CN107557385A - Acceptor class kinase gene AcSERK1 genetic transforming method and its application occurs for a kind of pineapple somatic embryo - Google Patents

Abstract

The invention discloses a kind of pineapple somatic embryo to occur acceptor class kinase geneAcSERK1Genetic transforming method and its application.The genetic transforming method of the present invention includes following steps：1）Build pFGC5941 35s AcSERK1 sense expression vectors；2）The preparation of callus and its tolerance test to PPT；3）The Agrobacterium to be suspended through MS infects the preparation of liquid；4）Preculture, infect, co-culture and PPT screening and culturings；5）PPT resistances seedling rooting and transplanting.The transfer-gen plant obtained using the genetic transforming method of the present invention is hybridized through PCR, Southern and RT PCR are detected, it was demonstrated thatAcSERK1Have been inserted into pineapple genomic DNA, and there occurs overexpression in pineapple somatic embryo.Functional verification shows, overexpressionAcSERK1The embryo callus subculture particle that is obtained after somatic embryo inducement of callus and somatic embryo number be significantly higher than wild type, this explanation overexpressionAcSERK1Formation and somatic embryo to pineapple cells,primordial have significant facilitation.

Description

Acceptor class kinase gene AcSERK1 genetic transformation occurs for a kind of pineapple somatic embryo Method and its application

Technical field

The present invention relates to a kind of pineapple somatic embryo occur acceptor class kinase gene AcSERK1 genetic transforming method and its Application in the generation of pineapple somatic embryo, belongs to biological technical field.

Background technology

Pineapple (Ananascomosus) pineapple is also known as, it is one of four big tropical fruit tree of the world, and the torrid zone that China is main Fruit tree, except collective fruits is edible outer, plant is available for viewing and admiring, the separable fiber of leaf.Edible pineapple kind all over the world amounts to more than 100 It is individual, it is divided into the major class of cacaine, queen, Spain and Puerto Rico etc. four.Pineapple is to be selfed not affine plant, and not only same kind is not It is affine, or even pollinated between different cultivars in same major class and can not all produce seed, after fruit harvesting need to inhale bud carry out it is numerous Grow renewal.Because most excellent every plant of cultivars can only be in 1 ~ 3 suction bud not of uniform size of Post flowering pumping, not only sprout Batch production is difficult, also often results in that the maturity period of progeny population etc. is inconsistent, seriously hinders the stock breeding of pineapple.Although spinach The organic regeneration technology of trailing plants cultured in vitro is more ripe, but cost is still too high, can't be as being equally perennial herb fruit tree Banana like that tissue culture commonly used in production.Therefore, pineapple somatic embryo (somatic embryo, SE) hair is studied The related gene and its function of raw early stage, high-frequency somatic embryo occur system is established by regulating and controlling this kind of gene expression, It is all significant to its genetic improvement, embryonic development study mechanism and industrial seedling rearing propulsion etc..

Somatic embryo originates from a non-zygote cell, have similar zygotic embryo growth course, genetically it is relatively stable, The features such as generating capacity is big, easy seedling, is the desirable route that Vitro Plant is quickly bred（Li Jun is bright etc., and 2005）.Embryogensis Life is a complicated gene expression process, and its Physiology and biochemistry and morphological change are regulated and controled by many genes, still only found so far SERK gene expressions are in non-cells,primordial（non-embryogenic cell）Played an important role into cells,primordial transition process (Schmidt et al., 2002； Thomas et al., 1993；Jianru et al., 2002), it is considered to be body is thin Marker gene that blastula occurs (Schmidt et al., 1997; Baudino et al. 2001; Quiroz-Figueroa et al., 2006).Carrot（Daucuscarota）DcSERKOnly expressed in cells,primordial to globular embryo period, and in non-embryo Property cell and globular embryo after somatic embryo in be not detected by expression, useDcSERKPromoter regulation luciferase （luciferase, LUC）The research discovery of reporter gene expression,LUCOnly with " the impression for developing into somatic embryo potentiality Detect in state cell ", and can't detect in the non-cells,primordial simply bred, andLUCExpression quantity with " competence is thin Born of the same parents " have develop between the potentiality of somatic embryo have close quantitative relation (（Schmidt et al., 1997).South will be intended Mustard（Arabidopsis thaliana）AtSERK1Overexpression is carried out in the case where being placed in 35S promoter, energy render transgenic plant is cured The incidence rate of somatic embryo of injured tissue improves 3 ~ 4 times, and without the phenotype for changing plant；AtSERK1Callus group after knockout The Somatic embryogenetic ability knitted is suppressed（Hecht et al., 2001）.Santos etc. also has foundTcSERKIt is cocoa （Theobroma cacao）The essential gene of somatic embryo occur, becauseTcSERKTranscription only in somatic embryo Detect, and can't detect in the callus simply bred（Santoset al., 2005）.Pineapple (Ananascomosus) in be isolated to 3AcSERKGene（It is respectively designated asAcSERK1、AcSERK2、AcSERK3）, but only AcSERK1 is at somatic embryo occur initial stage --- and non-cells,primordial is opened into cells,primordial transition process Raw high-level specifically expressing is originated, and maintains to arrive globular embryo period, is the marker gene that pineapple somatic embryo occurs（He Yehua Deng 2011；Horse is impartial, and 2011；Ma, et al.,2012）.But have not yet to see the report of gene function checking.This is The report of genetic transformation will be carried out from pineapple itself and related to somatic embryo occur gene first.

