CN101270353B - Method for quickly acquiring transgenic sugarcane by using pmi gene - Google Patents

Abstract

The invention provides a method for producing transgene sugarcane quickly with pmi genes. The method primarily comprises the steps of establishing a mannitose screening system; transformation of a gene gun; acquisition of resistant callus; differentiation and screening of the resistant callus; acquisition and rootage of resistant plantlets; molecule detection of resistant plantlets regenerated and detection of pmi gene expression by the chlorophenol red detection method. The transformation period is short, and the selection marker system has the advantages of safe product, simple selection procedure, remarkable effect of screening, no influence on metabolic balance of converted plants and so on. In addition, the detection method is quick, sensitive and reliable. With the method, a great many regenerated target transgene plantlets can be acquired in 12 weeks, and the safety problem of transgenes is solved.

Description

Utilize the pmi gene to obtain the method for transgenic sugarcane fast

Technical field the present invention relates to utilize the pmi gene to obtain the method for transgenic sugarcane fast.Contain the plant expression vector of pmi gene by structure, utilize seminose to obtain the methods of transgenic plant, belong to biological technical field as selective agent, dichlorophenol sulfonphthalein method rapid detection resistant plant.

The background technology sugarcane is a kind of asexually propagated crop of height heterozygosis, genetic background is very complicated, generally show as the aneuploid of allopolyploid and polyploid, its chromosome number is many, genome is huge, at not of the same race or widely different, from 2n=54 to 2n=128 with chromosome number between dissimilar in a kind of.The cultivar of sugarcane mostly from noble cane, cut the close kind of hand and India and several kinds species hybrid such as plant, be to have extensive heterogeneity and the unpaired chromosomal times combination that mixes, in fission process, behaviors such as chromosomal non homologous pairing, exchange, distribution all have special part, this brings very big blindness to breeding work, wants a plurality of good characters of parent are all combined very difficulty (Peng Shaoguang, 1990, cane breeding is learned, agriculture press).So want cross breeding method by routine and program in improved seeds, introduce the resistant gene time long, efficient is low.A brand-brand-new way has been opened up in reaching its maturity of transgenic technology in this respect, has great importance for the genetic improvement of sugarcane.

In the past, the genetic transformation of sugarcane is mainly still by particle gun transduction or agrobacterium-mediated transformation, utilize microbiotic such as Totomycin, G418 etc. to screen the acquisition resistant plant, detect and determine what transfer-gen plant was realized through biological and molecule (PCR, Southern and Northern hybridization) again, take a broad view of the application of domestic and international biotechnology on the improvement sugarcane, the regrowth acquisition cycle is long after transforming, efficient is low, is the subject matter of its development of restriction.Simultaneously, the screening system of using on plant transgene at present has a variety of, to transformant cell metabolism active pros and cons, is divided into forward screening system and negative sense screening system according to it.The mechanism that negative sense screening system makes plant produce resistance is that it has detoxification to antibiotics, classes of herbicides or other analogue, remove their toxicity to vegetable cell, thereby the render transgenic vegetable cell produces resistance to microbiotic etc., and not genetically modified vegetable cell is killed to sensitivity such as microbiotic.Negative sense screening system commonly used comprises antibiotic-screening system and classes of herbicides screening system etc.Though these negative sense screening systems are used more extensive at present, but also exist very important defective, as: selectable marker gene and goal gene are irrelevant, and materials such as use microbiotic have negative impact to plant-growth, and resistant maker gene has potential ecotope and edible safety hidden danger etc.In forward screening system, transformant can utilize the selective agent carbohydrate as main carbon source, can grow on screening culture medium, and non-transformed cell can not utilize the selective agent carbohydrate, is in starvation, and growth is suppressed but is not killed.In recent years, compare with negative sense screening system, the forward screen body ties up to the security aspect and has advantage, has therefore obtained fast development.

Seminose is exactly wherein a kind of forward selection system.The D-seminose as the Selecting Mechanism and Procedure (Joersbo and Okkels.1996) of selective agent is: the D-seminose changes 6-phosphomannose, reaction consumes ATP into by the endogenous hexokinase catalysis of vegetable cell; The phosphomannose isomerase of pmi genes encoding (PMI) is again with the metabolizable fructose-1, 6-diphosphate of 6-phosphomannose catalysis changing into vegetable cell.On the screening culture medium that contains the D-seminose, transformant constantly is transformed into fructose-1, 6-diphosphate with seminose, but non-transformed cell PMI for want of, can not continue metabolism, cause the accumulation of 6-phosphomannose in the cell, be accompanied by the mass consumption of ATP and phosphate, thereby growth is suppressed; (Sheu-Hwa C S, Lewis D H, Wakler D A.Stimulation of photosynthetic starchformation by sequestration of cytoplamic orthophosphate.NewPhytol, 1975,74:383-392) transformant then can change the 6-phosphomannose into fructose-1, 6-diphosphate, can continue metabolism and provide energy for cell, therefore avoid the accumulation of selective agent derivative 6-phosphomannose simultaneously, can on screening culture medium, utilize seminose to grow as carbon source and breed.

