DNA fragment miR156a, target gene ThSPL2 and its constituent miR156a-ThSPL2 module and their applications

Abstract

This invention discloses the DNA fragment miR156a, the target gene ThSPL2, and the miR156a-ThSPL2 module they constitute, as well as their applications, belonging to the field of plant genetic engineering technology. The nucleotide sequence of the DNA fragment miR156a is shown in SEQ ID NO.1, and the nucleotide sequence of the target gene ThSPL2 is shown in SEQ ID NO.2. The miR156a-ThSPL2 module they constitute can be used to regulate the adventitious root development of *Taxus chinensis* and for molecular breeding. This application cloned and identified the DNA fragment miR156a and the ThSPL2 gene in *Taxus chinensis*, clarified their core regulatory functions in promoting adventitious root development through regeneration, and confirmed the direct targeting and inhibition relationship between miR156a and ThSPL2. This provides molecular evidence for elucidating the age-dependent decline in rooting ability and the mechanism of regeneration recovery in *Taxus chinensis*, and has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a DNA fragment miR156a that regulates the development of adventitious roots of Metasequoia glyptostroboides, the target gene ThSPL2, the miR156a-ThSPL2 module composed of them, and their applications. Background Technology

[0002] *Taxodium hybrid 'Zhongshanshan'* is a general term for superior clones with certain super-parental traits, bred by the Jiangsu Institute of Botany, Chinese Academy of Sciences, from interspecific hybridization within the genus *Taxodium*. Currently, ten national and provincial-level superior forest tree varieties have been developed and widely promoted. Years of indoor evaluation trials, intermediate demonstration trials, and regional trials have shown that *Taxodium hybrid 'Zhongshanshan'* possesses advantages such as rapid growth, salt tolerance, waterlogging tolerance, wind resistance, disease resistance, and superior timber quality. It has broad application prospects and huge market demand in afforestation of lakes, wetlands, water networks, tidal flats, and plains in the Yangtze River Basin, southeastern coastal areas, and inland regions of China. Seedling propagation of *Taxodium hybrid 'Zhongshanshan'* mainly uses softwood cuttings. However, during the propagation process, it was found that the adventitious root regeneration capacity significantly declines with the physiological age of the clones, limiting the propagation and promotion of superior *Taxodium hybrid 'Zhongshanshan'* varieties.

[0003] The regenerative capacity of adventitious roots from cuttings is closely related to the physiological age of trees. Rejuvenation can partially or completely restore the characteristics of cells, tissues, or organs of mature trees to a juvenile state, restoring a higher capacity for adventitious root development and thus improving the survival rate of cuttings. This is an important measure for asexual reproduction of forest trees. Various rejuvenation methods can promote the restoration of mature trees to a juvenile state, such as continuous grafting, continuous cutting, pruning back, application of growth regulators, trunk burial, and etiolation. Currently, many trees have been restored from mature to juvenile status through rejuvenation measures, improving the regenerative capacity of adventitious roots. For example, pine trees have regained rooting vitality after continuous grafting. 'Zhongshanshan 302' is an excellent forest tree variety, possessing high ornamental value, superior wood quality, and tolerance to waterlogging and salinity. It can grow normally in soil conditions with pH < 8.5 and salinity < 0.3%. However, this variety faces a key bottleneck in large-scale propagation: as the physiological age of the mother plant increases, the adventitious root regeneration capacity of the cuttings significantly declines, making it difficult to efficiently propagate older, superior germplasm. Previous studies have found that grafting rejuvenation techniques can alter the endogenous hormone levels of cuttings and improve the adventitious root formation capacity of 'Zhongshanshan 302', but the molecular regulatory mechanism remains unknown.

