An Agrobacterium tumefaciens-mediated method for transforming oats with MIR

Abstract

The present invention belongs to the technical field of plant genetic transformation, and specifically relates to an Agrobacterium tumefaciens-mediated oat MIR transformation method. The method includes: Step 1: Introduce a plant recombinant expression vector containing an exogenous gene into Agrobacterium tumefaciens, and obtain positive genetic engineering Agrobacterium through colony identification; Step 2: Prepare a bacterial solution of positive genetic engineering Agrobacterium; Step 3: Genetic transformation of oat seedlings; Step 4: Co-culture the transformed oat stem segments; Step 5: Recovery culture; Step 6: Transplantation of the regenerated oat seedlings. The method of the present invention is a technical system that can stably, efficiently, and quickly deliver genes to different oat germplasms.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic transformation, and specifically relates to an Agrobacterium tumefaciens-mediated oat MIR transformation method. Background Art

[0002] Oat is an annual herbaceous plant of the genus Avena ( Poaceae ) in the family Poaceae ( Avena ). It is a food and forage (grass) crop widely cultivated worldwide. Based on the genome in oat cells and combined with its external morphological characteristics, the oats cultivated worldwide are currently divided into 30 species (Zheng Diansheng. 2010. Research Status and Prospect of Oat Germplasm Resources in China: Oat and Buckwheat Research and Development. / / Proceedings of the First and Second National Oat and Buckwheat Academic Seminars, Beijing: China Agricultural Science and Technology Press). Currently, genetic transformation systems for many cultivated oat species have been established using different genetic transformation methods. The progress of foreign transgenic oat research has been introduced in detail in the review published by Pathi and Sprink in 2023 (Pathi K.M., Sprink T. From Petri Dish to Field: Plant Tissue Culture and Genetic Engineering of Oats for Improved Agricultural Outcomes. Plants (Basel). 2023, 12(21): 3782). There are also relevant reports on transgenic oats in China (Zhang Yi (2007). Establishment of an Oat Genetic Transformation System and an Elymus dahuricus Regeneration System [D]. Sichuan University, 2007.; Wang Xunjing (2012). Establishment of an Oat Tissue Culture and Agrobacterium tumefaciens-mediated Oat Genetic Transformation System [D]. Northeast Normal University, 2012.; Zhang Lijun, Liu Longlong, Sun Yi, Zhang Jianzhen, Cui Lin. Study on an Agrobacterium tumefaciens-mediated Oat Wound Embryo Transformation System [J]. Journal of Hebei Agricultural Sciences, 2015, 19(3): 49 - 54.). However, due to the genetic germplasm diversity of oats, so far, no oat transgenic system that can efficiently and quickly perform genetic transformation on all oats cultivated worldwide has been established, which has made the transgenic breeding of oats lag far behind that of other major food crops (such as rice, wheat, corn, etc.). Therefore, the establishment of an efficient and quick transformation system for oats has become a bottleneck problem to be solved in oat germplasm innovation.

[0003] Based on the fact that dicotyledonous plants have the ability to be transformed by Agrobacterium rhizogenes ( Agrobacterium rhizogenesThe characteristics of forming hairy roots after infection and the phenomenon of root suckering (which refers to the development of adventitious buds from adventitious bud primordia located on the roots) in these hairy roots. The CDB (Cut-dip-budding) method for direct gene delivery in dicotyledonous plants without tissue culture has been reported (Cao X, Xie H, Song M, Lu J, Ma P, Huang B, Wang M, Tian Y, Chen F, Peng J, Lang Z, Li G, Zhu JK. Cut-dip-budding delivery system enables genetic modifications in plants without tissue culture. Innovation (Camb). 2022 Oct 25;4(1):100345.). The establishment of this genetic transformation system has greatly promoted the transgenic research process and its industrial application of dicotyledonous plants.

[0004] The establishment of the CDB gene delivery method is based on the long-term analysis by scientists of the molecular mechanism of crop pathogenicity caused by Agrobacterium.

[0005] Studies have found that the molecular mechanism by which Agrobacterium causes plants to form crown gall tumors or hairy roots is as follows: when different organs of dicotyledonous plants are damaged, the damaged parts secrete a chemical substance called acetosyringone; when there is Agrobacterium (including Agrobacterium tumefaciens and Agrobacterium rhizogenes) in the soil, once it senses acetosyringone, it will gather towards the damaged parts of the plant and, through a series of complex enzymatic reactions, finally transfer a fragment called T-DNA in Agrobacterium into the plant genome, causing the plant to exhibit the symptoms of crown gall tumors or hairy roots. The above pathogenic mechanism of Agrobacterium lays the molecular foundation for efficient and stable transformation of plants mediated by Agrobacterium.

[0006] Unlike dicotyledonous plants, monocotyledonous plants do not have the ability to secrete acetosyringone autonomously after being damaged. Therefore, for a long time, Agrobacterium-mediated plant genetic transformation was only limited to dicotyledonous plants. In 1993, it was found that when acetosyringone was added to the culture medium, Agrobacterium tumefaciens could also complete the genetic transformation of the monocotyledonous rice (Chan MT, Chang HH, Ho SL, Tong WF, Yu SM. Agrobacterium-mediated production of transgenic rice plants expressing a chimeric alpha-amylase promoter / beta-glucuronidase gene. Plant Mol Biol. 1993 Jun;22(3):491-506.). This enabled the use of immature embryos, mature embryos, and even intact seeds after maturity of food crops such as rice, corn, and wheat as materials to obtain embryogenic callus through tissue culture; then these embryogenic calli were transformed and screened; and the resistant calli obtained through screening were induced to form buds and roots, ultimately obtaining complete transgenic plants, which became the mainstream technology for the genetic transformation of these food crops. The reason is that compared with the genetic transformation of monocotyledonous plants using gene guns, the Agrobacterium tumefaciens-mediated transformation method has the characteristics of higher transformation efficiency, more exogenous genes being present in single-copy form, and stable genetic expression. However, the obvious disadvantage of this method is that it requires going through the time-consuming, laborious, and costly process of tissue culture. Moreover, determined by genetic germplasm differences, for the callus induction, regeneration, and subsequent genetic transformation system established for one material, when changing to another variety, the original system is no longer applicable. This severely limits the popularization and application of this technology in the field of transgenic breeding of monocotyledonous food crops.

