CN102433354B - A kind of screening technique that xylose isomerase gene is used for Semen arachidis hypogaeae genetic transformation - Google Patents
A kind of screening technique that xylose isomerase gene is used for Semen arachidis hypogaeae genetic transformation Download PDFInfo
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Abstract
The invention provides a kind of screening technique that xylose isomerase gene is used for Semen arachidis hypogaeae genetic transformation, it includes building the recombinant vector containing genes of interest；Recombinant vector is converted the outer implant of peanut embryo lobule；Outer for cotyledon Leaflet after conversion implant is transferred to the inducing culture being added with sucrose and xylose is cultivated, until inducing body embryo；Body embryo is transferred to germination medium is cultivated, until induction seedling；Regrowth is carried out PCR detection, it is thus achieved that transgenic positive plant；Transgenic positive plant is transplanted field by engrafting method.The present invention uses the culture medium of the xylose of sucrose and the 10g/L that with the addition of 5g/L to cultivate, non-transformed body can not seedling owing to xylose growth can not be utilized to be suppressed, transformant can regenerate seedling owing to can utilize xylose, therefore transfer-gen plant can be filtered out, it is to avoid use antibiotic to carry out screening the potential safety hazard being likely to result in；Transformation efficiency is high, is a kind of safe and efficient screening technique.
Description
Technical field
The present invention relates to peanut molecule breeding field, be specifically related to a kind of screening technique that xylose isomerase gene is used for Semen arachidis hypogaeae genetic transformation.
Background technology
Semen arachidis hypogaeae is the important oil crop of China and industrial crops, is respectively provided with critical role in agriculture or even whole national economy.But, owing to Among Cultivated Peanuts hereditary basis is narrow, resistance is poor, the especially multiple pest and disease damage of susceptible, have a strong impact on its yield and quality.Along with the development of biotechnology, transgenic technology is utilized to be subject to the extensive attention of researcher to improve Among Cultivated Peanuts.And utilize antibiotics gene and antiweed genoid to screen in Semen arachidis hypogaeae genetic transformation process, it is possible to environment and human health can be produced harmful effect and infringement.Therefore, utilize uncontested bio-safety marker gene to carry out construction of expression vector, be just increasingly becoming new study hotspot.Xylose is the key component of hemicellulose, nature also exists some and utilizes the filamentous fungi of xylose, yeast and antibacterial, but many plant cells can not utilize xylose, so being difficult to xylosyl because waiting safe marker gene to be used in plant to screen transformant.
Summary of the invention
Defect and the deficiency that screening exists for utilizing antibiotics gene and antiweed genoid to carry out in Semen arachidis hypogaeae genetic transformation process in prior art, the invention provides a kind of screening technique that xylose isomerase gene is used for Semen arachidis hypogaeae genetic transformation.The present invention constructs the plant expression vector pCAMBIA1301-xylA containing xylA gene, and by the outer implant of Agrobacterium-mediated transformation peanut embryo lobule, with the addition of the enterprising row filter of culture medium of sucrose (5g/L) and xylose (10g/L), induction planting percent reaches 15.25%, and transgenic positive rate reaches 77.27%.Present invention, avoiding the potential safety hazard utilizing antibiotic-screening to be likely to result in, and transformation efficiency is high, be a kind of safe and efficient screening technique.
For reaching to solve the purpose of above-mentioned technical problem, the present invention is achieved by the following technical solutions:
A kind of screening technique that xylose isomerase gene is used for Semen arachidis hypogaeae genetic transformation, it comprises the following steps:
(1) genes of interest is inserted in the plant expression vector containing xylose isomerase gene xylA, builds the recombinant vector containing genes of interest；
(2) recombinant vector is converted the outer implant of peanut embryo lobule；
(3) the outer implant of cotyledon Leaflet after converting is transferred to the inducing culture being added with sucrose and xylose is cultivated, after 4 ~ 5 weeks, induce body embryo；
(4) body embryo is transferred on the germination medium being added with sucrose and xylose and cultivate, after 4 ~ 5 months, induce seedling；
(5) regrowth is carried out PCR detection, it is thus achieved that transgenic positive plant；
(6) transgenic positive plant is transplanted field by engrafting method.
Further improvement to technical scheme: in described step (1), plant expression vector is pCAMBIA1301-xylA.