As it was previously stated, it is to improve pineapple somatic embryo formation frequency by studying and regulating and controlling body cell related gene expression occurs Molecular basis.But for some important model plants, the research in terms of the somatic embryo occur and genetic engineering of pineapple Report is also less.The external research to pineapple somatic embryo all concentrates on kinds of culture medium and Hormone change to somatic embryo occur Influence in terms of, and somatic embryo occur rate is always than relatively low（Less than 60%）（Sripaoraya et al., 2003； Suneerat et al., 2003；Firoozabady et al., 2004）.Some researchers using callus and leaf as Acceptor, foreign gene is imported in pineapple by agriculture bacillus mediated or particle gun（Firoozabady et al., 2006；Ko et al., 2006;Sripaoraya et al., 2001；Zhou et al., 2001；Espinosa et al., 2002）, but generally speaking genetic transformation efficiency is not high（Average 3%）, it is longer to obtain the time cycle of transfer-gen plant, at least needs Take more than 7 months（Firoozabady et al., 2006）；There is researcher to carry out bromelain enzyme gene and nifH genes Clone（Sawano et al., 2002；Ando et al., 2005；Leon et al., 2002）, but at present to from The gene functional research report of pineapple is still less, particularly has no and pineapple somatic embryo occurs related geneAcSERK1Work( The report being able to verify that.

The content of the invention

For above-mentioned deficiency, it is an object of the present invention to provide a kind of pineapple somatic embryo to occur acceptor class kinase geneAcSERK1Genetic transforming method and its application, beAcSERK1Gene is important using providing in the generation of pineapple somatic embryo Theoretical foundation.

Technical scheme is as follows：Acceptor class kinase gene AcSERK1 heredity occurs for a kind of pineapple somatic embryo Method for transformation, comprise the following steps：

（1）Build pFGC5941-35s-AcSERK1 sense expression vectors；

（2）The preparation of callus and its tolerance test to PPT；

（3）Agrobacterium infects the preparation of liquid；

（4）Preculture, infect, co-culture and screening and culturing：The agrobacterium liquid to be suspended through MS infects preculture 2d acceptor callus Tissue, contaminates 3 ~ 5min, after dark place culture 3d be respectively 2 containing PPT concentration, 3, carry out three in 4mg/L screening and culturing medium Wheel is screened, and PPT resistance seedlings are obtained after 50d；

（5）PPT resistances seedling rooting and transplanting.

The step（1）PFGC5941-35s-AcSERK1 sense expression vectors are built, are comprised the concrete steps that：According toAcSERK1CDNA sequence, design carriesNcoI andXbaThe gene specific primer of I restriction enzyme sites, with containingAcSERK1cDNA The pMD19-T of total length is template, and amplification containsAcSERK1ORF purpose fragment, PCR primer Takara QIAquick Gel Extraction Kits Reclaim purpose fragment；WithNcoI andXbaI carries out double digestion to the PCR primer and pFGC-5941 of recovery simultaneously, after being separately recovered It is attached, converts and bacterium solution PCR detections, positive colony carry out sequencing identification, confirm no frameshift mutation, obtains restructuring justice Expression vector, and pFGC5941-35s-AcSERK1 is named as, it is transferred to by freeze-thaw method in agrobacterium strains LBA4404, Preserved in -80 degree refrigerators.

The step（2）The preparation of callus acceptor material and its tolerance test to PPT, are comprised the concrete steps that：Choosing Eyelet stitch is taken, processing obtains sterilizable material, and callus acceptor material is obtained through culture；By callus acceptor material and not Adventitious bud with length is inoculated into containing PPT（Careless fourth phosphine）Differential medium in, observe its sensitiveness to PPT, with not plus Enter PPT processing as compareing, callus and adventitious bud death toll and the death rate under different PPT concentration are counted after 10d.

