CN103088058A - Genetic transformation method of Chinese fir - Google Patents
Genetic transformation method of Chinese fir Download PDFInfo
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- CN103088058A CN103088058A CN2013100356666A CN201310035666A CN103088058A CN 103088058 A CN103088058 A CN 103088058A CN 2013100356666 A CN2013100356666 A CN 2013100356666A CN 201310035666 A CN201310035666 A CN 201310035666A CN 103088058 A CN103088058 A CN 103088058A
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Images
Abstract
The invention discloses a genetic transformation method of a Chinese fir. The genetic transformation method comprises the steps of: establishment of a transformation receptor, preparation of agrobacterium liquid, infection, bacterium removal culture and screening culture. According to the genetic transformation method of the Chinese fir disclosed by the invention, a Chinese fir suspension cell system is taken as the receptor and a b-glucuronidase (GUS) gene is used as a report gene to implement a genetic transformation through an agrobacterium mediated method, and the plant regeneration is realized through a somatic embryogenesis way, and a stable and efficient Chinese fir genetic transformation system is established. Due to the utilization of the technical system, genetic improvement of the Chinese fir with comparatively low cost can be achieved; and anti-disease, stress-resistant, wood formation and other related genes obtained from a basic and theoretical research are subjected to the genetic transformation so as to obtain a high-quality Chinese fir variety having characteristics of rapid growth, strong anti-disease and stress-resistant performances, and the like.
Description
Technical field
The present invention relates to the China fir genetic transforming method, being specifically related to a kind of is the agrobcterium-mediated transformation of acceptor with the China fir suspension cell.
Background technology
China fir is the distinctive commerical tree species of China, is one of each most important reproducting tree species in provinces and regions of south.Growth of Chinese Fir is fast, and output is high, and the property material is good, and purposes is many, and timber is in demand, and its timber is the building material of high-quality, and decorative material is the most important merchantable timber seeds of China.Select that fast growth, reproductivity are high, the excellent strain of China fir strong stress resistance and clone be of great significance the production of forestry tool.
Yet the softwood tree growth cycle is long, the tree height is large, and due to long-term outcrossing, has the heterozygosity of height and a large amount of load.So the traditional breeding method cycle is long, is difficult to adapt to the needs of fast growing tree genetic improvement.The forest tree genetic genetically engineered is the Disciplinary Frontiers of forest biotechnology, and it not only opens up a new way for the germplasm innovation in the forest tree genetic improvement, is also simultaneously the important means of research gene function.Genetically engineered is combined with traditional breeding method, can greatly shorten the forest genetics cycle, accelerate breeding process.
Forest-tree Gene Engineering is by the gene transfer technique that is fit to, and imports useful foreign gene, obtains transfer-gen plant, carries out forest tree genetic improvement or relevant research.In recent years, Forest-tree Gene Engineering has been obtained larger progress.The field widespread uses such as at present, existing hundreds of kind of plant obtains genetic improvement, and is disease-resistant in high yield, cold-resistant.But be severely limited owing to lacking good method for transformation and regeneration system rapidly in softwood tree.For a long time, the host who it is believed that agrobacterium tumefaciens is mainly dicotyledons, but since D.Cleene and D.Ley reported first agrobacterium tumefaciens bacterial strain B6 also can infect the gymnosperm wounded tissue, and after the generation crown-gall nodule, many scholars begin to utilize agrobacterium tumefaciens to carry out the research of the especially acerose gene transformation of gymnosperm.And proved that foreign gene is expressed in the cell tissue of part softwood tree such as white spruce, Picea excelsa, torch pine and yellow fir etc.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the purpose of this invention is to provide a kind of China fir genetic transforming method, is acceptor with the China fir suspension cell, carries out agriculture bacillus mediated genetic transformation, to set up the China fir genetic conversion system of stability and high efficiency.
