PtoSAUR74 gene related to poplar adventitious root formation ability and application thereof

Abstract

This invention discloses a PtoSAUR74 gene related to the adventitious root formation ability of poplar and its applications. The nucleotide sequence of the coding region of the PtoSAUR74 gene disclosed in this invention is shown in SEQ ID NO.1, and the protein sequence is shown in SEQ ID NO.2. Overexpression of the PtoSAUR74 gene in poplar significantly inhibits the length, number, and growth rate of adventitious roots, indicating that the PtoSAUR74 gene is a key regulatory gene affecting the development of adventitious roots in poplar, providing a key target for precise molecular regulation of poplar root growth. This invention also discloses a method for regulating the adventitious root formation ability of poplar. By regulating the expression of the PtoSAUR74 gene, adventitious root formation can be directly inhibited, providing new technical means and theoretical basis for the genetic improvement of poplar.

Description

A PtoSAUR74 gene associated with the adventitious root formation ability of poplar and its application Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the PtoSAUR74 gene, which is related to the ability of poplar trees to form adventitious roots, and its application. Background Technology

[0002] Plant roots are vital organs for anchoring the plant and absorbing water and nutrients. Unlike lateral roots, which develop from the radicle, adventitious roots can arise from various non-root organs such as stems and leaves. This developmental plasticity is of great significance for plant environmental adaptation and asexual reproduction (such as cuttings). In agricultural and forestry production, the efficiency of cutting propagation directly depends on the explant's ability to produce adventitious roots.

[0003] The development of adventitious roots is primarily regulated by auxin. Auxin accumulates at the base and, through the TIR1 / AFB-Aux / IAA-ARF signaling pathway, activates a series of downstream response elements, such as ARF7, ARF19, and their target genes LBD16 and LBD29, collectively driving the initiation and development of adventitious root primordia. In recent years, the plant SAUR (Small Auxin-Up RNA) gene family, as one of the core gene families for early auxin response, has played a crucial role in plant root development, environmental adaptation, and cell elongation regulation. Existing research indicates that SAUR genes mainly function by regulating cell elongation and cell wall relaxation. Their mechanism involves SAUR proteins inhibiting plasma membrane-associated PP2C.D phosphatase activity, thereby activating the plasma membrane H+-ATPase proton pump, increasing the plasma membrane electrochemical potential, acidifying the apoplast, and ultimately promoting cell expansion. For example, in Arabidopsis thaliana, overexpression of AtSAUR41 leads to pleiotropic phenotypes, including increased hypocotyl elongation, nutrient biomass and lateral root development, petal enlargement, and inflorescence stem twisting. Similarly, overexpression of the cucumber CsSAUR31 gene also significantly promotes root and hypocotyl elongation.

[0004] However, a robust developmental system necessarily contains both positive "promotion" and cautious "inhibition" mechanisms. Currently, research on the regulation of adventitious root formation is significantly unbalanced: we have a good understanding of the positive linear pathway from auxin sensing (TIR1) to signal transduction (ARF) and then to cellular responses (such as SAUR), but we know very little about the negative regulatory links in this pathway, as well as the upstream inhibitory mechanisms of key effector factors such as SAUR. Therefore, creating novel forest tree genotypes with controlled root sucker and adventitious root development capabilities, significantly weakening or even eliminating their ability to reproduce asexually through root suckers and branch breakage in natural environments, will lay a solid foundation for achieving safer, more efficient, and more eco-friendly modern forestry. Summary of the Invention

[0005] To overcome the above problems, the inventors first identified a PtoSAUR74 gene and its encoded protein that are related to the adventitious root formation ability of poplar. By introducing this gene into poplar trees to obtain overexpressing transgenic plants, it was found that the number, length, and growth rate of adventitious roots in poplar trees overexpressing the PtoSAUR74 gene were significantly inhibited compared with WT, indicating that this gene can precisely regulate the formation of adventitious roots in poplar trees. This provides a key target for achieving precise molecular regulation of poplar root growth, and also provides a new theoretical basis for the genetic improvement of poplar trees, laying a solid foundation for achieving safer, more efficient, and more eco-friendly modern forestry, thus completing this invention.

[0006] Specifically, the object of the present invention is to provide the following aspects:

[0007] In one aspect, a PtoSAUR74 gene related to the adventitious root formation ability of poplar is provided, the nucleotide sequence of the coding region of the gene being shown in SEQ ID NO.1.

[0008] In a second aspect, a protein related to the adventitious root formation ability of poplar is provided, the protein being encoded by the PtoSAUR74 gene described in the first aspect, and its amino acid sequence is shown in SEQ ID NO.2.

[0009] Thirdly, the application of the PtoSAUR74 gene described in the first aspect or the protein described in the second aspect in regulating the formation of adventitious roots in poplar is provided.

[0010] Fourthly, a vector and / or strain comprising the PtoSAUR74 gene described in the first aspect is provided, the vector comprising an overexpression recombinant vector, and the strain comprising Escherichia coli and Agrobacterium.

