Research on the Establishment of TRV-VIGS System of Fruit Shape- Related Genes CRC in Fingered Citron and Hongkong Kumquat

doi:10.16420/j.issn.0513-353x.2023-0542

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (4): 715-726.doi: 10.16420/j.issn.0513-353x.2023-0542

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

Research on the Establishment of TRV-VIGS System of Fruit Shape- Related Genes CRC in Fingered Citron and Hongkong Kumquat

WEI Junhai¹^,², WU Zhongyuan¹^,², WANG Xiaoqing¹, MEI Yulin¹, CHEN Wenrong¹, LIAO Fanglei¹^,²^,^*(), GUO Weidong¹^,²^,^*()

¹ College of Life Sciences，Zhejiang Normal University，Jinhua，Zhejiang 321004，China
² Zhejiang Provincial Key Laboratory of Biotechnology on Specialty Economic Plants，Jinhua，Zhejiang 321004，China

Received:2023-11-01 Revised:2024-01-03 Online:2024-04-25 Published:2024-04-26
Contact: LIAO Fanglei, GUO Weidong

Abstract

Abstract:

Using fingered citron（Citrus medica L. var. sarcodactylis Swingle）and Hongkong kumquat（Fortunella hindsii Swingle）leaves from the Rutaceae family as materials，the role of CRC genes in fruit development were investigated. To achieve this，the tobacco rattle virus（TRV）to create a virus-induced gene silencing（VIGS）technology system were employed，which provided a simple and effective method for studying gene function. The silencing vector targeting the CRC gene was injected twice into the top leaves of fingered citron and Hongkong kumquat，with a time interval of 15-30 days. Between 25 and 115 days after the injections，a significant down-regulation of CmsCRC expression was observed in the neonatal leaves，neonatal flowers，and fruits of Fingered citron. The silenced plants exhibited clustered fruit shapes and an increased number of fruit fingers. The silencing effect of the CRC gene in fingered citron lasted for 25-115 days，while in Hongkong kumquat，it lasted for 25-105 days.

Key words: VIGS, fingered citron, Hongkong kumquat, CRC, fruit shape

WEI Junhai, WU Zhongyuan, WANG Xiaoqing, MEI Yulin, CHEN Wenrong, LIAO Fanglei, GUO Weidong. Research on the Establishment of TRV-VIGS System of Fruit Shape- Related Genes CRC in Fingered Citron and Hongkong Kumquat[J]. Acta Horticulturae Sinica, 2024, 51(4): 715-726.

Figures/Tables 8

Table 1 Primer used in this study

引物名称（用途） Primer name（usage）	上游引物序列（5′-3′） Forward primer sequence	下游引物序列（5′-3′） Reverse primer sequence	片段长度/bp Fragment length
CmsCRC（VIGS）	GAATTCAACTTCTGGGAGCAGCAG	GGATCCCAAGAAATTAGAGTACCATACATAT	311
CmsCRC（qRT）	CTCTCACCACTTCTCTCTTCTTC	AGAAGTTGCAGCGGACATAG	204
sjgCRC（VIGS）	GAATTCAACTTCTGGGAGCAGCAG	GGATCCCAAGAAATTAGAGTACCATACATAT	291
sjgCRC（qRT）	CTCTCACCACTTCTCTCTTCTTC	AGAAGTTGCAGCGGACATAG	204
pTRV1	ATTGAGGCGAAGTACGATGG	CCATCCACAATTATTTTCCGC	279
pTRV2	ATTCACTGGGAGATGATACGCT	GAATCTAAGTCCACTCGTCCGT	151

Fig. 1 Cloning of CRC gene from fingered citron or Hongkon kumquat and construction of VIGS vectors A：PCR results for CmsCRC and sjgCRC；B：Identification of pTRV2-CmsCRC and pTRV2-sjgCRC plasmids by EcoR I and BamH I double digestion；C：PCR products of Agrobacterium plasmids DNA；D，E：The schematic diagram of the VIGS vector containing pTRV2-CmsCRC or pTRV2-sjgCRC. Vf means verification fragment.

Fig. 2 TRV detection of the leaves of CRC gene silencing plants

Fig. 3 Comparison of gene silencing efficiency of fingered citron or Hongkong kumquat cultured under different temperatures after infiltration Different lowercase letters indicate significant differences between treatments. ** indicates a highly significant difference between treatment and control.

Fig. 4 Comparison of plant morphology and CRC expression level of fingered citron before and after pruning The percentage represents the rate at which gene expression is down-regulated in the treatment compared to the control. Asterisks indicate statistically significant differences compared to the plants with mock treatments（* P < 0.05，** P < 0.01）.

Fig. 5 The relative expression level of CRC gene in fingered-citron and Hongkong kumquat leaves（A，B）or fruits（C，D）after infiltration Asterisks indicate statistically significant differences compared to the plants with mock treatments（*P < 0.05，**P < 0.01）.

Fig. 6 Changes in the expression level of CRC in fingered-citron and Hongkong kumquat after infections twice Asterisks indicate statistically significant differences compared to the plants with mock treatments（* P < 0.05，** P <0.01）.

Fig. 7 Phenotype investigation in leaves and fruitlet after infiltration

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Research on the Establishment of TRV-VIGS System of Fruit Shape- Related Genes CRC in Fingered Citron and Hongkong Kumquat

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