香鳞毛蕨dfr-miR160a和靶基因DfARF10的生物信息学及表达模式分析

doi:10.11983/CBB23025

摘要/Abstract

摘要： 为深入探究miRNA参与调控香鳞毛蕨(Dryopteris fragrans)生长发育的分子机理, 依据实验室前期建立的miRNA数据库, 筛选出与非生物胁迫相关的差异表达dfr-miR160a前体(dfr-pri-mir160a), 预测其靶基因为DfARF10, 并通过本氏烟草(Nicotiana benthamiana)瞬时共转化GUS以及双荧光素酶(LUC)系统验证dfr-miR160a和DfARF10的靶向关系。结果显示, 共注射dfr-pri-mir160a和DfARF10的本氏烟草叶片中GUS活性和LUC活性明显降低; qRT-PCR分析显示, dfr-miR160a及其靶基因DfARF10在香鳞毛蕨的根、配子体、叶柄、叶片和孢子囊中均有表达, 在叶片中表达量最高, 在根中表达量最低; 通过qRT-PCR分析干旱、盐(NaCl)、高温和低温胁迫对dfr-miR160a及其靶基因DfARF10表达的影响。结果表明, 在干旱和高温处理下, dfr-miR160a的表达均上调, 但在NaCl处理下, dfr-miR160a的表达下调; 低温处理下, dfr-miR160a的表达在0-1小时下调, 在3-48小时上调。在NaCl、高温以及低温处理下DfARF10表达均上调; 但在干旱处理下, DfARF10表达下调, 与dfr-miR160a呈现相反的表达趋势。综上, dfr-miR160a靶向DfARF10基因且二者均能响应非生物胁迫。研究结果为从分子层面揭示香鳞毛蕨非生物胁迫抗性机制提供了新的科学依据。

关键词: 香鳞毛蕨, dfr-miR160a, DfARF10, 非生物胁迫

Abstract: To further understand the molecular mechanism underlying miRNA regulation of growth and development of Dryopteris fragrans, we screened the differentially expressed dfr-pri-mir160a through the miRNA database established earlier in the laboratory, and predicted its target gene as DfARF10. The target relationship between dfr-pri-mir160a and DfARF10 was verified by tobacco transient co-transformation, together with double luciferase (LUC) activity. The results showed that the GUS and LUC activity in tobacco leaves co-injected with dfr-pri-mir160a and DfARF10 decreased significantly. qRT-PCR analysis showed that dfr-miR160a and its target gene DfARF10 were expressed in the gametophytes, roots, petioles, leaves and sporangium of D. fragrans, with the highest expression in the leaves and the lowest in the roots. We analyzed the effects of drought, NaCl, high temperature and low temperature stress treatments on dfr-miR160a and its target gene DfARF10 through qRT-PCR. Under drought and high temperature treatment, the relative expression of dfr-miR160a was up-regulated, but under NaCl treatment, the expression of dfr-miR160a was down-regulated. Under low temperature treatment, the expression of dfr-miR160a was down-regulated at 0-1 h and was up-regulated at 3-48 h. The expression of DfARF10 was up-regulated under NaCl, high temperature and low temperature treatments. However, under drought treatment, the expression of DfARF10 decreased, distinct from dfr-miR160a. The above results indicated that target gene of dfr-miR160 was DfARF10, and both of them can respond to abiotic stress treatment. This study provides a new scientific basis for revealing the abiotic stress resistance mechanism of D. fragrans at the molecular level.

Key words: Dryopteris fragrans, dfr-miR160a, DfARF10, abiotic stress

仲昭暄, 张冬瑞, 李璐, 苏颖, 王黛宁, 王泽冉, 刘洋, 常缨. 香鳞毛蕨dfr-miR160a和靶基因DfARF10的生物信息学及表达模式分析. 植物学报, 2024, 59(1): 22-33.

