CN102851309A - Transgenic crowtoe plant cultivation and screening method - Google Patents
Transgenic crowtoe plant cultivation and screening method Download PDFInfo
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Abstract
The invention discloses a transgenic crowtoe plant cultivation and screening method which comprises the following steps: (1) constructing a recombinant plant expression carrier containing a marker gene and a target gene, and transforming the recombinant plant expression carrier into crowtoe to obtain a transformed explant; and (2) sequentially performing differentiation culture, selective culture and rooting culture regeneration on the transformed explant to obtain a transgenic crowtoe plant, wherein the marker gene is a phosphomannose isomerase (PMI) gene; and a culture medium for the selective culture or rooting culture contains mannose. The invention constructs the plant expression carrier taking PMI as the screening marker gene and transforms the plant expression carrier into crowtoe through an agrobacterium-mediated transformation method. Results show that the positive transformation rate obtained by taking PMI as the screening marker is 23%, and show that the bio-safe PMI/mannose screening system established by the invention can be used for transgenic crowtoe plant cultivation and screening instead of the traditional antibiotic resistance marker.
Description
Technical field
The present invention relates to cultivation and the screening method of transgenic plant, relate in particular to cultivation and the screening method of transgenosis Root or stem of Littleleaf Indianmulberry plant, belong to cultivation and the screening field of transgenosis Root or stem of Littleleaf Indianmulberry plant.
Background technology
Along with the in recent years application of transgenic technology on plant bioreactor, genetically modified biological safety problem has caused the extensive concern in the world.People are mainly derived from the resistance screening gene of using in the Genetic Transformation in Higher Plants to the food safety of genetically modified crops and the suffering of environmental safety.Up to the present, the selectable marker gene of using at Genetic Transformation in Higher Plants mainly is antibiotic resistance gene and anti-herbicide gene.These resistant maker genes probably can cause the gene contamination problem by the transgenosis drift, ecotope are caused seriously influence.The problem of resistant maker gene aspect food safety also certainly will have influence on the development of transgenic plant.How to remove the resistance screening marks such as microbiotic of transgenic plant, the transgenic plant of cultivating Biosafety have become the focus of current plant genetic engineering research, and one of approach that solves the resistance marker safety problem is exactly to develop the Biosafety marker gene.
Phosphomannose isomerase (PMI) gene is the safe selectable marker gene of a kind of new bio, extensively is present in the organic sphere outside the plant, has huge application potential.Phosphomannose isomerase can be converted into fructose-6-phosphate with Man-6-P, makes the transformant can be take seminose as only or main carbon source normal growth, and non-transformed cell is owing to not utilizing seminose to stop growing.With the PMI gene as selectable marker gene, be applied in the conversion of plant with the screening system of seminose as selective agent, screening procedure is simple, selective agent is cheap, and do not affect transformed plant metabolic balance and grow, unfavorable variation is few, screening effect is remarkable, and is to human body and environmental nonpollution, all applicable in monocotyledons and dicotyledons.In addition, the PMI gene not only can be used as screening-gene and replaces the resistant genes such as microbiotic, also can utilize the dichlorophenol sulfonphthalein colour developing as the reporter gene of transfer-gen plant, and reduces the false positive plant in conjunction with conventional Molecular Detection.Therefore, PMI/ seminose screening system will obtain using more widely in transgenic plant are cultivated, and replaces traditional resistance screening system, eliminates Biosafety hidden danger, becomes in the Genetic Transformation in Higher Plants process safely and effectively novel screening system.At present, this selection markers has been successfully applied to the plants such as beet, sugarcane, corn, cucumber.
PMI/ seminose screening system depends on Man-6-P, and this does not participate in metabolism and to the effective consumption of the hexose accumulation of the toxic effect of plant-growth and ATP and phosphate anion and realize the screening effect, thereby be subjected to easily the interference of the physiologic factors such as interior other carbohydrate level of substratum or explant, cause the susceptibility of seminose screening lower.Different plants is different to mannose-sensitive, when transgenic beet screens, in selecting substratum, add 30g/L sucrose, when mannose concentration reaches 2.5-3g/L, seedling regeneration is suppressed fully, the mannose concentration that obtains optimal conversion is (Joersbo et al about 1.25g/L, 1998) (Joersbo M, Donaldson I, Kreiberg J (1998) Analysis of mannose selection used for transformation of sugar beet[J] .Mol.Breed 4:111-117.).In the corn Study on Transformation, the optimum concn of seminose and sucrose then is respectively 5g/L and 10g/L (Wright M in the screening culture medium, Dawson J, Dunder E (2001) Efficient biolistic transformation of maize (Zeamays L.) and wheat (Triticumaestivum L.) using the phosphomannoseisomerase gene, pmi, as the selectable marker[J] .Plant Cell Rep 20:429-436.); The optimal concentration that seminose screening system is used for rape then is 4.5g/L(Wallbraun M, Sonntag K, Eisenhauer C (2009) Phosphomannose-isomerase (pmi) gene as a selectable marker for Agrobacterium-mediated transformation of rapeseed [J] .Plant CellTiss.Organ.Cult 99:345-351).
Point out Chinese cassia tree and some beans in the bibliographical information because to have an endogenous PMI active, thereby this screen body ties up on these plants and can not use, for the material that former studies not yet related to, need detect endogenous PMI activity first; In addition, as a kind of new screening system, its application in plant is still very limited, before using, need to the seminose in the screening culture medium and other sugar and and the factor such as phosphorus acid ion concentration do further optimization, also need simultaneously to consider the impact (Yang Li of genotype on transforming, Xu Changjie, Chen Kun pine .PMI/ seminose screen body ties up to the application in the plant transgene. forest-science .2005 May, the 3rd phase of the 41st volume).Joersbo etc. study discovery, select the character of carbon source in the substratum and the concentration of concentration and phosphate anion to become the key factor that restricts transformation efficiency, and genotype and optical density(OD) are less on the impact of screening system.
Root or stem of Littleleaf Indianmulberry (L.Corniculatus L.) has another name called five leaves grass (Herba Galii Bungei), bird foot beans, Birdfoot, it is perennial pulse family Lotus herbaceous plant, originate in the Eurasia Temperate Region in China, the ground such as Hebei China, Yunnan, Guizhou, Sichuan, Gansu all have wild species to distribute.After 1980, some scientific research departments of China introduce the Root or stem of Littleleaf Indianmulberry Cultivar from states such as Canada, are widely used in orchard ground cover, green manure, grassland improvement and herbage.The soft delicate succulence of its cauline leaf, good palatability, all kinds of domestic animals are all liked food, can modulate green hay, and processing grass meal and mixed fodder also can be used as and herd utilization.When being used for green grass or young crops and raising or herd, its dark green phase is long, and it is low to contain saponin, and anti-herding property is strong, can not cause the domestic animal bloat, for general leguminous forage can't be obtained.In addition, barren-resistant, the solid native antiscour ability of Root or stem of Littleleaf Indianmulberry is strong, is a kind of good soil conservation plant.
