PtoSAUR74 gene related to forming ability of adventitious roots of poplar and application of PtoSAUR74 gene

Abstract

The invention discloses a PtoSAUR74 gene related to the forming ability of adventitious roots of poplar and application of the PtoSAUR74 gene. A nucleotide sequence of a coding region of the PtoSAUR74 gene is shown as SEQ ID NO.1, and a protein sequence of the PtoSAUR74 gene is shown as SEQ ID NO.2. Through overexpression of the PtoSAUR74 gene in a poplar, the length, the number and the growth rate of adventitious roots can be remarkably inhibited, which indicates that the PtoSAUR74 gene is a key regulatory gene influencing the development of the adventitious roots of the poplar, and the PtoSAUR74 gene can be used for inhibiting the adventitious roots of the poplar. A key target spot is provided for realizing precise molecular regulation and control of poplar root system growth. The invention also discloses a method for regulating and controlling the forming ability of the adventitious root of the poplar, the generation of the adventitious root can be directly inhibited by regulating and controlling the expression of the PtoSAUR74 gene, and a new technical means and theoretical basis are provided for genetic improvement of the poplar.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a PtoSAUR74 gene related to the formation ability of poplar adventitious roots and application thereof. BACKGROUND

[0002] Plant root system is an important organ for fixing plants and absorbing water and nutrients. Unlike lateral roots developed from radicles, adventitious roots can be generated from various non-root organs such as stems and leaves, and this development plasticity has extraordinary significance for the environmental adaptation and vegetative propagation (such as cutting) of plants. In agricultural and forestry production, the efficiency of cutting propagation directly depends on the ability of the explant to produce adventitious roots.

[0003] The occurrence of adventitious roots is mainly regulated by auxin. Auxin accumulates at the base, activates a series of downstream response elements such as ARF7, ARF19 and their target genes LBD16 and LBD29 through the TIR1 / AFB-Aux / IAA-ARF signal pathway, and jointly drives the initiation and development of adventitious root primordia. In recent years, as one of the core gene families of early auxin response, the plant SAUR (Small Auxin-Up RNA) gene family plays a key role in the regulation of plant root development, environmental adaptation and cell elongation. Existing studies have shown that SAUR genes mainly play a role by regulating cell elongation and cell wall loosening process. Its mechanism of action involves that SAUR protein inhibits the activity of plasma membrane-related PP2C.D phosphatase, thereby activating the plasma membrane H+-ATPase proton pump, increasing the electrochemical potential of the plasma membrane, acidifying the apoplast, and ultimately promoting cell expansion. For example, in Arabidopsis, overexpression of AtSAUR41 leads to pleiotropic auxin-related phenotypes, including increased length of hypocotyls, development of lateral roots, expansion of petals and twisting of inflorescence stems. Similarly, overexpression of cucumber CsSAUR31 gene also significantly promotes the elongation of roots and hypocotyls.

[0004] However, a robust development system must contain both positive "promotion" and cautious "inhibition" mechanisms. At present, the regulation of adventitious root formation is obviously unbalanced: we have a better understanding of the positive linear pathway from auxin perception (TIR1) to signal transduction (ARF) to cell response (such as SAUR), but we still know little about the negative regulation links in this pathway and the upstream inhibition mechanism of key effectors such as SAUR. Therefore, creating new forest genotypes with controlled root sprouting and adventitious root development ability, and significantly weakening or even eliminating their ability to produce adventitious plants through root sprouting and broken branches for vegetative propagation in natural environments, will lay a solid foundation for realizing safer, more efficient and more ecologically friendly modern forestry. SUMMARY

[0005] In order to overcome the above problems, the present inventors first identify a PtoSAUR74 gene related to the adventitious root formation ability of poplar and its encoded protein, and find that the number, length and growth rate of adventitious roots of the poplar overexpressing the gene are significantly inhibited compared with WT, indicating that the gene can precisely regulate the formation of adventitious roots of poplar, providing a key target for precise molecular regulation of poplar root growth, and providing a new theoretical basis for genetic improvement of poplar, and laying a solid foundation for realizing modern forestry that is safer, more efficient and more ecological friendly, so as to complete the present application.

