外源miR3979处理水稻对拟禾本科根结线虫趋性、侵染和发育的影响

doi:10.16819/j.1001-7216.2025.240906

摘要/Abstract

摘要：

【目的】miR3979-3p参与水稻响应多种生物和非生物胁迫，在拟禾本科根结线虫(Meloidogyne graminicola)侵染的根组织中下调表达，因此探究miR3979-3p对根结线虫与水稻互作中的作用具有重要意义。【方法】利用人工合成的双链miR3979(ds-miR3979)浸泡水稻根组织，采用qPCR测量水稻内源miR3979-3p的表达水平，通过平板接种和组织染色观察二龄幼虫的趋性和侵染能力，结合盆钵接种评价ds-miR3979处理对水稻根结产生和线虫发育的影响。【结果】100~800 nmol/L ds-miR3979处理12~24 h对二龄幼虫的运动活性无显著影响；与ddH₂O处理的对照相比，400 nmol/L ds-miR3979浸泡根系12~24 h显著提高了立针期和三叶期水稻根系和叶片miR3979-3p的表达水平；连续显微观察2~8 h，发现二龄幼虫向水稻根尖移动趋性降低，集聚在根尖周围的线虫数量显著减少；同时，平板接种1~3 d侵入根内的线虫数量减少，盆钵接种7~21 d根系上的根结数目、根内雌虫数量和占比显著下降，且雌虫的发育进程延缓。【结论】人工合成的ds-miR3979可被水稻吸收并上调内源miR3979-3p表达，进而影响根结线虫的侵染和雌虫发育。

关键词: 小RNA, miR3979-3p, 水稻, 拟禾本科根结线虫, 侵染, 发育

Abstract:

【Objective】 miR3979-3p is involved in rice responses to multiple biotic and abiotic stresses. Its expression is downregulated in root tissues infected by the root-knot nematode Meloidogyne graminicola. Therefore, it is important to investigate the role of miR3979-3p in the interaction between M. graminicola and rice.【Method】 Rice roots were treated with artificially synthesized double-stranded miR3979 (ds-miR3979) via root soaking. Endogenous miR3979-3p expression levels were quantified using qPCR. Chemotaxis and infectivity of second-stage juveniles (J2s) were assessed through plate inoculation and tissue staining. The effects of ds-miR3979 treatment on root gall formation and nematode development were evaluated using pot inoculation assays.【Result】 Treatment with 100-800 nmol/L ds-miR3979 for 12-24 h had no significant effect on J2 motility. Compared with ddH₂O-treated controls, root soaking with 400 nmol/L ds-miR3979 for 12-24 h significantly upregulated miR3979-3p expression in both roots and leaves of rice seedlings at the seedling emergence and three-leaf stages. Continuous microscopic observation revealed reduced chemotactic movement of J2s toward root tips within 2-8 h, with significantly fewer nematodes aggregating around root tips. Plate inoculation showed a decrease in the number of nematodes penetrating roots at 1-3 days post-inoculation (dpi). Pot inoculation assays demonstrated significant reductions in root gall number, number of females within roots, and proportion of females at 7-21 dpi, along with delayed female development.【Conclusion】 Artificially synthesized ds-miR3979 can be taken up by rice plants, upregulating endogenous miR3979-3p expression, thereby inhibiting M. graminicola infection and female development.

Key words: small RNA, miR3979-3p, rice, Meloidogyne graminicola, infection, development

杨行洲, 崔苗苗, 魏利辉, 顾爱国, 李东霞, 乐秀虎, 冯辉. 外源miR3979处理水稻对拟禾本科根结线虫趋性、侵染和发育的影响[J]. 中国水稻科学, 2025, 39(5): 703-710.

YANG Xingzhou, CUI Miaomiao, WEI Lihui, GU Aiguo, LI Dongxia, LE Xiuhu, FENG Hui. Effects of Exogenous miR3979 on Chemotaxis, Infection and Development of Root Knot Nematode Meloidogyne graminicola in Rice[J]. Chinese Journal OF Rice Science, 2025, 39(5): 703-710.