Up to now, the selectable marker gene that adopts in transgenosis Root or stem of Littleleaf Indianmulberry Genetic Transformation in Higher Plants system mainly is antibiotic resistance gene or anti-herbicide gene, exist comparatively serious Biosafety hidden danger, demand cultivation and the screening method of the transgenosis Root or stem of Littleleaf Indianmulberry plant of a kind of Biosafety of needs urgently.
Summary of the invention
Technical problem to be solved by this invention is the defective that overcomes the existing Biosafety hidden danger of screening method of existing transgenosis Root or stem of Littleleaf Indianmulberry plant, cultivation and the screening method of a kind of new transgenosis Root or stem of Littleleaf Indianmulberry plant are provided, this screening method adopts the Biosafety selectable marker gene, and biological safety is good.
Technical problem to be solved by this invention is achieved through the following technical solutions:
The screening method of transgenosis Root or stem of Littleleaf Indianmulberry plant comprises: (1) makes up the recombinant plant expression vector that contains marker gene and goal gene, and the recombinant plant expression vector is transformed Root or stem of Littleleaf Indianmulberry, obtains to transform explant; (2) will transform explant and obtain transgenosis Root or stem of Littleleaf Indianmulberry plant by differentiation culture, selection cultivation and root culture regeneration successively; Wherein, the marker gene described in the step (1) is Phophomannose isomerase gene; Contain seminose in the substratum of the selection cultivation described in the step (2) or root culture.
Wherein, the substratum of described selection cultivation forms preferably: MS+6-BA0.1mg/L+Cef500mg/L+ sucrose 20g/L+ seminose 15-20g/L; Preferred, the substratum composition that described selection is cultivated is: MS+6-BA0.1mg/L+Cef500mg/L+ sucrose 20g/L+ seminose 20g/L.
The substratum of described root culture forms preferably: MS+6-BA0.1mg/L+Cef100mg/L+NAA0.2mg/L+ sucrose 20g/L+ seminose 10-20g/L;
MS+6-BA0.1mg/L+Cef100mg/L+NAA0.2mg/L+ sucrose 20g/L+ seminose 15g/L more preferably.
The present invention at first measures the Root or stem of Littleleaf Indianmulberry plant explants seminose is tolerated concentration, has designed 27 kinds of different seminose sugar and sucrose concentration proportioning, observes Root or stem of Littleleaf Indianmulberry explant differentiation situation in different concns gradient substratum.Behind the 30d, the explant fresh weight of measuring under the different concns gradient is depicted as typical curve.
Under same sucrose concentration, along with the rising of mannose concentration, the explant fresh weight trend that tapers off, after mannose concentration reached 20g/L, the fresh weight of explant had on average descended 80%.The situation of sprouting of explant under the statistics different concns gradient, under the same sucrose concentration, along with the rising of mannose concentration, the explant differentiation rate declines to a great extent, and the quantity of sprouting reduces, and the growth of bud is also because being suppressed growth retardation.Explant no longer breaks up after mannose concentration reaches 25g/L, and yellow is withered gradually.Under same mannose concentration, increase sucrose concentration, the bud ratio of explant is in rising trend, and bud length is sturdy dense.The cotyledon of relative Root or stem of Littleleaf Indianmulberry, root is comparatively responsive to seminose.The Root or stem of Littleleaf Indianmulberry seed is placed the substratum of different mannose concentration, find the increases along with mannose concentration after 2 weeks, percentage of germination reduces gradually.Taking root is suppressed, and root system shortens, and after mannose concentration reached 10g/L, taking root was suppressed, and mannose concentration reaches after the 20g/L, and taking root is suppressed fully.By measuring explant fresh weight, bud ratio, root system length etc., observing explant breaks up and the situation of taking root in different concns gradient substratum, determine that Root or stem of Littleleaf Indianmulberry is to the tolerance concentration of seminose, the result shows in the explant differentiation sprouts period, seminose tolerance concentration is 15-20g/L, is preferably 20g/L; Tolerating concentration in the stage of taking root is 10-20g/L, is preferably 15g/L; On this basis, the present invention has set up the Root or stem of Littleleaf Indianmulberry genetic conversion system take PMI as selection markers.
According to present bibliographical information, when transgenic beet screens, in substratum, add 30g/L sucrose, when mannose concentration reached 2.5-3g/L, seedling regeneration was suppressed fully, and the mannose concentration that obtains optimal conversion is (Joersbo et al about 1.25g/L, 1998), in the corn Study on Transformation, the optimum concn of screening culture medium seminose and sucrose then is respectively 5g/L and 10g/L (Wright et al, 2001).The optimal concentration that seminose screening system is used for rape then is seminose 4.5g/L(Wallbraun et al, 2009).The seminose tolerance concentration that the white arteries and veins root of the transgenosis that the present invention determines sprouts period in the explant differentiation is 15-20g/L, stage tolerance concentration is 10-20g/L taking root, obviously high more a lot of than the screening concentration in the bibliographical information, other plant is less to mannose-sensitive relatively for this explanation Root or stem of Littleleaf Indianmulberry.
The present invention has made up take PMI as the plant expression vector of selection markers gene and with it and has transformed Root or stem of Littleleaf Indianmulberry with agrobacterium-mediated transformation, screen by seminose, the resistant plant that obtains is carried out PCR, RT-PCR and Southern-blotting detection, and transfer-gen plant carried out enzyme activity determination, experimental result shows take PMI and obtains transformation efficiency as 23% as selection markers.The present invention has further made up the plant fusion expression vector take PMI as selection markers, this plant fusion expression vector merges avian influenza hemagglutinin antigen gene (MHA) and coli heat-sensitive toxin B subunit gene, by agrobacterium-mediated transformation this plant fusion expression vector is transformed Root or stem of Littleleaf Indianmulberry, obtain 78 strain resistant plants by the determined seminose screening concentration screening of the present invention, obtain to turn plant 37 strains of PMI gene through Molecular Identification, turn plant 28 strains of MHA-MLTB fusion gene, PMI gene and MHAMLTB fusion gene all are integrated into genomic plant 21 strains of Root or stem of Littleleaf Indianmulberry, show that the PMI/ seminose screening system of the Biosafety that the present invention sets up can replace traditional antibiotics resistance tag application in cultivation and the screening of transgenosis Root or stem of Littleleaf Indianmulberry plant.
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art and usually understand identical implication.