[0007] Oats are monocotyledonous plants with fibrous root systems. So far, no phenomenon of adventitious bud formation from single oat fibrous roots has been found. This indicates that the above-mentioned method of CDB gene delivery mediated by Agrobacterium rhizogenes for dicotyledonous plants is not suitable for the genetic transformation of oats. In addition, different from food crops such as rice, corn, wheat, sorghum, and barley, the chromosome ploidy in oat cells is complex, with natural diploids, tetraploids, and hexaploids, which results in up to more than 30 cultivated oat species worldwide. It can be seen that if a set of embryogenic callus induction and subsequent Agrobacterium tumefaciens-mediated genetic transformation technology systems are established separately for each type of oat, it is obviously impossible to meet the urgent needs of the high-quality development of the oat industry for oat germplasm innovation. Summary of the Invention

[0008] The genetic germplasm diversity of oats has led to the absence of a technical system that can stably, efficiently, and quickly deliver genes to different oat species planted worldwide. To solve the above technical problems, the present invention proposes a MIR gene delivery method for highly efficient, rapid, and stable genetic transformation of oats with different genotypes using Agrobacterium tumefaciens.

[0009] Based on the characteristic that oats can regenerate after mowing, the present invention proposes mowing oat seedlings at the age of 10 - 30 days (Mow), and then immersing the mowed oat materials with the genetically engineered Agrobacterium tumefaciens strain EHA105 carrying exogenous genes such as reporter genes EGFP (Enhanced Green FlorescentProtein), herbicide resistance genes OsAHASΔ548W ((Fang J, Wan C, Wang W, Ma L, Wang X, Cheng C, Zhou J, Qiao Y, Wang X. Engineering Herbicide-Tolerance Rice Expressing an Acetohydroxyacid Synthase with a Single Amino Acid Deletion. Int J Mol Sci. 2020 Feb 13;21(4):1265.), namely the acetolactate synthase gene from rice with a tryptophan deletion at position 548), and finally allowing the transformed oat materials to form transgenic regenerated seedlings through regeneration (Regeneration). This is the oat MIR transformation method.

[0010] The present invention specifically adopts the following technical solutions:

[0011] An Agrobacterium tumefaciens-mediated oat MIR transformation method, comprising:

[0012] Step 1: Introduce a plant recombinant expression vector containing an exogenous gene into Agrobacterium tumefaciens, and screen to obtain positive genetically engineered Agrobacterium.

[0013] Step 2: Under sterile conditions, add the activated positive genetically engineered Agrobacterium to a liquid medium to make the OD 600 value of the obtained genetically engineered Agrobacterium liquid medium range between 0.4 and 0.6; the specific liquid medium used is AAM medium. The specific operation for activating the genetically engineered Agrobacterium is: activate the genetically engineered Agrobacterium with YEB and AB solid plates in sequence; then prepare a transformation bacterial liquid with AAM liquid medium.

[0014] Step 3: Genetic transformation of oat seedlings:

[0015] Add acetosyringone to the genetic engineering Agrobacterium liquid; Cut the oat seedlings after 10 days of germination, and infect the cut oat seedlings with the genetic engineering Agrobacterium liquid containing acetosyringone; The cultivation of oat seedlings is as follows: When the oat seeds germinate to form adventitious roots and a true leaf extends from the coleoptile, transplant the seedlings into a container with vermiculite at the bottom of the bottle for cultivation; The thickness of vermiculite in the container is 2 - 3 cm; The vermiculite is moistened with 1 / 2 MS liquid medium.

[0016] During infection, the concentration of acetosyringone in the genetic engineering Agrobacterium liquid is 100 μM; The cutting is to cut off part of the stems and leaves of the oat seedlings, leaving a 2 - 5 cm stem segment of the seedling with roots; The infection time is 15 min.

[0017] Step 4: Co - culture of transformed oat stem segments:

[0018] Co - culture the transformed oat stem segments. The specific operation of the co - culture is as follows: Moisten the filter paper in the culture container with N6 - As liquid medium, drain the transformed oat stem segments of the bacterial liquid, place them on the filter paper, and then co - culture them in a plant artificial climate incubator at 24 °C in the dark for two days.

[0019] Step 5: Recovery culture:

[0020] Transfer the co - cultured oat materials to a container filled with vermiculite and conduct recovery culture in a light culture room. During this period, supplement nutrients with 1 / 2 MS liquid medium to allow the transgenic oat materials to recover;

[0021] Step 6: Transplanting of oat regenerated seedlings:

[0022] When the recovered oats regenerate into 2 - leaf and 1 - heart regenerated seedlings, conduct transplantation. The substrate used for transplantation is composed of nutrient soil and cultivation soil, and the volume ratio of nutrient soil to cultivation soil is 1:3.

[0023] Step 7: Identification of transgenic oats:

[0024] Molecular identification and phenotypic identification can be carried out on transgenic oats.