[0004] In current plant biology research, the combined application of multi-omics technologies has become the mainstream strategy for elucidating the molecular mechanisms of complex traits. In particular, combining transcriptomics, miRNA sequencing, and degradome sequencing can construct a complete network map from gene expression regulation to post-transcriptional regulation, thus profoundly revealing the molecular regulatory logic of plants in growth, development, and stress responses. In a study on adventitious root formation in American red maple, researchers identified over 80,000 DEGs using transcriptomics analysis, discovered 48 known miRNAs and 95 new miRNAs, and validated 172 target genes through degradome sequencing. Further functional studies showed that specific miR160a negatively regulates adventitious root induction by targeting ARF10. This discovery not only elucidates the core role of auxin signaling in adventitious root development but also verifies the importance of miRNA-mediated regulatory networks in plant organogenesis. Similar strategies have been applied to the study of woody plants such as apples. Through multi-omics integration, scientists discovered that strigolactone (SL) inhibits adventitious root formation by downregulating core pivot genes such as MdLAC3 and MdORE1. Further miRNA-degradome interaction analysis identified key negative regulatory pairs such as mdm-miR164b-MdORE1. The construction of this regulatory network not only revealed the complex interactions between plant hormones but also provided potential molecular breeding targets for improving woody crops that are difficult to root. In addition, using miRNA, transcriptome, and degradome sequencing, the critical periods of embryo abortion in cultivated jujube and wild jujube were analyzed, identifying 1142 DEGs targeted by 93 miRNAs. It was found that 21 days after pollination is the critical period for embryo abortion, at which time carbohydrate flow is biased towards the pulp. Further construction of a miRNA-target gene regulatory network revealed that transcription factors such as DELLA, TCP14, and bHLH93 may be involved in regulating embryo development. In summary, the combined analysis of transcriptomics, miRNA sequencing, and degradome sequencing, by constructing a multi-gene hierarchical regulatory network, systematically elucidated the molecular basis from overall expression patterns to key regulatory mechanisms, thus providing a clear technical path and important theoretical guidance for overcoming the rooting problem and improving the efficiency of tissue culture propagation.

[0005] The miR156-SPL targeting module, a classic and well-studied molecular module in plant developmental regulatory networks, precisely regulates the spatiotemporal expression abundance of the SPL transcription factor through base complementarity between miR156 and the SPL gene mRNA sequence, thereby achieving post-transcriptional splicing or translational repression of the target gene. This targeting relationship is highly conserved in terrestrial plants, present in bryophytes to angiosperms, indicating its important biological function in plant evolution. In stage transition regulation, the miR156-SPL module plays a central role: high levels of miR156 in the juvenile stage effectively suppress SPL gene expression, maintaining the plant's vegetative growth state; as plant development progresses, miR156 expression gradually decreases, relieving post-transcriptional repression of the SPL gene, allowing SPL protein to accumulate and activate the expression of downstream flowering-related genes, thus promoting the plant's entry into adulthood and reproductive growth stages. This regulatory mechanism has been studied in greater depth in perennial woody fruit trees. For example, in the study of citrus fruit ripening, researchers found that the CsmiR156k-CsTHSPL2 module regulates the ripening process of citrus fruit by regulating the expression of the ABA receptor gene PYL2, accompanied by changes in epigenetic modifications such as DNA and histone methylation. This provides a potential molecular target for fruit quality improvement and postharvest preservation.

[0006] In recent years, the function of the miR156-SPL module in adventitious root development has gradually attracted attention. However, whether this module participates in the process of rejuvenation and promotion of adventitious root development in *Taxus chinensis*, and its specific molecular mechanisms and physiological functions, remain unreported. Therefore, identifying relevant genes and elucidating the regulatory mechanism of the miR156-SPL module in promoting adventitious root development in *Taxus chinensis* is of significant theoretical and applied importance for the breeding and promotion of superior *Taxus chinensis* varieties. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a DNA fragment miR156a for regulating the development of adventitious roots of Metasequoia glyptostroboides, the target gene ThSPL2, the miR156a-ThSPL2 module composed of them, and their applications.

[0008] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0009] The DNA fragment miR156a used to regulate the development of adventitious roots of Metasequoia glyptostroboides has the nucleotide sequence shown in SEQ ID NO.1.

[0010] The DNA fragment miR156a used to regulate the expression level of the ThSPL2 gene of Taxodium alpinum has the nucleotide sequence shown in SEQ ID NO.1, and the CDS sequence of the ThSPL2 gene is shown in SEQ ID NO.2.

[0011] An overexpression vector or host bacterium containing the aforementioned DNA fragment miR156a.

[0012] The application of the DNA fragment miR156a in promoting the formation of adventitious roots in plants.

[0013] In the aforementioned application, the plant is either Metasequoia glyptostroboides or Populus tomentosa.

[0014] An overexpression vector or host bacterium containing the aforementioned Metasequoia glyptostroboides gene ThSPL2.

[0015] The application of the Metasequoia glyptostroboides gene ThSPL2 in promoting the formation of adventitious roots in plants.

[0016] In the aforementioned application, the plant is either Metasequoia glyptostroboides or Populus tomentosa.

