阅读说明：本技术 一种采用花序浸染快速高效遗传转化二穗短柄草的方法 (Method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing ) 是由 李鹏 储昭庆 于 2019-09-27 设计创作，主要内容包括：本发明公开了一种采用花序浸染快速高效遗传转化二穗短柄草的方法,包括：A、农杆菌EHA105的转化和阳性克隆的鉴定；B、农杆菌浸染二穗短柄草花序；C、转基因二穗短柄草T0代阳性植株获得。本发明通过农杆菌介导的浸染花序遗传转化方法,避免了愈伤组织的培养过程,而且可节省大量的时间,为作物的遗传改良提供了有效的快速获得转基因植株的方法。(The invention discloses a method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing, which comprises the following steps: A. transformation of agrobacterium EHA105 and identification of positive clones; B. the agrobacteria dip-dyeing of the inflorescence of the two-spike brachypodium; C. transgenic brachypodium distachyon T0 generation positive plants. The invention avoids the culture process of callus by the agrobacterium-mediated genetic transformation method of the dip-dyed inflorescence, can save a large amount of time, and provides an effective and rapid method for obtaining transgenic plants for genetic improvement of crops.)

1. A method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing is characterized by comprising the following steps:

A. agrobacterium transformation

Adding plasmid DNA into agrobacterium tumefaciens competent cells, carrying out ice bath for the first time, then carrying out liquid nitrogen treatment, and carrying out water bath treatment; adding the mixture into a YEB liquid culture medium after the second ice bath for shake culture, taking out the cultured bacterial liquid, coating the bacterial liquid on a solid YEB culture medium containing antibiotics, and performing inverted culture to obtain a positive agrobacterium tumefaciens single colony;

B. agrobacteria dip-dyeing brachypodium distachyon inflorescence

Inoculating single colony of positive agrobacterium tumefaciens, performing shake culture in YEB culture medium containing antibiotics, performing amplification culture until OD600 value reaches 0.8-1.2, centrifuging, and resuspending to obtain agrobacterium tumefaciens suspension; immersing the inflorescence of the brachypodium distachyon to be transformed in the agrobacterium tumefaciens suspension for dip dyeing, then carrying out dark culture, then carrying out normal environment culture, after a new spikelet grows out, dip dyeing by the same method, repeating for 2-3 times, and obtaining the dip-dyed brachypodium distachyon;

C. obtaining transgenic brachypodium distachyon T0 generation positive plants

And D, culturing the dip-dyed brachypodium distachyon obtained in the step B until seeds are mature, peeling off shells, sowing the seeds on the surface of an 1/2MS culture medium containing hygromycin after disinfection, transplanting seedlings into soil after the seedlings grow out, and normally culturing to obtain a transgenic brachypodium distachyon T0 generation positive plant.

2. The method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing according to claim 1, wherein in step A, the amount of the plasmid DNA added is 0.1-1 μ g/50 μ l of Agrobacterium tumefaciens competent cells;

the first ice-bath time is 30min, the liquid nitrogen treatment time is 5min, the water bath treatment is 37 ℃ water bath for 5min, and the second ice-bath time is 2-5 min.

3. The method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing according to claim 1, wherein in the step A, the shaking culture is as follows: carrying out shaking culture at 28 deg.C and 220rpm for 3-5 h;

the inverted culture comprises the following steps: culturing at 28 deg.C for 2-3 days.

4. The method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing according to claim 1, wherein in the step B, the shaking culture is as follows: shaking and culturing at 28 ℃ overnight;

the expanded culture comprises the following steps: inoculating the single colony of the agrobacterium after shaking culture to YEB culture medium containing antibiotics, enabling the initial OD600 value to be 0.2-0.6, and carrying out shaking culture at 28 ℃ and 220rpm for 3-4 h;

the centrifugation conditions were: centrifuging at 4000rpm for 15 min;

the heavy suspension comprises MS liquid culture medium, Silwet L-770.01-0.04%, acetosyringone 100-; the OD600 value of the obtained Agrobacterium suspension was 0.8-1.2.

5. The method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing according to claim 1, wherein in the step B, the dip-dyeing time is 10s, the dark culture time is 24h, and the culture is carried out for 7-10 days in a normal environment.

6. The method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing according to claim 1, wherein in the step C, the disinfection treatment specifically comprises the following steps: sterilizing with 75% ethanol for 2-10min, sterilizing with 30% sodium hypochlorite solution for 5-10min, and rinsing with sterile water for 3 times;

the seedlings have white long roots and 2-3 leaves.

7. The method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing according to claim 1, wherein in the step C, the hygromycin concentration in the 1/2MS culture medium is 20-80 mg/L.

