CN102124949B - Method for transferring total DNA of ginseng into single cell of potato through suspension co-culture - Google Patents
Method for transferring total DNA of ginseng into single cell of potato through suspension co-culture Download PDFInfo
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Abstract
The invention relates to a method for transferring the total DNA of a ginseng into single cells of a potato through suspension co-culture, comprising the following steps of: culturing a potato cell suspension culture; separating the single cells inside a suspension cell; carrying out the suspension co-culture on the total DNA of the ginseng and the single cells of the potato; acquiring test-tube plantlets, and culturing to obtain potato seeds. The offspring tuber of the potato obtained with the method provided by the invention contains ginsenoside RB1; the content of amino acids and proteins is greatly increased; and other characters are not changed along with the increase of the content and the quality of the proteins of the offspring tuber.
Description
Technical field:
The present invention relates to the crop field of molecular breeding, particularly relate to the molecular breeding method of potato, be specifically related to a kind of method that adopts the cultivation altogether that suspends the total DNA of genseng to be imported the unicellular acquisition of potato potato transfer-gen plant.
Background technology:
Plant molecular breeding is without sexual process, and foreign DNA is imported recipient plant, produces heritable variation, has the breeding technique (period-luminosity space etc., Chinese agriculture science and technology publishing house, 1993) of the improved seeds of purpose proterties with seed selection.It has overcome the limitation that the conventional breeding method cycle is long, foresight is poor, efficiency of selection is low, can break the species boundary, realizes excellent genes reorganization and polymerization, can make molecular breeding become the focus of breeding research to the new crop varieties directive breeding.
Genseng is applied to the history in existing more than 2,000 year of tcm clinical practice as one of valuable medicinal, has pharmacological action widely and medical application.Research shows that isolated ginsenoside is considered to one of main active ingredient of genseng from genseng, and wherein ginsenoside RB1 has strong active at anti-oxidant, aspects such as removing oxygen radical, cardiovascular system and reversing tumor cell resistance.Potato is one of whole world staple food crop, has short, characteristics such as output is high, adaptability is strong breeding time, more and more receives global extensive attention.But because potato common cultivation kind is an autotetraploid, the principal element of the narrow restriction potato breeding development of the complexity of genetic constitution and genetic background, conventional breeding is made slow progress.
1974, the period-luminosity space proposes " dna fragmentation hybridization " to be supposed, and simulation distant hybridization foreign DNA is cultivated into cotton and new rice variety through the pollen tube route.The methods such as " pollen tube passage method ", " soaking the seedling method " of using import plant with foreign DNA has become a kind of new effective genetic transforming method.Total dna direct introductory technique has been gone beyond the reproductive disorder in the conventional breeding; Remedied that cell engineering is loaded down with trivial details, plant regeneration frequency is low, progeny population is little; The defective that is difficult to screen; Generally acknowledged it is the effective way that realizes distant hybridization, and in various crop, obtained the excellent strain of ability genetic stability.Application such as Zhu Shengwei " are soaked the seedling method " and successfully foreign DNA are imported tobacco, import the offspring and have produced variation widely in form, quality with aspect becoming to grade.
Along with the development of technique for gene engineering, plant function Gene Isolation technology is also constantly perfect.Witty etc. have obtained the potato of commentaries on classics thaumatin T gene (thaumatin) with the method for transformation of agrobacterium rhizogenes mediation, and have detected the expression of active thaumatin T.Sheng Wanmin etc. are connected to 5 ' end promoter sequence (2.5ku) of the 240ku GFP of type I and Type II on beta-glucuronidase (GUS) gene of T plasmid binary vector PB1101.1; Change Agrobacterium strain LBA4404 over to; Potato is transformed, obtained the transfer-gen plant of protein high level expression.Show from these reports; Though adopt the gene of 1-2 known function to import protein content and the quality that potato can be improved stem tuber really; Also be merely 20% but the amplitude that complex steps, transformation efficiency are low, improve is the highest, and resistance selected marker such as antibiotic possibly retain also.
Hong Yahui etc. as acceptor, utilize static immersion training method altogether to import the total DNA of genseng with the small cell cluster of potato callus, though obtain the transformant plant, transformation efficiency is extremely low, is merely 2-3%, and the protein content of transformation generation does not improve.And adopt to suspend cultivate altogether with foreign DNA import single celled method improve crop quality operate in the potato aspect never the someone attempted.Therefore, still need a kind of new method that increases substantially potato protein content in this area.
