CN100354423C

CN100354423C - Method for culturing transgene aloes

Info

Publication number: CN100354423C
Application number: CNB2005101090720A
Authority: CN
Inventors: 何聪芬; 叶兴国; 董银卯; 张佳星; 陈杰; 王艳丽; 徐惠君; 杜丽璞; 赵华; 兰社益
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2005-10-17
Filing date: 2005-10-17
Publication date: 2007-12-12
Anticipated expiration: 2025-10-17
Also published as: CN1742565A

Abstract

The present invention discloses a method for culturing transgenic aloes. The method comprises the following steps: step 1, sterilized stem segments are used as explants and are cut into blocks, the stem blocks are soaked in agrobacterium liquid which contains target genes and is prepared by the suspension of a liquid culture medium for the co-culture of aloe agrobacteria at the temperature of 23 to 27 DEG C for 20 to 40 min for infection, and then the stem blocks are taked out and are further infected for 2 to 4 days under the conditions that the temperature is from 22 to 26 DEG C, and the light irradiation lasts 8 to 12 hours/day. step 2, the aloe stem blocks after the agrobacterium infection are placed in a recovery culture medium without selective agents, and the recovery culture lasts for 6 to 8 days; step 3, the aloe stem blocks after the recovery culture are placed in a cespitose bud inducing and sieving culture medium, and the culture lasts for 18 to 22 days under the conditions that the temperature is from 22 to 26 DEG C, and the light irradiation lasts 8 to 12 hours/day; step 4, calluses or fresh cespitose buds grown on the cespitose bud inducing and sieving culture medium are transplanted into a new cespitose bud inducing and sieving culture medium, and secondary culture and sieving culture are carried out under conditions the same as those in step 3 to obtain cespitose buds; step 5, transgenic aloes are obtained after seedling adaptation and rootage of the cespitose buds.

Description

A kind of method of cultivating transgene aloes

Technical field

The present invention relates to a kind of method of cultivating transgene aloes.

Background technology

Aloe (Aloe) belongs to the perennial monocotyledons of Liliaceae, has medical treatment, beauty treatment, health care, multiple function such as edible, is described as " the 21 century mankind's optimum medicines " by Food and Argriculture OrganizationFAO (FAO).Can be used for the good aloe kind of commercial cultivation, must possess three conditions simultaneously: 1) active constituent content height; 2) adaptability is strong, is convenient to enlarge cultivated area, and the development intensification is produced; 3) yield per unit height helps obtaining best economical efficiency.Yet aloe is not cold-resistant, though drought-enduring, poor growth in barren, arid soil, and yield poorly; Edible Aloe kind bitter, sense of taste are poor, and these unfavorable factors have hindered the extensive industrialization process of aloetic.Though aboundresources (having kind more than 500) in the Aloe, aloe blooms shaky, or setting percentage is extremely low, and seed seedling-raising technical sophistication, seedling rate low (Xiong Youqing, Yao Li, aloe, the China Agricultyre University Press, 62-65).In addition, the improvement of Part Traits can not reach by conventional breeding, need be by engineered means.At present people research lays particular emphasis on analysis and development and use to its effective constituent to aloetic.(red by continuing, the aloe Study on tissue culture is made progress, Chinese wild plant resource, 2001,2:10-11 though aloe tissue culture and fast breeding technique are ripe; Du Yongjun, Li Huizhi, Qi Yunzhi, the fast numerous and test-tube plantlet cultivation domestication research of aloe bud, northwest Botany Gazette, 2003,12 (1): 72-75), but the structure of aloe transformation system and the research of relevant transgenosis aspect thereof are not appeared in the newspapers as yet.

