Application of populus trichocarpa gene PtrMYBR069 in regulation and control of secondary xylem development

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a populus trichocarpa gene PtrMYBR069 to regulation and control of secondary xylem development. According to the invention, the genetic function of the PtrMYBR069 transcription factor is analyzed, and a foundation is laid for the improvement of the wood quality in the future. A PtrMYBR069 over-expressed populus trichocarpa transgenic plant is obtained through an agrobacterium tumefaciens-mediated populus trichocarpa genetic transformation system, and the phenotypes of the transgenic plant are that the number of secondary xylem fiber cell layers is increased, the secondary xylem is widened, the internode length is increased, and the stem node diameter is increased. Overexpression of PtrMYBR069 affects expression of a cellulose metabolic pathway gene, and the cellulose content of a transgenic plant is slightly increased. After the PtrMYBR069 performs heterologous expression on the arabidopsis thaliana, the basal stem diameter of the transgenic arabidopsis thaliana is increased. The results show that the PtrMYBR069 transcription factor has a regulation effect on the development of plant secondary xylem and the formation and development of wood. The research content provided by the invention has important theoretical guiding significance for improving new high-quality wood varieties through genetic engineering.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of the Populus trichocarpa gene PtrMYBR069 in regulating the development of secondary xylem. Background Art

[0002] Wood is an important renewable biological resource in the world, which plays an important role in human production and life and is widely used in various fields such as housing construction, furniture production, and papermaking. The important ecological value of trees and the important economic value of wood have prompted people to pay more attention to the genetic improvement of the quantity and quality of wood. The formation of wood (secondary xylem) is a continuous developmental process starting from the vascular cambium, and wood tissues are formed through cell division / differentiation, cell expansion, secondary cell wall deposition, and programmed cell death. The formation of wood originates from the differentiation of the vascular cambium, differentiating inward into secondary xylem (wood tissue) and outward into secondary phloem. After the division of cambial initial cells, the volume of secondary xylem cells expands, and then enters the stage of secondary cell wall thickening. During secondary growth, the cell wall components of the xylem change from the cellulose and pectin of the primary wall to the three substances of lignin, cellulose, and hemicellulose in the secondary wall. The end of the cell wall thickening process means the termination of the life activities of individual cells. After the continuous accumulation of dead cells, the wood shows elongation longitudinally and thickening transversely, and finally forms wood.

[0003] MYB transcription factors exist in plants and are widely involved in biological processes such as plant growth and development, metabolic processes, and stress responses. Among them, the single R-domain MYB-like transcription factors (MYBR) are a newly discovered class of transcription factors. MYBR transcription factors play multiple roles in plant growth and development, including regulating defense mechanisms, overcoming biotic and abiotic stresses, participating in growth and development, regulating hormone content changes, and regulating secondary growth. Among them, the expression level of the Populus trichocarpa PtrMYBR069 transcription factor gradually increases during stem segment development, but its genetic function has not been studied so far. Summary of the Invention

[0004] The problem to be solved by the present invention is to find a molecular mechanism for promoting the development of secondary xylem in woody plants and a solution for wood formation based on previous studies on the wood formation mechanism and influencing factors.

[0005] The application of the Populus trichocarpa gene PtrMYBR069 in regulating the development of secondary xylem, wherein the nucleotide sequence of the Populus trichocarpa gene PtrMYBR069 is as shown in SEQ ID No.1.

[0006] Further defined, the amino acid sequence of the protein encoded by the Populus trichocarpa gene PtrMYBR069 is as shown in SEQ ID No.2.

[0007] Further limitation is that the application is to increase the plant height, and / or thicken the basal stem of the plant, and / or increase the internode length, and increase the number of secondary xylem cell layers through overexpression of the PtrMYBR069 gene, resulting in the widening of the secondary xylem.

