CN1283784C - Process for construvting transgene teceptor system of rye grass and its application - Google Patents

Abstract

The present invention discloses a method for establishing a system of transgenic receptors system of rye grass and an application thereof. The method comprises the main step: taking sterile stem tips of seed seedlings of annual or perennial rye grass as materials, inducing the occurrence of multiple buds on an induction culture medium, uninterruptedly proliferating multiple bud blocks on a proliferation culture medium, transferring the multiple bud blocks on a root culture medium to generate whole plants, taking the multiple bud blocks in an optimum growth period from the proliferation culture medium, stripping small blades from the multiple buds to reveal growing points, transferring genes via a particle gun bombardment method or an agrobacterium mediating method to obtain transgenic plants, and using the treatment of surface acting agents Silwet L-77 and decompression treatment to effectively improve transformation frequency in the genetic transformation of agrobacterium mediation; consequently, a transgenic technology system of the rye grass with high efficiency and small restriction of genotypes is established.

Description

A kind of method and application thereof of setting up rye grass transgene receptor system

Technical field

The invention belongs to crop biotechnology breeding field or crop breeding field.Specifically, relate to a kind of method and application background technology of setting up the rye grass genetic conversion system.

Background technology

Rye grass is a Gramineae lolium plant, the branch of living one year rye grass and English ryegrass.English ryegrass cries the perennial root rye grass again.Annual ryegrass is Itanlian rye or Italian ryegrass again, originates in southwestern Europe, north African and southwest, Asia, and be introduced into China as high quality forage the 1950's.Rye grass tillers many, the output height, and the quality softness, the green phase is long, is good green feed; Its well developed root system in addition, suitable natural disposition is strong, and anti-trample has salt tolerant alkali potentiality, can increase the soil organism, improves Soil structure, prevent erosion, be again good turfgrass.English ryegrass is widespread use in urban afforestation.Yet most rye grass kind drought tolerances are relatively poor, and the salt tolerant degree does not reach the requirement in the saltings plantation yet.Rye grass is a cross pollinated plant, adopts the traditional breeding method cultivation salt tolerant drought-enduring variety cycle long, and difficulty is big.In order to adapt to the needs of rye grass breeding work, isolated culture of rye grass and transgenic technology are subjected to increasing attention.

The difficulty that good rye grass genetic conversion system is set up is bigger, mainly is because existing rye grass tissue culture and plant regeneration system can not satisfy genetically modified needs.Greemers Molenaar in 1988 is an explant with the inflorescence of immature English ryegrass and annual ryegrass, and evoked callus takes place, and has studied 2, and 4-D and donor plant environment have obtained regeneration plant to the influence of plant regeneration.But the rye grass callus induction rate is low, and embryo is lost easily in the succeeding transfer culture process, and somaclonal variation easily takes place regeneration plant.1994, Chen Wenpin was an explant with annual ryegrass and English ryegrass children fringe, without the callus stage, directly from ear differentiation Cheng Miao, but has only the annual ryegrass inductivity higher, has influenced further application.Though the genetic transformation work of rye grass beginning early, makes little progress.As far back as 1993, Hensgens etc. studied the instantaneous and stably express of fusion gene in English ryegrass of gusA and paddy gene.1994, Van der Maas etc. changed the gusA reporter gene over to the English ryegrass callus, and has studied its long-acting expression in cell.Nineteen ninety-five, Spangenberg etc. have carried out the work that utilizes the micropellet bombardment method to transform the embryonal suspension cell of English ryegrass.Ye in 1997 etc. use with quadrat method and transform annual ryegrass embryonal suspension cell acquisition transfer-gen plant.1998, Folling etc. studied the influence of the reduction of rye grass protoplastis nuclease to transformation efficiency.In the same year, Dalton etc. utilize silicon-carbon fiber mediated method to transform annual ryegrass and English ryegrass suspension cell line, obtain transfer-gen plant respectively.Dalton in 1999 etc. attempt utilizing the micropellet bombardment method to transform annual and English ryegrass again.Calendar year 2001, Ye etc. change the SacB gene over to annual ryegrass, have studied the variation of Polylevulosan in the transfer-gen plant.In the same year, Xu etc. have obtained the English ryegrass transfer-gen plant of mosaic disease resisting poison.2003, the Dalton laboratory attempt utilize agrobacterium-mediated transformation to transform annual ryegrass.In above work, the genetic transformation acceptor of selecting for use is generally callus or the suspended culture cell with regenerative power, because callus induction rate is low, subculture makes a variation easily, regenerative power is poor, have only several laboratories to obtain transgenosis rye grass plant, and frequency is lower, does not obtain transfer-gen plant after a lot of laboratories transform.Therefore find suitable receptor system significant to rye grass cell engineering and genetically engineered research.

