Agrobacterium rhizogenes-mediated papaya hairy root genetic transformation method and papaya transgenic positive plant

Abstract

The invention provides an agrobacterium rhizogenes-mediated papaya hairy root genetic transformation method and a papaya transgenic positive plant, and belongs to the technical field of plant genetic engineering. The invention provides a papaya genetic transformation method independent of tissue culture, which can directly utilize agrobacterium rhizogenes to treat explants for culture to obtain papaya transgenic positive plants without a series of tedious and complicated sterile processes such as callus induction and induced differentiation rooting. The problems of long tissue culture transformation period, large manpower and material resource consumption, low positive rate and the like are avoided. The constructed agrobacterium rhizogenes mediated genetic transformation system has the characteristics of high efficiency, stability and rapidness, the agrobacterium rhizogenes K599 cultured by using the solid TY culture medium can directly infect wounds, the operation is simple, the hair root incidence rate and the positive rate are relatively high, additional experimental equipment is not needed, and the method is suitable for most laboratories.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a papaya hairy root genetic transformation method mediated by Agrobacterium rhizogenes and a papaya transgenic male plant. Background Art

[0002] Papaya (Caricapapaya L.) is native to tropical America, belongs to the family Caricapa, the genus Carica, and is a perennial herb. Papaya in my country is mainly distributed in Guangdong, Guangxi Zhuang Autonomous Region, Hainan, Yunnan, Fujian, Taiwan and other places. It is listed as the "three major tropical herbaceous fruit trees" along with bananas and pineapples. Papaya fruits are rich in carbohydrates, proteins, minerals, vitamins, carotene, papain, papain, rennet, etc.; seeds are rich in benzyl isothiocyanate and papaya; leaves contain papain and papaya, choline, etc., which have extremely high medicinal and edible value and are widely used in food processing, medicine and health, beauty and health care and other industries. Therefore, it has the reputation of "Longevity Fruit", "King of Hundred Benefits Fruit", and "Lingnan Good Fruit" in southern my country. And papaya fruits, seeds and leaves can be used as medicine. Its fruit contains papain, papain and other ingredients, which have the effects of strengthening the spleen and digestion, fighting epidemics and killing insects, promoting lactation and anti-cancer. The seeds contain 25% fatty oil and can be pressed for oil. The latex of green fruit is rich in papain, which has the function of digesting protein and is widely used in industry.

[0003] Genetic transformation is an important tool for gene editing and genetic improvement of fruit trees. Agrobacterium-mediated genetic transformation is a technology that uses the natural genetic transformation system of Agrobacterium to introduce foreign genes into plant cells and integrate them into their genomes. It is also a common method for obtaining transgenic plants in papaya and other fruit trees. Papaya callus is obtained by tissue culture, and foreign genes are transferred to the callus through Agrobacterium-mediated transfer. Transgenic papaya plants can be obtained by sustainable aseptic culture. However, this method has problems such as long transformation cycle, low positive rate, and complex operation, and it is difficult to save once contaminated during tissue culture. After infecting plants, Agrobacterium rhizogenes can induce plants to produce a large number of highly branched adventitious roots, and its mediated genetic transformation system has the characteristics of high efficiency, stability, and rapidity. Therefore, the development of a new Agrobacterium rhizogenes-mediated papaya hairy root genetic transformation method is of great significance to papaya transgenic breeding. Summary of the invention

[0004] The purpose of the present invention is to provide a papaya hairy root genetic transformation method mediated by Agrobacterium rhizogenes and a papaya transgenic positive plant, which can quickly obtain papaya transgenic materials without going through a series of tedious and complicated aseptic processes such as inducing callus tissue and inducing differentiation and rooting, thereby avoiding the problems of long tissue culture transformation cycle, large consumption of manpower and material resources, and low positive rate.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for genetic transformation of papaya hairy roots mediated by Agrobacterium rhizogenes, comprising the following steps:

[0007] (1) Using the liquid nitrogen freeze-thaw method, the vector carrying the RUBY marker gene was transferred into the competent cells of Agrobacterium rhizogenes, and single colonies were obtained by streaking culture in antibiotic TY solid medium. The single colony was cultured and activated on a shaker in antibiotic TY liquid medium, and then inoculated on antibiotic TY solid medium;

[0008] (2) Explants were cut from papaya seedlings, and the incision was dipped into solid TY medium containing bacterial cells to obtain bacterial cells, and then placed in a tissue culture box for culture to obtain transgenic positive seedlings.