[0006] Specifically, the present application aims to provide the following aspects:

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

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

[0009] In a third aspect, the PtoSAUR74 gene of the first aspect or the protein of the second aspect is provided for use in regulating the formation of adventitious roots of poplar.

[0010] In a fourth aspect, a vector and / or strain comprising the PtoSAUR74 gene of the first aspect is provided, wherein the vector comprises an overexpression recombinant vector, and the strain comprises E. coli and Agrobacterium.

[0011] In a fifth aspect, the PtoSAUR74 gene of the first aspect, the protein of the second aspect or the vector and / or strain of the fourth aspect is provided for use in poplar breeding.

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

[0013] The present application has the following beneficial effects:

[0014] (1) The present application first identifies that the PtoSAUR74 gene has the function of significantly inhibiting the formation and rooting length of adventitious roots of poplar, and provides a key target for precise molecular regulation of poplar root growth;

[0015] (2) The application provides a method for regulating the formation ability of adventitious roots of poplar, and by regulating the expression of the PtoSAUR74 gene, the generation of adventitious roots can be directly inhibited, thereby providing a new technical means and theoretical basis for genetic improvement of poplar;

[0016] (3) The application discloses the direct application of the PtoSAUR74 gene and the coded protein thereof in regulating the formation of adventitious roots of poplar, and can be used for creating a new type of forest genotype with controlled development ability of root sprouts and adventitious roots, thereby significantly weakening or even eliminating the ability of the genotype to produce adventitious plants through root sprouting and broken branch in a natural environment, and laying a solid foundation for realizing safer, more efficient and more ecological friendly modern forestry;

[0017] (4) The application of the poplar variety with intervened development of adventitious roots provided by the application has clear regulation of gene function, and the cultivation method is quick and effective, and is beneficial to reducing breeding cost, improving breeding efficiency and shortening the generation time. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A structural schematic diagram of a PtoSAUR74-pBI121-EGFP overexpression vector constructed in Example 2 is shown;

[0019] Figure 2 RT-qPCR detection of the transcription level of PtoSAUR74 in overexpression plants (OE-3, OE-6) is shown;

[0020] Figure 3 The root morphological phenotype of wild type plants (WT-1, WT-2) and PtoSAUR74 gene overexpression plants (OE-3, OE-6) after growth in a tissue culture bottle 1 / 2MS medium for 0, 4, 5, 6 and 12 days is shown;

[0021] Figure 4 The total length of adventitious roots of wild type plants (WT-1, WT-2) and PtoSAUR74 gene overexpression plants (OE-3, OE-6) after growth in a tissue culture bottle for 12 days is shown;

[0022] Figure 5 The average length of adventitious roots of wild type plants (WT-1, WT-2) and PtoSAUR74 gene overexpression plants (OE-3, OE-6) after growth in a tissue culture bottle for 12 days is shown;

[0023] Figure 6 The number of adventitious roots of wild type plants (WT-1, WT-2) and PtoSAUR74 gene overexpression plants (OE-3, OE-6) after growth in a tissue culture bottle for 12 days is shown;

[0024] Figure 7The rooting ratio of adventitious roots of wild type plants (WT-1, WT-2), PtoSAUR74 gene overexpression plants (OE-3, OE-6) 12 days after cutting is shown. DETAILED DESCRIPTION

[0025] The application will be further described in detail below by preferred embodiments and examples. The features and advantages of the application will become more apparent through these descriptions.

[0026] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0027] In a first aspect of the application, a PtoSAUR74 gene related to the adventitious root formation ability of poplar is provided, and the nucleotide sequence of the coding region of the gene is shown in SEQ ID NO. 1.

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

[0029] In the application, the poplar is white poplar, preferably it is white poplar or 84k poplar.

