CN118006681A - Wheat genetic transformation method - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering and transgenosis, and particularly relates to a wheat genetic transformation method. The invention successfully creates a genetic transformation method by taking the mature wheat embryo as an explant, and greatly improves the convenience and the working efficiency of the genetic transformation of wheat.

Description

Wheat genetic transformation method

Technical Field

The invention belongs to the technical field of genetic engineering and transgenosis, and particularly relates to a wheat genetic transformation method.

Background

The plant genetic transformation technology is a key element of the research and development of transgenic plant products. Agrobacterium-mediated methods have become the most widely used genetic transformation technique in plant transgenic research today.

Wheat is a heterohexaploid monocot plant, is not a natural host of agrobacterium, and has the problems of complex genome and difficult regeneration of explants, and low transformation efficiency in agrobacterium-mediated genetic transformation. The morphogenic genes Bbm/Wus2, GRF4/GIF1 and the like play an important role in improving the genetic transformation efficiency of plants. Chinese patent CN 114134173A discloses that the genetic transformation efficiency of wheat can be improved to 19.0-39.5% by using Bbm/Wus2 auxiliary vector.

However, the genetic transformation method described above uses wheat embryo as explant. Young embryos need to be harvested at specific time points (e.g., 10-15 days after pollination), and because the size of the young embryo is too small (about 1 mm) a highly skilled technician is required to strip the embryo from the seed, these procedures can greatly reduce the efficiency of genetic transformation, increase labor and material planting costs. Thus, those skilled in the art have long desired the ability to use mature embryos as explants for genetic transformation. However, since agrobacterium is extremely difficult to infect wheat mature embryos, there has been no efficient genetic transformation method based on mature embryos in the art.

Disclosure of Invention

In order to solve the problems, the invention provides a wheat efficient genetic transformation method based on mature embryo.

The specific technical scheme is as follows:

the invention provides a method for infecting mature wheat embryo by agrobacterium, which is characterized by comprising the following steps:

1) Obtaining agrobacterium expressing three proteins of GRF4/GIF1, bbm and Wus 2;

2) Infecting the mature embryo of the wheat subjected to the preculture by using the agrobacterium tumefaciens of 1), and selecting a mature embryo which is successfully infected;

in some embodiments, the agrobacterium in step 1) above is obtained by mixing the agrobacterium expressing GRF4/GIF1 with the agrobacterium expressing Bbm and Wus simultaneously in a ratio of 10:1.

In some embodiments, the GRF4/GIF1 protein sequence is shown in SEQ ID NO.1, the Bbm protein sequence is shown in SEQ ID NO.2, and the Wus protein sequence is shown in SEQ ID NO. 3.

In some embodiments, the GRF4/GIF1 protein is expressed by an expression cassette formed by sequentially connecting a UBI promoter, a GRF4/GIF1 nucleic acid molecule and a Nos terminator, the Bbm protein is expressed by an expression cassette formed by sequentially connecting a corn PLTP promoter, a corn Bbm gene and a tT28 terminator, and the Wus protein is expressed by an expression cassette formed by sequentially connecting a corn AXIG1 promoter, a corn Wus2 gene and a tIN2-1 terminator.

In some embodiments, the sequences of the UBI promoter, GRF4/GIF1 nucleic acid molecule, nos terminator, corn PLTP promoter, corn Bbm gene, tT28 terminator, corn AXIG1 promoter, corn Wus2 gene, tIN2-1 terminator are shown in SEQ ID NO. 4-SEQ ID NO.12, respectively. The promoters, genes and terminators mentioned are well known to those skilled in the art, and the sequences of the above elements can be adjusted on the basis of their effect equivalency according to the actual need.

In some embodiments, the preculture is performed using MS medium at 25-28℃for 1-3 days.

In some embodiments, the preculture described above is performed using MS medium at 25℃for 2 days.