[0009] Preferably, the Agrobacterium rhizogenes in step (1) is K599.

[0010] Preferably, the antibiotics in step (1) are kanamycin and streptomycin, both at a concentration of 40-60 mg / L, and the TY liquid culture medium comprises 5 g / L tryptone, 3 g / L yeast extract, and 1 M calcium chloride aqueous solution; the TY solid culture medium comprises 5 g / L tryptone, 3 g / L yeast extract, 1 M calcium chloride aqueous solution, and agar.

[0011] Preferably, the papaya seedlings in step (2) are seedlings that are 10 to 20 days old from when the seeds turn white to when they are planted in the soil, and the explant is one of a cotyledon, a true leaf, and a stem.

[0012] Preferably, the tissue culture box in step (2) is prepared from vermiculite and water in a mass ratio of 1:1 to 3.

[0013] Preferably, the culture temperature in step (2) is 28-30° C., the culture time is 25-35 days, and the lighting conditions are: 16 hours of light and 8 hours of darkness.

[0014] The invention also provides a papaya transgenic positive plant obtained by a papaya hairy root genetic transformation method mediated by Agrobacterium rhizogenes.

[0015] The beneficial effects of the present invention compared with the prior art are:

[0016] The invention provides a papaya genetic transformation method that does not rely on tissue culture. The method can directly utilize Agrobacterium rhizogenes to treat explants for culture to obtain papaya transgenic positive plants without going through a series of tedious and complicated aseptic processes such as inducing callus tissue and inducing differentiation and rooting, thereby avoiding the problems of long tissue culture transformation cycle, large consumption of manpower and material resources, low positive rate, etc.

[0017] The Agrobacterium rhizogenes-mediated genetic transformation system constructed by the present invention has the characteristics of high efficiency, stability and rapidity. The Agrobacterium rhizogenes K599 cultured in solid TY medium can directly infect wounds. The operation is simple, the incidence rate and positive rate of hairy roots are high, and no additional experimental equipment is required, so the system is suitable for most laboratories.

[0018] By using the RUBY marker gene, positive roots can be seen with the naked eye, without the need for PCR testing, saving the time for DNA verification, and is suitable for large-scale transgenic identification work. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 The figure is a schematic diagram of the transformation process of papaya in the present invention (taking true leaves as an example); wherein A is a 20-day-old papaya seedling; B is Agrobacterium rhizogenes K599 activated on TY medium; C is a pretreated true leaf; D is a papaya true leaf infected with Agrobacterium rhizogenes K599; E is the induction of hairy root formation by co-cultivation in the greenhouse; F is the positive hairy root formed by observation under body posture;

[0021] Figure 2 This is a picture of the positive roots of papaya in Example 1 of the present invention; A is the hairy root of papaya cotyledon, B is the hairy root of papaya true leaf, and C is the hairy root of papaya stem

[0022] Figure 3 The effect of tissues of different parts of papaya on the transformation efficiency of papaya in Example 1 of the present invention; wherein A is the hairy root incidence rate; B is the positive rate;

[0023] Figure 4 The figure shows the effect of papaya seedlings at different stages on the transformation efficiency of papaya in Experimental Example 1 of the present invention; wherein A is the hairy root incidence rate; and B is the positive rate. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The present invention provides a method for genetic transformation of papaya hairy roots mediated by Agrobacterium rhizogenes (see Figure 1 ), comprising the following steps:

[0030] (1) Using liquid nitrogen freeze-thaw method, the vector carrying RUBY marker gene is transferred into Agrobacterium rhizogenes competent cells, streaked in antibiotic TY solid medium to obtain single colonies, and the single colonies are cultured and activated on a shaker in antibiotic TY liquid medium, and then inoculated in antibiotic TY solid medium; wherein the antibiotics are kanamycin and streptomycin, and the concentrations are both 40-60 mg / L, and the TY liquid medium includes 5 g / L tryptone, 3 g / L yeast extract, and 1M calcium chloride aqueous solution; the TY solid medium includes 5 g / L tryptone, 3 g / L yeast extract, 1M calcium chloride aqueous solution, and agar.

