A gene for regulating plant architecture and its application

Abstract

The application discloses a gene for regulating plant plant type and application thereof. The disclosed PopLBO1 gene has a coding region nucleotide sequence as shown in SEQ ID NO:1, and the amino acid sequence of the coded protein is as shown in SEQ ID NO:2. The gene can negatively regulate the branch number of a poplar tree, positively regulate the plant height and the first leaf node height of the poplar tree, and provides a key candidate gene and a theoretical basis for genetic improvement of the plant type of the poplar tree. The application further discloses a method for regulating the plant type of the poplar tree and improving the plant type of the poplar tree by using the PopLBO1 gene. By overexpressing or silencing expressing the PopLBO1 gene in the poplar tree, directional genetic improvement of the plant type of the poplar tree can be realized, and this is beneficial to fast breeding of excellent germplasm of the poplar tree suitable for different uses.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a gene that regulates plant architecture and its application, particularly the PopLBO1 gene that regulates plant architecture and its application. Background Technology

[0002] Poplar (Populus L.) is a general term for plants in the genus Populus of the family Salicaceae. It is characterized by rapid growth, strong adaptability, and wide range of uses, making it an important economic and ecological tree species globally. Poplar wood is light and soft, easy to process, and is an important raw material for papermaking and furniture making. In terms of ecological functions, it can also be used for windbreak and sand fixation and urban greening. However, the tree type characteristics of poplars (such as tree height, branching characteristics, and stem posture) directly affect their economic value and ecological benefits. For example, poplar varieties with tall trunks and few branches can significantly improve timber yield and quality, while poplar varieties with many branches are more conducive to windbreak and sand fixation. Therefore, optimizing tree type traits for different uses is of significant practical importance for achieving the efficient utilization of poplar resources.

[0003] Differences in poplar tree form are closely related to its branching characteristics. Increased branching leads to nutrient dispersion in the trunk, thus inhibiting vertical growth. This indicates that regulating branching can significantly affect poplar tree form. Studies have shown that plant branching is precisely regulated by various hormones, among which strigolactones (SLs) are key inhibitory hormones discovered in recent years that regulate plant branching. Increased SLs levels lead to reduced branching.

[0004] The lack of genes related to poplar tree type traits in existing technologies has slowed down the process of breeding poplar varieties with superior tree type traits. Therefore, it is necessary to develop key genes that affect tree type traits and to deeply analyze the functions of these genes in order to carry out targeted genetic improvement of poplar tree type and breed new poplar varieties adapted to different uses. Summary of the Invention

[0005] To overcome the above problems, the inventors, based on phenotypic data of a population of 303 Populus tomentosa germplasm resources, identified a key gene, PopLBO1, involved in the regulation of poplar tree type through genome-wide association genetics analysis. This gene negatively regulates the number of branches in poplar and positively regulates the plant height and the height of the first leaf node, providing a key candidate gene and theoretical basis for the genetic improvement of poplar tree type. By overexpressing or silencing the PopLBO1 gene in poplar, targeted genetic improvement of poplar tree type can be achieved, which is beneficial for the rapid selection of superior poplar germplasm adapted to different uses, thus completing this invention.

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

[0007] In a first aspect, a gene for regulating plant architecture is provided, the gene being the PopLBO1 gene, the nucleotide sequence of its coding region being shown in SEQ ID NO:1.

[0008] Secondly, a protein that regulates plant architecture is provided, the protein being encoded by the PopLBO1 gene, the amino acid sequence of which is shown in SEQ ID NO:2.

[0009] Thirdly, the application of the genes described in the first aspect or the proteins described in the second aspect in regulating plant architecture is provided.

[0010] Fourthly, a method for regulating poplar tree shape is provided. The method regulates the tree shape by controlling the expression of the PopLBO1 gene mentioned in the first aspect in poplar trees. The tree shape is defined as tree height, height of the first leaf node, and number of branches.

[0011] Fifthly, a method for improving poplar tree type using the PopLBO1 gene is provided, wherein the poplar tree type is defined as tree height, height of the first leaf node, and number of branches;

[0012] The improvements include: overexpressing the PopLBO1 gene in poplar to increase plant height and first leaf node height; or silencing the PopLBO1 gene in poplar to increase the number of branches.