The invention provides a genetic transformation method of a wheat mature embryo, which is characterized by further comprising the following steps on the basis of the method:

3) Carrying out heat shock treatment at 46 ℃ and ultrasonic treatment with 40% intensity on the mature embryo before infection, wherein the heat shock treatment time is between 30 and 40 minutes, and the ultrasonic treatment time is 6 minutes;

4) And (3) carrying out recovery culture, differentiation screening culture and rooting culture on the infected mature embryo until a positive transformation plant is obtained.

In some embodiments, the heat shock treatment described above is a 46 ℃ heat shock treatment for 40 minutes.

In some embodiments, the mature embryo at the time of the infection is from a seed that begins to germinate after preculture.

In some embodiments, the germinated seeds are seeds that begin to grow roots.

The invention has the advantages and beneficial effects as follows: the invention successfully realizes the wheat high-efficiency genetic transformation method based on mature embryo by using morphogenic genes and optimizing the operation links of preculture, heat shock, ultrasound and the like.

Drawings

FIG. 1 is a schematic diagram of a carrier structure.

Detailed Description

The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise indicated, terms are to be construed according to conventional usage by those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards, and other publications cited herein are incorporated by reference in their entirety.

As used herein, "wheat" is any wheat plant and includes all plant varieties that can be bred with wheat, including whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, whole plant cells in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like. Unless otherwise indicated, nucleic acids are written in the 5 'to 3' direction from left to right; the amino acid sequence is written in the amino to carboxyl direction from left to right. Amino acids may be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee. Likewise, nucleotides may be referred to by commonly accepted single letter codes. The numerical range includes the numbers defining the range.

The following examples are illustrative of the application and are not intended to limit the scope of the application. Modifications and substitutions to methods, procedures, or conditions of the present application without departing from the spirit and nature of the application are intended to be within the scope of the present application. Examples follow conventional experimental conditions, such as the molecular cloning laboratory manual of Sambrook et al (Sambrook J & Russell D W, molecular cloning: a laboratory manual, 2001), or conditions recommended by the manufacturer's instructions, unless otherwise indicated. Unless otherwise indicated, all chemical reagents used in the examples were conventional commercial reagents, and the technical means used in the examples were conventional means well known to those skilled in the art.

Examples

Example 1 transformation of wheat mature embryo Using Bbm/Wus2 morphogenic Gene

The inventors first attempted transformation of wheat mature embryos using the method disclosed in the CN 114134173A patent. Specific procedures reference is made to CN 114134173A patent example 4, except that the explant uses wheat mature embryo (receptor is Fielder): wheat seeds with full appearance and no obvious stains are selected, after sterilization, the mature embryo is taken out by a knife and forceps and immersed in a suspension culture medium, and the operation is the same as that of using the young embryo as an explant. As a result, it was found that the transformation efficiency of the method can reach 19.0% to 39.5% when a young embryo is used as an explant, but that positive plants can not be obtained at all when a mature embryo is used as an explant.

The inventor further optimizes the genetic transformation process, including adding a pre-culture link, and testing different heat shock time and ultrasonic treatment time during infection.

1) And adding a preculture link.

Wheat seeds with full appearance and no obvious stains are sterilized, then washed clean by sterile water, and the wheat seeds with the abdomen furrows facing downwards are placed in an MS culture medium for preculture. After the preculture is finished, removing the hypocotyl of the mature embryo by forceps, taking out the mature embryo by a knife and forceps, soaking for 10min by using agrobacterium tumefaciens bacteria liquid, uniformly spreading the mature embryo on a co-culture medium, drying the bacteria liquid, and carrying out dark culture for 3-4 days at 25 ℃ with scutellum facing downwards. Transferring to callus induction culture medium after co-culture, dark culturing at 28deg.C for 2-3 weeks to induce callus, and intermediate subculturing once. After the callus induction is finished, transferring the callus to a differentiation screening culture medium for culture, and regenerating plants. And finally transferring the regenerated seedlings to a rooting culture medium for culturing and hardening the seedlings.