The step（3）Agrobacterium infects the preparation of liquid, comprises the concrete steps that：It will containAcSERK1Agrobacterium line training Support on the YEB solid medium flat boards of the Km containing 50mg/L, in 28 DEG C, light culture 3d；Picking single bacterium colony, in containing 50mg/L In Km YEB fluid nutrient mediums, 200rpm concussion and cultivates about 36h at 28 DEG C, bacterium solution muddiness is in foresythia, OD₆₀₀For 0.8~ 1.0；1.0mL bacterium solutions are taken to be added to fresh YEB fluid nutrient mediums（Without antibiotic, contain 100 μm of ol/L AS）, concussion and cultivate 4 ~ 6h or so, make OD₆₀₀For 0.3 ~ 0.5, obtain expanding numerous bacterium solution, show slightly yellow, muddiness；The numerous bacterium solution of above-mentioned expansion is transferred to 50mL In sterile centrifugation tube, 5000rpm, 4 DEG C, 10min is centrifuged, removes supernatant, collects thalline；It is resuspended in sterilized MS liquid training Support base（pH7.0）In, it is diluted to OD₆₀₀For 0.4 or so, MS suspension agrobacterium liquids are obtained, it is standby.

The step（4）Preculture, infect, co-culture and screening and culturing, comprising the concrete steps that：By step（2）In be ready to Callus acceptor material be cut into about 2 ~ 3mm³Fritter, be inoculated on pre-culture medium, optical culture 2d, obtain by preculture Acceptor callus；By the acceptor callus Jing Guo preculture in step（4）In soak in the MS suspension agrobacterium liquids that prepare 3 ~ 5min is contaminated, bacterium solution unnecessary on acceptor callus is blotted with aseptic filter paper, is transferred to and co-cultures in culture medium, in dark condition Lower co-cultivation 3d, then it is transferred on screening and culturing medium 1 and carries out selective differentiation culture, it is after 8-10 d, the anti-PPT of green is indefinite Bud d of renewal cultivation 15 ~ 20 in the above-mentioned screening and culturing medium for do not contain PPT；The adventitious bud of recovered culture is continued to be transferred to sieve Select and carry out continuous 2 screenings on culture medium 2 and screening and culturing medium 3 again, screen 8-10d every time, count resistance rate；Will be anti-after 20d Property seedling be transferred on root media seedling of taking root, obtain PPT resistance seedlings.

Wherein, the composition of the precultivation medium is：MS + 2.0 mg/L 6-BA + 1.0 mg/L NAA；It is described The composition of screening and culturing medium 1 is：MS + 2.0 mg/L 6-BA + 1.0 mg/L NAA + 400 mg/L Carb + 2 mg/ L PPT；The composition of the screening and culturing medium 2 is：MS + 2.0 mg/L 6-BA + 1.0 mg/L NAA + 300 mg/L Carb + 3 mg/L PPT；The composition of the screening and culturing medium 3 is：MS + 2.0 mg/L 6-BA + 1.0 mg/L NAA + 200 mg/L Carb + 4 mg/L PPT；The composition of the root media is：MS + 1.0 mg/L NAA；The training altogether Support base composition be：MS + 2.0 mg/L 6-BA + 1.0 mg/L NAA + 100 μmol/L AS.

The step（5）PPT resistances seedling rooting and transplanting, are comprised the concrete steps that：By step（4）2 ~ the 3cm obtained PPT Resistance seedling goes to root induction in root media；After cultivating 60d, a length of 3 ~ 6cm of plant strain, obvious quickening of taking root, radical is 4-6 bars, 4 ~ 5cm of root long, carry out acclimatization and transplantses；During hardening, 2 ~ 3d of bottle cap is first opened in culturing room, then shifts tissue culture bottle Continue the 2~3d that uncaps to outdoor；During transplanting, the culture medium that adheres on root is gently cleaned with water, then is transplanted to that loose, humus is rich In rich soil, first time basin soil pours permeable, makes root definite value in soil；After transplanting, watering is moderate, is finally colonized in plastics In basin, the moderate location of ventilation, illumination is placed on, periodically grants suitable water and fertilizer management.

Using step（1）-（5）The genetic conversion system of optimization carries out functional verification to AcSERK1, and the transgenosis of acquisition is planted Strain detects through PCR, Southern hybridization and RT-PCR, it was demonstrated that and AcSERK1 is had been inserted into pineapple genomic DNA, and There occurs overexpression in pineapple somatic embryo.Functional verification shows that overexpression AcSERK1 callus is through somatic embryo The embryo callus subculture particle and somatic embryo number obtained after induction is higher than wild type by 74.0% and 97.5% respectively.