Plant transgene mainly adopts agrobacterium-mediated transformation and particle gun mediated method.Agrobacterium-mediated transformation is simple to operate because of having, cost is low, transformation efficiency is high, good reproducibility, can import large fragment DNA, and the gene that imports is generally single copy or low copy is integrated, can reduce advantages such as the reticent phenomenon of producer, be most widely used, in the improvement of dicotyledonous cash crop such as soybean, applied.Along with people's going deep into to agrobacterium-mediated transformation transformation mechanism understanding, method for transformation also constantly is improved, as select the bacterial strain of high invasiveness, super binary vector, suitable explant, promotor, suitable cotransformation substratum, suitable selective agent, the phenolic compound such as the Syringylethanone (acetoayringone of higher concentration efficiently for use, AS) processing waits (Kong Yingzhen, Zhou Gongke, Wang Genxuan, Wang Yafu, influence the factor of agrobacterium tumefaciens conversion and the application on monocot crops thereof, Chinese Journal of Applied Ecology, 2000,11 (5): 791-794).The agrobacterium-mediated transformation that utilizes improvement has also successfully obtained transfer-gen plant in paddy rice, corn, wheat, barley etc. have the monocotyledons of important value, and detailed molecular biology and heredity evidence (Hiei Y have been obtained, Komari T, Kabo T., Transformation of rice mediated by Agrobacteriumtumefaciens., Plant Mol Biol, 1997,35:205-218; Ishida Y, Saito H, Ohta S.High efficiency transformat ion of maize (Zea mays L) mediated by Agrobacteriumtumefaciens, Nat Biotech, 1996,14:745-750; Cheng Ming, E.F.Joyce, PangShengzhi et al., Genetic transformation of wheat mediated by Agrobacteriumtumefaciens, Plant Physiol, 1997,115:971-980).At present, people are devoted to widen the raising of monocotyledonous host range of Agrobacterium-mediated Transformation and transformation efficiency, set up agriculture bacillus mediated transgenosis scheme (the CHAI Bao-Feng of unifacial leaf annual bluegrass after deliberation as Chai Baofeng etc., LIANG Ai-Hua, WANG Wei, Hu Wei, Agrobacterium-mediated Transformation of Kentucky Bliuegrass, Acta BotanicaSinica, 2003,45 (8): 966-973), find that the principal element that influences transgene efficiency has: the embryo of callus, light application time, the cotransformation time, antibiotic concentration and selective pressure.The monocotyledonous example that successfully transforms that has obtained shows, the embryo callus that rataria and maturation or immature embryo produce through inducing is the basis (Yin Liqing that Agrobacterium-mediated Transformation and regeneration are succeedd, Zhang Jianjun, Wang Huimei, Cheng Lei, Wang Xinqi, Shen Gezhi improves the research that Agrobacterium tumefaciens mediated rice genetic transforms transformation frequency, Shanghai Communications University's journal (agricultural sciences), 2003,21 (supplementary issues): 43-47), in addition, culture condition comprises temperature altogether, time, medium component also is the principal element (Li Wei that influences Agrobacterium-mediated Transformation efficient, Guo Guangqin, Zheng state Chang, some new developments (commentary) of Agrobacterium tumefaciens mediated Study on Genetic Transformation, Science Bulletin, 2000,45 (8): 798-807).How to induce the non-embryonal connective tissue that produces suitable Agrobacterium-mediated Transformation, the suitable common culture condition of development is one of task of at present agriculture bacillus mediated monocotyledons gene transformation.

Summary of the invention

The purpose of this invention is to provide a kind of method of utilizing agrobacterium-mediated transformation to cultivate transgene aloes.

The method of cultivation transgene aloes provided by the present invention can may further comprise the steps:

1) be explant with stem section through sterilization, after being cut into fritter, under 23-27 ℃, soaking 20-40min with the bacterium liquid of cultivating the Agrobacterium that carries goal gene made from liquid nutrient medium (ACC) suspension through the aloe Agrobacterium altogether infects, take out the stem piece, under the condition of 22-26 ℃, illumination 8-12 hour/day, further infected 2-4 days again; It is to have added 6-benzyladenine (6-BA) on the basis of 1/8-1/12MS minimum medium that described aloe Agrobacterium is cultivated altogether with liquid nutrient medium, α-Nai Yisuan (NAA), and Syringylethanone (acetoayringone, AS), glucose, glutaminate and casein hydrolysate;

2) the Caulis Aloe piece that infects through Agrobacterium is placed on the recovery media (ARC) that does not add selective agent, under the condition of 22-26 ℃, illumination 8-12 hour/day, recover to cultivate 6-14 days; The described recovery media that does not add selective agent is the microbiotic that has added 6-benzyladenine and α-Nai Yisuan and Agrobacterium is had lethal effect on the basis of MS minimum medium;