Term " host cell " means to comprise the cell of polynucleotide of the present invention, and no matter use which kind of method to insert to produce recombinant host cell, for example directly absorb, other known method in transduction, f pairing or the affiliated field.Exogenous polynucleotide can remain the nonconformity carrier of plasmid for example or can be integrated in the host genome.
Term " polynucleotide " or " gene " mean deoxyribonucleotide, dezyribonucleoside, ribonucleoside or ribonucleotide and the polymkeric substance thereof of sub-thread or bifilar form.Unless specific limited, otherwise the nucleic acid of the known analogue that contains natural nucleotide contained in described term, described analogue have the binding characteristic that is similar to reference nucleic acid and carry out metabolism in the mode of the Nucleotide that is similar to natural generation.Unless other specific limited, otherwise described term also means oligonucleotide analogs, it comprises the PNA(peptide nucleic acid(PNA)), in antisense technology used DNA analogue (thiophosphatephosphorothioate, phosphamide acid esters etc.).Unless otherwise, otherwise specific nucleic acid sequence is also impliedly contained its conservative varient of modifying (including, but is not limited to degenerate codon replaces) and complementary sequence and the clear and definite sequence of appointment.Specific, can realize that through mixing sequence that base and/or Hypoxanthine deoxyriboside residue replace degenerate codon replaces (people such as Batzer, Nucleic Acid Res.19:5081 (1991) by producing one of them or selected more than one (or all) codon the 3rd; The people such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With the people such as Cassol, (1992); The people such as Rossolini, Mol Cell.Probes 8:91-98 (1994)).
Term " goal gene " refers to that this dna sequence dna belongs to external source to this specific host cell, if or from identical primary source but this original series has been carried out modifying or transforms.
Term " conversion ": the heterology dna sequence dna is incorporated into host cell or organic method.
Term " expression ": endogenous gene or transgenosis transcribing and/or translating in vegetable cell.
Term " encoding sequence ": the nucleotide sequence that is transcribed into RNA.
Term " plant expression vector ": one or more are used for realizing the dna vector of Plant Transformation; These carriers often are called as binary vector in this area.Binary vector is mostly to be usually used in agrobacterium-mediated conversion together with the carrier with helper plasmid.Binary vector generally includes: T-DNA shifts needed cis acting sequence, processes so that the selectable marker that can express heterology dna sequence dna to be transcribed etc. through through engineering approaches in vegetable cell.
Breviary term involved in the present invention:
PMI: phosphomannose isomerase; 6-BA:6-benzyl aminoadenine; Cef: cephamycin; NAA: naphthylacetic acid; Rif: Rifampin microbiotic; Kan: kantlex microbiotic.
Description of drawings
The typical curve that the fresh weight of the explant during each concentration gradient of Fig. 1 seminose is drawn; X-coordinate is each concentration of seminose; Ordinate zou is the fresh weight of explant; When S0 represents that sucrose concentration is 0g, along with the continuous increase of the mannose concentration influence curve to Root or stem of Littleleaf Indianmulberry explant fresh weight; When S10 represents that sucrose concentration is 10g, along with the continuous increase of the mannose concentration influence curve to Root or stem of Littleleaf Indianmulberry explant fresh weight; When S20 represents that sucrose concentration is 20g, along with the continuous increase of the mannose concentration influence curve to Root or stem of Littleleaf Indianmulberry explant fresh weight; When S30 represents that sucrose concentration is 30g, along with the continuous increase of the mannose concentration influence curve to Root or stem of Littleleaf Indianmulberry explant fresh weight.
The enzyme of Fig. 2 recombinant plasmid pMD-19T-PMI is cut qualification result; M:DNA Marker; 1, Xhol single endonuclease digestion; 2, pMD-19T plasmid.
The synoptic diagram of Fig. 3 recombinant plasmid pCAMBIA1302-PMI.
The enzyme of Fig. 4 recombinant plasmid pCAMBIA1302-PMI is cut qualification result; M:DNA Marker; 1, Xhol single endonuclease digestion; 2, Xhol single endonuclease digestion.
The PCR of Fig. 5 Root or stem of Littleleaf Indianmulberry resistant plant detects; M:DNA Marker; M:DNA Marker PC:pCAMBIA1302-PMI plasmid NC: wild- type 1,2,3,4,5,6,7,9,10,11: positive plant.
The PCR-Southern of Fig. 6 Root or stem of Littleleaf Indianmulberry resistant plant detects; The PC:pCAMBIA1302-PMI-MLTB-MHA-MARs plasmid; NC: wild-type P1, P2, P3, P4, P5, P6: positive plant.
The RT-PCR of Fig. 7 Root or stem of Littleleaf Indianmulberry plant detects; M:DNA Marker; 1-7: transfer-gen plant.
The enzyme of Fig. 8 recombinant vectors pMD-19T-MHA and pMD-19T-MLTB is cut qualification result.
The enzyme of Fig. 9 recombinant vectors pBI121-MLTB-MHA is cut qualification result.
The enzyme of Figure 10 recombinant vectors pCAMBIA1302-PMI-MLTB-MHA is cut qualification result.
The enzyme of Figure 11 recombinant plasmid pMD-19T-Mars and pCAMBIA1302-PMI-MLTB-MHA is cut qualification result.
Figure 12 recombinant plasmid pCAMBIA1302-PMI-MLTB-MHA-Mars enzyme is cut qualification result.
The structure synoptic diagram of Figure 13 recombinant plasmid pCAMBIA1302-PMI-MLTB-MHA-Mars.
Figure 14 pCAMBIA1302-PMI-MLTB-MHA-Mars changes Agrobacterium PCR qualification result over to.
The PCR detected result of Figure 15 PMI gene.
The PCR detected result of Figure 16 MHA gene.
The PCR detected result of Figure 17 MLTB gene.
The PCR-Southern detected result of Figure 18 MHA gene.
The PCR-Southern detected result of Figure 19 MLTB gene.
The RT-PCR detected result of Figure 20 PMI gene.
The RT-PCR detected result of Figure 21 MHA gene.
The RT-PCR detected result of Figure 22 MLTB gene.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
The foundation of the PMI/ seminose screening system of test example 1 transgenosis Root or stem of Littleleaf Indianmulberry plant
1 test materials
1.1 vegetable material
Root or stem of Littleleaf Indianmulberry (L.Corniculatus L.): inner Root or stem of Littleleaf Indianmulberry difficult to understand is available from Clover Seed ﹠ Turf Co..
1.2 bacterial classification and plasmid
1.2.1. bacterial classification
Agrobacterium tumefaciens lba4404 (Agrobacterium tumefaciens LBA4404) is preserved (agrobacterium tumefaciens lba4404 also can be bought by various biological reagent company and obtain) by inventor laboratory; Bacillus coli DH 5 (Escherichia coli DH5) is available from the full Shi Jin in Beijing biotech firm.
