Jujube genetic transformation method using anther-induced callus as receptor

Abstract

The invention discloses a jujube genetic transformation method using anther to induce callus as a receptor. The method comprises the following steps: culturing sterile anther to obtain callus; constructing a plant recombinant overexpression vector containing a target gene, and transferring into agrobacterium to obtain agrobacterium dip dyeing liquid; the callus is dip-dyed with an agrobacterium dip-dyeing solution, and then the dip-dyed callus is co-cultured under a dark condition; and then putting the callus on a culture medium containing hygromycin for screening, and screening and culturing for 20-30 days to obtain the homozygous resistant callus. According to the method, the callus induced by the Jing 39 jujube anther is used as a receptor for the first time, the method of the Jing 39 jujube genetic transformation system is established, the transformation frequency is high, meanwhile, screening can be assisted by means of a 35S:: EGFP fluorescent reporter gene marker, and convenience and rapidness are achieved. The method provides convenience for jujube gene function analysis and jujube genetic improvement.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for establishing a jujube genetic transformation system by using anther-induced callus as a receptor. Background Art

[0002] Jujube (Ziziphus jujuba Mil.) is native to China, with rich germplasm resources and a long history of cultivation. It has become the largest dry fruit tree species in my country and has important economic, medicinal and ornamental values. However, due to the high embryo abortion rate, small flowers and difficulty in emasculation, and severe flower and fruit drop, the application of hybrid breeding in the cultivation of new jujube varieties is greatly limited. To date, people still know little about the genetic background and gene function information of jujube trees.

[0003] In 2014, Liu Mengjun's research group at Hebei Agricultural University was the first in the world to complete the whole genome sequencing of jujube, assembling a genome of 437.65Mb and annotating 32,808 genes. Based on this, a large number of candidate key genes that regulate the formation of important traits have been reported one after another. However, compared with genetic transformation research on other plants, transgenic work on jujube trees is still in the exploratory stage, and there are few reports on stable genetic transformation systems.

[0004] Therefore, it is necessary to establish a set of efficient callus genetic transformation system methods to solve the problems in genetic improvement of jujube trees and identification of gene functions, so as to accelerate the research process of jujube functional genes. This is of great significance to the research on the expression system of relevant functional genes in jujube itself. Summary of the invention

[0005] In view of this, the present invention provides a method for establishing a jujube genetic transformation system using anther-induced callus tissue as a receptor. This method has a high transformation frequency and high repeatability, and uses a fluorescent labeling method to achieve rapid visual identification, which facilitates the analysis of jujube gene function and also provides a possibility for the genetic improvement of jujube.

[0006] The technical solution of the present invention is specifically as follows: A method for genetic transformation of jujube using anther-induced callus as a receptor comprises the following steps: (i) culturing sterile anthers to obtain callus tissue; (ii) constructing a plant recombinant overexpression vector containing the target gene and transferring it into Agrobacterium to obtain Agrobacterium infection solution; (iii) co-culturing the callus tissue with the Agrobacterium-infected solution in the dark, and then culturing the drained callus tissue in the dark; (iv) Placing the calli cultured in (iii) on hygromycin medium for screening and culture to obtain homozygous resistant calli.

[0007] Preferably, in the above-mentioned jujube genetic transformation method, the following steps are also included: (v) Using homozygous resistant calli, analyze the expression level and gene function of the target gene.

[0008] Preferably, in the above-mentioned jujube genetic transformation method, the jujube is Jing 39 jujube. Jing 39 jujube has moderate fruit size, thin skin, crisp flesh, rich juice and sweet taste, high yield, disease resistance, drought tolerance, and is an excellent jujube variety. In one embodiment of the present invention, an efficient Jing 39 jujube efficient genetic transformation system is established using the callus tissue induced by the anther of Jing 39 jujube as the receptor, filling the technical gap of the genetic transformation of the anther callus tissue of Jing 39 jujube.

[0009] Preferably, in the above-mentioned jujube genetic transformation method, the method for obtaining the sterile anthers in step (i) is: selecting flower buds from plants that are healthy and free of pests and diseases, treating the flower buds with 70% alcohol and 0.1% mercuric chloride in sequence, and then peeling off the petals.

[0010] Preferably, in the above-mentioned jujube genetic transformation method, the culturing method of step (i) is: inoculating sterile anthers onto an induction medium, culturing in the dark at 26°C for 30 days, and then transferring the successfully induced callus to a proliferation medium for culturing, and the light cycle is 16 / 8h, and the culture temperature is 26°C; wherein the induction medium (pH=5.8) is 4.43g / L 1 / 2MS+1.0mg / L2,4-dichlorophenoxyacetic acid+0.5mg / L thidiazuron+30g / L sucrose+6.0g / L agar; the proliferation medium (pH=5.8) is 4.43g / L MS+1.0mg / L 2,4-dichlorophenoxyacetic acid+0.5mg / L thidiazuron+30g / L sucrose+8.0g / L agar.

