A method for genetic transformation of jujube by using anther to induce callus as a receptor

Abstract

The application discloses a jujube genetic transformation method using anther-induced callus as a receptor, comprising the following steps: culturing sterile anthers to obtain callus; constructing a plant recombinant overexpression vector containing a target gene and transferring the plant recombinant overexpression vector into agrobacterium to obtain an agrobacterium immersion liquid; immersing the callus in the agrobacterium immersion liquid, and then co-culturing the immersed callus under dark conditions; and then placing the callus on a culture medium containing hygromycin for screening, so that a homozygous resistant callus can be obtained after 20-30 days of screening culture. The application firstly uses the anther-induced callus of Jing 39 jujube as a receptor to establish a method of the jujube genetic transformation system, the method has a high transformation frequency, and meanwhile, the method can be assisted by 35S::EGFP fluorescent reporter gene marking for screening, and is convenient and fast. The method provides convenience for jujube gene function analysis and jujube genetic improvement.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for establishing a jujube genetic transformation system by using anther-induced callus as a receptor. BACKGROUND

[0002] Jujube (Ziziphus jujuba Mil.) is originally from China, and has rich germplasm resources and a long history of cultivation. It has become the first large fruit tree species in China, and has important economic, medicinal and ornamental values. However, due to the high embryo abortion rate, small flowers and difficulty in emasculation, and serious flower and fruit drop of jujube trees, the application of hybridization breeding in the cultivation of new jujube varieties is greatly limited, and the genetic background and gene function information of jujube trees are still poorly understood.

[0003] In 2014, Liu Mengjun's research group of Hebei Agricultural University completed the whole genome sequencing of jujube first in the world, assembled a genome of 437.65 Mb, and annotated 32808 genes. Based on this, a large number of candidate key genes regulating important traits have been reported. However, compared with the genetic transformation research of other plants, the transgenic work of jujube is still in the exploratory stage, and there are few reports on stable genetic transformation system.

[0004] Therefore, it is necessary to establish a high-efficiency callus genetic transformation system method to solve the problems in jujube genetic improvement and gene function identification, so as to accelerate the research process of jujube functional genes. This is of great significance to the expression system research of jujube-related functional genes. SUMMARY

[0005] Therefore, the present application provides a method for establishing a jujube genetic transformation system by using anther-induced callus as a receptor. The method has high transformation frequency and high repeatability, and realizes visual rapid identification by means of fluorescent labeling method, which provides convenience for jujube gene function analysis, and also provides a possibility for jujube genetic improvement.

[0006] The technical scheme of the present application is specifically as follows:

[0007] A jujube genetic transformation method using anther-induced callus as a receptor, comprising the following steps:

[0008] (i) culturing sterile anthers to obtain callus;

[0009] (ii) constructing a plant recombinant overexpression vector containing a target gene and transferring it into Agrobacterium to obtain an Agrobacterium immersion solution;

[0010] (iii) co-culturing the callus with the Agrobacterium immersion solution in darkness, and then culturing the drained callus under dark conditions;

[0011] (iv) placing the callus of (iii) into a hygromycin medium screening culture to obtain homozygous resistant callus.

[0012] Preferably, the jujube genetic transformation method further comprises the following steps:

[0013] (v) using the homozygous resistant callus to analyze the expression amount and gene function of the target gene.

[0014] Preferably, in the jujube genetic transformation method, the jujube is Jing 39 jujube. Jing 39 jujube has moderate fruit size, thin skin, crisp and juicy flesh, sweet taste, high yield, disease resistance and drought resistance, and is a good jujube variety. In an embodiment of the present application, the anther-induced callus of Jing 39 jujube is used to establish a high-efficiency genetic transformation system of Jing 39 jujube, which fills the technical gap of genetic transformation of Jing 39 jujube anther callus.

[0015] Preferably, in the jujube genetic transformation method, the sterile anther is obtained by the following method: selecting a flower bud from a healthy and disease-free plant, sequentially treating the flower bud with 70% alcohol and 0.1% mercuric chloride, and then peeling off the petals to obtain the sterile anther.

