CN113832178A

CN113832178A - A method for establishing a genetic transformation system of vegetable pea mediated by Agrobacterium rhizogenes

Info

Publication number: CN113832178A
Application number: CN202111048944.2A
Authority: CN
Inventors: 冯志娟; 刘娜; 卜远鹏; 张古文; 王斌; 龚亚明
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2021-09-08
Filing date: 2021-09-08
Publication date: 2021-12-24

Abstract

The invention discloses a method for establishing a vegetable pea genetic transformation system mediated by Agrobacterium rhizogenes, comprising the following steps: 1) explant preparation; 2) cutting root infection; 3) hairy root formation. The invention adopts the mediation of Agrobacterium rhizogenes as the mediator to induce the formation of hairy roots of vegetable peas by one-step method. The convenience of experimental operation.

Description

Method for establishing agrobacterium rhizogenes-mediated vegetable pea genetic transformation system

Technical Field

The invention belongs to the technical field of plant genetic engineering, belongs to a method for establishing an agrobacterium rhizogenes-mediated efficient genetic transformation system for vegetable peas, and particularly relates to a method for establishing an agrobacterium rhizogenes-mediated efficient genetic transformation system for vegetable peas by a one-step method.

Background

Peas (pisum sativum L.) are the second largest edible legume crop in the world and can be classified into grain peas (dry peas), vegetable peas with edible tender bean granules (sweet peas, green peas) and vegetable peas with edible tender pods (sweet peas) according to their purposes. The total yield of green peas in China is the first in the world and plays an important role in the production of peas in the world. Peas have nitrogen fixation capacity, are environment-friendly crops, have the effects of losing weight, improving the efficiency, improving the soil and the like, and play an important role in the sustainable development agricultural system in China. In 2019 french scholars reported that the first annotated pea reference genome at the chromosome level, with a genome size of about 4.45Gb, consisting of 7 pairs of homologous chromosomes (2 n-2 x-14), would accelerate the exploitation and genetic improvement of functional genes.

The transgenic technology is an important means for researching plant gene function and mechanism of action, and is one of ways for character improvement, and the most widely applied transgenic method at present is mainly an agrobacterium-mediated method, including an agrobacterium tumefaciens-mediated method and an agrobacterium rhizogenes-mediated method. The reported transgenic transformation period of peas and other beans mediated by agrobacterium tumefaciens is long, the pollution rate is high, the transformation rate is low, the gene stability transformation difficulty is high, and therefore the strategy of forming hairy roots carrying target genes by utilizing agrobacterium rhizogenes induction is widely applied to leguminous crops such as soybeans, kidney beans and the like. The general principle of the technology is to use agrobacterium rhizogenes to induce the underground part of a plant to form transgenic hairy roots, so as to obtain composite seedlings (composite plants), wherein the underground part is a transformant, and the overground part is a non-transformant. The agrobacterium rhizogenes mediated method comprises a two-step method and a one-step method. It has been reported that transgenic hairy root combined seedlings are obtained by agrobacterium rhizogenes mediated pea hairy root genetic transformation technology, as shown in the attached specification, figure 1(Irina et al, 2019), and the method belongs to a two-step method. Compared with a two-step method, the one-step method further shortens the conversion period, further reduces the pollution rate and improves the convenience of experimental operation. One-step strategies are currently reported only on pears and soybeans (Mandal et al, 2019; Fan et al, 2020), and no relevant report has been found on peas. In addition, the cotyledons do not come out during the germination growth process of the pea seeds, unlike the cotyledons coming out during the germination growth process of the soybean seeds. Therefore, it is necessary to establish an efficient hairy root transgenic system for vegetable peas, and the efficient hairy root transgenic system is simple and convenient to operate, low in cost, high in transformation efficiency, good in repeatability, short in test period and low in pollution rate. The invention aims to establish a method for establishing a one-step agrobacterium rhizogenes-mediated efficient genetic transformation system for vegetable peas, and optimally combines the optimal cutting position, seedling age and culture time with higher transformation rate and rooting rate, so as to further save the experiment cost, shorten the experiment period, reduce the pollution rate and improve the convenience of experiment operation.