Summary of the invention

Deficiency at above-mentioned technology, the aseptic seed seedling stem apex that the present invention is bigenered with annual ryegrass and English ryegrass improved seeds or rye grass kind is a material, the induced bundle generation of sprouting on inducing culture, forward to then and produce the bud of growing thickly in a large number on the proliferated culture medium, genetically modified acceptor material is provided.Adopt particle gun blast technique or agrobacterium-mediated transformation that foreign gene is imported recipient cell, through selecting to obtain transformant and plant.On this basis, established in agriculture bacillus mediated genetic transformation, adopted tensio-active agent Silwet L-77 processing and reduced pressure treatment effectively to improve transformation frequency, thereby set up a little rye grass transgenic technology system of genotype restriction efficiently.

Key step of the present invention comprises: with ryegrass seed seedling stem apex is test materials, induces its bud of growing thickly, and the latter continues hyperplasia on proliferated culture medium, genetically modified acceptor material is provided.The utilization sprout tuber vegetative point of growing thickly is acceptor, adopts particle gun blast technique or agrobacterium-mediated transformation that foreign gene is imported recipient cell, through selecting to obtain transformant and plant, and transfer-gen plant is identified and selects.

Rye grass described in the present invention comprises Itanlian rye (annual ryegrass) kind, English ryegrass kind, rye grass species hybrid kind and rye grass and fescue grass hybrid variety.

The described bud of growing thickly is meant the budlet and the secondary culture thereof of the dense collection of one-tenth that explant produces on substratum.The sprout tuber that grows thickly is meant the tissue block and the secondary culture thereof of the grow thickly budlet and the not organized somatoblast formation of isolated culture.

Described transgene receptor is meant the sprout tuber vegetative point of growing thickly of the stem apex isolated culture generation of removing blade or bud scale.

Utilization is grown thickly bud when carrying out genetic transformation for acceptor, at first peels off grow thickly bud leaflet or leaf sheath, exposed vegetative point, and the latter is used for agroinfection or particle gun bombardment.

Selective agent among the present invention comprises weedicide (chlorsulfuron, careless fourth phosphine, careless dried phosphine etc.), microbiotic (Totomycin, kantlex etc.).The selective agent resistant gene has: herbicide resistance gene such as als (acetolactate synthase gene of sudden change), bar (gene) etc., antibiotics resistance gene such as hpt (the plain phosphoric acid transferase gene of damp enzyme), nptII (kalamycin resistance gene) etc.

Described culture medium prescription is:

Inducing clumping bud substratum: MS substratum, 200-500mg L ^-1Caseinhydrolysate (CH), sucrose 30-60gL ^-1, 6-BA 0.1-2.0mg.L ^-1, 2,4-D 0.01-0.5mg.L ^-1, agar 6.5g L ^-1, pH5.8-6.0.

Adventitious buds proliferation substratum: MS substratum, 200-500mg L ^-1Caseinhydrolysate (CH), sucrose 30-40g L ^-1, 6-BA0.1-2.0mg.L ^-1, 2,4-D 0-0.1mg.L ^-1, agar 6.5g L ^-1, pH5.8-6.0.

Seedling root media: 1/2MS substratum, 200mg L ^-1Caseinhydrolysate (CH), sucrose 20-40g L ^-1, NAA 0.2-2.5mg L ^-1, agar 6.5g L ^-1, pH5.8-6.0.