[0030] The 84k poplar is a clone variety generated by hybridization of Populus alba and Populus glandulosa (Populus alba x Populus glandulosa), and it belongs to the white poplar family, which is easy to root, grows fast, has good wood quality and wide adaptability. At the same time, due to its easier standardization of transformation conditions, it is often selected as an ideal model for genetic transformation.

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

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

[0033] The PtoSAUR74 gene provided by the application has a clear function and can accurately regulate the adventitious root formation ability of poplar, and deeply analyzes the molecular regulation mechanism of the development of adventitious roots of poplar, thereby providing an important theoretical basis for molecular breeding of poplar.

[0034] The PtoSAUR74 gene is first identified to have the function of significantly inhibiting poplar adventitious root formation and rooting length, and provides a key target for realizing precise molecular regulation of poplar root growth.

[0035] In a second aspect, the application provides a protein related to poplar adventitious root formation ability, which is a protein encoded by the PtoSAUR74 gene in the first aspect, and the amino acid sequence is shown as SEQ ID NO. 2.

[0036] In a third aspect, the application provides application of the PtoSAUR74 gene related to poplar adventitious root formation ability in the first aspect or the protein related to poplar adventitious root formation ability in the second aspect in regulating poplar adventitious root formation.

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

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

[0039] Preferably, the application is to inhibit the formation of poplar adventitious roots by increasing the expression of the PtoSAUR74 gene in the poplar individual.

[0040] More preferably, the application is implemented by overexpressing the PtoSAUR74 gene related to the poplar adventitious root formation ability in the poplar individual.

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

[0042] The application explicitly defines the direct application of the PtoSAUR74 gene and the protein encoded thereby in regulating poplar adventitious root formation, which can be used to create new forest genotypes with controlled root sprout and adventitious root development ability, significantly weaken or even eliminate the ability of the new forest genotypes to produce adventitious plants through root sprouting and broken branch in the natural environment, and lay a solid foundation for realizing safer, more efficient and more ecological friendly modern forestry.

[0043] In a fourth aspect, the application provides a vector and / or strain comprising the PtoSAUR74 gene in the first aspect, wherein the vector comprises an overexpression recombinant vector, and the strain comprises Escherichia coli and Agrobacterium.

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

[0045] More preferably, the Agrobacterium is GV3101.

[0046] In a fifth aspect, the present application provides the use of the gene of the first aspect or the protein of the second aspect or the vector and / or strain of the third aspect in poplar breeding.

[0047] Preferably, the poplar is white poplar, preferably, Populus tomentosa or 84K poplar, and the breeding is breeding of poplar varieties with inhibited adventitious root development.

[0048] More preferably, the breeding is overexpression of the PtoSAUR74 gene in poplar to breed poplar varieties with inhibited adventitious root development.

[0049] The use of the present application in poplar varieties with inhibited adventitious root development has clear gene function regulation, fast and effective breeding method, and is conducive to reducing breeding cost, improving breeding efficiency and shortening generation time.

[0050] In a sixth aspect, the present application provides a method for regulating the ability of poplar to form adventitious roots, the method comprising the step of overexpressing the PtoSAUR74 gene of the first aspect.

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

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

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

[0054] In a preferred embodiment, overexpression of the PtoSAUR74 gene is achieved by introducing a PtoSAUR74 gene overexpression vector into the body of poplar, wherein the PtoSAUR74 gene overexpression vector is obtained by constructing the CDS sequence of the PtoSAUR74 gene into a basic vector.

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

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

[0057] The pBI121-EGFP vector is 13629 bp, which contains a 35S strong promoter (CaMV35S) and a kanamycin (Kana) resistance for screening of transgenic plants, and can be single-digested by XbaI.

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

[0059] Step 1, constructing a recombinant vector for overexpression of the PtoSAUR74 gene.

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

[0061] Step 1-1, obtaining the coding region sequence of the PtoSAUR74 gene.

