Method for inducing apocarya stems to generate transgenic hairy roots

Abstract

The invention discloses a method for inducing apocarya stems to generate transgenic hairy roots, and belongs to the field of forest tree genetic engineering. According to the method, the semi-lignified or lignified stems of the apocarya are infected by utilizing the agrobacterium rhizogenes to obtain stable transgenic hairy roots, rooting can be induced within 1-2 months, the rooting rate is as high as 94.43%, and the transgenic positive rate can reach 98.38%. The invention provides an economical and efficient non-tissue culture dependent transgenic method, it is clear that transgenic roots can be used for gene function verification, the method breaks through the technical obstacle that stable genetic transformation of the carya illinoensis is difficult, and technical support is provided for research of functional genes.

Description

Technical Field

[0001] The invention relates to the field of forestry genetic engineering, in particular to a method for inducing Carya sylvestris stems to produce transgenic hairy roots. Background Art

[0002] At present, the main plant genetic transformation systems include Agrobacterium-mediated transformation, gene gun method, pollen tube channel method, protoplast transformation method, etc. Among them, the gene gun method has relatively low transformation efficiency and high equipment cost; the pollen tube channel method transformation is restricted by season and the efficiency is unstable; the protoplast transformation method involves protoplast culture and regeneration process, which is complicated to operate and has high technical requirements, and is only applicable to some plants. Agrobacterium-mediated transformation method uses T-DNA on Ti plasmid or Ri plasmid in Agrobacterium tumefaciens or Agrobacterium rhizogenes cells to integrate the target gene into the plant genome when Agrobacterium infects plant cells. It has the advantages of high transformation efficiency, simple operation and low cost. However, plant transgenic mediated by Agrobacterium rhizogenes usually uses sterile seedlings of plants and leaves or stem segments of sterile seedlings as explants, and co-cultivates with bacterial solution to obtain transgenic roots, and the experimental conditions are strict; Agrobacterium tumefaciens construction of transgenic plants requires a lot of time for callus induction, differentiation and subculture, which has a long cycle, cumbersome operation and low efficiency.

[0003] Pecan is an economic tree of the Juglandaceae family that is rich in phenolic substances. Its explants are prone to browning and difficult to induce differentiation. It is reported that the traditional tissue culture method using stem segments as explants can successfully induce plant regeneration, but the rooting rate is less than 50%, and it is limited to seedlings of 2 to 3 months old. Although a regeneration system using stem segments of Pecan has been constructed as explants, there has been no report on the relevant genetic transformation system. This may be because the tissue culture operation is cumbersome and the non-embryonic callus has a weak acceptance of exogenous DNA. Since embryonic cells have a strong acceptance of exogenous DNA, the reported genetic transformation systems of Pecan are mostly based on somatic embryos as explants, but the transformation rate is still less than 60%, and some reports are only 6.10%. Due to the shortcomings of cumbersome operation, long time consumption, and high experimental conditions in the somatic embryo induction process, the application of somatic embryos as transformation materials is still rare. At present, the method of ring cutting and transverse cutting has been used to induce thin-shelled pecan seedlings to produce hairy roots in the hypocotyl or stem, but the induction rate is still low, at 56.5%, and the positive rate of transgenic is only 45.20%. Usually, the hypocotyl or stem tends to differentiate into cortical tissue to heal the wound, while the wounds caused by ring cutting and transverse cutting are small, making it difficult to induce the stem to produce root tissue in a directional manner; and the hypocotyl tissue is relatively tender, and the stem tissue at the upper end of the incision is prone to wither and die. This may be the main reason for the low induction rate of transgenic hairy roots in hypocotyl or stem tissue. In view of the shortcomings of the existing thin-shelled pecan genetic transformation system, such as low transformation rate, cumbersome tissue culture operation, high cost, and low positive rate, it is still very necessary to establish an efficient and reliable thin-shelled pecan genetic transformation system. Summary of the invention

[0004] The purpose of the present invention is to provide a method for inducing thin-shell pecan stems to produce transgenic hairy roots, so as to solve the problems existing in the above-mentioned prior art. The method can simply and efficiently induce thin-core pecan to produce transgenic hairy roots.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for inducing walnut stems to produce transgenic hairy roots, comprising the following steps:

[0007] The binary vector plasmid containing the target gene is transferred into Agrobacterium rhizogenes to prepare the recombinant Agrobacterium rhizogenes;

[0008] The stems of Carya sylvestris were infected with the infection fluid of recombinant Agrobacterium rhizogenes and co-cultured, followed by rooting culture.

[0009] Optionally, the target gene includes the CiGATA12a gene.

