CN102517327A - Method for establishing agrobacterium tumefacien mediated castor genetic transformation system - Google Patents
Method for establishing agrobacterium tumefacien mediated castor genetic transformation system Download PDFInfo
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Abstract
The invention relates to a plant genetic transformation method in the field of bioengineering, and relates to a method for establishing an agrobacterium tumefacien mediated castor genetic transformation system. The method is characterized in that castor cotyledonary nodes are adopted as acceptors; culturing, agrobacterium tumefacien infecting, co-culturing, bacteria separating, screening and seedling rooting are carried out, such that castor progenies with descendible target genes are obtained. The method provided by the invention is advantaged in simple procedure, optimized parameters, convenient operations, and low cost. With the method, a castor genetic transformation system can be established in a relatively short period of time. According to PCR detections, the effects of the obtained resistant seedlings are good. Therefore, a basis is established for simple and highly efficient castor transgene researches.
Description
Technical field
The present invention relates to the plant gene method for transformation of bioengineering field, promptly a kind of method of setting up agriculture bacillus mediated castor-oil plant genetic conversion system.
Background technology
Research about castor-oil plant transgenic aspect is reported few at present both at home and abroad; Sujatha M etc. utilizes agrobacterium-mediated transformation that the castor-oil plant plumular axis is carried out genetic transformation reported first in 2005; On expression vector, made up herbicide resistance gene bar; With weedicide Basta is selective agent, has obtained transgenic castor-oil plant plant.MalathiB etc. obtain transfer-gen plant at report in 2006 with identical method.The shortcoming of this technology is that the needed time is longer.Mckeon T A etc. declared patent in 2003, infected whole strain, bud point and plumular axis, the bud of castor-oil plant with Agrobacterium.Wherein: it is when the long 20-25cm of arriving of plant is high that whole strain transforms, and its cotyledon and first group of true leaf are wiped out, and uproops, and is inverted in the beaker that fills the Agrobacterium penetrating fluid.This beaker places a vacuum drier, after the vacuum-treat plant is planted the lap waste basin again.Two week of processing, GUS dyeing detection was carried out to the leaf disk of second group of true leaf of this plant in the back, and discovery has positive reaction.The conversion of bud point and plumular axis is the Agrobacterium penetrating fluid to be instilled into the sharp and plumular axis surface of bud carry out conversion processing.Cultivate the sharp and plumular axis regeneration plant of the bud handled and obtain transgenic castor-oil plant plant.The conversion of bud mainly is that wound is marked with pin in the side of the unopened petal still of castor-oil plant female flower bud, then Agrobacterium LBA4404 penetrating fluid is instilled on the wound, and the bud that encases processing with plastics bag at last is to preserve moisture.Such processing can repeat, and is open up to female flower.The seed of the Hua Suojie of collection and treatment might obtain genetically modified castor-oil plant plant through screening.This method is simple, has avoided the complicated processes of tissue culture, is mostly mosaic but its shortcoming is the transfer-gen plant that is obtained.This shows that the genetically modified research of castor-oil plant also very not deeply, is not also set up efficient, a stable genetic conversion system, the establishment method that finds a kind of castor-oil plant genetic conversion system of comparative maturity more useless.
Summary of the invention
The purpose of this invention is to provide in the short period of time a kind ofly, set up the method for efficient, stable castor-oil plant genetic conversion system.
Above-mentioned purpose is realized by following technical scheme: a kind of method of setting up agriculture bacillus mediated castor-oil plant genetic conversion system is provided; Be characterized in: said method is to be the mediation carrier with the Agrobacterium; With the Semen Ricini leaf segment is acceptor; Infect through giving cultivation, Agrobacterium, cultivate altogether, bacterium removes, screening and blastogenesis root, obtain having can hereditary goal gene the castor-oil plant plant.
The choosing method of said Semen Ricini leaf segment is that the castor-oil plant seed is removed sterilization behind the exosper, inoculation, treat seed germination after, select plumular axis and cotyledon to be the germinated seeds in faint yellow period, cut cotyledonary node.