Disclosure of Invention

Aiming at the defects of the conventional two-step method pea hairy root genetic transformation system and the problems that the conventional system does not optimize and combine the hairy root rooting rate and the positive rate of vegetable peas, the optimal cutting position, the seedling age and the culture time, and the like, the invention provides a method for establishing an agrobacterium rhizogenes-mediated vegetable pea high-efficiency genetic transformation system by one-step method through inducing pea hairy root genetic transformation and optimizing and combining the cutting position, the seedling age and the culture time, and provides technical support for the functional genome research of vegetable peas.

In order to achieve the purpose, the invention adopts the following technical scheme: a method for establishing an agrobacterium rhizogenes-mediated vegetable pea genetic transformation system comprises the following steps:

1) obtaining explants, namely performing disinfection and aseptic treatment on vegetable pea seeds, sowing the seeds in a matrix, thoroughly pouring sterile water, and culturing the seeds in an illumination incubator; after 5-9 days of growth, cutting off the plant at the upper end or the lower end of the cotyledonary node of the plant along an angle of 45 degrees obliquely downwards to obtain an explant to be infected by the overground part;

2) infection: wrapping the wound generated by cutting the explant to be infected with bacteria by using bacteria paste, wherein the used bacteria is agrobacterium rhizogenes AR 1193; then, planting the explant wrapped with the bacteria on a substrate, and adding 5ml of liquid culture medium containing agrobacterium rhizogenes AR 1193;

3) hairy root induction and combined seedling formation: culturing the infected explants in dark for 24 h; then transferring to an illumination incubator for culturing, sealing and moisturizing by using a plastic cover in the whole process, and culturing for 10-18 days; then continuing to culture for two weeks, opening the vent holes on the plastic cover two days in advance to slowly reduce the humidity, and then completely removing the plastic cover to obtain the combined seedlings which grow vigorously. Where it is used to promote the production of hairy roots.

Preferably, the upper end of the cotyledon node of the plant in the step 1 refers to the upper end of the junction of the cotyledon of the plant; the lower end refers to the upper end close to the junction of the hypocotyl of the plant.

Preferably, the growth time in the step 1 is preferably 7 days; the culture time in the step 3) is preferably 18 days.

Preferably, the matrix in

step

1 or 2 is prepared by the following method: mixing vermiculite and turf according to a mass ratio of 3 to 1, and autoclaving at 121 ℃ for 20 min.

Preferably, the agrobacterium rhizogenes AR1193 in the step 2 carries a binary expression vector pCAMBIA3301 containing GUS reporter gene.

Preferably, the mush obtained in the step 2 is scraped from TY solid culture medium of agrobacterium rhizogenes AR 1193; the TY solid culture medium of the agrobacterium rhizogenes AR1193 is prepared by the following method: taking 200 mu L of agrobacterium rhizogenes strain AR1193 glycerol bacteria, streaking and coating the strains on a TY solid culture medium, and carrying out inverted culture in a constant temperature incubator at 28 ℃ for 48 h; wherein the TY solid culture medium contains 50mg/L Kan.

Preferably, the liquid medium containing agrobacterium rhizogenes AR1193 in step 2 is prepared by the following method: inoculating 10 mu L of Agrobacterium rhizogenes strain AR1193 glycerol strain to 1mL of TY liquid culture medium, and culturing for 12h in a constant temperature incubator at 28 ℃ at the rotating speed of 180 rpm; inoculating 200 mu L of agrobacterium rhizogenes bacterial liquid into 200mL TY liquid culture medium, shaking overnight on a shaking table at 28 ℃, and culturing until OD600 is 0.6-1.0; the TY liquid culture medium contains 50mg/L Kan.

Preferably, the sterile treatment in step 1 is to sterilize the vegetable pea seeds for 15min by using 75% sodium hypochlorite and rinse the vegetable pea seeds for 5-6 times by using sterile water.

