Populus tomentosa auxin efflux carrier ptraux6 and application thereof

Abstract

The application discloses a populus trichocarpa auxin internal transport factor PtrAUX6 and application thereof, and belongs to the technical field of plant genetic engineering. The application carries out amino acid synonymous mutation replacement on the specific binding site of the PtrAUX6 gene and miR7833 to obtain an optimized PtrAUX6 gene and constructs a plant overexpression vector. After populus tomentosa plants are transformed, the production speed of the xylem cells of the populus tomentosa can be effectively improved, and the fast-growing ability of the wood can be improved. The application provides a method and germplasm basis for breeding of excellent varieties of forest trees. The application is beneficial to improving the wood yield and economic benefits of economic forests, and provides materials for construction of protective forests and urban and rural greening.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the auxin transport factor PtrAUX6 in Populus tomentosa, and also to the application of PtrAUX6 in regulating the development of Populus tomentosa wood. Background Technology

[0002] Wood is one of the most important energy sources on Earth, with applications in every aspect of life, from large-scale production of energy and chemical products, furniture making, papermaking, and agricultural applications, to small-scale manufacturing of matches and stationery. Wood is also one of the few acceptable alternatives to fossil fuels for the future environment. The white poplar is one of the most widely planted forestry tree species in my country and an important source of timber. Developing fast-growing varieties of this species has significant application value.

[0003] Wood originates from the secondary xylem produced by the secondary development of stem vascular tissue, with the vascular cambium dividing and differentiating to form the secondary xylem. Therefore, to increase wood biomass, it is essential to first understand the molecular mechanisms of secondary vascular development. Compared to food crops, the mechanisms of wood formation in woody plants are still poorly understood, which greatly limits the progress of wood property improvement and molecular breeding of forest trees. Poplar, as a model plant for forest plant research, has seen some studies on the secondary development of stem vascular tissue. According to existing reports, this process is influenced by many factors, including intrinsic genetic factors and extrinsic environmental factors such as hormones, transcriptional regulators, epigenetic factors, and light, temperature, and humidity. Further research is needed to identify regulatory genes that can improve the growth rate of xylem cells in Populus tomentosa, and to regulate the expression levels of endogenous genes through molecular biology and genetic techniques to obtain Populus tomentosa germplasm with rapid wood growth. This is a pressing issue in this field.

[0004] Auxin, one of the most important hormones in plants, participates in almost all plant growth and development processes, as well as adaptations to complex environments. The key to its function lies in the precise regulation of cell growth. In woody plants, auxin has been shown to play a crucial regulatory role in secondary vascular development. Auxin transport in plants mainly relies on transporter families such as PIN, ABCB, AUX / LAX, and PIN-LIKES. Among them, Aux / IAA, as a key factor in auxin influx, not only controls auxin influx into cells but is also considered the starting point for auxin signal transduction, playing a vital role in auxin's control of cell behavior and its influence on development. However, whether this family of factors can regulate wood development remains unclear. In poplar, whether the Aux / IAA family genes can regulate wood development and enhance rapid growth has not been reported; the key members of the Aux / IAA family responsible for auxin influx in poplar wood cells and involved in the regulation of poplar wood development also need to be identified. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide a auxin transport factor PtrAUX6 in Populus tomentosa; a second objective of the present invention is to provide a gene encoding the auxin transport factor PtrAUX6 in Populus tomentosa; a third objective of the present invention is to provide an modified gene encoding the auxin transport factor PtrAUX6 in Populus tomentosa; a fourth objective of the present invention is to provide an expression vector containing the modified gene PtrAUX6; a fifth objective of the present invention is to provide the application of the modified gene PtrAUX6 or the expression vector in regulating the development of Populus tomentosa wood; and a sixth objective of the present invention is to provide a method for constructing a Populus tomentosa line with rapid xylem growth.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. PtrAUX6, an endogenous transport factor of Populus tomentosa, wherein the amino acid sequence of PtrAUX6 is shown in SEQ ID No. 1.

[0008] 2. A gene encoding the auxin transport factor PtrAUX6 of the poplar, wherein the nucleotide sequence of the PtrAUX6 gene is shown in SEQ ID No. 2.