The described genetic transforming method bud of can rye grass growing thickly is that acceptor adopts the agroinfection sprout tuber method of growing thickly.The sprout tuber that is about to grow thickly is put into bacterium liquid and contaminates also attached with 0.98-0.10 normal atmosphere (0.98 * 10 ⁵Pa-1 * 10 ⁴Pa) handle, contaminated 3-18 minute.Inhale with aseptic filter paper then and remove bacterium liquid, metainfective tissue changed over to cultivated altogether on the proliferated culture medium 2-3 days, change step sizing 3 generations (per generation 15-20 days) on the screening culture medium that is added with suitable concentration selective agent and 100-200ppm cephamycin again over to, obtain anti-selective agent seedling.The seedling of 2-3cm changed in the root media take root, obtain transformed plant.

The genetic transformation procedures of described Agrobacterium (Agrobacterium comprises A.tumefaciens and A.rhizogenes) mediation waits with stone field (Ishida, 1996) basically and reports.In transfection liquid, add Syringylethanone (acetosyringone, As) phenolic compound, tensio-active agent Silwet L-77 (concentration is 0-0.3%) and neutral monose (glucose, wood sugar) such as, the sprout tuber that grows thickly and exposed stem apex are placed in the transfection liquid of suspension Agrobacterium, at 0.98-0.10 normal atmosphere (0.98 * 10 ⁵Pa-1 * 10 ⁴Pa) contaminated 3-18 minute down, on substratum, cultivated altogether 2-15 days then.Transformant is containing on the suitable antibiotic substratum antibacterially, screens on the substratum that is added with the suitable concn selective agent, breaks up seedling then on proliferated culture medium.

Described particle gun blast technique (particle bomrdment; Microprojectile; Biolistics; Particle gun) being the high-speed metal particle of mat introduces a kind of genetic transformation technology in the viable cell with nucleic acid molecule.Be gunpowder particle gun and the transformation technology that U.S. Cornell University Sa volt (Sanford) etc. is succeeded in developing at first, after this several genes rifle that different researchers develops and the transformation technology of updating are arranged.Used particle gun is a U.S. Bio-RAD company product in the present invention's research, and model is: Biolistic PDS-1000/He Particle Delivery System.Press the working instructions operation.The bombardment parameter is: the distance that can split disk and carrier is 2.5 centimetres, carrier and stop that the distance of net is 0.8 centimetre, and helium pressure is 1100psi, vacuum tightness 28inchHg, little bullet flying distance value between 3-9 centimetre.

The inducing clumping bud system can seed seedling stem apex be an explant, be added with suitable concentration 6-BA and 2, cultivate on the inducing culture of 4-D, induced growth point and outer leaf sheath thereof expand, from the vegetative point surface of expanding budlet being taken place, cultivates and form the bud of being made up of 8-10 intensive budlet of growing thickly after 10-15 days.Concrete steps are: ryegrass seed is through 70% alcohol sterilization 1min, 0.2% mercuric chloride sterilization 5min, aseptic washing 4 times.Do not have on the hormone culture-medium at MS and to sprout 25 ± 2 ℃ of temperature under the dark condition.After 4-5 days, when plumule is stretched to 2-3cm, cut stem apex position (epicotyl of 2-3mm and stem apex), be inoculated into and contain different concns 6-BA and 2, on the inducing culture of 4-D as the explant that induced bundle is sprouted.Culture condition: 25 ± 2 ℃ of temperature, illumination 12 hours/day, light intensity 800-1000Lx.Form the sprout tuber that grows thickly after 15-20 days.The kind of exogenous hormone and concentration play a part very important to the grow thickly form of bud of rye grass.In no 6-BA and 2, the bud of not growing thickly on the substratum of 4-D takes place; Substratum does not contain 2,4-D, and budlet Cong Ye look dark green, segment glassization, there is macula lutea on the blade face; Substratum 2, the lower (0.01-0.2mg.L of 4-D concentration ^-1), inductive budlet blade often twists, and bastem portion is thinner, and inductivity is also lower; 2,4-D concentration is higher than 0.5mg.L ^-1Inducing culture on, bastem portion callusization is more serious, takes place on the sprout tuber than multiple-blade, the budlet number tails off, and bastem portion has a lot of roots to take place.Peel off Lao Ye with what succeeding transfer culture formed after 20 days from the piece of sprouting, be divided into simple bud or little sprout tuber (2 millimeters sizes) then and transfer on the proliferated culture medium and cultivate, per 20 days subcultures once.For most of genotype, proliferated culture medium is with MS+2.0mg.L ^-16-BA is suitable, the average bud number of each bud clump maximum (9.96) on this substratum, and the bud growing way of growing thickly is good, and after 3 generations, the bud hyperplasia is very vigorous at succeeding transfer culture, and a simple bud or little sprout tuber can produce 50 left and right sides budlets at succeeding transfer culture after 20 days.