Preferably, the culture temperature of the light incubator in the

step

1 or 2 is controlled at 22 ℃, and the photoperiod is 16h of light and 8h of dark.

Preferably, the vegetable pea seeds in the step 1 are mature seeds without plant diseases and insect pests.

Compared with the traditional two-step method agrobacterium rhizogenes mediated hairy root genetic transformation technology, the scheme shortens the test period, reduces the pollution rate, saves the test cost and ensures that the test operation is more efficient and quicker. In the invention, the hairy roots are induced to form by the vegetable peas by the one-step method, so that the test period is shortened to a great extent, the risk of pollution in the test process is greatly reduced, the cost of hairy root induction is saved, and the stability and the repeatability are higher.

The scheme optimizes and combines the hairy root induction conditions of the vegetable pea and Zhejiang pea material No. 1, and obtains the optimal cutting position, seedling age and culture time with higher rooting rate and conversion rate after comparison. The one-step agrobacterium rhizogenes-mediated vegetable pea high-efficiency genetic transformation system established for the first time lays a foundation for efficiently developing the research on the transgenic gene function of vegetable pea.

Therefore, the invention has the following beneficial effects: (1) the test period is shortened, the pollution rate is reduced, the convenience of the test operation is improved, and the test cost is saved; (2) optimal cutting positions, seedling ages and culture time with high conversion rate and rooting rate are optimally combined.

Drawings

FIG. 1 is a schematic representation of the induction of hairy roots in peas using Agrobacterium rhizogenes in a two-step process.

FIG. 2 is a schematic representation of the growth of vegetable pea plants of the invention in a substrate.

FIG. 3 is a schematic representation of the preparation, infestation and formation of hairy roots of combined shoots of the overground part explant of vegetable peas according to the present invention.

FIG. 4 is a schematic diagram showing RT-PCR detection results of GUS genes in hairy roots of vegetable peas after the vegetable peas are infected by Agrobacterium rhizogenes AR1193 (carrying a binary expression vector pCAMBIA3301 containing GUS reporter genes).

FIG. 5 is a schematic view showing the growth state of hairy roots obtained by culturing for 18 days after vegetable pea explants are infected at different cutting positions (upper end and lower end of cotyledon) at different seedling ages (5 days, 7 days and 9 days) according to the present invention.

Detailed Description

Embodiments of the present application will be described in detail by examples, so that how to apply technical means to solve technical problems and achieve technical effects of the present application can be fully understood and implemented.

The raw materials and equipment used in the present application are all common raw materials and equipment in the field, and are all from commercially available products, unless otherwise specified. The methods used in this application are conventional in the art unless otherwise indicated.

Examples

1) Preparing an overground part explant of vegetable peas;

selecting mature vegetable pea seeds without diseases and insect pests, sterilizing with 75% sodium hypochlorite for 15min, washing with sterile water for 5-6 times, sowing in a matrix (preparation of the matrix, mixing vermiculite and turf according to a ratio of 3: 1, sterilizing at 121 ℃,20 min under high pressure, and pouring the sterile water thoroughly), and culturing in an illumination incubator (22 ℃, 16h illumination/8 h dark photoperiod). Growing for about one week (5 days, 7 days and 9 days), cutting the plant at the upper end and the lower end close to the cotyledon of the plant respectively to obtain the overground part explant of the vegetable pea.

2) Activating and culturing an AR1193 agrobacterium rhizogenes strain;

taking 200 mu L of agrobacterium rhizogenes strain AR1193 glycerol strain (carrying binary expression vector pCAMBIA3301 containing GUS reporter gene) to streak and coat TY (containing 50mg/L Kan) solid culture medium again, and culturing in an inverted manner in a constant temperature incubator at 28 ℃ for 48h for later use.