[0011] Fifthly, the application of the PtoSAUR74 gene described in the first aspect, the protein described in the second aspect, or the vector and / or strain described in the fourth aspect in poplar breeding is provided.

[0012] In a sixth aspect, a method for regulating the adventitious root formation ability of poplar is provided, the method comprising the step of overexpressing or the PtoSAUR74 gene described in the first aspect.

[0013] The beneficial effects of this invention include:

[0014] (1) This invention is the first to identify that the PtoSAUR74 gene has the function of significantly inhibiting the formation of adventitious roots and the length of root growth in poplar, providing a key target for the precise molecular regulation of poplar root growth;

[0015] (2) The method for regulating the ability of poplar to form adventitious roots provided by the present invention can directly inhibit the formation of adventitious roots by regulating the expression of the PtoSAUR74 gene, providing a new technical means and theoretical basis for the genetic improvement of poplar.

[0016] (3) This invention clarifies the direct application of the PtoSAUR74 gene and its encoded protein in regulating the formation of adventitious roots in poplar trees. It can be used to create new tree genotypes with controlled root sucker and adventitious root development capabilities, significantly weakening or even eliminating their ability to reproduce asexually through root suckers and broken branches in natural environments, laying a solid foundation for achieving safer, more efficient, and more eco-friendly modern forestry.

[0017] (4) The application of poplar varieties that intervene in the development of adventitious roots provided by this invention has clear regulatory gene functions, and the cultivation method is fast and effective, which helps to reduce breeding costs, improve breeding efficiency, and shorten the growth period. Attached Figure Description

[0018] Figure 1 shows a schematic diagram of the structure of the PtoSAUR74-pBI121-EGFP overexpression vector constructed in Example 2;

[0019] Figure 2 shows the transcriptional level of PtoSAUR74 in overexpressing plants (OE-3, OE-6) as detected by RT-qPCR;

[0020] Figure 3 shows the root morphology phenotypes of wild-type plants (WT-1, WT-2) and PtoSAUR74 gene overexpressing plants (OE-3, OE-6) after 0, 4, 5, 6, and 12 days of growth in 1 / 2 MS medium in tissue culture flasks.

[0021] Figure 4 shows the total length of adventitious roots of wild-type plants (WT-1, WT-2) and PtoSAUR74 gene overexpressing plants (OE-3, OE-6) after 12 days of growth in tissue culture bottles.

[0022] Figure 5 shows the average length of adventitious roots of wild-type plants (WT-1, WT-2) and PtoSAUR74 gene overexpressing plants (OE-3, OE-6) after 12 days of growth in tissue culture bottles.

[0023] Figure 6 shows the number of adventitious roots in the root systems of wild-type plants (WT-1, WT-2) and PtoSAUR74 gene overexpressing plants (OE-3, OE-6) after 12 days of growth in tissue culture bottles.

[0024] Figure 7 shows the proportion of adventitious roots formed in wild-type plants (WT-1, WT-2) and PtoSAUR74 gene overexpressing plants (OE-3, OE-6) 12 days after cutting. Detailed Implementation

[0025] The present invention will be further described in detail below through preferred embodiments and examples. Through these descriptions, the features and advantages of the present invention will become clearer and more apparent.

[0026] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0027] In a first aspect, the present invention provides a PtoSAUR74 gene associated with the ability of poplar trees to form adventitious roots, the nucleotide sequence of the coding region of the gene being shown in SEQ ID NO.1.

[0028] Preferably, the adventitious root formation ability of poplar includes the number of adventitious roots, the length of adventitious roots, and the rooting rate. More preferably, the adventitious root formation ability of poplar is the number of adventitious roots, the length of adventitious roots, and / or the rooting rate.

[0029] In this invention, the poplar is white poplar, preferably hairy white poplar or 84k poplar.

[0030] Among them, the 84k poplar is an asexual variety produced by the hybridization of silver poplar and glandular poplar (silver poplar × glandular poplar, Populus alba × Populus glandulosa). It belongs to the preferred tree species of the poplar school. Due to its easy rooting, rapid growth, excellent wood quality and wide adaptability, and because its transformation conditions are easier to standardize, it is often selected as an ideal model for genetic transformation.

[0031] In a preferred embodiment, the PtoSAUR74 gene is located on chromosome CM031988.1 of the Populus tomentosa genome.

[0032] Preferably, the PtoSAUR74 gene is located on chromosome CM031988.1 of the Populus tomentosa genome (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_018804465.1 / ), with its CDS region starting at position 14068599 and ending at position 14068976.

[0033] The PtoSAUR74 gene provided by this invention has a well-defined function and can precisely regulate the adventitious root formation ability of poplar trees. It provides an in-depth analysis of the molecular regulatory mechanism of adventitious root development in poplar trees and provides an important theoretical basis for molecular breeding of poplar trees.