[0010] Optionally, the recombinant Agrobacterium rhizogenes infection solution is prepared by the following steps: a positive single colony of the recombinant Agrobacterium rhizogenes is cultured in LB medium containing antibiotics and acetosyringone until OD 600 The concentration of Agrobacterium tumefaciens is 0.5-0.7, and the cells are collected by centrifugation; the cells are resuspended with a resuspending liquid to obtain the recombinant Agrobacterium rhizogenes infection liquid.

[0011] Optionally, the concentration of antibiotics in the LB medium is 50 mM, and the concentration of acetosyringone is 15 μM; and / or the components of the resuspension are: an aqueous solution containing 10 mM MgCl2, 150 μM acetosyringone and 10 mM MES.

[0012] Optionally, the OD of the recombinant Agrobacterium rhizogenes infection solution is 600 It is 1.0-1.4, and the infection time is 24h.

[0013] Optionally, the method of infection and co-cultivation comprises the following steps:

[0014] Ring peel the 0.5-1 cm thin-shelled pecan stem cortex, and scrape off the remaining cambium tissue in the wood as the infection site;

[0015] The infected part is impregnated with the impregnation liquid and the infected part is sealed and wrapped, and then co-cultured in a light-proof environment at 20-28° C. for 24-48 hours, the sealed and wrapped materials are removed, and the infected part is covered with a culture matrix.

[0016] A more specific option is to dip the infection liquid with a medium such as paper towels or cotton, wrap the infected part, and wrap the infection medium with plastic wrap to prevent the evaporation of the bacterial liquid; finally, place the stem in a light-proof environment at 20-28°C, culture for 24-48 hours, remove the plastic wrap and infection medium, and cover the infected part with the culture matrix.

[0017] Optionally, the stem is a semi-lignified or lignified stem of the current year of a thin-shelled pecan seedling or variety seedling.

[0018] Optionally, the temperature of the rooting culture is 25-28°C.

[0019] Optionally, the Agrobacterium rhizogenes includes C58C1, K599 and MSU440.

[0020] The present invention discloses the following technical effects:

[0021] (1) High infection success rate: The percentage of plants with hairy roots after infection, i.e., the rooting rate, can be as high as 94.43%, much higher than the 15.0-56.5% mentioned in previous reports. In addition, among the hairy roots produced, the percentage of transgenic positive roots is relatively high, up to 98.38%.

[0022] (2) Short cycle: Depending on the age of the mother tree, the rooting time is between 1 and 2 months. Compared with traditional tissue culture transformation, the time to obtain transgenic tissue is greatly shortened.

[0023] (3) Simple operation: the materials used are not required to be sterile seedlings, and tissue culture is not required. Only the semi-lignified or lignified stems of seed seedlings or variety seedlings that have not been separated from the body need to be used for operation; the ring-barking and bacterial liquid wrapping method is easy to operate and does not require high experimental equipment; the present invention provides an economical and efficient non-tissue culture-dependent transgenic method, and it is clear that the transgenic roots can fully exert the gene function.

[0024] (4) It can be used to improve the traits of cultivated varieties: In view of the fact that the present invention can induce the production of transgenic hairy roots in the seedling stems of varieties, specific genes can be introduced to improve the resistance, quality, yield and other traits of existing thin-shelled pecan varieties. The traditional method of improving variety traits through hybridization has uncertainty, because the parental traits will be separated in the offspring, and it is often difficult to screen out offspring with excellent comprehensive traits, and the screening cycle can be as long as decades. The transgenic method provided by the present invention can not only retain the original traits of the cultivated variety, but also improve the existing undesirable traits, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The process diagram of the infection method; (a) ring-peeling; (b) wrapping the ring-peeling part with a paper towel dipped in infection liquid; (c) wrapping the paper towel with plastic wrap; (d) covering the ring-peeling part with soil; scale: 5 mm;

[0027] Figure 2 The dynamic changes of the infected part; (a) callus tissue was generated one week after infection; (b) rooting two weeks after infection; (c) rooting three weeks after infection; (d) rooting four weeks after infection; (e) rooting five weeks after infection; (f) rooting six weeks after infection; scale bar: 5 mm;

[0028] Figure 3 The results of gene conversion verification are shown in Figure 1. (a) GUS staining results; (b) electrophoresis diagram of gene conversion verification using intron-spanning primers;

[0029] Figure 4 H2O2 and O in pecan overexpressing CiGATA12a before and after treatment with methyl viologen (MV) 2- Content changes; (a) H2O2 content; (b) O 2- Content; Un-treatment refers to samples without MV treatment, MV treatment refers to samples treated with MV, different capital letters indicate that the content of the wild type is significantly different after MV treatment, different lowercase letters indicate that the content of 35S::CiGATA12a is significantly different after MV treatment, and asterisks indicate that the content of the wild type and 35S::CiGATA12a is significantly different. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The present invention provides a method for inducing transgenic hairy roots in the stem of Carya sylvestris, comprising the following steps: 1. preparing an in vitro stem; 2. constructing a binary vector plasmid containing a target gene and transferring it into Agrobacterium rhizogenes; 3. preparing an infection solution; 4. infection transformation and co-cultivation; 5. rooting culture and detecting positive hairy roots. The following is further described with specific examples.