[0013] Sixthly, a method for detecting PopLBO1 transgenic plants is provided.

[0014] The PopLBO1 transgenic plants include PopLBO1 gene overexpression plants and PopLBO1 gene silence expression plants.

[0015] The method includes the steps of using PCR amplification to detect the vector sequences and Agrobacterium sequences at both ends of the PopLBO1 gene CDS fragment and the PopLBO1 silencing fragment cloning site, respectively.

[0016] The beneficial effects of this invention include:

[0017] (1) The gene for regulating plant type provided by the present invention has a clear function and can precisely regulate the plant type (plant height, first leaf node height and number of branches) of plants, especially poplars, providing a key candidate gene for cultivating poplar varieties with excellent plant type traits.

[0018] (2) The application of PopLBO1 gene and its encoded protein provided by this invention in regulating plant architecture has deeply analyzed the function of the gene and found that PopLBO1 gene can negatively regulate the number of branches of poplar and positively regulate the height of poplar plant and the height of the first leaf node, providing a theoretical basis for the genetic improvement of poplar plant architecture.

[0019] (3) The method for regulating poplar tree shape and the method for improving poplar tree shape by using PopLBO1 gene provided by the present invention are achieved by overexpressing or silencing PopLBO1 gene in poplar trees, which is conducive to the rapid selection of superior poplar germplasm adapted to different uses.

[0020] (4) The method for detecting PopLBO1 transgenic plants provided by the present invention is simple to operate, has high accuracy, and can quickly and effectively identify PopLBO1 transgenic plants. Attached Figure Description

[0021] Figure 1 The image shows a gel electrophoresis diagram of the PopLBO1 gene CDS fragment and the silenced fragment in Example 1;

[0022] Figure 2 Image (A) shows the gel electrophoresis diagrams of the vectors at both ends of the cloning sites in PopLBO1 gene overexpressing plants and PopLBO1 gene silent expressing plants in Example 3. Figure 2 (B) shows the gel electrophoresis diagram of Agrobacterium sequences in PopLBO1 gene overexpressing plants and PopLBO1 gene silent expressing plants.

[0023] Figure 3 The relative expression levels of PopLBO1 gene overexpression and PopLBO1 gene silence in Example 3 are shown.

[0024] Figure 4 The phenotypic diagrams of wild-type plants, PopLBO1 gene overexpression plants, and PopLBO1 gene silence expression plants in Example 4 are shown.

[0025] Figure 5 The statistical results of plant height, first leaf node height, and number of branches of wild-type plants, PopLBO1 gene overexpression plants, and PopLBO1 gene silent expression plants in Example 4 are shown. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] Based on phenotypic data from a population of 303 Populus tomentosa germplasm resources, the inventors identified a key gene, PopLBO1, involved in the regulation of poplar tree architecture through genome-wide association genetics analysis. Extensive research revealed that LBO (Lateral Branching Oxidelide Ductase) is a key component of the strigolactone (SL) signaling pathway, influencing plant branching by regulating the synthesis of active strigolactones (SLs).

[0029] In a first aspect, the present invention provides a gene for regulating plant architecture, said gene being the PopLBO1 gene, the nucleotide sequence of its coding region being shown in SEQ ID NO:1.

[0030] Preferably, the plant is a poplar.

[0031] More preferably, the plant type is plant height, first leaf node height, and number of branches.

[0032] The first leaf node height refers to the height from the position of the first true leaf to the ground, and the branch refers to the lateral branch formed by the branching of the plant's main stem.

[0033] In a second aspect, the present invention provides a protein that regulates plant architecture, said protein being encoded by the PopLBO1 gene described in the first aspect.

[0034] Preferably, the amino acid sequence of the protein encoded by the PopLBO1 gene is shown in SEQ ID NO:2.

[0035] The protein has a molecular weight of 41.3 kDa and an isoelectric point of 6.04.

[0036] More preferably, the plant is a poplar.

[0037] A third aspect of the invention provides the use of the gene described in the first aspect or the protein described in the second aspect in regulating plant architecture.

[0038] Preferably, the plant is a poplar, and the regulation of the plant's plant shape is achieved by regulating the expression level of the PopLBO1 gene in the plant or regulating the content of the protein encoded by the PopLBO1 gene.