3) the Caulis Aloe piece through recover cultivating is placed on inducing clumping bud and the screening culture medium (ASC), cultivation is 18-22 days under the condition of 22-26 ℃, illumination 8-12 hour/day, the inducing and screening of the bud of growing thickly; Described inducing clumping bud and screening culture medium are to have added 6-benzyladenine on the basis of MS minimum medium, α-Nai Yisuan, glufosinate-ammonium (commercial by name Liberty, French Aventis crop science company) and the microbiotic that Agrobacterium is had lethal effect;

4) callus that will grow on inducing clumping bud and screening culture medium or the tender bud of growing thickly of children move to and carry out subculture and screening and culturing on new inducing clumping bud and the screening culture medium under the conditions identical with step 3), the acquisition bud of growing thickly;

5) will grow thickly after bud carries out strong sprout, takes root, obtain transgene aloes.

The carry out disinfection method of sterilization of Caulis Aloe section be can be: the Caulis Aloe section is earlier soaked 20-30min in 20%NaClO solution, in 0.1% mercuric chloride (HgCl) solution, soak 6-8min again.

The preparation method of bacterium liquid who carries the Agrobacterium of goal gene in the step 1) can be: with OD ₆₅₀Value is cultivated altogether with in the liquid nutrient medium and is got final product for the thalline in the Agrobacterium bacterium liquid that carries goal gene of 0.6-0.8 is suspended in the long-pending aloe Agrobacterium of 0.6-1 times of bacteria liquid.The Caulis Aloe piece can be placed on the filter paper of sterilization, under 22-26 ℃, illumination 8-12 hour/day condition, infect further.

For determining sterilising effect, before infecting with Agrobacterium, can earlier the Caulis Aloe piece through sterilization be placed on the inducing clumping bud substratum (AC), the pre-cultivation 5-6 days under the condition of 22-26 ℃, illumination 8-12 hour/day, preferred pre-culture condition is: 24 ℃ of temperature, illumination 10 hours/day; Described inducing clumping bud substratum is to have added 6-benzyladenine and α-Nai Yisuan on the basis of MS minimum medium.

The Agrobacterium that is used to infect in the described step 1) can be any one agrobacterium tumefaciens, as EHA101, C58c1 or LBA4404 etc., is preferably EHA101; The aloe Agrobacterium is cultivated altogether to be preferably on the basis of 1/10MS minimum medium with liquid nutrient medium and has added 6-benzyladenine, α-Nai Yisuan, Syringylethanone (acetoayringone, AS), glucose, glutaminate and casein hydrolysate; Microbiotic in step 3) inducing clumping bud and the screening culture medium can be any one microbiotic that Agrobacterium is had lethal effect, as cephamycin, vancomycin or Pyocianil etc.

The concentration of used culture medium additive is in above-mentioned method of cultivation: the concentration of 6-BA was 2.5-4mg/L during the aloe Agrobacterium was cultivated with liquid nutrient medium altogether, be preferably 3mg/L, the concentration of NAA is 0.15-0.2mg/L, be preferably 0.18mg/L, the concentration of AS is 180-220 μ M, be preferably 200 μ M, the mass percentage concentration of glucose is 0.8-1.2%, be preferably 1%, the concentration of glutaminate is 0.3-0.7g/L, be preferably 0.5g/L, the concentration of casein hydrolysate is 0.08-0.12g/L, is preferably 0.1g/L; The concentration of not adding 6-BA in the recovery media of selective agent is 2.5-4mg/L, is preferably 3mg/L, and the concentration of NAA is 0.15-0.2mg/L, is preferably 0.18mg/L; The concentration of 6-BA is 2.5-4mg/L in inducing clumping bud and the screening culture medium, be preferably 3mg/L, the concentration of NAA is 0.15-0.2mg/L, be preferably 0.18mg/L, the concentration of glufosinate-ammonium is 2-3mg/L, is preferably 2.5mg/L, microbiotic kind difference is the concentration difference then, as when selecting Pyocianil for use, concentration is 200-300mg/L, is preferably 250mg/L; The concentration of 6-BA is 2.5-4mg/L in the inducing clumping bud substratum, is preferably 3mg/L, and the concentration of NAA is 0.15-0.2mg/L, is preferably 0.18mg/L.