[0008] Further limitation is that the application is to construct a plant expression vector with the PtrMYBR069 gene, and then transfer the plant expression vector containing the PtrMYBR069 gene into a plant with secondary xylem to obtain transgenic plants, specifically including the following steps:

[0009] (1) Clone the PtrMYBR069 gene, and the nucleotide sequence of the PtrMYBR069 gene is shown as SEQ ID No.1;

[0010] (2) Use seamless cloning to ligate the PtrMYBR069 gene with the pCAMBIA1300 plant expression vector to obtain the pCAMBIA1300-35s::PtrMYBR069-3×Flag recombinant vector;

[0011] (3) Use the Agrobacterium-mediated transformation method to obtain transgenic plants with the pCAMBIA1300-35s::PtrMYBR069-3×Flag plasmid, and identify them through resistance screening to obtain transgenic positive plants.

[0012] Further limitation is that the plant with secondary xylem is a woody plant or a non-woody plant.

[0013] Further limitation is that the woody plant is Populus trichocarpa, and overexpression of the PtrMYBR069 gene affects the expression of cellulose metabolism genes in Populus trichocarpa, and the cellulose content of overexpressed transgenic plants increases.

[0014] Further limitation is that the non-woody plant is Arabidopsis thaliana.

[0015] In this invention, Populus trichocarpa transgenic plants overexpressing the MYB transcription factor PtrMYBR069 with a single R domain were successfully obtained through the Agrobacterium-mediated Populus trichocarpa genetic transformation system. Phenotypic analysis of the transgenic plants found that overexpression of the PtrMYBR069 transcription factor significantly increased the internode length and stem diameter of Populus trichocarpa plants, the radial size of the secondary xylem increased, the number of xylem fiber cell layers increased, affecting the development of the secondary xylem, and overexpression of PtrMYBR069 affected the expression of genes in the cellulose metabolism pathway of Populus trichocarpa, increasing the cellulose content. The research content of this invention has important theoretical value and application significance for cultivating high-quality wood by genetic engineering means. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, obtaining other drawings based on these drawings still falls within the scope of the present invention.

[0017] Figure 1 : Genetic transformation map of Populus trichocarpa with pCAMBIA1300-35s::PtrMYBR069-3xFlag; where A in the figure is the screening of resistant buds, and B is the rooting of resistant buds;

[0018] Figure 2 : Statistical analysis of the transcriptional expression levels of PtrMYBR069 in PtrMYBR069-Flag overexpressing plants; WT: wild type; PtrMYBR069-Flag1, PtrMYBR069-Flag3, and PtrMYBR069-Flag8: different line strains of Populus trichocarpa transgenic plants overexpressing the PtrMYBR069 gene, the same below;

[0019] Figure 3 : Comparative analysis of the growth phenotypes of PtrMYBR069-Flag overexpressing plants and wild type; A is the phenotype diagram of wild type and overexpressing plants at 2 months; B is the statistical result of the plant height of wild type and overexpressing plants; C is the statistical result of the number of stem nodes of wild type and overexpressing plants; D is the statistical result of the length of stem nodes of wild type and overexpressing plants; E is the statistical result of the diameter of stem nodes of wild type and overexpressing plants; WT: wild type, the error bars represent the SD values of three biological replicates, * indicates significance (*P<0.05, **P<0.01);

[0020] Figure 4 : Cross-section of the eighth stem node of PtrMYBR069 overexpressing Populus trichocarpa stained with toluidine blue result diagram;

[0021] Figure 5: Electron microscopy phenotypic analysis of cross-sections of stem segments of Populus trichocarpa plants overexpressing PtrMYBR069; A. Electron micrograph of stem segments of Populus trichocarpa plants overexpressing PtrMYBR069, A-1. Tissue morphology at 30× magnification, Bars = 1 mm; A-2. Tissue morphology at 150× magnification, Bars = 200 μm; A-3. Tissue morphology at 2000× magnification, Bars = 10 μm; B. Statistical result graph of the width of secondary xylem of Populus trichocarpa overexpressing PtrMYBR069; C. Statistical result graph of the number of layers of fiber cells in the secondary xylem of Populus trichocarpa overexpressing PtrMYBR069; D. Statistical result graph of the thickness of the secondary cell wall of Populus trichocarpa overexpressing PtrMYBR069. Error bars represent the SD values of three biological replicates, * indicates significance (*P < 0.05, **P < 0.01);