[0025] The beneficial effects of the present invention are as follows:

[0026] 1. In the present invention, the seedlings obtained from the natural germination of mature seeds of different species of oats are cut and directly transformed with the Agrobacterium tumefaciens liquid containing the target gene, thus avoiding the complicated plant tissue culture processes such as embryogenic callus induction, bud induction, and root induction. Therefore, compared with the traditional embryogenic callus genetic transformation system of oats, the present invention is more time - saving, cost - saving, and labor - saving;

[0027] 2. In a further embodiment, the present invention activates Agrobacterium tumefaciens in genetic engineering using two types of solid plates (YEB, AB); at the same time, an AAM liquid medium rich in various nutrients required for the growth of Agrobacterium tumefaciens and oat seedlings is used to prepare the Agrobacterium tumefaciens solution for transformation, instead of the conventional MS medium, thereby ensuring a high transformation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 They are the maps of pCUbi1390-EGFP (A) and pCUbi1390-OsAHASΔ548W (B).

[0029] Figure 2 For the transformation and activation of Agrobacterium tumefaciens in genetic engineering. A: Colony map of Agrobacterium tumefaciens EHA105 transformed with the recombinant expression vector; B - C: Streak culture maps of Agrobacterium tumefaciens in genetic engineering containing OsAHASΔ548W, EGFP genes respectively; D: Plate activation culture map of Agrobacterium tumefaciens AB.

[0030] Figure 3 For the colony PCR identification of Agrobacterium tumefaciens in genetic engineering. M: DL2000 DNA molecular standard; Lanes 1 and 6: Positive controls, using the pCUbi1390-OsAHASΔ548W plasmid as a template, and amplifying HPTⅡ , EGFP with the HPTⅡPf76+Pr985 and EGFP Pf+Pr primer pairs respectively, thereby obtaining target DNA bands with sizes of 910bp and 720bp respectively; Lanes 2 - 5: Four independent single colonies of Agrobacterium tumefaciens transformed with pCUbi1390-OsAHASΔ548W were picked to amplify HPTⅡ genes. Lanes 7 - 10: Four independent single colonies of Agrobacterium tumefaciens transformed with pCUbi1390-OsAHASΔ548W were picked to amplify the EGFP gene.

[0031] Figure 4 For the germination of oat seeds for transformation and the cultivation of seedlings. A: Seeds of three types of oats (HXh1, HXh2, HXh3, see Figure 4 A - a, 4A - b, 4A - c respectively) after 2 days of germination; B: Seedlings formed by HXh1 10 days after germination.

[0032] Figure 5 For the infection and co - culture of oat seedlings. A: Pretreatment for infecting oat seedlings: Oat seedlings germinated for 22 days, with leaves and part of the young roots cut off, and the remaining part cut into two sections for use as transformation materials; B. Infection of the root and stem segments; C. Co - culture of the infected materials (before cultivation); D. Co - culture of the infected materials (two days after cultivation). The red arrow indicates the new shoots growing from the rootless and stemless segments after co - culture.

[0033] Figure 6 Recovery and transplantation of transgenic oat seedlings. A: The oat stem segments with roots after transformation were transplanted into sterilized vermiculite for recovery culture (nourishing the seedlings with 1 / 2MS liquid medium); B: Transgenic oat seedlings grown in a seedling pot.

[0034] Figure 7 PCR identification of transgenic oats. M: DL2000 DNA molecular standard; Lane 1: DNA extracted from untransformed oat seedlings used as a template for PCR amplification HPTⅡ of the gene; Lanes 2 - 7: Results of PCR amplification of the gene from 6 transgenic oat seedlings. HPTⅡ

[0035] Figure 8 trans EGFP Fluorescence detection of transgenic oat seedlings. A, C: Bright - field observation of the roots and leaves of transgenic oat seedlings; B, D: Observation of the roots and leaves of transgenic oat seedlings in the EGFP channel.

[0036] Figure 9 Induction of embryogenic callus from mature oat seeds. A: Day 0 of oat callus induction; B: Day 15 of oat callus induction; C: Day 30 of oat callus induction; D: Sub - culture of the oat callus formed after 30 days of induction.

[0037] Figure 10 Induction of oat embryogenic callus and establishment of its regeneration system. A: Oat seeds; B: Oat embryogenic callus; C: Bud differentiation; D: Rooting of tissue - cultured seedlings; E: Regenerated plants.

[0038] Figure 11 Transformation and screening of embryogenic callus induced from oat seeds. A: Batch transformation of oat embryogenic callus; B: Screening and identification of resistant callus. Note: Figure 11 The untransgenic callus "killed" by hygromycin is shown by the blue circle in B; Figure 11 The resistant callus containing the hygromycin resistance gene and the target gene is shown by the red circle in B. Detailed implementation mode

[0039] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0040] Example 1

[0041] I. Experimental materials

[0042] 1. The plant materials used in this example, the cultivated oat varieties HXh1, HXh2, HXh3, and HXh4, are two varieties of hulled oats (HXh1, HXh4) and two varieties of naked oats (HXh2, HXh3) collected, sorted, and preserved by the Key Laboratory of Inner Mongolia Agricultural University for Germplasm Innovation and Utilization of Wheat and Barley, a provincial-level key laboratory of higher education institutions.

[0043] 2. The strains used in this example include Escherichia coli ( Escherichia coli ) DH5α strain and Agrobacterium tumefaciens ( A. tumefaciens ) EHA105 strain, which are the competent cell states of the above-mentioned strains purchased commercially.

