佛手和山金柑果形相关基因CRC的TRV-VIGS体系的构建研究

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

摘要/Abstract

摘要：

以芸香科的佛手（Citrus medica L. var. sarcodactylis Swingle）和山金柑（Fortunella hindsii Swingle）花期叶片为材料，以果实发育相关的CRC基因为对象，利用烟草脆裂病毒（tobacco rattle virus，TRV）研究构建病毒诱导的基因沉默（virus-induced gene silencing，VIGS）技术体系，为基因功能研究提供有效简便的方法。待沉默的佛手植株剪枝、抹除花芽后，将CRC基因的沉默载体两次注射于佛手和山金柑的顶部叶片，注射间隔15 ~ 30 d。在注射后25 ~ 115 d内，佛手新生叶、新生花、果中的CmsCRC表达量均显著下调，且沉默植株的果形聚拢，果实指状结构数增加。CRC基因在佛手中的沉默有效时间是侵染后25 ~ 115 d，而在山金柑中沉默有效时间是25 ~ 105 d。

关键词: VIGS, 佛手, 山金柑, CRC, 果形

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

尉俊海, 吴中原, 王小青, 梅煜琳, 陈文荣, 廖芳蕾, 郭卫东. 佛手和山金柑果形相关基因CRC的TRV-VIGS体系的构建研究[J]. 园艺学报, 2024, 51(4): 715-726.

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.

图/表 8

表1 本研究中所用引物

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

图1 佛手CmsCRC、山金柑sjgCRC的克隆及其VIGS载体构建 A：CmsCRC和sjgCRC的PCR结果；B：pTRV2-CmsCRC和pTRV2-sjgCRC质粒EcoR I和BamH I双酶切鉴定；C：农杆菌中含有CmsCRC和sjgCRC质粒DNA的PCR扩增；D、E：pTRV2-CmsCRC与pTRV2-sjgCRC的VIGS载体构建示意图。Vf：验证片段。

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.

图2 沉默CRC植株的叶片TRV检测

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

图3 佛手和山金柑VIGS处理后不同温度培养下CRC沉默效率比较不同小写字母表示各处理间差异显著。** 表示处理与对照差异极显著。

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.

图4 佛手剪枝前后的形态对比及CRC表达水平对比百分数表示该处理与对照相比基因表达下调的比率。星号表示处理与对照之间存在统计学上的显著差异（*P < 0.05，**P < 0.01）。

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）.

图5 佛手和山金柑VIGS处理后CRC基因在叶片（A、B）和果实（C、D）中的相对表达水平星号表示处理与对照之间存在统计学上的显著差异（*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）.

图6 佛手和山金柑两次VIGS处理后CRC表达水平的变化星号表示处理与对照之间存在统计学上的显著差异（* 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）.

图7 侵染后叶片与幼果观察

Fig. 7 Phenotype investigation in leaves and fruitlet after infiltration

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