CN102433354B - A kind of screening technique that xylose isomerase gene is used for Semen arachidis hypogaeae genetic transformation - Google Patents

Abstract

The invention provides a screening method for using xylose isomerase gene in peanut genetic transformation, which comprises constructing a recombinant vector containing the target gene; transforming the recombinant vector into peanut embryo leaflet explant; explanting the transformed embryo leaflet The somatic embryos were transferred to the induction medium supplemented with sucrose and xylose for cultivation until the somatic embryos were induced; the somatic embryos were transferred to the germination medium for cultivation until the seedlings were induced; the regenerated seedlings were detected by PCR and positive for the transgene was obtained Plants; the transgenic positive plants were transplanted into the field by grafting. The present invention uses the substratum that has added the sucrose of 5g/L and the xylose of 10g/L to cultivate, and non-transformant can't become seedling because can not utilize xylose growth to be suppressed, and transformant can regenerate seedling because of being able to utilize xylose, Therefore, transgenic plants can be screened out, avoiding potential safety hazards that may be caused by using antibiotics for screening; the transformation efficiency is high, and it is a safe and efficient screening method.

Description

A Screening Method for Genetic Transformation of Peanut Using Xylose Isomerase Gene

technical field

The invention relates to the field of peanut molecular breeding, in particular to a screening method for using xylose isomerase gene in peanut genetic transformation.

Background technique

Peanut is an important oil crop and economic crop in my country, and plays an important role in agriculture and even the entire national economy. However, due to the narrow genetic basis and poor stress resistance of peanut cultivars, they are particularly susceptible to various pests and diseases, which seriously affect their yield and quality. With the development of biotechnology, the use of transgenic technology to improve peanut cultivars has received extensive attention from researchers. The use of antibiotic genes and herbicide resistance genes for screening in the process of peanut genetic transformation may have adverse effects and damages on the environment and human health. Therefore, the use of uncontroversial biosafety marker genes to construct expression vectors has gradually become a new research hotspot. Xylose is the main component of hemicellulose. There are some filamentous fungi, yeasts and bacteria that utilize xylose in nature, but many plant cells cannot utilize xylose, so it is difficult to use safety marker genes such as xylose gene in plants to select transformants.

Contents of the invention

Aiming at the deficiencies and deficiencies in the prior art of using antibiotic genes and herbicide resistance genes for screening in peanut genetic transformation, the present invention provides a screening method for peanut genetic transformation using xylose isomerase gene. The present invention constructs the plant expression vector pCAMBIA1301-xylA containing the xylA gene, and transforms the peanut embryo leaflet explants through the Agrobacterium-mediated method, in the medium added with sucrose (5g/L) and xylose (10g/L) Screened on the above, the induced seedling rate reached 15.25%, and the transgenic positive rate reached 77.27%. The invention avoids potential safety hazards that may be caused by antibiotic screening, and has high transformation efficiency, and is a safe and efficient screening method.

In order to achieve the purpose of solving the above technical problems, the present invention adopts the following technical solutions to achieve:

A screening method for using xylose isomerase gene in peanut genetic transformation, which comprises the following steps:

(1) Inserting the target gene into a plant expression vector containing the xylose isomerase gene xylA to construct a recombinant vector containing the target gene;

(2) transforming the recombinant vector into peanut embryo leaflet explants;

(3) Transfer the transformed embryo leaflet explants to the induction medium added with sucrose and xylose for culture, and induce somatic embryos after 4 to 5 weeks;

(4) Transfer the somatic embryos to the germination medium added with sucrose and xylose for cultivation, and induce seedlings after 4 to 5 months;

(5) Carry out PCR detection to regenerated seedling, obtain transgenic positive plant;

(6) The transgenic positive plants are transplanted into the field by grafting.

Further improvement to the technical solution: the plant expression vector in the step (1) is pCAMBIA1301-xylA.