8. The method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing according to claim 1, wherein the ecotype of brachypodium distachyon is one of Bd21, Bd21-3, Bd1-1 and Bd2-3, and the agrobacterium strain is one of EHA105, LBA4404, EHA101, EHA105, GV3101 and AGL 1; the plasmid DNA is pCAMBIA1300 containing hygromycin resistance gene (HYG (R));

in the solid YEB culture medium containing the antibiotics and the YEB culture medium containing the antibiotics, the antibiotics are kanamycin and rifampicin, and the concentration of the antibiotics is 50 mg/L.

9. The method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing according to claim 1, wherein in the steps A and C, the PCR system for identifying the positive plants of the positive agrobacterium single colony and the transgenic brachypodium distachyon T0 generation is adopted: in a reaction system of 20 mul, 0.2-1.5U of Taq DNA polymerase, 0.2-0.5 mul mol/L of dNTP, 0.2-1.0 mul mol/L of primer and 10-150ng of template DNA; the adopted primers are shown as SEQ ID NO.1 and SEQ ID NO. 2;

the PCR reaction procedure used was: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 58 deg.C for 30s, extension at 72 deg.C for 1.5min, circulation for 30 times, extension at 72 deg.C for 10min, and storage at 4 deg.C.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing.

Background

Gramineae comprises over 600 genera of 10000 species, of which poaceae families, wheat, barley, rye and oats are among the most economically valuable groups. The Povidae annuae crops mostly have large and complex genomes, such as 5096Mb for barley, and x is 7; the genome of hexaploid common wheat is 17000Mb, and has A, B and D completely independent genomes, 3x is 21, so that the genome and functional genome research of wheat crops faces huge challenges.

Rice is the first gramineae to complete whole genome sequencing, but rice as a model plant has many insurmountable difficulties in studying temperate cereal crops. Firstly, the rice and the wheat plants have far genetic relationship, and are on different evolution branches with temperate cereal crops as early as 5 million years ago, and the colinearity among the gene groups is not ideal; in addition, the rice needs strict cultivation conditions, and the rice is tall and big, has long growth cycle and is not suitable for large-scale close planting under laboratory conditions.

Brachypodium (Brachypodium) genus Poaceae family (Pooideae) comprising about 10 species, wherein Brachypodium is perennial with a base chromosome number of 9, 2 n-2 x-18, 1C-470 Mb; the brachypodium distachyon is annual, the number of basic chromosomes is 5, different ploidy materials such as 2 ploid, 4 ploid and 6 ploid exist, the genome is about 272Mb, is 2 times of that of arabidopsis thaliana and is smaller than that of rice genome (441Mb), and the genome contains less than 15% of DNA repetitive sequences. The diploid ecotype Bd21 can flower and fruit without vernalization induction, and the life cycle is about 3 months. The brachypodium distachyon and grain crops of wheat, barley, oat, rye and the like belong to the poaceae, a chromosome doubling event occurs earlier than that of the gramineae, and the close phylogenetic relationship ensures that the brachypodium distachyon is more suitable to be used as a model plant of the wheat crops than the rice. Important grain crops in Poaceae, such as wheat, barley and the like, have huge and complex genomes due to wide gene recombination, and can be subjected to gold-silver recognition and genome analysis by comparing genomics by utilizing the genome sequence information of brachypodium distachyon; BAC (bacterial Artificial chromasomes) physical maps show that brachypodium distachyon is very similar to rice and sorghum in gene content and gene family structure, so that brachypodium distachyon can be used as a functional genome research model of all plants in the family of Gramineae.

In addition, the brachypodium distachyon as a model material has the following advantages: the plant type is small, the height of a mature plant is about 20cm, the plant is almost the same as that of arabidopsis thaliana, and high-throughput genetic operation, mutant screening and other work are easy to perform. Easy cultivation and reduced test and maintenance cost. The life cycle is short, the Bd21 ecotype only needs 3 months from seed to seed, and the flowering time of some diploid lines can be regulated and controlled through vernalization and illumination induction. The anther and stigma of the brachypodium distachyon are tightly wrapped by the palea and the palea, so that the self-pollination is performed in a natural state, and a self-crossing line and an inbreeding line are easily formed.