The method that in the step 1) Caulis Aloe piece is infected is preferably: will be after disinfectant Caulis Aloe section is cut into small pieces, earlier under 25 ℃, carry in the bacterium liquid of Agrobacterium of goal gene and soak 30min, take out the stem piece, again at 24 ℃, under 10 hours/day the condition of illumination, on the filter paper of sterilization, cultivated 3 days; Step 2) recovering culture condition on the recovery media that does not add selective agent through Caulis Aloe piece that Agrobacterium is infected in is preferably: cultivated 7 days under 24 ℃, 10 hours/day condition of illumination; Caulis Aloe piece through recover cultivating in the step 3) is preferably at the culture condition on inducing clumping bud and the screening culture medium: cultivation is 20 days under 24 ℃, 10 hours/day condition of illumination.

The invention provides a kind of method of utilizing agrobacterium-mediated transformation to cultivate transgene aloes, aspect practical application, this method will produce following positively effect: (1) quickens the transfer of good foreign gene in aloe; (2) for improving cold-resistant, the salt resistance ability of aloe growth phase, expansion aloetic planting range improves the output of unit surface aloe leaf and lays a good foundation; (3) for strengthening the effect and the stability of aloe medical treatment, healthcare product, improve the mouthfeel of aloe food, prolong the aloe food freshness date, improve the aloe makeup preserve moisture, prevent many improvement with reality and industrialization meaning such as ultraviolet effect everything goes well with your work realize laying the foundation, thereby quicken the aloetic industrialization process.In addition, the present invention understands fully the principal element that influences agriculture bacillus mediated fleshiness monocotyledons transformation system, excavates and also utilizes agriculture bacillus mediated widely transgene receptor resource also significant.

The present invention will be further described below in conjunction with specific embodiment.

Description of drawings

Fig. 1 is for being induced the bud of growing thickly of generation by the Caulis Aloe section

Figure 1A is the physical map of pPNT289

Figure 1B is the physical map of pPNT290

Fig. 2 is that the coloration result of X-gluc after cultivating 3 days altogether on solid medium and the filter paper is respectively infected after the Caulis Aloe section in the combination of different Agrobacteriums-plasmid vector

Fig. 3 is the growing state of Caulis Aloe piece after growing 14 days on the inducing clumping bud solid medium that is added with 1mg/L, 3mg/L, 5mg/L glufosinate-ammonium

Fig. 4 for the Caulis Aloe piece at the survivorship curve figure of growth after 14 days on the inducing clumping bud solid medium that is added with different concns (1,2,3,4,5mg/L) glufosinate-ammonium

Fig. 5 is the PCR detected result of transgene aloes

Fig. 6 is the ELISA detected result of transgene aloes

Fig. 7 is the aloe transgenic seedling

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment; Used percentage composition is the quality percentage composition.

Inducing clumping bud substratum (AC): on the basis of MS minimum medium, add 2.5mg/L 6-BA and 0.15mg/LNAA.

The aloe Agrobacterium is cultivated altogether with substratum (ACC): add 2.5mg/L 6-BA on the basis of 1/10MS substratum, 0.15mg/L NAA, 200 μ M AS, 1% glucose, 0.5g/L glutaminate, 0.1g/L casein hydrolysate (solid medium adds 7-8% agar again).

Aloe recovery media (ARC): on the basis of MS minimum medium, add 2.5mg/L 6-BA, 0.15mg/LNAA and 250mg/L Pyocianil.

Aloe inducing clumping bud and screening culture medium (ASC): on the basis of MS minimum medium, add 2.5mg/L6-BA, 0.15mg/L NAA, glufosinate-ammonium 2.5mg/L and 250mg/L Pyocianil.

The optimization of embodiment 1, aloe explant sterilization method

With the Caulis Aloe section is example, sterilising method to the aloe explant is optimized, concrete grammar is: the fritter that the Caulis Aloe section is cut into about 1cm * 1cm, after in 70% alcohol, soaking 30s, be divided into four groups, respectively through 0.1% mercuric chloride solution (HgCl) sterilization 8min, 0.1%HgCl solution sterilization 13min, 10% clorox (NaClO) solution sterilization 15min, 20%NaClO solution sterilization 25min, use rinsed with sterile water 3-4 time again, then the Caulis Aloe piece is placed on the inducing clumping bud substratum, at 24 ℃, cultivate under 10 hours/day the condition of illumination, statistics explant surviving rate and pollution ratio, the result is as shown in table 1, take into account surviving rate and sterilizing efficiency, determine the aloe explant is soaked 20-30min earlier in 20%NaClO solution, soaking 6-8min again in 0.1% mercuric chloride (HgCl) solution is preferred aloe explant sterilization method.