[0039] More preferably, the regulation of plant architecture is achieved by overexpressing or silencing the PopLBO1 gene in the plant.

[0040] In a preferred embodiment, the overexpression of the PopLBO1 gene in the plant is achieved by introducing an overexpression recombinant vector containing the PopLBO1 gene into the plant.

[0041] The silencing of the PopLBO1 gene in plants is achieved by introducing a silencing recombinant vector containing a silencing fragment of the PopLBO1 gene into the plant.

[0042] Preferably, the overexpression recombinant vector of the PopLBO1 gene is obtained by constructing the CDS sequence of the PopLBO1 gene into a base vector;

[0043] The PopLBO1 gene silencing recombinant vector was obtained by constructing a silencing fragment of the PopLBO1 gene into a base vector.

[0044] More preferably, the CDS sequence of the PopLBO1 gene is shown in SEQ ID NO:1; the sequence of the silenced fragment of the PopLBO1 gene is shown in SEQ ID NO:3.

[0045] In a preferred embodiment, the higher the expression level of the PopLBO1 gene in the plant, the higher the plant height and the height of the first leaf node; the lower the expression level of the PopLBO1 gene in the plant, the lower the plant height and the height of the first leaf node.

[0046] Preferably, compared with wild-type plants, the plant height and first leaf node height of PopLBO1 gene overexpression plants are increased; compared with wild-type plants, the plant height and first leaf node height of PopLBO1 gene silence expression plants are decreased.

[0047] In this invention, the PopLBO1 gene positively regulates the plant height and the height of the first leaf node of poplar.

[0048] In another preferred embodiment, the higher the expression level of the PopLBO1 gene in the plant, the fewer the number of branches; the lower the expression level of the PopLBO1 gene in the plant, the more the number of branches.

[0049] Preferably, compared with wild-type plants, PopLBO1 gene overexpression plants have fewer branches, while PopLBO1 gene silenced expression plants have more branches.

[0050] In this invention, the PopLBO1 gene can negatively regulate the number of branches in poplar trees and positively regulate the height of the plant and the height of the first leaf node, indicating that this gene is a key regulator of poplar tree type.

[0051] In a fourth aspect, the present invention provides a method for regulating the tree shape of poplar trees, wherein the method regulates the tree shape by controlling the expression of the PopLBO1 gene described in the first aspect in poplar trees.

[0052] Preferably, plant architecture is regulated by overexpressing or silencing the PopLBO1 gene in poplar trees.

[0053] More preferably, the plant type is plant height, first leaf node height, and number of branches.

[0054] In a preferred embodiment, when the PopLBO1 gene is overexpressed, the plant height and the height of the first leaf node of poplar trees increase; when the PopLBO1 gene is silenced, the plant height and the height of the first leaf node decrease.

[0055] In another preferred embodiment, when the PopLBO1 gene is overexpressed, the number of branches in poplar trees decreases; when the PopLBO1 gene is silenced, the number of branches in poplar trees increases.

[0056] In a preferred embodiment, the method for regulating poplar tree shape includes the following steps:

[0057] Step 1: Obtain the CDS fragment and silencing fragment of the PopLBO1 gene.

[0058] In a preferred embodiment, the CDS fragment of the PopLBO1 gene is obtained by amplification using overexpression primers PopLBO1-F and PopLBO1-R, wherein the sequence of primer PopLBO1-F is shown in SEQ ID NO:4 and the sequence of primer PopLBO1-R is shown in SEQ ID NO:5.

[0059] In a preferred embodiment, the silenced fragment of the PopLBO1 gene is obtained using the silence expression primers PopLBO1-RNAi-F and PopLBO1-RNAi-R, the sequence of which is shown in SEQ ID NO:6 and the sequence of which is shown in SEQ ID NO:7.

[0060] Preferably, the CDS fragment and the silenced fragment of the PopLBO1 gene are cloned from 84K silver adenophora.

[0061] More preferably, the sequence of the obtained PopLBO1 gene CDS fragment is shown in SEQ ID NO:1, and the sequence of the silenced fragment is shown in SEQ ID NO:3.

[0062] Step 2: Construct PopLBO1 gene overexpression recombinant vector and silent expression recombinant vector.