Zhaoxuan Zhong, Dongrui Zhang, Lu Li, Ying Su, Daining Wang, Zeran Wang, Yang Liu, Ying Chang. Bioinformatic and Expression Pattern Analysis of dfr-miR160a and Target Gene DfARF10 in Dryopteris fragrans. Chinese Bulletin of Botany, 2024, 59(1): 22-33.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB23025

https://www.chinbullbotany.com/CN/Y2024/V59/I1/22

图/表 10

图1 香鳞毛蕨幼苗 Bars=5 cm

Figure 1 Dryopteris fragrans seedling plant Bars=5 cm

表1 引物序列

Table1 Primer sequences

Primer name	Sequence (5′-3′)	Purpose
DfARF10F	ATGCCCGGCCCTTTATCAAC	Gene clone
DfARF10R	TCACCTTGTAATGTTTTCACCG	Gene clone
dfr-pri-mir160aF	AAAATCACTCTGCCTGGCTC	Gene clone
dfr-pri-mir160aR	AGCGAGAAACTCTGCGTGG	Gene clone
DfARF10F-EGFP	CTCGGTACCCGGGGATCCATGCCCGGCCCTTTATCAAC	Gene clone
DfARF10R-EGFP	GGTGTCGACTCTAGAGGATCCCCTTGTAATGTTTTCACCG	Gene clone
pCAMBIA2301-dfr-pri-mir160aF	GGGCATCGATACGGGATCCATAAAATCACTCTGCCTGGCTC	Gene clone
pCAMBIA2301-dfr-pri-mir160aR	TCGAGCTCGATGGATCCCGTAAGCGAGAAACTCTGCGTGG	Gene clone
pCAMBIA2301-DfARF10F-GUS	GGGCATCGATACGGGATCCATATGCCCGGCCCTTTATCAAC	Gene clone
pCAMBIA2301-DfARF10R-GUS	TCGAGCTCGATGGATCCCGTACCTTGTAATGTTTTCACCG	Gene clone
pGreenII-dfr-pri-mir160aF	CAGTGGTCTCACACC AAAATCACTCTGCCTGGCTC	Gene clone
pGreenII-dfr-pri-mir160aR	CAGTGGTCTCAAGCGAGCGAGAAACTCTGCGTGG	Gene clone
pGreenII-LUC-DfARF10F	CAGTGGTCTCAGATCTCCATGGCAAGTGGAGCTA	Gene clone
pGreenII-LUC-DfARF10R	CAGTGGTCTCAAATTGTTCACAAGGCTACCCATGTTA	Gene clone
Df18sRNAF	GCTTTCGCAGTAGTTCGTCTTTC	qRT-PCR
Df18sRNAR	TGGTCCTATTATGTTGGTCTTCGG	qRT-PCR
DfARF10F	GCAATGCGGCGGGAGATCTT	qRT-PCR
DfARF10R	CAGAGCTCGAGCGCAAAGCC	qRT-PCR
dfr-miR160aF	TGCCTGGCTCCCTGTATGCCA	qRT-PCR
dfr-miR160aR	mRQ 3′ Primer	qRT-PCR

图2 dfr-pri-mir160a和DfARF10基因的RT-PCR扩增(A)以及DfARF10蛋白多序列比对(B) Marker: DNA分子标准品

Figure 2 RT-PCR of dfr-pri-mir160a and DfARF10 gene (A) and multiple sequence alignment of DfARF10 proteins (B) Marker: DNA marker

图3 香鳞毛蕨DfARF10蛋白质保守基序分析 (A) DfARF10系统进化树; (B) DfARF10基序分析; (C) DfARF10保守结构域分析

Figure 3 Conservative motif analysis of DfARF10 protein in Dryopteris fragrans (A) Phylogenetic tree of DfARF10 protein; (B) Motif analysis of DfARF10; (C) Conservative domains analysis of DfARF10

表2 DfARF10基序的序列

Table 2 Motif sequences of DfARF10

Motif	Sequence	PFAM analysis
Motif1	WKFRHIYRGQPRRHLLTTGWSVFVNQKKLVAGDSVVFLRNENGELRVGIR	B3
Motif2	SFCKTLTASDTNNGGGFSVPRRCAETIFPPLDYSQDPPVQELVAKDVHG	Auxin-resp
Motif3	DPVRWPNSKWRMLQVGWDEPEALZRPKRVSPWZIEPVSAPP	-
Motif4	LWHACAGPLVSIPPVGSKVYYFPQGHAEQ	-
Motif5	GMRFKMAFETEESSRRRYFG	-
Motif6	FEVVYYPRASPSEFVVPAKKV	-
Motif7	PKILCRVLNVKLLADPETDEVYAKITLQP	-
Motif8	WQVVYVDAEGDILLVGDDPWSEFVKTVRRIKILSPEEVQKM	AUX-IAA
Motif9	VGRSLDLSKFSSYEELREELARMFGIEG	-
Motif10	MPSSVISSHSMHIGVLAAAAHAVATNTM	-