Acceptor class kinase gene AcSERK1 answering in the generation of pineapple somatic embryo occurs for the pineapple somatic embryo of the present invention It is with mode：AcSERK1 genes are used to convert pineapple, there occurs overexpression, overexpression in pineapple somatic embryo AcSERK1 promotes the formation of pineapple cells,primordial and the generation of somatic embryo.

The present invention builds pFGC5941- by selecting the plant expression vector pFGC5941 containing marker gene bar genes 35s-AcSERK1 sense expression vectors, optimize transformation system, be transferred to using agrobacterium-mediated transformation in Pineapple callus, TurnedAcSERK1Pineapple material, relative to having the beneficial effect that for prior art：

（1）The time that transformation system method of the present invention obtains pineapple transfer-gen plant（Since co-cultivation）Foreshorten to 3 ~ 4 months, and conversion ratio is 12%, effectively raises the genetic transformation efficiency of pineapple；

（2）The present invention is first by from pineapple itself and the AcSERK1 genetic transformation pineapple related to somatic embryo occur, it was demonstrated that Formation and somatic embryo of the overexpression AcSERK1 to pineapple cells,primordial have significant facilitation.

Primary Study of the present inventionAcSERK1It is further right to the influence of somatic embryo occur in the case of overexpression The gene carries out expression regulation and utilized to provide theoretical foundation.

Brief description of the drawings

Fig. 1 is pFGC5941-35s-AcSERK1 sense expression vectors structure figure；Fig. 2 is to turn through agriculture bacillus mediated The PPT screenings of AcSERK1 genetic materials；Fig. 3 is the Molecular Detection of transfer-gen plant；Fig. 4 is that wild type and transformant embryo are cured Hinder particle and somatic embryo statistical analysis and stereoscope shines.

Embodiment

The present invention is described in further details below by embodiment, these embodiments are only used for illustrating the present invention, and Do not limit the scope of the invention.

Embodiment realizes that acceptor class kinase gene occurs for pineapple somatic embryo using following stepsAcSERK1Genetic transformation

1st, plant sense expression vector pFGC5941-35s-AcSERK1 structure according toAcSERK1CDNA（GeneBank sequences Row number:HM236375）Sequence, design carryNcoI andXbaThe gene specific primer of I restriction enzyme sites（It is shown in Table 2）, with containingAcSERK1The pMD19-T of cDNA total lengths is template, and amplification containsAcSERK1ORF purpose fragment, PCR primer Takara QIAquick Gel Extraction Kit reclaims purpose fragment.WithNcoI andXbaI carries out double digestion to the PCR primer and pFGC-5941 of recovery simultaneously, Be attached after being separately recovered, convert and bacterium solution PCR detection（Vector specific primer is shown in Table 2）, positive colony serves the raw work in sea and surveys After sequence identification, no frameshift mutation is confirmed, obtains restructuring sense expression vector, and be named as pFGC5941-35s-AcSERK1, It is transferred in agrobacterium strains LBA4404 by freeze-thaw method, is preserved in -80 degree refrigerators.

2nd, the preparation of callus acceptor material and its to the test of PPT tolerance in fine day, choose growth conditions it is good, " refreshing gulf " pineapple in vegetative growth phase inhales bud, divests the weathered leaf sheath of basal part of stem completely in laboratory, will not The blade of aging removes from leaf base 1cm or so, with 0.1% mercuric chloride（HgCl）Top layer is cut after sterilization 10min, is then used again 0.1% mercuric chloride（HgCl）Sterilize 10min, after the processing of each disinfectant, with aseptic water washing 3 ~ 5 times, be finally cut into 1cm × 0.5cm fritter, it is inoculated in phyllopodium calli induction media（Medium component is shown in Table 1）On, each tissue culture bottle connects 1 explant Body.Can obtain sterilizable material after about 30d, by sterilizable material in above-mentioned culture medium squamous subculture, can obtain stable preservation more Injured tissue.

Plant expression vector in the method for transformation is because containing selectable marker gene bar genes so as to PPT（Careless fourth phosphine） With certain tolerance, it is therefore desirable to obtain suitable PPT screenings pressure in atomization.By about 2 ~ 3mm³The callus of size Be inoculated into the adventitious bud of different length containing PPT be respectively 0,5,10,20 mg/L differential medium in, observe it to PPT Sensitiveness, to select in atomization suitable screening pressure, and screening is further reduced on the basis of experimental result is drawn Scope, PPT concentration is set as 0,1,2,3,4,5mg/L, further filter out most suitable concentration.Not add PPT's Processing counts callus and adventitious bud death toll and the death rate under different PPT concentration as compareing after 10d.

