阅读说明：本技术 一种可视报告基因及其载体的应用 (Visual reporter gene and application of carrier thereof ) 是由 王文博 杨丽 马明茹 叶润文 孙晓妍 别之龙 于 2021-08-20 设计创作，主要内容包括：本发明涉及基因工程领域,具体涉及一种可视报告基因及其载体的应用。本发明首次将可视报告基因DsRed用于黄瓜的遗传育种中,经过研究发现,该基因可在黄瓜中稳定遗传且得到的黄瓜幼苗、植株、果实及种子均呈现出肉眼可见的红色表型,且该性状可以稳定遗传；将该基因通过特定的载体及农杆菌介导的方式转化到黄瓜中,转化效率可以高达0.25％。(The invention relates to the field of genetic engineering, in particular to a visual reporter gene and application of a carrier thereof. The invention uses the visible reporter gene DsRed in the genetic breeding of cucumber for the first time, and researches show that the gene can be stably inherited in the cucumber, and the obtained cucumber seedlings, plants, fruits and seeds all show a macroscopic red phenotype, and the character can be stably inherited; the gene is transformed into cucumber by a specific vector and an agrobacterium-mediated mode, and the transformation efficiency can reach 0.25 percent.)

1. Use of a visual reporter gene in cucumber genetic transformation, wherein the visual reporter gene is DsRed.

2. A vector or cell comprising the visual reporter gene of claim 1.

3. The visual reporter gene vector according to claim 2, wherein the vector contains a 35S DsRed-Terminal expression cassette.

4. The visual reporter gene vector according to claim 2 or 3, wherein the vector comprises the G10-90pro XVE-E9T fragment.

5. A method for preparing the carrier of claim 4, comprising the steps of:

inserting the DsRed-Terminal expression cassette obtained by amplification into a pBSE401 vector to obtain an intermediate vector pBSE 401R;

inserting the G10-90pro XVE-E9T fragment obtained by amplification into an intermediate vector pBSE401R to obtain the recombinant plasmid.

6. The method of claim 5, wherein the 35S: DsRed-Terminal expression cassette is amplified using pCAMBIA2300:35S: DsRed2 vector as a template.

7. The method of claim 5, wherein the 35S DsRed-Terminal expression cassette is inserted between EcoRI sites of the pBSE401 vector.

8. The method for preparing the vector according to claim 5, wherein the G10-90pro XVE-E9T fragment is amplified using pX6-GFP-AF330636 vector as a template.

9. The method for preparing the vector according to claim 5, wherein the fragment G10-90pro XVE-E9T is inserted between Hind III sites of the intermediate vector.

10. Use of a vector as claimed in claim 2 for cucumber breeding, characterized in that the vector is transformed into cucumber in an agrobacterium-mediated manner.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a visual reporter gene capable of being stably inherited in cucumber and application of a carrier thereof.

Background

The cucurbitaceae crops are important economic crops and also are model plants for researching biological problems of fruit enlargement, long-distance transport of vascular bundles, sex differentiation and the like. At present, genome sequencing of several important crops of cucurbitaceae, including cucumbers, melons, watermelons and pumpkins, a melon CRISPR/Cas9 gene editing system is established, and further, functional genes are mined by utilizing gene big data and combining with a gene editing tool, and an efficient genetic transformation system is required to be matched with the gene big data. However, genetic transformation of cucurbit crops is a worldwide problem, and although breakthrough is made in recent years, the method still faces the problems of low transformation efficiency, high technical difficulty, time and labor waste and the like, is difficult to develop on a large scale, and the lack of an efficient genetic transformation system still is one of the biggest technical bottlenecks which restrict biological research and molecular breeding of cucurbit crops.

In addition, after the transformation of the existing transformation system is successful, whether the transformation is successful is generally verified through sequencing, but the transformation efficiency of cucurbit crops is often very low and sometimes is even only a few ten-thousandths, so that the detection period of positive plants is longer, the cost is higher, the experiment period is further prolonged, and the experiment difficulty is increased. Therefore, the development of a visual reporter gene of cucurbit plants and the guarantee of good transformation efficiency of the visual reporter gene are of great significance for the research on genetic transformation of cucurbit plants.

Disclosure of Invention

One of the purposes of the present invention is to provide an application of a visual reporter gene in cucumber genetic transformation, wherein the difference is that the visual reporter gene is DsRed.