[0017] The miR156a-ThSPL2 module is used to study the development of adventitious roots in plants. The module contains a DNA fragment miR156a and its target gene ThSPL2. The nucleotide sequence of the DNA fragment miR156a is shown in SEQ ID NO.1, and the CDS sequence of the ThSPL2 gene is shown in SEQ ID NO.2.

[0018] Overexpression vectors or host bacteria containing the miR156a-ThSPL2 module and their application in plant cutting propagation.

[0019] The application of the miR156a-ThSPL2 module for regulating the development of adventitious roots of Metasequoia glyptostroboides is described above. The module is used to regulate the development of adventitious roots in plants, wherein miR156a negatively regulates the expression of ThSPL2 and promotes adventitious root development by inhibiting the activity of ThSPL2.

[0020] The application of the miR156a-ThSPL2 module for regulating the development of adventitious roots in Metasequoia glyptostroboides is described. This module is used in plant cutting propagation to promote the formation and development of adventitious roots by increasing the expression level of miR156a or inhibiting the expression level of ThSPL2.

[0021] This invention uses juvenile and mature cuttings of 'Zhongshanfeng 302' as materials to systematically compare the physiological and histological differences between the two during the rooting process. Through multi-omics analysis, the core regulatory module miR156a-ThSPL2 was screened and verified. Using transient co-transformation in tobacco and dual-luciferase reporter gene experiments, it was confirmed that miR156a can specifically target ThSPL2 and negatively regulate its expression. Overexpression of miR156a in 84K poplar significantly promoted adventitious root development, while overexpression of ThSPL2 significantly inhibited adventitious root development. Physiological analysis showed that in miR156a overexpressing lines, peroxidase (POD) and polyphenol oxidase (PPO) activities and starch content were significantly reduced, while soluble sugar content was significantly increased; while ThSPL2 overexpressing lines showed the opposite trend. This indicates that the miR156a-ThSPL2 module affects adventitious root development by regulating reactive oxygen species metabolism, phenolic oxidation, and carbon metabolism reprogramming.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0023] 1) This application is the first to clone and identify the DNA fragment miR156a and ThSPL2 genes in Metasequoia glyptostroboides, and to clarify their negative regulatory role in promoting adventitious root development during regeneration. It reveals the core regulatory function of the miR156a-ThSPL2 module in promoting adventitious root development during regeneration in woody plants, and confirms the direct targeting and inhibition relationship between miR156a and ThSPL2.

[0024] 2) This application provides key physiological data and histological support for elucidating the intrinsic relationship between age differences in woody plant cuttings and their ability to form adventitious roots. It offers new insights into the mechanisms of age-dependent decline and rejuvenation of rooting ability in Metasequoia glyptostroboides, and provides potential molecular targets and theoretical guidance for molecular breeding and efficient asexual reproduction of Metasequoia glyptostroboides and other woody plants that are difficult to root. Attached Figure Description

[0025] Figure 1 This is a phenotypic observation diagram of adventitious root development in mature and juvenile cuttings of 'Zhongshanfeng 302';

[0026] Figure 2 These are paraffin-stained sections of 'Zhongshanfeng 302' at the critical stage of adventitious root development in mature and juvenile cuttings.

[0027] Figure 3 This is a chart showing the physiological indicators measured during the critical period of adventitious root development in mature and juvenile cuttings of 'Zhongshanfeng 302'.

[0028] Figure 4 This is a graph showing the transcriptome sequencing results;

[0029] Figure 5 This is a graph showing the results of miRNA sequencing.

[0030] Figure 6 This is a graph showing the combined analysis results of multi-omics sequencing;

[0031] Figure 7 This is a diagram of 5 miRNA-transcription factor regulatory pairs obtained through joint analysis and screening;

[0032] Figure 8 This is a verification diagram of the miR156a-ThSPL2 interaction based on tobacco transient co-conversion and dual-luciferase gene reporter experiments;

[0033] Figure 9 This is a graph showing the phenotypic data, relative expression levels, and physiological parameters of miR156a-ThSPL2 overexpression. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. In the following embodiments, operations not described in detail are all routine biological experimental procedures and can be performed with reference to molecular biology experimental manuals and existing publicly available journal literature. Reagents (kits) whose sources are not specifically provided are also reagents commonly used in the art.