Further improvement on the technical solution: In the step (2), the recombinant vector is transformed into the peanut embryo leaflet explant by using the Agrobacterium-mediated method.

Further improvement to the technical solution: the induction medium in the step ( ₃ ) is MS-B5 medium containing 5g/L sucrose, 10g/L xylose, and 10mg/L 2,4-D.

Further improvement to the technical solution: the germination medium in the step (4) is the MS-B ₅ medium containing 5g/L sucrose, 10g/L xylose, and 4mg/LBAP.

Further improvement to the technical solution: the culture conditions in the steps (3) and (4) are 25° C., 3000 lx, and 13 hours of light per day.

Further improvement to the technical scheme: the xylA gene primers detected by PCR in the step (5) are:

The upstream primer is 5′-CTCGAGATGGAGTTCAATATGCAAGCCTA-3′,

The downstream primer is 5′-CTCGAGATTATTTGTCGAACAGATAATGGTTT-3′.

Compared with prior art, advantage and positive effect of the present invention are:

1. The screening method of using the xylose isomerase gene for peanut genetic transformation according to the present invention is cultivated in a medium supplemented with sucrose (5g/L) and xylose (10g/L), and the transformant is grown in xylose Under the catalysis of the isomerase gene xylA, xylose can be converted into xylulose, and then catabolized by the pentose phosphate pathway, which can be used for cell growth. On the medium with xylose as the main carbon source, the transformed cells can Utilizing xylose showed dominant growth, while non-transformants were inhibited in growth due to insufficient supply of carbon source and could not form seedlings. Therefore, transformants can be screened out, which avoids potential safety hazards caused by screening with antibiotics.

2. Cultivated in the medium added with sucrose (5g/L) and xylose (10g/L), the induced seedling rate reaches 15.25%, and the positive rate of the regenerated plants obtained by PCR is as high as 77.27%, which is a safe , Efficient screening method.

Description of drawings

Figure 1 shows the growth of peanut embryo leaflet explants on the medium added with sucrose (10g/L) in the present invention.

Figure 2 shows the growth of peanut embryo leaflet explants on the medium added with sucrose (5g/L) in the present invention.

Fig. 3 is the PCR detection result of the transgenic plants in the present invention after selection on the medium supplemented with sucrose (5g/L) and xylose (10g/L).

Fig. 4 is the field growth of grafted and transplanted regenerated transgenic plants in the present invention.

detailed description

The technical solution of the present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

Example 1. Establishment of a screening method using xylose isomerase gene for peanut genetic transformation

1. Cloning of xylA gene

Primers were designed according to the xylA gene sequence in GenBank (Accession No. X04691), and an Xho I restriction site (italic part) was added at the 5' end of the primer:

The upstream primer is 5′- CTCGAG ATGGAGTTCAATATGCAAGCCTA-3′,

The downstream primer is 5′- CTCGAG ATTATTTGTCGAACAGATAATGGTTT-3′.

The genomic DNA of Escherichia coli DH-5α strain was used as a template for PCR reaction to amplify the xylA gene and clone it into the pMD18-T vector (purchased from Treasure Biotech Dalian Engineering Co., Ltd.) to obtain the T-xylA recombinant plasmid.

2. Construction of plant expression vector pCAMBIA1301-xylA

The pCAMBIA1301 plasmid (purchased from Shanghai Xipiji Biotechnology Co., Ltd.) was digested with Xho Ⅰ, the hygromycin-B-phosphotransferase gene (hygromycin-B-phosphotransferase, Hpt) was removed, the T-xylA recombinant plasmid was digested with Xho Ⅰ, and Next xylA gene fragment. Recover the large fragment of pCAMBIA1301 plasmid and the small fragment of xylA gene, and use T ₄ DNA ligase to connect the xylA fragment to the pCAMBIA1301 plasmid that excised the Hpt gene to obtain the recombinant plasmid pCAMBIA1301-xylA.