图/表 6

表1 本研究所用引物

Table 1. Primers used in this study

引物名称 Primer name	序列(5’-3’) Sequence(5’-3’)	目的 Aim
miR3979-3p.RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTTCTCTCC	RT-PCR
miR390-5p.RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGCGCT	RT-PCR
miR3979-3p.F	TCCCGCCTTCGGGGGAGGAG	qPCR
miR390-5p.F	TGACCAAAGCTCAGGAGGGAT
Universal R	GTGCAGGGTCCGAGGT

图1 二龄幼虫在ds-miR3979溶液中的存活率

Fig. 1. Survival rate of J2s exposed to ds-miR3979 solution

图2 ds-miR3979处理后水稻内源miR3979-3p水平 A~B, 400 nmol/L ds-miR3979浸泡水稻根系12 h（A）或24 h（B）后, minR3979-3P相对表达量，以ddH2O处理作为对照。每个处理3个重复；**, P<0.01。

Fig. 2. Relative expression of miR3979-3p in rice plants treated with ds-miR3979 solution A-B, Relative expression of miR3979-3p in roots and shoots of rice treated with 400 nmol/L ds-miR3979 solution for 12 h (A) or 24 h (B). Each treatment repeated three times. **, P < 0.01.

图3 ds-miR3979处理的水稻对二龄幼虫的趋性影响 A, 线虫向水稻根尖迁移观察；B, 聚集在水稻根尖周围的线虫数量。截取用400 nmol/L ds-miR3979浸泡水稻根系12 h的2 cm长根段用于实验，以ddH2O处理作为对照，每个处理5个重复。图中标尺=1 mm；*, P<0.05。

Fig. 3. Chems tabis of J2 towards rice roots treated with ds-miR3979 solution A. Chemotaxis of J2s towards rice root tips. B. Number of J2s around rice root tips. Rice roots were soaked into 400 nmol/L ds-miR3979 solution or ddH2O for 12 h and then cut into 2 cm length for the assay. Each treatment repeated three times. Scale bar = 1 mm; *, P < 0.05.

图4 ds-miR3979处理水稻对二龄幼虫早期侵入的影响 A, 接种后1 d和3 d侵入根部线虫染色观察；B, 接种后1 d和3 d侵入根部的线虫数量调查。400 nmol/L ds-miR3979浸泡水稻根系12 h后接种线虫，同时以ddH2O处理作为对照。每个处理6个重复。图中标尺=1 mm；*, P<0.05。

Fig. 4. Early infection of J2 in rice roots treated with ds-miR3979 solution

图5 ds-miR3979处理水稻对拟禾本科根结线虫发育影响 A~B: 线虫接种不同时间诱导的根结数量(A)和根系线虫侵染数量(B); C: 根内线虫染色观察; D: 根内不同发育阶段线虫百分占比。J2和J3/4: 二龄和三/四龄幼虫; FM: 正在发育的雌虫; FML: 正在产卵的雌虫。400 nmol/L ds-miR3979浸泡水稻根系24 h后接种线虫，同时以ddH2O处理作为对照，每个处理6个重复。图中标尺=1 mm；不同小写字母表示同一接种时间同一龄期线虫百分占比的差异显著性(P<0.05)；ns表示无显著差异；*, P<0.05。

Fig. 5. Development of M. graminicola in rice treated with ds-miR3979 solution A and B, Number of galls (A) and nematodes (B) in roots at 7, 14, and 21 day after inoculation (dpi); C, Nematodes in the roots stained with acid fuchsin; D, percentage of different stages of nematodes in the roots. J2, Second stage juvenile; J3/4, Third/fourth stage juvenile; FM, Developing female, FML, Egg-caying female. Rice roots were soaked into 400 nmol/L ds-miR3979 solution or ddH2O for 24 h. Each treatment repeated six times. Scale = 1 mm. Different letters in a column (D) indicate significant difference for identical developmental stages between control and treatment at the same time point at P<0.05; ns, No significant difference; *, P<0.05.

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