Quercus fabri stem in-vitro regeneration system and construction method of Quercus fabri genetic transformation system

Abstract

The invention discloses a method for constructing a white oak stem in-vitro regeneration system, which comprises the following steps: taking a white oak stem as an explant, exploring the influence of different factors in white oak in-vitro regeneration on explant selection, browning degree control, fixed bud germination, adventitious bud proliferation and adventitious roots, and establishing the white oak regeneration system. The tissue culture seedling material with consistent genetic background and short culture period is obtained, and the problems that explants of oak trees are easy to brown and pollute are solved. The invention further discloses a combined culture medium for constructing the quercus alba stem segment in-vitro regeneration system. The combined culture medium comprises a fixed bud starting culture medium, an adventitious bud proliferation culture medium and an adventitious root induction culture medium. The invention also discloses a construction method of the Quercus fabri genetic transformation system, and the Quercus fabri PDS gene (QfPDS) is used as a reporter gene to construct a TRV-mediated VI GS system. By combining a tissue culture regeneration system and an instantaneous transformation system of the white oak, accurate analysis of gene functions of the white oak is realized, and a foundation is laid for promoting genetic improvement and molecular breeding of the white oak.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a method for constructing an in vitro regeneration system of a white oak stem segment and a white oak genetic transformation system. Background Art

[0002] The genus Quercus (Quercus L.) of the Fagaceae family is a plant with many species, strong adaptability and wide distribution. There are more than 400 species in total, distributed in North America, Europe, Africa and Asia, of which about 130 species are found in my country. White oak (Quercus fabri Hance) is a deciduous tree of the genus Quercus and is a native tree species unique to the subtropical regions of my country. The wood of white oak is hard and dense, and can be used for construction and furniture. Its fruit is rich in starch and can be used to make wine or tofu, vermicelli, etc. It can also be used as feed. White oak has a well-developed root system, strong resistance to adversity and adaptability, and is an excellent soil and water conservation tree species with significant ecological benefits.

[0003] Since white oak seeds vary greatly and are not resistant to storage, asexual reproduction is beneficial for controlling the quality of white oak seedlings and achieving rapid reproduction. In recent years, in vitro regeneration systems have been established for a variety of oak trees at home and abroad through different approaches: for example, European cork oak (Q.suber L.), North American red oak (Q.rubra L.), cork oak (Q.variabilis Blume) and Shuma oak (Q.shumardii Buckley) are regenerated through induced somatic embryogenesis; holly oak (Q.ilex L.), acutissima Carruth., oak (Q.dentata Thunb.), Mongolian oak (Q.mongolica Fisch.exLedeb.) and big-leaf oak (Q.griffithii Hook.f.&Thomson ex Miq.) are regenerated through direct or indirect organogenesis. However, in the process of establishing an in vitro regeneration system, one of the most common problems is that oak explants are prone to browning and contamination.

[0004] In addition, the traditional breeding cycle of white oak is long and lacks an efficient and stable genetic transformation technology system. Therefore, experiments related to gene function exploration can only be performed on model plants, which greatly limits the progress of molecular research on white oak. Virus-induced gene silencing (VIGS) is a transient transformation technology that is particularly suitable for species that lack a stable genetic transformation system. This technology is based on the plant's defense mechanism against RNA viruses. It infects plants with recombinant viruses that insert target gene fragments, induces plant endogenous genes to be silenced at the transcriptional level or post-transcriptional level, and then realizes the analysis of gene function. Compared with the construction of a stable genetic system, VIGS is simple to operate, has a short experimental cycle, and can simultaneously silence single or multiple gene family members. It is an indispensable method in reverse genetics. At present, VIGS has been widely used in plant gene function research and genetic improvement.

[0005] Phytoene desaturase (PDS) is a key rate-limiting enzyme in the β-carotene biosynthesis pathway. When the endogenous PDS gene in plants is silenced, the plants will show dwarfing or leaf photobleaching. This obvious phenotypic change makes the PDS gene often used as a reporter gene in the VIGS system. The success of the VIGS system construction has been verified by silencing the PDS gene in many woody plants such as Vernicia fordii (Hemsl.) Airy Shaw, Populus tomentosa Carrière, Camellia oleifera Abel, Eucommia ulmoides Oliv. and Populus euphratica Oliv.

[0006] It can be seen that the construction of the existing in vitro regeneration system of white oak and the genetic transformation system of white oak still has defects and needs to be further improved. How to create an efficient in vitro regeneration system of white oak stem segments and a construction method of white oak genetic transformation system is one of the important research and development topics at present. Summary of the invention

[0007] The first technical problem to be solved by the present invention is to provide a method for constructing an efficient in vitro regeneration system of white oak stem segments, so that tissue culture seedling materials with consistent genetic background and short culture cycle can be obtained, and the defects of easy browning and contamination of oak explants can be overcome.

[0008] In order to solve the above technical problems, the present invention provides a method for constructing an in vitro regeneration system of white oak stem segments, comprising:

[0009] (1) Collecting white oak stem segments:

[0010] The semi-lignified stem segments of the current year's branches of Quercus alba were collected from May to June;

[0011] (2) Flushing and disinfection:

[0012] Cut off the leaves from the white oak stem segment, and wash and disinfect it; the disinfection includes disinfection with mercuric chloride, using 0.1-0.2% mercuric chloride for 3.0-6.0 minutes;

[0013] (3) Bud germination:

[0014] The stem segments with axillary buds after washing and disinfection were cut into 2-3 cm pieces and inoculated into a budding initiation medium for budding germination; the budding initiation medium used WPM medium as the basic medium and added with 0.50-1.50 mg·L -1 6-BA and 0~0.10mg·L -1 NAA;

[0015] (4) Adventitious bud proliferation:

[0016] The stem segments with axillary buds and one bud point are inoculated into adventitious bud proliferation medium for adventitious bud proliferation; the adventitious bud proliferation medium uses WPM as the basic medium and adds 0.80-1.50 mg·L -1 6-BA and 0.01~0.10mg·L -1 NAA;

[0017] (5) Adventitious bud rooting:

[0018] Select the robust buds of Quercus alba and inoculate them into adventitious root induction medium to make adventitious buds root and obtain sterile tissue culture seedlings of Quercus alba. The adventitious root induction medium uses 1 / 4MS as the basic medium and adds 0-0.10 mg·L -1 NAA and 0~1.00mg·L -1 IBA;

[0019] The adventitious bud initiation medium, adventitious bud proliferation medium and adventitious root induction medium are additionally added with 25-35 g·L -1 Sucrose and 5-10 g·L -1 Agar; the bud start medium is also supplemented with ascorbic acid 60-90 mg·L -1 .

[0020] As a further improvement of the present invention, when collecting the white oak stem segments, the semi-lignified stem segments of the white oak branches of the current year are collected in May.

[0021] Furthermore, during the mercuric chloride disinfection, 0.1% mercuric chloride is used for disinfection, and the disinfection time is 4.5 minutes.

[0022] Furthermore, in the budding germination, the budding initiation medium is a WPM medium as a basic medium, supplemented with 0.50 mg·L -1 6-BA;

[0023] And / or, in the adventitious bud proliferation, the adventitious bud proliferation medium is a basic medium with WPM and 0.08-1.00 mg·L -1 6-BA and 0.01~0.03mg·L -1 NAA;

[0024] And / or, in the adventitious bud rooting, the adventitious root induction medium is a 1 / 4MS basic medium supplemented with 0-0.05 mg·L -1 NAA and 0~0.50mg·L -1 IBA;

[0025] The adventitious bud initiation medium, adventitious bud proliferation medium and adventitious root induction medium were each supplemented with 30 g·L -1 Sucrose and 7g·L -1 Agar; the bud starter medium is also supplemented with ascorbic acid 80 mg·L -1 .

[0026] Furthermore, in the adventitious bud proliferation medium, WPM is used as the basic medium and 1.00 mg·L -1 6-BA and 0.03mg·L -1 NAA;

[0027] In the adventitious root induction medium, 1 / 4MS was used as the basic medium and 0.05 mg·L -1 NAA and 0.50mg·L -1 IBA.

[0028] The second technical problem to be solved by the present invention is to provide a combined culture medium for constructing an efficient in vitro regeneration system of white oak stem segments, so that tissue culture seedling materials with a consistent genetic background and a short culture cycle can be obtained.

[0029] In order to solve the above technical problems, the present invention provides a combined culture medium for constructing an in vitro regeneration system of white oak stem segments, including a fixed bud initiation culture medium, an adventitious bud proliferation culture medium and an adventitious root induction culture medium;

[0030] The budding initiation medium uses WPM medium as the basic medium and adds 0.50-1.50 mg·L -1 6-BA and 0~0.10mg·L -1 NAA;

[0031] The adventitious bud proliferation medium uses WPM as the basic medium and adds 0.80-1.50 mg·L -1 6-BA and 0.01~0.10mg·L -1 NAA;

[0032] The adventitious root induction medium uses 1 / 4MS as the basic medium and adds 0-0.10 mg·L -1 NAA and 0~1.00mg·L -1 IBA;

[0033] The adventitious bud initiation medium, adventitious bud proliferation medium and adventitious root induction medium are additionally added with 25-35 g·L -1 Sucrose and 5-10 g·L -1 Agar; the bud start medium is also supplemented with ascorbic acid 60-90 mg·L -1 .

[0034] As a further improvement of the present invention, the budding initiation medium is a WPM medium as the basic medium, with 0.50 mg·L -1 6-BA;

[0035] The adventitious bud proliferation medium is a basic medium with WPM added with 0.08-1.00 mg·L -1 6-BA and 0.01~0.03mg·L -1 NAA;

[0036] The adventitious root induction medium is a 1 / 4MS basic medium supplemented with 0-0.05 mg·L -1 NAA and 0~0.50mg·L -1 IBA;

[0037] The adventitious bud initiation medium, adventitious bud proliferation medium and adventitious root induction medium were each supplemented with 30 g·L -1 Sucrose and 7g·L -1 Agar; the bud starter medium is also supplemented with ascorbic acid 80 mg·L -1 .

[0038] Furthermore, in the adventitious bud proliferation medium, WPM is used as the basic medium and 1.00 mg·L -1 6-BA and 0.03mg·L -1 NAA;

[0039] In the adventitious root induction medium, 1 / 4MS was used as the basic medium and 0.05 mg·L -1 NAA and 0.50mg·L -1 IBA.

[0040] The third technical problem to be solved by the present invention is to provide a method for constructing a genetic transformation system of white oak, so that it can provide an efficient instantaneous transformation system, which can be used to achieve functional analysis of unknown genes of white oak through reverse genetics methods.

[0041] In order to solve the above technical problems, the present invention provides a method for constructing a genetic transformation system of white oak, using the white oak PDS gene QfPDS as a reporter gene to construct a VIGS system mediated by tobacco rattle virus TRV;

[0042] Vacuum infiltration was used to infect leaves of sterile tissue culture seedlings of Quercus alba;

[0043] The white oak sterile tissue culture seedlings are obtained by constructing the white oak stem segment in vitro regeneration system according to any one of claims 1 to 5.