Technical scheme: in order to realize the foregoing invention purpose, the technical solution used in the present invention is as follows:
A kind of China fir genetic transforming method comprises the following steps:
(1) foundation of transformation receptor:
Get the China fir embryo callus cell in 3 weeks of subculture, be inoculated in the China fir liquid nutrient medium, 23 ℃, the constant-temperature table shaking culture of 85r/min; Every 7 days subcultures once namely can be used for for the third time Agrobacterium and infect after 3 days after subculture; Wherein, the China fir liquid nutrient medium is the DCR minimum medium, additional Vc 10 mg/L, GA 0.1-2 mg/L, ABA 2-5 mg/L, CH 0.5 g/L, maltose 30 g/L;
(2) Agrobacterium bacterium solution preparation:
Activation EHA105 bacterium liquid, picking list bacterium colony, activation again, 28 ℃, 220r/min vibration training liquid are supported to OD
600Be 0.6-0.8, with the resuspended thalline of China fir liquid subculture medium;
(3) infect:
Bacterium liquid is shaken up, get 3mL bacterium liquid and be added in the subculture China fir suspension cell of 3 days, after standing 1-3min, be placed in 23 ℃, the constant-temperature table of 85r/min continues shaking culture 16 ~ 36h;
(4) taking off bacterium cultivates:
Wash for several times bacterium with sterilized water, with the resuspended China fir callus of China fir liquid nutrient medium, the China fir callus after resuspended is transferred in the mode of paving plate on the body embryonal induction substratum that is added with cephamycin 500mg/L, wherein every ware 4-5mL; And put into 23 ℃ of constant incubators and secretly cultivate.Every 21 days subcultures once, induced 30 ~ 60 days; Body embryonal induction substratum is the 1/2DCR minimum medium, additional Vc 10 mg/L, GA 2-8 mg/L, inositol 5 g/L, CH 0.5 g/L, A C2 g/L, maltose 25 g/L, crystal agar 2.8 g/L;
(5) screening and culturing:
After China fir cotyledonary embryos maturation, it is chosen on the DCR minimum medium that is added with 200 mg/L cephamycins 23 ℃ of illumination cultivation.Cultivate after 21 days, transfer to and be added with 100 mg/L cephamycins, the enterprising row filter of the DCR minimum medium of 20 mg/L kantlex is cultivated; Every 21 days subcultures once.
In step (2), the resuspended thalline of China fir liquid subculture medium, the OD of bacterium liquid
600Be 0.5.
In step (4), contain the 500mg/L cephamycin in sterilized water.
Described EHA105 is with 35S:GUS gene and NPT-II gene.
In step (5), after subculture 2 times, just can be used for transplanting.
Beneficial effect: compared with prior art, China fir genetic transforming method of the present invention, be acceptor with the China fir suspension cell, carry out genetic transformation take gus gene as reporter gene by agriculture bacillus mediated method, realize plant regeneration by the somatic embryo occurring mode, set up the China fir genetic conversion system of stability and high efficiency.Utilize the present technique system, can carry out genetic improvement to China fir at lower cost, the relevant genes such as disease-resistant, degeneration-resistant, timber formation that obtain in fundamental research are carried out genetic transformation, obtain to have fast-growing, the high-quality China fir kind of the characteristics such as disease-resistant, strong stress resistance.
Description of drawings
Fig. 1 is China fir embryo callus subculture displaing micro photo figure;
Fig. 2 is China fir ripe cotyledon embryo figure;
Fig. 3 is China fir GUS Transient Expression detection figure.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
In following examples, the culture medium prescription that uses is as follows, does not list especially, is conventional medium.
In following examples, the DCR(Gupta and Durzan medium of use) formation and the standard recipe of minimum medium are: 340 mg/L KNO
3, 556 mg/L Ca (NO
3)
24H
20,400 mg/L NH
4NO
3, 170 mg/L KH
2PO
4, 85 mg/L CaC1
22H
20,370 mg/L MgSO
47H
20,37.3 mg/L Na
2-EDTAH
20,27.9 mg/L FeSO
47H
2O, 6.2 mg/L H
3BO
3, 22.3 mg/L MnSO
44H
20,8.6 mg/L ZnSO
47H
20,0.25 mg/L Na
2MoO
42H
2O, 0.25 mg/L CuSO
45H
2O, 0.83 mg/L KI, 0.025 mg/L CoC1
2, 200 mg/L MyO-Inositol, 2.0 mg/L Glycine, 1.0 mg/L ThiamineHCl, 0.5 mg/L Nictinicacid, 0.5 mg/L PyridoxineHCl.
LB culture medium prescription: 5 g/L Yeast extract, 10 g/L Tryptone, 5 g/L NaCl.
The foundation of embodiment 1 transformation receptor.