Result of the test shows（Table 3）：Pineapple callus has stronger sensitiveness to PPT, and 7 ~ 10 days i.e. it can be seen that bright Aobvious damage symptoms, it is mainly shown as flavescence, withered；As PPT concentration increases, the death toll and the death rate of adventitious bud significantly increase Add.When the concentration of the PPT in differential medium reaches more than 2mg/L, there is more than 97.8% callus dead；And work as and divide When the concentration for changing the PPT in culture medium is 1mg/L, 70.0% callus becomes yellowish-brown death, small part yellowish-brown callus Tissue, which has new growing point, to be occurred.In theory, the suitable PPT screening concentrations of Pineapple callus are 2 ~ 3 mg/L.

3rd, Agrobacterium infects the preparation of liquid and will containedAcSERK1Agrobacterium line be incubated at the YEB of the Km containing 50mg/L On solid medium flat board, in 28 DEG C, light culture 3d.Picking single bacterium colony, in the YEB fluid nutrient mediums containing 50mg/L Km In, 200rpm concussion and cultivates about 36h at 28 DEG C, bacterium solution muddiness is in foresythia, OD₆₀₀For 0.8~1.0.1.0mL bacterium solutions are taken to be added to Fresh YEB fluid nutrient mediums（Without antibiotic, contain 100 μm of ol/L AS）, 4 ~ 6h of concussion and cultivate or so, make OD₆₀₀For 0.3 ~ 0.5, now bacterium solution show slightly yellow, muddiness, for feeling bacterium and co-cultivation.

The influence that bacterium solution is broken up to callus：1）MS suspension agrobacterium liquids：By the numerous bacterium solution of above-mentioned expansion be transferred to 50mL without In bacterium centrifuge tube, 5000rpm, 4 DEG C, 10min is centrifuged, removes supernatant, collects thalline；It is resuspended in sterilized MS Liquid Cultures Base（pH7.0）In, it is diluted to OD₆₀₀It is standby for 0.4 or so；2）The agrobacterium liquid being resuspended without MS；3）Sterile pure water.

Result of the test shows（Table 4）：The Agrobacterium stoste processing being resuspended without MS significantly reduces callus adventitious bud Differentiation rate, and Pineapple callus adventitious buds differentiation is not made significant difference through MS suspensions agrobacterium liquid, therefore in the conversion journey MS suspension agrobacterium liquids are taken in sequence.

4th, preculture, infect, co-culture and ready callus in step 2 is cut into about 2 ~ 3mm by screening and culturing³ Fritter, be inoculated on precultivation medium, optical culture 2d, as acceptor material.By the acceptor callus Jing Guo preculture 3 ~ 5min is contaminated in the MS suspension agrobacterium liquids prepared in above-mentioned steps 4, it is unnecessary on acceptor material to be blotted with aseptic filter paper Bacterium solution, be transferred to co-cultivation culture medium（Medium component is shown in Table 1）In, co-culture 3d under dark condition.Then it is transferred to screening Culture medium 1（Medium component is shown in Table 1）Upper progress selective differentiation culture.After about 10 d, by the anti-PPT adventitious buds of green not The d of renewal cultivation 15 ~ 20 in above-mentioned culture medium containing PPT.

Due to the part false positive transformation bud that in primary screening process, can usually obtain, this part bud is particularly likely that The bud former base that Agrobacterium is just formed before infecting, the selection course of meeting severe jamming positive transformants bud stronger to PPT tolerances, Cause higher false positive rate.Therefore, during the second generation and the third generation are screened, PPT concentration is brought up to successively 3mg/L and 4mg/L, so in the second generation and third generation screening process most of false positive plant be reduced into withered and yellow plant and by Eliminate.

Therefore the adventitious bud of recovered culture is continued to be transferred to screening and culturing medium 2（Medium component is shown in Table 1）Trained with screening Support base 3（Medium component is shown in Table 1）On carry out again it is continuous 2 times screening, every time screen about 10d, count resistance rate；Will be anti-after 20d Property seedling is transferred to root media（Medium component is shown in Table 1）On take root seedling.

Test result indicates that the callus co-cultured after 3d cultivates 20 d in 2 mg/L PPT screening and culturing medium Afterwards, most of callus and adventitious bud are withered, have small part callus Surface Differentiation to go out less jade-green adventitious bud With larger adventitious bud (Fig. 2), the average resistance rate of adventitious bud is 48.33%.Adventitious bud is screened by the second generation and the third generation Average resistance rate is respectively 33.50%, 12.10%.The experiment has converted 200 pieces of callus, is repeated 3 times, by continuous three-wheel Screening, 72 are obtained altogether and is turnedAcSERK1PPT resistant plants, conversion ratio 12%.