[0009] 3. An engineered gene encoding the auxin transport factor PtrAUX6 of Populus tomentosa, obtained by replacing the amino acid synonyms of the PtrAUX6 gene with the specific binding site of miR7833, and its nucleotide sequence is shown in SEQ ID No. 9.

[0010] 4. An expression vector containing the modified PtrAUX6 gene.

[0011] 5. Application of the modified PtrAUX6 gene or the expression vector in regulating the development of Populus tomentosa wood.

[0012] The present invention preferably involves overexpressing the PtrAUX6 gene shown in SEQ ID No. 9 in Populus tomentosa to obtain transgenic Populus tomentosa lines with increased xylem cell production rate and improved wood growth capacity.

[0013] 6. A method for constructing a fast-growing xylem poplar line, comprising the following steps:

[0014] 1) The PtrAUX6 gene shown in SEQ ID No. 9 was ligated into a plant expression vector to obtain the 35S::mAUX6 overexpression vector;

[0015] 2) Transformation with Agrobacterium: The 35S::mAUX6 overexpression vector constructed in step (1) was introduced into wild-type Populus tomentosa leaves using the Agrobacterium-mediated leaf disc method.

[0016] 3) Through co-culture, selective culture, obtaining clustered shoots, obtaining rooted transgenic plants, and identifying positive plants, the Populus tomentosa strains that overexpress PtrAUX6 were finally obtained.

[0017] Preferably, the plant expression vector of the present invention is pCAMBIA1300-GFP.

[0018] Preferably, the Agrobacterium is strain GV3101.

[0019] The beneficial effects of this invention are as follows:

[0020] This invention screened and cloned the PtrAUX6 gene from the Aux / IAA auxin transporter family in poplar. High-throughput sequencing of the degradation genome of wild-type poplar revealed that the site where miR7833 specifically binds to PtrAUX6 mRNA was cleaved, leading to silencing of the exogenously introduced PtrAUX6 gene. Figure 1 Therefore, based on the characteristics of the cleavage site, an optimized mAUX6 sequence with a cleavage site mutation was designed—that is, an mAUX6 sequence that cannot be specifically cleaved by miR7833. Figure 1 B). Using genetic engineering methods, an overexpression vector (35S::mAUX6, carrying an optimized mAUX6 sequence) was constructed. Figure 1 C).

[0021] Genetic transformation of Populus tomentosa and screening for transgenic overexpression lines yielded two overexpression lines of PtrAUX6 with expression levels upregulated by 81.3-fold and 63.6-fold respectively compared to the wild type: 35S::mAUX6-L2 and 35S::mAUX6-L4. Figure 2 Further analysis of the wood development of the overexpression lines showed that, compared with the wild type, the 35S::mAUX6 transgenic Populus tomentosa lines grown for 3 months exhibited significantly increased cambium mitotic activity; the number of cambium cell layers in the 35S::mAUX6-L2 and 35S::mAUX6-L4 plants increased by 17.9% and 25.4% respectively compared with the wild type plants. Figure 3 Meanwhile, the xylem area of ​​the 35S::mAUX6 transgenic Populus tomentosa increased by 4.7% and 5.9% respectively in L2 and L4 plants compared to the wild type. Figure 4The differences between the two groups (A and C) were highly significant (p < 0.001); the number of xylem cell layers was also significantly increased in the 35S::mAUX6-L2 and 35S::mAUX6-L4 transgenic Populus tomentosa compared with the wild type, increasing by 13.1% and 17.7% respectively. Figure 4 B).

[0022] In summary, the mAUX6 gene and vector optimized by this invention can effectively increase the rate of xylem cell production and the rapid growth of wood after transforming Populus tomentosa plants. This provides a method and germplasm basis for the breeding of superior forest tree varieties, which is conducive to increasing the timber yield and economic benefits of economic forests. It also provides materials for the creation of protective forests and urban and rural greening. Attached Figure Description

[0023] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0024] Figure 1 Methods for modifying the PtrAUX6 gene sequence and constructing the 35S::mAUX6 overexpression vector;

[0025] A. Results of degradome sequencing analysis of Populus tomentosa; B. Optimized design of mAUX6 sequence; C. Construction of 35S::mAUX6 recombinant plasmid.

[0026] Figure 2 Screening and identification of transgenic Populus tomentosa 35S::mAUX6 plants;

[0027] A. Identification of transgenic positive lines; B. Screening of overexpression lines.