[0044] 3. The plant binary expression vector pCUbi1390-EGFP used in this example was kindly provided by Professor Xia Lanqin of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. The vector map is as shown in Figure 1 Figure A; while the pCUbi1390-OsAHASΔ548W expression vector containing the herbicide resistance gene is a vector constructed and preserved independently, and the vector map is as shown in Figure 1 Figure B.

[0045] 4. The conventional chemical reagents required in this example are: LB solid and liquid media (for the culture of Escherichia coli DH5α strain and the extraction of expression vectors), AB and YEB solid / liquid media (for the culture and activation of EHA105), AAM liquid medium (for the suspension of EHA105), 1 / 2MS liquid medium (for the hydroponics of oat seedlings), 2N6-AS liquid medium (for the co-culture of oat seedlings after infection); the biochemical reagents required are: kanamycin (CAS No.: 25389-94-0), ticarcillin (CAS No.: 4697-14-7), acetosyringone (CAS No.: 2478-38-8).

[0046] 5. The nutrient soil used in this example was purchased from Inner Mongolia Zhaochen Biotechnology Co., Ltd.

[0047] 6. The formula of the medium used is as follows:

[0048] (1). Preparation of AB solid plate (for the activation of Agrobacterium)

[0049] (1) Preparation of 20×AB buffer stock solution (Buffer): Weigh the compounds that make up the buffer, dissolve them in deionized water, make up the volume, and then sterilize them separately.

[0050] Volume 1L

[0051] K2HPO4 60g

[0052] 20 g of NaH₂PO₄

[0053] (2) Preparation of 20×AB basic salt stock solution (Salts) (The preparation process is the same as above).

[0054] 20 g of NH₄Cl

[0055] 6 g of MgSO₄·7H₂O

[0056] 3 g of KCl

[0057] 0.2 g of CaCl₂

[0058] 50 mg of FeSO₄·7H₂O

[0059] (3) Preparation of 1 L of AB solid medium

[0060] 5 g of Glucose

[0061] 15 g of Agar

[0062] 50 mL of AB Buffer

[0063] 50 mL of AB Salts

[0064] H₂O (dissolved with deionized water), made up to 1 L.

[0065] According to the above composition, add each component in turn; for the prepared medium, autoclave at 121 °C under high pressure for 30 min; when the temperature of the medium drops below 60 °C, add 50 mg / L of kanamycin sterilized by filtration through a 0.22 μm filter membrane; mix quickly, and then pour it into a sterilized and dried glass petri dish with a diameter of 10 cm. After the medium solidifies, seal it with PARAFILM membrane and store it in a 4 °C refrigerator for later use.

[0066] (II) Preparation of AAM liquid medium (for preparing Agrobacterium tumefaciens transformation solution)

[0067] (1) 50 mL of 1000×AA-1 (trace elements)

[0068] 1.25 mg of CuSO₄·5H₂O

[0069] 1.25 mg of CoCl₂·6H₂O

[0070] 12.5 mg of Na₂MoO₄·2H₂O

[0071] 37.5 mg of KI

[0072] 100 mg of ZnSO₄·7H₂O

[0073] 150 mg of H3BO4

[0074] 416 mg of MnSO4·H2O

[0075] (2) 50 mL of 1000×AA-2 (CaCl2·2H2O)

[0076] 7.5 g of CaCl2·2H2O

[0077] (3) 50 mL of 1000×AA-3

[0078] 12.5 g of MgSO4·7H2O

[0079] (4) 1000 mL of 1000×AA-4 (iron salt)

[0080] 2.78 g of FeSO4·7H2O

[0081] 3.73 g of Na2·EDTA

[0082] Note: For the above two solutions, first dissolve them separately in 400 mL of deionized water, then combine the dissolved solutions in a 1 L beaker, make up to 1 L, and finally pour into a brown reagent bottle.

[0083] (5)50 mL of 1000×AA-5

[0084] 7.5 g of NaH2PO4·2H2O

[0085] (6) 100 mL of 1000×AA-6

[0086] 1000× B5 vitamin stock solution

[0087] Note: The B5 vitamin stock solution is a commercially available product.

[0088] (7)50 mL of 1000×AA-Sol

[0089] 88.3 mg of Arg (arginine)

[0090] 38 mg of Gly (glycine)

[0091] For the prepared AAM stock solutions above, except for AA-Sol which is filtered and sterilized with a 0.22 μm filter membrane, the others are autoclaved at 121 °C under high pressure for 30 min; after cooling, store at room temperature for use.

[0092] (8)Preparation of AAM liquid medium: 400 mL

[0093] 400 μL of 1000×AA-1 stock

[0094] 1000×AA-2 stock 400μL

[0095] 1000×AA-4 stock 400μL

[0096] 1000×AA-5 stock 400μL

[0097] 1000×AA-Sol stock 400μL

[0098] Casamino Acid 0.2 g

[0099] Sucrose 27.4g

[0100] Glucose 14.4g

[0101] Asp 0.12g

[0102] Inositol 0.04g

[0103] KCl 1.2g

[0104] Take 300 mL of deionized water, add the above reagents and drugs in sequence, stir and dissolve, adjust the pH value to 5.2, make up the volume to 400 mL, and autoclave at 121 °C under high temperature and high pressure for 30 min; after it cools down to room temperature, add 400 μL of AA-3 in the laminar flow hood and mix well. The prepared AAM liquid medium is stored in the refrigerator at 4 °C for standby.

