Bioinformatic and Expression Pattern Analysis of dfr-miR160a and Target Gene DfARF10 in Dryopteris fragrans

doi:10.11983/CBB23025

Abstract

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

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[J]. Chinese Bulletin of Botany, 2024, 59(1): 22-33.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB23025

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

Figures/Tables 10

Figure 1 Dryopteris fragrans seedling plant Bars=5 cm

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

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

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

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	-

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

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).

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

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.

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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