The genetic transformation method for obtaining transgenic material is a necessary means for analyzing gene function and improving crop traits. At present, the genetic transformation of brachypodium distachyon is realized by inducing callus through immature embryos or mature embryos, infecting the callus through agrobacterium tumefaciens and further inducing the callus to differentiate into buds and roots, so that a transgenic plant is obtained through a complicated procedure, the whole process also needs 3-4 months at the fastest speed, and certain somatic mutation exists. The transformation method of the agrobacterium-infected inflorescence provided by the invention is simple and feasible, omits complicated processes such as callus induction, culture and differentiation, avoids high requirements on experimental equipment and experimental technology in tissue culture, accurately adjusts the hormone proportion, saves a large amount of time, manpower and material resources, and greatly saves the experimental cost; the tissue culture stage is avoided, so that somatic cell variation caused by tissue culture can be eliminated; the transgenic plant obtained by the method has good genetic stability, accords with Mendelian genetic rule, and can directly harvest seeds for further screening homozygote.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing. The method has the advantages of simple and convenient operation, short transformation period, high transformation efficiency, low false positive and the like.

The purpose of the invention is realized by the following technical scheme:

the invention provides a method for rapid and efficient genetic transformation of brachypodium distachyon by inflorescence dip-dyeing, which comprises the following steps:

A. agrobacterium transformation

Adding plasmid DNA into agrobacterium tumefaciens competent cells, carrying out ice bath for the first time, then carrying out liquid nitrogen treatment, and carrying out water bath treatment; adding the mixture into a YEB liquid culture medium after the second ice bath for shake culture, taking out the cultured bacterial liquid, coating the bacterial liquid on a solid YEB culture medium containing antibiotics, and performing inverted culture to obtain a positive agrobacterium tumefaciens single colony;

B. agrobacteria dip-dyeing brachypodium distachyon inflorescence

Inoculating single colony of positive agrobacterium tumefaciens, performing shake culture in YEB culture medium containing antibiotics, performing amplification culture until OD600 value reaches 0.8-1.2, centrifuging, and resuspending to obtain agrobacterium tumefaciens suspension; immersing the inflorescence of the brachypodium distachyon to be transformed in the agrobacterium tumefaciens suspension for dip dyeing, then carrying out dark culture, then carrying out normal environment culture, after a new spikelet grows out, dip dyeing by the same method, repeating for 2-3 times, and obtaining the dip-dyed brachypodium distachyon;

C. obtaining transgenic brachypodium distachyon T0 generation positive plants

And D, culturing the dip-dyed brachypodium distachyon obtained in the step B until seeds are mature, peeling off shells, sowing the seeds on the surface of an 1/2MS culture medium containing hygromycin after disinfection, transplanting seedlings into soil after the seedlings grow out, and normally culturing to obtain a transgenic brachypodium distachyon T0 generation positive plant.

Preferably, in step A, the plasmid DNA is added in an amount of 0.1-1. mu.g/50. mu.l Agrobacterium-infected competent cells;

the first ice-bath time is 30min, the liquid nitrogen treatment time is 5min, the water bath treatment is 37 ℃ water bath for 5min, and the second ice-bath time is 2-5 min.

Preferably, in step a, the shaking culture is: carrying out shaking culture at 28 deg.C and 220rpm for 3-5 h;

the inverted culture comprises the following steps: culturing at 28 deg.C for 2-3 days.

Preferably, in step B, the shaking culture is: shaking and culturing at 28 ℃ overnight;

the expanded culture comprises the following steps: inoculating the single colony of the agrobacterium after shaking culture to YEB culture medium containing antibiotics, enabling the initial OD600 value to be 0.2-0.6, and carrying out shaking culture at 28 ℃ and 220rpm for 3-4 h;

the centrifugation conditions were: centrifuging at 4000rpm for 15 min;

the heavy suspension comprises MS liquid culture medium, Silwet L-770.01-0.04%, acetosyringone 100-; the OD600 value of the obtained Agrobacterium suspension was 0.8-1.2. Wherein Silwet L-77 is a surfactant, which can improve the transformation efficiency by reducing the surface tension of liquid and being beneficial to the adsorption of agrobacterium, but too high concentration can cause toxicity to plants and even lead the plants to wither; acetosyringone is used as a suspension ingredient, and the use concentration thereof is crucial. The acetosyringone activates the expression of other genes on the toxic region by inducing the activation of vir genes in the genome of the agrobacterium, so that the T-DNA is successfully introduced, and the transformation efficiency is improved. The concentration is too low, and the conversion efficiency is reduced; too high a concentration can have a toxic effect on the plant.

More preferably, the initial OD600 value before the expansion culture is 0.2-0.3, and after the expansion culture is carried out, the OD600 value is 0.8-1.0; the OD600 value of the obtained Agrobacterium suspension was 0.8-1.0.

Most preferably, the initial OD600 value before the expansion culture is 0.3, and after the expansion culture is carried out, the OD600 value reaches 0.8; the resulting Agrobacterium suspension had an OD600 value of 0.9. (ii) a

The heavy suspension comprises Silwet L-770.02%, acetosyringone 200 mu mol/L, and pH 5.8.