The comparative result of the different sterilising method effects of table 1 aloe stem explants

	The explant sum	Microbiological contamination explant number not	Not microbiological contamination ratio (%)	Survive the explant number	Survive ratio (%)
	The explant sum	Microbiological contamination explant number not	Not microbiological contamination ratio (%)	Survive the explant number	Survive ratio (%)	0.1% mercuric chloride soaked 13 minutes	139	121	87.1	90	42.7
0.1% mercuric chloride soaked 8 minutes	137	90	65.7			0.1% mercuric chloride soaked 13 minutes	139	121	87.1
0.1% mercuric chloride soaked 8 minutes	137	90	65.7	10% clorox soaked 15 minutes	59	0	0.0	179	82.5
20% clorox soaked 25 minutes	289	217	75.1	10% clorox soaked 15 minutes	59	0	0.0

Embodiment 2, be used for the preferred of aloe explant that Agrobacterium infects

After the aloe sterilization, respectively blade, leaf sheath, stem section are cut into the fritter of 1cm * 1cm, place on the inducing clumping bud substratum, under 24 ℃, 10 hours/day condition of illumination, cultivate 4-5 week, observe the regeneration situation of explant, statistics survival number and survival ratio, the result is as shown in table 2, determine that aloetic stem section is to be most appropriate to the explant that Agrobacterium is infected, grow thickly bud as shown in Figure 1 through what induce generation by it.

Table 2 aloe explant upgrowth situation comparative result

Explant type	The explant number	The survival number	Survival ratio %
Explant type	The explant number	The survival number	Survival ratio %	Blade	224	0	0.0
Leaf sheath	140	31	22.1	Blade	224	0	0.0
Leaf sheath	140	31	22.1	Stem	148	103	69.6

The comparison that embodiment 3, different Agrobacterium fungus strains infect effect

With plasmid vector pPNT289 (physical map is shown in Figure 1A), pPNT290 (physical map is shown in Figure 1B), transform Agrobacterium EHA101 respectively, carried the recon of pPNT289, pPNT290, called after EHA101/pPNT289 and EHA101/pPNT290 respectively through screening; With plasmid pPNT289, pPNT290 being transformed Agrobacterium C58c1 respectively, carried the recon of pPNT289, pPNT290, called after C58c1/pPNT289, C58c1/pPNT290 respectively through screening with quadrat method.Collect above-mentioned four kinds of OD ₆₅₀Value is the thalline of 0.8 reorganization Agrobacterium bacterium liquid, thalline is suspended in the long-pending aloe Agrobacterium of 0.8 times of above-mentioned bacteria liquid cultivates altogether with in the liquid nutrient medium (ACC), obtains Agrobacterium bacterium liquid.To infect 30min to the Caulis Aloe section with four kinds of Agrobacterium bacterium liquid that the suspension of ACC liquid nutrient medium is made then, again respectively on ACC solid medium and aseptic blank filter paper at 24 ℃, cultivated altogether 3 days under 10 hours/day the condition of illumination, after cultivating end, it is carried out X-Gluc dyeing, coloration result as shown in Figure 2, the transient expression rate of gus gene in the explant of calculating through infecting, and be analyzed, the result is as shown in table 3, the effect that infects of EHA101/pPNT289 obviously is better than other combination, the transient expression rate of gus gene reaches about 80% in the explant after EHA101 infects, and the transient expression rate of gus gene only has an appointment 30% in the explant of C58c1 after infecting, the integral body that shows the EHA101 fungus strain infects effect and is better than the c58c1 fungus strain, and EHA101 is defined as preferably being used to make up the starting strain that the aloe explant infects bacterial strain.

