Creating High Lycopene Fruit Using CRISPR/Cas9 Technology in Tomato

doi:10.16420/j.issn.0513-353x.2023-0782

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (2): 253-265.doi: 10.16420/j.issn.0513-353x.2023-0782

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

Creating High Lycopene Fruit Using CRISPR/Cas9 Technology in Tomato

YANG Liang¹^,²^,³^,⁴, LIU Huan¹^,³^,⁴, MA Yanqin¹^,²^,³^,⁴, LI Ju¹^,²^,³^,⁴, WANG Hai'e¹^,⁴, ZHOU Yujie¹^,³^,⁴, LONG Haicheng¹^,³^,⁴, MIAO Mingjun¹^,²^,³^,⁴, LI Zhi¹^,²^,³^,⁴^,^*(), CHANG Wei²^,³^,⁴^,⁵^,^*()

¹ Horticulture Research Institute，Sichuan Academy of Agricultural Sciences，Chengdu 610066，China
² Sichuan Province Engineering Technology Research Center of Vegetables，Pengzhou，Sichuan 611934，China
³ Vegetable Germplasm Innovation and Variety Improvement Key Laboratory of Sichuan Province，Chengdu 610066，China
⁴ Key Laboratory of Horticultural Crops Biology and Germplasm Enhancement in Southwest Regions，Ministry of Agriculture and Rural Affairs，Chengdu 610066，China
⁵ Sichuan Institute of Edible Fungi，Chengdu 610066，China

Received:2023-12-07 Revised:2024-01-06 Online:2024-02-25 Published:2024-02-26
Contact: LI Zhi, CHANG Wei

Abstract

Abstract:

The large-fruited cultivated tomato resource“T048”was used as the material for targeted editing of the tomato stay-green gene SlSGR1 using CRISPR/Cas9 technology. Sequence analysis of the target sites of 19 positive transgenic plants revealed that a total of ten plants had editing events，with an editing efficiency of 52.6%. The types of editing events involved deletions，insertions and substitutions of bases，most of which occurred 3-4 bp upstream of the PAM sequence. A total of 565 bp of sequence inversion between the two editing sites was also detected. Phenotypic analysis revealed that the homozygous edited plants in the T1 generation showed slower senescence of the leaves and rusty red color fruits，with significantly higher content of lycopene，chlorophyll and β-carotenoid，compared with unedited fruits. Analysis of the key genes for carotenoid synthesis revealed that the expression of the SGR1 in the fruit of the homozygous edited plants was significantly lower than that of the unedited plants，whereas the expression of the PSY1 was significantly higher than that of the unedited one. This study demonstrated that targeted editing of the SlSGR1 gene by CRISPR/Cas9 technology can effectively enhance the content of carotenoids such as lycopene and β-carotene in tomato fruits，and thus provide new germplasm for tomato nutritional quality breeding.

Key words: tomato, geno editing, lycopene, carotenoid, SlSGR1

YANG Liang, LIU Huan, MA Yanqin, LI Ju, WANG Hai'e, ZHOU Yujie, LONG Haicheng, MIAO Mingjun, LI Zhi, CHANG Wei. Creating High Lycopene Fruit Using CRISPR/Cas9 Technology in Tomato[J]. Acta Horticulturae Sinica, 2024, 51(2): 253-265.

Figures/Tables 9

Table 1 The primers used in this study

用途 Aim	引物名称 Primer name	引物序列（5′-3′） Primer sequence
载体构建Construction vector	SGR1-target1-F	ATATATGGTCTCGTTTGAGATGAAGTTGTTGCAGAGGTTTTAGAGCTAGAAATAG
载体构建Construction vector	SGR1-target2-R	ATTATTGGTCTCGAAACCCAGTGAGTGTTATGCCTTCCAAACTACACTGTTAGATTC
阳性克隆验证Identification of the positive clones	M13-F	GTAAAACGACGGCCAGT
阳性克隆验证Identification of the positive clones	SGR1-target2	CCAGTGAGTGTTATGCCTT
转基因株验证Identification of the transformation plants	Cas9-F	AGGTCACGGTTAAGCAGCTC
转基因株验证Identification of the transformation plants	Cas9-R	CCCTTCTGCGTGGTCTGATT
靶位点检测 Detection target	SGR1-Seqtarget-F	GTTTGATTTGCAGTTGCAAGGTT
靶位点检测 Detection target	SGR1-Seqtarget-R	AATGCTGCTTCCACAAACCC
Real-time PCR	SlACTIN-RT-F	GGGATGGAGAAGTTTGGTGGTGG
	SlACTIN-RT-R	CTTCGACCAAGGGATGGTGTAGC
	SGR1-RT-F	GGGTCCACTCAGAGATGCAA
	SGR1-RT-R	ACTCACTGGGGGAAAGCAAC
	PDS-RT-F	AAGGCGCTGTCTTATCAGGAAA
	PDS-RT-R	TAAACTACGCTTGCTTCCGACA
	ZDS-RT-F	ACCGTACAACTACGCTACAATGG
	ZDS-RT-R	CATCTGGCGTATAGAGGAGATTG
	PSY1-RT-F	TATTTGCTGGAAGGGTGACC
	PSY1-RT-R	TAGCTGAGCTCAATTCTGTC

Fig. 1 Schematic representation of SlSGR1 with target sites（A）and the pKSE401-SGR1 vector（B）

Fig. 2 Identification of positive transgenic lines in SlSGR1 edited T0 generation plants M：DL2000 DNA marker；1-26：Rooted plants；WT：Wild type；+：Positive control.

Fig. 3 Statistics of editing frequency and pattern in Target1 and Target2 loci of SlSGR1 edited plants

Fig. 4 Mutation sequence analysis of ten SlSGR1 edited T0 generation plants Ho：Homozygous mutation；He：Heterozygous mutation；Bi：Biallelic mutation；Ch：Chimeric mutation；WT：Wild type；d：Base deletion；i：Base insertion；s：Base substitution.

Fig. 5 Screening of T1 exogenous T-DNA-free homozygous lines in SlSGR1 edited plants （sgr1-4, sgr1-12 and sgr1-22）（A）and sequence analysis of their target sites（B）

Fig. 6 Phenotypes of leaves and fruits in SlSGR1 edited plants（sgr1-4-10，sgr1-12-1 and sgr1-22-12）

Table 2 Content of carotenoids，chlorophyll and nutritional quality traits in SlSGR1 edited plants

基因编辑株系 Genome editing plant		叶绿素/（μg • g^-1） Chlorophyll	β-胡萝卜素/（μg • g^-1） β-carotenoids	抗坏血酸/（μg · g^-1） Vitamin C	可溶性固形物/% Soluble solids
野生型Wild type	53.70 ± 9.54 b	4.28 ± 1.24 b	38.44 ± 3.98 b	251.64 ± 11.58 a	4.77 ± 0.40 a
sgr1-4-10	73.21 ± 9.56 a	29.60 ± 1.50 a	53.32 ± 2.74 a	246.43 ± 17.73 a	5.13 ± 0.85 a
sgr1-22-12	75.08 ± 9.11 a	37.20 ± 6.30 a	59.71 ± 1.13 a	254.73 ± 7.27 a	4.87 ± 0.15 a

Fig. 7 Relative expression of key genes of carotenoid synthesis pathway in four fruit development stages of different genome editing tomato plants MG：Mature green stage；Br：Break stage；Br + 4：Four days after break stage；Br + 10：Ten days after break stage.Columns with different lowercase letters indicate significant differences among lines in the same stage（P < 0.05）.

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Creating High Lycopene Fruit Using CRISPR/Cas9 Technology in Tomato

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