1.2.2 plasmid and gene
PCAMBIA1302 is available from the prosperous Bioisystech Co., Ltd of Beijing ancient cooking vessel state;
The pNOV2804 plasmid (carries the PMI gene; The accession number of PMI gene on GeneBank is GeneID:944840) be so kind as to give by teacher Shen Shihua of Institute of Botany, Chinese Academy of Sciences;
1.3 substratum
MS solid medium (1L): MS macroelement 50mL, MS calcium salt 50mL, MS organic element 5mL, MS molysite 5mL, MS trace element 5mL, MS inositol 5mL, sucrose 30g, plant gel 0.5g/200mL; Be settled to 1L with deionized water, transfer pH to be about 5.86, autoclaving 15min with NaOH.
B5 solid medium (1L): B5 macroelement 50mL, B5 calcium salt 50mL, B5 organic element 5mL, molysite 5mL, B5 trace element 5mL, inositol 5mL, plant gel 0.5g/200mL; Be settled to 1L with deionized water, transfer pH to be about 5.86, autoclaving 15min with NaOH.
LB substratum (1L): Tryptones 10g, yeast extract 5g, NaC l 10g, agar powder 1.70g/200mL; Deionized water is settled to 1L, and autoclaving (121 ℃, 1.034 * 10
5Pa) 15 minutes.
YEB substratum (1L): Tryptones 10g, yeast extract 1g, MgSO
47H
2O 0.5g, sucrose 5g, agar powder 1.7g/200mL; Deionized water is settled to 1L, autoclaving 15min.
2 test methods
2.1 Root or stem of Littleleaf Indianmulberry is to the mensuration of mannose-sensitive
2.1.1PMI the preparation of the different seminose gradient of screening system substratum
Because seminose is different to different plant poisoning degree, sucrose was also different from the seminose optimum proportion when different vegetable cell obtained optimal conversion, thus the different proportion design of this experimental evidence sucrose and seminose 27 concentration gradients carry out the test of mannose-sensitive degree.Being formulated as follows shown in table 1, table 2, table 3 and the table 4 of seminose different concns gradient substratum.
Table 1
| 10:0 | 10:5 | 10:10 | 10:15 | 10:20 | 10:25 | 10:30 | |
| The MS macroelement | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L |
| The MS calcium salt | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L |
| The MS molysite | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS organic element | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS trace element | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS inositol | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| Sucrose | 10g/L | 10g/L | 10g/L | 10g/L | 10g/L | 10g/L | 10g/L |
| Seminose | 0g | 5g/L | 10g/L | 15g/L | 20g/L | 25g/L | 30g/L |
| Plant gel | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L |
| pH | 5.86 | 5.86 | 5.86 | 5.86 | 5.86 | 5.86 | 5.86 |
Table 2
| 20:0 | 20:5 | 20:10 | 20:15 | 20:20 | 20:25 | 20:30 | |
| The MS macroelement | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L |
| The MS calcium salt | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L |
| The MS molysite | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS organic element | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS trace element | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS inositol | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| Sucrose | 20g/L | 20g/L | 20g/L | 20g/L | 20g/L | 20g/L | 20g/L |
| Seminose | 0g | 5g/L | 10g/L | 15g/L | 20g/L | 25g/L | 30g/L |
| Plant gel | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L |
| pH | 5.86 | 5.86 | 5.86 | 5.86 | 5.86 | 5.86 | 5.86 |
Table 3
| 30:0 | 30:5 | 30:10 | 30:15 | 30:20 | 30:25 | 30:30 | |
| The MS macroelement | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L |
| The MS calcium salt | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L |
| The MS molysite | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS organic element | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS trace element | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS inositol | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| Sucrose | 30g/L | 30g/L | 30g/L | 30g/L | 30g/L | 30g/L | 30g/L |
| Seminose | 0g | 5g/L | 10g/L | 15g/L | 20g/L | 25g/L | 30g/L |
| Plant gel | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L |
| pH | 5.86 | 5.86 | 5.86 | 5.86 | 5.86 | 5.86 | 5.86 |
Table 4
| 10:0 | 24:6 | 15:15 | 6:24 | 0:10 | 0:0 | |
| The MS macroelement | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L |
| The MS calcium salt | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L | 50ml/L |
| The MS molysite | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS organic element | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS trace element | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| The MS inositol | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L | 5ml/L |
| Sucrose | 10g | 24g | 15g | 6g | 0g | 0g |
| Seminose | 0g | 6g | 15g | 24g | 10g | 0g |
| Plant gel | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L | 0.5g/L |
| pH | 5.86 | 5.86 | 5.86 | 5.86 | 5.86 | 5.86 |
2.1.2 seminose is on the impact of explant differentiation
Inner Root or stem of Littleleaf Indianmulberry seed difficult to understand is put into triangular flask, with aseptic water washing 2 times, in Bechtop with 75% alcohol-pickled 10min.Again seed is moved on in the triangular flask of the bacterium of going out, then seed is cleaned 6-8 time with aqua sterilisa, seed is moved on to allow its suck dry moisture on the filter paper again.At last seed is moved into the B5(sugar-free) in the substratum, put into illumination box and grow.After planting during 7d, select the good aseptic seedling of growth conditions, its cotyledon and outer plumular axis are downcut respectively, put into the substratum of different gradients, with the blade adaxial and its surface of clip down.20 explants of each gradient, and select explant big or small, solid colour, observe its growing state, determine the best screening concentration of seminose by the fresh weight of measuring 30d.
2.1.3 the impact that seminose is taken root on Root or stem of Littleleaf Indianmulberry
The Root or stem of Littleleaf Indianmulberry seed is put into triangular flask, with aseptic water washing 2 times, in Bechtop with 75% alcohol-pickled 10min.Again seed is moved on in the triangular flask of the bacterium of going out, then seed is cleaned 6-8 time with aqua sterilisa, again seed is moved on to and allow its suck dry moisture on the filter paper, at last seed is moved in the substratum that contains respectively 0g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L seminose (only contain seminose and do not contain sucrose), put into illumination box and grow, observe its percentage of germination and the situation of taking root.
2.2pCAMBIA1302-PMI the acquisition of Agrobacterium-mediated Transformation Root or stem of Littleleaf Indianmulberry and regeneration plant
2.2.1 the acquisition of Root or stem of Littleleaf Indianmulberry aseptic seedling
In Bechtop, with the Root or stem of Littleleaf Indianmulberry seed with 75% alcohol-pickled 10min, use again aseptic water washing 5-8 time, use the aseptic filter paper suck dry moisture, be inoculated on the B5 solid medium, put into illumination box and grow, illumination 16h/ days, 25 ℃ of cultivations, after planting during about 8-12d, seed germination grows cotyledon.
