Soybean hairy root genetic transformation method

Abstract

The invention belongs to the technical field of biology, and particularly relates to a soybean hairy root genetic transformation method which comprises the following steps: taking soybean cotyledons as explants, infecting wounds of the explants by utilizing agrobacterium rhizogenes carrying reporter genes, transferring the infected explants into vermiculite, culturing, rooting, and screening positive roots. According to the method disclosed by the invention, a simple and efficient soybean hairy root genetic transformation system is established, tissue culture is not needed, and the problems of long transformation period, high cost and low transformation rate in the prior art are solved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a soybean hairy root genetic transformation method. Background Art

[0002] Soybean (Glycine max (L.) Merr.) is an important economic crop. Its seeds contain a large amount of unsaturated fatty acids, multiple trace elements, vitamins and high-quality proteins, providing an important source of nutrition for humans. Due to obstacles such as hybrid incompatibility and gene linkage effects between species, traditional breeding methods often find it difficult to optimize excellent gene combinations. In recent years, significant progress has been made in the fields of soybean genomics, genome-wide association studies (GWAS), and gene cloning. However, with the rapid development of biological technologies such as transgenic technology and gene editing, the functional annotation of a large number of candidate genes, the integration of exogenous genes into the soybean genome, and the creation of new soybean varieties through gene editing, the improvement of nutritional quality, and the increase in yield have become important technical means for soybean molecular breeding. However, to carry out the above technical research, a simple and efficient genetic transformation method is the primary condition.

[0003] In soybean, stable genetic transformation has low efficiency, long cycle and is restricted by genotype. The root hair transformation method mediated by Agrobacterium rhizogenes has been widely used in a variety of plants as an efficient instant tool in the fields of gene function research, gene editing development and other technical fields, including plants that are difficult to operate with traditional genetic transformation methods. At present, the root hair transformation methods reported in soybean include syringe injection of hypocotyls, hypocotyl cuttings, and sterile tissue culture technology. The needle injection method requires the use of a syringe to manually inject Agrobacterium rhizogenes into the hypocotyls of soybean seedlings. This method has low operating efficiency and low transformation efficiency, and is difficult to be widely used; the hypocotyl cutting technology cuts the hypocotyls of the seedlings and applies Agrobacterium on the cut surface for cutting. This method needs to ensure the survival of the seedlings after cutting. In addition, the positive rate of root hairs induced by a single seedling is low; the sterile tissue culture technology relies on a sterile environment and has high requirements for technical operations. The growth of root hairs requires the configuration of corresponding culture medium, and the transformation efficiency is not high. The technologies that have been reported have problems such as long conversion cycle, high cost and low conversion rate. Therefore, it is necessary to improve and innovate the above shortcomings. Summary of the invention

[0004] The purpose of the present invention is to provide a "easy-to-operate, high-efficiency, short-cycle" soybean hairy root genetic transformation method, break through the limitations of genotypes, expand the scope of application, so as to achieve genetic transformation of conventional cultivated varieties and difficult-to-transform germplasm, and apply molecular biology technologies such as gene editing to agricultural production.

[0005] The present invention adopts the following technical solution:

[0006] A soybean hairy root genetic transformation method uses soybean cotyledons as explants, uses rhizogenes carrying reporter genes and target genes to infect explant wounds, transfers the infected explants to vermiculite for culture, and screens positive roots after hairy roots are produced.

[0007] Preferably, the explant is a single cotyledon of a mature soybean seed after imbibition.

[0008] Preferably, the swelling time is 45-50 hours.

[0009] Preferably, the explant wound is formed by cutting off the proximal end of the cotyledon.

[0010] Preferably, the infection method is wound application infection.

[0011] Preferably, the infection is performed once.

[0012] Preferably, the reporter gene carried is green fluorescent protein.

[0013] Preferably, the Agrobacterium is Agrobacterium rhizogenes K599.

[0014] Preferably, the soybean variety is Heihe 43, Zhonghuang 301 or Chengdou 17.

[0015] Preferably, the positive root screening is performed using a LUYOR-3415 handheld dual-wavelength fluorescent protein excitation light source detector.