Said Agrobacterium is LBA4404, includes pBI-121-RTA-RTA and repeats expression vector, the last kalamycin resistance gene that has of pBI-121.
The pre-incubated substratum of said Semen Ricini leaf segment is 1/8MS+ZT (8mg/L)+NAA (1mg/L)+sucrose (20g/L)+agar powder (10g/L), and incubation time is set at 2 days in advance.
It is to use OD that said Agrobacterium is infected
600Be 0.5 Agrobacterium LBA4404, infect the cotyledonary node after the preparatory cultivation, time of infection is 20min.
The said cultivation altogether is the cotyledonary node that has infected with Agrobacterium to be inoculated in the substratum of 1/8MS+ZT (8mg/L)+NAA (1mg/L)+sucrose (20g/L)+agar powder (10g/L)+Syringylethanone (100 μ M/L) carry out common cultivation; Incubation time was made as 5 days altogether, till the visible single bacterial plaque of Agrobacterium on the substratum.
Said degerming is that the cotyledonary node after cultivating altogether is inoculated on the substratum of 1/8MS+ZT (8mg/L)+NAA (1mg/L)+sucrose (20g/L)+agar powder (10g/L)+23mg/L kantlex, adds the cephalo that concentration is 1.2g/L simultaneously.
Said screening is to be that the Semen Ricini leaf segment of 23mg/L kantlex after to degerming cultivated processing with concentration, and screening time is 30 days.
The said stage substratum of taking root is 1/16MS+ sucrose (20g/L)+agar powder (10g/L), adds the NAA that concentration is 1.0mg/L simultaneously, after the resistant buds root development is good, tames transplanting.
The invention has the beneficial effects as follows: process is simple; Parameter optimization; Easy to operate, with low cost, can accomplish the foundation of castor-oil plant genetic conversion system in the short period of time; Resulting resistance seedling detects through PCR and obtains satisfied effect, thereby lays a good foundation for carrying out the castor-oil plant transgenic research simply, efficiently.
Description of drawings
Fig. 1 is that pBI-121-RTA-RTA repeats the expression vector collection of illustrative plates;
Fig. 2 is that the castor-oil plant genetic system is set up schema.
Embodiment
The total design of the present invention is: to the problem of prior art existence; Using that still unworn Semen Ricini leaf segment is an acceptor in the castor-oil plant transgenic experiments instead, is mediation with the Agrobacterium, is selective agent with the kantlex; With the cephalo is degerming agent; Through preparatory cultivation, Agrobacterium infect, cultivate altogether, processes such as bacterium removes, screening and blastogenesis root, obtain having can hereditary goal gene the castor-oil plant plant, particularly can be used for the material system of castor-oil plant transgenic research.
Around above-mentioned design,, found the specific practice of each step through a large amount of experiments.Introduce relevant implementation process and experimental technique below:
One, the preparation of experiment material:
Vegetable material is planted industry ltd for leading to castor No. 5 available from the logical section in academy of agricultural sciences, Tongliao City;
Agrobacterium LBA4404 includes pBI-121-RTA-RTA and repeats expression vector, and the last kalamycin resistance gene that has of pBI-121, pBI-121-RTA-RTA repeat the expression vector collection of illustrative plates and see Fig. 1, are preserved by the laboratory.
LA Taq archaeal dna polymerase, DL2000 Marker, primer, kantlex, Rifampin, Vetstrep etc. are all purchased the company in TaKaRa; Cephalo is that Shanghai Xinxianfeng Pharmaceutical Co., Ltd. produces; Zein (ZT), Syringylethanone are the import analytical pure, and it is the home-made analytical pure that naphthylacetic acid (NAA) waits other pharmaceutical chemicalss.