[0016] Preferably, in the jujube genetic transformation method, the culture method of step (i) is as follows: inoculating the sterile anther into an induction medium, culturing in the dark at 26°C for 30 days, and then transferring the successfully induced callus to a proliferation medium, 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 / L 2,4-dichlorophenoxyacetic acid+0.5mg / L thidiazuron+30g / L sucrose+6.0g / L agar; and 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.

[0017] Preferably, in the jujube genetic transformation method, the plant recombinant overexpression vector is a pH7WG2D plasmid containing a target gene, and the pH7WG2D plasmid carries a 35S promoter, an EGFP tag, spectinomycin and a hygromycin resistance gene. Experiments show that this plasmid can significantly improve the callus genetic transformation rate compared with other plasmids. For example, when using a pK7WG2D plasmid (carrying a 35S promoter, an EGFP tag, spectinomycin and kanamycin resistance genes), the callus genetic transformation rate is basically 0.

[0018] Preferably, in the jujube genetic transformation method, the Agrobacterium immersion liquid in step (ii) 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.

[0019] Preferably, in the jujube genetic transformation method, the culture mode in step (iii) is that the anther callus drained of the Agrobacterium immersion liquid is placed on a solid MS co-culture medium and co-cultured in the dark for 2-3 days; 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.

[0020] Preferably, in the jujube genetic transformation method, the screening culture mode in step (iv) is that the callus is cultured with a medium containing hygromycin. The screening 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.

[0021] Preferably, in the jujube genetic transformation method, the method for obtaining the homozygous resistant callus in step (iv) is that the callus with fluorescent labeling is placed in the screening medium alone, and after 2-3 generations of screening culture, each generation for 3-4 weeks, the homozygous resistant callus is obtained.

[0022] The jujube genetic transformation method of the present application has the following beneficial effects:

[0023] The present application uses the anther callus induced by Jingtai 39 jujube flowers as the receptor to establish a method for the genetic transformation system of Jingtai 39 jujube, and perfects the genetic transformation research of jujube anther callus. Moreover, the transformation frequency of the method is high, the repeatability is high, the operation steps are simple, and the screening is facilitated by the 35S::EGFP fluorescent reporter gene marker. The Jingtai 39 jujube genetic transformation system established by the present application provides convenience for jujube gene function analysis, and also accelerates the research process of jujube functional genes. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The structure diagram of the recombinant overexpression vector constructed for the embodiment of the present application.

[0025] Figure 2 The growth chart of Jingtai 39 anther callus and related infection chart; wherein, A is anther callus induction, B is anther callus propagation, C is to select anther callus with good growth state for dark culture, D is anther callus after 15 days of dark culture, E is a fluorescent labeling chart after 30 days of anther callus infection by Agrobacterium, and F is a fluorescent labeling chart of propagation of homozygous callus.

[0026] Figure 3 Figure 1 is a statistical result chart of genetic transformation rate of Jujube anther callus.

[0027] Figure 4 Figure 2 is a chart of homozygous callus ZjHXK1 Figure 3 is a chart of anther glue map; in the chart, OE1-11 are 11 randomly selected homozygous positive calluses carrying the target gene, WT1-3 are randomly selected anther calluses without soaking, - (EV) are randomly selected homozygous calluses carrying empty plasmids, and + is a plasmid containing the target gene.

[0028] Figure 5 Figure 4 is a chart of homozygous callus ZjHXK1 Figure 5 is a chart of detection results of gene expression amount.

[0029] Figure 6 Figure 6 is 35S::ZjHXK1 Figure 7 is a chart of detection results of sucrose, glucose and fructose content of callus. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be described clearly and completely below in conjunction with examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The term "comprising" and any variation thereof in the specification and claims of the present application is intended to cover not exclusively including.

[0032] In the following examples, if not specifically stated, they are all conventional methods; the reagents and materials, if not specifically stated, can be obtained from commercial channels.

[0033] Example 1

[0034] In this example, Ziziphus jujuba (common jujube) hexokinase-1-like gene (candidate hexokinase gene (ZjHK1) is used as the target gene, and a genetic transformation system of Jujube is established, which specifically includes the following steps: ZjHXK1

[0035] (1) Obtaining of genetic transformation receptor.