Wherein, the pre-culture step uses different treatments such as culturing for 1-3 days, at 25 ℃ or 28 ℃ with or without light.

As a result, it was found that positive transformants were not obtained by various preculture treatments.

2) Different heat shock and sonication times were tested.

Many researches show that the short heat shock and ultrasonic treatment for 3-5min are helpful for the infection and transformation of agrobacterium, and the inventor further tests the heat shock (46 ℃) for 3min or 5min and the ultrasonic treatment (40% power) for 3min or 6min on the basis of adding a pre-culture link, so that the improvement measures can not realize the successful transformation of the mature wheat embryo.

This shows the difficulty of transformation of wheat mature embryos and even after the BBM/WUS2 morphogenic gene is used and the procedure is optimized, successful transformation is not achieved.

Example 2 further increasing morphogenic genes

Since GRF4/GIF1 was also a morphogenic gene, the inventors tried to further increase the GRF4/GIF1 gene on the basis of BBM/WUS2 to see if it could promote transformation of mature wheat embryo.

The inventors constructed two morphogenic gene-containing expression vectors (see FIG. 1 for vector structure) in which UBI:: GRF4/GIF1 and UBI:: GFP were expressed in tandem in p 203162. In p203163, the tandem expression of BBM and WUS2 was driven by specific promoters PLTP (promoter of phosphotransferase protein gene) and Axig1 (auxin-inducible promoter), respectively. The constructed vectors are respectively transformed into agrobacterium EHA105 strain for standby. The stored glycerol bacteria were activated on YP plates and streaked on AB plates the day before infection. The OD of the bacterial liquid at the infection is 0.3. At the time of infection, the p203162 bacterial liquid and the p203163 bacterial liquid are mixed in a ratio of 10:1. Due to the replacement of the vector protocol, young wheat embryos were transformed synchronously as parallel controls when the mature wheat embryos were transformed using the combination of p203162 and p203163 vectors.

Since p203162 contains GFP reporter gene, the inventors first compared the effect of different pretreatment conditions on the infection effect of mature embryos by fluorescence signal. For specific procedures, reference is made to example 1, and the fluorescence signal of the embryo is checked after the end of co-cultivation, and the results are shown in Table 1. Infection of mature embryos with Agrobacterium can be successfully achieved using a combination of GRF4/GIF1 and Bbm/Wus2 in combination with a pre-culture treatment. Wheat seeds are pre-cultured for 2 days at 25 ℃ before infection, and then infection is carried out, so that the instantaneous transformation efficiency of the vector is highest.

TABLE 1 infection effects of different preculture conditions on mature wheat embryo

Example 3 test on Heat shock and sonication

The inventors further tested the effect of different heat shock and sonication on the transformation effect of mature embryos using the vector combinations of p203162 and p 203163. In combination with the test results of example 2, this example uses heat shock and sonication of explants pre-incubated under light for 2d (25 ℃) with the explants immersed in the embryo collection solution.

Since heat shock times of 3-5min are optimal, too long time is likely to lead to death of the explant, and treatment test results in young embryos also confirm this conclusion. However, the inventors have not successfully achieved transformation after brief heat and sonication of mature embryo explants. Surprisingly, however, the mature embryo was transformed initially when the heat shock treatment time was increased to 30min and the sonication time was increased to 6min, the transformation being best when heat shock was applied for 40min and sonicated for 6min (see Table 2 for specific results).

TABLE 2 effect of pWMDR002 and p193412 vectors on Mixed rotation Zheng 58

The heat shock treatment temperature is 46 ℃; the ultrasound intensity was 40% power.

Example 4 Effect of seed germination status on transformation efficiency

Based on the test results, the inventor further tests the influence of different germination states of the mature embryo seeds on the transformation efficiency. After the mature embryo seeds are pre-cultured for 2 days under the light, the seeds can be in three states of long roots, sprouting dew white and non-sprouting, and the test shows that the non-sprouted seeds cannot realize transformation, the transformation efficiency of the seeds with sprouting rooting (long roots) is highest, the seeds with sprouting dew white can be transformed, but the transformation efficiency is lower. This helps the person skilled in the art to pick the seeds in the long root state after the end of the preculture for subsequent transformation operations, thus improving the working efficiency.