The cucumber seedlings transformed by the visible gene not only show a red phenotype, but also show red phenotypes of fruits and seeds, and the phenotype can be observed by naked eyes, so that the positive transformed seedlings can be easily judged.

The second object of the present invention is to provide a vector and a cell containing the above-mentioned visual reporter gene.

In one embodiment of the invention, the vector contains a 35S DsRed-Terminal expression cassette.

In one embodiment of the invention, the vector comprises the fragment G10-90pro XVE-E9T.

The invention also aims to provide a preparation method of the carrier, which comprises the following steps: inserting the DsRed-Terminal expression cassette obtained by amplification into a pBSE401 vector to obtain an intermediate vector pBSE 401R; inserting the G10-90pro XVE-E9T fragment obtained by amplification into an intermediate vector pBSE401R to obtain the recombinant plasmid.

In one embodiment of the present invention, the expression cassette 35S: DsRed-Terminal is obtained by amplification using pCAMBIA2300:35S: DsRed2 vector as a template.

In one embodiment of the invention, the 35S: DsRed-Terminal expression cassette is inserted between EcoRI cleavage sites of the pBSE401 vector.

In one embodiment of the invention, the G10-90pro XVE-E9T fragment is amplified using pX6-GFP-AF330636 vector as a template.

In one embodiment of the invention, the G10-90pro XVE-E9T fragment is inserted between HindIII sites in the intermediate vector.

The fourth purpose of the present invention is to provide the application of the above-mentioned vector in cucumber breeding, i.e. the vector can be transferred into cucumber by means of agrobacterium-mediated transformation.

The invention uses the visible reporter gene DsRed2 in the genetic breeding of cucumber for the first time, and researches show that the gene can be stably inherited in cucumber, and the obtained cucumber seedlings, plants, fruits and seeds all show macroscopic red phenotypes, and the traits can be stably inherited; the gene is transformed into cucumber by a specific vector and an agrobacterium-mediated mode, and the transformation efficiency can reach 0.25 percent.

Drawings

FIG. 1 is a set of Vector P Vector expression elements;

FIG. 2 shows a stable transformed cucumber seedling and cucumber buds and cucumber tendrils with red fluorescence under a fluorescence microscope, wherein A is the red fluorescence seedling in a milky tea cup, B is the cucumber bud transformed with Vector P observed under a fluorescence microscope, and C is the cucumber tendrils transformed with Vector P observed under a fluorescence microscope under different light sources;

FIG. 3 shows red cucumber fruits and seeds harvested from stably transformed cucumber seedlings, wherein A is a cucumber fruit and B is a cucumber seed;

FIG. 4 is a plasmid map of vector P.

Detailed Description

The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.

The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.

The invention has the following inventive concept:

a melon CRISPR/Cas9 gene editing system is established, and functional genes are further mined by utilizing gene big data and combining a gene editing tool, and an efficient genetic transformation system is matched with the gene big data. However, the genetic transformation of cucurbit crops is always a difficult problem worldwide, and an efficient genetic transformation system is still one of the biggest technical bottlenecks which restrict the biological research and molecular breeding of cucurbit crops. After the transformation of the existing transformation system is successful, whether the transformation is successful is generally verified through sequencing, but the transformation efficiency of cucurbitaceae crops is often very low and sometimes is even only a few ten-thousandths, so that the detection period of positive plants is longer, the cost is higher, the experiment period is further prolonged, and the experiment difficulty is increased. Therefore, the inventors tried to develop a visual reporter gene of cucurbitaceae plants while ensuring good transformation efficiency.

The inventor firstly collects a plurality of vectors of red light genes of RFP, m Cherry and DsRed for transformation experiments, finally finds that the red light expression of the RFP and the m Cherry red light vectors after transformation is weak, needs expensive instruments to observe and is still difficult to distinguish from the background material of the plant, and the red fluorescence intensity form expressed by the pCAMBIA2300:35S: DsRed2 vector in the healing of cucumber is very obvious.