Further improvement to technical scheme: utilize agrobacterium-mediated transformation that recombinant vector converts the outer implant of peanut embryo lobule in described step (2).
Further improvement to technical scheme: in described step (3) inducing culture be containing 5g/L sucrose, 10g/L xylose, 10mg/L2,4-D MS-B5Culture medium.
Further improvement to technical scheme: in described step (4) germination medium be containing 5g/L sucrose, 10g/L xylose, 4mg/LBAP MS-B5Culture medium.
Further improvement to technical scheme: the condition of culture in described step (3) and (4) is 25 DEG C, 3000lx, illumination every day 13h.
Further improvement to technical scheme: in described step (5), the xylA gene primer of PCR detection is:
Forward primer is 5-CTCGAGATGGAGTTCAATATGCAAGCCTA-3,
Downstream primer is 5-CTCGAGATTATTTGTCGAACAGATAATGGTTT-3.
Compared with prior art, advantages of the present invention and having the benefit effect that
1, the screening technique that xylose isomerase gene is used for Semen arachidis hypogaeae genetic transformation of the present invention, use with the addition of the culture medium of sucrose (5g/L) and xylose (10g/L) and cultivates, xylose can be xylulose by transformant under the catalysis of xylose isomerase gene xylA, then through phosphopentose pathway catabolism, utilized by Growth of Cells, with xylose for the culture medium of primary carbon source, converting cell and present dominant growth because utilizing xylose, non-transformed body then makes growth be suppressed because carbon source is under-supply can not seedling.Therefore transformant can be filtered out, it is to avoid use antibiotic to carry out screening the potential safety hazard being likely to result in.
2, cultivating in the culture medium adding sucrose (5g/L) and xylose (10g/L), induction planting percent reaches 15.25%, and obtained regeneration plant up to 77.27%, is a kind of safe and efficient screening technique through PCR Positive rate.
Accompanying drawing explanation
Fig. 1 is peanut embryo lobule outer implant growing state in the culture medium adding sucrose (10g/L) in the present invention.
Fig. 2 is peanut embryo lobule outer implant growing state in the culture medium adding sucrose (5g/L) in the present invention.
Fig. 3 be in the present invention transfer-gen plant add sucrose (5g/L) and xylose (10g/L) culture medium on screen after PCR testing result.
Fig. 4 is transgenic regenerated plant graft and transplantation field growing situation in the present invention.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, technical scheme is described in further detail.
Xylose isomerase gene is used for the screening technique of Semen arachidis hypogaeae genetic transformation by embodiment 1, foundation
1, the clone of xylA gene
Design primer according to xylA gene order (accession number No.X04691) in GenBank, and add at primer 5 endXhoI restriction enzyme site (italicized item):
Forward primer is 5-CTCGAGATGGAGTTCAATATGCAAGCCTA-3,
Downstream primer is 5-CTCGAGATTATTTGTCGAACAGATAATGGTTT-3ˊ。
Carrying out PCR reaction with escherichia coli DH-5 α strain gene group DNA for template, amplification xylA gene is also cloned on pMD18-T carrier (purchased from precious biological Dalian Engineering Co., Ltd), obtains T-xylA recombiant plasmid.
2, the structure of plant expression vector pCAMBIA1301-xylA
WithXhoI enzyme action pCAMBIA1301 plasmid (purchased from Shanghai Xi Piji Bioisystech Co., Ltd), removes hygromycin phosphotransferase gene (hygromycin-B-phosphotransferase, Hpt), usesXhoI enzyme action T-xylA recombiant plasmid, cuts xylA genetic fragment.Reclaim pCAMBIA1301 plasmid large fragment and xylA gene small fragment, use T4XylA fragment is connected on the pCAMBIA1301 plasmid of excision Hpt gene by DNA ligase, obtains recombiant plasmid pCAMBIA1301-xylA.
3, expression vector is converted Semen arachidis hypogaeae, comprises the following steps:
A, the preparation of Agrobacterium recombinant bacterial strain, activation and bacterium solution preparation: pCAMBIA1301-xylA recombiant plasmid utilizes frozen-thawed method convert agrobacterium strains EHA105(purchased from sky, Beijing bounties Gene Tech. Company Limited) competent cell, filter out the recombinant bacterial strain containing recombiant plasmid.Picking recombinant bacterial strain list bacterium colony, is inoculated into YEB(rifampicin 50mg/L, kanamycin 50mg/L) in fluid medium, 28 DEG C, 180rpm be cultured to OD600When=0.5 ~ 0.8, take 2mL bacterium solution and transfer to 50mLYEB(rifampicin 50mg/L, kanamycin 50mg/L) in culture medium, cultivate OD600=0.6 ~ 0.8.By bacterium solution after the centrifugal 15min of 5000rpm, with the liquid MS-B of same volume5Suspend standby.