(1) the China fir embryo callus cell of getting 3 weeks of subculture is 5mL approximately, it is inoculated in the aseptic triangular flask of 250mL, and strikes gently loosely at the bottle wall, and purpose is that cell is disperseed.The Photomicrograph of China fir embryo callus cell as shown in Figure 1.
(2) measure 50mL China fir liquid nutrient medium and add in triangular flask, material is placed in 23 ℃, the constant-temperature table shaking culture of 85r/min.The China fir liquid nutrient medium is the DCR minimum medium, additional 10mg/L VC, 0.1～2.0mg/L GA, 2 ~ 5mg/L ABA, 0.5g/L CH, 30g/L maltose.
(3) every 7 days subcultures once, for the third time after subculture after 3 days (namely since the 4th generation the 3rd day) namely can be used for Agrobacterium and infects.
Receptor system in the plant gene conversion process refers to for the explant that transforms by tissue culture approach or other non-tissue culture approach, the clone of efficiently, stably regenerating of setting up, and can accept foreign DNA and integrate, to transforming the regeneration system rapidly of selecting antibiotic sensitive.At first successful gene transformation depends on the foundation of good Plant host systems.So far, a large amount of work has been carried out in the systematic research of plant gene transformation receptor, successively set up multiple effective receptor system, be adapted to the requirement of different method for transformation and different conversion purposes.The receptor system of now having set up comprises callus regeneration system, directly differentiation and regeneration system, protoplast regeneration system, somatic embryogenesis system, sexual cell regeneration system rapidly.
At present, more and more come into one's own as receptor system with plant materials embryo generation system or body embryo.This system mainly contains following characteristics: it is very capable that the cells,primordial of one, composition embryoid is accepted foreign DNA, be desirable gene transformation competent cell, and these cell proliferation amounts is large, and synchronism is good, so transformation efficiency is very high; Two, between the embryoid individuality, genetic background is consistent, and somaclonal variation is little, and seedling is fast, and quantity is many, but also can make synthetic seed, is conducive to production and the popularization of transfer-gen plant; Three, embryoid mostly is unicellular origin, transforms the transfer-gen plant mosaic that obtains few; Four, embryoid has the two poles of the earth, can break up simultaneously in growth course and sprout and root, forms complete plant, has reduced the difficult problem of the difficulty of taking root in the uncertain buds growth process.Ripe China fir body embryo generation system has been set up in this laboratory.
The present invention is the basis with China fir body embryo artificial body for generating, is acceptor material with the China fir suspension cell, sets up the China fir genetic transformation system.
Embodiment 2 Agrobacterium bacterium solution preparations.
(1) melting on ice with 35S:GUS gene (be used for transforming and detect) and the EHA105 bacterium liquid of NPT-II gene (being used for screening)-70 ℃ of preservations, dip a small amount of bacterium liquid with transfering loop, be inoculated into and contain appropriate microbiotic (kantlex 50mg/L, Streptomycin sulphate 30mg/L) on LB substratum, be placed in 28 ℃ of constant incubators and cultivate.
(2) after it grows single bacterium colony, with transfering loop picking list bacterium colony, be inoculated on the LB substratum that contains appropriate microbiotic (kantlex 50mg/L, Streptomycin sulphate 30mg/L), then reactivate once is placed in 28 ℃ of constant incubators and cultivates.
(3) after it grows single bacterium colony, get single colony inoculation with the rifle choicest of the bacterium of going out and be added with in the triangular flask of appropriate antibiotic LB liquid nutrient medium to containing 10mL, 28 ℃, 220r/min shaking culture.
(4) approximately after 20h, draw 2mL bacterium liquid, be inoculated into and contain in the triangular flask of LB liquid nutrient medium that 50mL is added with appropriate microbiotic (kantlex 50mg/L, Streptomycin sulphate 30mg/L), 28 ℃, 280r/min shaking culture are to OD
600Be 0.6-0.8.
(5) treat that Agrobacterium cultivates OD
600During for 0.6-0.8, get 25mL bacterium liquid in the 50mL centrifuge tube, in 5000r/min, 4 ℃ of centrifugal 10min remove supernatant, collect thalline.
(6) with the resuspended thalline of China fir liquid subculture medium, bacterial concentration is diluted to OD
600It is 0.5 left and right.