[0044] As a further improvement of the present invention, it includes:

[0045] (1) The VIGS system mediated by tobacco rattle virus TRV is constructed by using the Quercus alba PDS gene QfPDS as a reporter gene, and comprises:

[0046] Using Seq ID No: 1 and 2 as primers, the TRV2-QfPDS-1 fragment of the QfPDS gene of Quercus alba was cloned;

[0047] Using Seq ID No: 3 and 4 as primers, the TRV2-QfPDS-1 fragment of the QfPDS gene of Quercus alba was cloned;

[0048] Using Seq ID No: 5 and 6 as primers, the TRV2-QfPDS-1 fragment of the QfPDS gene of Quercus alba was cloned;

[0049] The TRV2-QfPDS-1 fragment, the TRV2-QfPDS-2 fragment and the TRV2-QfPDS-3 fragment were inserted into TRV2 as target genes to obtain the pTRV2-QfPDS-1, pTRV2-QfPDS-2 and pTRV2-QfPDS-3 recombinant vectors;

[0050] (2) The vacuum infiltration method for infecting the leaves of the sterile tissue culture seedlings of Quercus alba comprises:

[0051] Agrobacterium carrying the auxiliary plasmid pTRV1 and pTRV2-QfPDS-1, pTRV2-QfPDS-2 and pTRV2-QfPDS-3 were mixed in a ratio of 3:1:1:1, and then the leaves of sterile tissue culture seedlings of Quercus alba were infected by vacuum infiltration.

[0052] The present invention first uses the stem segments of white oak as explants to explore the effects of different factors on explant selection, browning degree control, bud germination, adventitious bud proliferation and adventitious roots in the in vitro regeneration of white oak, and establishes a regeneration system for direct organ regeneration of white oak, thereby obtaining tissue culture seedling materials with consistent genetic background and short culture cycle, and overcoming the problem that oak explants are easy to brown and contaminated. On this basis, the present invention uses the white oak PDS gene (QfPDS) as a reporter gene to construct a VIGS system mediated by tobacco rattle virus (TRV). By combining the tissue culture regeneration system of white oak with the transient transformation system, the accurate analysis of the gene function of white oak is achieved, laying the foundation for promoting genetic improvement and molecular breeding of white oak. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0054] Figure 1 This is a flow chart of infection of seedlings regenerated from in vitro stem segments of Quercus alba;

[0055] Figure 2 The figure shows the effect of different explants and disinfection time on bud initiation, including: (a) bud initiation rate of stem segments under different 0.1% mercuric chloride disinfection time; (b) contamination rate of stem segments under different 0.1% mercuric chloride disinfection time; (c) browning rate of stem segments under different 0.1% mercuric chloride disinfection time; Note: Data are mean ± standard error, different lowercase letters indicate significant differences among different treatments (P<0.05);

[0056] Figure 3 is a flow chart of tissue culture of Quercus alba, where: (a) stem segment inoculated with adventitious bud initiation medium for 20 days; (b) stem segment just inoculated with adventitious bud proliferation medium; (c) stem segment inoculated with adventitious bud proliferation medium for 15 days; (d) stem segment inoculated with adventitious bud proliferation medium for 30 days; (e) intact plant inoculated with adventitious root induction medium for 40 days;

[0057] Figure 4 The phenotypes of plants regenerated from in vitro stem segments of white oak with VIGS silencing QfPDS are shown in Figure 1, including: (a) white oak plants inoculated with a blank vector; (b) (c) (d) white oak plants inoculated with pTRV-QfPDS in three biological replicates;

[0058] Figure 5 This is the relative expression level change of QfPDS in the Quercus white oak VIGS system. Note: Data are mean ± standard deviation, WT: inoculated blank vector; *** indicates extremely significant difference (P<0.001). DETAILED DESCRIPTION

[0059] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the biochemical reagents, carriers, consumables, etc. used in the examples are all commercially available products.

[0060] 1 Materials and methods

[0061] 1.1 Test materials and treatments

[0062] Robust, disease-free one-year-old white oak seedlings were selected from the experimental base of the Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang District, Hangzhou City, Zhejiang Province. In May and June 2024, the tender shoots and semi-lignified stem segments of the current year were collected and named May young stem segments, May semi-lignified stem segments, June young stem segments and June semi-lignified stem segments, respectively.

[0063] Cut off the leaves from the stem segment and rinse it under running tap water for 2 hours. Then, in the clean bench, use 75% (volume ratio) ethanol to disinfect the stem segment for 30 seconds and rinse it with sterile water once; use 0.1% (mass fraction) HgCl2 solution (add 1-2 drops of Tween-80) to disinfect the explants for 3.0 minutes, 4.5 minutes and 6 minutes respectively, and rinse it with sterile water 5 times. Cut the stem segment with axillary buds into 2-3 cm (the length from the base to the axillary bud occupies 2 / 3 of the total length of the stem segment) and inoculate it into WPM medium (WPM+0.50mg·L -1 6-BA). Six stem segments were inoculated in each treatment and repeated 10 times. After 20 days, the bud initiation rate, contamination rate and browning rate of explants were counted.

[0064] 1.2 Medium formulation and culture conditions

[0065] All culture media were supplemented with sucrose 25-35 g L -1 , in this embodiment, 30g·L is preferred -1 , agar 5-10 g·L -1 , this embodiment preferably uses 7g·L -1 , pH was adjusted to 5.80. The culture conditions were: temperature 25°C, relative humidity 70%, light intensity 3000 lx, and light-dark cycle of 14 h light / 10 h dark.

[0066] 1.2.1 Bud starter culture medium

[0067] The sterilized white oak stem segments were inoculated into different basal culture media (WPM and 1 / 4MS) and culture media with different mass concentrations of 6-benzylaminopurine (6-BA) and naphthaleneacetic acid (NAA), and the culture media were all supplemented with ascorbic acid 60-90 mg·L -1 , in this embodiment, 80 mg·L is preferred -1 Ten stem segments were inoculated with each medium formulation and replicated three times.