Remove bud point leaf sheath on every side in the succeeding transfer culture process bud of growing thickly is played an important role, the leaf sheath parcel can make the bud of growing thickly be obstructed.In the succeeding transfer culture process, if 2,4-D and 6-BA excessive concentration can cause the budlet callusization, finally cause the albefaction seedling to produce increase frequency.The 6-BA excessive concentration (surpasses 2.0mg.L ^-1) time, the blade tip jaundice appears in budlet and the chlorisis striped appears in blade.In addition, the 6-BA excessive concentration can influence and take root.

The described genetic transforming method sprout tuber that can rye grass grows thickly is that acceptor imports recipient cell with the plasmid that the particle gun blast technique will carry goal gene and selective agent resistant gene, specific procedure is: extract plasmid and the purifying that carries goal gene and selective agent resistant gene from the host bacterium, dilution DNA concentration is 1 μ g/ μ l; Take by weighing bronze and thoroughly washing; Disperse bronze and add plasmid DNA, CaCl successively ₂And spermidine, vortex limit, limit application of sample reaches with DNA and wraps up little bullet; Little bullet drops on the carrier with the particle gun bombardment, and the distance that can split disk and carrier is 2.5m, carrier and stop that the distance of net is 0.8m, little bullet flying distance 6-9m; Tissue block after the bombardment was recovered to cultivate 2-3 days darkling, then they are changed in the new substratum of components unchanged and cultivated for 3 weeks, the gene that changes over to is given full expression to, then they are changed over to step sizing three generations on the proliferated culture medium that is added with selective agent (microbiotic or weedicide), per generation 15-20 days; Pick out tissue block that the three generations screens back survival and transfer to the subculture of no selective agent and become on the clump substratum induced bundle formation of sprouting, and pass through subculture on proliferated culture medium

The present invention can obtain a large amount of grow thickly sprout tuber and aseptic seedling with very strong plant regeneration ability, can efficiently obtain transfer-gen plant from most genotype, and the regeneration plant somaclonal variation is little, and most vine growth and developments are normal.Characteristics of the present invention are 1) reduce genotype barrier significantly, can induce most genotypic rye grass stem apexs to produce the bud of growing thickly; 2) transformation frequency is higher; 3) draw materials and be not subject to seasonal restrictions; 4) the most features that keep the protogene type of transfer-gen plant.

Embodiment

Embodiment 1, employing agrobacterium-mediated transformation obtain the rye grass transfer-gen plant

Inducing clumping bud and succeeding transfer culture vegetable material are English ryegrass kind Tai Chuilaite.Ryegrass seed is through 70% alcohol sterilization 1min, and 0.2% mercuric chloride sterilization 5min after the aseptic washing 4 times, is sprouting 25 ± 2 ℃ of temperature under the dark condition on the no hormone culture-medium.Behind the 4-5d, when plumule is stretched to 2-3cm, cut be about 2-3mm the stem apex position as explant, be inoculated into and contain 2.0mg.L ^-16-BA and 0.5mg.L ^-12, on the inducing culture of 4-D.Culture condition: 25 ± 2 ℃ of temperature, illumination 12h/d, light intensity 800-1000Lx.Form the sprout tuber that grows thickly after 15-20 days.The sprout tuber that will grow thickly is peelled off older blade, is divided into simple bud or little sprout tuber (2 millimeters sizes) and transfers to 6-BA2.0mg.L ^-1Proliferated culture medium on cultivate, under same light and temperature condition, cultivating, per 20 days succeeding transfer culture once.