[0036] Example 1

[0037] In this example, the stems of Hickory nut were used to induce hairy roots overexpressing CiGATA12a and verify its function. The specific steps are as follows:

[0038] 1. Prepare semi-lignified stems

[0039] Soak fresh thin-shelled pecan seeds for 5-6 days, changing the water every day. Then, bury the seeds in perlite and place them in an incubator at 24-35℃ for germination. Water them in time to maintain humidity during this process. After the seeds are germinated, cracked, and roots and buds grow, transplant them into a container containing a sterilized substrate (a mixture of garden soil: perlite: vermiculite = 3:1:1 (volume ratio)) and water them thoroughly. Cultivate them under light conditions at 25-28℃ until the base of the stem is semi-lignified.

[0040] 2. Preparation of 35S::CiGATA12a vector for transformation of Agrobacterium rhizogenes

[0041] RNA was extracted from the leaves of Hickory 'Ponyi', and the first strand of cDNA was synthesized after passing the quality inspection. The CDS amplification primers of CiGATA12a gene were designed according to the reference genome. The upstream primer was: 5'-ATGGAGGCACCTGAGTTTT-3' (SEQ ID No.1); the downstream primer was: 5'-GATCAGCTGCCTGAAATC-3' (SEQID No.2). The synthesized cDNA was used as a template for PCR amplification. The product size was 1050 bp, and the sequence was shown in SEQ ID No.3. The CDS of CiGATA12a was connected to the pCAMBIA2301 vector by homologous recombination, and Escherichia coli was transformed. The positive single clone was picked, and the plasmid was extracted after amplification and culture. The plasmid was transferred into Agrobacterium rhizogenes C58C1 using thermal stimulation.

[0042] The sequence shown in SEQ ID No.3 is:

[0043]

[0044] 3. Prepare infection solution

[0045] Pick a single clone of 35S::CiGATA12a rhizogenes Agrobacterium and place it in 1 mL of LB liquid medium containing 50 mM kanamycin (Kan), and shake it at 28°C and 200 rpm for 24 h. Transfer 1 mL of the Agrobacterium culture to 20 mL of LB medium containing 50 mM Kan antibiotic and 15 μM acetosyringone (AS), and shake it at 28°C and 200 rpm until the OD 600 The cells were then centrifuged at 6000 rpm for 10 min to collect the cells, and the Agrobacterium cells were resuspended in a resuspension solution (containing 10 mM MgCl2, 150 μM AS and 10 mM MES aqueous solution) to an OD of about 0.6. 600 = 1. Leave to stand at room temperature for 2-3 hours (2 hours is used in this experiment) and set aside.

[0046] 4. Infection, transformation and co-cultivation

[0047] Use a blade to ring the semi-lignified stems of the thin-shelled pecan seedlings with a ring width of 0.5-1 cm (0.5 cm was used in this experiment), and scrape off the remaining cambium in the xylem ( Figure 1 (a)); Use a paper towel to dip in the bacterial solution and wrap the ring-barking part ( Figure 1 (b)), and then wrap with plastic wrap to prevent the bacterial solution from evaporating ( Figure 1 After culturing in the dark for 24 h, remove the plastic wrap and paper towels, and use a matrix (peat: perlite: vermiculite = 3:1:1 (volume ratio)) to cover the infected area ( Figure 1 (d)).

[0048] 5. Rooting culture and detection of positive hairy roots

[0049] Seedlings were cultured at 25-28℃, and callus appeared one week after infection ( Figure 2 Middle (a)), root tissue appeared two weeks after infection ( Figure 2 Middle (b)), more hairy roots differentiated three weeks after infection ( Figure 2 Middle (c)), four weeks after infection, the roots rapidly elongated ( Figure 2 Middle (d)), capillary roots appeared five weeks after infection ( Figure 2 Middle (e)), six weeks after infection, the base of some roots began to lignify ( Figure 2 Middle (f). The positive roots were detected by GUS staining, and the results showed that the hairy roots were all stained, while the wild-type WT without vector transformation did not show blue ( Figure 3(a)). The intron amplification primers were designed for positive detection. The upstream primer was 5'-ACCTTTGTGAGCCGTATGACG-3' (SEQ ID No. 4); the downstream primer was 5'-CTAGATCAGCTGCCTGAAATCG-3' (SEQ ID No. 5). The PCR amplification results showed that the brightness of the transgenic band was much higher than that of the WT ( Figure 3 Middle (b)) further proves that the hairy roots are positive.