Preferably, in the step B, the soaking time is 10s, the dark culture time is 24h, and the culture is carried out for 7-10 days in a normal environment.

Preferably, in step C, the sterilization treatment is specifically: sterilizing with 75% ethanol for 2-10min, sterilizing with 30% sodium hypochlorite solution for 5-10min, and rinsing with sterile water for 3 times;

the seedlings have white long roots and 2-3 leaves.

More preferably, the sterilization treatment is in particular: sterilizing with 75% ethanol for 5min, sterilizing with 30% sodium hypochlorite solution for 10min, and rinsing with sterile water for 3 times;

preferably, in step C, the hygromycin concentration in the 1/2MS medium is 20-80 mg/L. More preferably, the hygromycin concentration is 50 mg/L.

Preferably, the ecotype of the brachypodium distachyon is one of Bd21, Bd21-3, Bd1-1 and Bd2-3, but is not limited thereto; the agrobacterium strain is one of EHA105, LBA4404, EHA101, EHA105, GV3101 and AGL1, but is not limited thereto; the plasmid DNA is pCAMBIA1300 containing hygromycin resistance gene (HYG (R)), or other plant binary expression vectors containing hygromycin resistance gene (HYG (R)); the nucleotide sequence of the hygromycin resistance gene is shown as SEQ ID NO. 3;

in the solid YEB culture medium containing the antibiotics and the YEB culture medium containing the antibiotics, the antibiotics are kanamycin and rifampicin, and the concentration of the antibiotics is 50 mg/L.

Preferably, in the steps A and C, the PCR system for identifying positive plants of the positive agrobacterium single colony and the transgenic brachypodium distachyon T0 generation is as follows: in a reaction system of 20 mul, 0.2-1.5U of Taq DNA polymerase, 0.2-0.5 mul mol/L of dNTP, 0.2-1.0 mul mol/L of primer and 10-150ng of template DNA; the adopted primers are shown as SEQ ID NO.1 and SEQ ID NO. 2;

the sequences of the upstream primer and the downstream primer are 5 'ATGAAAAAGCCTGAACTCACCGCGA 3' (SEQ ID NO.1) and 5 'CTATTTCTTTGCCCTCGGACGAGTG 3' (SEQ ID NO.2), respectively;

the PCR reaction procedure used was: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 58 deg.C for 30s, extension at 72 deg.C for 1.5min, circulation for 30 times, extension at 72 deg.C for 10min, and storage at 4 deg.C.

More preferably, the PCR system is: in each 20. mu.L reaction system, 1.0U of Taq DNA polymerase, 0.25. mu. mol/L of dNTP, 0.5. mu. mol/L of primer and 50ng of template DNA are added.

YEB culture medium and MS culture medium used in the invention are all conventional products sold in the market.

Compared with the prior art, the invention has the following beneficial effects:

in the traditional method for genetic transformation of brachypodium distachyon, the tissue culture operation is complex, the technology is high, and certain somatic mutation exists. The invention utilizes the genetic transformation method of the agrobacteria-mediated dip-dyeing inflorescence, avoids the processes of tissue culture and regeneration, can directly harvest seeds, greatly shortens the transformation period, and avoids the high requirements on experimental equipment and experimental technology in the tissue culture and the accurate adjustment of hormone proportion; the tissue culture stage is avoided, so that somatic cell variation caused by tissue culture can be eliminated; the method is simpler and more convenient, has stronger operability and higher transformation efficiency, and the transgenic plant obtained by the method has good genetic stability, accords with Mendelian genetic law, and can directly harvest seeds for further screening homozygotes.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a map of plasmid pCAMBIA 1300;

FIG. 2 is an electrophoretogram of colony PCR for identifying Agrobacterium, M: standard molecular weight DNA, the same below, 1-8:8 Agrobacterium single colonies;

FIG. 3 is a diagram showing the growth state of the selected strains of positive seedlings obtained by hygromycin screening; wherein, FIG. 3a shows the overground part growth state of the positive seedling, and FIG. 3b shows the root growth state of the positive seedling;

FIG. 4 is a comparison graph of growth states of hygromycin screening non-positive seedlings and positive seedling candidate plants, wherein three plants on the left side are untransformed and three plants on the right side are positive seedling candidate plants obtained by transformation;

FIG. 5 is the PCR identification electrophoresis of the positive seedling candidate, WT: wild type, 1-8: and selecting positive seedlings.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.

Unless otherwise stated, the experimental materials and biological products of the present invention are commercially available products, and the biological means used are commonly performed by those skilled in the art.