[0068] After 15 days, the bud initiation rate, bud initiation time and growth status of the stem segments were counted.

[0069] γ=(n i / N)×100%, where: γ is the budding rate, ni is the number of explants that initiate budding, and N is the total number of inoculated explants.

[0070] 1.2.2 Adventitious bud proliferation medium

[0071] Take WPM as the basic culture medium and add 0.8-1.5 mg·L -1 6-BA and 0.01~0.1mg·L -1 NAA, a completely randomized experiment was conducted. The stem axillary buds were cut into 1 cm segments with one bud point and inoculated into bud proliferation medium. The medium was changed every 15 days.

[0072] The differentiation rate, proliferation coefficient and average elongation of adventitious buds were statistically analyzed at 30 days.

[0073] ε=(n s / N)×100%, where: ε is the germination rate of adventitious buds, n s is the number of explants with adventitious buds, N is the total number of explants inoculated; ρ = (n p / N p )×100%, where: ρ is the proliferation coefficient, n p is the number of new adventitious buds, N p is the original number of buds in the inoculated stem segment; l b =∑(L b ) / n p , where: l b is the average elongation of adventitious buds, l b New is the elongation of each adventitious bud, n p It is the number of adventitious buds with new buds (bud elongation ≥ 0.5 cm).

[0074] 1.2.3 Adventitious root induction medium

[0075] Take 1 / 4MS as the basic culture medium and add 0-0.1mg·L -1 NAA and 0~1mg·L -1Indoleacetic acid (IBA) was used to conduct a completely randomized experiment. The rooting rate, average root number, average root length and growth status were counted on day 40.

[0076] δ=(n δ / N)×100%, where: δ is the rooting rate, n δ is the number of rooted explants, N is the total number of inoculated explants; l r =∑(L r ) / n r , where: l r is the average elongation of adventitious roots, L r Xin is the average elongation of adventitious roots of each plant, n r is the number of rooted explants. After 40 days, sterile tissue culture seedlings of Quercus alba were obtained for the construction of the VIGS system in the later stage.

[0077] 1.3 Establishment of the Quercus alba VIGS system

[0078] 1.3.1 Cloning of QfPDS gene fragment

[0079] Based on the previous transcriptome data of Quercus alba tissues (NCBI accession number: SRR22801261-SRR22801278; BioProject: PRJNA913393), the complete gene coding sequence (CDS) fragment annotated as PDS was selected, and the specific primers of QfPDS gene were designed using Primer 3.0. The study adopted Nimble Cloning (NC cloning) technology, adding 20bp sequences at both ends of the primer as linker sequences (Table 1). Among them, TRV2-QfPDS-1, TRV2-QfPDS-2 and TRV2-QfPDS-3 are three different fragments of QfPDS gene, which will be inserted into TRV2 as target genes and constitute a recombinant vector.

[0080] Table 1 Primer information

[0081]

[0082] Note: The lowercase letters in the primer sequences of TRV2-QfPDS-1, TRV2-QfPDS-2 and TRV2-QfPDS-3 are NC cloning adapter sequences, and the upstream and downstream primer sequences correspond to Seq ID No: 1-6 respectively;.

[0083] RNA was extracted using HiPure HP Plant RNA Mini Kit (Meiji, Guangzhou) according to the instructions, and then its integrity was checked by 1.0% agarose gel electrophoresis. RNA was reverse transcribed into cDNA using PrimeScripTM RTreagent Kit with gDNA Eraser from TAKARA.

[0084] The above cDNA was used as a template and I-5 2×High-Fidelity Master Mix (Klauning, Beijing) was used to amplify the QfPDS gene. The PCR amplification system was as follows: 2×High-Fidelity Master Mix 25μL, upstream primer 2μL, downstream primer 2μL, cDNA 2μL and ddH2O 19μL. PCR amplification program: 98℃ pre-denaturation for 2min; 98℃ denaturation for 10s, 55℃ annealing for 15s, 72℃ extension for 30s, 32 cycles; 72℃ final extension for 5min; 4℃ storage. 1.0% agarose gel electrophoresis was used to test the PCR amplification product and the integrity and accuracy of the bands. The recovered product was connected to the TRV2 vector using the Nimble Cloning kit (Nixing, Hainan) and transformed into DH5α competent cells (Weidi, Shanghai). After kanamycin sulfate resistance screening, single colonies were picked for PCR detection and the positive bacterial solution was sent to Youkang Biotechnology (Hangzhou) Co., Ltd. for sequencing. Finally, the recombinant plasmids pTRV2-QfPDS-1, pTRV2-QfPDS-2 and pTRV2-QfPDS-3 were obtained.

[0085] 1.3.2 Preparation of infection solution and infection of Quercus alba

[0086] A single colony of GV3101 Agrobacterium (Weidi, Shanghai) that had successfully transformed the QfPDS target vector plasmid and TRV1 auxiliary plasmid was picked and inoculated into 1 mL of 50 mg·L -1 Kanamycin sulfate and 20mg·L -1 Rifampicin dual-antibody LB liquid medium, 28 ° C, 200 r min -1 Small-scale shaking culture was performed until the OD600 value of the bacterial solution was between 0.8 and 1.0; then 1 mL of the bacterial solution was inoculated into 100 mL containing 50 mg·L -1 In LB liquid medium containing kanamycin sulfate, 28°C, 200 r·min -1 Large-scale shaking culture was performed until the OD600 value of the bacterial solution was between 0.8 and 1.0. The bacterial solution was rotated at a speed of 6000 r / min -1 Centrifuge for 5 min. Use 10 mmol·L -1Magnesium chloride (Mgcl2·6H2O) + 10mmol·L -1 2-Morpholineethanesulfonic acid (MES) + 200mg·L -1 Acetosyringone (AS) was added and the pH was adjusted to 5.2 to prepare the infection solution. The bacteria were resuspended in the infection solution to an OD of 600 =0.8, let stand at room temperature for 3 h, and wait for Agrobacterium to recover.