At present, in plant genetic engineering research, agriculture bacillus mediated transgenic method is that transformation mechanism is the clearest, most widely used general, a kind of method that success ratio is the highest.In conversion process, the cultivation of Agrobacterium, growth conditions and purity have vital role to transformation efficiency.If the poor growth of Agrobacterium own, its infection ability descends greatly.Therefore prepare that purity is high, Agrobacterium that growth is vigorous, infection ability is strong is infected the key that liquid is conversion.This bacterium liquid that infects is also referred to as engineering bacteria liquid.
The cultivation of Agrobacterium can be divided into the solid plate cultivation and liquid oscilaltion is cultivated.Solid culture generally needs 2～3 days, and the liquid culture growth is faster, generally needs 1～2 day.After Agrobacterium is seeded in liquid nutrient medium, do not begin immediately propagation, generally needed just begin to divide in 1～2 hour.After the growth beginning, number of bacteria just stops propagation with a constant index rate multiplication until medium component changes and supports when lacking.When reaching the logarithm index, bacterial growth speed is referred to as the logarithmic growth state.The Agrobacterium infection ability that is in the logarithmic growth state is the strongest.
Studies show that, when Agrobacterium is cultured to OD
600During for 0.6-0.8, thalline is in logarithmic phase, and this moment, infection ability was the strongest.
Embodiment 3 infects.
Bacterium liquid is shaken up, and the bacterium liquid of drawing the 3mL dilution is added in the subculture China fir suspension cell of 3 days (embodiment 1), after standing 1-3min, is placed in 23 ℃, and the constant-temperature table of 85r/min continues shaking culture, namely cultivates altogether.
Infect and refer to engineering bacteria is inoculated into the acceptor material surface, method is exactly that the Agrobacterium bacterium liquid that will prepare adds in the acceptor material nutrient solution, after soaking certain hour, carries out common cultivation.GPRS time of infection in infection processs helps to reduce the pollution that may cause in the later stage culturing process, and can alleviate bacterium to the toxic action of plant.Immerged time is too short, can not make abundant Agrobacterium be attached to the explant wound, thereby reduces the frequency of genetic transformation.Immerged time is long, easily causes the anaphylaxis of explant, may cause Agrobacterium to pollute simultaneously in follow-up culturing process, finally causes brownization of converting material dead.
Explant after the inoculation thalline is in cell fission, growth, and Agrobacterium is in explant cut sides also proliferate, and the process of these both co-cultivation is called common cultivation.It is very important link in whole conversion process that Agrobacterium and explant are cultivated altogether, because Agrobacterium adheres to, the transfer of T-DNA and integration are all completed within this period.Studies show that, during Agrobacterium-mediated Transformation, not " intrusion " in vegetable cell, but T-DNA is transferred to vegetable cell.After adhering to, Agrobacterium can not transform immediately, the ability of the bacterial strain after wound site existence 16h induced tumor only, and this section period is called " Cell regulate phase ".Therefore, incubation time must be longer than 16h altogether.And incubation time is oversize altogether, and due to the hypertrophy of Agrobacterium, vegetable cell is dead because being poisoned.In this experiment, the China fir Suspension Cells is after Agrobacterium is infected, and it is cultivated altogether still and carries out in liquid medium within.
Embodiment 4 takes off bacterium and cultivates.
(1) will cultivate altogether China fir suspension cell after about 36h rock several under, then be poured in the 100mL graduated cylinder, standing 1-2min, after precipitation of material, remove supernatant, then material is refunded in triangular flask, and measure the sterilized water that about 50mL is added with cephamycin 100mg/L and pour in triangular flask, rock again several under, pour in the 100mL graduated cylinder; Repeat 3-4 time, until supernatant liquor is limpid.This process is for washing bacterium.
(2) with the China fir liquid nutrient medium, that the China fir callus in graduated cylinder is resuspended, and pour in the 250mL triangular flask.
(3) the China fir callus after resuspended is transferred in the mode of paving plate on the body embryonal induction substratum that is added with cephamycin 500mg/L, wherein every ware 4-5mL.Body embryonal induction substratum is the 1/2DCR minimum medium, additional Vc 10mg/L, GA 2-8mg/L, inositol 2-10g/L, CH 0.5g/L, AC 2g/L, maltose 25g/L, crystal agar 2.8g/L.
(4) every 21 days subcultures once.
(5) the body embryonal induction approximately after 30 days, can be seen the column embryo, approximately can see cotyledonary embryos after 60 days, China fir ripe cotyledon embryo figure, as shown in Figure 2.