[0105] (III). Preparation of 2N6-As liquid medium (for co-culturing transformed callus)

[0106] Volume 500mL

[0107] N6D basal Salt (Cas number, C416, Phyto Technology) 1.99g

[0108] Casamino Acid 0.15g

[0109] Prolamine 1.439g

[0110] Sucrose 15g

[0111] Take 400 mL of deionized water, sequentially add the above-mentioned reagents and drugs, stir and dissolve them, adjust the pH value to 5.8 - 6.0, and perform autoclaving at 121 °C under high temperature and high pressure for 30 min; after it cools down to room temperature, add 500 μL of 1000×N6 Vitamin Stock Solution sterilized by filtration through a 0.22 μm filter membrane and 1 mL of 1 mg / mL 2,4-D in a super clean bench; before use, add 200 mM acetosyringone solution to a final concentration of 100 μM.

[0112] Note: Unless otherwise specified, the conventional chemical drugs, biochemical reagents, consumables, nutrient soil, etc. used in the present invention are all purchased from Inner Mongolia Zhaochen Biotechnology Co., Ltd.

[0113] 7. The primers used in the examples are as follows:

[0114] Table 1 Primer Information

[0115] 。

[0116] II. Test methods, procedures and results

[0117] Step 1. Transformation of pCUbi1390-EGFP and pCUbi1390-OsAHASΔ548W:

[0118] Respectively take 1 μg of the above-mentioned plant binary expression vectors pCUbi1390-EGFP and pCUbi1390-OsAHASΔ548W, and use the heat shock transformation method (heat shock at 42 °C for 90 sec, then ice-water bath for 5 min) to introduce the above vectors into 50 μL of DH5α competent cells (Beijing Coolaber Technology Co., Ltd., Cat# CC501-20×100 μL). After resuscitation culture (add 500 μL of LB liquid medium, shake culture at 37 °C and 80 rpm for 45 minutes), take 300 μL of the bacterial solution and spread it on an LB solid plate containing 50 mg / L kanamycin, and culture it overnight in a 37 °C constant temperature incubator.

[0119] Step 2. Extraction of the recombinant expression vector:

[0120] From the plates prepared in Step 1, pick two single colonies containing the plant binary expression vector pCUbi1390-EGFP and two single colonies containing the plant binary expression vector pCUbi1390-OsAHASΔ548W respectively, and inoculate them into 20 mL of LB liquid medium containing 50 mg / L kanamycin, and shake culture overnight at 37 °C and 180 rpm; for the above-prepared bacterial solution, use the plasmid mini-prep kit (Code#EM101-01) of Beijing TransGen Biotech Co., Ltd. to complete the plasmid extraction of the above plant binary expression vector according to its instructions.

[0121] Step 3: Preparation of genetically engineered Agrobacterium tumefaciens:

[0122] Take 1 μg each of the pCUbi1390-EGFP and pCUbi1390-OsAHASΔ548W plasmids prepared in Step 2, and mix them with 50 μL of EHA105 competent cells (Beijing Coolaber Technology Co., Ltd., Cat#CC403-10×100 μL) respectively. According to the instruction manual of the competent cells, use the liquid nitrogen quick-freezing - heat shock method (the competent cell - plasmid mixture is sequentially left standing on ice for 5 min, quick-frozen in liquid nitrogen for 5 min, heat-shocked in a 37°C water bath for 5 min, and finally ice-bathed for 5 min) to complete the introduction of the above vectors into EHA105, and thus prepare genetically engineered Agrobacterium tumefaciens containing the above binary expression vectors respectively. Spread the two obtained genetically engineered Agrobacterium tumefaciens on YEB solid plates. The colonies after transformation are shown in Figure 2 A.

[0123] Step 4: Colony PCR identification of genetically engineered Agrobacterium tumefaciens:

[0124] Perform colony PCR identification on the single colonies formed on the two YEB plates in Step 3 respectively.

[0125] (1) For the genetically engineered Agrobacterium tumefaciens transformed with pCUbi1390-EGFP, complete the PCR amplification detection of the reporter gene EGFP in the vector according to the following reaction system and PCR amplification conditions. Reaction system: 5 μL of 2×ExTaq Premix [Takara Bio Inc. (Dalian), Cat#PR902A], 1 μL of EGFP Pf_ BamH I+Pr_ SmaI (Pf, Pr each at 5 μM), 4 μL of sterilized deionized water; pick an appropriate amount of bacterial cells with a sterilized pipette tip and add them to the above colony PCR amplification premix. PCR amplification conditions: Step 1, pre-denaturation at 94°C for 5 min; Step 2, denaturation at 94°C for 30 sec; Step 3, annealing at 60°C for 1 min; Step 4, extension at 72°C for 1 min; Step 5, extension at 72°C for 10 min; among them, Steps 2 - 4 are cycled 30 times.

[0126] (2) For the genetically engineered Agrobacterium tumefaciens containing pCUbi1390-OsAHASΔ548W, perform PCR amplification detection of the target gene OsAHAS Δ548W and PCR amplification detection of the plant selection marker gene HPTⅡ (hygromycin resistance gene). The amplification systems used for the above OsAHASΔ548W, HPTⅡ genes, except that the primer pairs are OsAHASPf+Pr and HPTⅡPf76+Pr985 respectively, are the same as EGFPPCR detection amplification system; The amplification conditions used for PCR amplification of OsAHASΔ548W are the same as those of EGFP except that the annealing temperature is 50 °C. HPTⅡ The amplification conditions are the same as those of EGFP except that the annealing temperature is 55 °C.