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

[0063] The sequence of the primer PtoSAUR74-F is agaacacgggggactATGAAGAAGATCAACTTGATACTAA, and the sequence of the primer PtoSAUR74-R is: acccccgggggatcctGAACATGTAGAGATCAGCTAATTCT.

[0064] In the sequence, the lower case part is the vector homologous arm sequence, and the upper case part is the gene sequence.

[0065] Step 1-2, linking the coding region sequence of the PtoSAUR74 gene to a basic vector for transformation.

[0066] Preferably, the basic vector is a pBI121-EGFP vector, and XbaI is used for single digestion.

[0067] The linked vector is transformed into E. coli DH5α competent cells.

[0068] Step 1-3, identifying the linked recombinant vector.

[0069] Preferably, the E. coli bacterial liquid is subjected to PCR identification, and the reaction conditions are as follows: 94℃ for 5 min; (94℃ for 30 s; 58℃ for 30 s; 72℃ for 70 s) for 35 cycles; 72℃ for 10 min; 4℃ for ∞.

[0070] Step 2, transforming the constructed overexpression recombinant vector into Agrobacterium.

[0071] Step 3, infecting, proliferating, differentiating and rooting culturing the poplar material by the Agrobacterium-mediated method to obtain a transgenic plant.

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

[0073] Step 4, identifying the obtained transgenic plants.

[0074] Preferably, the identification comprises PCR identification and RT-PCR (fluorescent quantitative PCR) identification.

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

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

[0077] More preferably, the PCR identification result has a specific band, and the RT-PCR result shows that the PtoSAUR74 gene overexpression plant has a significantly increased transcription level of the PtoSAUR74 gene.

[0078] The above PCR identification primers and reaction programs selected in the present application are determined through a large number of experimental researches, and can accurately identify the transgenic plants.

[0079] In a preferred embodiment, the PtoSAUR74 gene overexpression plant is subjected to adventitious root phenotype analysis, and compared with the wild type plant, the number of adventitious roots, the length of adventitious roots and the rooting rate of the overexpression plant are significantly reduced.

[0080] Preferably, after being cultured in 1 / 2MS medium for 12 days, the total length of adventitious roots of the PtoSAUR74 gene overexpression plant is reduced by 8.21 cm compared with the wild type plant;

[0081] After being cultured in 1 / 2MS medium for 12 days, the average length of adventitious roots of the PtoSAUR74 gene overexpression plant is reduced by 1.55 cm compared with the wild type plant;

[0082] After being cultured in 1 / 2MS medium for 12 days, the number of adventitious roots of the PtoSAUR74 gene overexpression plant is reduced by 3.17 compared with WT;

[0083] The WT strain had some individuals start rooting at day 4, and the PtoSAUR74 gene overexpression strain did not root at this time; by day 6, the rooting rate of the WT strain reached 60%, and only a few PtoSAUR74 gene overexpression strains rooted; by day 7-8, the WT strain had completed rooting, and the PtoSAUR74 gene overexpression strain completed rooting by day 11-12.

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

[0085] Embodiment

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

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

[0088] Example 1 Cloning of PtoSAUR74 gene

[0089] The cds_from_genomic.fna file was downloaded from the public website https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_018804465.1 / , and the KAG6756651.1 was searched to obtain the CDS sequence of the PtoSAUR74 gene of P. tomentosa, which is located on chromosome CM031988.1, with a start position of 14068599 and an end position of 14068976, and a total length of 378 bp.

[0090] Referring to the P. tomentosa genome, and considering various principles of primer design, the primer blast tool (NCBI, https: / / blast.ncbi.nlm.nih.gov) was used to design the amplification primers PtoSAUR74-F and PtoSAUR74-R of the PtoSAUR74 gene CDS sequence. The sequence of the primer PtoSAUR74-F is: agaacacgggggactATGAAGAAGATCAACTTGATACTAA; and the sequence of the primer PtoSAUR74-R is: acccccgggggatcctGAACATGTAGAGATCAGCTAATTCT.