Summary of the invention:
Technical problem to be solved by this invention is: to the deficiency of above-mentioned prior art, provide a kind of suspension quick and easy and simple to handle to cultivate altogether the total DNA of genseng is imported the single celled method of potato, can improve the nutritional quality of potato.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopted is: a kind of suspension is cultivated altogether the total DNA of genseng is imported the single celled method of potato, and this method comprises the steps:
(1), the cultivation of potato cell suspension culture: will grow for being inoculated into to put after the sterilization of the potato haulm section of 0.8-1.2cm has in the triangular flask of inducing culture; Every bottle graft kind 6-7 piece; This inducing culture is MS+NAA 0.4mg/L+6-BA2mg/L+ sucrose 30g/L+ agar 8g/L, and condition of culture is: temperature 25-28 ℃, and illumination 3000Lux; Light application time 12-14h/d cultivated 10 days; Successive transfer culture is 5 days again, and subculture number is 1 time, choose that stem section both sides are expanded significantly, stem section middle part increase thick, outward appearance is loose, color is jonquilleous callus as suspension cell; Transfer to the liquid suspension medium: among the MS+NAA0.4mg/L+6-BA2mg/L+ sucrose 30g/L, shaking table 100-120 commentaries on classics/min, 25-28 ℃; Under the dark condition; Cultivate after 3-5 days, the cell on the triangular flask wall is all scraped down, being moved into volume ratio is the liquid suspension medium of 2: 1 new and old mixing: among the MS+NAA 0.4mg/L+6-BA 2mg/L+ sucrose 30g/L; Suspension culture 2-4 days; Subculture 1 time (through scraping the subculture material that the cell of getting on bottle wall obtains the potato cell suspension culture, and observe through microscopy, the cell of screening good dispersion is as successive transfer culture);
(2), unicellular in the separate out suspended cell: selecting the aperture earlier for use is that 30 purpose cells sieve removes by filter the big callus in the cell suspension culture; Selecting the aperture for use according to the cell size of microscopy observation again is that small cell cluster is removed in the sieving of 60-120 purpose second layer cell; Selecting the aperture then for use is the single-cell suspension liquid that 300 purposes the 3rd confluent monolayer cells sieve obtains disperseing; Selecting the aperture at last for use is that 400 purpose cells sieve removes by filter the impurity in the suspension, and it is unicellular to obtain potato;
(3), total DNA of genseng and potato single-cell suspension are cultivated altogether: obtain the total DNA of genseng, the concentration of the total DNA of genseng is 350-400 μ g/ml; Total DNA of genseng and potato single-cell suspension are cultivated altogether, and condition of culture is shaking table 120-150 commentaries on classics/min, and temperature 25-28 ℃, culture fluid is MS+NAA 0.5mg/L+6-BA 2mg/L+ sucrose 40g/L, dark culturing 6-8 days;
(4), obtain test-tube plantlet, through cultivating the acquisition potato seed: the unicellular MS+NAA of forwarding to the 0.05mg/L+6-BA of the potato 2.0mg/L+GA after will cultivating altogether
35.0mg/L+ induce unicellular differentiation adventitious buds on the differential medium of sucrose 30g/L+ agar 8g/L, condition of culture is: temperature 25-28 ℃, illumination 3000Lux; Light application time 14-16h/d forwards the indefinite bud that differentiates on the root media of 1/2MS+NAA 0.2mg/L+ sucrose 30g/L+ agar 8g/L root induction and cultivates, and condition of culture is: temperature 25-28 ℃; Illumination 3000Lux; Light application time 14-16h/d obtains test-tube plantlet, carries out the RAPD Molecular Detection; Filter out the test-tube plantlet that contains potato contrast body and genseng donor same strap and make explant evoked callus and differentiation again, culture of rootage Cheng Miao, through cultivating the acquisition potato seed.
The above-mentioned potato haulm section is sterilized is meant the ethanol disinfection 30s of the potato haulm section of rinsing well with volumetric concentration 75%, with 0.1% mercuric chloride solution sterilization 7-8min, uses rinsed with sterile water at last 3 times.
The total DNA of above-mentioned genseng can from ginseng tissue such as root, stem, leaf, acquisition.The method that from ginseng tissue, obtains the total DNA of genseng is well known to those of ordinary skill in the art; For example can adopt (Bendich A.J; Plant Physiology; 1967,42 (7): 959-967) method of the rapid extraction plant tissue DNA of report prepares the total DNA of genseng, and only needing the concentration of the total DNA of genseng of acquisition is that 350-400 μ g/ml gets final product.