Summary of the invention the objective of the invention is to set up a kind of method that obtains transgenic sugarcane efficiently, fast, thereby realizes the efficient gene engineering breeding of sugarcane, so that improve the resistance of sugar cane breed from now on from many aspects and improve its quality.

The present invention utilizes the pmi gene to obtain the method for transgenic sugarcane fast, the pmi gene (GenBank:accession M15380) that is used in intestinal bacteria makes up plant expression vector, having set up is the transgenosis genetic transformation screening system of selective agent with the seminose, and set up a whole set of transgenosis detection technique fast and efficiently, increased substantially the pick-up rate of sugarcane genetically modified regeneration plant, for the genetic engineering breeding of sugarcane has been established solid basis.

The present invention utilizes the pmi gene to obtain the method for transgenic sugarcane fast, may further comprise the steps:

1, the acquisition of resistant plant

Cutting sterile-processed back diameter is 0.4～0.8cm, and thick is the sugarcane young leaves of 1～2mm, is inoculated on the inducing culture, cultivates in the darkroom, and culture temperature is 25 ± 1 ℃, and every 15d subculture once; Select well-grown sugarcane embryo callus as transgene receptor,, utilize particle gun transduction method to transform sugarcane cell the plant expression vector that contains the pmi gene that builds; The composition of described inducing culture is: MS+2,4-D 3.0mgL ^-1Described pUP carrier refer to utilize Ubi as promotor, no as terminator, the pmi plant expression vector that the conventional molecular biology method of gene makes up that serves as a mark; The MS substratum is the collective media of plant tissue culture.

Embryo callus after the particle gun bombardment is put into height oozes substratum and change subculture medium after 10～20 hours over to and cultivated 2～10 days, after to change total carbon source over to be to screen containing in the seminose subculture medium of 10g, 26～28 ℃ of dark culturing 10～20 days; Afterwards well-grown callus is transferred to and broke up in the division culture medium for 6～8 weeks, cultivate 12hd ^-1, illuminance 1000～2000Lux, culture temperature is 26～28 ℃; Treat to be transferred to when seedling grows to 6～8cm short root cultivation in the root media, obtain resistant plant; Described height oozes medium component: MS+2,4-D 2.0mgL ^-1+ 0.2molL ^-1N.F,USP MANNITOL+0.2molL ^-1Sorbyl alcohol; Described succeeding transfer culture based component is: MS+2,4-D2.0mgL ^-1The described seminose subculture medium that contains refers to add seminose and sucrose in subculture medium, and the seminose span is 2gL ^-1～8gL ^-1, the sucrose span is 8gL ^-1～2gL ^-1, seminose and sucrose summation are 10gL ^-1Described differentiation culture based component is: MS+6-BA 1.0mgL ^-1NAA 0.1mgL ^-1+ KT 2.0mgL ^-1+ seminose 3～4gL ^-1+ sucrose 7～6gL ^-1+ agar 7gL ^-1PH5.8; The composition of described root media is: 1/2MS+NAA 3.0mgL ^-1+ 6-BA0.1mgL ^-1+ sucrose 40gL ^-1PH5.8.

2, the rapid detection of transfer-gen plant:

Adopt dichlorophenol sulfonphthalein (chlorophenol red, CPR) method, tissue to be measured is inoculated in the MS liquid nutrient medium that contains seminose and dichlorophenol sulfonphthalein, under dark condition, behind 20～35 ℃ of cultivation 1～5d, observe the substratum colour-change, substratum color yellowing or orange, then the pmi gene successfully imports.In the described MS liquid nutrient medium that contains seminose and dichlorophenol sulfonphthalein, mannose content is 2～5gL ^-1, dichlorophenol sulfonphthalein content is 30～60mgL ^-1

The embodiment example is in order to explain that in more detail the present invention utilizes the pmi gene to obtain the method for transgenic sugarcane fast, but the present invention is not limited to this.

Embodiment: utilize the pmi gene to obtain the method for transgenic sugarcane fast, may further comprise the steps:

1, is used for the acquisition of genetic transformation acceptor

Experimental cultivar is the new variety of sugarcane good fortune farming 95-1702 that sugarcane institute of University Of Agriculture and Forestry In Fujian cultivates.Eugonic good fortune farming 95-1702 taper 30cm is chosen at first disinfection, removes its blade and top 5cm.Behind 70% alcohol surface sterilization, 2～3min, peel off its outermost layer on the Bechtop, again with peelling off time skin behind 70% alcohol surface sterilization, 1～2min, carefully cutting diameter then is 0.4～0.8cm, thick is the young leaves of 1～2mm, be inoculated on the inducing culture, cultivate in the darkroom, culture temperature is (25 ± 1 ℃).Subculture was once in subculture medium in per 15 days.Choose well-grown, be rich in gloss, the color cadmium yellow, the embryo callus subculture of tissue block particle densification is the acceptor material of genetic transformation.

