Enhancing Ascorbic Acid Content in Non-heading Chinese Cabbage Using Gene Editing Technology

doi:10.16420/j.issn.0513-353x.2025-0036

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (5): 1317-1325.doi: 10.16420/j.issn.0513-353x.2025-0036

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

Enhancing Ascorbic Acid Content in Non-heading Chinese Cabbage Using Gene Editing Technology

WANG Wenlong¹, LIU Zhaokun², LU Wenjun¹, WANG Yaolong¹, LI Xiaofeng³, ZHU Hongfang³, LIU Tongkun¹, LI Ying¹, HOU Xilin¹, ZHANG Changwei¹^,^*()

¹ State Key Laboratory of Crop Genetics and Germplasm Innovation，College of Horticulture，Nanjing Agricultural University，Nanjing 210095，China
² Suzhou Academy of Agricultural Sciences，Suzhou，Jiangsu 215000，China
³ Institute of Horticulture，Shanghai Academy of Agricultural Sciences，Shanghai 201106，China

Received:2025-02-28 Revised:2025-04-01 Online:2025-05-23 Published:2025-05-21
Contact: ZHANG Changwei

Abstract

Abstract:

To increase the ascorbic acid content in non-heading Chinese cabbage，the variety‘Yellow Rose’was used as the experimental material. Targeting the uORF sequence of the GGP gene in non-heading Chinese cabbage，a CRISPR/Cas9 genome editing vector was constructed. Through Agrobacterium rhizogenes-mediated genetic transformation，four different gene-edited types of the GGP gene uORF in‘Yellow Rose’were obtained. The ascorbic acid content was measured in the T₁ generation homozygous green plants of the regenerated edited lines，showing a 26.2% to 67.1% increase compared to empty vector transgenic regenerated plants. This study provides a foundation for applying the CRISPR/Cas9 system in gene editing and molecular breeding research in non-heading Chinese cabbage.

Key words: non-heading Chinese cabbage, upstream open reading frame, genetic transformation, ascorbic acid, gene editing

WANG Wenlong, LIU Zhaokun, LU Wenjun, WANG Yaolong, LI Xiaofeng, ZHU Hongfang, LIU Tongkun, LI Ying, HOU Xilin, ZHANG Changwei. Enhancing Ascorbic Acid Content in Non-heading Chinese Cabbage Using Gene Editing Technology[J]. Acta Horticulturae Sinica, 2025, 52(5): 1317-1325.

Figures/Tables 6

Fig. 1 WIP-RUBY-cas9 gene editing vector

Table 1 Primer sequences

引物名称Primer name	引物序列（5′-3′）Primer sequence
靶点sgRNA序列 Target sgRNA sequence	GGAGCTCTGCCGTCTGAAGG
RUBY-F	TGGATCATGCGACCCTCGCC
RUBY-R	GAACACGGCGATCAGCTTGT
Cas9-F	GACAAGAAGTACTCCATCGG
Cas9-R	CTCGATCTTCTTGAAGTAGT
Bra026398-F	AAGCCATTCAATCTCCCCGAATC
Bra026398-R	TCTCAGACAGACAGAAAGACTTC

Fig. 2 Agrobacterium rhizogenes-mediated growth process of transgenic‘Yellow Rose’plants A：Hypocotyl preparation；B：Bud induction；C：Seedling cultivation；D：Transplant；E：Bolting；F：Flowering and seed setting；G：Harvesting；H：Sowing and germination；I：T1 generation plants

Fig. 3 Cas9 fusion gene PCR detection M：DL 2000 marker；N：Water；WT：Wild type；V：Empty vector；P：Positive plasmid；1-10：Resistant plant

Fig. 4 Types of uORFBrGGP mutations induced by CRISPR/Cas9 A：The dotted red box represents the base substitution，the black box with the arrow represents the base deletion（uORFBrGGP1 ~ uORFBrGGP3），the same base sequence is framed with the black box，and the missing part is marked with a red underline and arrow line（uORFBrGGP4）；B：Blue represents the uORF sequence，red represents base substitution，hyphen represents base deletion，and underlined sgRNA；Hi-TOM sequencing results are shown on the right，D：Base deletion；SNP：Base substitution. WT：Wild type

Fig. 5 Comparison of ascorbic acid content in new leaves of T1 generation homozygous green plants. WT was wild-type，EV was an unloaded transgenic plant，M0 was a CRISPR/Cas9 vector positive unedited plant，M1-M4 was a CRISPR/Cas9 vector positive mutant plant uORFBrGGP1-uORFBrGGP4，compared to empty vector transgenic plants，***P < 0.001，ns：no significant difference by two-tailed t-test

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Enhancing Ascorbic Acid Content in Non-heading Chinese Cabbage Using Gene Editing Technology

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