The separation of the outer implant of b, peanut embryo lobule: cut by the peanut embryo lobule together with plumular axis, soaks 10 ~ 20s in 70% ethanol, soaks 8min in 0.1% mercuric chloride, and soaked overnight after aseptic water washing, plumular axis is cut and isolates cotyledon Leaflet by next day, puts into MS-B5Preculture 3 days in culture medium, condition of culture is 25 DEG C, 3000lx, illumination every day 13h.
C, Agrobacterium-mediated genetic transformation: outer for the cotyledon Leaflet after preculture 3 days implant is dipped in the Agrobacterium bacterium solution got ready, 28 DEG C, the concussion of 90rpm gentleness infect 15min.With aseptic filter paper, residual bacterium solution is blotted, be inoculated into MS-B5After culture medium co-cultures 3 days, transfer to the body embryonal induction culture medium (MS-B adding Carbenicillin (500mg/L)5, add 10mg/L2,4-D) and middle cultivation, induce body embryo after about 4 ~ 5 weeks.Condition of culture is 25 DEG C, 3000lx, illumination every day 13h.
4, the foundation of xylose screening system, comprises the following steps:
A, minimal medium and condition of culture: culture medium is MS-B5, add the sucrose/xylose concentration ratio of variable concentrations, 0.8% agar, pH is 5.8,121 DEG C, 105Kpa when sterilizing 20min.Condition of culture is 25 DEG C, 3000lx, illumination every day 13h.
B, the suitableeest sucrose concentration determination: take peanut varieties " flower educate 22 " mature seed 24, be divided into 4 groups, often group 6.Separate the outer implant (method is ibid) of cotyledon Leaflet, 0.1% mercuric chloride sterilization 10min, it is then transferred into MS-B5Culture medium is cultivated, and sucrose concentration is 5g/L respectively, 10g/L, 20g/L, 30g/L.Statistical analysis is carried out after cultivating 4 ~ 5 months.Result shows that, when sucrose concentration is when 10g/L and concentrations above, outer implant can seedling differentiation (as shown in Figure 1)；When sucrose concentration is 5g/L, outer implant rests on the body embryo stage, it is impossible to seedling differentiation (as shown in Figure 2).So finally determining that 5g/L is the minimum critical concentration of sucrose.
C, the suitableeest xylose concentration determination: utilize agrobacterium-mediated transformation that recombiant plasmid pCAMBIA1301-xylA converts the outer implant of peanut embryo lobule, it is then transferred into the addition of sucrose (5g/L) and variable concentrations xylose (5g/L, 10g/L, 20g/L, 30g/L) body embryonal induction culture medium on cultivate.The body embryo induced forwards to the addition of and cultivates in the body embryo germination culture medium of sucrose (5g/L) and variable concentrations xylose (5g/L, 10g/L, 20g/L, 30g/L), carries out statistical analysis after about 4 ~ 5 months induction seedlings.Result is as shown in table 1, and along with the rising of xylose concentration, induction planting percent declines to some extent；When xylose concentration is 5g/L, induction planting percent the highest (17.54%), but plant strain growth is more weak；When xylose concentration is 10g/L, induction planting percent higher (15.25%), plant strain growth is healthy and strong, and graft survival rate is high；So finally determining that 10g/L is the suitableeest xylose concentration.
Table 1: the planting percent of outer implant and ratoon growth situation under different xylose concentrations
| Xylose concentration | Outer implant number | Number of seedling | Planting percent | Ratoon growth situation |
| 5g/L | 285 | 50 | 17.54% | Growing more weak, graft survival rate is low |
| 10g/L | 282 | 43 | 15.25% | Growth is normal, and graft survival rate is high |
| 20g/L | 272 | 29 | 10.66% | Growth is normal, and graft survival rate is high |
| 30g/L | 302 | 18 | 5.96% | Growth is normal, and brownization mortality rate is high |
4, the PCR detection of transfer-gen plant
Extract the genomic DNA of regeneration plant, utilize above-mentioned xylA gene primer to carry out pcr amplification.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.Transgenic positive rate reaches 77.27%, and PCR electrophoresis pattern is as shown in Figure 3.