[0011] Preferably, in the above-mentioned jujube genetic transformation method, the plant recombinant overexpression vector is a pH7WG2D plasmid containing the target gene, and the pH7WG2D plasmid carries a 35S promoter, an EGFP tag, and spectinomycin and hygromycin resistance genes. Experiments have shown that this plasmid can significantly improve the callus genetic transformation rate compared to other plasmids. For example, when the pK7WG2D plasmid (carrying a 35S promoter, an EGFP tag, spectinomycin and kanamycin resistance genes) is used, the callus genetic transformation rate is basically 0.

[0012] Preferably, in the above-mentioned jujube genetic transformation method, the Agrobacterium infection solution in step (ii) consists of Agrobacterium carrying a plant recombinant overexpression vector and a resuspension solution, wherein the resuspension solution is: 4.43 g / L MS+30 g / L sucrose+100 mM acetosyringone.

[0013] Preferably, in the above-mentioned jujube genetic transformation method, the culturing method of step (iii) is: placing the callus tissue from which the Agrobacterium-infected solution has been drained on a solid MS co-culture medium and co-culturing for 2-3 days in the dark; wherein the solid MS co-culture medium is 4.43 g / L MS + 1.0 mg / L 2,4-dichlorophenoxyacetic acid + 0.5 mg / L thidiazuron + 30 g / L sucrose + 8 g / L agar + 100 mM acetosyringone.

[0014] Preferably, in the above-mentioned jujube genetic transformation method, the screening culture method in step (iv) is: callus culture is performed using a culture medium containing hygromycin. The screening culture medium is 4.43g / L MS+1.0mg / L 2,4-dichlorophenoxyacetic acid+0.5mg / L thidiazuron+30g / L sucrose+8g / L agar+50mg / L hygromycin.

[0015] Preferably, in the above-mentioned jujube genetic transformation method, the method for obtaining homozygous resistant callus in step (iv) is: placing the fluorescently labeled callus tissue alone in a screening medium, and obtaining the homozygous resistant callus after 2-3 generations, each generation for 3-4 weeks.

[0016] The beneficial effects of the present invention are: The present invention uses the callus tissue induced by the anther of Jing 39 jujube as a receptor, establishes a method for the genetic transformation system of Jing 39 jujube, and improves the genetic transformation research of the anther callus tissue of jujube. In addition, the method of the present invention has a high transformation frequency, high repeatability, simple operation steps, and is convenient and fast to assist screening with the help of 35S::EGFP fluorescent reporter gene marker. The Jing 39 jujube genetic transformation system established by the present invention provides convenience for the analysis of jujube gene functions, and also accelerates the research process of jujube functional genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the structure of the recombinant overexpression vector constructed in an embodiment of the present invention.

[0018] Figure 2 These are the anther callus growth pictures and related infection pictures of Jing 39; among them, A is the anther induced callus, B is the anther callus propagation, C is the selection of anther callus with good growth status for dark culture, D is the anther callus after 15 days of dark culture, E is the fluorescence labeling picture of anther callus 30 days after Agrobacterium infection, and F is the fluorescence labeling picture of propagated homozygous callus.

[0019] Figure 3 This is the statistical result of genetic transformation rate of anther callus of Jing 39 jujube.

[0020] Figure 4 Homozygous callus HkDJPositive test gel image; in the image, OE1-11 are 11 randomly selected homozygous positive calli carrying the target gene, WT1-3 are randomly selected uninfected anther calli, -(EV) are randomly selected homozygous calli carrying empty plasmids, and + are plasmids containing the target gene.

[0021] Figure 5 Homozygous callus HkDJ Gene expression detection results.

[0022] Figure 6 for 35S::ZjHXK1 The results of the test of sucrose, glucose and fructose content in callus. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The term "comprises" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0025] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0026] Example 1 In this case, the Ziziphus jujuba (common jujube) hexokinase-1-like gene (candidate hexokinase gene ( HkDJ )) as the target gene, a genetic transformation system of Jing 39 jujube was established, which specifically includes the following steps: (1) Acquisition of genetic transformation receptors.