2.2.2pCAMBIA1302-PMI Agrobacterium-mediated Transformation Root or stem of Littleleaf Indianmulberry
Picking list bacterium colony on the flat board of 28 ℃ of cultivation 2d is inoculated into 2mL YEB liquid nutrient medium, and 48h are cultivated in 28 ℃ of shaking tables concussions.Get the above-mentioned bacterium liquid of 500 μ l, add in the 50ml YEB liquid culture, 28 ℃ of shaking table shaking culture make bacterium reach logarithmic phase (OD 600 is 0.5-0.6) with the activation thalline.The centrifugal 15min of 10000rpm, thalline is resuspended with the MS liquid nutrient medium, makes OD 600 be 0.5-0.6.
(1) shakes bacterium
The day before yesterday Agrobacterium is met 1ml in the YEB+RiF30mg/L+Kan50mg/L of about 50ml nutrient solution infecting, then put into 28 ℃ of incubated overnight of shaking table.
(2) centrifugal
The bacterium liquid that being of incubated overnight is yellow is poured in the centrifuge tube of 50ml, and in whizzer 4 ℃, 8000 turn, centrifugal 5min.Then supernatant bacterium liquid is outwelled.Remaining precipitation is mixed into the 60-80ml bacterium liquid that is creamy white with liquid MS medium with precipitation, and these bacterium liquid can be contaminated 2 explants in the culture dish.
(3) infect
Get after planting about 8 days Root or stem of Littleleaf Indianmulberry seedling, shear at the cotyledon petiole position as explant with the Root or stem of Littleleaf Indianmulberry cotyledon, put into culture dish; Mixed uniform Agrobacterium bacterium liquid is poured in the culture dish of putting cotyledon, soaked 20min, then will cotyledon wherein change in the culture dish of filter paper and dry.
(4) the dark cultivation
After blotting bacterium liquid on the explant with filter paper, cotyledon is put into the not MS substratum of added with antibiotic one by one, will put into a filter paper on the substratum and be used for interval insulant and directly contact with substratum, at last culture dish is put into dark, 25 ℃ of dark cultivations 3-4 days.
2.2.3 the acquisition of Root or stem of Littleleaf Indianmulberry regeneration plant
(1) differentiation culture
Callus after secretly cultivating is transferred to (MS+BA0.1mg/L+Cef500mg/L) cultivates in the division culture medium of added with antibiotic.Put into about 1 week of illumination box.
(2) select to cultivate
The well-grown cotyledon callus in one all left and right sides is entered to select the substratum (MS substratum+sucrose 20g/L+ seminose 20g/L+6-BA 0.1mg/L+ cephamycin 500mg/L) from the culture dish transfer.Approximately per two weeks change once new selection substratum.When turning seedling, withered and yellow cauline leaf is cut, brownization and loose softening callus are cut.Numerous such as the need expansion, partly be cut into several with scissors at callus, be divided into several young plants and put into new substratum.
(3) root culture
To selecting well-grown aseptic seedling on the substratum, transfer on the root media of MS+6-BA0.1mg/L+Cef100mg/L+NAA0.2mg/L+ sucrose 20g/L+ seminose 15g/L.Per two weeks are changed once new substratum, and Rapid Rooting is until seedling.
3 test results and analysis
3.1 Root or stem of Littleleaf Indianmulberry is to the tolerance concentration of seminose
3.1.1 the impact that seminose is taken root on Root or stem of Littleleaf Indianmulberry
The impact of Root or stem of Littleleaf Indianmulberry being taken root in order to study seminose in the substratum of seminose and the different proportionings of sucrose, behind the 14d, finds that mannose concentration increases with the Root or stem of Littleleaf Indianmulberry seed, and the Seed germination rate reduces, and taking root is suppressed, and root system shortens.
3.1.2 seminose is on the impact of explant differentiation
Because different plant is different to the tolerance degree of seminose, therefore according to the sucrose proportion design different with seminose the substratum of 27 kinds of different concns gradients, to measure the suitableeest screening concentration.Found that the increase along with mannose concentration, the number of explant formation bud is fewer and feweri, and the explant differentiation that high mannose concentration is processed is slow.After mannose concentration reached 25g/L, explant no longer broke up, and along with the prolongation of time, these explants are gradually by green flavescence, and are withered at last.Even other explant differentiation is arranged when seminose is 25g/L, sprouts but but can not continue differentiation.The explant differentiation rate can be slowed down seminose to the toxicity (table 5) of explant along with the rising of mannose concentration is more and more lower and increase sucrose concentration as can be seen from Table 5.In addition, along with the rising of mannose concentration, the number that the explant differentiation generates bud obviously reduces, and blastogenesis is long slow, and during 30d, the bud quantity that the explant that the high density seminose is processed differentiates reduces, and bud length is short, and growing obviously is suppressed.
Explant differentiation rate under the different mannose concentration of table 5
The fresh weight drawing standard curve (Fig. 1) of the explant of each concentration gradient during according to 30d, can find out when mannose concentration is 20g/L, the fresh weight of explant has on average descended 80%, and root be grown in 15g/L the time almost completely be suppressed, this explanation root more responsive to seminose.
Finding out according to above these data, sprout period in the explant differentiation that seminose tolerance concentration is 15-20g/L, is preferably 20g/L, is 10-20g/L in the stage tolerance concentration of taking root, and is preferably 15g/L.
3.2pCAMBIA1302-PMI the structure of plant expression vector
3.2.1pMD-19T-PMI Vector construction
From pNOV2804 carrier amplification to two ends with the PMI gene fragment of Xhol restriction enzyme site (accession number of PMI gene on GeneBank is Gene ID:944840), be connected to cloning vector pMD-19T multiple clone site place.Extract the plasmid of PCR positive colony, carry out enzyme and cut evaluation, enzyme is cut qualification result and is seen Fig. 2, will identify that correct clone checks order.
3.2.2pCAMBIA1302-PMI the structure of plant expression vector
Cut the correct pMD-19T-PMI plasmid of order-checking with the Xhol enzyme, reclaim small segment.Simultaneously, cut the pCAMBIA1302 plasmid with the Xhol enzyme that spends the night, reclaim large fragment.Through T
4Ligase enzyme connects, and the PMI small segment that reclaims is spent the night with the pCAMBIA1302 large fragment is connected (Fig. 3).To connect product and transform DH5 α competent cell, extraction positive colony plasmid carries out enzyme and cuts evaluation, and enzyme is cut qualification result and seen Fig. 4, will identify that correct plasmid checks order.