[0028] Figure 3 Analysis of vascular cambium mitotic activity in the 35S::mAUX6 transgenic line of Populus tomentosa;

[0029] A. Transverse section imaging of the vascular cambium (intersegment 8); B. Analysis of vascular cambium cell division activity; C. Statistical analysis of the number of vascular cambium cell layers; different letters indicate p<0.05.

[0030] Figure 4 Phenotypic analysis of xylem development regulation in the 35S::mAUX6 transgenic line of Populus tomentosa;

[0031] A. Xylem cross-section imaging (8th internode); B. Xylem cell layer count (***, p<0.001); C. Xylem area percentage count; "***" indicates p<0.001). Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0033] Example 1: Cloning of the PtrAUX6 gene in Populus tomentosa

[0034] Based on amino acid sequence homology, the amino acid sequence of the endogenous auxin factor PtrAUX6 in *Populus hairy fruit* (serial number Potri.009G132100, as shown in SEQ ID No. 1) and the CDS sequence of its encoding gene PtrAUX6 (as shown in SEQ ID No. 2) were identified in the phytozome database (https: / / phytozome-next.jgi.doe.gov / ). Based on the CDS sequence of AUX6, upstream and downstream primers for the complete coding frame were designed and synthesized as follows:

[0035] Upstream primer F-PtrAUX6: 5'-ATGGCTACTGATAAGGTCG-3' (SEQ ID No. 3);

[0036] Downstream primer R-PtrAUX6: 5'-TCATGGGCTGTGAGTAAGG-3' (SEQ ID No. 4).

[0037] Using the cDNA sequence of *Populus hairy fruit* as a template, the PtrAUX6 gene was amplified by PCR using 5×PrimeSTAR Buffer Mix, and the sequence accuracy and integrity were verified by sequencing. The PCR reaction conditions were: 98℃ for 3 min; 98℃ for 10 sec, 56℃ for 20 sec, extension time of 15 sec / kb at 72℃, for a total of 35 cycles; 72℃ for 10 min.

[0038] Agarose gel electrophoresis showed that the gene band of the target size was obtained, and sequencing results also verified that we had cloned the correct PtrAUX6 gene.

[0039] Example 2: Sequence optimization of the PtrAUX6 gene

[0040] Based on our degradome sequencing results from Populus tomentosa, we found that the site where endogenous AUX6 mRNA specifically binds to miR7833 was cleaved, resulting in degradation fragments. This indicates that endogenous miR7833 in poplar can induce silencing of the AUX6 gene expression. Figure 1 A).

[0041] Therefore, genetic transformation of the PtrAUX6 gene requires artificial sequence modification to ensure normal expression of the exogenously introduced PtrAUX6 gene. To this end, the specific binding site between the two needs to be identified: 5'-UCAUUUG. UAGUAGUGUG GGUGCUAAUUG UUGGG-3' (underlined to indicate the binding site region, SEQ ID No. 5), and then designed to perform amino acid synonymous mutation substitutions on several bases at the binding site, so that the specific binding site sequence becomes: 5'-UCAUUUG UGGUCGUCUGGGUA CUGAUCG UUGGG-3' (SEQ ID No. 6), thereby designing the optimized mAUX6 sequence (such as...). Figure 1 (As shown in B). The specific method is as follows:

[0042] 1) Design primers for site mutation sequences:

[0043] Upstream primer F-mAUX6-overlap:5'-TGTGGTCGTCTGGGTACTGATCGTT-3' (SEQ ID No. 7);

[0044] Downstream primer R-mAUX6-overlap:5'-CGATCAGTACCCAGACGACCACAA-3' (SEQ ID No. 8).

[0045] 2) First, the front CDS sequence mAUX6-1, containing the mAUX6 mutation site, was amplified from the PtrAUX6 sequence using upstream primer F-PtrAUX6 and downstream primer R-mAUX6-overlap. This amplification was performed by PCR using 5×PrimeSTAR Buffer Mix, followed by sequencing verification. Simultaneously, the rear CDS sequence mAUX6-2, containing the mAUX6 mutation site, was amplified from the PtrAUX6 sequence using upstream primer F-mAUX6-overlap and downstream primer R-PtrAUX6. This amplification was performed by PCR using 5×PrimeSTAR Buffer Mix, followed by sequencing verification.