[0087] like Figure 1 As shown in the figure, the leaves of the regenerated plants from the in vitro stem segments of white oak were infected by vacuum infiltration. First, Agrobacterium carrying pTRV1 (helper plasmid), pTRV2-QfPDS-1, pTRV2-QfPDS-2 and pTRV2-QfPDS-3 were mixed in a ratio of 3:1:1:1, so that the three pTRV2 recombinant plasmids acted together on the three sites of the QfPDS gene to improve the silencing efficiency. The infection solution was poured into a 50mL syringe with the needle removed and the interface sealed. Then, the complete white oak plant with the base callus removed was placed in the syringe, and the piston was repeatedly pushed and pulled to achieve a vacuum state to ensure that the resuspension fully penetrated into the leaves. After the surface of the plant was naturally dried, it was transferred to a syringe containing 300mg·L -1 The culture medium containing cephalosporin was continued for 22 days at a temperature of 25°C, a relative humidity of 70%, a light intensity of 3000 lx and a light-dark cycle of 14 h / 10 h.

[0088] 1.3.3 Analysis of PDS gene expression levels

[0089] Leaves of white oak with albino phenotype were collected 22 days after virus inoculation, and the total RNA was extracted and reverse transcribed into cDNA. The cDNA was amplified by RT-PCR using the corresponding primers (Table 1). The amplified cDNA was detected by qRT-PCR using QuantStudio 7 fluorescence quantitative PCR instrument, and the white oak Actin gene (QfActin) was used as the internal reference gene to analyze the relative expression of the QfPDS gene. The relative expression was based on 2 -ΔΔCt Calculation rules.

[0090] 1.4 Data Processing

[0091] Microsoft Excel 2016 was used to organize the data, SPSS Statistics 25 was used for one-way analysis of variance and multiple comparisons (Duncan's method), and GraphPad Prism 9.5 was used for graphics.

[0092] 2 Results and analysis

[0093] 2.1 Effects of different explants and disinfection time on budding initiation

[0094] like Figure 2 As shown in the data, under the same disinfection time of 0.1% mercuric chloride, there was no significant difference in the contamination rate of explants in May and June (P>0.05); when disinfected for 3min and 4.5min, the bud initiation rate and browning rate of explants in May were higher than those in June. When disinfected with 0.1% mercuric chloride for 4.5min, the bud initiation rate of semi-lignified stem segments collected in May reached a maximum of 65%, which was significantly higher than other disinfection times (P<0.05). Since the browning of white oak explants is unlikely to cause complete death of the plant, it will only delay the bud initiation time. Therefore, considering the bud initiation rate, contamination rate and browning rate, it is advisable to collect semi-lignified stem segments in May and set the 0.1% mercuric chloride disinfection time to 4.5min.

[0095] 2.2 Screening of budding initiation culture medium

[0096] The explants of white oak stem segments collected from the greenhouse were inoculated into the culture medium with the formulas shown in the table (Table 2) to compare their budding and germination conditions. There was no significant difference in the budding and germination rates of the five formulas, all reaching more than 75%; No. V (WPM + 0.05 mg·L - 1 NAA+1.00mg·L -1 6-BA) had the fastest bud start time of 8.15 days, which was significantly higher than that of Ⅰ, Ⅱ and Ⅲ (P<0.05). -1 6-BA) The buds have large elongation, new leaves are produced and the buds have good growth, so they are suitable for later propagation and proliferation.

[0097] Therefore, it is advisable to choose No. Ⅳ (WPM+0.50mg·L -1 6-BA) is the best bud initiation medium for stem segments of white oak explants.

[0098] Table 2 Effects of different culture medium formulas on budding initiation of Quercus alba

[0099]

[0100] Note: Data are mean ± standard deviation, different lowercase letters indicate significant differences among different treatments;

[0101] P<0.05).

[0102] 2.3 Effects of different plant growth regulator combination concentrations on adventitious bud proliferation

[0103] Cut the budding stem segments of white oak and inoculate them with 0.01-0.10 mg·L -1 NAA and 0.80~1.50mg·L -1 6-BA WPM medium, and the germination rate, proliferation coefficient and average bud elongation of the stem segments were measured after 30 days (Table 3).

[0104] From the 7th day after inoculation, the base of the stem segment began to swell and gradually formed clusters of buds. The experimental results showed that within the test concentration range, all combinations of NAA and 6-BA could effectively promote the occurrence rate of adventitious buds to 60% or more, and the significant differences between the numbered culture media were small. Among them, only in the culture media numbered S6 and S7, the germination rate of adventitious buds of white oak reached the optimal 90%, which was significantly higher than S8 and S10 (P < 0.05).

[0105] The proliferation coefficient and average elongation of adventitious buds of Quercus alba showed a trend of increasing first and then decreasing with the gradual increase of 6-BA and NAA concentrations. -1 NAA+1.00mg·L -1 6-BA), the proliferation coefficient of adventitious buds of Quercus alba reached a maximum of 3.22; when S6 medium (0.03 mg·L -1 NAA+1.00mg·L -1 When the concentration of NAA was 0.10 mg·L -1 All culture medium formulations (P<0.05).

[0106] One-way analysis of variance (Table 4) on the proliferation coefficient showed that different concentrations of NAA had extremely significant differences in the proliferation of adventitious buds (P < 0.001), and different concentrations of 6-BA had relatively significant differences (P < 0.01).

[0107] In addition, when the 6-BA concentration was 0.80 mg·L -1 When the concentration of 6-BA was 1.50 mg·L -1 At this time, the number of nodes of the white oak stem segments was large, the leaves were small and a large number of calluses were induced at the base. At this time, the adventitious buds were more suitable to be cut into stem segments and inoculated into bud proliferation medium for propagation.