Two, experiment situation:
1. the screening of the righttest kalamycin resistance concentration of Semen Ricini leaf segment: owing to adopt kantlex is selective agent, at first needs to be grasped the kalamycin resistance index of Semen Ricini leaf segment self, and concrete experimentation and result are following:
The castor-oil plant seed is sterilized after removing exosper, inoculation.After treating seed germination, select plumular axis and cotyledon to be the germinated seeds in faint yellow period, cut cotyledonary node.Cotyledonary node is inoculated on the substratum of 1/8MS+ZT 8.0mg/L+NAA1.0mg/L+ sucrose 20g/L+ agar powder 10g/L pH value 5.8 and cultivates.After 7 days, under the situation of other components unchanged, on the substratum that adds kantlex, cotyledonary node is carried out the screening of resistance concentration, the long bud in leaf segment place is the pairing kantlex concentration of white bud and is suitable kalamycin resistance concentration.Screening experiment divides and carries out for 4 times:
Screening for the first time: the concentration gradient of kantlex is 0,20mg/L, 40mg/L, 60mg/L, 80mg/L, 100mg/L, and statistics is seen table 1:
Table 1
From table 1, can find out; When being 20mg/L, kantlex still have 1 cotyledonary node explant to grow green bud; When kantlex is 40mg/L, do not grow green bud on the cotyledonary node explant, the suitable kantlex concentration of this explanation should be between 20mg/L to 40mg/L; Therefore data are accurate inadequately, also will screen.
Programmed screening: according to the first time shaker test result confirm that concentration gradient is 0,5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L, 30mg/L, 35mg/L, 40mg/L, statistics is seen table 2:
Table 2
Can find out that from table 2 suitable kantlex concentration should also should further be screened between 15mg/L to 30mg/L.
Screening for the third time: confirm that according to the programmed screening test-results screening concentration gradient is 18mg/L, 19mg/L, 20mg/L, 21mg/L, 22mg/L, 23mg/L, 24mg/L, 25mg/L, 26mg/L, 27mg/L, 28mg/L, statistics is seen table 3:
Table 3
Can find out that from table 3 suitable kantlex concentration should also should further be screened between 19mg/L to 23mg/L.
The 4th screening: according to the concentration gradient of the definite screening of shaker test for the third time is 19mg/L, 20mg/L, 21mg/L, 22mg/L, 23mg/L, and statistics is seen table 4:
Table 4
Annotate: green bud rate (%)=green bud number/total bud is counted the white bud rate of x100% (%)=white bud number/total bud and is counted x100%
Can find out that by table 4 when the concentration of kantlex is 23mg/L, when cotyledonary node was inoculated on the resistance screening substratum about 30 days, white bud rate reached 100%.So confirming cotyledonary node is 23mg/L to kalamycin resistance screening ultimate density, screening time is 30 days.
2, the best screening of incubation time in advance of cotyledonary node:
The pre-incubated substratum of cotyledonary node is 1/8MS+ZT (8mg/L)+NAA (1mg/L)+sucrose (20g/L)+agar powder (10g/L), and the MS here is that Murashige and Skoog cultivated the substratum of design for tobacco cell in 1962.In advance incubation time be set at 1 day, 2 days, 3 days and 4 days, totally 4 processing, infecting the back cotyledonary node with Agrobacterium is that the number of individuals of green is a statistical indicator with the green bud rate in kalamycin resistance screening back, confirms the preparatory incubation time of the best of cotyledonary node.The screening situation is seen table 5.
The different incubation times in advance of table 5 are to the influence of cotyledonary node
Annotate: the green rate (%) of cotyledonary node=green cotyledon joint number/cotyledonary node sum x100%
Can find out that from table 5 different incubation times in advance have certain influence to cotyledonary node.Green rate of 1 day cotyledonary node of cultivation and green bud rate are starkly lower than 2 days, 3 days, 4 days processing in advance; And 2 days, 3 days, 4 days processing is not having notable difference aspect green rate of cotyledonary node and the green bud rate.Usually, at the non-transgenic state, the preparatory incubation time of cotyledonary node is long more, and is strong more to the resistance capacity of kantlex; And when utilizing agrobacterium-mediated transformation that acceptor is infected, require acceptor material also to be in and receive the macromolecular state of external source the most easily.Handled in 2 days, 3 days, 4 days and do not have difference; Explain that the cotyledonary node of cultivating 2 days in advance receives the external source macromole the most easily; The probability that makes the pBI-121 carrier that has kalamycin resistance gene get into the cotyledonary node wound strengthens; The cotyledonary node in 2 day treatment time strengthens kalamycin resistance, thereby confirms that cultivation was the righttest preparatory incubation time in 2 days in advance.