[0035] Example 1: Comparison of the rooting ability of 'Taxus chinensis 302' cuttings

[0036] Using juvenile and mature (30-year-old) cuttings of 'Zhongshanfeng 302' grown at the same time as materials, cuttings were collected at three key stages of adventitious root development (induction, initiation, and rooting stages) to compare their differences in rooting ability. The mature cuttings at the three stages were named MS1, MS2, and MS3, respectively, and the juvenile cuttings were named RS1, RS2, and RS3, respectively. Basal stem cortex and root tissues were collected and frozen at -80℃ for subsequent multi-omics and physiological index determinations. The remaining samples were fixed with FAA for paraffin sectioning. Juvenile cuttings were obtained from the cutting orchard of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, and mature cuttings were obtained from the National Zhongshanfeng Forest Seedling Base in Jingjiang City, Jiangsu Province.

[0037] The cutting experiment was conducted in late June 2024. Semi-lignified cuttings were selected, pruned to 10-12 cm, retaining 3-5 compound leaves, with the upper cut horizontal and the lower cut oblique. The base was soaked in 2000 mg / L NAA solution for 2 min, and after moisturizing treatment, they were planted at a spacing of 4 cm × 7 cm, at a depth of 4-5 cm. The substrate was perlite and peat moss (volume ratio 1:1), which was disinfected with 50% carbendazim at 800 times dilution before planting. Moisture was regulated by an automatic sprayer to maintain relative humidity at 80%-90%. Each treatment had 3 replicates, with 400 cuttings per replicate. Observations and data were collected every 7 days, and samples were taken at 3 key periods. The results showed that after 21 days of planting, more than 60% of the young cuttings formed callus tissue, and the base swelled (…). Figure 1 (a) ; Only 27.1% of mature cuttings formed callus, while the rest mostly showed blackening at the base ( Figure 1 (a)). After 63 days, the rooting rate of the regenerated cuttings reached 52.8% ( Figure 1 (a) and (b)); the rooting rate of mature cuttings was only 2.9%, 70.4% were stuck in the callus stage, and 26.7% showed no signs of rooting. Figure 1 (a) and (b)). It was confirmed that the rejuvenation treatment significantly improved the rooting ability of 'Zhongshanfeng 302'.

[0038] Example 2: Results of paraffin section staining of rooted cuttings of 'Zhongshanfeng 302'

[0039] The fixed stem cortex and root tissues (RS3 roots were directly embedded) were dehydrated with graded ethanol, cleared with xylene, and impregnated with paraffin before embedding. 8-10 μm serial sections were prepared using a rotary microtome, stained with toluidine blue, and mounted for observation. Results showed significant cytological differences between juvenile and mature cuttings during adventitious root development. Figure 2 In the early stages of induction (MS1 and RS1), both cell lines were tightly packed with no significant difference. Figure 2 (a) and (d)). 21 days after cutting, the vascular system of mature cuttings (MS2) showed cell structure damage, increased chromatin condensation in the nuclei, and programmed cell death characteristics, consistent with the basal browning phenotype. Figure 2 (b)); while the cells of the rejuvenated cuttings (RS2) enter an active dedifferentiation state, their volume expands, and they form typical callus cell clusters ( Figure 2 (e)). 63 days after cutting, the cell volume of mature cuttings (MS3) became heterogeneous, and callus tissue continued to proliferate. Figure 2 (c)); The root cap progenitor cells of the rejuvenated cuttings (RS3) showed localized active division, with dense cytoplasm, ultimately initiating the morphogenesis of adventitious root primordia ( Figure 2 (f)

[0040] Example 3: Determination of physiological indicators during the critical period of adventitious root development in mature and juvenile cuttings.

[0041] To assess the changes in relevant physiological indicators of mature and juvenile shoots of 'Zhongshanfeng 302' during three key stages of adventitious root development, the contents of malondialdehyde (MDA), peroxidase (POD), superoxide dismutase (SOD), soluble sugars, and soluble proteins were determined using kits from Nanjing Comerson Biotechnology Co., Ltd. The measurement procedures were performed according to the provided instruction manual, and each treatment included three biological replicates.