[0022] Figure 6 : Effects of overexpression of PtrMYBR069-Flag on cellulose biosynthesis genes in Populus trichocarpa; among them, WT: wild type, error bars represent the SD values of three biological replicates, * indicates significance (*P < 0.05);

[0023] Figure 7 : Overexpression of PtrMYBR069 affects the cellulose content in Populus trichocarpa plants. WT: wild type, error bars represent the SD values of three biological replicates;

[0024] Figure 8 : Analysis of the transcriptional expression levels of Arabidopsis thaliana plants transformed with PtrMYBR069; WT: wild type, OE8, OE9, 0E14: different line strains of Arabidopsis thaliana transformed with Populus trichocarpa PtrMYBR069 gene;

[0025] Figure 9 : Heterologous transformation of Arabidopsis thaliana with PtrMYBR069 affects the diameter length at the base of the main stem; WT: wild type, OE8, OE9, 0E14: different line strains transformed with PtrMYBR069 gene. Detailed implementation manners

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] To make the above objectives, features, and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Unless otherwise specified in the following embodiments, they are all conventional methods. The materials, reagents, enzymes, competent cells, plasmids, etc. used, unless otherwise specified, can all be obtained from commercial sources. The present invention will be described in detail below through specific embodiments.

[0028] Example 1: Construction of the pCAMBIA1300-35s::PtrMYBR069-3×Flag Plant Expression Vector (1) Obtaining the PtrMYBR069 Gene

[0029] The total RNA of Populus trichocarpa plants was extracted using pBIOZOL Plant Total RNA Extraction Reagent (BSC55S1). The RNA was reverse transcribed into cDNA using the Takara reverse transcription kit (RR047Q), and the system is shown in Table 1.

[0030] Table 1 cDNA synthesis reaction system

[0031]

[0032]

[0033] According to the sequence information of the PtrMYBR069 gene (Potri.007G122800.1) provided by the Phytozme website, the upstream and downstream primer sequences for cloning the PtrMYBR069 gene are shown in Table 2 and were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0034] Table 2 Upstream and downstream primer sequences for cloning the PtrMYBR069 gene

[0035]

[0036] Note: The underlined part is the specific sequence added to the upstream primer to facilitate the subsequent ligation to the TOPO vector

[0037] The full-length CDS of the PtrMYBR069 gene was amplified using the high-fidelity KOD Plus enzyme (F0934K) from TOYOBO. The reaction system for amplifying the target gene is as follows:

[0038]

[0039] The reaction program was: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing at 60°C for 30 s, extension at 68°C for 30 s, and 35 cycles of amplification; finally, extension at 68°C for 10 min and storage at 16°C. The agarose gel block containing the target fragment was recovered using the Silica Bead DNA Gel Extraction Kit (D0056) from Thermo Fisher Scientific.

[0040] (2) Ligation of the PtrMYBR069 Gene to the TOPO Vector

[0041] Use the pENTR from Thermo Fisher ScientificTM / SD / D-TOPO TM For the ligation reaction in the cloning kit (K240020), the 2.5 μL ligation reaction system is as follows:

[0042]

[0043] Incubate in a 25 °C water bath overnight for ligation.

[0044] Transform the ligation product, TOPO-PtrMYBR069 plasmid, into competent E. coli cells. After correct identification and sequencing, it is reserved for use.

[0045] (3) Construction of the pCAMBIA1300-35s::PtrMYBR069-3×Flag vector

[0046] Design upstream and downstream primers for the full-length PtrMYBR069 gene containing homologous recombination fragments.

[0047] Use PCR to amplify the PtrMYBR069 sequence containing homologous recombination fragments. The reaction system is shown in Table 3, the reaction program is shown in Table 4, and the primer sequences are shown in Table 5.

[0048] Table 3 Reaction system for PCR amplification of the full length of PtrMYBR069

[0049]

[0050]

[0051] Table 4 Reaction program for PCR amplification

[0052]

[0053] Table 5 Primer sequences

[0054]

[0055] Recover the PCR product from the gel. Use SacI and XbaI to perform double digestion on the pCAMBIA1300-35s-3xFlag vector. The digestion system (40 μL) is shown in Table 6.