The determining of the selective agent concentration bud of will growing thickly is cut into simple bud and is inoculated on the subculture medium that is added with the different concns Totomycin, per 15 days subcultures once, 3 generations of succeeding transfer culture altogether.8 selective agent concentration are got in inoculation seedling 100-300 strain usually on every kind of screening culture medium.Concentration and concentration gradient change because of the selective agent kind is different with the acceptor material genotype.Step sizing is added up the seedling survival rate after 3 generations.Selective agent concentration with the not approaching whole death of transgenosis seedling are the screening concentration of transformant.The Totomycin concentration of determining is 10mg.L ^-1

The agrobacterium tumefaciens LBA4404 that agriculture bacillus mediated genetic transformation will have plant expression vector pCAMBIA1300-hpt-betA is placed on 28 ℃ of concussion cultivations down in the additional antibiotic LB substratum, makes bacterium be in logarithmic phase (OD ₆₀₀=0.4-0.6).Centrifugal then 10 minutes, abandon supernatant liquor.Thalline washs centrifugal again collection with the liquid-based basal culture medium of 1/2 concentration.Again thalline is used and added Syringylethanone (acetosyringone, As) 100 μ mol L ^-1Suspend with the liquid-based basal culture medium of 1/2 concentration of tensio-active agent Silwet L-77 0.02%, dilution 10-20 doubly is used for transforming.8 days the sprout tuber that grows thickly of succeeding transfer culture is cut to the exposure sharp vegetative point that sprouts, put into bacterium liquid and contaminated 6-8 minute, and attached with 0.95-0.10 normal atmosphere (0.95 * 10 ⁵Pa-1 * 10 ⁴Pa) negative pressure is handled.Change proliferated culture medium (6-BA 2.0mg L after tissue block blots with aseptic filter paper after the transfection over to ^-1) cultivated 2-4 days, change over to then and be added with Totomycin 10mg.L ^-1With step sizing 3 generations (20 days per generations) on the screening culture medium of 100ppm cephamycin, obtain anti-selective agent seedling.The seedling of 2-3cm changed in the root media take root.On root media, when the seedling root is grown to 2-3cm, be placed under the natural light and cultivated 1-2 days, remove then and sealed the film hardening 1-2 days, be transplanted to flowerpot or field early morning or dusk.The seedling of transplant survival is got blade and is used for molecular Biological Detection.Analyze through PCR and Southern blotting, transformation efficiency (producing sprout tuber number * 100 of the sprout tuber number/agroinfection of transfer-gen plant) is about 6%.

Example 2, employing particle gun blast technique obtain the rye grass transfer-gen plant

Definite acceptor of inducing clumping bud and succeeding transfer culture and selective agent concentration is an annual ryegrass bay kind, and other is with example 1.

Plasmid is pCAMBIA1300-hpt-antiport.Extract and purifying employing standard program and method (referring to " molecular cloning ").The plasmid quality is that OD260/OD280 is between 1.7-1.8.The plasmid DNA dilution is 1 μ g/ μ l.

DNA wraps up little bullet 1), take by weighing the bronze of 3mg 1.0 μ m sizes, put into an Eppendorf pipe, added 1ml 70% ethanol violent vortex 3-5 minute, left standstill then 15 minutes, 15000rpm removes supernatant liquor after centrifugal 5 seconds under the room temperature.2), add the 1ml sterilized water, violent vortex 1 minute left standstill 1 minute, 15000rpm removes supernatant liquor after centrifugal 5 seconds under the room temperature.This step repeats 3 times.3) bronze after washing, for the third time, (final concentration is the little bullet of 60mg/ml to add 50 μ l, 50% sterile glycerol.This preparation liquid chamber temperature can store for 2 weeks).Vortex was broken the bronze aggegation in 5 minutes when 4), using.5), disperse the back bronze to add 5 μ l plasmid DNA (1 μ g/ μ l), 50 μ l 12.5M CaCl2,20 μ l 0.1M spermidines, vortex limit, limit application of sample successively.6), continued vortex 2-3 minute, left standstill 1 minute.7), 15000rpm abandons supernatant liquor after centrifugal 2 seconds, adds 140 μ l, 70% ethanol, staticly settles, 15000rpm abandons supernatant liquor after centrifugal 2 seconds.8), add 48 μ l dehydrated alcohols, flick tube wall for several times, vortex 2-3 second under the low speed, sampling is added on little missile-borne body then.Little bullet consumption is every bullet 0.5mg.