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

[0092] The RNA of Populus tomentosa was extracted by the plant RNA extraction kit of Yeasen Biotechnology Co., Ltd. The RNA was reversely transcribed into cDNA by HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novogene Bio- tech Co., Ltd.), and the obtained genomic cDNA was used as a template for PCR amplification by using primers PtoSAUR74-F and primers PtoSAUR74-R, and 2xVazyme LAmp Master Mix (Dye Plus) of Nanjing Vazyme Co., Ltd. was used. The PCR experimental reaction system (50 μL) is shown in Table 1:

[0093] Table 1

[0094]

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

[0096] The product of the PCR reaction was subjected to agarose gel electrophoresis, and after detection, the DNA Clean-up Kit reagent box of Jiangsu Kangweishijishe Biological Technology Co., Ltd. was used for purification. After purification, the purity and concentration of the DNA purification product were determined by using the instrument. Finally, the CDS sequence of the PtoSAUR74 gene was obtained, as shown in SEQ ID NO. 1.

[0097] Example 2 Construction of PtoSAUR74 gene overexpression vector

[0098] (1) Vector enzyme digestion

[0099] The selected overexpression vector is pBI121-EGFP vector, which is purchased from Mingsheng Plasmid Platform. The vector is 13629 bp, which contains 35S strong promoter (CaMV35S), and the resistance in transgenic plant screening is kanamycin (Kana), which can be digested by XbaI. The enzyme digestion reaction system is shown in Table 2:

[0100] Table 2

[0101]

[0102]

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

[0104] The product of the PCR reaction was subjected to agarose gel electrophoresis, and after correct detection, a DNA Clean-up Kit reagent box from Jiangsu Kangwei Shijis Century Biotechnology Co., Ltd. was used for purification. After purification, the purity and concentration of the DNA purified product were determined using an instrument. Finally, the pBI121-EGFP vector of the digested gene was obtained.

[0105] (2) Ligation transformation

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

[0107] 50 μl of DH5α competent cells were aspirated, 5 μl of ligation product was added, mixed by flicking, and placed on ice for half an hour. After ice bath, 42°C water bath heat shock for 45 s, then quickly cool on ice for 2 min to avoid shaking and reduce transformation efficiency. Add 700 μl of sterile LB liquid medium without antibiotics, mix well after blowing, and then place in a 37°C, 200 rpm shaker for 15 min to recover the bacterial cells. After recovery, centrifuge at 6000 rpm for 1 min to collect the bacterial cells, resuspend the bacterial cells with 100 μl of supernatant, and spread on LB solid plate medium containing kanamycin (100 mg / ml), and incubate at 37°C for 14-16 h. The final vector structure constructed is shown in Figure 1 .

[0108] (3) Bacterial liquid PCR identification of positive clones

[0109] A sterile gun head was used to pick single colonies from the plate in 250 μl of LB liquid medium containing kana, and incubated at 37°C, 200 rpm / min for about 3 h as an amplification template. The gene PCR purified product and ddH2O (double distilled water) were used as templates, and positive and negative controls were set up, respectively. Taq Plus Master Mix from Nanjing Vazyme Company was used for PCR amplification. The PCR reaction system is shown in Table 3:

[0110] Table 3

[0111]

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

[0113] Reaction conditions: 94°C 5 min; (94°C 30 s; 58°C 30 s; 72°C 70 s) 35 cycles; 72°C 10 min; 4°C ∞.

[0114] The PCR product was detected by 1% agarose gel electrophoresis, and the colonies that could amplify the same band as the positive control were positive clones.

[0115] (4) Positive clone plasmid extraction

[0116] The positive clone detected by PCR was taken to 6 ml LB liquid medium containing spectinomycin, and cultured at 37°C, 200 rpm / min overnight. The plasmid was extracted using the plasmid extraction kit from Beijing Kangwei Reagent Co., Ltd. and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. After the sequence was determined and compared correctly, the vector construction was completed.