[0108] In summary, it is advisable to choose S6 (WPM + 0.03mg L -1 NAA+1.00mg·L -1 6-BA) is the best white oak bud

[0109] Proliferation medium.

[0110] Table 3 Effects of different combinations of plant growth regulators on adventitious bud proliferation and growth

[0111]

[0112] Note: Data are mean ± standard deviation, different lowercase letters indicate significant differences among different treatments (P<0.05).

[0113] Table 4 Analysis of variance on the proliferation and growth results of adventitious buds of Quercus alba

[0114]

[0115]

[0116] Note: * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01), *** indicates extremely significant difference (P<0.001).

[0117] 2.4 Effects of different plant growth regulator combination concentrations on adventitious bud rooting

[0118] Select the robust buds of Quercus alba and inoculate them with 0-0.10 mg·L -1 NAA and 0~1.00mg·L -1 1 / 4MS medium with IBA. Since the 12th day after inoculation, adventitious buds have gradually formed adventitious roots. The test results (Table 5) show that all tested combinations of NAA and IBA at different concentrations can effectively induce the occurrence of adventitious roots, and their rooting rates are all over 50%, and there is no significant difference among the formulations (P>0.05).

[0119] As the concentrations of NAA and IBA gradually increased, the rooting rate and average root number generally showed a trend of first increasing and then decreasing. -1 With IBA concentration of 0.50 mg·L -1 When the rooting rate of Quercus alba reached a maximum of 92.86%, the average number of roots was 4.57, which was significantly higher than that of all media except R8 (P < 0.05). In addition, the plants with this auxin ratio had a strong root system, well-developed fibrous roots, and excellent overall growth.

[0120] On the other hand, when the auxin concentration was maintained at a low level, the induced roots showed an advantage in average length. In the medium without NAA and IBA, the average root length of the plants reached a maximum of 5.82 cm, which was significantly higher than the average root length of all mediums except R2 (P < 0.05). However, the roots of the white oak under this formula were slender and weak, lacking the fibrous roots necessary for growth.

[0121] The single factor ANOVA was performed on the average root length and average root number of adventitious roots (Table 6), indicating that NAA and IBA had significant differences in the rooting of adventitious buds (P < 0.01). In particular, different concentrations of IBA had extremely significant differences in the number and elongation of adventitious roots (P < 0.001).

[0122] In summary, it is advisable to choose R11 (1 / 4MS + 0.05mg·L -1 NAA+0.50mg·L -1 IBA) was the best medium for inducing adventitious roots of Quercus alba.

[0123] Table 5 Effects of different combinations of plant growth regulators on rooting of adventitious buds

[0124]

[0125]

[0126] Note: Data are mean ± standard deviation, different lowercase letters indicate significant differences among different treatments (P<0.05).

[0127] Table 6 Analysis of variance of rooting results of adventitious buds of Quercus alba

[0128]

[0129] Note: * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01), *** indicates extremely significant difference (P<0.001).

[0130] The tissue culture process of Quercus alba Figure 3 shown.

[0131] 2.5 Analysis of the silencing effect of QfPDS in Quercus alba

[0132] To verify whether the VIGS system of Quercus alba can successfully silence the reporter gene QfPDS, the plant morphology was observed 22 days after silencing. Figure 4 The leaves of the plants shown all showed different degrees of chlorosis. Compared with the unloaded plants (a), the leaves of some plants became lighter or yellow, showing chlorosis; some plants showed partial bleaching along the veins to the leaf margins.

[0133] The expression level of QfPDS gene was analyzed in the leaves of Quercus alba 22 days after inoculation. The results showed that ( Figure 5 ), compared with the plants inoculated with the blank vector, the relative expression levels of the indicator genes in the plants inoculated with the TRV virus showed a very significant decrease (P < 0.001), indicating that QfPDS had been efficiently silenced.

[0134] 3 Discussions

[0135] 3.1 Effects of explant treatment on bud germination

[0136] The biggest difficulty in the in vitro culture of white oak tissue is that the explants are prone to contamination and browning. On the one hand, oak trees are rich in phenolic substances such as lignin, pigments and tannins. The accumulation and oxidation of these phenolic compounds make oak species more prone to browning than other plants. On the other hand, the twigs of white oak are densely covered with pubescence, which makes it easier for microorganisms to attach and colonize.

[0137] In the experiment, as the disinfection time of mercuric chloride prolonged, the rate of budding started to increase first and then decrease. When the disinfection time was insufficient, the microorganisms that were not completely eliminated hindered the growth, development and regeneration of the plants; when the disinfection time was too long, the browning degree of the white oak explants increased significantly, which delayed the start of budding. At this time, the young stem segments were more sensitive to mercuric chloride because of their cell structure, and their browning rate was generally higher than that of semi-lignified stem segments.

[0138] The sampling time also affects the budding of the white oak stem segments. In spring, the white oak is in a period of vigorous growth, and the metabolism in the body may be more intense. The activity of enzymes related to enzymatic browning, such as polyphenol oxidase (PPO), is relatively high. Collecting explants makes the enzymes easier to activate. Therefore, the budding initiation rate and browning rate of white oak explants collected in May are generally higher than those in June. In addition, high temperature and high humidity environments are conducive to the reproduction and spread of bacteria or fungi. Therefore, the explant contamination rate in summer is usually higher than that in spring. At this time, the disinfection time can be extended accordingly.

[0139] 3.2 Establishment of adventitious bud regeneration system of Quercus alba

[0140] The germination of fixed buds is an important step in plant regeneration. Through the germination of fixed buds or axillary buds, complete, uncontaminated young stem segments are obtained, thereby efficiently achieving the proliferation of adventitious buds in white oak stem segments. In this experiment, a lower concentration of 6-BA is more suitable for the natural growth of plants, and the new leaves make the fixed buds grow more robustly through photosynthesis.