[0063] In a preferred embodiment, the overexpression recombinant vector is obtained by constructing a CDS fragment of the PopLBO1 gene onto a base vector;

[0064] The silenced expression recombinant vector was obtained by constructing a silenced fragment of the PopLBO1 gene into a base vector.

[0065] Preferably, the base vector is pBI121-eGFP, which drives the expression of the target gene with a strong 35S promoter and carries a GFP tag, facilitating molecular identification and gene function studies.

[0066] More preferably, the CDS fragment of the PopLBO1 gene is forward-ligated into the pBI121-eGFP vector to obtain the PopLBO1 gene overexpression recombinant vector; the silent fragment of the PopLBO1 gene is reverse-ligated into the pBI121-eGFP vector to obtain the pBI121-eGFP vector to obtain the silent expression recombinant vector.

[0067] In this invention, the CDS fragment and the silencing fragment are respectively forward and reverse ligated to the plant pBI121-eGFP vector, so that it is expressed under the drive of a strong promoter.

[0068] Step 3: Perform genetic transformation to identify PopLBO1 gene overexpression lines and silent expression lines.

[0069] In a preferred embodiment, the successfully constructed overexpression recombinant vector and silent expression recombinant vector are transformed into Agrobacterium, and then the recombinant vector is transferred into poplar trees through Agrobacterium-mediated leaf disc genetic transformation to obtain complete plants.

[0070] Preferably, the poplar is 84K silver gland poplar.

[0071] In a preferred embodiment, the identification of PopLBO1 gene overexpression lines and silent expression lines includes identification at the DNA level and identification at the transcriptional level.

[0072] Preferably, the DNA level identification includes the steps of detecting the vector sequences and Agrobacterium sequences at both ends of the PopLBO1 gene CDS fragment and the PopLBO1 silencing fragment cloning site using PCR amplification.

[0073] More preferably, the primers for detecting the vector sequences at both ends of the PopLBO1 gene CDS fragment and the PopLBO1 silencing fragment cloning site are pBI121-F and pBI121-R, whose nucleotide sequences are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0074] The primers for detecting Agrobacterium sequences are GV3101-VirD2-F and GV3101-VirD2-R, whose nucleotide sequences are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0075] In a preferred embodiment, if the PCR detection results of the PopLBO1 gene overexpression line show that the band size of the vector sequence at both ends of the cloning site is 1342bp and the Agrobacterium sequence does not amplify the target band of 220bp, then it is identified as a positive PopLBO1 gene overexpression line.

[0076] In the PCR detection results of PopLBO1 gene silencing expression lines, if the band size of the vector sequence at both ends of the cloning site is 484bp and the Agrobacterium sequence does not amplify the target band of 220bp, then it is identified as a positive PopLBO1 gene silencing expression line.

[0077] In a preferred embodiment, the identification of the transcription level includes the step of detecting the expression level of the PopLBO1 gene in overexpressing poplar lines and silent expression poplar lines using real-time quantitative PCR (RT-PCR).

[0078] Preferably, the primers for the real-time quantitative PCR are PopLBO1-qPCR-F and PopLBO1-qPCR-R, and their nucleotide sequences are shown in SEQ ID NO:12 and SEQ ID NO:13, respectively.

[0079] In a preferred embodiment, the method for regulating poplar tree type further includes: step 4, performing phenotypic analysis on overexpression lines and silent expression lines.

[0080] Preferably, the above-identified PopLBO1 gene overexpression plants, silent expression plants, and wild-type 84K poplar trees are selected and planted under the same conditions. Phenotypic tests are performed on the plants to observe the changes in plant type between the transgenic plants and the wild-type plants.

[0081] According to a preferred embodiment of the present invention, compared with wild-type plants, plants overexpressing the PopLBO1 gene have higher plant height and first leaf node height, while plants with silent PopLBO1 gene expression have lower plant height and first leaf node height.

[0082] Preferably, compared with wild-type plants, PopLBO1 gene-silenced plants have more branches.

[0083] The method for regulating poplar tree structure provided by this invention is achieved by overexpressing or silencing the PopLBO1 gene in poplar trees, which can rapidly select superior poplar varieties adapted to different uses.