[0056] Table 6 Digestion system for pCAMBIA1300-35s-3xFlag

[0057]

[0058] Flick to mix well, incubate in a 37 °C water bath overnight for digestion. Pipette 1 μL of the digested solution and electrophorese it with the original plasmid to compare the sizes. After correct identification by PCR reaction (primer sequences are shown in Table 5), electrophorese the digested fragment and recover it from the gel.

[0059] Using the seamless cloning kit (638947) from Clontech, the following 10 μL ligation system was established:

[0060]

[0061] Carefully mix with a pipette tip and incubate at 50 °C for 1 h. The ligation product was transformed into Escherichia coli competent cells and used after correct identification by PCR reaction (primer sequences are shown in Table 5).

[0062] Example 2: Obtaining of transgenic Populus trichocarpa plants overexpressing pCAMBIA1300-35s::PtrMYBR069-3×Flag

[0063] (1) Obtaining of transgenic Populus trichocarpa plants overexpressing PtrMYBR069

[0064] The pCAMBIA1300-35s::PtrMYBR069-3×Flag plasmid constructed in Example 1 was transformed into Agrobacterium tumefaciens GV3101 as follows:

[0065] 1) 1-2 μL (100 ng) of the plasmid of the constructed expression vector was added to the Agrobacterium competent cells and incubated on ice for 30 min;

[0066] 2) The competent cells containing the plasmid were quickly frozen in liquid nitrogen for 1 min, taken out with forceps, incubated in a water bath at 37 °C for 3 min, and then kept on ice for 2 min;

[0067] 3) 500-1000 μL of YEP or LB liquid medium was added, and the mixture was placed in an incubator at 28 °C and cultured at 200 rpm for 2-3 h;

[0068] 4) Centrifuge at 10000 g at room temperature for 15 s. In a laminar flow hood, 300 μL of the supernatant was discarded, and the remaining medium was pipetted to resuspend the precipitated bacteria, which were evenly spread on solid YEP medium with three resistances (containing Gen, Rif, and Kan antibiotics, the same below), and cultured at 28 °C in an inverted position for 48 h;

[0069] 5) Single colonies were picked for colony PCR and electrophoresis identification;

[0070] 6) The correctly identified single colonies were picked into YEP liquid medium containing resistance, and the OD 600 was measured at 1.0 for bacterial preservation, and stored at -80 °C.

[0071] (2) Transforming wild-type Populus trichocarpa using the conventional Agrobacterium-mediated method

[0072] The Agrobacterium stored at -80°C was streaked and activated on YEP solid medium containing triple antibiotics, and cultured in an inverted position at 28°C for 48 h. Colonies of appropriate size were picked and inoculated into YEP liquid medium containing triple antibiotics. After about 14 h, the culture was shaken until the cell density reached OD 600 = 0.6. The cells were centrifuged at 3500 rpm for 10 min at 4°C, resuspended with the suspension sterilized by moist heat, and used to infect the stem segments of wild-type Populus trichocarpa. The stem segments of wild-type Populus trichocarpa were generally selected from the 2nd to 5th segments of healthy Populus trichocarpa that had grown for 4 - 5 weeks, cut to about 0.8 cm in length, and infected for 20 min with occasional shaking. After infection, the stem segments were placed in a plate containing 400 μM acetosyringone (As), wrapped tightly with tinfoil, and cultured in the dark for 48 h. The dark-cultured stem segments were placed on a resistance screening plate (10 mg / L Hyg) in a laminar flow hood and cultured under normal photoperiod for 8 weeks. Then, the stem segments with bud points were placed on a resistance screening plate (5 mg / L Hyg) for further culture. After a period of time, the bud points differentiated and grew ( Figure 1 A). At this time, they were transferred to a rooting culture flask containing (5 mg / L Hyg) and further screened ( Figure 1 B). Subsequently, PCR method (the primers used were hygromycin resistance sequence primers, see Table 7) was used for identification, indicating that resistant seedlings were successfully obtained and finally confirmed.