[0027] In mid-May, flower buds were selected from healthy and pest-free plants. The flower buds were first soaked in 70% alcohol for 10 seconds, then rinsed with sterile distilled water 3 times, then soaked in 0.1% mercuric chloride solution for 10 minutes, and finally rinsed with sterile distilled water 3 times. The petals were peeled off with tweezers to remove the sterile anthers and inoculated on the culture dish. After culturing in the dark at 26°C for 30 days, the successfully induced callus was transferred to the proliferation medium as the genetic transformation material; the subsequent culture conditions were light intensity 2000Lx, light-dark cycle 16 / 8h, and culture temperature 26°C.

[0028] The induction medium used is 4.43g / L 1 / 2MS+1.0mg / L 2,4-dichlorophenoxyacetic acid+0.5mg / L thidiazuron+30g / L sucrose+6.0g / L agar. The proliferation medium used is 4.43g / L MS+1.0mg / L 2,4-dichlorophenoxyacetic acid+0.5mg / L thidiazuron+30g / L sucrose+8.0g / L agar. The pH of the induction medium and the proliferation medium are both 5.8. The culture medium and plant growth regulators are sterilized by high temperature and high pressure; active ingredients such as antibiotics are sterilized by filtration and added after the culture medium is sterilized.

[0029] (2) Clone the target gene to be verified.

[0030] According to the jujube genome database, the sequence information of the target gene is shown in SEQ ID NO. 1. Primer A is designed based on the sequence information of the target gene, wherein primer A includes upstream primer A and downstream primer A, and the sequence is specifically as follows: Upstream primer A: 5′-atgggaaaggtggcagtggga-3′ (SEQ ID NO. 2); Downstream primer A: 5'-ttaggattcctcaactccaag -3' (SEQ ID NO. 3).

[0031] RNA was extracted from the tissues and organs of Jing 39 jujube, and cDNA was obtained by reverse transcription as a template for PCR amplification. PCR amplification was performed based on the above primer A. The PCR amplification system was: PCR Mastermix 24.5μL (Tiangen Biochemical Technology (Beijing) Co., Ltd.), upstream primer A 2μL, downstream primer A 2μL, cDNA template 1μL and double distilled water 10μL. Positive bands were obtained by agarose gel electrophoresis (the molecular weight of the specific band in this case was about 1500bp), and the positive band DNA was recovered by gel excision, and then sequenced to verify the candidate target gene sequence.

[0032] (3) Construction of recombinant overexpression vector.

[0033] The cloned target gene (SEQ ID NO.1) was integrated into a plant overexpression vector to construct a plant recombinant overexpression vector, and the plant recombinant overexpression vector was transferred into Agrobacterium EHA105 to obtain Agrobacterium carrying a recombinant plasmid. The specific operation process is as follows: The plant overexpression binary vector is pH7WG2D plasmid, which carries 35S strong promoter, EGFP tag, spectinomycin and hygromycin resistance genes ( Figure 1). For specific recombinant plasmid methods, please refer to the one-step Gateway reaction method for constructing expression vectors and its application studied by Yu Deshui et al. (CN201910677970.8).

[0034] After the recombinant plasmid obtained above was transferred into DH5a competent cells by heat shock transformation method, LB liquid culture was added and activated at 200 rpm in a 37°C shaker for 40 min, and then evenly spread on solid LB culture medium containing 50 μg / mL spectinomycin for resistance screening and cultured at 37°C overnight; several positive single colonies were picked into LB liquid culture medium containing spectinomycin resistance, and after overnight culture at 37°C shaker at 150 rpm, 500 μL of bacterial solution sample was taken, and the bacterial solution was sequenced and verified. If the verification was correct, the bacterial solution with the recombinant plasmid was obtained.

[0035] The recombinant plasmids were extracted from the bacterial solution enriched with the recombinant plasmids obtained above using the plasmid rapid extraction kit of Tiangen Company, and then the extracted recombinant plasmids were transferred into the competent state of EHA105, and then added to the LB liquid culture medium and activated at 200rpm in a 28℃ shaker for 4h, and then evenly coated on a solid LB medium containing 50mg / L rifampicin and 50mg / L spectinomycin for resistance screening, and cultured at 28℃ for 48h; then the positive single colony was picked and cultured in the LB liquid containing rifampicin and spectinomycin resistance, and the culture was shaken at 200rpm at 28℃ overnight until the bacterial solution was turbid, and the Agrobacterium carrying the recombinant plasmid was obtained. The Agrobacterium was obtained and stored in sterilized glycerol with a final concentration of 40%, stored at -80℃, and recorded as pH7WG2D-HXK1-EGFP.

[0036] (4) Cultivation of Agrobacterium and preparation of infection solution.