In the methods of the invention, the RAPD Molecular Detection can adopt this area routine techniques (Wang Xiuling etc., Nankai University's journal, 2003,36 (2): 37-40) carry out.
In the methods of the invention, the cultivation of potato callus induction, successive transfer culture, differentiation culture and culture of rootage method are well-known to those skilled in the art, for example can be referring to (Lan S.L, Am Potato J, 1977, method 54:575-580).In the methods of the invention; After obtaining seed potato, can adopt conventional method (Sun Huisheng, Chinese agriculture publishing house; 2003; 283-340) make seed potato growth and cultivation obtain offspring's stem tuber, select the individual plant that protein increases then and sow, consecutive numbers obtains the stem tuber of genetic stability after generation.The available this area of mensuration routine techniques of the protein content of potato tubers, amino acid content and genseng RB1 carries out (Samakana KI, Chem Pharm Bull, 1995,43 (1): 137-141).
Genseng and potato used in the inventive method are not limited to any specific kind (being), but for potato protein content is greatly improved, should select the higher strain of protein content for use.In addition, selecting the potato strain, should select preferably potato kind (being) as acceptor according to proterties such as potato disease resistance, output.
The present invention has set up a kind of method of improvement potato nutritional quality quick and easy and simple to handle, potato first generation stem tuber the highest can comparison respectively according to improving 47.96%, 46.13%, 86.54% on protein, total amino acid, lysine content of utilizing this method to obtain.And; Through to the phenotype analytical in generation thereafter; Do not find other obvious phenotypes variation phenomenon, proterties such as its high yield, disease resistance do not change because of the raising of stem tuber protein content and quality, the offspring can obtain can genetic stability the high protein strain.
Description of drawings:
Fig. 1 is the chromatogram of ginsenoside RB1 among the offspring that cultivates altogether of total DNA of genseng of the present invention and potato single-cell suspension.
Fig. 2 is the chromatogram of saponin(e RB1 in the samples of Ginseng of the present invention.
Embodiment:
To do explanation further to the present invention with instantiation below:
Embodiment 1
In the present embodiment, the total DNA donor of external source genseng is fresh genseng, the high yield that the acceptor potato is provided by Hunan Province's potato Engineering Technical Research Centre, the Atlantic Ocean that disease resistance is good.
1,,, uses rinsed with sterile water at last 3 times with 0.1% mercuric chloride solution sterilization 7-8min with the ethanol disinfection 30s of the potato haulm section of rinsing well with volumetric concentration 75%; Be inoculated in the inducing culture, every bottle graft kind 6-7 piece explant, this inducing culture are MS+NAA 0.4mg/L+6-BA 2mg/L+ sucrose 30g/L+ agar 8g/L;, condition of culture is: temperature 25-28 ℃, and illumination 3000Lux; Light application time 12-14h/d; Cultivated 10 days, successive transfer culture 5 days (subculture 1 time) again, choose that stem section both sides are expanded significantly, stem section middle part increase thick, outward appearance is loose, color is jonquilleous callus as suspension cell culture.
2, the condition of potato cell suspension culture is shaking table 100-120 commentaries on classics/min; 25-28 ℃, after dark culturing 3-5 days the cell on the triangular flask wall is all scraped down, move into the liquid suspension medium (MS+NAA0.4mg+6-BA 2mg/L+ sucrose 30g/L) of new and old mixing; Its volume ratio is 2: 1; Suspension culture 2-4 days, subculture 1 time (observe through microscopy, the cell of screening good dispersion is as successive transfer culture).
3, adopt the filtration of four confluent monolayer cells sieve to obtain unicellular; At first selecting the aperture for use is that 30 purpose cells sieve removes by filter the big callus in the cell suspension culture; Selecting the aperture for use according to the cell size of microscopy observation again is that small cell cluster is removed in 120 purpose second layer cell sievings; Selecting the aperture then for use is the single-cell suspension liquid that 300 purposes the 3rd confluent monolayer cells sieve obtains disperseing, and selecting the aperture at last for use is that 400 purpose cells sieve removes by filter the impurity in the suspension, and it is unicellular to obtain potato through the microscopy observation.