[0141] The results of the bud germination test showed that WPM medium was suitable for the growth of white oak buds. Therefore, WPM medium was still selected in the subsequent adventitious bud proliferation, and high concentrations of cytokinins and low concentrations of auxins were applied to promote the proliferation of white oak buds. In this study, 6-BA and NAA were selected as plant growth regulators. When the medium formula was WPM+0.03mg·L -1 NAA+1.00mg·L -1 When 6-BA was used, the adventitious buds showed high proliferation coefficient, high elongation and excellent growth state in the proliferation stage, indicating that this formula was beneficial for the white oak to maintain a stable growth rate and good growth state.

[0142] Inducing plant adventitious root germination usually requires high concentrations of auxin accumulation and low concentrations of inorganic salt medium. This experiment selected 1 / 4MS medium and different concentrations of NAA and IBA to explore their effects on adventitious root germination of white oak. The results showed that when the medium formula was 1 / 4MS + 0.05mg·L -1 NAA+0.50mg·L -1 IBA, at this time the generated root coefficient is the largest.

[0143] However, the increase in the number of roots may consume more nutrients and energy, thus limiting the elongation of the roots. Although the root system is shorter, the overall nutrient absorption area of ​​the root system with developed fibrous roots is still large enough, so the plant growth is still good. In contrast, the culture medium without auxin has the longest root length. But at this time, the growth of the white oak plants is very poor, indicating that under the action of only endogenous auxin, the growth of the roots is promoted while the growth of the stems is relatively inhibited.

[0144] 3.3 Preliminary establishment of the Quercus alba VIGS system

[0145] In the case of transient transformation of plants, common leaf infection methods include injection penetration, friction inoculation, vacuum penetration, high-pressure spray gun, root absorption, etc. Since the leaves of white oak are thick, the secondary cell walls are thick and the vascular bundles are strong, it is difficult to use a syringe without a needle to penetrate the leaves with the infection solution, so the vacuum penetration method can be selected.

[0146] VIGS does not rely on a stable genetic transformation system. It can quickly produce phenotypes and continuously and efficiently silence genes. It is a convenient instantaneous transformation system. However, due to differences in plant species, individuals, tissues and organs, the start time and duration of silencing also vary.

[0147] In this experiment, in the real-time fluorescence quantitative analysis, the QfPDS silenced leaves 22 days after infection showed a very significant decrease in expression level compared with the blank vector leaves in the control group.

[0148] 4 Conclusion

[0149] In tissue culture, the explant stem segments of white oak are prone to browning and contamination. At this time, the browning can be slowed down by selecting explant samples, disinfection time or adding anti-browning agents. It is best to collect relatively mature semi-lignified stem segments of white oak in early summer. At this time, the browning rate, contamination rate and germination rate of the sample are better.

[0150] White oak has strong adaptability and shows excellent regeneration ability in most of the tested culture media. Among them, WPM medium is suitable for the germination and proliferation of white oak buds, and 1 / 4MS medium is suitable for inducing white oak adventitious roots. The cytokinins (6-BA) and auxins (NAA, IBA) used are suitable for the growth of white oak. During the process of adventitious bud proliferation, white oak showed a high sensitivity to NAA: only 0.10 mg·L -1 When NAA was added, the growth rate of white oak was inhibited; however, the concentration range of 6-BA suitable for the growth of white oak was not significant enough. The concentration of 6-BA could be optimized in the later stage to improve the proliferation ability of white oak adventitious buds.

[0151] The initially established Quercus alba VIGS system showed extremely significant differences in silencing efficiency (P < 0.001) in the analysis of gene expression levels.

[0152] By combining the above tissue culture regeneration system and transient transformation system, the functional analysis of unknown genes of white oak can be achieved through reverse genetics methods, laying the foundation for promoting genetic improvement and molecular breeding of white oak.

[0153] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art may make some simple modifications, equivalent changes or modifications using the technical contents disclosed above, which all fall within the protection scope of the present invention.

Claims

1. A method for constructing an in vitro regeneration system of white oak stem segments, characterized in that: include: (1) Collecting white oak stem segments: The semi-lignified stem segments of the current year's branches of white oak were collected from May to June; (2) Flushing and disinfection: Cut off the leaves from the white oak stem segment, and wash and disinfect it; the disinfection includes disinfection with mercuric chloride, using 0.1-0.2% mercuric chloride for 3.0-6.0 minutes; (3) Bud germination: The stem segments with axillary buds after washing and disinfection were cut into 2-3 cm pieces and inoculated into a budding initiation medium for budding germination; the budding initiation medium used WPM medium as the basic medium and added with 0.50-1.50 mg·L -1 6-BA and 0~0.10mg·L -1 NAA; (4) Adventitious bud proliferation: The stem segments with axillary buds and one bud point are inoculated into adventitious bud proliferation medium for adventitious bud proliferation; the adventitious bud proliferation medium uses WPM as the basic medium and adds 0.80-1.50 mg·L -1 6-BA and 0.01~0.10mg·L -1 NAA; (5) Adventitious bud rooting: Select the robust buds of Quercus alba and inoculate them into adventitious root induction medium to make adventitious buds root and obtain sterile tissue culture seedlings of Quercus alba. The adventitious root induction medium uses 1 / 4MS as the basic medium and adds 0-0.10 mg·L -1 NAA and 0~1.00mg·L -1 IBA; The adventitious bud initiation medium, adventitious bud proliferation medium and adventitious root induction medium are additionally added with 25-35 g·L -1 Sucrose and 5-10 g·L -1 Agar; the bud start medium is also supplemented with ascorbic acid 60-90 mg·L -1 .