The coloration result of X-gluc after cultivating 3 days altogether on solid medium and the filter paper is respectively infected after the Caulis Aloe section in the combination of different Agrobacteriums of table 3-plasmid vector

Fungus strain-carrier combinations	Co-culture method	The explant number that X-gluc detects	Express the explant number of GUS gene	Dyeing ratio (%)
Fungus strain-carrier combinations	Co-culture method	The explant number that X-gluc detects	Express the explant number of GUS gene	Dyeing ratio (%)	EHA101/pPNT289	Filter paper		30	20	66.7
Solid medium (ACC)	31	8	25.8			Filter paper		30	20	66.7
Solid medium (ACC)	31	8	25.8	EHA101/pPNT290		Filter paper	36	2	5.6
Solid medium (ACC)	33	1	3.0			Filter paper	36	2	5.6
Solid medium (ACC)	33	1	3.0		C58c1/pPNT290	Filter paper	33	0	0.0
Solid medium (ACC)	34	0	0.0			Filter paper	33	0	0.0
Solid medium (ACC)	34	0	0.0	C58c1/pPNT289		Filter paper	29	1	3.5
Solid medium (ACC)	34	0	0.0			Filter paper	29	1	3.5

The cultivation of embodiment 4, transgene aloes and detection

One, the cultivation of transgene aloes

With Agrobacterium EHA101 is starting strain, utilizes agrobacterium-mediated transformation to cultivate transgene aloes, and concrete cultivating process may further comprise the steps:

1, Agrobacterium infects

With the Caulis Aloe section is explant, after cleaning, the Caulis Aloe section is soaked 25min earlier in 20%NaClO solution, soaks 8min again and sterilize in 0.1% mercuric chloride solution.Caulis Aloe section after will sterilizing with the 15# scalpel is cut into the fritter of 1cm * 1cm.With OD ₆₅₀Value is that the thalline that carries among 0.7 the embodiment 3 in the Agrobacterium EHA101/pPNT289 bacterium liquid of goal gene is suspended in the long-pending ACC liquid nutrient medium of 1 times of bacteria liquid, obtains EHA101/pPNT289 bacterium liquid.Under 25 ℃, the EHA101/pPNT289 bacterium liquid that will place the suspension of ACC liquid nutrient medium to make through the Caulis Aloe piece of sterilization soaks 30min and infects, take out the stem piece, place on the aseptic blank filter paper, under 24 ℃, 10 hours/day condition of illumination, cultivate altogether again and further infected in 3 days.

2, recover to cultivate

The Caulis Aloe piece through Agrobacterium is infected that step 1 is obtained places on the recovery media that does not add the antibiotic-screening agent, under 24 ℃, 10 hours/day condition of illumination, recovers to cultivate 7 days.

3, grow thickly the determining of the inducing of bud, screening and selective agent suitable concentration

1) definite Caulis Aloe piece through recovering to cultivate that step 2 is obtained of selective agent glufosinate-ammonium suitable concentration places and is added with different concns (1mg/L in inducing clumping bud and the screening culture medium, 2mg/L, 3mg/L, 4mg/L, 5mg/L) in the inducing clumping bud solid medium of selective agent glufosinate-ammonium, at 24 ℃, cultivate under 10 hours/day the condition of illumination, observe growth of aloe explant and bud and induce situation, the Caulis Aloe piece of cultivating after 14 days is being added with 1mg/L, 3mg/L, growing state on the inducing clumping bud solid medium of 5mg/L glufosinate-ammonium as shown in Figure 3, the stem hop count and the survival ratio of statistics survival, statistics is as shown in table 4, be depicted as graphic representation (Fig. 4 according to statistics, X-coordinate is a selective agent concentration, ordinate zou is the survival ratio), selective agent concentration during with surviving rate 50% is decided to be its suitable concentration, thus determine aloe transform in the suitable concentration of selective agent glufosinate-ammonium in inducing clumping bud and the screening culture medium (with the bar gene as selective marker) be 2.5mg/L.