[0127] The results of colony PCR identification are shown in detail in Figure 3 . It can be seen from Figure 3 A (Lane7-Lane10) that for the genetically engineered Agrobacterium tumefaciens transformed from pCUbi1390-EGFP, using the EGFP Pf+Pr primer pair, a DNA band of about 750 bp was amplified ( EGFP The actual size of the gene is 720 bp + 16 bp _ restriction enzyme sites added on the primer and 4 protective bases); It can be seen from Figure 3 A (Lane2-Lane5) and Figure 3 B (Lane1-Lane4) that for the genetically engineered Agrobacterium tumefaciens transformed from pCUbi1390-OsAHASΔ548W, amplified with primer pairs HPTⅡPf76+Pr985 and OsAHASPf+Pr respectively, the results showed that DNA bands of 900 bp ( Figure 3 A Lane2-Lane5), about 1900 bp ( Figure 3 B, Lane1-4) were amplified, which is consistent with the HPTⅡ hygromycin resistance gene (910 bp), OsAHASΔ548W gene (1932 bp) sizes carried on the pCUbi1390-OsAHASΔ548W vector.

[0128] Step 5: Preservation of genetically engineered Agrobacterium tumefaciens:

[0129] Pick 2 single colonies of the genetically engineered Agrobacterium tumefaciens with positive colony PCR amplification each, inoculate them into 20 mL of liquid YEB medium containing 50 mg / L carbenicillin, and culture them on a shaker at 28 °C with an oscillation speed of 180 rpm for 36 - 48 h (OD 600 value 0.6 - 0.8); Aliquot the above cultured bacterial solution into sterilized 1.5 mL centrifuge tubes at 1 mL / tube, then add 500 μL of sterilized glycerol (60%), mix well, quickly freeze in liquid nitrogen, and then store the bacterial strain in a -80 °C ultra-low temperature refrigerator.

[0130] Step 6: Activation of genetically engineered Agrobacterium tumefaciens:

[0131] Take one glycerol bacterium of genetically engineered Agrobacterium prepared in step 5 and place it on crushed ice; then, in a laminar flow hood, use an inoculation needle to pick a small amount of solid bacteria and streak it on a plate containing YEB solid medium (abbreviated as YEB, containing 50 mg / L carbenicillin), and then incubate it in an incubator at 28 °C until clear single colonies are formed, as shown in 2B-C; pick a single colony on the YEB plate and streak it on a plate containing AB solid medium (abbreviated as AB plate), and incubate it in an incubator at 28 °C for 2 - 3 days (until the bacteria between the streaks are connected into a sheet), and complete the activation of genetically engineered Agrobacterium, as shown in Figure 2 D.

[0132] Note: Based on the number of oat seedlings to be transformed, prepare AB plates according to one single colony corresponding to one AB plate, one AB plate corresponding to the amount of bacteria used for one transformation (OD 600 value = 0.4 - 0.6, 30 mL of bacterial solution), and the amount of bacteria used for one transformation corresponding to 20 oat seedlings.

[0133] Step 7: Preparation of AAM bacterial solution of genetically engineered Agrobacterium for transformation:

[0134] In a laminar flow hood, use a sterilized medicine spoon to scrape an appropriate amount of activated genetically engineered Agrobacterium on the AB plate in portions and add it to a 50 mL centrifuge tube containing 30 mL of liquid AAM medium. After adding bacteria each time, measure the OD 600 , when the OD 600 value is between 0.4 and 0.6, stop adding bacteria; if it exceeds 0.6, add an appropriate amount of AAM medium to the tube and adjust the OD 600 value back to the above range.

[0135] Step 8: Preparation of oat seedlings for transformation:

[0136] (1) Germination of oat seeds

[0137] Complete the germination of oat seeds using the petri dish - filter paper method. Specifically: Take a petri dish with a diameter of 10 cm, place 2 - 3 filter papers of corresponding size at the bottom of the dish; moisten the filter papers with tap water. Take an appropriate amount (30 - 35 seeds) of pre - transformed oat seeds (in this example, 3 oat varieties were selected, namely HXh1, HXh2, and HXh3, among which the first two are forage hulled oat varieties and the latter is a grain naked oat variety). Arrange the seeds evenly spaced on the filter paper, cover the petri dish lid, wrap it with tin foil, and then place it in a plant artificial climate incubator at 24 °C for cultivation.

[0138] The germination and seedling growth of different oat seeds are as shown in Figure 4 shown.

[0139] (2) Transplanting of germinated seedlings

[0140] When oat seeds germinate in a culture dish to form adventitious roots and a true leaf extends from the coleoptile, transplant the seedlings into a 300 mL glass tissue culture bottle with 2 - 3 cm of vermiculite at the bottom of the bottle; moisten the vermiculite with 1 / 2 MS liquid medium; in the first few days after transplantation, cover the tissue culture bottle cap; when the seedling leaves grow to the position of the tissue culture bottle cap, open the cap for cultivation, and pay attention to watering in a timely manner; every other week, supplement and water with 1 / 2 MS liquid medium once.

[0141] Step 9. Genetic transformation of oat seedlings:

[0142] Take 30 mL of the genetically engineered Agrobacterium AAM bacterial liquid prepared in step 7 into a 300 mL glass tissue culture bottle, and add 15 μL of 200 mM acetosyringone solution to the bacterial liquid to a final concentration of 100 μM. Take 20 seedlings of HXh1 that have germinated for 10 days, reserve a 2 - 5 cm root-bearing seedling stem segment, cut off the other parts of the stem and leaves with a sterilized surgical scissors, and soak the remaining root-bearing oat stem segments in the above-prepared genetically engineered Agrobacterium AAM bacterial liquid containing 100 μM acetosyringone for 15 minutes, gently shaking the bacterial liquid during this period to allow the oat stem segments to come into full contact with the bacterial liquid (if necessary, use forceps to press the part exposed above the bacterial liquid into the bacterial liquid). Complete the genetic transformation of all materials according to the above process.