2. The method for constructing an in vitro regeneration system of white oak stem segments according to claim 1, characterized in that: When collecting the white oak stem segments, semi-lignified stem segments of the white oak branches of the current year are collected in May.

3. The method for constructing an in vitro regeneration system of white oak stem segments according to claim 1, characterized in that: During the mercuric chloride disinfection, 0.1% mercuric chloride is used for disinfection, and the disinfection time is 4.5 minutes.

4. The method for constructing an in vitro regeneration system of white oak stem segments according to any one of claims 1 to 3, characterized in that: In the budding germination, the budding initiation medium is a WPM medium as a basic medium, supplemented with 0.50 mg·L -1 6-BA; And / or, in the adventitious bud proliferation, the adventitious bud proliferation medium is a basic medium with WPM and 0.08-1.00 mg·L -1 6-BA and 0.01~0.03mg·L -1 NAA; And / or, in the adventitious bud rooting, the adventitious root induction medium is a 1 / 4MS basic medium supplemented with 0-0.05 mg·L -1 NAA and 0~0.50mg·L -1 IBA; The adventitious bud initiation medium, adventitious bud proliferation medium and adventitious root induction medium were each supplemented with 30 g·L -1 Sucrose and 7g·L -1 Agar; the bud starter medium is also supplemented with ascorbic acid 80 mg·L -1 .

5. The method for constructing an in vitro regeneration system of white oak stem segments according to claim 4, characterized in that: In the adventitious bud proliferation medium, WPM was used as the basic medium and 1.00 mg·L -1 6-BA and 0.03mg·L -1 NAA; In the adventitious root induction medium, 1 / 4MS was used as the basic medium and 0.05 mg·L -1 NAA and 0.50mg·L - 1 IBA.

6. A combined culture medium for constructing an in vitro regeneration system of Quercus alba stem segments, characterized in that: It includes adventitious bud initiation medium, adventitious bud proliferation medium and adventitious root induction medium; The budding initiation medium uses WPM medium as the basic medium and adds 0.50-1.50 mg·L -1 6-BA and 0~0.10mg·L -1 NAA; The adventitious bud proliferation medium uses WPM as the basic medium and adds 0.80-1.50 mg·L -1 6-BA and 0.01~0.10mg·L -1 NAA; The adventitious root induction medium uses 1 / 4MS as the basic medium and adds 0-0.10 mg·L -1 NAA and 0~1.00mg·L -1 IBA; The adventitious bud initiation medium, adventitious bud proliferation medium and adventitious root induction medium are additionally added with 25-35 g·L -1 Sucrose and 5-10 g·L -1 Agar; the bud start medium is also supplemented with ascorbic acid 60-90 mg·L -1 .

7. The combined culture medium for constructing an in vitro regeneration system of white oak stem segments according to claim 6, characterized in that: The budding initiation medium is a WPM medium as the basic medium, supplemented with 0.50 mg·L -1 6-BA; The adventitious bud proliferation medium is a basic medium with WPM added with 0.08-1.00 mg·L -1 6-BA and 0.01~0.03mg·L -1 NAA; The adventitious root induction medium is a 1 / 4MS basic medium supplemented with 0-0.05 mg·L -1 NAA and 0~0.50mg·L -1 IBA; The adventitious bud initiation medium, adventitious bud proliferation medium and adventitious root induction medium were each supplemented with 30 g·L -1 Sucrose and 7g·L -1 Agar; the bud starter medium is also supplemented with ascorbic acid 80 mg·L -1 .

8. The combined culture medium for constructing an in vitro regeneration system of white oak stem segments according to claim 7, characterized in that: In the adventitious bud proliferation medium, WPM was used as the basic medium and 1.00 mg·L -1 6-BA and 0.03mg·L -1 NAA; In the adventitious root induction medium, 1 / 4MS was used as the basic medium and 0.05 mg·L -1 NAA and 0.50mg·L - 1 IBA.

9. A method for constructing a genetic transformation system of Quercus alba, characterized in that: The Quercus alba PDS gene QfPDS was used as a reporter gene to construct a VI GS system mediated by tobacco rattle virus TRV. Vacuum infiltration was used to infect leaves of sterile tissue culture seedlings of Quercus alba; The white oak sterile tissue culture seedlings are obtained by constructing the white oak stem segment in vitro regeneration system according to any one of claims 1 to 5.

10. The method for constructing a genetic transformation system of white oak according to claim 9, characterized in that: include: (1) The VIGS system mediated by tobacco rattle virus TRV is constructed by using the Quercus alba PDS gene QfPDS as a reporter gene, and comprises: Using Seq ID No: 1 and 2 as primers, the TRV2-QfPDS-1 fragment of the QfPDS gene of Quercus alba was cloned; Using Seq ID No: 3 and 4 as primers, the TRV2-QfPDS-1 fragment of the QfPDS gene of Quercus alba was cloned; Using Seq ID No: 5 and 6 as primers, the TRV2-QfPDS-1 fragment of the QfPDS gene of Quercus alba was cloned; The TRV2-QfPDS-1 fragment, the TRV2-QfPDS-2 fragment and the TRV2-QfPDS-3 fragment were inserted into TRV2 as target genes to obtain the pTRV2-QfPDS-1, pTRV2-QfPDS-2 and pTRV2-QfPDS-3 recombinant vectors; (2) The vacuum infiltration method for infecting the leaves of the sterile tissue culture seedlings of Quercus alba comprises: Agrobacterium carrying the auxiliary plasmid pTRV1 and pTRV2-QfPDS-1, pTRV2-QfPDS-2 and pTRV2-QfPDS-3 were mixed in a ratio of 3:1:1:1, and then the leaves of sterile tissue culture seedlings of Quercus alba were infected by vacuum infiltration.

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