图4 香鳞毛蕨dfr-miR160a保守基序分析 (A) dfr-miR160a系统进化树; (B) dfr-miR160a二级茎环结构 (黑线代表成熟miR160a序列); (C) dfr-miR160a成熟序列碱基保守性分析

Figure 4 Conservative motif analysis of dfr-miR160a in Dryopteris fragrans (A) Phylogenetic tree of dfr-miR160a; (B) Stem-loop structure of dfr-miR160a (black line represents the mature miR160a sequence); (C) Conservative base analysis of dfr-miR160a mature sequence

图5 香鳞毛蕨不同组织中dfr-miR160a (A)及其靶基因DfARF10 (B)的相对表达量 P值用One-way ANOVA计算, 不同小写字母表示不同组织间差异显著(P<0.05)。

Figure 5 Relative expression of dfr-miR160a (A) and target gene DfARF10 (B) in different tissues of Dryopteris fragrans P value is calculated with One-way ANOVA, different lowercase letters indicate significant differences among different tissues (P<0.05).

图6 不同处理时间下非生物胁迫对香鳞毛蕨叶片dfr-miR160a (A)及其靶基因DfARF10 (B)相对表达量的影响在不同处理时间下, 将4种胁迫下检测的表达量分别与对照(WT)相比较。P值(以野生型为对照)用One-way ANOVA计算; * P<0.05; ** P<0.01

Figure 6 Effects of abiotic stress on the relative expression level of dfr-miR160a (A) and target gene DfARF10 (B) in the leaves of Dryopteris fragrans under different treatment times The expression level under four kinds of stresses were compared with control (WT) at different treatment times. P value (wild type as control) is calculated with One-way ANOVA; * P<0.05; ** P<0.01

图7 农杆菌介导瞬时共转本氏烟草GUS以及双荧光素酶(LUC)活性验证dfr-miR160a靶向切割DfARF10 (A) DfARF10基因结构中dfr-miR160a的切割位点; (B) 35S::dfr-pri-mir160a和35S::DfARF10-GUS瞬时共转本氏烟草组织化学染色分析(+、++、+++分别代表不同浓度的35S::DfARF10-GUS (bars=5 mm)); (C) 35S::dfr-pri-mir160a和35S::DfARF10-GUS瞬时共转本氏烟草的GUS活性(将GUS活性标准化为本氏烟草的表达水平, +、++、+++分别代表不同浓度的35S::DfARF10-GUS); (D) 双荧光素酶验证系统通过LUC活性可视化dfr-miR160a的靶向降解能力(bars=3 cm); (E) 用LUC法定量计算荧光发光强度。

Figure 7 Agrobacterium mediated transient co-transformation of tobacco and double luciferase (LUC) activity to verify that dfr-miR160a targeted cleavage of DfARF10 (A) Cutting site of dfr-miR160a in DfARF10 gene structure; (B) β-glucuronidase (GUS) phenotype observed by histochemical staining analysis of 35S::dfr-pri-mir160a and 35S::DfARF10-GUS (+, ++, and +++ represent different concentrations of 35S::DfARF10-GUS, respectively (bars=5 mm)); (C) Co-expression of the constructs containing 35S::DfARF10-GUS and 35S::dfr-pri-mir160a in tobacco leaves (GUS activity were normalized to the expression levels of tobacco, +, ++, and +++ represent different concentrations of 35S::DfARF10-GUS, respectively); (D) The dual luciferase validation system visualizes the targeted degradation ability of dfr-miR160a through LUC activity (bars=3 cm); (E) Quantitative calculate intensity of fluorescence by LUC method.

图8 香鳞毛蕨DfARF10蛋白质在本氏烟草叶片中的亚细胞定位 DAPI的蓝色荧光指示细胞核(bars=10 µm)。

Figure 8 Subcellular localization of DfARF10 proteins of Dryopteris fragrans in tobacco leaf Blue fluorescence of DAPI indicate cell nucleus (bars=10 µm).

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