Under different concns glufosinate-ammonium selective agent, the survive statistics of ratio of table 4 Caulis Aloe section

Selective agent concentration (mg/L)	Caulis Aloe section sum	Survival stem hop count	Survival ratio (%)
Selective agent concentration (mg/L)	Caulis Aloe section sum	Survival stem hop count	Survival ratio (%)	1	43	34	79.1
2	38	24	63.2	1	43	34	79.1
2	38	24	63.2	3	40	17	42.5
4	39	15	38.5	3	40	17	42.5
4	39	15	38.5	5	39	11	28.2

2) grow thickly the inducing, screen of bud

Caulis Aloe piece through recover cultivating is placed on the inducing clumping bud and screening culture medium that contains 2.5mg/L glufosinate-ammonium, and cultivation is 20 days under 24 ℃, 10 hours/day condition of illumination, the inducing and screening of the bud of growing thickly.

4, the grow thickly acquisition of bud

Callus that step 3 is grown on inducing clumping bud and screening culture medium or the tender bud of growing thickly of children move to and carry out subculture and screening and culturing on new inducing clumping bud and the screening culture medium under the conditions identical with step 3, the acquisition bud of growing thickly.

5, the acquisition of transgene aloes

The bud of growing thickly that step 4 is obtained carries out strong sprout, take root after, obtain transgene aloes.

Two, the detection of transgene aloes

1, the PCR of transgene aloes detects

The transgene aloes that step 1 is obtained detects with the method for PCR, designs primer according to NPT II gene order, and primer sequence is as follows:

Primer 1 (upstream primer): 5 '-TGTTCCGGCTGTCAGCGCAG-3 '

Primer 2 (downstream primer): 5 '-TCGGCAAGCAGGCATCGCCA-3 '

Adopt the SDS method extract total DNA of aloe regrowth and with this as template, under the guiding of primer 1 and primer 2, the NPTII gene in the transfer-gen plant is carried out pcr amplification, the PCR reaction conditions is: 94 ℃ of 5min earlier; 94 ℃ of 30s then, 58 ℃ of 30s, 72 ℃ of 90s, totally 30 circulations; Last 72 ℃ of 10min.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis to be detected, detected result is (swimming lane M:DNA MarkerDL2000 as shown in Figure 5, swimming lane 1-8: different plant to be measured, swimming lane 9: negative control, swimming lane 10: positive control), can amplify size and be the positive transfer-gen plant of 476bp dna fragmentation.

2, the ELISA of transgene aloes detects

Detect the proteic expression of NPT II in the transfer-gen plant with the ELISA method, adopt the Pathoscreen kit for neomyc in phosphotransferase II test kit and the reference reagent box specification sheets of agdia company to operate, detected result is (1-4:PCR male plant to be measured as shown in Figure 6; 5: negative control; 6-7: do not carry out the plant to be measured that PCR detects; 8: positive control), obtained positive transgene aloes (Fig. 7) through step 1 PCR detection and step 2 ELISA detection.

Claims

1, a kind of method of cultivating transgene aloes is characterized in that may further comprise the steps:

1) be explant with stem section through sterilization, after being cut into fritter, under 23-27 ℃, soaking 20-40min with the bacterium liquid of cultivating the Agrobacterium that carries goal gene made from the liquid nutrient medium suspension through the aloe Agrobacterium altogether infects, take out the stem piece, under the condition of 22-26 ℃, illumination 8-12 hour/day, further infected 2-4 days again; It is that to have added concentration on the basis of 1/8-1/12 MS minimum medium be the 6-benzyladenine of 2.5-4mg/L that described aloe Agrobacterium is cultivated altogether with liquid nutrient medium, concentration is the α-Nai Yisuan of 0.15-0.2mg/L, concentration is the Syringylethanone of 180-220 μ M, mass percentage concentration is the glucose of 0.8-1.2%, and concentration is that glutaminate and the concentration of 0.3-0.7g/L is the casein hydrolysate of 0.08-0.12g/L;

2) the Caulis Aloe piece that infects through Agrobacterium is placed on the recovery media that does not add selective agent, under the condition of 22-26 ℃, illumination 8-12 hour/day, recover to cultivate 6-14 days; The described recovery media that does not add selective agent is that to have added concentration on the basis of MS minimum medium be the 6-benzyladenine of 2.5-4mg/L, the α-Nai Yisuan that concentration is 0.15-0.2mg/L and the microbiotic that Agrobacterium is had lethal effect;