3.2.3pCAMBIA1302-PMI plant expression vector transforms Root or stem of Littleleaf Indianmulberry
Choose the good 7d seedling age Root or stem of Littleleaf Indianmulberry aseptic seedling of growth conditions, the clip cotyledon infects 20min as explant with preprepared pCAMBIA1302-PMI Agrobacterium bacterium liquid.Blot the bacterium liquid of explant with aseptic filter paper, put on the MS substratum, with an aseptic filter paper interval, put into growth cabinet and carry out common cultivation, dark culturing 3d between substratum and the explant.Explant after secretly cultivating is transferred to (MS+6-BA0.1mg/L+Cef500mg/L) cultivation in the MS division culture medium that does not add selective agent.Put into an about week of artificial climate incubator.The well-grown cotyledon explant in one all left and right sides is moved in the selection substratum that adds the 20g/L seminose (MS+6-BA0.1mg/L+Cef500mg/L+ sucrose 20g/L), per two weeks change once in the new substratum, and the toxic substance of avoiding producing affects the differentiation of explant.When transplanting seedlings, the callus of withered and yellow leaf and brownization is cut.After about 8 weeks, will select well-grown aseptic seedling on the substratum, transfer in the root media that adds the 15g/L seminose (MS+6-BA0.1mg/L+Cef100mg/L+NAA0.2mg/L+ sucrose 20g/L).Per 2 weeks are changed once new substratum, and Rapid Rooting is until seedling.After about 3 weeks, the seedling of having taken root is changed in the compost, and cover one deck preservative film at Culture basin, and tear an osculum, so that growing environment had both kept was moistening, maintain the circulation of air again.About hardening will be cultivated in its immigration greenhouse after 1 week.
3.2.4pCAMBIA1302-PMI the Molecular Identification of transfer-gen plant
3.2.4.1PCR detect
When treating the positive seedling length of Root or stem of Littleleaf Indianmulberry to the 6cm left and right sides, the small portion tissue of clip seedling extracts DNA and carries out PCR detection (Fig. 5).
3.2.4.2 extracting genome DNA and PCR-Southern detect
In the plant of PCR test positive, choose at random plant and carry out the PCR-Southern detection, hand over take the fragment of the middle 532bp of PMI gene as the probe impurity elimination, found that these several plant of choosing at random are all positive, simultaneously, Southern-Blotting detects the reliability (Fig. 6) of also having verified PCR.
3.2.4.3RNA extract and the RT-PCR detection
Choose the positive plant that PCR-Southern detects, extract total RNA, 28S and 18S band are obvious as a result, and 28S is clear brighter than 18S band, meets the requirement of carrying out follow-up test fully.The total RNA that extracts is carried out reverse transcription become cDNA, then the PMI gene in the plant is carried out RT-PCR and detect, the result is (Fig. 7) as shown in the figure.
3.2.5 dichlorophenol sulfonphthalein detects (Chlorphenol red assay)
Choose at random PCR, RT-PCR, PCR-Southern and detect 5 of all positive plant, and in the PCR-Southern test positive, but RT-PCR detects in the negative plant and get at random 3 strains, carry out dichlorophenol sulfonphthalein and detect.The PCR that chooses at random, PCR-Southern, RT-PCR detect 5 all positive plant, and their tissue all can make medium acidification, thereby makes developer dichlorophenol sulfonphthalein become yellow by redness, illustrate that the PMI that expresses in the tissue has activity.Yet, reach the PCR-Southern test positive of choosing at random at negative control, but RT-PCR detecting the not variable color of substratum of 3 negative plant, this explanation does not detect PMI.
This test utilizes agrobacterium-mediated transformation, and the plant expression vector pCAMBIA1302-PMI take PMI as selection markers is changed in the Root or stem of Littleleaf Indianmulberry cotyledon explant, through selecting Screening of Media, obtains 78 strains of resistance seedling; The resistance seedling detects through PCR, RT-PCR equimolecular, obtains positive seedling 18 strains, and wherein the positive seedling ratio that accounts for the resistance seedling that obtains reaches 23%.
Test example 2 turns cultivation and the screening of avian influenza hemagglutinin antigen gene and coli heat-sensitive toxin B subunit fusion gene Root or stem of Littleleaf Indianmulberry plant
1, the structure of recombinant plasmid pCAMBIA1302-PMI-MLTB-MHA-Mars
1.1 the structure of recombinant vectors pMD-19T-MHA and pMD-19T-MLTB
Avian influenza hemagglutinin gene (HA) derives from Harbin veterinary drug institute of the Chinese Academy of Agricultural Sciences, the present invention is according to the hemagglutinin aminoacid sequence of this high pathogenic avian influenza strain A/Goose/Guangdong/3/96 (H5N1), artificial reconstructed according to the leguminous plants preference codon, the avian influenza hemagglutinin gene of suitable expression of plants has been synthesized in design, and (illustrate: the nucleotide sequence of described " avian influenza hemagglutinin gene " has been CN 100410378C at Granted publication number, open in the patent of invention document of denomination of invention for " gene and plant expression vector and the application of coding avian influenza hemagglutinin ") and be kept in the pMD-MHA plasmid.Pass through pcr amplification, the pMD-MHA that preserves from the laboratory increases two ends respectively with the restriction enzyme site MHA sequence of BamH I and Sac I, be connected on the pMD19-T carrier, choose positive colony, carrying out enzyme cuts, enzyme is cut and be the results are shown in Figure 8, enzyme is cut the correct plasmid company that takes away check order recombinant products called after pMD-19T-MHA.
According to the MLTB primer of design, one section removal TAA terminator, the upstream and downstream of increase is respectively with the MLTB fragment of Xba I and BamH I restriction enzyme site.Amplified fragments pMD-19T carrier is connected, and the picking positive colony carries out enzyme and cuts, and enzyme is cut and be the results are shown in Figure 8, enzyme is cut the correct plasmid company that takes away check order recombinant products called after pMD-19T-MLTB.
1.2 the structure of recombinant vectors pBI121-MLTB-MHA
With Xba I and BamH I digestion with restriction enzyme pMD-19T-MLTB carrier, reclaim the small segment of 400bp; With Xba I and BamH I digestion with restriction enzyme pBI121 carrier, reclaim large fragment.With small segment and the pBI121 large fragment of the 400bp that reclaims, through T
4Ligase enzyme connects.The picking positive colony carries out PCR and enzyme and cuts evaluation.Recombinant products called after pBI121-MLTB.
With BamH I and Sac I digestion with restriction enzyme pMD-19T-MHA carrier, reclaim the fragment of 1700bp; With BamH I and Sac I digestion with restriction enzyme pBI121-MLTB carrier, reclaim large fragment.With 1700bp fragment and the pBI121-MLTB large fragment that reclaims, through T
4Ligase enzyme connects.The picking positive colony carries out PCR and enzyme and cuts evaluation, and qualification result as shown in Figure 9.Recombinant products called after pBI121-MLTB-MHA.