[0016] By implementing the above technical solution, the present invention has the following advantages:

[0017] The present invention establishes a non-tissue culture, simple and efficient soybean hairy root genetic transformation system. It has the following characteristics:

[0018] (1) The soybean materials used can be the conventional cultivars of my country such as Heihe 43, Zhonghuang 301, Chengdou 17, etc. The cotyledons of mature soybeans are used as explants. The swollen soybean seeds are peeled off, divided into two halves along the midline, and the proximal end of the cotyledons is removed. The explants are simple and easy to obtain;

[0019] (2) Cotyledons were selected as explants. Hairy roots were directly induced at the infected site of the cotyledons without the need for seedlings. Positive GFP signals could be observed 7 days after the seeds were soaked. A large number of positive hairy roots were produced in 12 days, with a positive rate of 90%-100%;

[0020] (3) Cultivation in vermiculite does not require a sterile environment, thus reducing contamination;

[0021] (4) No need for tissue culture, which simplifies the operation process, saves manpower and material resources, and reduces production costs;

[0022] (5) Spreading Agrobacterium K599 carrying the reporter gene on a solid culture plate for culture, directly spreading it on the cotyledon incision, without the need to culture the bacteria in liquid, prepare an infection solution, perform ultrasonic treatment, vacuumize, and a series of other currently commonly used Agrobacterium infection steps;

[0023] (6) Using GFP as a reporter gene and a handheld fluorescence detector, rapid observation and batch screening of positive hairy roots can be performed. Compared with reporter genes such as GUS, there is no need to take samples for chemical staining, which reduces the workload and saves time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a plasmid map containing the GFP reporter gene;

[0025] Figure 2 is a flow chart of the genetic transformation method of the present invention;

[0026] Figure 3 This is a picture of the positive hairy root test results;

[0027] Figure 4 PCR detection of the reporter gene GFP;

[0028] Figure 5 Analysis of transformation efficiency of different main cultivated varieties. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are described clearly and completely below through the accompanying drawings. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The experimental methods used in the following examples are all public conventional methods unless otherwise specified; the materials, reagents, instruments and equipment used are all commercially available unless otherwise specified.

[0031] Example 1

[0032] Construction of vector containing reporter gene GFP

[0033] The pCAMBIA3301 vector backbone was used and restriction endonucleases NcoI and Pml I from New England Biolabs were used for double digestion. The digested vector was separated by 1% agarose gel electrophoresis, and the target fragment was recovered and purified using a gel recovery kit from Novozymes. The concentration of the purified fragment was measured by Nanodrop nucleic acid quantitative analyzer and then used for later use. Specific primers were used to amplify the eGFP fragment from the pAN580 vector, and the recovered fragment was ligated to pCAMBIA3301 to form the recombinant plasmid pCAMBIA3301-GFP.

[0034] For the construction of vectors of other target genes, conventional molecular techniques can be used to construct the target genes into the pCAMBIA3301 vector.

[0035] Example 2

[0036] (1) Transformation of K599 Agrobacterium with pCAMBIA3301-GFP

[0037] The pCAMBIA3301-GFP plasmid DNA obtained in Example 1 was extracted and purified, and its concentration and purity were determined to ensure that it was suitable for subsequent transformation experiments. The pCAMBIA3301-GFP plasmid was mixed with K599 competent cells, placed on ice for 30 min, treated with liquid nitrogen for 45 s, and heat-shocked at 42°C for 5 min. The cells were transferred to ice for 3 min, 200 μL of LB liquid culture medium was added, and the cells were activated on a shaker at 28°C and 200 rpm for 20 min. The cells were then spread on a solid culture medium containing 50 mg / L of kanamycin and spectinomycin, and cultured at 28°C for 2 days. A single clone was picked, and 400 μL of the corresponding LB liquid culture medium was added after correct PCR identification. The cells were cultured on a shaker until OD600 = 0.8, 200 μL of 50% glycerol was added, and the cells were stored in a -80°C refrigerator for later use.