5th, PPT resistances seedling rooting is with transplanting the PPT resistance seedlings of above-mentioned acquisition（About 2 ~ 3cm）Go in root media （Medium component is shown in Table 1）Root induction.After cultivating 30d, take root obvious；After 60d, the long about 3 ~ 6cm of plant strain, take root obvious Accelerate, radical is 5 or so, root long about 4 ~ 5cm, can now carry out acclimatization and transplantses（Fig. 2）.Hardening is wanted before transplanting, is first being cultivated 2 ~ 3d of indoor opening bottle cap, tissue culture bottle is then transferred to outdoor and continues the 2~3d that uncaps.During transplanting, root is gently cleaned with water The culture medium of upper attachment, then be transplanted in loose, the more rich soil of humus, first time basin soil pour it is permeable, make root definite value in In soil.After transplanting, watering is moderate.Finally it is colonized in plastic tub, is placed on the moderate location of ventilation, illumination, periodically grants Suitable water and fertilizer management.After general summer transplanting, transformed plant normal growth, survival rate is more than 70%.

Since co-cultivation, the time that pineapple transfer-gen plant obtains greatly shortened to 3 ~ 4 months above-mentioned genetic method, and Conversion ratio is 12%, effectively raises the genetic transformation efficiency of pineapple.

The AcSERK1 gene applications of embodiment 2：AcSERK1 gene pairs pineapple somatic embryos are transferred to using following steps checking The influence of generation.

1st, the Molecular Detection PCR detection method of transfer-gen plant：Transformed plant leaves genomic DNA is extracted, it is non-transformed Plant（Wild type）Leaf DNA is negative control, pFGC5941-AcSERK1DNA is positive control, and primer is bar bases Because of primer（It is shown in Table 2）And vector specific primer（It is shown in Table 2）.Prepare pcr amplification reaction liquid (25 μ L systems)：10×PCR Buffer (Mg²⁺) 2.5 μ L, dNTP (2.5mM) 2 μ L, P1 (2.5mM) 1 μ L, P2 (2.5mM) 1 μ L,TaqEnzyme0.2 μ L, Template DNA1 μ L, ddH₂O adds to 25 μ L.Reaction condition is：94 DEG C of min of pre-degeneration 5；94 DEG C of 30 s of denaturation, 58 DEG C are moved back 30 s of fire, 72 DEG C of 1 min of extension, 30 circulate；72 DEG C of 10 min of extension.1.0% agarose gel electrophoresis of pcr amplification product Detection.

Southern hybridization checks：According to expression vector pFGC5941-35s-AcSERK1 plasmid map, selectEcoRI Digestion, electrophoresis, transferring film are carried out to the genomic DNA of PCR tests positive plant；Because target gene fragment in itself be present in pineapple Expression, so selection carrier sequence onbarSegments as probes.Southern hybridization checks are marked using special primer method Carry out Southern hybridization（Enter by the Dig-Labeling and Detecting Kit kit operation instructions of Roche companies OK）, it is exposed, develops and is fixed after washes film.

PCR and Southern hybridization checks result such as Fig. 4.The part of above-mentioned acquisition is turnedAcSERK1PPT resistant plants PCR is detected, and as a result has 3 plants to be positive, and 460bp is arrived in amplification（Bar genes）And 2000bp（AcSERK1 genes）Purpose bar Band, tentatively showAcSERK1It may be incorporated into pineapple genome；Further Southern hybridization checks, as a result 3 plants of PCR sun There is obvious hybridization signal in property plant, and this further demonstrates that target gene has been successfully plugged into pineapple gene DNA.Its In, No. 3 strains are three copies, and 4 and No. 5 strains are single copy.

2nd, AcSERK1 gene overexpressions are carried out to the influence that pineapple somatic embryo occurs by taking No. 5 strains singly copied as an example Functional verification, the AcSERK1 expression conditions in transgenic line are detected using RT-PCR.Using wild type as control, Ac β- Actin extracts total serum IgE from the non-embryo of pineapple and embryo callus using TRIZOL methods, synthesized as reference gene The laggard performing PCR amplifications of cDNA.Amplimer is respectively Ac β-actin special primers and AcSERK1 special primers（Table 2）, PCR expansions Increase reaction solution (25 μ L systems)：1 μ gcDNA, 0.2 μM of special primer, 200 μM of dNTP, 1 U TaqDNA polymerase (Takara), 2 10 × PCRbuffer of μ l of；Reaction condition is：94°C /3min；94 °C/30s, 53 °C/30s, 72 °C / 1 min, 30 circulations；72°C /5 min.RT-PCR testing results such as Fig. 4 shows, non-embryo of the AcSERK1 genes in wild type Expression is nearly no detectable in property callus, and has expression in the non-embryo and embryo callus of transgenic line, And expressed in its embryo callus most by force, this explanation causes it in pineapple somatic embryo by being transferred to AcSERK1 genes There occurs overexpression.