[0042] The results are as follows Figure 3 As shown, mature cuttings showed high MDA accumulation at the MS2 stage, indicating significant oxidative damage to the cell membrane system; while the MDA content of rejuvenated cuttings decreased at the RS3 stage, indicating that rejuvenation treatment could alleviate membrane lipid peroxidation in the later stages of rooting. Figure 3 (a)). POD activity ( Figure 3 In the middle (b) stage, POD activity significantly increased to a peak during the rejuvenating RS2 stage, possibly promoting root primordia differentiation; however, POD activity decreased during the mature cutting MS2 stage, indicating impaired antioxidant metabolism. SOD activity ( Figure 3 (c) showed a stress-induced peak at the mature MS2 stage, indicating that it suffered from strong oxidative stress; the SOD of the rejuvenated cuttings continued to rise at the RS2 stage, indicating that it could initiate an orderly ROS scavenging mechanism earlier. Soluble sugar content ( Figure 3 The soluble protein content of the cuttings increased during the mature MS3 stage and peaked during the rejuvenated RS3 stage, suggesting that sugar metabolism plays an important role in callus and adventitious root formation. (The text also mentions soluble protein content in rejuvenated cuttings, but this seems unrelated to the main topic of sugar metabolism.) Figure 3 The values ​​of the middle (e) materials were consistently higher than those of mature materials from the RS2 to RS3 stages, and peaked at the RS3 stage, indicating that rejuvenation can promote protein synthesis, enhance metabolism and resistance, and is beneficial to the formation and development of adventitious roots.

[0043] Example 4: Transcriptome sequencing of key genes for rejuvenating and improving adventitious root development of 'Zhongshanfeng 302' based on RNA-seq.

[0044] Total RNA was extracted from each sample, and after quality control, mRNA enrichment, and fragmentation, double-stranded cDNA was synthesized by reverse transcription. Fragments of 370-420 bp were selected to construct libraries, and PE150 paired-end sequencing was performed on the Illumina Novaseq™ 6000 platform. The raw data underwent quality control to obtain high-quality clean reads, which were then aligned to the *Cephalotaxus fortunei* reference genome for gene expression calculation, DEGs screening, and functional enrichment analysis. Transcript assembly and quality assessment were performed using Trinity and BUSCO.

[0045] PCA analysis shows that ( Figure 4In (a), there was significant differentiation between samples from different treatment groups, and the reproducibility was reliable. Based on the phenotypes of juvenile and mature cuttings at the three key stages, four principal comparison groups were set up, and DEGs were statistically analyzed and Venn diagram analysis was performed. Figure 4 (b) and (c)). Mfuzz clustering was used to divide the 21353 DEGs of 18 samples into 10 clusters ( Figure 4 (d) KEGG pathway enrichment analysis was performed on each cluster, and the results were summarized in a bubble chart. Figure 4 (e)). The results showed that clusters 2 and 5 exhibited an increasing trend during the critical period of adventitious root development. Both clusters were enriched in plant hormone signal transduction and ABC transporter pathways; cluster 2 was also enriched in phenylpropanoid biosynthesis and brassinolide biosynthesis pathways; while cluster 5 was enriched in plant-pathogen interactions, histidine metabolism, and plant MAPK signaling pathways (e). Figure 4 (e)). The pathways described above may represent important changes in the developmental stages of adventitious roots and are of great significance for subsequent joint analysis.

[0046] Example 5: Identification of miRNAs related to adventitious root development in 'Taxus chinensis 302'

[0047] miRNA sequencing and degradome sequencing were performed on the base of juvenile and mature cuttings to systematically identify miRNAs and their target genes related to adventitious root development. RNA extraction was performed as described in Example 4. Small RNA libraries were purified and enriched by PAGE and then SE50 sequencing was performed on an Illumina Hiseq 2000 / 2500 platform. For degradome sequencing, equal amounts of mixed RNA samples were used, and mRNA fragments containing PolyA tails were enriched by Oligo(dT). After adding 5' adapters, reverse transcription was performed to construct 200-400 bp libraries, and 50 bp single-end sequencing was performed on an Illumina Hiseq 2500 platform. RT-qPCR was used to validate nine differentially expressed miRNAs, with U6 as an internal control. Primer design is shown in Table 1.

[0048] Table 1. RT-qPCR primers for differentially expressed miRNAs

[0049]

[0050] from Figure 5 It can be observed that the miRNA sequence length is mainly concentrated in the range of 18-24 nt, with 21 nt having the highest proportion. Figure 5 (a)). Statistical analysis of differentially expressed miRNAs in the four comparison groups showed ( Figure 5In (b), RS3 vs RS2 contained the most differentially expressed miRNAs (68), followed by RS3 vs MS3 (59). Heatmap analysis of the two key comparison groups, RS3 vs MS3 and RS2 vs MS2, identified known conserved miRNAs such as miR156a, miR319a, and miR396b, as well as several novel miRNAs. RT-qPCR validation results showed a high degree of consistency with the expression trends in sequencing data, confirming the reliability of the sequencing results. GO enrichment analysis of degradome target genes showed that they were mainly enriched in processes such as biosynthesis, DNA transcriptional regulation, and protein construction. Combined analysis of transcriptome and miRNA results, along with degradome data to screen for negative regulatory relationships, yielded 21 pairs of miRNA-mRNAs in the RS3 vs MS3 and RS2 vs MS2 comparison groups. Figure 6 (a) involves 16 miRNAs regulating 19 mRNAs. Expression profiling analysis ( Figure 6 (b) and Figure 6 The regulatory patterns in (a) are basically the same. Further screening of target genes as transcription factor interaction pairs ultimately yielded 5 miRNA-transcription factor regulatory pairs. Figure 7 Among them, the TD11C4443 (ThSPL2) gene splicing site was classified as category 0. Figure 7 (a) shows the strongest supporting evidence. Therefore, the miR156a-ThSPL2 module was selected for subsequent functional verification.