[0034] This invention is the first to identify that the PtoSAUR74 gene has the function of significantly inhibiting the formation of adventitious roots and the length of root growth in poplar trees, providing a key target for the precise molecular regulation of poplar root growth.

[0035] In a second aspect, the present invention provides a protein related to the ability of poplar trees to form adventitious roots, which is a protein encoded by the PtoSAUR74 gene described in the first aspect, and the amino acid sequence is shown in SEQ ID NO.2.

[0036] A third aspect of the present invention provides the application of the PtoSAUR74 gene, which is associated with the ability of poplar to form adventitious roots as described in the first aspect, or the protein, which is associated with the ability of poplar to form adventitious roots as described in the second aspect, in regulating the formation of adventitious roots in poplar.

[0037] Preferably, the adventitious root formation capacity of the poplar tree is defined as the number of adventitious roots, the length of the adventitious roots, and / or the rooting rate.

[0038] In a preferred embodiment, the application is carried out by regulating the expression of the PtoSAUR74 gene in poplar individuals or by regulating the activity of proteins related to the adventitious root formation ability of poplar individuals.

[0039] Preferably, the application involves inhibiting the formation of adventitious roots in poplar trees by increasing the expression of the PtoSAUR74 gene in individual poplar trees.

[0040] More preferably, the application is carried out by overexpressing the PtoSAUR74 gene, which is associated with the adventitious root formation ability of poplar trees, in poplar individuals.

[0041] The PtoSAUR74 gene negatively regulates the number of adventitious roots, the length of adventitious roots, and the rooting rate of poplar adventitious roots.

[0042] This invention clarifies the direct application of the PtoSAUR74 gene and its encoded protein in regulating the formation of adventitious roots in poplar trees. It can be used to create new tree genotypes with controlled root sucker and adventitious root development capabilities, significantly weakening or even eliminating their ability to reproduce asexually through root suckers and broken branches in natural environments, thus laying a solid foundation for achieving safer, more efficient, and more eco-friendly modern forestry.

[0043] A fourth aspect of the invention provides a vector and / or strain comprising the PtoSAUR74 gene described in the first aspect, the vector comprising an overexpression recombinant vector, and the strain comprising Escherichia coli and Agrobacterium.

[0044] Preferably, the overexpression recombinant vector is obtained by constructing the CDS sequence of the PtoSAUR74 gene into the pBI121-EGFP vector.

[0045] More preferably, the Agrobacterium is GV3101.

[0046] A fifth aspect of the invention provides the application of the gene described in the first aspect, the protein described in the second aspect, or the vector and / or strain provided in the third aspect in poplar breeding.

[0047] Preferably, the poplar is a white poplar, more preferably a hairy white poplar or 84K poplar, and the breeding is: to cultivate poplar varieties that inhibit the development of adventitious roots.

[0048] More preferably, the breeding involves overexpressing the PtoSAUR74 gene in poplar trees to cultivate poplar varieties with inhibited adventitious root development.

[0049] The application of poplar varieties that intervene in adventitious root development provided by this invention has clearly defined gene functions, and the cultivation method is rapid and effective, which helps to reduce breeding costs, improve breeding efficiency, and shorten the growth period.

[0050] A sixth aspect of the present invention provides a method for regulating the adventitious root formation ability of poplar trees, the method comprising the step of overexpressing the PtoSAUR74 gene described in the first aspect.

[0051] Preferably, the regulation is to reduce the ability of poplar trees to form adventitious roots.

[0052] The adventitious root formation ability includes the number of adventitious roots, the length of adventitious roots, and / or the rooting rate.

[0053] Preferably, when the PtoSAUR74 gene is overexpressed, the number of adventitious roots, the length of adventitious roots, and the rooting rate of poplar trees decrease.

[0054] In a preferred embodiment, overexpression of the PtoSAUR74 gene is achieved by introducing a PtoSAUR74 gene overexpression vector into poplar trees. The PtoSAUR74 gene overexpression vector is obtained by constructing the CDS sequence of the PtoSAUR74 gene onto a base vector.

[0055] Preferably, the CDS sequence of the PtoSAUR74 gene is shown in SEQ ID NO.1.

[0056] More preferably, the base vector is pBI121-EGFP.

[0057] The pBI121-EGFP vector is 13629 bp in length and contains a strong 35S promoter (CaMV35S). The resistance in the transgenic plant screening is kanamycin (Kana), which can be digested with XbaI enzyme.

[0058] In a preferred embodiment, the method for regulating the adventitious root formation ability of poplar trees includes the following steps:

[0059] Step 1: Construct a recombinant vector for overexpression of the PtoSAUR74 gene.

[0060] Preferably, step 1 includes the following sub-steps:

[0061] Step 1-1: Obtain the coding region sequence of the PtoSAUR74 gene.

[0062] Preferably, the coding region sequence of the PtoSAUR74 gene is obtained by amplification using primers PtoSAUR74-F and PtoSAUR74-R.