The thick substratum of 0.4cm is poured in particle gun bombardment in the culture dish of diameter 9cm, the sprout tuber that will grow thickly then (2-3mm size, behind the succeeding transfer culture 6-9 days) high-density is put into culture dish, bombards with particle gun.Every ware bombardment 1-2 time.The bombardment parameter is got: the distance that can split disk and carrier is 2.5cm, carrier and stop that the distance of net is 0.8cm, little bullet flying distance 8 or 9cm.Other parameter is pressed the working instructions value.

The cultivation and the plant regeneration bombardment back tissue block that transform the back tissue block were recovered to cultivate 2-3 days darkling, then they were changed in the proliferated culture medium and cultivated for 3 weeks.This phase sprout tuber well-grown of growing thickly, the target gene that changes over to is given full expression to.The sprout tuber that grows thickly after will recovering then to cultivate changes over to and is added with Totomycin 10mg.L ^-1The enterprising row filter of proliferated culture medium cultivate.3 generations of step sizing, 20 days per generations.Pick out tissue block that the three generations screens back survival and transfer to the seedling of regenerating on the proliferated culture medium of no Totomycin.

Transform the taking root of seedling, transplanting and molecular Biological Detection with embodiment 1.

Claims (4)

1, a kind of method of setting up rye grass transgene receptor system, step comprises: stem apex isolated culture under 800-1000Lx illumination on the inducing culture of getting the high seed seedling of 2-3cm of axenic germination, the induced bundle generation of sprouting, wherein said inducing culture consists of: MS substratum, 200-500mgL ^-1Caseinhydrolysate, sucrose 30-60gL ^-1, 6-BA 0.1-2.0mgL ^-1, 2,4-D 0.01-0.5mgL ^-1, agar 6.5gL ^-1, pH5.8-6.0; The bud of will growing thickly is peelled off old blade, is divided into the little sprout tuber of simple bud or 2mm and transfers to succeeding transfer culture on the adventitious buds proliferation substratum, and described adventitious buds proliferation substratum consists of: MS substratum, 200-500mgL ^-1Caseinhydrolysate, sucrose 30-40gL ^-1, 6-BA 0.1-2.0mgL ^-1, 2,4-D 0-0.1mgL ^-1, agar 6.5gL ^-1, pH5.8-6.0; With succeeding transfer culture 7-12 days the sprout tubers that grow thickly was that acceptor carries out genetic transformation, and converting material is at additional 10mgL ^-1In 3 generations of step sizing on the adventitious buds proliferation substratum of Totomycin, per generation 15-20 days, the sprout tuber that grows thickly of survival was transferred to root induction on the root media, and described root media consists of: 1/2MS substratum, 200mgL ^-1Caseinhydrolysate, sucrose 20-40gL ^-1, NAA 0.2-2.5mgL ^-1, agar 6.5gL ^-1, pH5.8-6.0; Transfer-gen plant is determined in laggard performing PCR of seedling transplant survival and the Southern hybridization of taking root.

2, the method for claim 1 is used for the application of genetic transformation, it is characterized in that, described genetic transformation is one of particle gun blast technique and agrobacterium-mediated transformation.

3, application as claimed in claim 2 is characterized in that, described agrobacterium-mediated transformation is 0.98 * 10 ⁵-1 * 10 ⁴Carry out genetic transformation under the Pa, adding volume percent in bacterium liquid is the tensio-active agent Silwet L-77 of 0-0.3%.

4, the method for claim 1 is used for the application of genetic transformation, it is characterized in that, described rye grass is one of annual ryegrass kind, English ryegrass kind, rye grass species hybrid kind and rye grass and fescue grass hybrid variety.