Secondly, inoculating 10 mu L of agrobacterium rhizogenes strain AR1193 glycerol (carrying a binary expression vector pCAMBIA3301 containing GUS reporter genes) to 1mL of TY (containing 50mg/L Kan) liquid culture medium, and culturing for 12h in a constant temperature incubator at 28 ℃ at the rotating speed of 180 rpm; then 200. mu.L of Agrobacterium rhizogenes bacterial liquid is inoculated into 200mL TY (containing 50mg/L Kan) liquid culture medium, and the mixture is shaken on a shaking table at 28 ℃ overnight and cultured until OD600 is 0.6-1.0 for standby.

3) Infecting the overground part explants of the vegetable peas and obtaining combined seedlings;

scraping the overground part explants of the vegetable peas in the step 1 from the TY solid culture medium in the step 2 to obtain bacterial paste on the surfaces, and carrying out bacteria wrapping on wounds generated by cutting; then, planting the explants wrapped with bacteria on a substrate (the preparation of the substrate comprises mixing vermiculite and turf according to the proportion of 3: 1, sterilizing at 121 ℃ for 20min under high pressure, and pouring sterile water thoroughly), pouring 5ml of TY liquid culture medium in the step 2, and operating the whole process under a sterile environment; then, culturing for 24h in dark in the dark, transferring to an illumination incubator, culturing at 22 ℃ for 16h illumination/8 h dark light period, and sealing and moisturizing by using a plastic cover in the whole process; after culturing for about two weeks (10 days, 14 days, 18 days), the number of hairy roots was observed and GUS staining was carried out. After two weeks, the humidity was slowly reduced by opening the vent holes on the plastic cover for the first two days, and then the plastic cover was completely removed to obtain well-grown combined seedlings.

Hairy root induction is carried out on the vegetable pea Zhejiang pea No. 1 material according to the steps, the average root number and the positive rate of different cutting positions, seedling ages and culture time are counted, difference significance analysis is carried out, and the optimal cutting position, seedling age and culture time for inducing hairy formation are screened out after comprehensive comparison. The results are shown in table 1 and fig. 1 to 5.

Table 1 shows the analysis results of the total hairy root number, the rooting induction rate and the positive rate obtained by cultivating vegetables with different seedling ages (5 days, 7 days and 9 days) at different cutting positions (upper end and lower end of cotyledon) infected by pea explants for different times (10 days, 14 days and 18 days).

TABLE 1 analysis of total hairy root number and root induction rate obtained by infecting vegetable with pea explants at different cutting positions of different seedling ages and culturing for different time

Note: the representation of the same column of data without the same lower case letters in the table was significantly different (P < 0.05).

TABLE 2 GUS detection primer sequences

Primer name	Primer sequences	Size of primer
			GUSF	GCTATACGCCATTTGAAGCC	20bp
GUSR	TTGACTGCCTCTTCGCTGTA	20bp

Table 2 shows the primer sequences used when RT-PCR was used to detect the GUS gene in hairy roots after infection.

FIGS. 1 and 2 are schematic diagrams of a two-step process and Agrobacterium rhizogenes induced pea hairy root formation and pea plant of the present invention, respectively.

In FIG. 3, A is the growth status of the vegetable pea plant at the best seedling age (7 days) and the mode of obtaining the overground part explant (the upper arrow indicates the upper cutting position of the cotyledon, and the lower arrow indicates the lower cutting position of the cotyledon); b is obtained by solid plate of Agrobacterium rhizogenes AR1193 (carrying binary expression vector pCAMBIA3301 containing GUS reporter gene); c is the obtained bacterial liquid of agrobacterium rhizogenes AR1193 (carrying binary expression vector pCAMBIA3301 containing GUS reporter gene); d is a bacterium wrapping mode for cutting the overground part explant of the vegetable pea to generate a wound; e, sowing the wrapped explants in a sterile matrix and then pouring the explants; f, sealing and moisturizing the vegetable pea explant by using a plastic cover after infection; g is hairy root formed by vegetable pea combined seedlings; h is a GUS staining result of hairy roots after being infected by agrobacterium rhizogenes AR1193 (carrying a binary expression vector pCAMBIA3301 containing GUS reporter genes).