[0063] The sequence of primer PtoSAUR74-F is agaacacgggggactATGAAGAAGATCAACTTGATACTAA; the sequence of primer PtoSAUR74-R is acccccggggatcctGAACATGTAGAGATCAGCTAATTCT.

[0064] In this sequence, the lowercase part is the vector homologous arm sequence, and the uppercase part is the gene sequence.

[0065] Steps 1-2: Ligate the coding region sequence of the PtoSAUR74 gene to the basic vector for transformation.

[0066] Preferably, the base vector is the pBI121-EGFP vector, which is digested with XbaI.

[0067] The ligated vector was transformed into Escherichia coli DH5α competent cells.

[0068] Steps 1-3: Identify the recombinant vector after ligation.

[0069] Preferably, the Escherichia coli bacterial culture is identified by PCR under the following conditions: 94℃ for 5 min; (94℃ for 30 s; 58℃ for 30 s; 72℃ for 70 s) for 35 cycles; 72℃ for 10 min; 4℃ for ∞.

[0070] Step 2: Transform the constructed overexpression recombinant vector into Agrobacterium.

[0071] Step 3: Poplar materials were infected, proliferated, differentiated, and rooted using Agrobacterium-mediated transformation to obtain transgenic plants.

[0072] Preferably, the poplar material is 84K poplar.

[0073] Step 4: Identify the obtained transgenic plants.

[0074] Preferably, the identification includes PCR identification and RT-PCR (quantitative real-time PCR) identification.

[0075] In a preferred embodiment, the primers used in the RT-PCR reaction are PtoSAUR74-qPCR-F and PtoSAUR74-qPCR-R, whose nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0076] Preferably, the RT-PCR reaction program is as follows: 95℃ for 3 min; (95℃ for 5 sec; 60℃ for 30 sec; 72℃ for 15 sec) for 40 cycles; 72℃ for 5 min; 4℃ for ∞.

[0077] More preferably, plants overexpressing the PtoSAUR74 gene are those where PCR identification results show specific bands and RT-PCR results show a significant increase in the transcription level of the PtoSAUR74 gene.

[0078] The PCR identification primers and reaction procedures selected in this invention were determined through extensive experimental research and are capable of accurately identifying transgenic plants.

[0079] In a preferred embodiment, adventitious root phenotypic analysis was performed on plants overexpressing the PtoSAUR74 gene. Compared with wild-type plants, the number of adventitious roots, adventitious root length, and rooting rate of the overexpressing plants were significantly reduced.

[0080] Preferably, after culturing in 1 / 2 MS medium for 12 days, the total length of adventitious roots of PtoSAUR74 gene overexpressing plants was reduced by 8.21 cm compared with wild-type plants;

[0081] After 12 days of culture in 1 / 2 MS medium, the average length of adventitious roots in PtoSAUR74 gene-overexpressing plants was reduced by 1.55 cm compared to wild-type plants.

[0082] After 12 days of culture in 1 / 2 MS medium, the number of adventitious roots in PtoSAUR74 gene-overexpressing plants was reduced by 3.17 compared to WT.

[0083] Some individuals of the WT strain began to root on day 4, while none of the PtoSAUR74 overexpression strains had rooted by this time. By day 6, the rooting rate of the WT strains had reached 60%, while only a few of the PtoSAUR74 overexpression strains had rooted. By days 7-8, all WT strains had completed rooting, while the PtoSAUR74 overexpression strains only completed rooting on days 11-12.

[0084] The method for regulating the adventitious root formation ability of poplar provided by this invention can directly inhibit the formation of adventitious roots by regulating the expression of the PtoSAUR74 gene, providing a new technical means and theoretical basis for the genetic improvement of poplar.

[0085] Example

[0086] The present invention is further described below through specific examples; however, these examples are merely exemplary and do not constitute any limitation on the scope of protection of the present invention.

[0087] Unless otherwise specified, the reagents involved in the following examples are all commercially available conventional reagents, and the methods used are all methods commonly used in this technical field.

[0088] Example 1: Cloning of the PtoSAUR74 gene

[0089] Download the cds_from_genomic.fna file from the public URL https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_018804465.1 / , and find KAG6756651.1 to obtain the CDS sequence of the PtoSAUR74 gene of Populus tomentosa. Its location is: chromosome CM031988.1, start position 14068599, end position 14068976, totaling 378bp.

[0090] Based on the Populus tomentosa genome and considering various primer design principles, primers PtoSAUR74-F and PtoSAUR74-R for amplifying the CDS sequence of the PtoSAUR74 gene were designed using the Primer BLAST tool (NCBI, https: / / blast.ncbi.nlm.nih.gov). The sequence of primer PtoSAUR74-F is: agaacacgggggactATGAAGAAGATCAACTTGATACTAA; the sequence of primer PtoSAUR74-R is: acccccggggatcctGAACATGTAGAGATCAGCTAATTCT.

[0091] In this sequence, the lowercase part is the vector homologous arm sequence, and the uppercase part is the gene sequence.