4, total DNA of genseng and potato single-cell suspension are cultivated altogether; Condition of culture is shaking table 120-150 commentaries on classics/min altogether; 25-28 ℃, the concentration of the total DNA of genseng is 350-400 μ g/ml, and medium is MS+NAA 0.5mg/L+6-BA 2mg/L+ sucrose 40g/L; After dark culturing 6-8 days, forward MS+NAA 0.05mg/L+6-BA 2.0mg/L+GA to
35.0mg/L+ induce unicellular differentiation adventitious buds on the differential medium of sucrose 30g/L+ agar 8g/L, condition of culture is: temperature 25-28 ℃, illumination 3000Lux; Light application time 14-16h/d forwards the indefinite bud that differentiates on the root media of 1/2MS+NAA 0.2mg/L+ sucrose 30g/L+ agar 8g/L root induction and cultivates, and condition of culture is: temperature 25-28 ℃; Illumination 3000Lux; Light application time 14-16h/d obtains test-tube plantlet, carries out the RAPD Molecular Detection; Filter out the test-tube plantlet that contains potato contrast body and genseng donor same strap and make explant evoked callus and differentiation again, culture of rootage Cheng Miao, through cultivating the acquisition potato seed.Wherein, test-tube plantlet can be cultivated into 215 strain whole plants, and through detecting, transfer-gen plant is 26 strains, and conversion ratio is 12.1%.
5, from first generation material, filter out the higher individual plant of protein content and sow respectively, obtain second generation potato tubers.Rescreen and select the higher individual plant of protein content and continue plantation respectively and obtain third generation potato tubers, same operation results the 4th generation stem tuber.
6, as shown in table 1; To transforming 5 individual plant stem tubers of arbitrary extracting in the successful first generation potato offspring stem tuber; Its protein content comparison is according to being significantly improved; Variance analysis shows with control group extremely significant difference (P＜0.01) is arranged all, and the highest comparable contrast of first generation stem tuber protein content improves 47.96%.Respectively conversion and unconverted potato, samples of Ginseng are carried out in the mensuration of ginsenoside RB1 again; Detecting to transform in potato and the samples of Ginseng has identical ginsenoside RB1 spike, and in unconverted potato, does not detect this spike (seeing Fig. 1 and Fig. 2).
On the basis of detecting first generation potato tubers protein content, the stem tuber of high protein individual plant is carried out analysis of amino acids (result sees table 1).Mensuration result shows that when high protein variation offspring increased along with the stem tuber protein content, stem tuber total amino acid and various amino acid content also were significantly improved.Analyze to show the potato offspring stem tuber total amino acid content relation that also all is proportionate through linear correlation.Contrast stem tuber lysine content is 0.52%, cultivates the high protein potato tubers lysine content peak that the back obtains altogether through the total DNA of genseng and potato single-cell suspension and reaches 0.97%, compares photograph and has improved 86.54%.First generation high protein material continues plantation screening through the three generations again, has obtained the high protein strain system that protein content can genetic stability in the 4th generation.
The analysis result in comprehensive four generations can learn that adopting suspends cultivates the protein content and the quality that the single celled method of the total DNA importing potato of genseng can be improved stem tuber altogether, and effect is fine.
Though importing total DNA of genseng is not a high protein gene that purpose is very clear and definite, and owing to do not have resistant maker gene to assist screening, thereby the workload of later stage screening increased.But consider from the safety perspective of food; Many popularizations that possibly retain the genetically modified crops of resistance selectable marker genes such as antibiotic at present have been restricted; Do not have any unfavorable gene that possibly influence animal or human's class and adopt among the present invention to suspend to cultivate altogether in the high protein potato that the single celled method of genseng total DNA importing potato is cultivated, so its popularization can not be restricted.
In implementation process of the present invention; Offspring to cultivating altogether through the total DNA of genseng and potato single-cell suspension carries out phenotype analytical; Do not find other obvious phenotypes variation phenomenon; Proterties such as its high yield, disease resistance do not change because of the raising of stem tuber protein content and quality, thus the offspring can obtain can genetic stability the high protein strain.Successfully cultivating high protein potato strain in the present invention shows; Total DNA of genseng and the common cultured method of potato single-cell suspension not only can fast and effeciently be improved the protein content and the quality of potato tubers, and can also overcome the negative correlation between potato high yield and the high protein.