TABLE 3 conversion efficiency of seed in different germination states

Uniformly treating with 46 ℃ heat shock for 40min, and adding 40% power for ultrasonic treatment for 6min.

Example 5 wheat genetic transformation method based on mature embryo

Based on the test results, the specific steps of genetic transformation of the mature wheat embryo are summarized as follows:

1. selection of materials: selecting mature wheat seeds with full seeds and no obvious stains.

2. Explant preparation:

the first day: in the super clean bench, the selected seeds are transferred to a sterilized container. Sterilizing with 75% alcohol for 4-5min, and pouring out alcohol; cleaning with sterile water, adding sodium hypochlorite stock solution, sterilizing for 4-5min, and pouring out the solution; finally, the seeds are soaked in sterile water and stored in a 4-DEG refrigerator for standby after being washed for 4-5 times by the sterile water.

The following day: taking out mature wheat seeds soaked overnight, sterilizing with 75% alcohol for 45s, pouring out alcohol, washing with sterile water once, adding 10% sodium hypochlorite solution (for use in preparation), sterilizing for 15min, washing with sterile water for 4-5 times, placing immature seeds in MS culture medium with abdominal cavity facing downwards, and pre-culturing in light room at 25deg.C for 2d.

4. Preparing a fungus plate: after p203162 and p203163 plasmids are constructed, the EHA105 agrobacterium strain is transformed, and the glycerinum is preserved in an ultralow temperature refrigerator for standby. The p203162 and p203163 strains were activated on Kana-resistant YP plates two days before infection (one day of incubation at 28 ℃), and the cells on YP plates were scraped onto AB plates one day before infection and incubation was continued at 28 ℃.

5. Preparing bacterial liquid: scraping the bacteria in the fresh AB plate into embryo collection liquid, sucking and uniformly mixing, and adjusting the OD600 to 0.3. After mixing p203162 and p203163 in a ratio of 10:1 prior to infestation, 200. Mu.M AS and 0.05% silwet-L77 were added to the solution according to the volume of the solution.

6. Mature embryo explant acquisition: on the fourth day, the mature wheat seeds that have germinated long roots are removed and a scalpel and forceps (preferably, the fresh blades are sharpened) are prepared. The forceps are held by the left hand to fix seeds, the knife is held by the right hand, the embryo axis of the mature embryo is removed by the knife blade, and then the scutellum of the mature embryo is peeled off by the knife blade to the embryo collecting liquid. About 20 embryos per tube were placed.

7. Pretreatment before infection: after the embryo is peeled off, the embryo collecting liquid is replaced once, 200ul embryo collecting liquid is added, and the mature embryo is subjected to heat shock treatment at 46 ℃ for 40 minutes and ultrasonic treatment for 6 minutes.

8. Infection: sucking out the embryo collecting liquid in the super clean bench after the treatment, adding the bacterial liquid, and infecting for 10min at room temperature. After infection, pouring the mature embryo into a co-culture medium with filter paper placed in advance, sucking the redundant bacterial liquid by a 1mL gun after the whole culture medium is filled with the bacterial liquid, turning the embryo by forceps after drying the bacterial liquid, and blowing the embryo upwards until the surface of the scutellum is free of liquid. And (3) placing the culture dish at 23 ℃ for dark culture for 3-4 days after sealing by the paraffin sealing film.

9. Recovery culture: after the co-cultivation, the mature embryo scutellum was transferred to a callus induction medium (scutellum placed upwards) and dark cultivated at 28℃for 3 weeks. Intermediate subculture once.