[0092] RNA was extracted from *Populus tomentosa* using a plant RNA extraction kit from Yeasen Biotechnology. RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novizan Biotechnology Co., Ltd.). Using primers PtoSAUR74-F and PtoSAUR74-R, the obtained genomic cDNA was used as a template for PCR amplification using 2×Vazyme LAmp Master Mix (Dye Plus) from Nanjing Vazyme. The PCR reaction volume (50 μL) is shown in Table 1.

[0093] Table 1

[0094]

[0095] The PCR amplification program was as follows: 95℃ for 5 min; (95℃ for 30 s; 50℃ for 30 s; 72℃ for 30 s, 35 cycles; 72℃ for 5 min; 4℃ for ∞).

[0096] The PCR product was subjected to agarose gel electrophoresis. After verification, it was purified using the DNA Clean-up Kit from Jiangsu Kangwei Century Biotechnology Co., Ltd. Following purification, the purity and concentration of the purified DNA product were determined using an instrument. The final CDS sequence of the PtoSAUR74 gene was obtained, as shown in SEQ ID NO.1.

[0097] Example 2: Construction of PtoSAUR74 gene overexpression vector

[0098] (1) Vector enzyme digestion

[0099] The selected overexpression vector was pBI121-EGFP, purchased from the Miaoling plasmid platform. The vector is 13629 bp in length and contains a strong 35S promoter (CaMV35S). The resistance used in the transgenic plant selection was kanamycin, which can be digested with XbaI. The enzyme digestion reaction system is shown in Table 2.

[0100] Table 2

[0101]

[0102]

[0103] The enzyme digestion reaction conditions are: incubation at 37℃ for 7-9 hours, and storage at -20℃.

[0104] The PCR product was subjected to agarose gel electrophoresis. After verification, it was purified using the DNA Clean-up Kit from Jiangsu Kangwei Century Biotechnology Co., Ltd. Following purification, the purity and concentration of the purified DNA product were determined using an instrument. The resulting pBI121-EGFP vector containing the digested gene was then obtained.

[0105] (2) Connection transformation

[0106] PtoSAUR74 was constructed into the enzyme-digested pBI121-EGFP overexpression vector using the Uniclone One Step Seamless Cloning Kit from Beijing Jinsha Biotechnology Co., Ltd., to obtain the recombinant vector. Ligation was performed at 50℃ for 10 min, and the ligation product was used to transform *E. coli* DH5α competent cells.

[0107] Take 50 μl of DH5α competent cells, add 5 μl of ligation product, gently tap to mix, and incubate on ice for half an hour. After the ice bath, heat shock in a 42°C water bath for 45 seconds, then rapidly cool on ice for 2 minutes to avoid shaking and reducing transformation efficiency. Add 700 μl of antibiotic-free sterile LB liquid medium, mix well by pipetting, and incubate at 37°C with shaking at 200 rpm for 15 minutes to allow the cells to recover. After recovery, centrifuge at 6000 rpm for 1 minute to collect the cells, resuspend the cells in 100 μl of supernatant, and spread onto LB agar plates containing kanamycin (100 mg / ml). Incubate upside down at 37°C for 14–16 hours. The final vector structure is shown in Figure 1.

[0108] (3) Identification of positive clones by bacterial culture PCR

[0109] Single colonies from the plate were picked sequentially with sterilized pipette tips and added to 250 μl of LB broth containing kanamycin. The culture was incubated at 37°C with shaking at 200 rpm for approximately 3 hours to serve as amplification templates. Positive and negative controls were set up using the purified gene PCR product and ddH2O (double-distilled water) as templates. PCR amplification was performed using Taq Plus Master Mix from Nanjing Vazyme. The PCR reaction system is shown in Table 3.

[0110] Table 3

[0111]

[0112] The sequence of pBI121-R is: 5'-gtgcagatgaacttcagggtc-3';

[0113] The reaction conditions were: 94℃ for 5 min; (94℃ for 30 s; 58℃ for 30 s; 72℃ for 70 s) for 35 cycles; 72℃ for 10 min; 4℃ for ∞.

[0114] PCR products are detected by 1% agarose gel electrophoresis. Colonies that amplify a band of the same size as the positive control are considered positive clones.

[0115] (4) Extraction of positive clone plasmids

[0116] PCR-positive clones were aspirated into 6 ml of LB liquid medium containing spectinomycin and incubated overnight at 37°C with shaking at 200 rpm. Plasmids were extracted using a plasmid miniprep kit from Beijing Kangwei Reagent Co., Ltd., and sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Vector construction was completed after sequence alignment was confirmed.

[0117] Example 3: Genetic transformation of the PtoSAUR74 gene

[0118] (1) Transformation of Agrobacterium tumefaciens with recombinant plasmid

[0119] (1.1) Take about 1 μg of the overexpression vector plasmid of PtoSAUR74 and add it to 100 μL of Agrobacterium GV3101 competent cells and mix gently.