Explant and Agrobacterium cultivate altogether after for some time that in its surface and shallow layer tissue, symbiosis has a large amount of Agrobacteriums, must take off bacterium for the growth of killing and suppress Agrobacterium and cultivate, thereby explant is grown better.It is the vegetable material after cultivating altogether to be transferred to contain on antibiotic substratum cultivate that what is called is taken off the bacterium cultivation.Antibiosis commonly used have Pyocianil, cephamycin and ticarcillin.These microbiotic not only kill and wound or restraining effect Agrobacterium, and vegetable cell is had certain biological effect equally.The growth working concentration that uses cephamycin to kill in the present embodiment and suppress Agrobacterium is 500mg/L.
Embodiment 5 screening and culturing.
After China fir cotyledonary embryos maturation, it is chosen on the DCR minimum medium, carry out illumination cultivation under 23 ℃, be added with the 200mg/L cephamycin in substratum; Transfer to after 21 days and be added with the 100mg/L cephamycin, the enterprising row filter of the DCR minimum medium of 20mg/L kantlex is cultivated, and every 21 days subcultures once.After general subculture 2 times, just can be used for transplanting.
The cell of selecting conversion is also an important step in conversion process.The cell that transforms and non-transformed cell exist competition in growth and development process, if transformant can not be grown, transforming just can not be successful.Therefore select to cultivate the step that is absolutely necessary.
Select microbiotic to bring Selection In after substratum, the growth of cell is produced a kind of selective action, be referred to as to select to press.What in the present embodiment, selectable marker gene was used is NPT-II (neomycin phosphotransferase).The cell of expression NPT-II gene shows the resistance to the microbiotic kantlex.Therefore press as selecting with kantlex in embodiment.
According to selecting to press the difference in period that adds, can be divided into three kinds, namely selection in early stage, the delayed selection culture and later stage are selected.Select early stage is after acceptor material is cultivated altogether, is changing the pressure that brings Selection In at the very start of callus or adventitious bud inducing over to, namely first selects to regenerate afterwards; Later stage is selected namely first to regenerate to select afterwards.Because unconverted cell is containing on the substratum of select pressing and can not grow, the transformed cell growth ability also is difficult to growth a little less than too and forms transformant; And can grow equally without non-transformed cell in select pressing substratum, and to the transformant Yellow Gentian Extract, thereby be conducive to the growth of transformant, so the present embodiment is selected the later stage selection.
Embodiment 6 GUS cytology detect
Gus gene is present in some bacterial body, coding β-glucuronidase (β-glucuronidase, GUS), and this enzyme is a kind of lytic enzyme, the hydrolysis of the many beta-glucoside Esters of energy catalysis.Do not exist endogenous GUS active in most vegetable cells, thereby gus gene is widely used as the reporter gene of transgenic plant.
Using cytology to detect gus gene is to be substrate by X-Gluc, observes directly the activity of gus gene in histoorgan by color reaction.The method need not extract enzyme from tissue, but substrate is entered among tested plant tissue, cell or protoplastis.Detected material is immersed in the damping fluid that contains substrate is incubated, if the gus gene conversion has successfully occured material, give expression to GUS, this enzyme can generate blue material with the X-Gluc hydrolysis under optimum conditions, can with the naked eye or examine under a microscope.
Getting the body embryo seedling that plant is about 2cm is the GUS coloring material.Ready body embryo seedling is immersed in dye liquor, in 37 ℃ of insulation 2h to spending the night.The GUS formula for dye liquor:
(1) 50mmol/L buffer solution of sodium phosphate (pH7.0): A liquid: take NaH
2PO
42H
2O 3.12g is dissolved in sterilized water, is settled to 100ml.B liquid: take NaH
2PO
412H
2O 7.17g is dissolved in sterilized water, is settled to 100ml.Getting 39ml A liquid mixes with 61ml B liquid;
(2) staining fluid: 7.1ml 50mmol/L buffer solution of sodium phosphate, 0.2ml 10mmol/L Na
2EDTA, 0.2ml 5mmol/L K
4[Fe(CN)
6], 0.2ml K
3[Fe(CN)
6], 0.05ml 0.05%(v/v) Triton-100,2ml 20% methyl alcohol, 0.25ml 0.5mg/ml X-Gluc.Preparation 10mlGUS dye liquor is standby.