[0046] 3) The site mutations in the mAUX6-1 and mAUX6-2 sequences overlap. Therefore, the complete and optimized mAUX6 sequence can be amplified from templates mAUX6-1 and mAUX6-2 using overlap PCR with primers F-PtrAUX6 and R-PtrAUX6. The PCR reaction conditions were: 98℃ for 3 min; 98℃ for 10 sec, 56℃ for 20 sec, and an extension time of 15 sec / kb at 72℃, for a total of 35 cycles; 72℃ for 10 min.

[0047] 4) The sequence correctness was verified by sequencing to obtain the optimized mAUX6 sequence of PtrAUX6 (as shown in SEQ ID No. 9).

[0048] Example 3: Construction of a plant overexpression vector for AUX6 with sequence modification (35S::mAUX6)

[0049] To construct the mAUX6 overexpression vector, the pCAMBIA1300-GFP vector with the CaMV 35S promoter was first selected. The vector was then double-digested with restriction endonucleases BamHI and XbaI. After digestion at 37°C overnight, the vector was subjected to agarose gel electrophoresis. The linearized vector bands were excised and recovered using a Biospin gel recovery kit to obtain the digested linearized vector fragments.

[0050] Subsequently, primers for the mAUX6 gene with vector homologous arms were designed at the vector restriction sites, namely:

[0051] Upstream primer mAUX6-F-Bam HI: 5'-actgagctcggtacccgggATGGCTACTGATAAGGTCG-3' (SEQ ID No. 10);

[0052] Downstream primer mAUX6-R-Xba I: 5'-ctcaccatgtcgactctagaTGGGCTGTGAGTAAGGTTG-3' (SEQ ID No. 11).

[0053] Using mAUX6 amplified in Example 2 as a template, the mAUX6 gene with homologous arms of the pCAMBIA1300 vector was amplified. After amplification, the agarose gel was recovered using the Biospin gel recovery kit, and finally the mAUX6 linearized gene fragment with homologous arms was obtained.

[0054] The enzyme-digested vector fragment and the mAUX6 fragment with a homologous arm were homologously ligated using the DNA Ligation Kit LONG to obtain the ligated pCAMBIA1300 vector containing the mAUX6 fragment. The ligation product was transformed into *E. coli* using competent cells, and the resulting single-clone colonies were detected by PCR. Positive colonies were inoculated onto LB broth and incubated overnight at 37°C with a shaker. The vector plasmid was then obtained using the Biospin plasmid extraction kit.

[0055] The plasmid was successfully constructed by enzyme digestion and sequencing verification, yielding the 35S::mAUX6 recombinant plasmid. Figure 1 C).

[0056] Example 4: Agrobacterium tumefaciens-mediated 35S::mAUX6 genetic transformation of Populus tomentosa

[0057] In this study, wild-type Populus tomentosa was used for genetic transformation, and Agrobacterium-mediated leaf disc staining was employed for the genetic transformation of Populus tomentosa.

[0058] 1) Agrobacterium-mediated transformation

[0059] The 35S::mAUX6 plasmid was introduced into Agrobacterium GV3101 strain using a heat shock method (37℃, 5 min + liquid nitrogen, 2 min; repeated three times). Positive clones were selected by Kan resistance screening. Colony PCR (using F-PtrAUX6 primers and R-PtrAUX6 primers) was used to identify Agrobacterium GV3101 colonies containing the recombinant plasmid. The strain was then preserved.

[0060] 2) Culture of Agrobacterium

[0061] Agrobacterium GV3101 containing the recombinant plasmid was streaked onto YEP solid medium (containing 20 mg / ml Rif and 50 mg / ml Kan) and incubated upside down at 28°C. Single clones were picked and inoculated into YEP liquid medium containing the corresponding antibiotics and cultured at 28°C with shaking until the logarithmic growth OD600 reached 0.6-0.8. Then, activated Agrobacterium was inoculated into the same 50 ml YEP liquid medium at a ratio of 1:100 to 1:50 and cultured at 28°C until the OD600 reached 0.6-0.8. 600 To obtain a concentration of 0.6–0.8, add the viable Agrobacterium bifidum culture to a 50 ml centrifuge tube, centrifuge at 4000 rpm for 10 min at 4 °C, collect the bacterial pellet, add 10 ml of WPM resuspension (WPM powder + 2.14 g + 30 g sucrose + 100 μmol AS), mix well with a pipette, add 20–30 ml of WPM resuspension, transfer to a round-mouth bottle, and incubate at 28 °C and 200 rpm with shaking for 40 min–1 h.