[0031] (2) Explants were cut from papaya seedlings, and the incision was dipped into solid TY medium containing bacterial cells to obtain bacterial cells, and then placed in a tissue culture box for culture to obtain transgenic positive seedlings.

[0032] Among them, the papaya variety is medium white.

[0033] The papaya seedlings are seedlings that are planted in the soil for 10-20 days from the time the seeds turn white. The explants are one of cotyledons, true leaves, and stems. The cotyledons are directly cut from the stems using a sharp knife to obtain the experimental explants; the true leaves are cut off from half of the leaves to obtain the experimental explants; the stems are removed of excess leaves and buds, leaving the young and tender leaves at the top, and the experimental explants are obtained by obliquely cutting at 45 degrees from 1 cm from the top.

[0034] The tissue culture box is prepared from vermiculite and water in a mass ratio of 1:1 to 3, more preferably 1:2.

[0035] The culture temperature is 28-30° C., more preferably 28° C.; the culture time is 25-35 days, more preferably 30 days; and the lighting conditions are: 16 hours of light and 8 hours of darkness.

[0036] Preparation of TY liquid culture medium: 5 g / L tryptone, 3 g / L yeast extract, sterilized at 121°C for 20 minutes; prepare 1M calcium chloride aqueous solution, sterilized at 121°C for 20 minutes; add 10 mL calcium chloride aqueous solution to each 1 L sterilized TY liquid nutrient solution to obtain TY liquid culture medium, add 50 mg / L kanamycin and 50 mg / L streptomycin and mix;

[0037] Preparation of TY solid medium: Add 15 g of agar powder to TY liquid medium;

[0038] Example 1

[0039] Example 1 of the present invention provides a method for genetic transformation of papaya hairy roots mediated by Agrobacterium rhizogenes, and detects the effects of tissues in different parts of papaya on the transformation efficiency of papaya. The specific steps are as follows:

[0040] (1) Using the liquid nitrogen freeze-thaw method, the vector carrying the RUBY marker gene was transferred into the competent cells of Agrobacterium rhizogenes K599, centrifuged at 5000 rpm, 100 μL of the bacterial solution was streaked on a solid TY medium containing 50 mg / L kanamycin and 50 mg / L streptomycin, and cultured at 28°C for 36-48 h to obtain a single colony; a single colony was picked and placed in a liquid TY medium containing 50 mg / L kanamycin and 50 mg / L streptomycin, and activated by shaking culture at 28°C and 200 rpm for 12-16 h, and 50% glycerol was added according to a volume ratio of glycerol: bacterial solution = 1:1, and stored at -80°C for use; the bacterial solution after shaking culture activation was aspirated and applied to a solid TY medium containing 50 mg / L kanamycin and 50 mg / L streptomycin, and cultured at 28°C overnight;

[0041] (2) Select papaya seedlings that are 10-20 days old from the time the seeds turn white to the time they are planted in the soil as experimental donors; select papaya cotyledons, true leaves, and stems as explants, of which the cotyledons are taken from 10-day-old papaya seedlings, and the true leaves and stems are taken from 20-day-old papaya seedlings. The cotyledons are directly cut from the stems using a sharp knife to obtain the experimental explants; the true leaves are cut off from half of the leaves to obtain the experimental explants; the stems are removed of excess leaves and buds, leaving the young and tender leaves at the top, and cut at a 45° angle from 1 cm from the top to obtain the experimental explants.

