黄瓜卷须缠绕过程中小G蛋白活性变化

doi:10.11983/CBB22058

摘要/Abstract

摘要： 攀爬植物的卷须是对机械刺激极为敏感的器官。拟南芥(Arabidopsis thaliana)小G蛋白能够响应机械刺激调控自身发育, 但小G蛋白信号是否在卷须感受机械刺激发生缠绕过程中发挥作用目前尚不清楚。该研究以黄瓜(Cucumis sativus)为实验材料, 对其卷须受机械刺激缠绕过程中小G蛋白活性进行了探讨。ROP活性检测结果表明, 在卷须缠绕过程中小G蛋白CsROP6的活性显著增强; 同时, 质谱鉴定结果显示, 卷须缠绕过程中生长素的含量明显增加。进一步的实验表明, 外源生长素处理能明显增强CsROP6的活性, 暗示黄瓜卷须在缠绕过程中受机械刺激可能通过生长素增强ROP活性, 且机械刺激激活小G蛋白信号在不同植物中存在一定的保守性。

关键词: 生长素, 黄瓜卷须, 机械信号, 小G蛋白

Abstract: Tendrils of climbing plants are highly sensitive to mechanical stimuli. Small GTPases signals can be activated by mechanical stimulation which regulate development in Arabidopsis, but whether small GTPases signals play a role in the entanglement of tendrils response to mechanical stimuli is unknown. In this study, the activity of small GTPases in cucumber tendril after mechanical stimulation was investigated using Cucumis sativus, and ROP activity detection results showed that the activity of small GTPases CsROP6 was significantly enhanced during tendril winding. Meanwhile, mass spectrometry revealed that auxin content increased significantly during tendril winding. Further experiments showed that exogenous auxin treatment increased the activity of CsROP6, suggesting mechanical stimulation might activate ROP6 activity through auxin during tendril winding. This study suggests that mechanical stimulation of small GTPases signals may be conserved in different plants.

Key words: auxin, cucumber tendrils, mechanical signal, small GTPases

李彬琪, 闫佳慧, 李豪, 辛伟, 田云鹤, 杨贞标, 唐文鑫. 黄瓜卷须缠绕过程中小G蛋白活性变化. 植物学报, 2022, 57(3): 299-307.

Binqi Li, Jiahui Yan, Hao Li, Wei Xin, Yunhe Tian, Zhenbiao Yang, Wenxin Tang. Changes of Small GTPases Activity During Cucumber Tendril Winding. Chinese Bulletin of Botany, 2022, 57(3): 299-307.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22058

https://www.chinbullbotany.com/CN/Y2022/V57/I3/299

图/表 7

图1 黄瓜卷须的机械触碰反应 (A) 自然缠绕: 白色箭头表示不同时间点卷须触碰木棍并发生缠绕的过程; 20:00、20:30、21:00和22:00表示观察当天的时间点(bars=3 cm); (B) 人工触碰缠绕: 卷须经小木棍触碰30秒(30下)后, 每隔30秒拍照记录, 3分钟时发生约180°卷曲; 白色箭头表示木棍触碰点(bars=2 cm)。

Figure 1 Mechanical contact response of cucumber tendril (A) Natural winding: the white arrows show the winding process of the tendril after touching the stick; 20:00, 20:30, 21:00, and 22:00 indicate the time points of the observation day (bars=3 cm); (B) Winding after manual touching: after 30 seconds’ (30 touches) touch with a wooden stick, tendrils were photographed and recorded every 30 seconds, and tendrils were curled at about 180° at 3 minutes; white arrow indicates the stick touch point (bars=2 cm).

图2 黄瓜中ROP家族成员的进化分析与序列比对 (A) 黄瓜和拟南芥ROPs的系统进化树; (B) 用DNAMAN软件对黄瓜和拟南芥ROP6的氨基酸序列进行比对。

Figure 2 Evolutionary analysis and sequence alignment of ROP family members in cucumber (A) The phylogenetic tree of ROPs in cucumber and Arabidopsis thaliana; (B) Comparison of amino acid sequences of ROP6 between cucumber and Arabidopsis thaliana by DNAMAN.