[0051] Example 6: Interaction verification of miR156a-ThSPL2 modules

[0052] In this embodiment, *Nicotiana Benthamiana* Dominica was used as the material for the transient co-transformation and dual-luciferase detection system. The test seeds were preserved by our research group. The pBI121 vector was purchased from Chuzhou General Biotechnology Co., Ltd., and the pGreen-0800-Luc and pGreenll-62-SK vectors were purchased from Wuhan Boyuan Biotechnology Co., Ltd. *Escherichia coli* DH5α competent cells and *Agrobacterium tumefaciens* GV3101 competent cells were purchased from Beijing Coollab Technology Co., Ltd.

[0053] Given the limited length of miRNA precursors, directly constructing expression vectors presents technical challenges. Furthermore, to maintain the integrity of their natural stem-loop structure, this embodiment aligns the miR156a precursor sequence with a reference sequence from the *Taxus chinensis* genome to determine its precise location in the transcript. Then, it extends 300 bp upstream and downstream of this region, ultimately yielding a 724 bp DNA fragment (SEQ ID NO. 1), which completely contains the miR156a precursor sequence. Subsequently, the pBI121 vector is double-digested (XbaI-SacⅠ) using homologous recombination. The stop codon is removed from the ThSPL2 CDS sequence, yielding a 1692 bp sequence (SEQ ID NO. 2). This 1692 bp sequence is then ligated into the pBI121 linearized vector to construct a 35S::ThSPL2 overexpression vector. The recombinant plasmids pBI121-miR156a and pBI121-ThSPL2 were transformed into Agrobacterium GV3101 using the freeze-thaw method. The interaction between miR156a and ThSPL2 was verified in Agrobacterium-mediated transient co-transformation of tobacco by constructing miR156a overexpression vectors and ThSPL2-GUS fusion vectors. Figure 8 (a)

[0054] After transient conversion, tobacco leaves were thoroughly rinsed with ddH2O and transferred to 50 mL centrifuge tubes. GUS staining working solution was then immediately prepared: X-Gluc concentrate (50×) and GUS buffer were mixed at a volume ratio of 1:50 and thoroughly shaken. All reagents were purchased from the ready-to-use GUS detection kit of Beijing Solarbio Science & Technology Co., Ltd. The prepared GUS staining solution was injected into 50 mL centrifuge tubes, ensuring the tobacco leaves were completely submerged. The centrifuge tubes were wrapped with aluminum foil and placed in a 37℃ constant-temperature shaker at 100 rpm for 24-48 h. After staining, the samples were transferred to a solution containing 70% ethanol and decolorized at 37℃ for 6-8 h (if decolorization was inadequate, the solution could be replaced with 90% ethanol and treatment continued until the pigment in the leaves was completely removed). Finally, the decolorized samples were observed and image data recorded. Simultaneously, GUS activity in the same treated tobacco leaves was detected using a GUS gene quantification kit (Beijing Cooler Master Technology Co., Ltd.).

[0055] Experimental results show that ( Figure 8In (b), transient expression of 35S::miR156a in tobacco leaves resulted in white leaves with no GUS protein accumulation. In contrast, transformation with either 35S::GUS or 35S::ThSPL2 alone resulted in a larger and darker blue area in the leaves, confirming efficient transcription of the GUS gene. These control results verified that the GUS sequence in 35S::miR156a was successfully excised, while GUS in the 35S::ThSPL2 vector was normally expressed. When 35S::miR156a and 35S::ThSPL2 were co-transformed, the blue area in the leaves significantly decreased and the color became lighter, indicating a negative regulatory interaction between 35S::miR156a and 35S::ThSPL2. GUS activity in tobacco leaves was further detected using a GUS gene quantification kit. The results showed ( Figure 8 In (c), when 35S::GUS or 35S::ThSPL2 were transformed alone, the GUS activity was not significantly different; when 35S::miR156a was transformed, no GUS activity was generated; and when 35S::miR156a and 35S::ThSPL2 were co-transformed, the GUS activity was significantly reduced. These results are consistent with... Figure 8 The observed phenomena are consistent with those in (b), indicating that there is indeed a negative regulatory relationship between 35S::miR156a and the target gene 35S::ThSPL2.