[0036] ​In the middle of May, flower buds were selected from healthy and pest-free plants. The flower buds were first soaked in 70% alcohol for 10s, then rinsed with sterilized distilled water for 3 times, then soaked in 0.1% mercuric chloride solution for 10min, and finally rinsed with sterilized distilled water for 3 times. The petals were peeled off with tweezers to obtain an aseptic anther, which was inoculated on a culture dish. After 30d of dark culture at 26℃, the induced callus was transferred to the proliferation medium as the genetic transformation material. The subsequent culture conditions were light intensity of 2000Lx, light and dark cycle of 16 / 8h, and culture temperature of 26℃.

[0037] The induction medium used was 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 was 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 was 5.8. The medium and the plant growth regulators were sterilized at high temperature and high pressure; the active ingredients such as antibiotics were filtered and sterilized, and added after the medium was sterilized.

[0038] (2) Cloning of the target gene to be verified.

[0039] According to the jujube whole genome database, the sequence information of the target gene is shown as SEQ ID NO. 1. The primer A is designed according to the sequence information of the target gene, wherein the primer A includes an upstream primer A and a downstream primer A, and the sequence is specifically as follows:

[0040] Upstream primer A: 5'-atgggaaaggtggcagtggga-3' (SEQ ID NO. 2);

[0041] Downstream primer A: 5'-ttaggattcctcaactccaag-3' (SEQ ID NO. 3).

[0042] The RNA of Jingtong 39 jujube tissue organs was extracted, and the cDNA was obtained by reverse transcription as the template for PCR amplification. Based on the above primer A, PCR amplification was carried out. The PCR amplification system is as follows: 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. By agarose gel electrophoresis, a positive band (the molecular weight of the specific band in this example is about 1500bp) was obtained. After cutting and recovering the DNA of the positive band, the sequence of the candidate target gene was verified by sequencing.

[0043] (3) Construction of recombinant overexpression vector.

[0044] The cloned target gene (SEQ ID NO. 1) is integrated into a plant overexpression vector to construct a plant recombinant overexpression vector, and the plant recombinant overexpression vector is transformed into Agrobacterium EHA105 to obtain Agrobacterium carrying the recombinant plasmid. The specific operation process is as follows:

[0045] The plant overexpression binary vector is pH7WG2D plasmid, and the pH7WG2D plasmid vector carries a 35S strong promoter, an EGFP label, a spectinomycin and a hygromycin resistance gene Figure 1 ). The specific recombinant plasmid method can refer to the method of constructing an expression vector by one-step Gateway reaction and application (CN201910677970.8) by De Shui et al.

[0046] After the recombinant plasmid obtained above is transformed into DH5a competent cells by heat shock transformation, LB liquid culture is added and activated at 37°C on a shaker at 200 rpm for 40 min, then uniformly coated on solid LB medium containing 50 μg / mL spectinomycin for resistance screening, and cultured at 37°C overnight; several positive single colonies are picked into LB liquid medium containing spectinomycin resistance, and cultured at 37°C on a shaker at 150 rpm overnight, then 500 μL of bacterial liquid sample is taken for sequencing verification of the bacterial liquid, and the correct verification is obtained, i.e. the bacterial liquid with the recombinant plasmid.

[0047] The recombinant plasmid in the bacterial liquid enriched with the recombinant plasmid obtained above is extracted using the plasmid rapid extraction kit of Tiangen company, then the extracted recombinant plasmid is transformed into EHA105 competent cells, LB liquid culture is added and activated at 28°C on a shaker at 200 rpm for 4 h, then uniformly coated on solid LB medium containing 50 mg / L rifampicin and 50 mg / L spectinomycin for resistance screening, and cultured at 28°C for 48 h; positive single colonies are picked into LB liquid medium containing rifampicin and spectinomycin resistance, and cultured at 28°C on a shaker at 200 rpm overnight until the bacterial liquid is turbid, i.e. Agrobacterium carrying the recombinant plasmid is obtained. The Agrobacterium is stored in sterilized glycerol with a final concentration of 40% at -80°C, and is recorded as pH7WG2D-HXK1-EGFP.

[0048] (4) Agrobacterium culture and preparation of immersion liquid.