3, the screening of the best time of infection of Agrobacterium:
With strain culturing stage OD
600Be 0.6, infect stage OD
600Be 0.5 Agrobacterium LBA4404; Infect the cotyledonary node after the preparatory cultivation; Time of infection is provided with 10min, 15min, 20min, 25min, 30min; Totally 5 times handle, and infecting the green bud rate of number of individuals and kalamycin resistance screening back that the back cotyledonary node is a green with Agrobacterium is statistical indicator, confirms the best time of infection of Agrobacterium to cotyledonary node.The screening situation is seen table 6.
The different times of infection of table 6 Agrobacterium are to the influence of cotyledonary node
Can find out that from table 6 different times of infection have certain influence to cotyledonary node.Prolongation along with the Agrobacterium time of infection; The green rate of cotyledonary node obviously descends; But green bud rate is handled apparently higher than other when infecting 20min and handle, explain time of infection long cotyledonary node is injured bigger, thereby definite Agrobacterium the righttest to the time of infection of cotyledonary node be 20min.
4, the optimum screening of incubation time altogether:
To be inoculated into the cotyledonary node that Agrobacterium has been infected in the substratum of 1/8MS+ZT (8mg/L)+NAA (1mg/L)+sucrose (20g/L)+agar powder (10g/L)+Syringylethanone (100 μ M/L) and carry out common cultivation; It is thus clear that time is provided with 3 gradients, is respectively not see on the substratum on the single bacterial plaque of Agrobacterium (3 days), the substratum on the visible single bacterial plaque of Agrobacterium (about 5 days) and the substratum one deck bacterium liquid (more than 7 days).White bud rate and green bud rate with after the kalamycin resistance screening are statistical indicator, confirm the optimum incubation time altogether of Agrobacterium and cotyledonary node.The selection result is seen table 7.
The different incubation times altogether of table 7 are to the influence of cotyledonary node
Can find out that from 7 tables along with the prolongation of incubation time altogether, the total bud number of regenerated reduces on the cotyledonary node, the total bud number of processing of cultivating visible one deck bacterium liquid especially altogether obviously descends; And cultivate the processing of visible single bacterial plaque altogether, and green bud rate is apparently higher than other two processing, and what green bud rate was minimum is the processing of cultivating visible one deck bacterium liquid altogether.Thereby optimum synchronic of definite Agrobacterium and cotyledonary node are visible single bacterial plaque.
5, the optimum cephalo removes the screening of bacteria concentration:
Cotyledonary node after cultivating altogether is inoculated on the substratum of 1/8MS+ZT (8mg/L)+NAA (1mg/L)+sucrose (20g/L)+agar powder (10g/L)+23mg/L kantlex, adds the cephalo of different concns simultaneously.After 30 days, be statistical indicator with the severity of bacterial plaque on the substratum and the white bud rate and the green bud rate of regeneration bud, the optimum cephalo concentration when confirming degerming.The screening situation is seen table 8.
The different cephalo concentration of table 8 are to the Agrobacterium bacteria-eliminating efficacy at Semen Ricini leaf segment position
Can find out that from table 8 along with the increase of cephalo concentration, the pollution rate of cotyledonary node obviously descends, green bud rate is on a declining curve substantially; The pollution rate of cotyledonary node is 0 when cephalo concentration is 1.2g/L, and green bud rate descends to some extent, but very not remarkable.Thereby optimum cephalo concentration is 1.2g/L during definite degerming.
This substratum can carry out two steps of degerming and screening simultaneously, and cephalo plays degermation, and kantlex plays the screening effect.The selection result according to the righttest kalamycin resistance concentration of front statement; Kantlex concentration is 23mg/L; Continue 30 days, begin to calculate from the substratum that is inoculated into 1/8MS+ZT (8mg/L)+NAA (1mg/L)+sucrose (20g/L)+agar powder (10g/L)+1.2g/L cephalo+23mg/L kantlex.