[0143] Regarding the transformation of oat seedlings 15 days and 30 days after germination, the steps are the same as those of oat seedlings 10 days after germination.

[0144] Step 10. Co-culture of transformed oat stem segments:

[0145] Take 1 sterilized glass culture dish with a diameter of 10 cm, place 2 sterilized filter papers in the dish; moisten the filter papers with an appropriate amount of 2N6-As liquid medium, drain the bacterial liquid from the oat stem segments transformed in step 9, place them on the filter papers, and then wrap them with tin foil (simulating the dark environment of Agrobacterium in the soil), and co-culture in a 24 °C plant artificial climate incubator for two days.

[0146] Note: According to the principle of placing one type of transformed material in one culture dish, complete the co-culture of all transformed materials.

[0147] The pre-treatment, infection and subsequent co-culture of oat seedlings infected are as Figure 5 shown. As shown by the red arrow, when using the stem segments of oats for transformation, after two days of co-culture, seedlings also grow out from the coleoptiles of the transformed stem segments. However, in the subsequent regeneration culture, due to its inability to absorb nutrients from the medium or soil by itself, the seedling eventually withers (see step 11 for details). This result also shows that the CDB gene delivery method applicable to dicotyledonous plants is not applicable to monocotyledonous gramineous plants such as oats.

[0148] Step 11. Recovery culture of transformed materials:

[0149] In the early stage, the transgenic oat stem segments after co-culture were directly transplanted into the oat field cultivation soil that was not sterilized and contained a certain proportion of nutrient soil (nutrient soil: cultivation soil = 1:3, volume ratio). Since the oat seedlings had low vitality after processes such as mowing, Agrobacterium infection, and co-culture, the ratio of transgenic oat seedlings formed by the regeneration of the transformed oat stem segments was low.

[0150] To improve the survival rate of transgenic oat seedlings, a recovery culture step was added to the above-mentioned transformed oat stem segments in this example. Specifically: Take a seedling-growing plug tray (5×6 = 30 holes), fill the sterilized vermiculite according to the number of transformed oat stem segments (each hole corresponds to one transformed oat stem segment); Take the co-cultured oat materials in step 10, rinse the Agrobacterium attached to the surface of the materials under running water, and then plant the transformed oat materials into the plug tray. After all the transformed materials are planted into the plug tray, place the plug tray in a tray and pour 2 - 5 cm of tap water into the tray; In addition, pour 10 mL of 1 / 2 MS liquid medium into the holes with the transformed materials, cover the lid, and place the materials in a light culture room for cultivation (pay attention to replenishing water during the cultivation period, and add 1 / 2 MS nutrient solution every 7 days). The oat seedlings after recovery culture are as Figure 6 shown in A.

[0151] Step 12. Transplanting of the regenerated seedlings of transformed oats:

[0152] When the transformed oats in step 11 regenerate into 2-leaf and 1-heart regenerated seedlings, transplant them into an oat cultivation bucket. Fill 2 - 3 cm of absorbent cotton at the bottom of the bucket, and cover it with oat cultivation nutrient soil (nutrient soil: cultivation soil = 1:3, volume ratio). Place the oat cultivation bucket with the transplanted seedlings into the tray supporting the oat cultivation bucket containing 2 - 5 cm of tap water. The transgenic oats transplanted into the cultivation bucket are as Figure 6 shown in B.

[0153] Note: During the growth and development process of the transgenic oat seedlings after transplantation, replenish water in a timely manner; Add 1 / 2 MS nutrient solution in a timely manner based on their growth and development conditions.

[0154] Step 13. Molecular and phenotypic identification of transgenic oats:

[0155] (1) Molecular identification of oats with two transgenes ( EGFP gene, OsAHASΔ548W gene)

[0156] Appropriately take the leaves of each transgenic oat seedling (30 - 60 mg), and according to the instructions of the plant genomic DNA extraction and purification kit of Beijing TransGen Biotech Co., Ltd. (Cat#EE111 - 01 / 11), complete the extraction of genomic DNA from different transgenic oat plants. Using the extracted transgenic oat genomic DNA as a template, according to the reaction system: 2×ExTaq Premix 10 μL, gDNA (20 ng), HPTⅡ Pf76 + Pr985 (Pf and Pr are each 5 μM) 2 μL, supplement sterilized deionized water to 20 μL; use HPTⅡ the gene PCR amplification program to complete the PCR identification of the selectable marker gene in transgenic oat plants HPTⅡ . The electrophoresis results of the PCR amplification products are as Figure 7 shown. HPTⅡ The PCR detection primers for the gene are Pf76 and Pr985, and a 910 bp PCR product is expected to be amplified; from Figure 7 the electrophoresis results, it can be seen that the actual size of the amplified PCR product is between 750 bp and 1000 bp, which is consistent with the expected size, indicating that through MIR transformation, the foreign gene has been introduced into the oat genome.

[0157] (2) EGFP Phenotypic identification of gene - transformed oat plants

[0158] When transplanting transgenic oat seedlings, for the oat regenerated seedlings transformed by Agrobacterium containing the pCUbi1390 - EGFP plant expression vector, cut a 3 - 5 cm leaf segment and a 3 - 5 cm root tip respectively. Place the above materials under a fluorescence dissection microscope (Zeiss), set the EGFP observation parameters, and complete the phenotypic identification of transgenic oat materials.

[0159] From Figure 8 the results, it can be known that relatively strong EGFP fluorescence signals are detected in both the roots and leaves of the transgenic oat seedlings to be tested; especially the fluorescence signal detected in the roots, which rules out the suspicion of the autofluorescence of chlorophyll in the leaves. This indicates that the foreign gene has not only been integrated into the oat genome, but has also been expressed and thus endows the transgenic oat seedlings with new genetic phenotypes.