[0117] Example 3 Genetic transformation of PtoSAUR74 gene

[0118] (1) Recombinant plasmid transformation of Agrobacterium

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

[0120] (1.2) In turn, stand on ice for 5 min, freeze in liquid nitrogen for 1 min, immediately put into 37°C water bath for 5 min, and ice bath for 5 min.

[0121] (1.3) Add 700 μL of YEP liquid medium without antibiotics to the bacterial solution, mix thoroughly, and incubate at 28°C, 200 rpm for 2-3 hours. After recovery culture, centrifuge the bacterial solution at 6000 rpm for 1 minute, discard part of the supernatant, and reserve 100 μL of supernatant mixed with the bacterial solution. Use a disposable sterile coating rod to evenly coat the bacterial solution on the surface of YEP solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin, then place it in a 28°C incubator and incubate it upside down for 72-90 hours.

[0122] (1.4) After single colonies grow, use a sterile gun head to pick several single colony spots and place them in a 2 mL centrifuge tube containing 250 μL of YEP liquid medium (1:1000 addition of Rif, Kana), and incubate at 30°C, 200 rpm on a shaker for 2 hours. After the culture is completed, the bacterial solution is taken for PCR identification. The correct positive bacterial solution is added with 50% glycerol and frozen in liquid nitrogen, and stored in a -80°C refrigerator for subsequent genetic transformation experiments.

[0123] (2) Activation of Agrobacterium

[0124] (2.1) Take Agrobacterium liquid containing overexpression vector plasmid from -80°C refrigerator, streak inoculate to YEP solid medium (1:1000 add Rif, Kana) respectively, seal the plate, and invert culture in 28°C dark incubator for 2-3 days.

[0125] (2.2) Use sterile gun head to pick single colony from the plate and inoculate into 3 mL YEP liquid medium (1:1000 add Rif, Kana), and place in a shaker at 30°C, 200 rpm for overnight culture. Take 1 mL bacterial liquid and transfer into 100 mL YEP liquid medium (1:1000 add Rif, Kana) in a 250 mL sterile conical flask, and place in a shaker at 30°C, 200 rpm for 4-5 h until OD600 is 0.3-0.5.

[0126] (2.3) Transfer 100 mL bacterial liquid to two 50 mL sterile centrifuge tubes in a super-clean bench, centrifuge at 2560 g, 4°C for 20 min. Collect the bacterial bodies. In the super-clean bench, discard the supernatant, resuspend the bacterial bodies in a sterile wide-mouth bottle with 100 mL resuspension liquid. The bacterial liquid is used for subsequent infection experiments. The composition of the resuspension liquid (1 L) is: WPM 2.37 g + VB1 0.9 mg + MES 0.5 g + sucrose 20 g + 2,4-D 0.1 mg + AS 200 mM + ddH2O to 1 L.

[0127] (3) Callus method infection

[0128] Select the leaves of healthy and sterile seedlings (dark green leaves, thick texture), use a sterile scalpel to remove the petiole, the petiole can be left to grow callus, and 2-3 horizontal wounds are made on the main veins of the leaves, which are placed face down on callus medium (CIM) and placed in a 25°C environment in the dark. After the leaves grow for 20-30 days, white and loose callus will grow at the wound, the callus is peeled off from the leaves, divided into soybean-sized pieces, and transferred into new callus medium. The prepared callus is transferred into a sterile wide-mouth bottle containing bacterial liquid, and placed in a shaker at 28°C, 160 rpm for 15-20 min. The composition of the callus medium (1 L) is: WPM 2.37 g + 2,4-D 0.1 mg + KT 0.1 mg + MES 0.5 g + agar 7.5 g + ddH2O to 1 L.

[0129] (4) Co-culture

[0130] In a clean bench, the callus was taken out from the bacteria solution with tweezers, and the extra bacteria solution was absorbed with filter paper. The infected callus was spread on the co-culture medium and cultured in dark at 25°C for 2 days. The composition of the co-culture medium (1L) was as follows: WPM 2.37g + VB1 0.9mg + sucrose 20g + MES 0.5g + AS 200mM + agar 7.5g + ddH2O to 1L.