In FIG. 4, A is the GUS staining of all hairy roots formed by the vegetable pea combined seedlings; in B, M is D2000 DNA Marker of Tiangen Biochemical technology Co., Ltd, 1-5 is transgenic hairy root after infection of AR1193-pCAMBIA3301, 6 is AR1193-pCAMBIA3301 strain, 7-9 is non-transgenic hairy root after infection of AR1193-pCAMBIA3301, 10 is AR1193 strain, 11 is H₂O。

The above experiment results revealed that the optimum cutting position was the lower end of the cotyledon, the seedling age was 7 days and the cultivation time was 18 days, the hairy root induction rate was 100%, the average number of the grown roots was 14.4, and the hairy root positive rate was 35.2%.

There are many other possible embodiments of the present invention, which are not listed here, and the embodiments claimed in the claims of the present invention can be implemented.

The details not described in the specification of the present application belong to the common general knowledge of those skilled in the art.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect.

It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a commodity or system that includes the element.

The foregoing description shows and describes several preferred embodiments of the present application, but as aforementioned, it is to be understood that the application is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the application, which is to be protected by the claims appended hereto.

Claims

1. A method for establishing an agrobacterium rhizogenes-mediated vegetable pea genetic transformation system is characterized by comprising the following steps:

3) hairy root induction and combined seedling formation: culturing the infected explants in dark for 24 h; then transferring to an illumination incubator for culturing, sealing and moisturizing by using a plastic cover in the whole process, and culturing for 10-18 days; then continuing to culture for two weeks, opening the vent holes on the plastic cover two days in advance to slowly reduce the humidity, and then completely removing the plastic cover to obtain the combined seedlings which grow vigorously.

2. The method according to claim 1, wherein the upper end of the cotyledonary node of the plant of step 1 is the upper end of the junction between the cotyledons of the plant; the lower end refers to the upper end close to the junction of the hypocotyl of the plant.

3. The method according to claim 1, wherein the growth time in step 1 is preferably 7 days; the culture time in the step 3) is preferably 18 days.

4. The method of claim 1, wherein the matrix is formulated in step 1 or 2 by: mixing vermiculite and turf according to a mass ratio of 3 to 1, and autoclaving at 121 ℃ for 20 min.

5. The method as claimed in claim 1, wherein Agrobacterium rhizogenes AR1193 in step 2 carries binary expression vector pCAMBIA3301 containing GUS reporter gene.

6. The method of claim 1, wherein the mush of step 2 is obtained by scraping TY solid medium from agrobacterium rhizogenes AR 1193; the TY solid culture medium of the agrobacterium rhizogenes AR1193 is prepared by the following method: taking 200 mu L of agrobacterium rhizogenes strain AR1193 glycerol bacteria, streaking and coating the strains on a TY solid culture medium, and carrying out inverted culture in a constant temperature incubator at 28 ℃ for 48 h; wherein the TY solid culture medium contains 50mg/L Kan.

7. The method according to claim 1, wherein the liquid medium comprising Agrobacterium rhizogenes AR1193 of step 2 is prepared by: inoculating 10 mu L of Agrobacterium rhizogenes strain AR1193 glycerol strain to 1mL of TY liquid culture medium, and culturing for 12h in a constant temperature incubator at 28 ℃ at the rotating speed of 180 rpm; inoculating 200 mu L of agrobacterium rhizogenes bacterial liquid into 200mL TY liquid culture medium, shaking overnight on a shaking table at 28 ℃, and culturing until OD600 is 0.6-1.0; the TY liquid culture medium contains 50mg/L Kan.

8. The method as claimed in claim 1, wherein the sterilization and disinfection treatment of step 1 is to sterilize the vegetable pea seeds with 75% sodium hypochlorite for 15min and rinse with sterile water for 5-6 times.

9. The method according to claim 1, wherein the cultivation temperature of the light incubator in step 1 or 2 is controlled at 22 ℃ and the photoperiod is 16h light and 8h dark.

10. The method of claim 1, wherein said vegetable pea seeds of step 1 are mature pest-free seeds.