3. Transforming the expression vector into peanuts, comprising the following steps:

a. Preparation and activation of Agrobacterium recombinant strains and preparation of bacteria solution: The pCAMBIA1301-xylA recombinant plasmid was transformed into competent cells of Agrobacterium strain EHA105 (purchased from Beijing Tianenze Gene Technology Co., Ltd.) by liquid nitrogen freeze-thaw method, and the cells containing Recombinant strains with recombinant plasmids. Pick a single colony of the recombinant strain, inoculate it into YEB (rifampicin 50mg/L, kanamycin 50mg/L) liquid medium, cultivate at 28°C and 180rpm until OD ₆₀₀ =0.5~0.8, then take 2mL of bacterial liquid and transfer Put them into 50mL YEB (rifampicin 50mg/L, kanamycin 50mg/L) medium and culture to OD ₆₀₀ =0.6~0.8. After centrifuging the bacterial solution at 5000rpm for 15min, suspend it with the same volume of liquid MS-B ₅ for use.

b. Isolation of peanut embryo leaflet explants: cut off the peanut embryo leaflet together with the hypocotyl, soak in 70% ethanol for 10-20 seconds, soak in 0.1% mercury liter for 8 minutes, rinse with sterile water and soak overnight, and the next day The hypocotyls were cut off to separate the embryo leaflets, and put into MS-B ₅ medium for pre-cultivation for 3 days. The culture conditions were 25°C, 3000 lx, and 13 hours of light per day.

c. Genetic transformation mediated by Agrobacterium: immerse the embryo leaflet explants pre-cultured for 3 days in the prepared Agrobacterium solution, and infect with gentle shaking at 90 rpm for 15 minutes at 28°C. Use sterile filter paper to blot the residual bacterial solution dry, inoculate into MS-B ₅ medium and co-cultivate for 3 days, then transfer to somatic embryo induction medium (MS-B ₅ , add 10 mg/L) with carbenicillin (500 mg/L). L2, 4-D) were cultured, and somatic embryos were induced after about 4 to 5 weeks. The culture conditions are 25°C, 3000 lx, and 13 hours of light per day.

4. The establishment of xylose screening system comprises the following steps:

a. Basic medium and culture conditions: the medium is MS-B ₅ , added with different concentrations of sucrose/xylose concentration ratio, 0.8% agar, the pH is 5.8, and sterilized at 121°C and 105Kpa for 20 minutes. The culture conditions are 25°C, 3000 lx, and 13 hours of light per day.

b. Determination of the optimum sucrose concentration: 24 mature seeds of the peanut variety "Huayu 22" were taken and divided into 4 groups with 6 seeds in each group. The embryonic leaflet explants were isolated (the same method as above), sterilized with 0.1% mercuric chloride for 10 minutes, and then transferred to MS-B ₅ medium for cultivation, with sucrose concentrations of 5g/L, 10g/L, 20g/L, and 30g/L, respectively. Statistical analysis was performed after 4-5 months of culture. The results showed that when the sucrose concentration was 10g/L and above, the explants could differentiate into seedlings (as shown in Figure 1); when the sucrose concentration was 5g/L, the explants stayed at the somatic embryo stage and could not differentiate into Seedlings (as shown in Figure 2). So finally determine 5g/L as the minimum critical concentration of sucrose.

c. Determination of the optimal xylose concentration: the recombinant plasmid pCAMBIA1301-xylA was used to transform peanut embryo leaflet explants by Agrobacterium-mediated method, and then transferred to the explants with sucrose (5g/L) and different concentrations of xylose (5g/L). , 10g/L, 20g/L, 30g/L) cultured on somatic embryo induction medium. The induced somatic embryos were transferred to somatic embryo germination medium supplemented with sucrose (5g/L) and different concentrations of xylose (5g/L, 10g/L, 20g/L, 30g/L) for culture, about 4~ Statistical analysis was performed after 5 months of seedling induction. The results are shown in Table 1. With the increase of xylose concentration, the induced seedling rate decreased; when the xylose concentration was 5g/L, the induced seedling rate was the highest (17.54%), but the plant growth was weak; When the xylose concentration was 10g/L, the induced seedling rate was higher (15.25%), the plants grew robustly, and the survival rate of grafting was high; so 10g/L was finally determined as the optimal xylose concentration.