5, transgenic positive plant is carried out graft and transplantation
For stock with " flower educates 20 " aseptic seedling of 12 ~ 15d seedling age, excising the stem part saving more than 2cm from cotyledon, vertically rived stock upper end with scalpel, otch is 0.5 ~ 1cm deeply about.When transfer-gen plant grows to about 2 ~ 3cm, cutting regeneration seedling from bud clump base portion and do scion, the V-arrangement wound being about 0.5 ~ 1cm is cut in lower end, and otch is smooth.Scion is inserted in stock, make the cambium layer of stock and scion be in close contact, be then wound around interface with sealed membrane, degree of tightness appropriateness.Grafting is placed in MS-B5Aseptic culture 3 ~ 4 days in culture medium；Then transplant and in the seedling medium of sterilizing, (include Vermiculitum, turfy soil and perlite) carry out domestication 3 weeks, be transplanted to field afterwards.Transfer-gen plant is acted normally in field growing situation, as shown in Figure 4.
Embodiment 2, utilize xylose screening technique inspection glucanase gene Glu conversion
The screening technique that xylose isomerase gene is used for Semen arachidis hypogaeae genetic transformation of the present invention specifically includes following steps:
1, the recombiant plasmid containing genes of interest is built: by genes of interest β-1,3-glucanase gene (Glu) (accession number M58462.1) 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 (method for transformation is with example 1) of peanut embryo lobule.
3, outer for cotyledon Leaflet after conversion implant is transferred to cultivation in body embryonal induction culture medium
Outer for cotyledon Leaflet after conversion implant is transferred to the body embryonal induction culture medium (MS-B that with the addition of sucrose (5g/L) and xylose (10g/L)5, add 10mg/L2,4-D) on cultivate, obtain body embryo after about 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 transfers to the body embryo germination culture medium (MS-B containing sucrose (5g/L) and xylose (10g/L)5, add 4mg/LBAP) on cultivate, after about 4 ~ 5 months induce seedling.Concrete condition of culture is 25 DEG C, 3000lx, 13h illumination every day.
5, the statistics of regrowth and PCR detection
After 4 ~ 5 months cultivate, 213 outer implant of cotyledon Leaflet there are 32 regeneration plants, induces planting percent 15%.
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
Adopt sterile grafting method as described in Example 1 that transfer-gen plant is carried out grafting, transplant field afterwards.Transfer-gen plant is acted normally in field growing situation.
Present invention escherichia coli xylose isomerase gene xylA replaces the hygromycin phosphotransferase gene in pCAMBIA1301, build plant expression vector pCAMBIA1301-xylA, expression vector is proceeded to agrobacterium strains EHA105, utilize agrobacterium-mediated transformation that pCAMBIA1301-xylA imports the outer implant of peanut embryo lobule, transfer to the body embryonal induction culture medium (MS-B that with the addition of sucrose (5g/L) and xylose (10g/L)5, add 10mg/L2,4-D) and body embryo germination culture medium (MS-B5, add 4mg/LBAP) on cultivate, condition of culture is 25 DEG C, 3000lx, illumination every day 13h.After transformed plant is cultivated 4 months, induction planting percent reaches 15.25%, and transgenic positive rate reaches 77.27%.
Above example is only in order to illustrate technical scheme, but not is limited；Although the present invention being described in detail with reference to previous embodiment, for the person of ordinary skill of the art, still the technical scheme described in previous embodiment can be modified, or wherein portion of techniques feature is carried out equivalent replacement；And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of present invention technical scheme required for protection.
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.
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| CN105103859B (en) * | 2015-08-17 | 2017-10-13 | 青岛农业大学 | A kind of method for transplanting of peanut tissue culture seedlings |
| CN108148859B (en) * | 2018-03-05 | 2019-05-31 | 山东省农业科学院生物技术研究中心 | A method of utilizing expression vector screening transgenic peanut of the building containing △ 12- fatty acid dehydrogenase gene |
| CN115433740B (en) * | 2022-10-19 | 2024-09-20 | 连云港市农业科学院 | Method for easily obtaining positive callus and rapidly identifying genetic transformation of camellia oleifera |
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