表1 黄瓜RT-PCR扩增引物序列

Table 1 Primer sequences for RT-PCR in cucumber

Primer name	Forward primer (5′→3′)	Reverse primer (5′→3′)
Cucsa.322750	CAAATGTAGTTGTGAATGGGAGC	TTCCAACCAGAACAATAGGCACT
Cucsa.393880	TTTCCCACGGACTACGTGCCTAC	AACTGCTTGTCATCCCGAAGATC
Cucsa.234270	AGGCTTGTGGGATACTGCTGGTC	CTTCTTTCGCTTCTTCCGCTTTG
Cucsa.309440	TTCAGTGCTAATGTGGTGGTGGA	GATGGCTAGTCAGAAATTGCTTGTCGT
Cucsa.046650	TCAATCTTGGCTTATGGGACACT	GGAGCTGCATTCTATGTATGTAATT
Cucsa.197080	TTCGTTAGTTAGCCGAGCGAGTT	TGATTTCACATTCTGCTGGGTTT
Cucsa.256740	TGGTTAATGGGCAGTCTGTGAAT	TGGTCCCTACAAGAATAATGGGT
Cucsa.332390	TGTGGCTTTCTCGTTAATCAGCA	GTTCCTCCTCTTCCTTGGCATCT
Cucsa.116450	TACTGTTGGAGATGGAGCGGTTGG	TTAGCATATCGACGGTCCTCACG
Cucsa.322750	CAAATGTAGTTGTGAATGGGAGC	TTCCAACCAGAACAATAGGCACT

图3 黄瓜ROPs基因的组织表达模式 (A) RT-PCR检测ROPs在黄瓜卷须、茎、叶、果实和花中的表达丰度; (B) qRT-PCR检测黄瓜ROPs在卷须中的表达水平(n=4)。

Figure 3 Tissue expression pattern of ROPs gene in cucumber (A) Detect the expression abundance of ROPs in cucumber tendrils, stems, leaves, fruits and flowers by RT-PCR; (B) Detect the expression of ROPs in cucumber tendrils by qRT-PCR (n=4).

图4 黄瓜卷须缠绕过程中ROP活性变化 0C、1C和2C分别表示缠绕0、1和2圈。n=5; 差异显著性分析采用Student’s t test。**P<0.01

Figure 4 Changes of ROP activity during cucumber tendril winding 0C, 1C and 2C represent 0, 1 and 2 cycle of winding, respectively. n=5; significance analysis was performed by Student’s t test. **P<0.01

图5 生长素处理明显增强黄瓜卷须机械刺激的敏感性 (A) 液相质谱法测定卷须缠绕前后生长素含量的变化(0C、0.5C、1C和2C分别表示缠绕0、0.5、1和2圈); (B), (C) 生长素对黄瓜卷须机械触碰敏感性影响的鉴定(bars=3 cm); n=10; 角度显著性分析采用One-way ANOVA; *P<0.05; **P<0.01; NS: 无显著差异

Figure 5 Auxin treatment significantly enhanced mechanical stimulation sensitivity of cucumber tendrils (A) Determination of auxin content before and after tendril winding by liquid mass spectrometry (0C, 0.5C, 1C, 2C represent 0, 0.5, 1 and 2 cycles of winding, respectively); (B), (C) The effects of auxin on mechanical sensitivity of cucumber tendril (bars=3 cm); n=10; significance analysis was performed using One-way ANOVA; *P<0.05; **P<0.01; NS: No significant difference

图6 生长素诱导黄瓜卷须中ROP6活性增强 n=5; 显著性分析采用Student’s t test。**P<0.01

Figure 6 Auxin induces the increase of ROP6 activity in cucumber tendrils n=5; significance analysis was performed by Student’s t test. **P<0.01

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