[0056] To further verify the direct regulatory relationship between miR156a and the target gene ThSPL2, an interaction verification system using a dual-luciferase reporter gene assay was employed. The experimental vector construction procedure is as follows ( Figure 8 (d) First, a precursor fragment containing the mature miR156a sequence and its flanking sequences was amplified and directionally cloned into the pGreenII 62-SK vector to construct the effector vector pGreenII 62-SK-miR156a. Simultaneously, the 3'UTR region of the ThSPL2 gene (containing the miR156a predicted binding site) was amplified and directionally inserted into the downstream multiple cloning site of the firefly luciferase gene in the pGreen0800-LUC vector to construct the reporter vector pGreen0800-ThSPL2. The correctly constructed recombinant plasmids were transformed into Agrobacterium GV3101 competent cells. After verification by bacterial PCR, positive clones were picked and cultured overnight at 28°C with shaking in YEB liquid medium (containing the corresponding antibiotic). The next day, the bacterial cells were collected and resuspended in infection buffer to OD. 600= 0.6-0.8, incubate at room temperature for 2-3 h. Mix effector and reporter bacterial solutions at an appropriate volume ratio and inject into tobacco leaves. After injection, tobacco plants are cultured normally in a 25°C light incubator for 48-72 h, maintaining appropriate humidity. Each treatment has at least 3 biological replicates, with empty vectors pGreenII62-SK + 35S::LUC, pGreenII 62-SK + 35S::LUC-ThSPL2, and pGreenII 62-SK-miR156a + 35S::LUC as relevant controls. Finally, using the luciferase luminescence value of each sample as an internal reference, the relative activity of firefly luciferase, i.e., the LUC / REN ratio, is calculated. The results show ( Figure 8 In (e), when pGreenII 62-SK + 35S::LUC, pGreenII 62-SK + 35S::LUC-ThSPL2, or pGreenII 62-SK-miR156a + 35S::LUC were transformed alone, a high LUC / REN ratio was detected; however, when the miR156a effector was co-transformed with the target vector, the LUC / REN ratio decreased significantly, by as much as 63%. This result indicates that miR156a can specifically recognize ThSPL2 and inhibit its expression, further confirming a direct negative regulatory relationship between the two at the quantitative level.

[0057] Example 7: Phenotypic data, relative expression levels, and physiological parameters of miR156a-ThSPL2 overexpression.

[0058] Poplar 84K was used as the overexpression receptor for miR156a and ThSPL2 genes. Poplar materials were purchased from Wuhan Boyuan Technology Co., Ltd. Samples used for physiological parameter determination and RT-qPCR analysis were all taken from transgenic poplar lines overexpressing miR156a and ThSPL2.

[0059] Genetic transformation of poplar was performed using Agrobacterium-mediated leaf disc transformation. The constructed miR156a and ThSPL2 overexpression vectors were transformed into Agrobacterium EHA105 competent cells, and positive clones were identified by PCR before being used for infection. Leaves from sterile 84K poplar seedlings with uniform leaf color and 1-3 months of growth were selected, and after removing the edges, even incisions were made on both sides of the midrib. The activated Agrobacterium culture was diluted with infection solution to OD0.05. 600= 0.3-0.4, immerse the leaves in the bacterial solution for 15 min, shaking continuously during the process. Remove the leaves and tear them into small pieces, inoculate them onto a co-culture medium, and incubate in the dark at 25℃ for 2 days. After co-culture, transfer the leaves to induction and selection medium for callus induction, and incubate in the dark for about 2 weeks; after callus growth, subculture every 2 weeks for a total of 3 subcultures (maximum 45 days). Transfer the induced callus to differentiation medium for adventitious shoot differentiation, subculture approximately every 3 weeks until the callus turns green and hard. When the regenerated seedlings grow to 1-3 cm, cut them off and inoculate them onto a rooting selection medium to induce rooting. Finally, extract DNA from the resistant regenerated seedlings for PCR detection to identify positive transgenic poplar lines. For the phenotypic data of 35S::miR156a and the target gene 35S::ThSPL2 screened from poplar, count the number of adventitious roots and the maximum length of adventitious roots, and count 5 biological replicates for each line. The contents of POD, polyphenol oxidase (PPO), soluble sugars, and starch were further determined using kits from Nanjing Comerson Biotechnology Co., Ltd. The measurement procedures were performed according to the provided instruction manual, and each treatment included three biological replicates.