[0073] Table 7 Primer sequences for hygromycin identification

[0074]

[0075] (3) Detection of the expression level of overexpressed PtrMYBR069 Populus trichocarpa transgenic plants by fluorescence quantitative PCR method

[0076] Furthermore, transgenic plant lines Line1 (PtrMYBR069-Flag1), Line3 (PtrMYBR069-Flag3) and Line8 (PtrMYBR069-Flag8) were selected, and the overexpression of transgenic plants was detected using the fluorescence quantitative kit (RR820A) from Takara Bio. The fluorescence quantitative PCR system and procedure are shown in Table 8 and Table 9, and the internal reference primers and quantitative primers for the target gene used are shown in Table 10.

[0077] Table 8 qPCR reaction system

[0078]

[0079] Table 9 qPCR reaction procedure

[0080]

[0081] Table 10 Primers required for the identification of the expression level of PtrMYBR069 transgenic Populus trichocarpa

[0082]

[0083] Compared with wild - type Populus trichocarpa plants, the expression levels of the PtrMYBR069 gene in three transgenic independent resistant lines of transgenic Populus trichocarpa plants overexpressing PtrMYBR069, namely PtrMYBR069 - Flag1, PtrMYBR069 - Flag3, and PtrMYBR069 - Flag8, were significantly increased ( Figure 2 ), indicating that transgenic plants with over - expression of the PtrMYBR069 gene were successfully obtained.

[0084] Example 3: Application of the Populus trichocarpa PtrMYBR069 gene in regulating secondary xylem development and wood formation

[0085] (1) Effects of over - expression of the PtrMYBR069 gene on the phenotype of Populus trichocarpa plants

[0086] Select the identified over - expression lines 1, 3, and 8 of pCAMBIA1300 - PtrMYBR069 - 3×Flag in Example 2 as research materials. The wild - type and over - expression plants that had grown in tissue culture bottles for one month were transplanted into soil in the greenhouse. They were of the same growth height and thickness. Three plants of each line were planted in a tray to ensure the same growth environment and conditions. They were cultured in the greenhouse for 2 months, during which the growth phenotypes were observed and measured.

[0087] Comparing and observing the wild - type Populus trichocarpa plants and transgenic plants overexpressing the PtrMYBR069 gene cultured in the greenhouse, it was found that plants overexpressing the PtrMYBR069 gene showed obvious growth phenotype changes (such as Figure 3 A).

[0088] Compared with wild - type plants, the transgenic plants showed a significant increase in plant height phenotype ( Figure 3 B). The results of measurement and statistical analysis showed that: PtrMYBR069 - Flag1 had a height increase of more than 30% compared with the wild - type (p < 0.05), PtrMYBR069 - Flag3 had a height increase of more than 33% compared with the wild - type (p < 0.05), and PtrMYBR069 - Flag8 had a height increase of more than 34% compared with the wild - type (p < 0.05).

[0089] Compared with wild - type plants, although the number of stem nodes of the transgenic plants did not change ( Figure 3 C).

[0090] Compared with wild - type plants, the internode lengths of the 4th, 8th, and 11th nodes of the transgenic plants increased, and the internode lengths of the 4th and 8th nodes showed significant changes ( Figure 3D). Compared with the length of the stem of the wild-type plants at the 4th node, PtrMYBR069-Flag1 was 2 times that of the wild type (p < 0.01), PtrMYBR069-Flag3 was 2.2 times that of the wild type (p < 0.01), and PtrMYBR069-Flag8 was 2.3 times that of the wild type (p < 0.01); compared with the length of the stem of the wild type at the 8th node, PtrMYBR069-Flag1 was 1.6 times that of the wild type, PtrMYBR069-Flag3 was 1.8 times that of the wild type (p < 0.05), and PtrMYBR069-Flag8 was 1.8 times that of the wild type (p < 0.05); compared with the length of the stem of the wild type at the 11th node, PtrMYBR069-Flag1 was 1.1 times that of the wild type, PtrMYBR069-Flag3 was 1.5 times that of the wild type, and PtrMYBR069-Flag8 was 1.2 times that of the wild type.