[0084] In a fifth aspect of the present invention, there is a method for improving the plant type of poplar trees using the PopLBO1 gene, wherein the plant type of poplar trees is defined as plant height, height of the first leaf node, and number of branches.

[0085] Preferably, the improvement includes increasing the height of the poplar tree and the height of the first leaf node to enhance its economic benefits.

[0086] More preferably, the improvement also includes increasing the number of branches of the poplar tree to enhance its ecological benefits.

[0087] In a preferred embodiment, the improvement includes:

[0088] Overexpression of the PopLBO1 gene in poplar trees can increase plant height and first leaf node height; or silence expression of the PopLBO1 gene in poplar trees can increase the number of branches.

[0089] In this case, the overexpression or silencing of the PopLBO1 gene in poplar trees is carried out according to the method described in the third or fourth aspect.

[0090] The method for improving poplar tree type using the PopLBO1 gene provided by this invention can obtain PopLBO1 overexpressing poplar plants with significantly increased plant height and first leaf node height, as well as PopLBO1 silent expressing poplar plants with significantly increased branching number, which is beneficial for rapidly breeding superior poplar germplasm adapted to different uses.

[0091] In a sixth aspect, the present invention provides a method for detecting PopLBO1 transgenic plants, wherein the PopLBO1 transgenic plants include PopLBO1 gene overexpression plants and PopLBO1 gene silent expression plants.

[0092] The PopLBO1 transgenic plant is preferably obtained according to the method described in the fourth aspect.

[0093] Preferably, the method includes the steps of using PCR amplification to detect the vector sequences and Agrobacterium sequences at both ends of the PopLBO1 gene CDS fragment and the PopLBO1 silencing fragment cloning site, respectively.

[0094] More preferably, the primers for detecting the vector sequences at both ends of the PopLBO1 gene CDS fragment and the PopLBO1 silencing fragment cloning site are pBI121-F and pBI121-R, whose nucleotide sequences are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0095] The primers for detecting Agrobacterium sequences are GV3101-VirD2-F and GV3101-VirD2-R, whose nucleotide sequences are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0096] In a preferred embodiment, if the PCR detection results of PopLBO1 gene overexpressing plants show that the band size of the vector sequences at both ends of the cloning site is 1342bp and the Agrobacterium sequence does not amplify the target band of 220bp, then the plant is identified as a positive PopLBO1 gene overexpressing plant.

[0097] In the PCR detection results of PopLBO1 gene-silenced plants, if the size of the vector sequence at both ends of the cloning site is 484 bp and the Agrobacterium sequence does not amplify the target band of 220 bp, then the plant is identified as a positive PopLBO1 gene-silenced plant.

[0098] The method for detecting PopLBO1 transgenic plants provided by this invention is simple to operate, highly accurate, and can quickly and effectively identify PopLBO1 transgenic plants.

[0099] Example

[0100] 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.

[0101] 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.

[0102] Example 1: Construction of PopLBO1 gene overexpression recombinant vector and silencing recombinant vector

[0103] (1) Based on the phenotypic data of 303 Populus tomentosa germplasm resources, a key gene, PopLBO1, involved in the regulation of poplar tree architecture was identified through genome-wide association genetics analysis. Using Populus tomentosa 84K as material, the full-length CDS sequence of this gene was cloned, and its nucleotide sequence is shown in SEQ ID NO:1. A silenced fragment of this gene was also cloned using Populus tomentosa 84K as material, and its nucleotide sequence is shown in SEQ ID NO:3. Gel electrophoresis images of the CDS fragment and the silenced fragment are shown in [image missing]. Figure 1 As shown.

[0104] The full-length CDS of the PopLBO1 gene is 1095 bp, encoding a protein composed of 364 amino acids. The amino acid sequence of the protein encoded by the PopLBO1 gene is shown in SEQ ID NO:2. The protein has a molecular weight of 41.3 kDa and an isoelectric point of 6.04.

[0105] Specifically: Leaves of 84K *Populus silveraefolia* were thoroughly ground in a sterile, enzyme-free mortar. Total RNA was extracted from the leaves using a total RNA extraction kit. DNase I was used to remove genomic DNA contamination. Subsequently, Promega GoScript was used to extract the RNA. TMThe Reverse Transcription System reverse transcribes the DNA into cDNA.