1.3 the structure of recombinant vectors pCAMBIA1302-PMI-MLTB-MHA
With Hind III, EcoR I digestion with restriction enzyme pBI121-MLTB-MHA plasmid, reclaim small segment; With Hind III, EcoR I digestion with restriction enzyme pCAMBIA1302-PMI vector plasmid, reclaim large fragment.With small segment and the pCAMBIA1302-PMI large fragment of the pBI121-MLTB-MHA that reclaims, use T
4Ligase enzyme connects, and the picking positive colony carries out PCR and enzyme and cuts evaluation, and qualification result as shown in figure 10.Recombinant products called after pCAMBIA1302-PMI-MLTB-MHA.
1.4 the structure of recombinant vectors pMD-19T-Mars
Pass through pcr amplification, obtain two ends with the MAR sequence of HindIII restriction enzyme site from the carrier pGMET-MAR amplification of preserving the MAR sequence, be connected on the pMD19-T carrier, its positive colony plasmid of picking, the HindIII enzyme is cut, enzyme is cut, and enzyme is cut correct plasmid order-checking, recombinant products called after pMD-19T-MARs.
1.5 the structure of recombinant vectors pCAMBIA1302-PMI-MLTB-MHA-Mars
With Hind III restriction enzyme single endonuclease digestion pCAMBIA1302-PMI-MLTB-MHA plasmid, reclaim large fragment; With Hind III restriction enzyme single endonuclease digestion pMD-19T-Mars(purpose fragment two ends with Hind III restriction enzyme site), reclaim small segment.With the large fragment of the pCAMBIA1302-PMI-MLTB-MHA plasmid that reclaims and the small segment of pMD-19T-Mars, use T
4Ligase enzyme connects.The picking positive colony carries out PCR and enzyme and cuts evaluation, and the enzyme of recombinant plasmid pMD-19T-Mars and pCAMBIA1302-PMI-MLTB-MHA is cut qualification result as shown in figure 11.Recombinant products called after pCAMBIA1302-PMI-MLTB-MHA-Mar.
With EcoR I restriction enzyme single endonuclease digestion pCAMBIA1302-PMI-MLTB-MHA-Mar plasmid, reclaim large fragment; With EcoR I restriction enzyme single endonuclease digestion pMD-19T-Mars(purpose fragment two ends with EcoR I restriction enzyme site), reclaim small segment.With the large fragment of the pCAMBIA1302-PMI-MLTB-MHA-Mar plasmid that reclaims and the small segment of pMD-19T-Mars, use T
4Ligase enzyme connects.The picking positive colony carries out PCR and enzyme and cuts evaluation, and qualification result as shown in figure 12.Recombinant products called after pCAMBIA1302-PMI-MLTB-MHA-Mars.
The structure synoptic diagram of recombinant plasmid pCAMBIA1302-PMI-MLTB-MHA-Mars is seen Figure 13.
2, pCAMBIA1302-PMI-MLTB-MHA-Mars changes Agrobacterium PCR evaluation over to
The picking regular shape, single bacterium colony of the same size is inoculated in 400 μ l and contains in the corresponding antibiotic LB liquid nutrient medium, puts 37 ℃ of shaking table 200rpm, and then incubation 45min carries out bacterium colony PCR, picking positive colony, order-checking.The PCR reaction conditions is: at first 94 ℃, 5min, and 94 ℃ of sex change 45sec then, 55.4 ℃ of annealing 45sec, 72 ℃ are extended 1min, 30 circulations, last 72 ℃ are extended 10min.The PCR reaction system is as shown in table 6 below.
Table 6PCR reaction system
The PCR qualification result is seen Figure 14.
3, Root or stem of Littleleaf Indianmulberry genetic transformation
(1) acquisition of Root or stem of Littleleaf Indianmulberry aseptic seedling
In Bechtop, the Root or stem of Littleleaf Indianmulberry seed with 75% alcohol-pickled 10min, is used aseptic water washing 5-8 time again, use the aseptic filter paper suck dry moisture, be inoculated on the B5 solid medium, put into illumination box and grow, illumination 16h/ days, 25 ℃ of cultivations.After planting during about 8-12d, seed germination grows cotyledon.
(2) restructuring Agrobacterium-mediated Transformation Root or stem of Littleleaf Indianmulberry
Picking list bacterium colony on the flat board of 28 ℃ of cultivation 2d is inoculated into 2mL YEB liquid nutrient medium, and 48h are cultivated in 28 ℃ of shaking tables concussions.Get the above-mentioned bacterium liquid of 500 μ l, add in the 50ml YEB liquid culture, 28 ℃ of shaking table shaking culture make bacterium reach logarithmic phase (OD 600 is 0.5-0.6) with the activation thalline.The centrifugal 15min of 10000rpm, thalline is resuspended with the MS liquid nutrient medium, makes OD 600 be 0.5-0.6.
A. shake bacterium
The day before yesterday Agrobacterium is met 1ml in the YEB+RiF30mg/L+Ka50mg/L of about 50ml nutrient solution infecting, then put into 28 ℃ of incubated overnight of shaking table.
B. centrifugal
The bacterium liquid that being of incubated overnight is yellow is poured in the centrifuge tube of 50ml, and in whizzer 4 ℃, 8000 turn, centrifugal 5min.Then supernatant bacterium liquid is outwelled.Remaining precipitation is mixed into the 60-80ml bacterium liquid that is creamy white with liquid MS medium with precipitation, and this bacterium liquid can be contaminated 2 explants in the culture dish.
C. infect
Get after planting about 8 days Root or stem of Littleleaf Indianmulberry seedling, shear at the cotyledon petiole position as explant with the Root or stem of Littleleaf Indianmulberry cotyledon, put into culture dish.Mixed uniform bacterium liquid is poured in the culture dish of putting cotyledon, soaked 20min.Then cotyledon that will be wherein changes in the culture dish of filter paper and dries.
D. secretly cultivate
After blotting bacterium liquid on the explant with filter paper, cotyledon is put into the not MS substratum of added with antibiotic one by one, will put into a filter paper on the substratum and be used for interval insulant and directly contact with substratum.At last culture dish is put into dark, 25 ℃ of dark cultivations 3-4 days.
(3) acquisition of Root or stem of Littleleaf Indianmulberry regeneration plant
A. differentiation culture
Callus after secretly cultivating is transferred to (MS+BA0.1++Cef500mg/L) cultivates in the division culture medium of added with antibiotic.Put into about 1 week of illumination box.
B. select to cultivate
The well-grown cotyledon callus in one all left and right sides is entered to select the substratum (MS substratum+6-BA 0.1mg/L+ cephamycin 500mg/L+ sucrose 20g/L+ seminose 20g/L) from the culture dish transfer.Approximately per two weeks change once new selection substratum.When turning seedling, withered and yellow cauline leaf is cut, brownization and loose softening callus are cut.Numerous such as the need expansion, partly be cut into several with scissors at callus, be divided into several young plants and put into new substratum.