Table 1: Seedling formation rate of explants and growth of regenerated seedlings under different xylose concentrations

Xylose concentration Number of explants Number of seedlings Seedling rate Growth of regenerated seedlings 5g/L 285 50 17.54% Weak growth, low graft survival 10g/L 282 43 15.25% Normal growth, high graft survival rate 20g/L 272 29 10.66% Normal growth, high graft survival rate 30g/L 302 18 5.96% Normal growth, high browning mortality

4. PCR detection of transgenic plants

Genomic DNA of regenerated plants is extracted, and PCR amplification is carried out by using the above-mentioned xylA gene primers. The PCR reaction program is: 95°C, 5min; 95°C for 50s, 56°C for 50s, 72°C for 70s, 30 cycles; 72°C, 10min. The positive rate of transgene reached 77.27%, and the PCR electrophoresis pattern is shown in Figure 3.

5. Grafting and transplanting transgenic positive plants

Use the aseptic seedlings of "Huayu 20" with a seedling age of 12-15 days as the rootstock, cut off the main stem part that is more than 2cm away from the cotyledons, and split the upper end of the rootstock vertically with a scalpel, and the incision is about 0.5-1cm deep. When the transgenic plant grows to about 2-3 cm, the regenerated seedlings are cut from the base of the bud cluster to make a scion, and the lower end is cut into a V-shaped wound about 0.5-1 cm long, and the incision is smooth. Insert the scion into the rootstock so that the cambium of the rootstock and the scion are in close contact, and then wrap the joint with a parafilm with a moderate degree of tightness. The grafted seedlings were cultured aseptically in MS-B ₅ medium for 3-4 days; then transplanted into sterile seedling-raising substrates (including vermiculite, peat soil and perlite) for 3 weeks of acclimatization, and then transplanted into field. The transgenic plants grew normally in the field, as shown in Figure 4.

Embodiment 2, utilizing the xylose screening method to test the transformation of the glucanase gene Glu

The screening method using xylose isomerase gene of the present invention for peanut genetic transformation specifically comprises the following steps:

1. Construction of a recombinant plasmid containing the target gene: Insert the target gene β-1,3-glucanase gene (Glu) (accession number M58462.1) into the plant expression vector pCAMBIA1301-xylA to obtain a recombinant plasmid containing the target gene Glu pCAMBIA1301-xylA-Glu.

2. Transform the recombinant plasmid containing the target gene into peanut

The obtained recombinant plasmid pCAMBIA1301-xylA-Glu was transformed into peanut embryo leaflet explants by the Agrobacterium-mediated method (the transformation method was the same as in Example 1).

3. Transfer the transformed embryo leaflet explants to somatic embryo induction medium for culture

Transfer the transformed embryo leaflet explants to the somatic embryo induction medium (MS-B ₅ , supplemented with 10mg/L2,4-D) supplemented with sucrose (5g/L) and xylose (10g/L) for culture , somatic embryos were obtained after about 4 to 5 weeks. The specific culture conditions are 25°C, 3000lx, and 13h light per day.

4. Transfer the induced somatic embryos to the somatic embryo germination medium for culture

The induced somatic embryos were transferred to the somatic embryo germination medium (MS-B ₅ , supplemented with 4mg/LBAP) containing sucrose (5g/L) and xylose (10g/L) for culture, and after about 4 to 5 months Induce seedlings. The specific culture conditions are 25°C, 3000lx, and 13h light per day.

5. Statistics and PCR detection of regenerated seedlings

After 4-5 months of cultivation, 32 regenerated plants were obtained from 213 embryonic leaflet explants, and the induced seedling rate was 15%.