[0106] Primers were designed based on the CDS sequence and silencing fragment of the PopLBO1 gene, and amplification was performed using a high-fidelity enzyme. The primers for amplifying the CDS fragment of the PopLBO1 gene were PopLBO1-F and PopLBO1-R, with sequences shown in SEQ ID NO:4 and SEQ ID NO:5, respectively. The primers for amplifying the silencing fragment of the PopLBO1 gene were PopLBO1-RNAi-F and PopLBO1-RNAi-R, with sequences shown in SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0107] (2) The amplification products (CDS fragment and silent fragment) were ligated to the pBI121-eGFP vector in the forward and reverse directions respectively by homologous recombination. The pBI121-eGFP vector drives the expression of the target gene with a strong 35S promoter and carries a GFP tag, which facilitates molecular identification and gene function research.

[0108] The ligation products were heat-shocked into *E. coli* DH5α competent cells, and single colonies were picked and sequenced after plating. The correctly sequenced plasmids were then heat-shocked into *Agrobacterium* GV3101 cells, plated on LB agar containing 17 mg / L rifampin and 50 mg / L kanamycin, and incubated overnight at 28°C. Single colonies with the correct bands were identified by PCR and stored in 100 mL LB liquid medium (17 mg / L rifampin and 50 mg / L kanamycin) by shaking. The PCR program was as follows: 95°C denaturation for 5 minutes; (95°C, denaturation for 30 seconds; 56°C annealing for 30 seconds; 72°C, extension for 90 seconds) 35 cycles; 72°C, extension for 5 minutes.

[0109] After the above steps, the PopLBO1 gene overexpression recombinant vector and the silent expression recombinant vector were constructed.

[0110] Example 2: Obtaining PopLBO1 gene overexpression lines and silent expression lines

[0111] (1) Pretreatment: Take leaves of 84K tissue culture seedlings in good growth condition, make wounds on the leaves with a sterile scalpel, and place the wounded leaves in 84K differentiation medium (MS + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 7 g / L agar + 30 g / L sucrose) without antibiotics for 2-3 days for pretreatment.

[0112] (2) Shaking culture: Pick a single colony of activated Agrobacterium from the plate and inoculate it into 100 mL of liquid LB medium (25 mg / L kanamycin, 25 mg / L rifampin). Mix well and incubate at 28°C with shaking at 180 rpm for 24 h. When the OD600 is 0.6-0.8, it can be used for Agrobacterium transformation.

[0113] (3) Infection: Pour the Agrobacterium tumefaciens bacterial suspension into a 50ml sterile centrifuge tube, centrifuge at 3500rpm-5000rpm for 7-10 minutes at room temperature to collect the bacterial suspension, and then gently shake the centrifuge tube with an equal volume of sterile resuspension to make the bacteria evenly suspended in the liquid. Further immerse the pretreated leaves in the resuspension of Agrobacterium tumefaciens bacterial suspension for 10-15 minutes. Place the infected leaves on a symbiotic medium (MS + 0.5mg / L 6-BA + 0.05mg / L NAA + 7g / L agar + 30g / L sucrose + 100uM AS) and incubate in the dark at 25℃ for 3-4 days.

[0114] (4) Resistance culture: After 3 days of dark treatment, the co-cultured leaves were placed on sterile filter paper to remove excess bacteria, and then transferred to selection medium (MS + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 7 g / L agar + 30 g / L sucrose + 25 mg / L kanamycin + 200 mg / L termethin) for selection culture. The photoperiod was 16 h light / 8 h dark, and the temperature was set at 25℃. The selection medium was changed every 10 days until resistant buds appeared.

[0115] (5) Rooting culture: When the adventitious buds grow to 1-2cm, use sterile tweezers or a scalpel to cut off the adventitious buds individually and place them in rooting medium (1 / 2MS + 0.05mg / L IBA + 0.05mg / L NAA + 7g / L agar + 20g / L sucrose + 25mg / L kanamycin + 200mg / L termethin) for rooting culture. The adventitious buds will grow roots in about 10 days.

[0116] After the roots have developed, propagation can be carried out to obtain complete plants.