C. root culture
To selecting well-grown aseptic seedling on the substratum, transfer on the root media of MS+6-BA0.1mg/L+Cef100mg/L+NAA0.2mg/L+ sucrose 20g/L+ seminose 15g/L.Per two weeks are changed once new substratum, and Rapid Rooting is until seedling.
4, the Molecular Identification of transfer-gen plant
4.1 the PCR of transfer-gen plant detects
The PCR detected result of PMI gene is seen Figure 15, and the PCR detected result of MHA gene is seen Figure 16, and the PCR detected result of MLTB gene is seen Figure 17.
4.2 the PCR-Southern of transfer-gen plant detects
The PCR-Southern detected result of MHA gene is seen Figure 18, and the PCR-Southern detected result of MLTB gene is seen Figure 19.
4.3 the RT-PCR of transfer-gen plant detects
The RT-PCR detected result of PMI gene is seen Figure 20, and the RT-PCR detected result of MHA gene is seen Figure 21, and the RT-PCR detected result of MLTB gene is seen Figure 22.
5. dichlorophenol sulfonphthalein detects
Choose at random PCR, PCR-Southern, RT-PCR and detect all positive Root or stem of Littleleaf Indianmulberry plant 5 strains, choose PCR, PCR-Southern test positive, but RT-PCR detects negative plant 3 strains, carry out dichlorophenol sulfonphthalein and detect.Result: PCR, PCR-Southern, RT-PCR the detection all tissue of 5 positive strain Root or stem of Littleleaf Indianmulberry all can make medium acidification, make developer dichlorophenol sulfonphthalein become yellow by redness, illustrate that the PMI of tissue expression has activity.And the Root or stem of Littleleaf Indianmulberry of negative control and RT-PCR feminine gender can not make the dichlorophenol sulfonphthalein variable color.
This test has made up the plant fusion expression vector pCAMBIA1302-PMI-MHA-MLTB-MARs that fusion take PMI as the selection markers gene has antigen gene and coli heat-sensitive toxin B subunit, constructed plant fusion expression vector is transformed Root or stem of Littleleaf Indianmulberry, the PMI/ seminose of establishing by test example 1 screens system, through selecting Screening of Media, obtain 95 strains of resistance seedling; The resistance seedling detects through PCR, RT-PCR equimolecular, obtains positive seedling 21 strains, and wherein the ratio of the resistance seedling of the shared acquisition of positive seedling can reach 22.1%.
Claims (8)
1. cultivation and the screening method of transgenosis Root or stem of Littleleaf Indianmulberry (L.Corniculatus L.) plant comprise: (1) makes up the recombinant plant expression vector that contains marker gene and goal gene, and the recombinant plant expression vector is transformed Root or stem of Littleleaf Indianmulberry, obtains to transform explant; (2) will transform explant and obtain transgenosis Root or stem of Littleleaf Indianmulberry plant by differentiation culture, selection cultivation and root culture regeneration successively; It is characterized in that: the described marker gene of step (1) is Phophomannose isomerase gene; Contain seminose in the substratum of the selection cultivation described in the step (2) or root culture.
2. in accordance with the method for claim 1, it is characterized in that the substratum composition that described selection is cultivated is: MS+6-BA0.1mg/L+Cef500mg/L+ sucrose 20g/L+ seminose 15-20g/L.
3. in accordance with the method for claim 2, it is characterized in that the substratum composition that described selection is cultivated is: MS+6-BA0.1mg/L+Cef500mg/L+ sucrose 20g/L+ seminose 20g/L.
4. in accordance with the method for claim 1, it is characterized in that the substratum composition of described root culture is: MS+6-BA0.1mg/L+Cef100mg/L+NAA0.2mg/L+ sucrose 20g/L+ seminose 10-20g/L.
5. in accordance with the method for claim 4, it is characterized in that the substratum composition of described root culture is: MS+6-BA0.1mg/L+Cef100mg/L+NAA0.2mg/L+ sucrose 20g/L+ seminose 15g/L.
6. it is characterized in that in accordance with the method for claim 1: described Root or stem of Littleleaf Indianmulberry is bird foot Root or stem of Littleleaf Indianmulberry.
7. it is characterized in that in accordance with the method for claim 6: described bird foot Root or stem of Littleleaf Indianmulberry is inner Austria " Leo ".
8. it is characterized in that in accordance with the method for claim 1: described goal gene comprises avian influenza hemagglutinin antigen gene or coli heat-sensitive toxin B subunit.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103141392A (en) * | 2013-03-19 | 2013-06-12 | 辽宁大学 | Method for rapid and vegetative propagation of crowtoe |
| CN103757050A (en) * | 2014-01-22 | 2014-04-30 | 深圳市农科集团有限公司 | Trivalence HA-LTB (hemagglutinin-leukotrienes B4) fusion expression vector of lotus corniculatus specific expression H5N1 antigen protein |
| CN103865953A (en) * | 2014-01-22 | 2014-06-18 | 中国农业科学院生物技术研究所 | Trivalent HA-2HA1-LTB fusion expression vector of lotus corniculatus specifically-expressed H5N1 antigen protein |
| CN109234306A (en) * | 2018-09-25 | 2019-01-18 | 四川农业大学 | A kind of method for building up of diploid crowtoe transformation system |
-
2012
- 2012-08-08 CN CN2012102807711A patent/CN102851309A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 刘发涛等: "里奥百脉根的有点与利用评价", 《中国草地》 * |
| 张鸭关等: "云南引进帝国百脉根的研究", 《四川草原》 * |
| 郭倩倩: "PMI筛选体系的建立及MHA-MLTB融合基因在百脉根中的表达", 《中国优秀硕士学位论文全文数据库，农业科学辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103141392A (en) * | 2013-03-19 | 2013-06-12 | 辽宁大学 | Method for rapid and vegetative propagation of crowtoe |
| CN103141392B (en) * | 2013-03-19 | 2015-04-08 | 辽宁大学 | Method for rapid and vegetative propagation of crowtoe |
| CN103757050A (en) * | 2014-01-22 | 2014-04-30 | 深圳市农科集团有限公司 | Trivalence HA-LTB (hemagglutinin-leukotrienes B4) fusion expression vector of lotus corniculatus specific expression H5N1 antigen protein |
| CN103865953A (en) * | 2014-01-22 | 2014-06-18 | 中国农业科学院生物技术研究所 | Trivalent HA-2HA1-LTB fusion expression vector of lotus corniculatus specifically-expressed H5N1 antigen protein |
| CN109234306A (en) * | 2018-09-25 | 2019-01-18 | 四川农业大学 | A kind of method for building up of diploid crowtoe transformation system |
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Application publication date: 20130102 |