[0049] The above obtained and stored at -80°C pH7WG2D-HXK1-EGFP Agrobacterium EHA105 and the laboratory stored pH7WG2D Agrobacterium EHA105 are respectively coated on LB plates containing hygromycin and rifampicin, and cultured at 28°C for 48h, single colonies are picked and transferred into 1mL LB liquid medium containing spectinomycin and rifampicin, and cultured in a 28°C constant temperature incubator at a rotation speed of 200rpm for 12h, then liquid bacterial PCR detection is performed, the liquid bacteria with positive detection results are streaked, 40uL of liquid bacteria are taken to LB plates containing hygromycin and rifampicin, and cultured in a 28°C constant temperature incubator for 40h-48h, the liquid bacteria are washed with resuspension liquid, and finally 50mL of resuspension liquid is added to resuspend the bacterial bodies, which are adjusted to a liquid concentration OD 600 =0.6, and the Agrobacterium infiltration liquid is obtained for standby use. The resuspension liquid is 4.43g / L MS+30g / L sucrose+100mM acetosyringone, and the pH is 5.8.

[0050] (5) Callus infiltration, co-culture and selection culture.

[0051] The Agrobacterium infiltration liquid prepared in step (4) and the original Agrobacterium EHA105 infiltration liquid are respectively infiltrated with Jing39 jujube callus in a constant temperature shaker at 28°C and 200rpm in the dark for 30min, and the infiltrated Jing39 jujube callus is obtained.

[0052] The above obtained infiltrated Jing39 jujube callus is drained of the infiltration liquid on filter paper and placed in solid MS co-culture medium, and co-cultured in the dark for 3 days. The solid MS co-culture medium is 4.43g / L MS+1.0mg / L 2,4-dichlorophenoxyacetic acid+0.5mg / L thidiazuron+30g / L sucrose+8g / L agar+100mM acetosyringone, and the pH is 5.8.

[0053] (6) Selection culture

[0054] The Jing39 jujube callus obtained in step (5) is placed in solid MS selection medium (4.43g / L MS+1.0mg / L 2,4-dichlorophenoxyacetic acid+0.5mg / L thidiazuron+30g / L sucrose+8g / L agar+50mg / L hygromycin) for selection culture for 30d, the callus with fluorescent markers is separately placed in solid MS selection medium, and after 3 generations of selection culture, each generation for 3-4 weeks, the homozygous Jing39 jujube resistant callus is obtained, and the culture condition is: dark culture, temperature 26°C.

[0055] Figure 2Figure 1 shows the growth of Jingtai 39 anther callus and the related pictures of infection. With the help of fluorescence labeling screening, the genetic transformation rate of Jingtai 39 anther callus under this method was counted in this example. Specifically, a total of 234 dishes of Jingtai 39 anther callus were immersed and infected, and 130 dishes of transgenic Jingtai 39 anther callus with green fluorescence were obtained, accounting for 55.85%, i.e., the genetic transformation rate of transgenic Jingtai 39 anther callus was 55.85% (see Figure 3 , 78 dishes were a repeat, and there were three repeats in total).

[0056] Example 2

[0057] Using the Jingtai 39 genetic transformation system established in Example 1, the expression amount and function of the target gene were further verified and analyzed in this example, as follows:

[0058] (1) The Jingtai 39 resistant callus was detected by RT-PCR method, and the integration and expression of the target gene were determined according to whether there was a band of the target gene. Specifically, the Jingtai 39 callus positive callus after subculture for 15 days was uniformly sampled, and then RNA extraction, agarose gel electrophoresis and fluorescent quantitative PCR detection were performed.

[0059] ① According to the design of positive detection primer B of expression vector pH7WG2D-HXK1-EGFP, the positive detection primer B includes upstream primer B and downstream primer B, and the specific sequences are as follows:

[0060] Upstream primer B: 5'-gacgcacaatcccactatcc-3' (SEQ ID NO. 4);

[0061] Downstream primer B: 5'-tgtacaagaaagctgggtccccttaggattcctcaactccaag-3' (SEQ ID NO. 5).

[0062] The detection results based on the positive primer B are shown in Figure 4 .