[0117] Example 3: Identification of PopLBO1 gene overexpression lines and silent expression lines

[0118] (1) DNA level identification

[0119] Genomic DNA was extracted from PopLBO1 overexpressing and silent Poplar trees using the FastPurePlant DNAIsolation Mini Kit from Nanjing Novizan Biotechnology Co., Ltd. PCR amplification was used to detect the vector sequences and Agrobacterium sequences at both ends of the cloning site of the PopLBO1 gene CDS fragment and the PopLBO1 silent fragment.

[0120] The primers for detecting the vector sequences at both ends of the cloning site of the PopLBO1 gene CDS fragment and the PopLBO1 silencing fragment are pBI121-F and pBI121-R, and their nucleotide sequences are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively; the primers for detecting the Agrobacterium sequence are GV3101-VirD2-F and GV3101-VirD2-R, and their nucleotide sequences are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0121] In PCR detection of PopLBO1 gene overexpression lines, if the vector sequence band size at both ends of the cloning site is 1342 bp and the Agrobacterium sequence does not amplify the target band of 220 bp, then it is identified as a positive PopLBO1 gene overexpression line (PopLBO1-OE). In PCR detection of PopLBO1 gene silencing lines, if the vector sequence band size at both ends of the cloning site is 484 bp and the Agrobacterium sequence does not amplify the target band of 220 bp, then it is identified as a positive PopLBO1 gene silencing line (PopLBO1-RNAi).

[0122] The results of the identification are as follows Figure 2 As shown in (A) and (B), (A) represents the gel electrophoresis results of the vectors at both ends of the cloning site in plants overexpressing and silencing the PopLBO1 gene; (B) represents the gel electrophoresis results of the Agrobacterium sequences in plants overexpressing and silencing the PopLBO1 gene. Figure 2 As can be seen, the PopLBO1 gene overexpressing poplar plants (numbered #1 to #10) and PopLBO1 gene silent expression plants (numbered #1 to #10) obtained in this embodiment showed the presence of vector sequences at both ends of the cloning site by PCR detection, with correct band lengths (1342bp and 484bp, respectively), and the absence of Agrobacterium sequence bands. This indicates that both PopLBO1 overexpressing poplar lines #1 to #10 and silent expression poplar lines #1 to #10 are positive transgenic plants.

[0123] (2) Identification of transcription level

[0124] Total RNA was extracted from poplar trees using the Novozymes RNA Extraction Kit (a centrifuge column kit). cDNA was then synthesized using the Reverse Transcription cDNA First-Strand Synthesis Kit (R312-01 / 02). The expression levels of the PopLBO1 gene in overexpressing and silenced poplar lines were subsequently detected by real-time quantitative PCR. The procedure is as follows:

[0125] ① Design RT-qPCR primers

[0126] The designed primers are PopLBO1-qPCR-F and PopLBO1-qPCR-R, and their nucleotide sequences are shown in SEQ ID NO:12 and SEQ ID NO:13, respectively.

[0127] ② The PopLBO1 overexpressing poplar lines #1 to #10 and the silent expression poplar lines #1 to #10 obtained above were used as experimental materials and labeled as OE-1, OE-2, OE-3, OE-4, OE-5, OE-6, OE-7, OE-8, OE-9 and OE-10, respectively; RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6, RNAi-7, RNAi-8, RNAi-9 and RNAi-10. RNA was extracted from the above positive seedlings and reverse transcribed into cDNA.

[0128] ③ Measure the cDNA concentration and dilute it to 200 ng / μl.

[0129] ④ Prepare the PCR reaction system on ice, as shown in Table 1.

[0130] Table 1

[0131]

[0132] ⑤ Using QuantStudio 6Flex Real-Time PCR System software, with 18S RNA as an internal control, RT-qPCR was performed in triplicate for each sample. The PCR reaction program is shown in Table 2.

[0133] Table 2

[0134]

[0135]

[0136] Test results as follows Figure 3 As shown, the PopLBO1 gene expression level of overexpressing poplar lines OE-1 to OE-10 was significantly higher than that of wild-type poplar (WT), while the PopLBO1 gene expression level of silent expression poplar lines RNAi-1 to RNAi-10 was significantly lower than that of wild-type poplar (WT).

[0137] Based on the above DNA level identification (PCR amplification) and transcription level identification (real-time quantitative PCR), the PopLBO1 gene overexpressing poplar lines were obtained as #1 to #10, and the PopLBO1 gene silent expression poplar lines were obtained as #1 to #10.