[0131] (5) Differentiation culture

[0132] After the dark culture, the appropriate plant resistance was selected according to the carrier, and the differentiation medium containing the corresponding antibiotic was prepared.

[0133] The callus was transferred to the differentiation medium, and the medium was changed once in about 20 days for the first time, and then once in 10 days. During this period, the callus would turn green, harden, and then partially turn red. This stage lasted about two months, and the whole process was cultured in a 25°C light incubator. The composition of the co-culture medium (1L) was as follows: WPM 2.37g + VB1 0.9mg + sucrose 20g + MES 0.5g + AS 200mM + 6-BA 0.5mg + NAA 0.1mg + TDZ 0.002mg + TMT 250mg + Cef 250mg + kana 20mg + agar 7.5g + ddH2O to 1L.

[0134] (6) Cluster shoot induction rooting

[0135] After the adventitious shoots grew to about half a centimeter, sterile tweezers or a scalpel were used to cut off the adventitious shoots (attention was paid to the growth point of the adventitious shoots to avoid selecting adventitious shoots differentiated from the same cell), which were then placed in the shoot elongation medium for growth. After a period of elongation culture, when the adventitious shoots grew to 1-2 centimeters, sterile tweezers or a scalpel were used to cut off the adventitious shoots individually, which were then placed in the rooting medium for rooting culture. After about 10 days, the adventitious shoots would grow roots, and the rooting culture would become a complete plant.

[0136] The composition of the elongation medium (1L) was as follows: WPM 2.37g + IBA 0.2mg + sucrose 20g + 6-BA 0.5mg + TMT 250mg + Cef 250mg + agar 7.5g + ddH2O to 1L.

[0137] The composition of the rooting medium (1L) was as follows: 1 / 2MS + sucrose 20g + IBA 0.05mg + NAA 0.05mg + agar 7g + TMT 250mg + Cef 250mg + kana 20mg + ddH2O to 1L.

[0138] Example 4 Identification of PtoSAUR74 gene overexpression plants

[0139] (1) Wild type 84K poplar and PtoSAUR74 gene overexpression plant DNA crude extraction:

[0140] (i) Take a piece of the sample leaf to be extracted and place it in a 2 mL centrifuge tube and add 1 grinding bead, freeze in liquid nitrogen and crush with a tissue crusher for 5 min.

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

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

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

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

[0145] (vi) Place in a 40°C oven for 2 h.

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

[0147] (2) Identification of PtoSAUR74 gene overexpression plants

[0148] (2.1) DNA level identification: Use the crude extracted transgenic plant gDNA as a template, use Taq Plus Master Mix for PCR identification, the PCR reaction system is referred to Table 3 in Example 2, directly run the PCR product for electrophoresis, check if the band is correct, and if correct, it is a PtoSAUR74 gene overexpression plant.

[0149] (2.2) Transcription level identification: RT-qPCR is used to detect the transcription level of PtoSAUR74 in overexpression plants.

[0150] (i) Extract the root tissue RNA to be detected, and reverse transcribe the template cDNA according to the method described in the kit in Example 1 to extract the RNA and reverse transcribe the cDNA.

[0151] (ii) RT-qPCR experiment was performed using 2x ChamQ SYBR Color qPCR Master Mix, and the fluorescent quantitative PCR (polymerase chain reaction) primers were designed according to the PtoSAUR74 gene sequence of 84K poplar, as shown below, and the primer names were PtoSAUR74-qPCR-F and PtoSAUR74-qPCR-R, and the nucleotide sequences were shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively. The reaction system (20 μL) is shown in Table 4.

[0152] Table 4

[0153]

[0154]

[0155] The primer sequence of Actin-F is shown in SEQ ID NO. 5, and the primer sequence of Actin-R is shown in SEQ ID NO. 6.