6, the green resistant buds screening of stage optimum NAA concentration of taking root
The stage substratum of taking root is 1/16MS+ sucrose (20g/L)+agar powder (10g/L), adds the NAA of different concns simultaneously.After 30 days, statistics different concns NAA handles the rooting rate of green resistant buds.After the resistant buds root development is good, tame transplanting.The screening situation is seen table 9.
Table 9 different concns NAA is to inducing the influence of green resistant buds rooting efficiency
Can find out that from table 9 green resistant buds root induction rate was the highest when NAA concentration was 1.0mg/L, can reach 89%, thereby confirm that the green resistant buds optimum NAA concentration of stage of taking root is 1.0mg/L.
7. the PCR of resistance seedling detects
Repeat the expression vector structure according to the pBI-121-RTA-RTA in the Agrobacterium LBA4404; Design the detection primer; The upstream primer sequence is 5 '-GGTCTAGACGAGCAATAATCTCCAGG-3 ', and the downstream primer sequence is 5 '-GGCCCGGGCACCAAAGGCGAATGTA-3 '.The PCR reaction system is: 10 * LA PCR BufferII, 5 μ l, dNTPs (each 2.5mM) 8 μ l, Mgcl
2(25mM) 5 μ l, upstream primer (10pmol) 1 μ l, downstream primer (10pmol) 1 μ l, resistance seedling genomic dna 1 μ l to be checked, LA Taq (5U/ μ l) 0.5 μ l, ddH
2O 28.5 μ l.The touchdown PCR reaction conditions is:
During design of primers, upstream primer is in the 35S promoter position design that repeats expression vector, and downstream primer is in the design of RTA gene locus, owing to repeating on the expression vector 2 RTA genes are arranged, so can detect 2 purpose bands during PCR.6 resistance seedlings are carried out PCR detect, obtaining 4 resistance seedlings is that PCR is positive.Through order-checking, further verified the exactness that PCR detects.Thereby explain that with the Semen Ricini leaf segment be acceptor, the genetic conversion system of being optimized can be used for the transgenic research of castor-oil plant.
Claims (9)
1. method of setting up agriculture bacillus mediated castor-oil plant genetic conversion system; It is characterized in that: said method is to be the mediation carrier with the Agrobacterium; With the Semen Ricini leaf segment is acceptor; Infect through giving cultivation, Agrobacterium, cultivate altogether, bacterium removes, screening and blastogenesis root, obtain having can hereditary goal gene the castor-oil plant plant.
2. a kind of method of setting up agriculture bacillus mediated castor-oil plant genetic conversion system according to claim 1; It is characterized in that: the choosing method of said Semen Ricini leaf segment is to sterilize, inoculate after the castor-oil plant seed is removed exosper; After treating seed germination; Select plumular axis and cotyledon to be the germinated seeds in faint yellow period, cut cotyledonary node.
3. a kind of method of setting up agriculture bacillus mediated castor-oil plant genetic conversion system according to claim 1, it is characterized in that: said Agrobacterium is LBA4404, includes pBI-121-RTA-RTA and repeats expression vector, the last kalamycin resistance gene that has of pBI-121.
4. a kind of method of setting up agriculture bacillus mediated castor-oil plant genetic conversion system according to claim 1; It is characterized in that: the pre-incubated substratum of said Semen Ricini leaf segment is 1/8MS+ZT (8mg/L)+NAA (1mg/L)+sucrose (20g/L)+agar powder (10g/L), and incubation time is 2 days in advance.
5. a kind of method of setting up agriculture bacillus mediated castor-oil plant genetic conversion system according to claim 1 is characterized in that: it is to use OD that said Agrobacterium is infected
600Be 0.5 Agrobacterium LBA4404, infect the cotyledonary node after the preparatory cultivation, time of infection is 20min.