[0160] Comparative example

[0161] Establishment of the embryogenic callus induction and regeneration system from mature oat seeds, including:

[0162] Step 1: Induction of primary callus:

[0163] Using the improved N6D medium (N6 basal medium + 2,4 - D 4 mg / L, proline 2878 mg / L, casein hydrolysate 300 mg / L), induce primary callus from mature oat seeds, asFigure 9 as shown

[0164] Step 2: Obtaining and regeneration of embryogenic callus:

[0165] The primary callus induced from mature oat seeds can form embryogenic callus (light yellow, loose, granular callus, Figure 10 shown by the red line in B) after subculture; the embryogenic callus can be induced to form adventitious buds, adventitious roots, and then seedlings. As Figure 10 shown

[0166] On the basis of establishing the system for inducing embryogenic callus from mature oat seeds and its regeneration system, this example attempts to complete the genetic transformation of oats and create transgenic oat seedlings using this system. The results show that when the embryogenic callus induced from mature seeds of two oats ("HXh1", "HXh2") is transformed and cultured in N6D medium containing 50 mg / L hygromycin, 212 / 219 resistant calli of "HXh1" / "HXh2" are respectively screened. After PCR identification, the target gene can be amplified in these resistant calli; however, when these resistant calli are subjected to subsequent differentiation culture, they fail to successfully differentiate into seedlings, as HPTⅡ shown Figure 11 as shown

[0167] The results of this example show that without going through Agrobacterium infection and subsequent screening, oat embryogenic callus can successfully differentiate into regenerated seedlings; while after Agrobacterium infection of the callus and screening, resistant callus can be obtained, but the resistant callus fails to successfully differentiate into transgenic seedlings.

[0168] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the present invention in each example.

Claims

1. An Agrobacterium tumefaciens-mediated oat MIR transformation method, characterized in that, Including: Step 1: Introduce the plant recombinant expression vector containing the exogenous gene into Agrobacterium tumefaciens, and screen to obtain positive genetically engineered Agrobacterium tumefaciens; Step 2: Prepare the positive genetically engineered Agrobacterium tumefaciens bacterial liquid; Step 3: Genetic transformation of oat seedlings: Add acetosyringone to the genetically engineered Agrobacterium tumefaciens bacterial liquid; Cut the oat seedlings after 10 days of germination, and infect the cut oat seedlings with the genetically engineered Agrobacterium tumefaciens bacterial liquid containing acetosyringone; The cutting is to cut off part of the stems and leaves of the oat seedlings, leaving a 2-5 cm stem segment with roots; Step 4: Co-culture the transformed oat stem segments; Step 5: Resuscitation culture of the co-cultured oat materials; The resuscitation culture is specifically as follows: Transplant the co-cultured oat materials into a container filled with vermiculite, and carry out resuscitation culture in a light culture room. During this period, supplement nutrition with 1 / 2MS liquid medium to resuscitate the transgenic oat materials; Step 6: Transplant the oat regenerated seedlings; When the resuscitated oats regenerate into two-leaf and one-heart regenerated seedlings, carry out transplantation; Step 7: Identify the transgenic oats.

2. The Agrobacterium tumefaciens-mediated oat MIR transformation method according to claim 1, characterized in that, In Step 2, the preparation of the positive genetically engineered Agrobacterium tumefaciens bacterial liquid is as follows: Under sterile conditions, add the activated genetically engineered Agrobacterium tumefaciens to the liquid medium to obtain it; The liquid medium is AAM liquid medium.

3. The Agrobacterium tumefaciens-mediated oat MIR transformation method according to claim 2, wherein, The specific operation of activating the genetically engineered Agrobacterium tumefaciens is as follows: Activate the genetically engineered Agrobacterium tumefaciens with YEB and AB solid plates in sequence; Then prepare the transformation bacterial liquid with AAM liquid medium.

4. A method for Agrobacterium tumefaciens-mediated transformation of oats MIR according to claim 1, characterized in that, In step 2, the OD of the obtained positive genetic engineering Agrobacterium liquid 600 value is between 0.4 and 0.

6.

5. A method for Agrobacterium tumefaciens-mediated transformation of oat MIR according to claim 1, characterized in that, In Step 3, the cultivation of oat seedlings is specifically as follows: When the oat seeds germinate to form adventitious roots and a true leaf extends from the coleoptile, transplant the seedlings into a container with vermiculite at the bottom of the bottle for cultivation, and the vermiculite is moistened with 1 / 2MS liquid medium.

6. The Agrobacterium tumefaciens-mediated oat MIR transformation method according to claim 5, characterized in that, The thickness of the vermiculite in the container is 2-3 cm.

7. A method for Agrobacterium tumefaciens-mediated transformation of oat MIR according to claim 1, characterized in that In Step 3, during the infection, the concentration of acetosyringone in the genetically engineered Agrobacterium tumefaciens bacterial liquid is 100 μM.

8. The Agrobacterium tumefaciens-mediated oat MIR transformation method according to claim 1, characterized in that, In Step 4, the specific operation of the co-culture is as follows: Moisten the filter paper in the culture container with N6-As liquid medium, drain the transformed oat stem segments of the bacterial liquid, place them on the filter paper, and then co-culture them in a plant artificial climate incubator at 24 °C in the dark for two days.

9. The Agrobacterium tumefaciens-mediated oat MIR transformation method according to claim 1, characterized in that, In Step 6, the substrate used for transplantation is composed of nutrient soil and cultivation soil, and the volume ratio of nutrient soil to cultivation soil is 1:3.

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