[0038] (2) Activation of recombinant Agrobacterium K599

[0039] Take out K599 Agrobacterium from the -80℃ refrigerator, add 5mL of liquid culture medium with corresponding resistance and culture until OD600=0.6, aspirate 600μL, spread on solid culture medium containing corresponding resistance, and culture overnight at 28℃ until the bacteria cover the entire solid culture medium.

[0040] Example 3

[0041] (1) Treatment of soybean explants, infection with Agrobacterium, and observation of results

[0042] 50 full soybean seeds (Heihe 43, Zhonghuang 301, Chengdou 17) were selected and placed in a 250 ml triangular flask, and an appropriate amount of tap water was added to immerse the seeds. The seeds were left standing overnight at room temperature. The tap water in the bottle was poured out, and the seeds were spread on a culture dish containing filter paper and cultured at room temperature for 24 h. The seed coat was peeled off, the two cotyledons were separated along the midline, the proximal part of the cotyledons was cut off, and the Agrobacterium on the solid culture medium in Example 2 was inoculated at the wound using an inoculation loop or a 10 μL gun tip. The cotyledons were inserted into vermiculite and cultured in the dark for 2 days, and then transferred to light for culture. The culture parameters were as follows: light time 12 / d, intensity 800 μmol m -2 s -1 , the temperature is 25℃, and a moisturizing, transparent culture box is used to ensure the survival rate.

[0043] (2) Observation of transgenic positive hairy roots

[0044] Five days after sowing, the infected cotyledons were taken out and the swollen callus and induced hairy roots were visible to the naked eye. Using the LUYOR-3415 handheld dual-wavelength fluorescent protein excitation light source detector and wearing special fluorescence observation glasses, the positive fluorescent signal could be observed. After 10 days of continued growth, a large number of positive hairy roots induced by the cotyledons could be observed.

[0045] Example 4

[0046] PCR Identification of Transgenic Positive Hairy Roots in Example 3

[0047] The DNA of hairy roots was extracted by CTAB method. The specific experimental method is as follows: Place the hairy roots in a 2mL centrifuge tube, add 2 sterilized steel beads, freeze in liquid nitrogen, and grind them using a ball mill. Add 800μL CTAB extraction buffer, and place in a 65℃ water bath for 1 hour. During this period, invert and mix several times every 15 minutes. The formula of CTAB extraction buffer is: 2% (M / V) CTAB, 1.4M NaCl, 100mM Tris-Cl (pH 8.0), 20mM EDTA, 2% (V / V) β-mercaptoethanol (added before use). Incubate at 65℃ for 30 minutes, and gently shake and mix every 3-4 minutes. Then centrifuge at low temperature and 12000rpm for 5 minutes, and transfer the supernatant to a new centrifuge tube. Add an equal volume of 24:1 chloroform / isoamyl alcohol extraction homogenate, invert to mix thoroughly, centrifuge at 4℃ and 7500g for 5 minutes, and recover the supernatant. The recovered supernatant was extracted with an equal volume of chloroform / isoamyl alcohol, mixed, centrifuged, and the supernatant was recovered. 0.6 volumes of isopropanol were added, mixed thoroughly, and centrifuged at 4°C and 7500g for 15 minutes. The precipitate was washed with 80% ethanol, dried, and resuspended with TE (0.1-0.5 mL per gram of starting material). The GFP marker gene was PCR amplified using primers (GFP-F: 5'-CCAGCTTGTGCCCCAGG-3'; GFP-R: 5'-GTGTCCGGCGAGGGCG-3'). The reaction system was as follows: 2×TaqMaster Mix 5μL, forward primer (10μM) 2μL, reverse primer (10μM) 2μL, DNA template 1μL.

[0048] PCR reaction procedure: (1) 98℃5min; (2) 98℃15s, 55℃15s, 72℃30s, 30-35 cycles; (3) 72℃10min; (4) 4℃1h. The results showed that the hairy roots with fluorescent signals could amplify a 340bp band, while the hairy roots without fluorescent signals could not amplify a band of the same size, which further confirmed the positive hairy roots.

[0049] After calculation, the corresponding conversion rates of Heihe 43, Zhonghuang 301 and Chengdou 17 soybean varieties were 96%, 92% and 91% respectively.