[0062] 3) Agrobacterium-mediated leaf disc infection

[0063] Take sterile leaves from tissue culture seedlings and cut them into 0.5×0.5cm pieces on a clean bench. 2 Place the leaf discs of the desired size into the pre-suspended Agrobacterium bacillus solution and immerse them for 10 minutes. Gently shake the solution every 2-3 minutes to ensure the leaf discs are fully immersed.

[0064] 4) Co-cultivation of Populus tomentosa

[0065] The infected leaf discs were removed with sterilized tweezers, placed on sterilized filter paper, and the bacterial solution was blotted dry. The leaf discs were then laid flat on WPM co-culture medium (WPM powder 2.14 g / L + sucrose 30 g / L + 100 μmol AS + 1.0 mg / L NAA + 2.0 mg / L ZT) and incubated in the dark at 25°C for 2 days.

[0066] 5) Selection and cultivation of Populus tomentosa

[0067] After co-culturing for 2 days, the transformed explants were transferred to a selective medium for inducing callus (WPM powder 2.14 g / L + sucrose 30 g / L + Hyg 9 mg / L + NAA 1.0 mg / L + ZT 2.0 mg / L + Cef 400 mg / L) and cultured in the dark at 25°C for 3–5 weeks, with the selective medium being replaced approximately every 5 days.

[0068] 6) Populus tomentosa bud induction culture

[0069] When white, loose callus tissue appears around the leaf margin, transfer the callus tissue to WPM budding medium (WPM powder 2.14 g / L + sucrose 30 g / L + Hyg 9 mg / L + NAA 0.1 mg / L + ZT 2.0 mg / L + Cef 400 mg / L) on a clean bench and culture at 25°C under light for about 4 to 5 weeks, replacing the WPM budding medium every 10 days.

[0070] 7) Rooting culture of Populus tomentosa

[0071] When the adventitious buds grow to about 5cm, they are transferred to WPM rooting medium (WPM 2.14g / L + sucrose 30g / L + Hyg 9mg / L + NAA 0.1mg / L + Cef 400mg / L) containing the appropriate antibiotics to induce rooting.

[0072] 8) Transplanting of white poplar

[0073] When the seedlings have a well-developed root system, remove them, rinse off the agar from the roots, transplant them into a greenhouse for cultivation, and cover them with plastic wrap to keep them warm and moist. Remove the film after one week of growth.

[0074] For every liter of YEP medium: 10g yeast extract, 10g peptone, 5g NaCl, pH 7.0, autoclaved at 121°C for 20 minutes. For solid medium, add 10-12g of agar powder before sterilization.

[0075] For each liter of LB medium: 5g yeast extract, 10g peptone, 10g NaCl, pH 7.0, autoclaved at 121°C for 20 minutes. For solid medium, add 10-12g of agar powder before sterilization.

[0076] Example 5: Screening and identification of transgenic lines of Populus tomentosa 35S::mAUX6 with heterologous overexpression of PtrAUX6

[0077] 1. Identification of 35S::mAUX6 transgenic positive plants

[0078] After tissue culture, the transgenic plants obtained need to have their genomic DNA extracted for positive identification to confirm whether the vector has been successfully transferred into the poplar plants.

[0079] The CTAB method for extracting genomic DNA from poplar trees is as follows:

[0080] 1) Take a 10mL centrifuge tube, add 3mL CTAB and 90μL beta-mercaptoethanol, and preheat in a 65℃ water bath;

[0081] 2) Take about 0.6g of fresh Populus tomentosa leaves, grind them into powder in liquid nitrogen, transfer them into the above CTAB extraction solution, vortex and mix well, or place the leaves in a 2mL centrifuge tube, add CTAB extraction solution and small steel balls (3 balls) and then crush them with a tissue homogenizer.

[0082] 3) Preheat in a 65℃ water bath for 45 minutes, mixing thoroughly with three (vigorous) vibrations during the process;

[0083] 4) After the time is up, remove it and cool it for 5 minutes, then centrifuge it at 12000 rpm for 10 minutes;

[0084] 5) Carefully aspirate the supernatant into another clean 1.5mL centrifuge tube, let it stand at room temperature for 5min, then add an equal volume of chloroform:isocyanate (24:1), mix by inverting, emulsify by laying flat for 10min, and then centrifuge at 12000rpm for 10min at 18℃.