[0037] The pH7WG2D-HXK1-EGFP Agrobacterium EHA105 obtained and stored at -80°C and the pH7WG2D Agrobacterium EHA105 stored in the laboratory were spread on LB plates containing hygromycin and rifampicin, cultured at 28°C for 48 h, single colonies were picked, transferred to 1 mL of LB liquid culture medium containing spectinomycin and rifampicin, cultured at 200 rpm in a 28°C constant temperature incubator for 12 h, and then PCR detection of the bacterial solution was performed. The bacterial solution with a positive test result was streaked and cultured, 40 uL of the bacterial solution was aspirated onto the LB plate containing hygromycin and rifampicin, and cultured inverted in a 28°C constant temperature incubator for about 40 h-48 h, the bacterial solution was washed with a resuspension solution, and finally 50 mL of the resuspension solution was added to resuspend the bacteria and the concentration of the bacterial solution was adjusted to OD 600 =0.6, and the Agrobacterium infiltration solution is obtained for use. The resuspended solution is 4.43 g / L MS+30 g / L sucrose+100 mM acetosyringone, and its pH is 5.8.

[0038] (5) Callus infection, co-culture and screening culture.

[0039] The Agrobacterium infection solution and native Agrobacterium EHA105 infection solution prepared in step (4) were co-cultured with Jing 39 jujube callus in a constant temperature shaker at 28° C. and 200 rpm in the dark for 30 minutes to obtain infected Jing 39 jujube callus.

[0040] The above-obtained callus of Jing 39 jujube was placed on filter paper to drain the impregnation solution and then placed on solid MS co-culture medium for 3 days under dark conditions. The solid MS co-culture medium was 4.43 g / L MS + 1.0 mg / L 2,4-dichlorophenoxyacetic acid + 0.5 mg / L thidiazuron + 30 g / L sucrose + 8 g / L agar + 100 mM acetosyringone, and the pH was 5.8.

[0041] (6) Screening and cultivation The Jing 39 jujube callus obtained in step (5) was placed on a solid MS screening medium (4.43 g / L MS + 1.0 mg / L 2,4-dichlorophenoxyacetic acid + 0.5 mg / L thidiazuron + 30 g / L sucrose + 8 g / L agar + 50 mg / L hygromycin) for screening and culture for 30 days, and then the fluorescently labeled callus was placed separately on the solid MS screening medium. After three generations of screening and culture for 3-4 weeks each, homozygous Jing 39 jujube resistant callus was obtained. The culture conditions were: dark culture at a temperature of 26°C.

[0042] Figure 2 The following are the growth diagrams of Jing 39 anther callus and infection-related pictures. With the help of fluorescent marker screening, this example counted the genetic transformation rate of Jing 39 jujube anther callus under this method. Specifically, a total of 234 dishes of Jing 39 jujube anther callus were infected, and 130 dishes of transgenic Jing 39 jujube anther callus emitted green fluorescence, accounting for 55.85%, that is, the genetic transformation rate of transgenic Jing 39 jujube anther callus was 55.85% (see Figure 3 , 78 dishes as one replicate, three replicates in total).

[0043] Example 2 Using the Jing 39 jujube genetic transformation system established in Example 1, this example further verified and analyzed the expression level and function of the target gene, as follows: (1) The resistant callus of Jing 39 jujube was tested by RT-PCR, and the integration and expression status of the target gene was determined based on whether there was a band of the target gene. Specifically, the positive callus of jujube subcultured for 15 days was uniformly sampled, and RNA was extracted, agarose gel electrophoresis and fluorescence quantitative PCR were performed subsequently.

[0044] ① Design positive detection primer B according to the expression vector pH7WG2D-HXK1-EGFP, the positive detection primer B includes upstream primer B and downstream primer B, and the specific sequence is as follows: Upstream primer B: 5′-gacgcacaatcccactatcc-3′ (SEQ ID NO. 4); Downstream primer B: 5′-tgtacaagaaagctgggtccccttaggattcctcaactccaag-3′ (SEQ ID NO. 5).

[0045] The test results based on positive primer B are as follows Figure 4 shown.

[0046] ② According to the target gene (candidate hexokinase gene, HkDJ ) sequence to design qPCR specific primer C, the primer C includes upstream primer C and downstream primer C, and the specific sequence is as follows: Upstream primer C: 5′-cgccaccgaatgaagagc-3′ (SEQ ID NO. 6); Downstream primer C: 5′-gtctgccacctgtctgagtttac-3′ (SEQ ID NO. 7).