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

[0157] The detection results of RT-qPCR are shown in Table 5. Figure 2 As can be seen, the expression of the overexpression plants numbered OE-3 and OE-6 is higher, which is 107.1 times and 119.6 times of the wild type plant (WT), respectively.

[0158] Example 5 Analysis of adventitious root phenotype of PtoSAUR74 gene overexpression plants

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

[0160] Specifically, Figure 3 The root morphological phenotype of wild type plants (WT-1, WT-2) and PtoSAUR74 gene overexpression plants (numbered OE-3, OE-6) after 0, 4, 5, 6, 12 days of growth in tissue culture bottle 1 / 2MS medium is shown. The results show that compared with WT, the number and length of adventitious roots of the overexpression plants are significantly reduced.

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

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

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

[0164] Figure 7 Rooting time analysis showed that some individuals of WT strain had started rooting at day 4, while PtoSAUR74 gene overexpression strains (OE-3, OE-6) had not yet rooted at this time; by day 6, the rooting rate of WT strain had reached 60%, while PtoSAUR74 gene overexpression strains had only a few roots; by day 7-8, the WT strain had completed rooting, while PtoSAUR74 gene overexpression strains had only 11-12 days of rooting rate. It can be seen that the rooting time and rate of overexpression strains are delayed compared with wild type.

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

[0166] In summary, overexpression of PtoSAUR74 gene can significantly inhibit the length, number and rooting rate of poplar adventitious roots, and can be used for optimizing poplar seedling propagation efficiency and root architecture design, and has important scientific research and industrialization value.

[0167] The above detailed description of the present application is combined with specific embodiments and exemplary examples, but these descriptions cannot be understood as limitations of the present application. Those skilled in the art understand that the technical solutions and embodiments of the present application can be variously replaced, modified or improved without departing from the spirit and scope of the present application, and these all fall within the scope of the present application.

Claims

1. A PtoSAUR74 gene related to the formation ability of poplar adventitious roots, characterized in that, The coding region nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. The gene of claim 1, wherein The adventitious root formation ability of the poplar includes the rooting number, length and rooting rate of the adventitious roots.

3. A protein associated with poplar adventitious root formation, wherein the protein is a protein of claim 1 or 2. The protein is the protein encoded by the PtoSAUR74 gene of claim 1, and the amino acid sequence is shown as SEQ ID NO.

2. ​ 4. Application of the PtoSAUR74 gene of claim 1 or 2 or the protein of claim 3 in regulating the formation of adventitious roots of poplar.

5. Use according to claim 4, characterized in that, The application is implemented by regulating the expression of the PtoSAUR74 gene in the poplar individual or by regulating the activity of the protein related to the adventitious root formation ability of the poplar individual.

6. A vector and / or strain comprising the PtoSAUR74 gene according to claim 1 or 2, characterized in that, The vector includes an overexpression recombinant vector, and the strain includes E. coli and Agrobacterium.

7. Application of the PtoSAUR74 gene of claim 1 or 2, the protein of claim 3 or the vector and / or strain of claim 6 in poplar breeding.

8. A method for modulating the ability of poplar to form adventitious roots, characterized in that, The method includes the step of overexpressing the PtoSAUR74 gene of claim 1 or 2.

9. The method of claim 8, wherein, The method includes the following steps: Step 1, constructing an overexpression recombinant vector of the PtoSAUR74 gene; Step 2, transforming the constructed overexpression recombinant vector into Agrobacterium; Step 3, performing infection, proliferation, differentiation and rooting culture on the poplar material by Agrobacterium-mediated method to obtain a transgenic plant; Step 4, identifying the obtained transgenic plant.

10. The method of claim 9, wherein, Step 1 includes the following sub-steps: Step 1-1, obtaining the coding region sequence of the PtoSAUR74 gene; Step 1-2, connecting and transforming the coding region sequence of the PtoSAUR74 gene with a basic vector; Step 1-3, identifying the connected recombinant vector.

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