The primers for PCR detection are:

The upstream primer is 5′-CTCGAGATGGAGTTCAATATGCAAGCCTA-3′,

The downstream primer is 5′-CTCGAGATTATTTGTCGAACAGATAATGGTTT-3′.

The PCR reaction program is: 95°C, 5min; 95°C for 50s, 56°C for 50s, 72°C for 70s, 30 cycles; 72°C, 10min. The test showed that the positive rate of transgene reached more than 75%.

6. Grafting and transplanting the transgenic positive seedlings into the field

Transgenic plants were grafted using the aseptic grafting method shown in Example 1, and then transplanted into the field. The transgenic plants grew normally in the field.

The present invention replaces the hygromycin phosphotransferase gene in pCAMBIA1301 with xylA of E. coli xylose isomerase gene xylA, constructs the plant expression vector pCAMBIA1301-xylA, transfers the expression vector into Agrobacterium strain EHA105, utilizes Agrobacterium-mediated method to transform pCAMBIA1301-xylA was introduced into peanut embryo leaflet explants, and transferred to somatic embryo induction medium supplemented with sucrose (5g/L) and xylose (10g/L) (MS-B ₅ , supplemented with 10mg/L2, 4-D) and somatic embryo germination medium (MS-B ₅ , supplemented with 4mg/LBAP), and the culture conditions were 25°C, 3000lx, and 13h of light per day. After the transformed plants were cultured for 4 months, the induced seedling rate reached 15.25%, and the transgenic positive rate reached 77.27%.

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still understand the foregoing embodiments. The recorded technical solutions are modified, or some of the technical features are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed in the present invention.

Claims (1)

1. the screening technique that xylose isomerase gene is used for Semen arachidis hypogaeae genetic transformation, it is characterised in that it comprises the following steps:

(1) recombiant plasmid containing genes of interest is built: by the genes of interest β-1 of accession number M58462.1,3-glucanase gene Glu inserts plant expression vector pCAMBIA1301-xylA, obtains the recombiant plasmid pCAMBIA1301-xylA-Glu containing genes of interest Glu；

(2) by the recombinant plasmid transformed Semen arachidis hypogaeae containing genes of interest

Utilize agrobacterium-mediated transformation that the recombiant plasmid pCAMBIA1301-xylA-Glu obtained converts the outer implant of peanut embryo lobule；

(3) outer for cotyledon Leaflet after conversion implant is transferred to cultivation in body embryonal induction culture medium

Transferring to the addition of by outer for cotyledon Leaflet after conversion implant and cultivate in the body embryonal induction culture medium of sucrose 5g/L and xylose 10g/L, body embryonal induction culture medium is add the MS-B of 10mg/L2,4-D₅, obtain body embryo after 4～5 weeks, concrete condition of culture is 25 DEG C, 3000lx, 13h illumination every day；

(4) the body embryo induced is transferred to and is cultivated in body embryo germination culture medium

The body embryo induced is transferred to and is cultivated in the body embryo germination culture medium containing sucrose 5g/L and xylose 10g/L, and body embryo germination culture medium is add the MS-B of 4mg/LBAP₅, induce seedling after 4～5 months, concrete condition of culture is 25 DEG C, 3000lx, 13h illumination every day；

(5) statistics of regrowth and PCR detection

The primer of PCR detection is:

Forward primer is 5 '-CTCGAGATGGAGTTCAATATGCAAGCCTA-3 ',

Downstream primer is 5 '-CTCGAGATTATTTGTCGAACAGATAATGGTTT-3 '；

PCR response procedures is: 95 DEG C, 5min；95 DEG C of 50s, 56 DEG C of 50s, 72 DEG C of 70s, 30 circulations；72 DEG C, 10min, detection shows that transgenic positive rate reaches more than 75%；

(6) by transgenic positive Seedling graft and transplantation field.