[0120] (1.2) Place on ice for 5 min, freeze in liquid nitrogen for 1 min, and immediately place in a 37℃ water bath for 5 min, and then in an ice bath for 5 min.

[0121] (1.3) Add 700 μL of antibiotic-free YEP liquid medium to the bacterial culture, mix thoroughly, and incubate at 28°C and 200 rpm with shaking for 2–3 hours. After the incubation period, centrifuge the bacterial culture at 6000 rpm for 1 minute, discard part of the supernatant, and mix 100 μL of the supernatant with the bacterial culture. Using a disposable sterile spreader, evenly spread the bacterial culture onto the surface of YEP solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin, and then incubate upside down in a 28°C incubator for 72–90 hours.

[0122] (1.4) After single colonies have grown, use a sterile pipette tip to pick up several single-clone plaques and place them in a 2 mL centrifuge tube containing 250 μL of YEP liquid medium (1:1000 with added Rif and Kana). Incubate at 30°C and 200 rpm for 2 h on a shaker. After incubation, aspirate the bacterial solution for PCR identification. Correctly identified positive bacterial solutions are added to 50% glycerol, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent genetic transformation experiments.

[0123] (2) Activation of Agrobacterium

[0124] (2.1) Take out the Agrobacterium liquid containing the overexpression vector plasmid from the -80℃ freezer, streak it onto YEP solid medium (1:1000 with added Rif and Kana), seal the plate, and incubate upside down in a dark incubator at 28℃ for 2-3 days.

[0125] (2.2) Using a sterile pipette tip, pick a single colony from the plate and inoculate it into 3 mL of YEP liquid medium (1:1000 with added Rif and Kana). Incubate overnight at 30°C and 200 rpm with shaking. Transfer 1 mL of the bacterial culture to a 250 mL sterile Erlenmeyer flask containing 100 mL of YEP liquid medium (1:1000 with added Rif and Kana). Incubate at 30°C and 200 rpm with shaking for 4–5 h until the OD600 reaches 0.3–0.5.

[0126] (2.3) In a laminar flow hood, transfer 100 mL of bacterial culture to two 50 mL sterile centrifuge tubes and centrifuge at 2560 g, 4 °C for 20 min. Collect the bacterial cells. In the laminar flow hood, discard the supernatant and resuspend the bacterial cells in 100 mL of resuspension in a sterile wide-mouth bottle. This bacterial culture will be used for subsequent infection experiments. The resuspension (1 L) consists of: WPM 2.37 g + VB1 0.9 mg + MES 0.5 g + sucrose 20 g + 2,4-D 0.1 mg + AS 200 mM + ddH2O, and bring the volume to 1 L.

[0127] (3) Healing method of infection

[0128] Select leaves from healthy, sterile seedlings (dark green leaves with thicker texture). Use a sterile scalpel to remove the petioles, leaving callus tissue. Make 2-3 horizontal incisions along the main vein of the leaf. Lay the leaves face down on callus medium (CIM) and incubate in the dark at 25°C. After 20-30 days, loose, white callus will grow from the incision sites. Peel the callus off the leaves, divide it into soybean-sized pieces, and place them in fresh callus medium. Transfer all the prepared callus to sterile wide-mouth bottles containing bacterial suspension and incubate on a shaker at 28°C and 160 rpm for 15-20 minutes. The callus medium (1L) consists of: WPM 2.37g + 2,4-D 0.1mg + KT 0.1mg + MES 0.5g + agar 7.5g + ddH2O, bringing the volume to 1L.

[0129] (4) Co-cultivation

[0130] In a clean bench, the callus was removed from the bacterial solution using tweezers, and excess bacterial solution was blotted dry with filter paper. The infected callus was then spread evenly on a co-culture medium and incubated in the dark at 25°C for 2 days. The co-culture medium (1L) consisted of: WPM 2.37g + VB1 0.9mg + sucrose 20g + MES 0.5g + AS 200mM + agar 7.5g + ddH2O, and the volume was adjusted to 1L.

[0131] (5) Differentiation culture

[0132] After dark culture, select appropriate plant resistance based on the vector and prepare differentiation medium containing the corresponding antibiotics.

[0133] The callus was transferred to differentiation medium, with the medium changed approximately every 20 days initially, and then every 10 days thereafter. During this period, the callus will turn green, harden, and then partially turn red. This stage takes about two months, and the entire process is carried out in a 25°C light incubator. The co-culture medium (1L) consisted of: WPM 2.37g + VB1 0.9mg + sucrose 20g + MES 0.5g + AS 200mM + 6-BA 0.5mg + NAA 0.1mg + TDZ 0.002mg + TMT 250mg + Cef 250mg + kana 20mg + agar 7.5g + ddH2O to a final volume of 1L.