[0050] Example 5

[0051] Analysis of transformation efficiency of soybean hairy roots from different conventional varieties

[0052] At present, genetic transformation of soybean has only been successfully achieved in a limited number of varieties, and conventional cultivars that are difficult to transform still face great challenges. This example uses Heihe 43, Zhonghuang 301, and Chengdou 17, conventional cultivars with large domestic planting areas, as research materials, and uses the same method described in Examples 1-4 to conduct hairy root transformation experiments on their cotyledons. The results show that the transformation rates of Heihe 43, Zhonghuang 301, and Chengdou 17 are as high as 96%, 92%, and 91%, respectively. This further proves that the present invention has great promotion potential in terms of improving conventional varieties using molecular biology techniques.

[0053] The above results consistently show that it is feasible to use K599 Agrobacterium without tissue culture and natural conditions, using the cotyledons of conventional soybean cultivars that are difficult to transform as explants, and through simple technical operations, to obtain a large number of positive hairy roots in only 12 days. The present invention selects conventional cultivars that are difficult to transform, expands the research scope, and improves the application value; shortens the transformation time to 12 days, which is the fastest genetic transformation method reported so far; increases the transformation rate to more than 90%; simplifies the test process, does not require a culture medium, and can be cultured in vermiculite, reducing production costs; uses GFP fluorescent protein as a label, and can quickly and efficiently screen positive hairy roots.

[0054] Comparative Example

[0055] The difference from Example 3 is that the inoculation method of Agrobacterium is different. The method of using the Agrobacterium infection liquid for infection in the comparative example of the present invention is to inoculate the Agrobacterium in Example 2 at the wound. The specific operation is: add 200 μL LB liquid culture medium to the Agrobacterium on the solid culture medium in Example 2, activate it on a shaker at 28°C and 200 rpm for 20 minutes, apply it on a solid culture medium containing 50 mg / L kanamycin and spectinomycin, culture it at 28°C for 2 days, pick a single clone, add 400 μL of the corresponding LB liquid culture medium after PCR identification is correct, and culture it on a shaker until OD600=0.6, which is used as the infection liquid for standby use.

[0056] The treated soybean explants were placed in the infection solution for infection. The infection conditions were as follows: temperature 25°C, standing for 30 minutes, and cultured in vermiculite.

[0057] The conversion rates of soybeans in the coating method in the embodiment and the impregnation liquid method in the comparative example were counted respectively. The results are shown in Figure 5 The results show that whether it is Heihe 43, Zhonghuang 301 or Chengdou 17, the coating method has a higher conversion rate than the dyeing liquid method.

Claims

1. A method for genetic transformation of soybean hairy roots, characterized in that: Soybean cotyledons were used as explants, and Agrobacterium rhizogenes carrying a reporter gene was used to infect the wounds of the explants. The infected explants were transferred to vermiculite for culture, and positive roots were screened after roots were generated.

2. A soybean hairy root genetic transformation method according to claim 1, characterized in that: The explant is a single cotyledon of mature soybean seeds after imbibition.

3. A soybean hairy root genetic transformation method according to claim 2, characterized in that: The swelling time is 45-50h.

4. A soybean hairy root genetic transformation method according to claim 2, characterized in that: The explant wound is formed by excising the proximal end of the cotyledon.

5. A soybean hairy root genetic transformation method according to claim 1, characterized in that: The infection method is wound spreading infection.

6. A soybean hairy root genetic transformation method according to claim 1, characterized in that: The number of times of infection is 1 time.

7. A soybean hairy root genetic transformation method according to claim 1, characterized in that: The reporter gene carried is green fluorescent protein.

8. A soybean hairy root genetic transformation method according to claim 1, characterized in that: The Agrobacterium is Agrobacterium rhizogenes K599.

9. A soybean hairy root genetic transformation method according to claim 1, characterized in that: The soybean variety is Heihe 43, Zhonghuang 301 or Chengdou 17.

10. A soybean hairy root genetic transformation method according to claim 1, characterized in that: The positive root screening was performed using a LUYOR-3415 handheld dual-wavelength fluorescent protein excitation light source detector.

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