[0091] Compared with the wild-type plants, the diameters of the stems at the 4th, 8th, and 11th nodes of the transgenic plants showed an increasing trend ( Figure 3 E). Compared with the diameter of the stem of the wild type at the 4th node, PtrMYBR069-Flag1 was 1.2 times that of the wild type, PtrMYBR069-Flag3 was 1.5 times that of the wild type (p < 0.05), and PtrMYBR069-Flag8 was 1.3 times that of the wild type; compared with the diameter of the stem of the wild type at the 8th node, PtrMYBR069-Flag1 was 1.4 times that of the wild type, PtrMYBR069-Flag3 was 1.5 times that of the wild type, and PtrMYBR069-Flag8 was 1.4 times that of the wild type; compared with the length of the stem of the wild type at the 11th node, PtrMYBR069-Flag1 was 1.4 times that of the wild type, PtrMYBR069-Flag3 was 1.4 times that of the wild type, and PtrMYBR069-Flag8 was 1.3 times that of the wild type.

[0092] These phenotypic changes in the transgenic plants indicate that overexpression of the PtrMYBR069 gene affects the growth and development of Populus trichocarpa plants.

[0093] (2) Effects of PtrMYBR069 overexpression on the phenotype of the secondary xylem of Populus trichocarpa

[0094] Select the PtrMYBR069-Flag1 and PtrMYBR069-Flag3 overexpressing plants and wild type that were cultured in the greenhouse for 2 months in Example 2. Take the 8th stem segment, after paraffin embedding, sectioning, dewaxing and rehydration, observe by toluidine blue staining, and compare the effect of overexpressing PtrMYBR069 on the morphology of the secondary xylem of the stem. The results show that compared with wild-type Populus trichocarpa, the overexpressing PtrMYBR069 transgenic plants show a continuous vascular cambium, an increase in the radial width of the xylem, and an increase in the number of transverse cell layers of the xylem fiber cells in the overexpressing PtrMYBR069 transgenic plants( Figure 4 ).

[0095] Furthermore, using the conventional freehand sectioning method, the effect of overexpressing the PtrMYBR069 gene on the secondary xylem was observed using a scanning electron microscope (NeoScope, JCM5000, Japan)( Figure 5 A).

[0096] The thickness of the secondary xylem of the 8th stem segment of wild-type Populus trichocarpa is about 400 μm, the thickness of the secondary xylem of the 8th stem segment of PtrMYBR069-Flag1 is about 500 μm, and the thickness of the secondary xylem of the 8th stem segment of PtrMYBR069-Flag3 is about 475 μm( Figure 5 B).

[0097] Statistical analysis was performed on the number of fiber cell layers in the secondary xylem. As Figure 5 shown in C, the number of wild-type fiber cell layers is 35 - 38 layers, the number of xylem fiber cell layers of PtrMYBR069-Flag1 is about 45 layers (P < 0.01), and the number of xylem fiber cell layers of PtrMYBR069-Flag3 is about 43 layers (P < 0.01).

[0098] Compared with the wild type, there is no significant difference in the secondary cell wall thickness of the fiber cells in the 8th stem segment of the plants overexpressing the PtrMYBR069 gene( Figure 5 D).

[0099] (3) Overexpression of PtrMYBR069 affects the expression of cell wall cellulose metabolism genes in Populus trichocarpa

[0100] Collect the 7th stem segments of wild-type Populus trichocarpa and PtrMYBR069 gene overexpressing plants grown in the greenhouse for RNA extraction, reverse transcription, and RT-qPCR to detect the expression levels of genes in the cellulose metabolism pathway in transgenic plants. The specific method refers to Example 1, and the primer sequences used are shown in Table 11.

[0101] Table 11 Primer sequences for analyzing the effect of PtrMYBR069 transgenic Populus trichocarpa on cellulose synthesis

[0102]

[0103] Further analysis of the effect of overexpression of the PtrMYBR069 gene on genes in the cellulose metabolic pathway of Populus trichocarpa was carried out, and the expression levels of 8 cellulose metabolic genes were detected by qPCR method ( Figure 6 ). The results showed that: compared with the wild type, in the PtrMYBR069 overexpressing transgenic plants, except for the insignificant change in PtrCSLAl, the cellulose synthesis genes PtrCesA7A, PtrCesA4, PtrCesA7B, PtrCesA8A, PtrCesA8B, PtrCesA17, and PtrCesA18 genes all showed an upward trend, and among them, the expression levels of the three genes PtrCesA7B, PtrCesA8B, and PtrCesA18 were significantly enhanced.