[0050] 6. Identification of CiGATA12a function using hairy roots

[0051] Transgenic hairy roots were immersed in 5 mM methyl viologen (MV) and the H2O2 and O 2- The results showed that MV treatment induced H2O2 and O in the hairy roots of WT and CiGATA12a. 2- Compared with the control, the H2O2 and O 2- The contents were significantly lower than those in the wild type ( Figure 4 ). The results showed that transgenic hairy roots can be used to identify gene functions and that CiGATA12a can scavenge reactive oxygen species caused by stress.

[0052] Example 2 Effects of different factors on the induction of hairy roots

[0053] As shown in Table 1, different factors were evaluated, including the Agrobacterium rhizogenes strains (K599, C58C1, MSU440), the concentration of the infection fluid (OD 600 =0.6, 1.0, 1.4) and infection time (12h, 24h, 36h) on the rooting rate and positive rate of stem induction. A three-factor three-level orthogonal design L9 (3 3 ) were tested (Table 2). Agrobacterium was transformed with the pCAMBIA2301 vector carrying 35S::GUS. After preparing the infection solution, the semi-lignified stems of the seedlings (about 2 months old) were ring-barked and infected. After 1.5 months of culture, the rooting rate and positive rate were statistically analyzed. The results showed that the rooting rate under different factors and levels was 85.01-94.43%, and the positive rate was 94.37-98.38%. The order of factors affecting the induction of transgenic hairy roots was bacterial solution concentration > bacterial type > infection time, and the best combination was C58C1, OD 600 =1.0, infection time 24h, under this condition the rooting rate of plants was 94.43%, and the positive roots of all rooted plants were 98.38% (Table 2).

[0054] Table 1 Three factors and three levels

[0055]

[0056] Table 2 Three-factor three-level orthogonal experimental design and results

[0057]

[0058] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for inducing transgenic hairy roots in walnut stems, characterized in that: The following steps are involved: The binary vector plasmid containing the target gene is transferred into Agrobacterium rhizogenes to prepare the recombinant Agrobacterium rhizogenes; The stems of Carya sylvestris were infected with the infection fluid of recombinant Agrobacterium rhizogenes and co-cultured, followed by rooting culture.

2. The method according to claim 1, characterized in that The target gene includes the CiGATA12a gene.

3. The method according to claim 1, characterized in that The recombinant Agrobacterium rhizogenes infection solution is prepared by the following steps: a positive single colony of the recombinant Agrobacterium rhizogenes is cultured in an LB medium containing antibiotics and acetosyringone until the OD 600 The concentration of Agrobacterium tumefaciens is 0.5-0.7, and the cells are collected by centrifugation; the cells are resuspended with a resuspending liquid to obtain the recombinant Agrobacterium rhizogenes infection liquid.

4. The method according to claim 3, characterized in that The concentration of antibiotics in the LB medium is 50 mM, and the concentration of acetosyringone is 15 μM; and / or the components of the resuspension are: an aqueous solution containing 10 mM MgCl2, 150 μM acetosyringone and 10 mM MES.

5. The method according to claim 1, characterized in that The OD of the recombinant Agrobacterium rhizogenes infection solution 600 It is 1.0-1.4, and the infection time is 24h.

6. The method according to claim 1, characterized in that The method of infection and co-cultivation comprises the following steps: Ring peel the 0.5-1 cm thin-shelled pecan stem cortex, and scrape off the remaining cambium tissue in the wood as the infection site; The infected part is impregnated with the impregnation liquid and the infected part is sealed and wrapped, and then co-cultured in a light-proof environment at 20-28° C. for 24-48 hours, the sealed and wrapped materials are removed, and the infected part is covered with a culture matrix.

7. The method according to claim 1, characterized in that The stem is a semi-lignified or lignified stem of the current year of the walnut seed seedling or variety seedling.

8. The method according to claim 1, characterized in that The temperature of the rooting culture is 25-28°C.

9. The method according to claim 1, characterized in that The Agrobacterium rhizogenes include C58C1, K599 and MSU440.

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