6. a kind of method of setting up agriculture bacillus mediated castor-oil plant genetic conversion system according to claim 1; It is characterized in that: the said cultivation altogether is the cotyledonary node that has infected with Agrobacterium to be inoculated in the substratum of 1/8MS+ZT (8mg/L)+NAA (1mg/L)+sucrose (20g/L)+agar powder (10g/L)+Syringylethanone (100 μ M/L) carry out common cultivation; Incubation time is 5 days altogether, till the visible single bacterial plaque of Agrobacterium on the substratum.
7. according to claim 1 and 3 described a kind of methods of setting up agriculture bacillus mediated castor-oil plant genetic conversion system; It is characterized in that: said degerming is that the cotyledonary node after cultivating altogether is inoculated on the substratum of 1/8MS+ZT (8mg/L)+NAA (1mg/L)+sucrose (20g/L)+agar powder (10g/L)+23mg/L kantlex with screening, adds the cephalo that concentration is 1.2g/L simultaneously.
8. a kind of method of setting up agriculture bacillus mediated castor-oil plant genetic conversion system according to claim 1 is characterized in that: said screening is to be that the Semen Ricini leaf segment of 23mg/L kantlex after to degerming handled with concentration, and screening time is 30 days.
9. a kind of method of setting up agriculture bacillus mediated castor-oil plant genetic conversion system according to claim 1; It is characterized in that: the said stage substratum of taking root is 1/16MS+ sucrose (20g/L)+agar powder (10g/L); Add the NAA that concentration is 1.0mg/L simultaneously, after the resistant buds root development is good, tame transplanting.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105039394A (en) * | 2015-09-08 | 2015-11-11 | 内蒙古民族大学 | Method for obtaining new castor-oil plant materials with improved ricinoleic acid content |
| CN108546710A (en) * | 2018-04-20 | 2018-09-18 | 刘寒冬 | A kind of castor-oil plant genetic transforming method |
| CN111100871A (en) * | 2020-01-09 | 2020-05-05 | 天津科技大学 | Method for reducing browning and improving genetic transformation rate of agrobacterium-mediated castor |
| CN114107365A (en) * | 2021-09-02 | 2022-03-01 | 长江大学 | Construction method of castor instantaneous expression system for researching phloem conductivity of carrier pesticide |
| CN114807219A (en) * | 2022-04-15 | 2022-07-29 | 河南省农业科学院芝麻研究中心 | Agrobacterium-mediated sesame cotyledon transgenic method |
| CN115088623A (en) * | 2022-07-21 | 2022-09-23 | 南开大学 | Establishment method of tissue culture system for inducing castor bean leaf node cluster buds to generate at high frequency |
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- 2011-12-22 CN CN2011104346715A patent/CN102517327A/en active Pending
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039394A (en) * | 2015-09-08 | 2015-11-11 | 内蒙古民族大学 | Method for obtaining new castor-oil plant materials with improved ricinoleic acid content |
| CN108546710A (en) * | 2018-04-20 | 2018-09-18 | 刘寒冬 | A kind of castor-oil plant genetic transforming method |
| CN111100871A (en) * | 2020-01-09 | 2020-05-05 | 天津科技大学 | Method for reducing browning and improving genetic transformation rate of agrobacterium-mediated castor |
| CN114107365A (en) * | 2021-09-02 | 2022-03-01 | 长江大学 | Construction method of castor instantaneous expression system for researching phloem conductivity of carrier pesticide |
| CN114807219A (en) * | 2022-04-15 | 2022-07-29 | 河南省农业科学院芝麻研究中心 | Agrobacterium-mediated sesame cotyledon transgenic method |
| CN114807219B (en) * | 2022-04-15 | 2024-02-09 | 河南省农业科学院芝麻研究中心 | Agrobacterium-mediated sesame cotyledon transgenic method |
| CN115088623A (en) * | 2022-07-21 | 2022-09-23 | 南开大学 | Establishment method of tissue culture system for inducing castor bean leaf node cluster buds to generate at high frequency |
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Application publication date: 20120627 |