[0134] (6) Inducing rooting from clustered buds

[0135] After the adventitious buds have grown to about half a centimeter, use sterile forceps or a scalpel to cut them off (carefully observe the growth point of the adventitious buds and avoid selecting adventitious buds differentiated from the same cell type), and place them in a bud elongation medium for growth. After a period of elongation culture, when the adventitious buds have grown to 1-2 centimeters, use sterile forceps or a scalpel to cut them off individually and place them on a rooting medium for rooting culture. The adventitious buds will grow roots in about 10 days, and the rooted buds will develop into complete plants.

[0136] The elongation medium (1L) consists of: WPM 2.37g + IBA 0.2mg + sucrose 20g + 6-BA 0.5mg + TMT 250mg + Cef 250mg + agar 7.5g + ddH2O to a final volume of 1L.

[0137] The rooting medium (1L) consists of: 1 / 2 MS + 20g sucrose + 0.05mg IBA + 0.05mg NAA + 7g agar + 250mg TMT + 250mg cephalosporin + 20mg kana + ddH2O to a final volume of 1L.

[0138] Example 4: Identification of plants overexpressing the PtoSAUR74 gene

[0139] (1) Crude extraction of DNA from wild-type 84K poplar and PtoSAUR74 gene overexpressing plants:

[0140] (i) Take a leaf sample to be extracted, place it in a 2mL centrifuge tube and add a grinding bead. After quick freezing with liquid nitrogen, use a tissue homogenizer to homogenize for 5 minutes.

[0141] (ii) After the mixture is broken into powder, add 500 μL of TBS buffer to the centrifuge tube, mix well, and then heat in a 65°C water bath for 10 min.

[0142] (iii) Centrifuge at 12000 rpm for 15 min, transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, and allow to settle at room temperature for 30 min. A white flocculent precipitate will be visible.

[0143] (iv) Centrifuge at 12000 rpm for 15 min, slowly discard the supernatant, and add 1 ml of 75% ethanol to wash the precipitate.

[0144] (v) Centrifuge at 7500 rpm for 10 min, slowly pour off the supernatant and remove the excess liquid with a pipette.

[0145] (vi) Place in a 40℃ oven for 2 hours.

[0146] (vii) Add 50 μL of ddH2O to the centrifuge tube to dissolve the precipitate and store it at -20°C.

[0147] (2) Identification of plants overexpressing the PtoSAUR74 gene

[0148] (2.1) DNA level identification: The crude extracted transgenic plant gDNA was used as a template and PCR identification was performed using Taq Plus Master Mix. The PCR reaction system was as shown in Table 3 of Example 2. The PCR products were directly run by electrophoresis to check whether the bands were correct. If they were correct, the plant was an overexpression of the PtoSAUR74 gene.

[0149] (2.2) Identification of transcription level: RT-qPCR was used to detect the transcription level of PtoSAUR74 in overexpressing plants.

[0150] (i) RNA was extracted from the root tissue to be tested and reverse transcribed to obtain template cDNA. RNA extraction and reverse transcription of cDNA were performed according to the method described in the kit in Example 1.

[0151] (ii) RT-qPCR experiments were performed using 2×ChamQ SYBR Color qPCR Master Mix. In this example, primers for real-time PCR (polymerase chain reaction) were designed based on the PtoSAUR74 gene sequence of 84K Yang, as shown below. The primer names are PtoSAUR74-qPCR-F and PtoSAUR74-qPCR-R, and their nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The reaction system (20 μL) is shown in Table 4.

[0152] Table 4

[0153]

[0154]

[0155] The primer sequences for Actin-F are shown in SEQ ID NO.5, and the primer sequences for Actin-R are shown in SEQ ID NO.6.

[0156] The PCR reaction program was as follows: 95℃ for 3 min; (95℃ for 5 sec; 60℃ for 30 sec; 72℃ for 15 sec) for 40 cycles; 72℃ for 5 min; 4℃ for ∞.

[0157] The results of RT-qPCR detection are shown in Figure 2. It can be seen that the overexpression plants numbered OE-3 and OE-6 have higher expression levels, which are 107.1 times and 119.6 times higher than those of wild-type plants (WT), respectively.

[0158] Example 5: Phenotypic Analysis of Adventitious Roots in Plants Overexpressing the PtoSAUR74 Gene

[0159] By comparing plants overexpressing the PtoSAUR74 gene (OE) and WT plants, it was found that the gene has a significant inhibitory effect on root development of Populus 84K.

[0160] Specifically, Figure 3 shows the root morphology of wild-type plants (WT-1, WT-2) and PtoSAUR74 gene overexpressing plants (numbered OE-3, OE-6) after 0, 4, 5, 6, and 12 days of growth in 1 / 2 MS medium in tissue culture flasks. The results showed that the number and length of adventitious roots of the overexpressing plants were significantly reduced compared with WT.

[0161] The quantitative analysis in Figure 4 further shows that after 12 days of culture in 1 / 2 MS medium, the total length of adventitious roots of PtoSAUR74 gene overexpressing plants (OE-3, OE-6) was reduced by 8.21 cm compared with WT.

