Escaping endogenous miRNA post-transcriptional silencing of JrGRF4b enhanced transformation efficiency in woody plants

Abstract

The stable expression of transgenes was critically influenced by post-transcriptional regulatory mechanisms in transgenic plants. In this study, we investigated the influence of endogenous miRNA-mediated silencing on heterologous gene expression by introducing walnut (Juglans regia L.)-derived Growth-Regulating Factors 4 (JrGRF4b), disrupting miR396-mediated silencing of replace-JrGRF4b (rJrGRF4b), and Jr-miR396a into birch (Betula platyphylla Suk.). While JrGRF4b overexpression showed no significant improvement in transformation efficiency due to Bp-miR396-mediated suppression, transgenic lines expressing rJrGRF4b exhibited a 2.53% increase in transformation efficiency, along with significantly enhanced callus diameter, adventitious bud height, root elongation, cellular expansion, and shoot primordia proliferation compared to control (**p<0.01). In contrast, Jr-miR396a-overexpressing plants displayed growth inhibition through suppression of endogenous BpGRFs. The results showed that escaping endogenous miRNA regulation by targeted site modification of rJrGRF4b significantly improved transgene performance in woody plants. Thus, comprehensive evaluation of post-transcriptional epigenetic regulation between transgenes and endogenous miRNAs in recipient plants was demonstrated to be important, and targeted escape from such miRNA-mediated suppression was shown to ensure stable and high-efficiency transgene expression.

Keywords: MiR396; birch; growth-regulating factors; post-transcriptional regulatory; transgene; walnut.

Figure 1

Walnut JrGRFs family classification and expression. (A) Phylogenetic analyses of the walnut, Arabidopsis, rice, and birch GRFs families. (B) Transcript levels of JrGRFs genes during the organogenesis of walnut. (C) Multiple sequence alignment of JrGRF4a and JrGRF4b with AtGRF4 and OsGRF4 proteins. Conserved QLQ and WRC domains in GRF family proteins were highlighted using black boxes. (D) The expression of JrGRF4a and JrGRF4b during the organogenesis of walnut. LF, leaf; MF, male flower; GH, green husk; RT, root; SM, stem; KL: kernel; FF, female flower; EB, embryo. The error bars represent the SE of three independent biological replicates (t-test: **p < 0.01).

Figure 2

Walnut miR396 identification and expression. (A) Secondary structures of miR396 stem-loop precursors. The minimum free energy of the RNA secondary structure was calculated as ΔG (kcal/mol). (B) The miR396a binding site of JrGRFs genes. The miR396a binding site is marked in orange. The value on the right represents the binding energy of miR396 with JrGRFS mRNA. (C) The relative expression level of Jr-MIR396a. (D) The relative expression level of mature Jr-miR396a. LF, leaf; MF, male flower; GH, green husk; RT, root; SM, stem; KL: kernel; FF, female flower; EB, embryo. a, b, c, d, and e indicated extremely significant differences among the groups. The error bars represent the SE of three independent biological replicates (t-test: **p < 0.01).

Figure 3

rJrGRF4b accelerated the birch transformation process. (A) Schematic representation of JrGRF4b and rJrGRF4b gene structure showing the BpmiR396a target site. The Bp-miR396a-resistant rJrGRF4b version was introduced mutations (in red) to reduce interactions with Bp-miR396a. The Bp-miR396a seed region (nucleotides 2-8 from 5' to 3') was indicated by the blue highlighted line. (B) Schematic construction of the vectors. The expression of Jr-miR396a, JrGRF4b and rJrGRF4b were driven by the Cauliflowever mosaic virus 35S promoter (35Sp). The Basta represents the expression cassette for the Basta gene, which serves as a selection marker for transgenic lines. LB and RB, T-DNA left and right borders. (C) Overexpression of rJrGRF4b increased birch transformation efficiency.

Figure 4

Statistical analysis and morphology of callus cells of transformed with different vectors in birch transformation process. (A) The diameter of callus. 30 callus were selected for each period. (B) The height of adventitious buds. 30 callus were selected for each period. (C) Height of rooted seedings. 30 seedings were selected for each genotype. (D) The rooting rates. 30 seedings were selected for each genotype. (E) The number of roots. 30 seedings were selected for each genotype. (F) The length of roots. 30 seedings were selected for each genotype. (G) Histological analysis of callus cells at 30 DAYs. 20× Scale bars = 50 μm, 5×Scale bars = 200 μm. The blastemates were marked by a red arrow. (H) The areas of cell. 30 biological replicates were selected for each genotype. (I) The number of blastemates. 30 biological replicates were selected for each genotype. (J) Relative expression level of BpGRFs. The error bars represent the SE of three independent biological replicates (t-test: **p < 0.01).