[0104] (4) Overexpression of PtrMYBR069 affects the cellulose content in the cell wall of Populus trichocarpa

[0105] The cellulose content of the 9th stem segment of wild type plants and PtrMYBR069-Flag1, PtrMYBR069-Flag3, and PtrMYBR069-Flag8 was measured by the conventional Updegraff method. As Figure 7 shown, the cellulose content of wild type plants was about 41%, the cellulose content of PtrMYBR069-Flag1 was about 42%, the cellulose content of PtrMYBR069-Flag3 was about 43%, and the cellulose content of PtrMYBR069-Flag8 was about 43%. The results showed that: the cellulose content of PtrMYBR069 overexpressing plants increased slightly compared with wild type plants, and it is a potential excellent gene for improving wood, and excellent wood with higher strength and durability can be obtained.

[0106] In summary, the overexpression of the PtrMYBR069 gene positively activates the expression of genes in the cellulose metabolic pathway, affects the number of layers and width of secondary xylem fiber cells, and further affects the plant height, internode length, and stem diameter of Populus trichocarpa plants, and can be used as a target gene for genetic improvement of wood to meet the requirements of wood quantity and quality.

[0107] Example 4: Heterologous transformation of Arabidopsis thaliana with PtrMYBR069 affects the basal diameter of Arabidopsis thaliana

[0108] (1) Obtaining of pGWB2-PtrMYBR069 heterologous transgenic Arabidopsis thaliana

[0109] Based on the TOPO-PtrMYBR069 plasmid constructed in Example 1, Gateway of Thermo Fisher Scientific was used TM LR Clonase TMII enzyme (11791100) was used to construct the pGWB2-PtrMYBR069 plant overexpression vector through homologous recombination reaction with the plant expression vector pGWB2. The reaction system was as follows:

[0110]

[0111] After mixing, it was placed for 2 min and vortexed 2 times, 2 s each time. The reaction was carried out at 25 °C for 2 h. Then, 0.5 μL of Protein K was added to the mixture, briefly vortexed, and incubated at 37 °C for 10 min. The method for transformation and identification of competent cells was the same as that in Example 1.

[0112] The wild-type Arabidopsis thaliana was transformed by the agrobacterium-mediated floral dipping method. The method was as follows:

[0113] The competent cells containing the pGWB2-PtrMYBR069 recombinant plasmid were streaked on a triple-antibiotic YEP solid medium containing Kan\Gen\Rif (kanamycin / gentamicin / rifampicin, the same below), and cultured in an inverted position for 48 h. Colonies of appropriate size were picked and cultured in a small shaker in 20 ml of Yep liquid medium containing the triple antibiotics Kan\Gen\Rif at 28 °C and 1800 rpm for about 14 - 16 h until the OD600 was between 0.8 and 1.0. The cells were centrifuged at 4000 rpm for 10 min at 4 °C, the supernatant was discarded, and the precipitated cells were fully suspended in 250 mL of fresh transformation solution.

[0114] The preparation method of 2 L of the transformation solution was as follows: 4.4 g of MURASHIGE&SKOOG BASAL SALT MIXTURE, 100 g of sucrose, and 1 g of MES were added, the pH was adjusted to 5.7, and finally 200 μL of Silwet-77 was added.

[0115] The suspended bacterial solution was used to soak the flower buds of wild-type Arabidopsis thaliana for 5 min, the excess transformation solution was blotted off with absorbent paper, the infected part was wrapped with plastic cling film, then wrapped with a black plastic bag, and incubated in the dark for 24 h. The cling film was gently removed, and the transformed Arabidopsis thaliana plants were fixed with bamboo sticks to prevent lodging. The plants were cultured under normal light conditions to avoid sterility of the plants and reduce the seed harvest. Within 10 days after transformation, the supply of water and nutrients was enhanced. Three weeks later, only the pods remained on the flowers of Arabidopsis thaliana. At this time, the plants were fixed and watered less, and waited for the seeds to mature, and the transgenic progeny were harvested.