Take gus gene as reporter gene, detect the Transient Expression, long-term expression of gus gene, Efficient Conversion by definite foreign genes such as molecular biology method detections by the cytology method.As shown in Figure 3, naked eyes or microscopically are observed, and the blueness in material is the GUS expression sites, and as seen, foreign gene is by Efficient Conversion.
Claims (5)
1. a China fir genetic transforming method, is characterized in that, comprises the following steps:
(1) foundation of transformation receptor:
Get the China fir embryo callus cell in 3 weeks of subculture, be inoculated in the China fir liquid nutrient medium, 23 ℃, the constant-temperature table shaking culture of 85r/min; Every 7 days subcultures once namely can be used for for the third time Agrobacterium and infect after 3 days after subculture; Wherein, the China fir liquid nutrient medium is the DCR minimum medium, additional 10 mg/L Vc, 0.1-2.0 mg/L GA, 2-5mg/L ABA, 0.5g/L CH, 30g/L maltose;
(2) Agrobacterium bacterium solution preparation:
Activation EHA105 bacterium liquid, picking list bacterium colony, activation again, 28 ℃, 220r/min vibration training liquid are supported to OD
600Be 0.6-0.8, with the resuspended thalline of China fir liquid subculture medium;
(3) infect:
Bacterium liquid is shaken up, get 3mL bacterium liquid and be added in the subculture China fir suspension cell of 3 days, after standing 1-3min, be placed in 23 ℃, the constant-temperature table of 85r/min continues shaking culture 16 ~ 36h;
(4) taking off bacterium cultivates:
Wash for several times bacterium with sterilized water, with the resuspended China fir callus of China fir liquid nutrient medium, the China fir callus after resuspended is transferred in the mode of paving plate on the body embryonal induction substratum that is added with cephamycin 500mg/L, wherein every ware 4-5mL; And put into 23 ℃ of constant incubators and secretly cultivate; Every 21 days subcultures once, induced 30 ~ 60 days; Body embryonal induction substratum is the 1/2DCR minimum medium, additional Vc 10 mg/L, GA 2-8 mg/L, inositol 2-10g/L, CH 0.5g/L, AC 2g/L, maltose 25g/L, crystal agar 2.8g/L;
(5) screening and culturing:
After China fir cotyledonary embryos maturation, it is chosen on the DCR minimum medium, carry out illumination cultivation under 23 ℃, be added with the 200mg/L cephamycin in substratum; Transfer to after 21 days and be added with the 100mg/L cephamycin, the enterprising row filter of the DCR minimum medium of 20mg/L kantlex is cultivated, and every 21 days subcultures once.
2. China fir genetic transforming method according to claim 1, is characterized in that, in step (2), and the resuspended thalline of China fir liquid subculture medium, the OD of bacterium liquid
600Be 0.5.
3. China fir genetic transforming method according to claim 1, is characterized in that, in step (4), contains the 500mg/L cephamycin in sterilized water.
4. China fir genetic transforming method according to claim 1, it is characterized in that: described EHA105 is with 35S:GUS gene and NPT-II gene.
5. China fir genetic transforming method according to claim 1 is characterized in that: in step (5), after subculture 2 times, just can be used for transplanting.
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| CN109874675A (en) * | 2019-04-09 | 2019-06-14 | 南京林业大学 | A method of promote China fir body embryo to occur using deionized formamide |
| CN110194724A (en) * | 2019-01-07 | 2019-09-03 | 广州同隽医药科技有限公司 | A kind of compound containing diphenyl-methane structure and its application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106222181A (en) * | 2016-08-11 | 2016-12-14 | 南京林业大学 | Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α CLPSK2 gene and application thereof |
| CN106244595A (en) * | 2016-08-11 | 2016-12-21 | 南京林业大学 | Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α CLPSK1 gene and application thereof |
| CN106222181B (en) * | 2016-08-11 | 2019-05-07 | 南京林业大学 | China fir phytosulfokine-α CLPSK2 gene and its application |
| CN110194724A (en) * | 2019-01-07 | 2019-09-03 | 广州同隽医药科技有限公司 | A kind of compound containing diphenyl-methane structure and its application |
| CN109874675A (en) * | 2019-04-09 | 2019-06-14 | 南京林业大学 | A method of promote China fir body embryo to occur using deionized formamide |
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