvPDS基因的研究

doi:10.16420/j.issn.0513-353x.2024-0612

摘要/Abstract

摘要：

八氢番茄红素脱氢酶（phytoene desaturase，PDS）是类胡萝卜素生物合成的关键酶，其编码的PDS基因参与并调控植物类胡萝卜素的累积，PDS基因突变会使植物产生肉眼可见的白化表型，常用来作为检测基因编辑成功与否的标记基因。选取森林草莓FvPDS基因作为靶标，利用CRISPR/Cas9系统构建双靶点基因编辑载体，农杆菌介导法稳定遗传转化森林草莓种质HLJ-005（红果）、HLJ-004（白果），通过TA克隆测序法分析基因编辑的类型。遗传转化HLJ-005约2 250块叶片外植体，筛选到具有卡那霉素抗性的植株147株，通过PCR鉴定，24株为阳性材料，阳性率为16.32%，最后测序获得了5株具有编辑的材料，编辑效率为20.83%；遗传转化HLJ-004约2 305块叶片外植体，获得具有卡那霉素抗性的植株234株，PCR检测为6株阳性材料，阳性率为2.56%，测序只有3株为具有编辑的材料，编辑效率为50%。两个编辑位点都产生了突变，分别是单碱基或多个碱基的缺失，但是这两份种质的突变类型不尽相同。结果表明，利用CRISPR/Cas9系统可以通过稳定表达，HLJ-004、HLJ-005两个森林草莓实现双靶点同时敲除，HLJ-004、HLJ-005可作为草莓基因编辑的遗传转化材料，并验证了适合草莓基因编辑的CRISPR/Cas9系统。

关键词: 草莓, CRISPR/Cas9, FvPDS, 基因编辑

Abstract:

The phytoene desaturase gene（FvPDS）in Fragaria vesca was selected as the marker gene. This gene encodes a key enzyme in carotenoid biosynthesis. Because of its mutants with visible albino phenotypes，FvPDS has been served as a model gene for CRISPR/Cas9 mediated targeted gene editing. In this study a gene knockout vector was constructed using the CRISPR/Cas9 system，different types of mutations were detected in F. vesca through stable transformation of strawberries leaves including HLJ-005 with red fruits and HLJ-004 with white fruits. Through Agrobacterium mediated transformation 2 250 explants from HLJ-005 were transformed，147 kanamycin resistant lines were obtained through resistance screening. Of theses，24 transgenic lines（16.32%）were confirmed as positive transgenic lines by T-DNA specific PCR. Sequencing detection revealed that 5 transgenic lines had different types of mutations at the two target sites with an editing efficiency of 20.8%. For the other F. vesca HLJ-004，2 305 leaves were genetically transformed，234 kanamycin resistant lines were screened and 6 transgenic lines（2.56%）were confirmed as positive transgenic lines by T-DNA specific PCR. Sequencing detection showed that 3 lines had different types of mutations with an editing efficiency of 50%. Among these，mutations at two editing sites resulted in the deletion of single or multiple amino acids，leading to albino and dwarf phenotypes. These results demonstrate that the CRISPR/Cas9 system enables simultaneous double-site knockout in the woodland strawberry varieties HLJ-004 and HLJ-005 via stable expression. HLJ-004 and HLJ-005 can serve as suitable genetic transformation materials for strawberry gene editing，and the study validated a CRISPR/Cas9 system appropriate for editing strawberry genes.

Key words: strawberry, CRISPR/Cas9, FvPDS, gene editing

刘丽锋, 宋艳红, 赵霞, 李刚, 周厚成. 利用CRISPR/Cas9编辑草莓FvPDS基因的研究[J]. 园艺学报, 2025, 52(7): 1687-1695.

LIU Lifeng, SONG Yanhong, ZHAO Xia, LI Gang, and ZHOU Houcheng. Analysis of FvPDS Gene Using CRISPR/Cas9 System in Strawberry[J]. Acta Horticulturae Sinica, 2025, 52(7): 1687-1695.

图/表 6

表1 本研究中的引物名称及序列

Table 1 The prime names and sequences

引物名称 Prime name	引物序列（5′-3′） Sequences of primes
U626F	TGTCCCAGGATTAGAATGATTAGGC
U629R	AGCCCTCTTCTTTCGATCCATCAAC
FvPDS-F	GCATTATCTTCGTCTTGCGTGAT
FvPDS-R	GACTCCTCAATCTAGCCTAAACAC
FvPDS-BsF	ATATATGGTCTCGATTGTTCTGCCAGCACCCTTAAAGTT
FvPDS-F0	TGTTCTGCCAGCACCCTTAAAGTTTTAGAGCTAGAAATAGC
FvPDS-R0	AACAGAATTGCAGGCACAAGTCCAATCTCTTAGTCGACTCTAC
FvPDS-BsR	ATTATTGGTCTCGAAACAGAATTGCAGGCACAAGTCCAA

图1 FvPDS基因双靶点选择（a）和pKSE401表达盒（b）

Fig. 1 Schematic representation of FvPDS with two target sites（a）and pKSE401 expression cassettes（b）

图2 农杆菌介导的森林草莓遗传转化过程 A：叶片在诱导培养基上预培养；B：叶片在筛选培养基上诱导不定芽；C：丛生芽在筛选培养基上伸长；D：1个月后丛生芽在筛选培养基上继续生长；E：嵌合白化植株

Fig. 2 Agrobacterium-mediated genetic transformation of the Fragaria vesca A：The leaves were pre-cultured on induction medium；B：The adventitious buds were on the screening medium；C：The cluster buds were elongated on the screening medium；D：After 1 month，the cluster buds continued to grow on the screening medium；E：Chimeric albino plant were cultured on the screening medium

图3 HLJ-005（A）和HLJ-004（B）阳性植株PCR鉴定结果 M：Marker；WT：野生型；P：阳性质粒对照

Fig. 3 The positive plants were identified by pCR of HLJ-005（A）and HLJ-004（B） M：Marker；WT：Wild type；P：Plasmid used as positive control

表2 CRISPR/Cas9介导的编辑FvPDS草莓突变类型

Table 2 CRISPR/Cas9 mediated editing of FvPDS in strawberry mutant types

种质 Germplasm	外植体数 Number of explants	抗性植株 Kan-resistant plants	转化植株 Transgenic plan		突变植株 Mutant plant		杂合突变 M-he		等位突变 M-bi
HLJ-005	2 250	147		24（16.32）		5（20.83）		3（12.50）	5（20.83）
HLJ-004	2 305	234		6（2.56）		3（50.00）		1（16.67）	3（50.00）

图4 CRISPR/Cas9介导两份种质不同的编辑类型 A：PDS第一个靶位点突变类型；B：PDS第二个靶位点突变类型

Fig. 4 Different types of mutations in the two edited germplasms after CRISPR/Cas9 mediated gene editing A：Mutation types at the first target site of PDS gene；B：Mutation types at the second target site of PDS gene

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