[0138] Example 4: Phenotypic analysis of PopLBO1 gene overexpression lines and silent expression lines

[0139] PopLBO1 gene-overexpressing plants, silenced expression plants, and wild-type 84K poplar trees were simultaneously planted in a greenhouse in three biological replicates. After two months of culture, phenotypic measurements and photographs were performed to observe the changes in plant morphology between the transgenic plants and the wild-type plants. The results are as follows: Figure 4 As shown.

[0140] Figure 4 In the middle, from left to right, are the phenotypes of PopLBO1 gene overexpression lines #1 to #3 (denoted as OE-1, OE-2, and OE-3, respectively), wild-type 84K poplar (denoted as WT), and PopLBO1 gene silent expression lines #1 to #3 (denoted as RNAi-1, RNAi-2, and RNAi-3, respectively) obtained in Example 3.

[0141] Furthermore, the plant height, first leaf node height, and number of branches of PopLBO1 gene-overexpressing plants, wild-type 84K poplar, and PopLBO1 gene-silenced plants were statistically analyzed. The results are as follows: Figure 5 As shown.

[0142] Depend on Figure 4 and Figure 5 It can be seen that, compared with wild-type 84K poplar, the plant height and first leaf node height of the PopLBO1 gene overexpression plants are significantly increased, and the number of branches of the PopLBO1 gene silence expression plants is significantly increased.

[0143] 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. A gene that regulates plant architecture, characterized in that, The gene in question is the PopLBO1 gene, and its coding region nucleotide sequence is shown in SEQ ID NO:

1.

2. A protein that modulates plant architecture, characterized in that, The protein is encoded by the PopLBO1 gene, and its amino acid sequence is shown in SEQ ID NO:

2.

3. Use of a gene according to claim 1 or a protein according to claim 2 for modulating plant architecture, characterized in that, The plant in question is a poplar.

4. Use according to claim 3, characterized in that, The regulation of plant architecture is achieved by adjusting the expression level of the PopLBO1 gene in the plant or by adjusting the content of the protein encoded by the PopLBO1 gene.

5. Use according to claim 3, characterized in that, The regulation of plant architecture is achieved by overexpressing or silencing the PopLBO1 gene within the plant.

6. A method of modulating plant type in a poplar tree, the method comprising, The method regulates plant type by controlling the expression of the PopLBO1 gene as described in claim 1 in poplar trees. The plant type is defined as plant height, height of the first leaf node, and number of branches.

7. The method of claim 6, wherein, When the PopLBO1 gene was overexpressed, the plant height and first leaf node height of poplar trees increased; when the PopLBO1 gene was silenced, the plant height and first leaf node height decreased; and / or When the PopLBO1 gene is overexpressed, the number of branches in poplar trees decreases; when the PopLBO1 gene is silenced, the number of branches in poplar trees increases.

8. The method of claim 6, wherein, The method for regulating poplar tree shape includes the following steps: Step 1: Obtain the CDS fragment and silencing fragment of the PopLBO1 gene; Step 2: Construct PopLBO1 gene overexpression recombinant vector and silencing recombinant vector; Step 3: Perform genetic transformation to identify PopLBO1 gene overexpression lines and silent expression lines.

9. A method for improving the plant type of Populus by using PopLBO1 gene, characterized in that, The poplar tree type is defined by its height, the height of the first leaf node, and the number of branches. The improvements include: overexpressing the PopLBO1 gene in poplar to increase plant height and first leaf node height; or silencing the PopLBO1 gene in poplar to increase the number of branches; The nucleotide sequence of the coding region of the PopLBO1 gene is shown in SEQ ID NO:

1.

10. A method for detecting PopLBO1 transgenic plants, characterized in that, The PopLBO1 transgenic plants include PopLBO1 gene overexpression plants and PopLBO1 gene silence expression plants. The method includes the steps of using PCR to detect the vector sequences and Agrobacterium sequences at both ends of the cloning site of the PopLBO1 gene CDS fragment and the PopLBO1 silencing fragment, respectively. The genetically modified plant was a genetically modified poplar. The nucleotide sequence of the coding region of the PopLBO1 gene is shown in SEQ ID NO:1.

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