In order to further verify pineapple somatic embryo occurs AcSERK1 gene overexpressions influence, respectively from wild Type, turn to cut phyllopodium in AcSERK1 aseptic seedling, through phyllopodium calli induction media（Medium component is shown in Table 1）Induction, obtain The consistent non embryogenic callus of raised growth developmental condition.These non embryogenic callus are cut into 0.3cm × 0.3cm respectively Fritter, per 250mL blake bottles（25mL culture mediums）6 pieces of callus are accessed, are repeated 4 times.In somatic embryo inducement culture medium（Training Foster based component is shown in Table 1）Being transferred to after upper light culture 30d under illumination condition induces 20d to obtain embryo callus, observes and counts embryo Property callus particle generating capacity etc..Culture after Induce of embryoid is transferred to somatic embryo development culture medium（Medium component is shown in Table 1）On, 5 pieces of embryo callus of every bottle of access, it is repeated 5 times, after optical culture 30d, observes and count and induce embryogensis Raw amount.

As a result show（Fig. 4）：Callus is after the d of Fiber differentiation about 50, it is observed that outside divided under stereoscope Flaxen embryo callus subculture particle is dissolved, statistics is found, average every gram turnsAcSERK1Callus can obtain 9.22 embryos and be cured Hinder particle, 74.0% is higher by than wild type；Embryo callus is after illumination cultivation 30d, it can be seen that the body sprouted under stereoscope Blast, further statistics shows, turnsAcSERK1Callus can obtain 17.12/g somatic embryos, be higher by than wild type 97.5%, this tentatively showsAcSERKThe generation of somatic embryo can be promoted under overexpression.Result of study is further to the gene Carry out expression regulation and utilize to provide theoretical foundation.

Note：Using SPSS18.0 according to one-way analysis of variance, different letters are indicated after same column mean and are represented through SNK ratios Compared with significant difference

Claims (8)

1. acceptor class kinase gene AcSERK1 genetic transforming method occurs for a kind of pineapple somatic embryo, it is characterised in that：Including Following steps：

（1）Build pFGC5941-35s-AcSERK1 sense expression vectors；

（2）The preparation of callus and its tolerance test to PPT；

（3）Agrobacterium infects the preparation of liquid；

（4）Preculture, infect, co-culture and screening and culturing：The agrobacterium liquid to be suspended through MS infects preculture 2d acceptor callus Tissue, contaminates 3 ~ 5min, after dark place culture 3d be respectively 2 containing PPT concentration, 3, carry out three in 4mg/L screening and culturing medium Wheel is screened, and PPT resistance seedlings are obtained after 50d；

（5）PPT resistances seedling rooting and transplanting.

2. genetic transforming method according to claim 1, it is characterised in that：The step（1）Build pFGC5941-35s- AcSERK1 sense expression vectors, are comprised the concrete steps that：According toAcSERK1CDNA sequence, design carriesNcoI andXbaI digestions The gene specific primer in site, with containingAcSERK1The pMD19-T of cDNA total lengths is template, and amplification containsAcSERK1ORF Purpose fragment, PCR primer with Takara QIAquick Gel Extraction Kits reclaim purpose fragment；WithNcoI andXbaI is simultaneously to the PCR of recovery Product and pFGC-5941 carry out double digestion, are attached, convert and bacterium solution PCR detections, positive colony are surveyed after being separately recovered Sequence is identified, confirms no frameshift mutation, is obtained restructuring sense expression vector, is named as pFGC5941-35s-AcSERK1, passes through Freeze-thaw method is transferred in agrobacterium strains LBA4404, is preserved in -80 degree refrigerators.

3. genetic transforming method according to claim 1, it is characterised in that：The step（2）Callus acceptor material Preparation and its PPT tolerance is tested, comprise the concrete steps that：Eyelet stitch is chosen, processing obtains sterilizable material, is obtained through culture Obtain callus acceptor material；The adventitious bud of callus acceptor material and different length is inoculated into the differentiation containing PPT to train Support in base, observe its sensitiveness to PPT, not add PPT processing as control, statistics callus and indefinite after 10d Bud death toll and death rate under different PPT concentration.