[0162] The quantitative analysis in Figure 5 further shows that after 12 days of culture in 1 / 2 MS medium, the average length of adventitious roots of PtoSAUR74 gene overexpressing plants (OE-3, OE-6) was reduced by 1.55 cm compared with WT.

[0163] The quantitative analysis in Figure 6 further showed that after 12 days of culture in 1 / 2 MS medium, the number of adventitious roots in the PtoSAUR74 gene overexpressing plants (OE-3, OE-6) was reduced by 3.17 compared with WT.

[0164] The rooting time analysis in Figure 7 shows that some individuals of the WT lines began to root on day 4, while the PtoSAUR74 overexpression lines (OE-3 and OE-6) had not yet rooted at this time. By day 6, the rooting rate of the WT lines had reached 60%, while only a few of the PtoSAUR74 overexpression lines had rooted. By days 7-8, all WT lines had completed rooting, while the PtoSAUR74 overexpression lines only completed rooting on days 11-12. Therefore, the rooting time and rate of the overexpression lines were both delayed compared to the wild type.

[0165] The results in Figures 3-7 indicate that the PtoSAUR74 gene negatively regulates the formation of adventitious roots in 84K poplar.

[0166] In summary, overexpression of the PtoSAUR74 gene can significantly inhibit the length, number, and rooting rate of adventitious roots in poplar trees. This can be used to optimize the propagation efficiency and root system design of poplar seedlings, and has significant scientific research and industrialization value.

[0167] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the present invention, and all such modifications and improvements fall within the scope of the present invention.

Claims

1. The application of the PtoSAUR74 gene or protein, which is related to the ability of poplar to form adventitious roots, in regulating the formation of adventitious roots in poplar, characterized in that, The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.1, and the protein is encoded by the PtoSAUR74 gene, the amino acid sequence of which is shown in SEQ ID NO.

2. The adventitious root formation ability of poplar includes the number of adventitious roots, the length of adventitious roots, and the rooting rate. The application is to inhibit the formation of adventitious roots in poplar by increasing the expression of the PtoSAUR74 gene in individual poplars. When the PtoSAUR74 gene is overexpressed, the number of adventitious roots, the length of adventitious roots, and the rooting rate of poplars decrease. The overexpression of the PtoSAUR74 gene is achieved by introducing a PtoSAUR74 gene overexpression vector into poplars. The PtoSAUR74 gene overexpression vector is obtained by constructing the CDS sequence of the PtoSAUR74 gene onto a base vector. The base vector is pBI121-EGFP. The PtoSAUR74 gene overexpression vector is introduced into poplars using Agrobacterium, specifically GV3101.

2. The application of the PtoSAUR74 gene, protein, or vector and / or strain containing the PtoSAUR74 gene, which are related to the adventitious root formation ability of poplar, in poplar breeding, characterized in that... The nucleotide sequence of the coding region of the PtoSAUR74 gene is shown in SEQ ID NO.1, and the protein is the protein encoded by the PtoSAUR74 gene, the amino acid sequence of which is shown in SEQ ID NO.

2. The vector includes an overexpression recombinant vector, and the strains include Escherichia coli and Agrobacterium. The overexpression vector is obtained by constructing the CDS sequence of the PtoSAUR74 gene onto a base vector. The base vector is pBI121-EGFP, and the Agrobacterium is GV3101. The adventitious root formation ability of poplar includes the number of adventitious roots, the length of adventitious roots, and the rooting rate. The breeding method is to overexpress the PtoSAUR74 gene in poplar to cultivate poplar varieties with inhibited adventitious root development.

3. A method for regulating the adventitious root formation ability of poplar trees, characterized in that, The method includes the step of overexpressing the PtoSAUR74 gene, which is associated with the adventitious root formation ability of poplar trees; the nucleotide sequence of the coding region of the PtoSAUR74 gene is shown in SEQ ID NO.1; the adventitious root formation ability of poplar trees includes the number of adventitious roots, the length of adventitious roots, and the rooting rate; when the PtoSAUR74 gene is overexpressed, the number of adventitious roots, the length of adventitious roots, and the rooting rate of poplar trees decrease.

4. The method according to claim 3, characterized in that, The method includes the following steps: Step 1, constructing an overexpression recombinant vector of the PtoSAUR74 gene; Step 2, transferring the constructed overexpression recombinant vector into Agrobacterium; Step 3, infecting, proliferating, differentiating, and rooting poplar materials using Agrobacterium-mediated transformation to obtain transgenic plants; the poplar material is 84K poplar; Step 4, identifying the obtained transgenic plants.

5. The method according to claim 4, characterized in that, Step 1 includes the following sub-steps: Step 1-1, obtaining the coding region sequence of the PtoSAUR74 gene; Step 1-2, ligating the coding region sequence of the PtoSAUR74 gene to the base vector for transformation; Step 1-3, identifying the ligated recombinant vector.

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