[0063] ② According to the sequence of the target gene (candidate hexokinase gene, ZjHXK1 ), qPCR specific primer C was designed, which includes upstream primer C and downstream primer C, and the specific sequences are as follows:

[0064] Upstream primer C: 5'-cgccaccgaatgaagagc-3' (SEQ ID NO. 6);

[0065] Downstream primer C: 5'-gtctgccacctgtctgagtttac-3' (SEQ ID NO. 7).

[0066] The detection results based on primer C are shown in Figure 5As shown, the expression level of the hexokinase gene in transgenic jujube callus tissue increased sharply.

[0067] (2) Measurement of relevant physiological indicators.

[0068] To verify the target gene ( ZjHXK1 This example demonstrates the function of [missing information - likely a function or method], and measures 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 transgenic jujube callus was significantly reduced.

[0069] The above results demonstrate that the candidate hexokinase gene has the function of reducing sucrose, glucose and fructose content.

[0070] In summary, this invention successfully established a method for the genetic transformation of jujube 'Jing 39' using callus induced by the anthers of 'Jing 39' jujube as the recipient. Furthermore, this method is simple to operate, has a high transformation rate, and can be utilized with the help of… 35S::EGFP Fluorescent reporter gene markers enable visual screening, which is convenient and rapid. The system established in this invention provides a new approach for the functional analysis of jujube genes and the genetic improvement of jujube.

[0071] The above description is a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention. It should be noted that any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for genetic transformation of Zizyphus jujuba Mill. using anther induced callus as a recipient, characterized by, The jujube is Jing 39 jujube, and the genetic transformation method comprises the following steps: (i) culturing sterile anthers to obtain callus; (ii) constructing a plant recombinant overexpression vector containing a target gene and transferring the plant recombinant overexpression vector into Agrobacterium to obtain Agrobacterium immersion liquid; (iii) immersing the callus in the Agrobacterium immersion liquid, and then culturing the immersed callus under dark conditions; (iv) culturing the callus in (iii) in a screening medium to obtain homozygous resistant callus; The culturing mode in step (i) is that the sterile anthers are inoculated on an induction medium, cultured in dark at 26°C for 30 days, and then the callus obtained by induction is transferred to a proliferation medium for culture, and the light cycle is 16 / 8 hours, and the culture temperature is 26°C; wherein the induction medium is 4.43 g / L 1 / 2MS+1.0 mg / L 2,4-dichlorophenoxyacetic acid+0.5 mg / L thidiazuron+30 g / L sucrose+6.0 g / L agar; and the proliferation medium is 4.43 g / L MS+1.0 mg / L 2,4-dichlorophenoxyacetic acid+0.5 mg / L thidiazuron+30 g / L sucrose+8.0 g / L agar; The plant recombinant overexpression vector is a pH7WG2D plasmid containing a target gene, and the pH7WG2D plasmid carries a 35S promoter, an EGFP label, spectinomycin and hygromycin resistance genes; The culturing mode in step (iii) is that the callus drained of the Agrobacterium immersion liquid is placed on a solid MS co-culture medium and co-cultured in dark for 2-3 days; 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; The screening medium culture method in step (iv) is that the callus is cultured in a screening medium containing hygromycin; and 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.

2. The jujube genetic transformation method according to claim 1, characterized in that, The method further comprises the following step: (v) using the homozygous resistant callus to analyze the expression amount of the target gene and the gene function.

3. The method of claim 1, wherein the method is performed on a date palm (Phoenix dactylifera) plant. The sterile anthers are obtained by the following method: selecting a flower bud from a healthy and pest-free plant, sequentially treating the flower bud with alcohol and mercuric chloride, and then peeling off the petals.

4. The method of claim 1, wherein the method is performed on a date palm (Phoenix dactylifera). The Agrobacterium immersion liquid in step (ii) is composed of Agrobacterium carrying the plant recombinant overexpression vector and a resuspension liquid, and the resuspension liquid is 4.43 g / L MS+30 g / L sucrose+100 mM acetosyringone. ​ 5. The method of claim 1, wherein the method is performed on a date palm (Phoenix dactylifera). The method for obtaining the homozygous resistant callus in step (iv) is that the callus with fluorescent labeling is placed in a solid MS screening medium alone, and after 2-3 generations of screening culture, each generation for 3-4 weeks, the homozygous resistant callus is obtained.

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