What should explain at last is: above embodiment is the unrestricted technical scheme of the present invention in order to explanation only; Although the present invention is specified with reference to the foregoing description; Those of ordinary skill in the art is to be understood that; Still can make amendment or be equal to replacement the present invention, and not break away from any modification or the local replacement of the spirit and scope of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Each amino acid of first generation potato offspring stem tuber, total amino acid and protein content (mg/100mg) that total DNA of table 1 genseng and potato single-cell suspension are cultivated altogether
Claims (2)
1. the cultivation altogether that suspends imports the single celled method of potato with the total DNA of genseng, and it is characterized in that: this method comprises the steps:
(1), the cultivation of potato cell suspension culture: will grow for being inoculated into to put after the sterilization of the potato haulm section of 0.8-1.2cm has in the triangular flask of inducing culture; Every bottle graft kind 6-7 piece; This inducing culture is MS+NAA 0.4mg/L+6-BA 2mg/L+ sucrose 30g/L+ agar 8g/L, and condition of culture is: temperature 25-28 ℃, and illumination 3000Lux; Light application time 12-14h/d cultivated 10 days; Successive transfer culture is 5 days again, and subculture number is 1 time, choose that stem section both sides are expanded significantly, stem section middle part increase thick, outward appearance is loose, color is jonquilleous callus as suspension cell; Transfer to the liquid suspension medium: among the MS+NAA0.4mg/L+6-BA2mg/L+ sucrose 30g/L, shaking table 100-120 commentaries on classics/min, 25-28 ℃; Under the dark condition; Cultivate after 3-5 days, the cell on the triangular flask wall is all scraped down, being moved into volume ratio is the liquid suspension medium of 2: 1 new and old mixing: among the MS+NAA 0.4mg/L+6-BA 2mg/L+ sucrose 30g/L; Subculture suspension culture 2-4 days, subculture number are 1 time;
(2), unicellular in the separate out suspended cell: selecting the aperture earlier for use is that 30 purpose cells sieve removes by filter the big callus in the cell suspension culture; Selecting the aperture for use according to the cell size of microscopy observation again is that small cell cluster is removed in the sieving of 60-120 purpose second layer cell; Selecting the aperture then for use is the single-cell suspension liquid that 300 purposes the 3rd confluent monolayer cells sieve obtains disperseing; Selecting the aperture at last for use is that 400 purpose cells sieve removes by filter the impurity in the suspension, and it is unicellular to obtain potato;
(3), total DNA of genseng and potato single-cell suspension are cultivated altogether: obtain the total DNA of genseng, the concentration of the total DNA of genseng is 350-400 μ g/ml; Total DNA of genseng and potato single-cell suspension are cultivated altogether, and condition of culture is shaking table 120-150 commentaries on classics/min, and temperature 25-28 ℃, culture fluid is MS+NAA 0.5mg/L+6-BA 2mg/L+ sucrose 40g/L, dark culturing 6-8 days;
(4), obtain test-tube plantlet, through cultivating the acquisition potato seed: the unicellular MS+NAA of forwarding to the 0.05mg/L+6-BA of the potato 2.0mg/L+GA after will cultivating altogether
35.0mg/L+ induce unicellular differentiation adventitious buds on the medium of sucrose 30g/L+ agar 8g/L, condition of culture is: temperature 25-28 ℃, illumination 3000Lux; Light application time 14-16h/d forwards the indefinite bud that differentiates on the medium of 1/2MS+NAA 0.2mg/L+ sucrose 30g/L+ agar 8g/L root induction and cultivates, and condition of culture is: temperature 25-28 ℃; Illumination 3000Lux; Light application time 14-16h/d obtains test-tube plantlet, carries out the RAPD Molecular Detection; Filter out the test-tube plantlet that contains potato contrast body and genseng donor same strap and make explant evoked callus and differentiation again, culture of rootage Cheng Miao, through cultivating the acquisition potato seed.
2. suspension as claimed in claim 1 is cultivated altogether the total DNA of genseng is imported the single celled method of potato; It is characterized in that: in the said step (1) sterilization of potato haulm section is meant the ethanol disinfection 30s of the potato haulm section of rinsing well with volumetric concentration 75%; With 0.1% mercuric chloride solution sterilization 7-8min, use rinsed with sterile water at last 3 times.
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| 丁国华等.农业分子育种中外源DNA导入技术的研究与发展.《生物学教学》.2000,第25卷(第11期),2-3. * |
| 杨景成等.外源DNA导入植物技术的发展及其在作物育种中的应用.《核农学报》.2002,第17卷(第1期),79-84. * |
| 洪亚辉等.人参总DNA导入马铃薯及其变异后代检测的研究.《马铃薯产业与粮食安全（2009）》.2009,219-223. * |
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