[0047] The detection results based on primer C are as follows Figure 5 As shown, the expression level of hexokinase gene in transgenic jujube callus increased sharply.

[0048] (2) Determination of relevant physiological indicators.

[0049] To verify the target gene ( HkDJ ) function, this example measured the changes in the content of key sugar components in jujube callus. The results are as follows Figure 6 As shown: The content of key sugar components in the callus tissue of transgenic jujube was significantly reduced.

[0050] The above results prove that the candidate hexokinase gene has the function of reducing the content of sucrose, glucose and fructose.

[0051] In summary, the present invention uses the callus tissue induced by the anther of Jing 39 jujube as the receptor and successfully establishes a method for genetic transformation of Jing 39 jujube. The method has simple operation steps and high transformation rate. 35S::EGFP Fluorescent reporter gene markers can realize visual screening, which is convenient and quick. The system established by the invention provides a new approach for the analysis of jujube gene functions and genetic improvement of jujube.

[0052] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for genetic transformation of jujube using anther-induced callus as a receptor, characterized in that: The following steps are involved: (i) culturing sterile anthers to obtain callus tissue; (ii) constructing a plant recombinant overexpression vector containing the target gene and transferring it into Agrobacterium to obtain Agrobacterium infection solution; (iii) infecting the callus tissue with an Agrobacterium infection solution, and then culturing the infected callus tissue in a dark environment; (iv) Placing the callus from (iii) on a screening medium to obtain homozygous resistant callus.

2. The method for genetic transformation of jujube according to claim 1, characterized in that: The following steps are also included: (v) Using homozygous resistant calli, analyze the expression level and gene function of the target gene.

3. The method for genetic transformation of jujube according to claim 1, characterized in that: The method for obtaining the sterile anthers is as follows: selecting flower buds from plants that are healthy and free of diseases and insect pests, treating the flower buds with alcohol and mercuric chloride in sequence, and then peeling off the petals to obtain the sterile anthers.

4. The method for genetic transformation of jujube according to claim 1, characterized in that: The culture method of step (i) is: inoculating sterile anthers onto an induction medium, culturing at 26°C in the dark for 30 days, and then transferring the successfully induced callus onto a proliferation medium for culture at a light cycle of 16 / 8h and a culture temperature of 26°C; Wherein, the induction medium is 4.43g / L 1 / 2MS+1.0mg / L 2,4-dichlorophenoxyacetic acid+0.5mg / L thidiazuron+30g / L sucrose+6.0g / L agar; the proliferation medium is 4.43g / L MS+1.0mg / L 2,4-dichlorophenoxyacetic acid+0.5mg / L thidiazuron+30g / L sucrose+8.0g / L agar.

5. The method for genetic transformation of jujube according to claim 1, characterized in that: The plant recombinant overexpression vector is a pH7WG2D plasmid containing a target gene, and the pH7WG2D plasmid carries a 35S promoter, an EGFP tag, and spectinomycin and hygromycin resistance genes.

6. The jujube genetic transformation method according to claim 1, characterized in that: In step (ii), the Agrobacterium infection solution is composed of Agrobacterium carrying the plant recombinant overexpression vector and a resuspension solution, wherein the resuspension solution is 4.43 g / L MS+30 g / L sucrose+100 mM acetosyringone.

7. The jujube genetic transformation method according to claim 1, characterized in that: The culture method of step (iii) is: placing the callus tissue from which the Agrobacterium-infected liquid has been drained on a solid MS co-culture medium and co-culturing for 2-3 days in the dark; wherein the solid MS co-culture medium is 4.43 g / L MS + 1.0 mg / L 2,4-dichlorophenoxyacetic acid + 0.5 mg / L thidiazuron + 30 g / L sucrose + 8 g / L agar + 100 mM acetosyringone.

8. The jujube genetic transformation method according to claim 1, characterized in that: The method of culturing the screening medium in step (iv) is: culturing with a screening medium containing hygromycin; the screening co-culture medium is 4.43 g / L MS + 1.0 mg / L 2,4-dichlorophenoxyacetic acid + 0.5 mg / L thidiazuron + 30 g / L sucrose + 8 g / L agar + 50 mg / L hygromycin.

9. The method for genetic transformation of jujube according to claim 1, characterized in that: The method for obtaining homozygous resistant callus in step (iv) is: placing the fluorescently labeled callus tissue alone in a solid MS screening medium, and after 2-3 generations of screening and culture, each generation lasting 3-4 weeks, the homozygous resistant callus can be obtained.

10. The method for genetic transformation of jujube according to claim 1, characterized in that: The jujube described is Beijing 39 jujube.

Images