The inventor further takes a pCAMBIA2300:35S: DsRed2 vector as a template to amplify a 35S: DsRed-Terminal expression frame, and inserts the DsRed-Terminal expression frame between EcoRI enzyme cutting sites of a pBSE401 vector to obtain an intermediate vector pBSE 401R; then, a G10-90pro: XVE-E9T fragment is amplified by taking a pX6-GFP-AF330636 Vector as a template, LoxP sites in the fragment are removed, and the fragment is inserted between HindIII enzyme cutting sites of a pBSE401R Vector through Infusion to obtain a Vector P (Vector P: pBSE401-Cas 9-XVE-dsRed). Vector P is transformed into agrobacterium and cucumber is transformed by adopting a direct cotyledon regeneration method, DsRed fluorescence is brightly visible under a body type fluorescence microscope, non-transformation background can be well distinguished, and the transformation rate can reach 0.25%.

Example 1

The construction method of the vector P comprises the following specific steps:

using pCAMBIA2300:35S: DsRed2 vector as a template to amplify the 35S: DsRed-Terminal expression frame by PCR, and inserting the DsRed-Terminal expression frame between EcoRI enzyme cutting sites of the pBSE401 vector to obtain an intermediate vector pBSE 401R;

a G10-90pro XVE-E9T fragment is amplified by adopting a pX6-GFP-AF330636 Vector as a template through adopting overlap PCR, LoxP sites in the fragment are removed, and the fragment is inserted between HindIII enzyme cutting sites of a pBSE401R Vector through Infusion to obtain a Vector P (Vector P: pBSE401-Cas 9-XVE-dsRed).

Example 2

The Vector P obtained in the example 1 is transferred into an escherichia coli competent cell DH5 alpha, plasmids are extracted, and then the plasmid of the Vector P is transferred into agrobacterium to prepare for a subsequent cucumber genetic transformation infection experiment.

(1) The method for transforming the Escherichia coli comprises the following steps:

a. taking 1 mu L of vector P plasmid from a clean bench, adding the vector P plasmid into 100 mu L of escherichia coli competent cell DH5 alpha, and gently and uniformly mixing;

b. carrying out ice bath for 5min, putting the mixture into a water bath kettle at 42 ℃ for heat shock for 90s, and immediately putting the mixture into an ice box for 2-3 min after the heat shock is finished;

c. adding 1mL of LB solution into a clean bench, and uniformly mixing; shaking-culturing at 37 deg.C and 200rpm for 30 min;

d. uniformly coating about 100-;

e. after the single bacterial colony grows out, the single bacterial colony is picked and inoculated into a liquid LB culture medium (containing 50mg/L kanamycin) to be subjected to shake cultivation at 37 ℃, PCR identification is carried out, and bacterial liquid of the identified target band is added with isometric glycerol to be stored in a refrigerator at minus 80 ℃.

(2) And (3) plasmid extraction: in an ultraclean bench, 2. mu.L of the above E.coli suspension was taken and put into a conical flask containing 20mL of LB + kana liquid medium, cultured overnight in a shaker at 37 ℃ and then Plasmid was extracted using TIANPrep Mini Plasmid Kit Plasmid minikit Kit (purchased from TIANGEN, DP180123), and the Plasmid concentration in each tube was measured and then stored under a label.

(3) The agrobacterium transformation method comprises the following steps:

a. 2 mu L of plasmid is taken out from a superclean workbench, added into 100 mu L of EHA105 agrobacterium-mediated cells, and flicked and mixed evenly;

b. ice-cooling for 5min, freezing in liquid nitrogen for 1min, and rapidly placing in 37 deg.C water bath for 5 min;

c. adding 800 μ L LB culture medium, culturing for 2h on a shaking table at 200 rpm;

d. centrifuging at 12000rpm for 1min to concentrate bacterial liquid, adding 200 μ L LB liquid culture medium for resuspension, mixing, spreading on LB + Kana solid culture medium, and culturing in 28 deg.C incubator for 2 d;

e. picking out the monoclonal bacterial plaque of the agrobacterium, shaking the bacterial plaque at 28 ℃ and 200rpm overnight, and carrying out PCR and agarose gel electrophoresis identification;

f. and (4) placing the bacterium liquid identified as positive in a refrigerator at the ultralow temperature of-80 ℃ for storage for later use.

The Agrobacterium transformation vector P (containing Cas9-dsRED-XVE element) obtained above was inherited by the cotyledon direct regeneration method.