[0042] (3) Dip the explant cut into the solid TY medium containing the bacteria to pick up the bacteria, then insert the explant into a tissue culture box with air holes containing 50 g of vermiculite and 100 mL of water, cover it with a lid, and place it in a greenhouse at 28°C with 16 h of light and 8 h of darkness per day for 30 days to obtain transgenic positive seedlings;

[0043] (4) The explants cultured in moist vermiculite for 30 days were taken out, the surface vermiculite was removed, and bright rose-red hairy roots were observed with the naked eye, i.e., transgenic positive seedlings. The hairy root incidence of different explants was tested. The results were as follows: Figure 2 , 3 shown.

[0044] Figure 3 The results showed that the incidence of hairy roots in cotyledons was 60.42% and the positive rate was 23.28%; the incidence of hairy roots in true leaves was 81.25% and the positive rate was 23.08%; the incidence of hairy roots in stems was 81.25% and the positive rate was 1.92%. It can be seen that the positive rate of stems was the lowest, and the positive rates of cotyledons and true leaves were both good, but the incidence of hairy roots in cotyledons was lower, so true leaves were the best choice for genetic transformation of papaya hairy roots.

[0045] Transformation with a vector carrying the RUBY marker gene avoids the tedious process of traditional DNA / RNA extraction and identification.

[0046] Test Example 1

[0047] Test Example 1 of the present invention detected the effect of papaya seedlings at different stages on the transformation efficiency of papaya, and the specific steps were as follows:

[0048] As shown in Example 1, the positive rates of cotyledons and true leaves are not much different, and the hairy root incidence rate of true leaves is higher. Therefore, in order to study the difference in genetic transformation efficiency of papaya seedlings at different stages, papaya medium white varieties were selected from the time when the seeds were white to the time when they were planted in the soil for 10d, 15d, 20d, 30d, and 45d, and their true leaves were taken, and half of the leaves were cut off to obtain experimental explants;

[0049] The method of Example 1 was used to cultivate transgenic positive seedlings, and the effect of papaya seedlings at different stages on the transformation efficiency of papaya was detected. The experimental results are as follows: Figure 4 shown.

[0050] Figure 4 The results showed that the incidence rate and positive rate of hairy roots of 20-day-old true leaves were the best compared with those at other periods, so 20-day-old true leaves were the best choice as experimental explants.

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for genetic transformation of papaya hairy roots mediated by Agrobacterium rhizogenes, characterized in that: The steps include: (1) Using the liquid nitrogen freeze-thaw method, the vector carrying the RUBY marker gene was transferred into the competent cells of Agrobacterium rhizogenes, and single colonies were obtained by streaking culture in antibiotic TY solid medium. The single colony was cultured and activated on a shaker in antibiotic TY liquid medium, and then inoculated on antibiotic TY solid medium; (2) Explants were cut from papaya seedlings, and the incision was dipped into solid TY medium containing bacterial cells to obtain bacterial cells, and then placed in a tissue culture box for culture to obtain transgenic positive seedlings.

2. The method for genetic transformation of papaya hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The Agrobacterium rhizogenes described in step (1) is K599.

3. The method for genetic transformation of papaya hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The antibiotics in step (1) are kanamycin and streptomycin, both at a concentration of 40-60 mg / L. The TY liquid culture medium comprises 5 g / L tryptone, 3 g / L yeast extract, and 1 M calcium chloride aqueous solution; the TY solid culture medium comprises 5 g / L tryptone, 3 g / L yeast extract, 1 M calcium chloride aqueous solution, and agar.

4. The method for genetic transformation of papaya hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The papaya seedlings in step (2) are seedlings that are 10 to 20 days old from when the seeds turn white to when they are planted in the soil, and the explant is one of cotyledons, true leaves, and stems.

5. The method for genetic transformation of papaya hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The tissue culture box in step (2) is prepared from vermiculite and water in a mass ratio of 1:1 to 3.

6. The method for genetic transformation of papaya hairy roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The culture temperature in step (2) is 28-30° C., the culture time is 25-35 days, and the lighting conditions are: 16 hours of light and 8 hours of darkness.

7. A papaya transgenic positive plant obtained by the papaya hairy root genetic transformation method mediated by Agrobacterium rhizogenes according to any one of claims 1 to 6.

Images