3) the Caulis Aloe piece through recover cultivating is placed on inducing clumping bud and the screening culture medium, cultivation is 18-22 days under the condition of 22-26 ℃, illumination 8-12 hour/day, the inducing and screening of the bud of growing thickly; Described inducing clumping bud and screening culture medium are that to have added concentration on the basis of MS minimum medium be the 6-benzyladenine of 2.5-4mg/L, the α-Nai Yisuan that concentration is 0.15-0.2mg/L, the glufosinate-ammonium that concentration is 2-3mg/L and the microbiotic that Agrobacterium is had lethal effect;

2, method according to claim 1 is characterized in that: in the described step 1) to the carry out disinfection method of sterilization of Caulis Aloe section be: the Caulis Aloe section is earlier soaked 20-30min in 20%NaClO solution, soak 6-8min again in 0.1% mercuric chloride solution.

3, method according to claim 1 is characterized in that: carrying the bacterium liquid of the Agrobacterium of goal gene in the described step 1), is with OD ₆₅₀Value is cultivated with making in the liquid nutrient medium altogether for the thalline in the Agrobacterium bacterium liquid that carries goal gene of 0.6-0.8 is suspended in 0.6-1 times of bacteria liquid long-pending aloe Agrobacterium; The Caulis Aloe piece under 22-26 ℃, illumination 8-12 hour/day condition, is advanced-goes on foot to infect and be to carry out on the filter paper of sterilization.

4, according to claim 1 or 2 or 3 described methods, it is characterized in that: before infecting, will place on the inducing clumping bud substratum pre-the cultivation 5-6 days under the condition of 22-26 ℃, illumination 8-12 hour/day earlier through the Caulis Aloe piece of sterilization with Agrobacterium; Described inducing clumping bud substratum is that to have added concentration on the basis of MS minimum medium be that 6-benzyladenine and the concentration of 2.5-4mg/L is the α-Nai Yisuan of 0.15-0.2mg/L.

5, method according to claim 4 is characterized in that: describedly the Caulis Aloe piece is carried out pre-incubated condition be: 24 ℃ of temperature, illumination 10 hours/day.

6, according to claim 1 or 2 or 3 described methods, it is characterized in that: the Agrobacterium starting strain that is used to infect in the described step 1) is EHA101 or C58c1; The aloe Agrobacterium is cultivated with liquid nutrient medium altogether to have added 6-benzyladenine on the basis of 1/10 MS minimum medium, α-Nai Yisuan, Syringylethanone, glucose, glutaminate and casein hydrolysate; Microbiotic in step 3) inducing clumping bud and the screening culture medium is cephamycin, vancomycin or Pyocianil.

7, method according to claim 6 is characterized in that: the Agrobacterium starting strain that is used to infect in the described step 1) is EHA101.

8, according to claim 1 or 2 or 3 described methods, it is characterized in that: the concentration that described aloe Agrobacterium is cultivated altogether with 6-BA in the liquid nutrient medium is 3mg/L, the concentration of NAA is 0.18mg/L, the concentration of AS is 200 μ M, the mass percentage concentration of glucose is 1%, the concentration of glutaminate is 0.5g/L, and the concentration of casein hydrolysate is 0.1g/L; The concentration of not adding 6-BA in the recovery media of selective agent is 3mg/L, and the concentration of NAA is 0.18mg/L; The concentration of 6-BA is 3mg/L in inducing clumping bud and the screening culture medium, and the concentration of NAA is 0.18mg/L, and the concentration of glufosinate-ammonium is 2.5mg/L.

9, according to claim 1 or 2 or 3 described methods, it is characterized in that: the method that in the described step 1) Caulis Aloe piece is infected is: will be through disinfectant Caulis Aloe piece earlier under 25 ℃, soak 30min with the bacterium liquid of cultivating the Agrobacterium that carries goal gene of making through the aloe Agrobacterium altogether with the liquid nutrient medium suspension, take out the stem piece, again at 24 ℃, under 10 hours/day the condition of illumination, cultivated 3 days; Described step 2) recovering culture condition on the recovery media that does not add selective agent through Caulis Aloe piece that Agrobacterium is infected in is: cultivated 7 days under 24 ℃, 10 hours/day condition of illumination; Caulis Aloe piece through recover cultivating in the described step 3) at the culture condition on inducing clumping bud and the screening culture medium is: cultivation is 20 days under 24 ℃, 10 hours/day condition of illumination.