4. genetic transforming method according to claim 1, it is characterised in that：The step（3）Agrobacterium infects the system of liquid It is standby, comprise the concrete steps that：It will containAcSERK1Agrobacterium line be incubated at the YEB solid medium flat boards of the Km containing 50mg/L On, in 28 DEG C, light culture 3d；Picking single bacterium colony, in the YEB fluid nutrient mediums containing 50mg/L Km, 200rpm at 28 DEG C Concussion and cultivate 36h, bacterium solution muddiness are in foresythia, OD₆₀₀For 0.8~1.0；Take 1.0mL bacterium solutions to be added to without antibiotic, contain 100 μm ol/L AS fresh YEB fluid nutrient mediums, 4 ~ 6h of concussion and cultivate, make OD₆₀₀For 0.3 ~ 0.5, obtain expanding numerous bacterium solution, slightly Displaing yellow, muddiness；The numerous bacterium solution of above-mentioned expansion is transferred in 50mL sterile centrifugation tubes, 5000rpm, 4 DEG C, centrifugation 10min, in removal Clear liquid, collect thalline；It is resuspended in sterilized, pH7.0 MS fluid nutrient mediums, is diluted to OD₆₀₀For 0.4, MS suspension agriculture bars are obtained Bacterium solution, it is standby.

5. genetic transforming method according to claim 1, it is characterised in that：The step（4）Preculture, infect, train altogether Foster and screening and culturing, is comprised the concrete steps that：By step（2）In ready callus acceptor material be cut into about 2 ~ 3mm³It is small Block, it is inoculated on precultivation medium, optical culture 2d, obtains the acceptor callus by preculture；By by preculture by Body callus is in step（4）In contaminate 3 ~ 5min in the MS suspension agrobacterium liquids that prepare, blot acceptor with aseptic filter paper and be cured Unnecessary bacterium solution on injured tissue, it is transferred to and co-cultures in culture medium, 3d is co-cultured under dark condition, is then transferred to screening and culturing medium Selective differentiation culture is carried out on 1, after 8-10 d, by the anti-PPT adventitious buds of green in the screening and culturing medium 1 for do not contain PPT it is extensive 15 ~ 20 d are cultivated again；The adventitious bud of recovered culture is continued to be transferred on screening and culturing medium 2 and screening and culturing medium 3 and connected again Continuous 2 screenings, screen 8-10d, count resistance rate every time；Resistance seedling is transferred on root media seedling of taking root after 20d, obtained Obtain PPT resistance seedlings.

6. genetic transforming method according to claim 1, it is characterised in that：The step（5）PPT resistances seedling rooting is with moving Plant, comprise the concrete steps that：By step（4）2 ~ the 3cm obtained PPT resistance seedlings go to root induction in root media；Culture After 60d, a length of 3 ~ 6cm of plant strain, obvious quickening of taking root, radical is 4-6 bars, 4 ~ 5cm of root long, carries out acclimatization and transplantses；During hardening, 2 ~ 3d of bottle cap is first opened in culturing room, tissue culture bottle is then transferred to outdoor and continues the 24 ~ 3d that uncaps；During transplanting, with water gently The culture medium that adheres on root is cleaned, then is transplanted in soil loose, that humus is abundant, first time basin soil pours permeable, determines root It is worth in soil；After transplanting, watering is moderate, is finally colonized in plastic tub, is placed on the moderate location of ventilation, illumination, periodically Grant suitable water and fertilizer management.

7. genetic transforming method according to claim 5, it is characterised in that：Wherein, the composition of the precultivation medium For：MS + 2.0 mg/L 6-BA + 1.0 mg/L NAA；The composition of the screening and culturing medium 1 is：MS + 2.0 mg/L 6- BA + 1.0 mg/L NAA + 400 mg/L Carb + 2 mg/L PPT；The composition of the screening and culturing medium 2 is：MS + 2.0 mg/L 6-BA + 1.0 mg/L NAA + 300 mg/L Carb + 3 mg/L PPT；The screening and culturing medium 3 into It is divided into：MS + 2.0 mg/L 6-BA + 1.0 mg/L NAA + 200 mg/L Carb + 4 mg/L PPT；It is described to take root The composition of culture medium is：MS + 1.0 mg/L NAA；It is described co-culture base composition be：MS + 2.0 mg/L 6-BA + 1.0 mg/L NAA + 100 μmol/L AS。

8. applications of the acceptor class kinase gene AcSERK1 in the generation of pineapple somatic embryo occurs for a kind of pineapple somatic embryo, its It is characterised by：Using the step described in claim 1（1）-（5）, AcSERK1 genes are used to convert pineapple, gene is in pineapple There occurs overexpression, overexpression AcSERK1 in somatic embryo to promote the formation of pineapple cells,primordial and the hair of somatic embryo It is raw.