[0060] To understand the roles of 35S::miR156a and 35S::ThSPL2 in the development of adventitious roots in poplar, the phenotypic development of poplar was visually demonstrated through observation. The results showed that ( Figure 9 In the study (a), compared with WT, 35S::miR156a significantly promoted adventitious root development, resulting in a significant increase in plant height, adventitious root number, and maximum adventitious root length; while overexpression of 35S::ThSPL2 inhibited poplar adventitious root development, with both the number of adventitious roots and the maximum adventitious root length being significantly lower than WT (a). Figure 9 (b) and (c)). The above results indicate that miR156a and ThSPL2 exhibit opposite functional patterns in poplar adventitious root development. Combined with the previous dual-luciferase validation results of miR156a's targeted inhibition of ThSPL2, this further demonstrates that the negative regulatory relationship of the miR156a-ThSPL2 module is manifested at the functional level.

[0061] Subsequently, RT-qPCR was used to detect the expression levels of miR156a and SPL2 in each transgenic line, with WT poplar as a control. The results showed that in the miR156a line, miR156a expression was significantly upregulated to 5.4-fold that of WT, while SPL2 expression was significantly downregulated. Figure 9 (d) further confirmed the targeted inhibitory effect of miR156a on SPL2. In the SPL2 strain, SPL2 expression was significantly upregulated to 6.5-fold of WT, while miR156a expression showed no significant difference. Figure 9(e) indicates that SPL2 was successfully expressed, and as a transcription factor, it did not show any feedback inhibition on upstream miR156a. The results of poplar physiological index measurements showed that ( Figure 9 In the middle (f)-(i) sections, compared with WT, the 35S::miR156a line showed significantly decreased POD activity, PPO activity, and starch content, while soluble sugar content was significantly increased; conversely, in the 35S::ThSPL2 line, POD activity, PPO activity, and starch content were significantly increased, while soluble sugar content was significantly decreased. This result suggests that miR156a overexpression may maintain a higher IAA level by reducing POD activity, while PPO-mediated phenolic metabolism may be involved in regulating adventitious root development, and its low activity is beneficial for maintaining cell division activity, thereby promoting rooting. Simultaneously, soluble sugars, as energy sources and osmotic regulators, provide the carbon skeleton and energy for adventitious root development, while starch is hydrolyzed into soluble sugars for energy. ThSPL2 overexpression, however, exhibits the opposite physiological characteristics, leading to inhibited rooting ability.

Claims

1. A DNA fragment miR156a for regulating the development of adventitious roots of Metasequoia glyptostroboides, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

2. The application of the DNA fragment miR156a according to claim 1 in regulating the expression level of the *Taxus chinensis* gene ThSPL2, characterized in that, in, The CDS sequence of the ThSPL2 gene is shown in SEQ ID NO.

2.

3. An overexpression vector or host bacterium containing the DNA fragment miR156a as described in claim 1.

4. The application of the DNA fragment miR156a as described in claim 1 in promoting the formation of adventitious roots in plants.

5. The application according to claim 4, characterized in that, The plant in question is either Metasequoia glyptostroboides or Populus tomentosa.

6. An overexpression vector or host bacterium containing the CDS sequence of the ThSPL2 gene of Taxodium distichum, as shown in SEQ ID NO.

2.

7. Application of the ThSPL2 gene of Metasequoia glyptostroboides with a CDS sequence as shown in SEQ ID NO.2 in promoting the formation of adventitious roots in plants.

8. The application according to claim 7, characterized in that, The plant in question is either Metasequoia glyptostroboides or Populus tomentosa.

9. A miR156a-ThSPL2 module for studying adventitious root development in plants, characterized in that, The module contains a DNA fragment miR156a and its target gene ThSPL2; the nucleotide sequence of the DNA fragment miR156a is shown in SEQ ID NO.1, and the CDS sequence of the ThSPL2 gene is shown in SEQ ID NO.

2.

10. An overexpression vector or host bacterium containing the miR156a-ThSPL2 module of claim 9 and its application in the study of plant cutting propagation.

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