[0085] 6) Repeat step 5 once to better remove the protein;

[0086] 7) Transfer the supernatant to another clean 10mL centrifuge tube, add an equal volume of -20℃ pre-cooled isopropanol, invert and mix until a white flocculent precipitate is visible, centrifuge at 12000rpm for 10min.

[0087] 8) Rinse twice with 500 μL of 75% (V / V) ethanol, then rinse once more with 500 μL of anhydrous ethanol. Use a pipette to remove the ethanol, and dry the precipitate in a 37°C oven until transparent.

[0088] 9) Dissolve the precipitate in 50-100 μL of sterile water containing RNase A to obtain the genomic DNA of Populus tomentosa.

[0089] Next, using genomic DNA from each line as a template and the wild type as a control, PCR amplification of specific fragments from the recombinant plasmid was performed. The amplification products were detected by agarose gel electrophoresis, observing the presence or absence of bands at corresponding positions. The results showed that multiple regenerated lines contained the exogenously introduced target gene fragment, proving that the 35S::mAUX6 transgenic Populus tomentosa line was successfully obtained through genetic transformation. Figure 2 A).

[0090] 2. Screening of 35S::mAUX6 overexpression lines

[0091] To screen transgenic lines overexpressing PtrAUX6, we plan to use quantitative PCR (qRT-PCR) to screen plants overexpressing AUX6 from 35S::mAUX6 transgenic positive lines for subsequent analysis of wood development.

[0092] First, total RNA was extracted from transgenic positive Populus tomentosa plants with 35S::mAUX6 using the Biospin Polysaccharide and Polyphenol Plant Total RNA Extraction Kit. The specific steps are as follows:

[0093] (1) Add 490 μL of BG Lysis and 10 μL of β-mercaptoethanol to a 1.5 mL RNase-Free EP tube to a final concentration of 2%, and mix well.

[0094] (2) Liquid nitrogen grinding: After grinding an appropriate amount of material into powder in liquid nitrogen, weigh 50-100mg of the powder and put it into the centrifuge tube containing 1.5mL of BGLysis and β-mercaptoethanol. Immediately shake vigorously until there are no obvious particles.

[0095] (3) Centrifuge the above sample in a centrifuge at 12,000 rpm for 5 minutes, and carefully aspirate the supernatant into a new RNase-free 1.5 mL centrifuge tube.

[0096] (4) Estimate the volume of the supernatant of the lysate more accurately, add 0.5 times the volume of anhydrous ethanol. Precipitation may occur at this time, but it will not affect the extraction process. Shake and mix immediately.

[0097] (5) Aspirate the entire mixture into the Spin column, centrifuge at 12,000 rpm for 1 minute, and discard the liquid in the receiving tube.

[0098] (6) Add 600 μL of PG Bufer to the Spin column, centrifuge at 12,000 rpm for 30 seconds, and discard the liquid in the receiving tube.

[0099] (7) Add 500 μL of Wash Buffer to the Spin column, centrifuge at 12,000 pm for 30 seconds, and discard the liquid in the receiving tube. Then add 250 μL of Wash Buffer and repeat the washing once.

[0100] (8) Then the empty column is centrifuged at 12,000 rpm for 1 minute to remove as much Wash Buffer as possible, so as to prevent the residual ethanol in the Wash Buffer from inhibiting the downstream reaction.

[0101] (9) Transfer the Spin column to a new 1.5 mL RNase-Free centrifuge tube, add 25 μL of RElution Buffer to the center of the membrane, let stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute, add the first elution buffer back onto the Spin column membrane, let stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute to obtain total RNA.

[0102] Next, cDNA was obtained through reverse transcription, following the instructions for the long RNA reverse transcription kit provided by TaKaRa. The specific steps are as follows:

[0103] (1) Add 2 μL of 5×gDNA Eraser Buffer and 1 μL of gDNA Eraser to the EP tube, then add RNA, the amount of RNA is calculated according to 1000 ng / concentration, and finally add RNase Free ddH2O to make up to 10 μL, and mix well;

[0104] (2) After keeping it at 42℃ for 2 minutes, quickly place it on ice to cool;

[0105] (3) Add 4 μL of 5×Prime Script Buffer 2, 1 μL of Prime Script RT Enzyme Mix I, 0.5 μL each of specific stem-loop primer and U6 RT primer, 1 μL of RT Primer Mix, and 4 μL of RNase Free ddH2O to the EP tube in step 2 and mix well.