[0116] (2) Identification and phenotype of pGWB2-PtrMYBR069 heterologous transgenic Arabidopsis thaliana

[0117] The method was the same as that in Example 1. Arabidopsis thaliana lines 8, 9, and 14 (named OE-8, OE-9, and OE-14) heterologously transfected with the PtrMYBR06 gene were selected and used to identify the transgenic Arabidopsis thaliana with the primer sequences in Table 12. The results are shown inFigure 8 All plants heterologously expressed the PtrMYBR069 gene. Taking the expression level of the PtrMYBR069 gene in the heterologous expression plant OE-8 as a reference, the expression level of the PtrMYBR069 gene in the heterologous expression plant OE-9 was more than 3-fold.

[0118] Table 12 Upstream and downstream primer sequences for the identification of PtrMYBR069 transgenic Arabidopsis thaliana

[0119]

[0120] Cultured under the same environment, when comparing wild-type and transgenic Arabidopsis thaliana plants heterologously overexpressing PtrMYBR069, the basal diameter lengths of the main stems of the two overexpression transgenic plants OE8 and OE9 were significantly higher than that of WT (P<0.05)( Figure 9 ). The results suggest that the PtrMYBR069 gene can also increase the biomass of herbaceous plants.

[0121] Based on all the above results, the PtrMYBR069 gene is involved in the development of secondary xylem in poplar, affects the number of fiber cell layers in secondary xylem, overexpression can increase the thickness of secondary xylem, increase plant height, affect the expression of genes in the cellulose metabolic pathway, and increase lignin content. In addition, the PtrMYBR069 gene also affects the basal stem length of herbaceous plants. Therefore, this gene can be used for the genetic improvement of woody plants to increase the quantity and quality of wood.

[0122] The above-disclosed are only the preferred embodiments of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. Application of the Populus trichocarpa gene PtrMYBR069 in regulating the development of secondary xylem, characterized in that: The nucleotide sequence of the Populus trichocarpa gene PtrMYBR069 is shown in SEQ ID No.

1.

2. The use of the Populus trichocarpa gene PtrMYBR069 in regulating the development of secondary xylem according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the Populus trichocarpa gene PtrMYBR069 is shown in SEQ ID No.

2.

3. The use of the Populus trichocarpa gene PtrMYBR069 in regulating the development of secondary xylem according to claim 1, characterized in that: The application is to increase the height of the plant, and / or thicken the base stem of the plant, and / or increase the length between stems, and / or increase the number of secondary xylem cell layers through overexpression of the PtrMYBR069 gene, thereby causing the secondary xylem to become wider.

4. The use of the Populus trichocarpa gene PtrMYBR069 in regulating the development of secondary xylem according to claim 1, characterized in that: The application is to construct a plant expression vector through the PtrMYBR069 gene, and then transfer the plant expression vector containing the PtrMYBR069 gene into a plant with secondary xylem to obtain a transgenic plant.

5. The use of the Populus trichocarpa gene PtrMYBR069 in regulating the development of secondary xylem according to claim 4, characterized in that: The plant with secondary xylem is a woody plant.

6. The use of the Populus trichocarpa gene PtrMYBR069 in regulating the development of secondary xylem according to claim 5, characterized in that: The woody plant is Populus trichocarpa. Overexpression of the PtrMYBR069 gene affects the expression of cellulose metabolism genes of Populus trichocarpa, and the cellulose content of the overexpressed transgenic plants is increased.

7. The use of the Populus trichocarpa gene PtrMYBR069 in regulating the development of secondary xylem according to claim 4, characterized in that: The plant with secondary xylem is a non-woody plant.

8. The use of the Populus trichocarpa gene PtrMYBR069 in regulating the development of secondary xylem according to claim 7, characterized in that: The non-woody plant is Arabidopsis thaliana.

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