10. Differentiation screening culture: transferring the callus after recovery culture into a differentiation screening culture medium, and carrying out dark culture screening at 28 ℃, wherein if green buds grow on the callus during screening, a culture dish is required to be transferred to a light room for culture. The green spots can be differentiated generally for about 1-2 weeks and transferred to the light chamber (at this time, all materials should be transferred to the light chamber, and later care should be taken to observe the callus differentiation).

11. Rooting: transferring the callus which is transferred to the light chamber for 1-2 weeks and has differentiated green spots into a new screening culture medium, arranging seedlings after the seedlings grow up, transferring the seedlings into a rooting culture medium, continuously culturing in the light chamber until complete plants grow, and then transplanting, detecting and hardening the seedlings.

The person skilled in the art can follow the above operation steps, on the premise of ensuring the important operation links disclosed in the invention

General applicability of the procedure of example 6

The present invention tested the transformation effect of other representative wheat varieties using the method of example 5. The results show that the method disclosed by the invention can realize high-efficiency genetic transformation of mature embryos of common wheat varieties such as Kenong, yangmai 158, jimai 22 and the like (Table 4), and shows that the universality of the method can overcome the barrier of genotype limitation of wheat.

TABLE 4Kenong199 conversion efficiencies

The heat shock treatment temperature is 46 ℃; the ultrasound intensity was 40% power.

TABLE 5Kenong199 transformation efficiency of Yangmai 158 and Jimai 22

The heat shock treatment temperature is 46 ℃; the ultrasound intensity was 40% power.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. A method for infecting mature wheat embryo by agrobacterium, comprising the following steps:

1) Obtaining agrobacterium expressing three proteins of GRF4/GIF1, bbm and Wus 2;

2) Infecting the mature embryo of the wheat subjected to the preculture with the agrobacterium of 1), and selecting the mature embryo which is successfully infected.

2. The method according to claim 1, wherein the agrobacterium of step 1) is obtained by mixing agrobacterium expressing GRF4/GIF1 with agrobacterium expressing Bbm and Wus simultaneously in a ratio of 10:1.

3. The method of any one of claims 1-2, wherein the GRF4/GIF1 protein sequence is shown in SEQ ID No.1, the Bbm protein sequence is shown in SEQ ID No.2, and the Wus protein sequence is shown in SEQ ID No. 3.

4. The method of claim 3, wherein the GRF4/GIF1 protein is expressed by an expression cassette comprising a UBI promoter, a GRF4/GIF1 nucleic acid molecule, and a Nos terminator, the Bbm protein is expressed by an expression cassette comprising a corn PLTP promoter, a corn Bbm gene, and a tT28 terminator, and the Wus2 protein is expressed by an expression cassette comprising a corn AXIG1 promoter, a corn Wus2 gene, and a tIN2-1 terminator.

5. The method of claim 4, wherein the UBI promoter, GRF4/GIF1 nucleic acid molecule, nos terminator, maize PLTP promoter, maize Bbm gene, tT28 terminator, maize AXIG1 promoter, maize Wus2 gene, tIN2-1 terminator are each shown in SEQ ID No.4 to SEQ ID No. 12.

6. The method according to claim 1, wherein the preculture is performed using an MS medium at 25 to 28℃for 1 to 3 days.

7. The method of claim 6, wherein the preculture is performed using MS medium at 25℃for 2 days.

8. A method for genetic transformation of mature wheat embryo, characterized by further comprising the steps of, on the basis of the method according to any one of claims 1-7:

3) Carrying out heat shock treatment at 46 ℃ and ultrasonic treatment with 40% intensity on the mature embryo before infection, wherein the heat shock treatment time is between 30 and 40 minutes, and the ultrasonic treatment time is 6 minutes;

4) And (3) carrying out recovery culture, differentiation screening culture and rooting culture on the infected mature embryo until a positive transformation plant is obtained.

9. The method of claim 8, wherein the heat shock treatment is a heat shock treatment at 46 ℃ for 40 minutes.

10. The method of claim 8, wherein the mature embryo at the time of infestation is from a seed that begins to germinate after preculture; optionally, the germinated seeds are seeds with long roots.