Example 3

The embodiment provides a cucumber genetic transformation method, the cucumber material used is Cucumis sativus L germplasm CU2 of Cucumis of Cucurbitaceae, Cucumis, and the specific steps are as follows:

1. sowing: collecting CU2 cucumber seeds with full seeds and uniform size, soaking in warm water at 55 deg.C for more than half an hour, and removing seed coat. In a clean bench, the sample was washed with 75% ethanol for 30s, then soaked in 0.3% NaClO solution for 15min, gently shaken during the soaking, and finally washed with sterile water for 5 times. The sterilized seeds were transferred to a seed germination medium prepared in advance, and approximately 30 seeds were sown per dish. Culturing in 28 deg.C oven for 24h, and cutting when the seed has just a little edge and is not whitened;

2. preparing an explant: taking germinated seeds, cutting off cotyledons with the far end being about 1/3 in a super clean bench, removing hypocotyls, separating two cotyledons, forming a U-shaped wound on the near end of each cotyledon, and dotting one point at the U-shaped wound to obtain an explant;

(3) infection of agrobacterium: the successfully transformed Vector P Agrobacterium, stored at-80 ℃ was removed and inoculated into 2mL LB liquid medium (containing 50mg/L Kana and 50mg/L Rif) and shaken at 200rpm at 28 ℃ overnight. Then diluted at a ratio of 1:500, and added to fresh 50mL LB liquid medium (containing 50mg/L Kana and 50mg/L Rif), shaking overnight at 200rpm at 28 ℃. The concentration of the bacteria liquid reaches OD_600＝Centrifuging at 6000rpm for 8min at 0.4-0.8, removing supernatant, collecting thallus, resuspending thallus with IM liquid culture medium, and diluting to OD₆₀₀Is 0.2. Taking out the piston core rod of the injector, adding the cotyledon explant into a needle cylinder of a 20mL injector, slightly sleeving the piston core rod, emptying redundant air in the injector as much as possible, sucking agrobacterium liquid with the volume larger than 10mL from a needle hole, and slightly pushing the piston forwards to discharge bubbles and redundant liquid from the needle hole of the injector until the piston is pushed to a scale position with the volume of 10mL and stays. The head pinhole of the syringe is sealed by a rubber plug, the piston core rod is pulled slowly and forcefully backwards, the needle hub is shaken slightly to ensure that all explants are stressed uniformly, and the explants are kept for 1.5min when the piston stays at the scale of 20mL, thereby achieving the purpose of applying vacuum negative pressure. The hand is slightly loosened, the piston slowly returns to the 10mL scale position, and then the vacuum negative pressure is repeatedly applied for 1.5 min/time;

(4) co-culturing: after infection, the explants were spread on filter paper and the attached bacterial liquid was slightly blotted dry, and then the explants were transferred to a co-cultivation medium padded with a layer of filter paper. Co-culturing at 24 deg.C in dark for 4 days. Observing the red light emitting condition of the DsRed under a fluorescent body type microscope, and evaluating the infection efficiency;

(5) and (3) recovery culture: washing the explant 7-8 times with sterile water, drying the liquid attached to the surface with sterilized absorbent paper, and obliquely inserting the explant on a recovery culture medium at 30 degrees or 45 degrees;

(6) differentiation culture: culturing under light for 7d, changing to a differentiation culture dish, culturing under light for three weeks, selecting cotyledons containing DsRed fluorescent buds under a body type fluorescence microscope, and subculturing to a tissue culture bottle containing a regeneration culture medium or an elongation culture medium;

(7) elongation: when DsRed fluorescent bud is larger than 1cm, cutting off cotyledon explant, false positive escaping leaf and bud, culturing fluorescent bud in tissue culture bottle containing elongation culture medium under light;

(8) rooting: when the DsRed fluorescent bud is larger than 4cm, subculturing the DsRed fluorescent bud to a rooting culture medium to induce rooting;

(9) moving and cutting: transplanting the transgenic positive seedlings with good rooting into a sterilized substrate, hardening seedlings in an illumination incubator, squatting the seedlings for one month, and detecting.

The results of the DsRed test indicate: the vector P can be smoothly transformed into wild cucumber, about 2000 explants are transformed to obtain 5 red fluorescent seedlings, and the genetic transformation efficiency is about 0.25%.

The fluorescent seedlings are transplanted to a greenhouse for cultivation, and a large number of seeds are bred, and fruits with red epidermis are produced, which indicates that the obtained transformed plants are ideal transformed plants. The stably transformed cucumber seedlings and the microscopic cucumber buds and cucumber tendrils with red fluorescence are shown in fig. 2, and the red cucumber fruits and seeds harvested from the stably transformed cucumber seedlings are shown in fig. 3.

It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.