[0106] (4) 42℃, 15min; 85℃, 5sec;

[0107] (5) Finally, store in a refrigerator at -20℃.

[0108] Finally, specific quantitative primers were designed for qRT-PCR amplification using reverse-transcribed poplar cDNA as a template. The amplification was performed according to a quantitative real-time PCR kit. This guide explains how to use the qPCR Master Mix to perform RT-qPCR to detect the expression level of the PtrAUX6 gene. The PCR reaction conditions are: 95℃, 30 sec; 40 cycles: 95℃, 5 sec; 60℃, 1 min; 95℃, 15 sec; 60℃, 30 sec; 95℃, 15 sec.

[0109] The quantitative primers are as follows:

[0110] AUX6-qPCR-F: 5'-ATGGGGAGATGGGTAGGGA-3' (SEQ ID No. 12);

[0111] AUX6-qPCR-R: 5'-GTTGTGTGGGTGGTGGAGAG-3' (SEQ ID No. 13).

[0112] Experimental results showed that among the transgenic Populus tomentosa lines with 35S::mAUX6, multiple lines significantly overexpressed AUX6 compared to the wild type. Among them, L2 and L4 plants showed the highest expression levels, reaching 81.3 times and 63.6 times that of the wild type, respectively. Figure 2 B).

[0113] Example 6: Analysis of the wood development regulation function of the Populus tomentosa 35S::mAUX6 transgenic line

[0114] To investigate the regulation of secondary vascular development in poplar by mAUX6 overexpression, two lines with the highest relative expression levels were selected for subsequent experiments: the L2 and L4 lines under a wild-type background. To elucidate the function of AUX6 in secondary stem vascular development, the selected positive plants were cultured for approximately three months, sectioned, stained with toluidine blue, and the development of the cambium and xylem was observed. ImageJ software was used to analyze relevant indicators.

[0115] The results are as follows Figure 3 and Figure 4 As shown, the 35S::mAUX6 transgenic Populus tomentosa line, after 3 months of growth, exhibited significantly increased cambium mitotic activity compared to the wild type. Figure 3 The cambium cell layers of the 35S::mAUX6-L2 and 35S::mAUX6-L4 plants were 17.9% and 25.4% higher than those of the wild-type plants, respectively. Figure 3 (A and C). Meanwhile, the xylem area of ​​the 35S::mAUX6 transgenic Populus tomentosa increased by 4.7% and 5.9% respectively in L2 and L4 plants compared to the wild type. Figure 4The differences between A and C were both highly significant (p<0.001); the number of xylem cell layers was also significantly increased in transgenic Populus tomentosa plants of 35S::mAUX6-L2 and 35S::mAUX6-L4 compared to wild-type plants, increasing by 13.1% and 17.7% respectively. Figure 4 B).

[0116] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. PtrAUX6 The application of genes in regulating the development of Populus tomentosa wood is characterized by: Overexpression of the expression shown in SEQ ID No. 9 in Populus tomentosa PtrAUX6 Genes were used to obtain transgenic Populus tomentosa strains with increased xylem cell production rate and improved wood growth rate.

2. A method for constructing a fast-growing xylem poplar line, comprising the following steps: 1) The one shown in SEQ ID No. 9 PtrAUX6 The gene was ligated into a plant expression vector to obtain the 35S::mAUX6 overexpression vector; 2) Transformation with Agrobacterium: The 35S::mAUX6 overexpression vector constructed in step (1) was introduced into wild-type Populus tomentosa leaves using the Agrobacterium-mediated leaf disc method. 3) After co-culture, selection culture, obtaining clustered shoots, obtaining rooted transgenic plants and identifying positive plants, the Populus tomentosa strains that overexpress PtrAUX6 were finally obtained.

3. The method according to claim 2, characterized in that, The plant expression vector was pCAMBIA1300-GFP.

4. The method according to claim 2, characterized in that, The Agrobacterium is GV3101 strains.

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