2, particle bombardment genetic transformation sugarcane embryo callus subculture

Bombard at particle gun and earlier acceptor material to be seeded in height in preceding 4 hours and to ooze on the substratum and carry out.CaCl ₂, spermidine and tungsten powder respectively get 1 eppendof pipe and be put on ice, gets solid CaCl ₂0.4～0.6g is in the little culture dish of sterilizing (fully grinding the autoclaving oven dry), and the filter paper that shears is put thereon, then the metal ring that contains carrier film is placed on the filter paper.The tungsten powder pipe is put in the ultrasonic oscillator and fully vibrates (if tungsten powder sticks on the pipe, centrifugal being sunken to managed at the end in whizzer earlier) be put in the centrifugal 30sec of 8000g in the whizzer again, remove supernatant, again with isopyknic sterilized water vibration 1min, the centrifugal 1min of 8000g removes supernatant liquor.Repeat flushing once with sterilized water, add 60 μ L sterilized waters vortex on the vortex instrument at last and keep adding successively in the vibration (by the consumption of handling 10 samples): 10 μ LDNA (1 μ g μ L ^-1), 50 μ L2.5molL ^-1CaCl ₂, 20 μ L0.1molL ^-1Spermidine vibrates mixture behind the 2min, ice bath 5min; 8, the centrifugal 5sec of 000g abandons supernatant; With 200 μ L70% alcohol flushings precipitation once; 8, the centrifugal 5sec of 000rpm abandons supernatant, and 200 μ L dehydrated alcohols are suspended particle again; 8, the centrifugal 5sec of 000rpm abandons supernatant; Add 140 μ L dehydrated alcohols suspended particle again at last, mixing.Inhale 10 μ L in not stopping the above-mentioned suspension of vortex, evenly coat on the area of slide glass central diameter 1cm rapidly with the rifle head, cover it immediately after being coated with, put 10min, do not stop vibrations therebetween and prevent caking, smear should use in 2h.

3, the screening of transgenic sugarcane and cultivation

Callus after the bombardment is still put back to height and is oozed continuation processing 18h on the substratum, inserts MS+2 thereafter, 4-D 2mgL ^-1Substratum on, 25 ± 1 ℃ of dark 5～8d that cultivate.Callus after the recovery 25 ± 1 ℃ of dark cultivations the in the subculture screening culture medium of transferring, subculture screening 15 days selects well-grown callus to be linked in the division culture medium thereafter, places illumination box to cultivate (12hd ^-1, illuminance 1000～2000Lux), culture temperature are (27 ± 1) ℃, change once new substratum in per 15 days.Treat to be transferred to when seedling grows to 6～8cm short root cultivation in the root media.

4, the rapid detection of transgenic sugarcane

Obtain the resistant plant blade under aseptic condition, put in the MS liquid nutrient medium that contains seminose and dichlorophenol sulfonphthalein and develop the color, 29 ℃ of dark cultivations 2 days, observe its change in color, substratum color yellowing or orange be transfer-gen plant, if color still be purple or red be the non-transgenic plant.Preparation contains the MS liquid nutrient medium of seminose and dichlorophenol sulfonphthalein, promptly adds seminose and dichlorophenol sulfonphthalein in the MS liquid nutrient medium, and making mannose content is 2～5gL ^-1, dichlorophenol sulfonphthalein content is 30～60mgL ^-1

Claims (1)

1. method of utilizing the pmi gene to obtain transgenic sugarcane fast, it is characterized in that may further comprise the steps: the acquisition of (1) resistant plant: cutting sterile-processed back diameter is 0.4～0.8cm, thick is the sugarcane young leaves of 1～2mm, be inoculated on the inducing culture, in the darkroom, cultivate, culture temperature is 25 ± 1 ℃, and every 15d subculture once; Select well-grown sugarcane embryo callus as transgene receptor,, utilize particle gun transduction or agrobacterium-mediated transformation to transform sugarcane cell the plant expression vector pUP that contains the pmi gene that builds; The composition of described inducing culture is: MS+2,4-D 3.0mgL ^-1Described pUP carrier refer to utilize Ubi as promotor, no as terminator, the pmi plant expression vector that the conventional molecular biology method of gene makes up that serves as a mark; The MS substratum is the collective media of plant tissue culture;

Embryo callus after the particle gun bombardment is put into height oozes substratum and change subculture medium after 10～20 hours over to and cultivated 2～10 days, after to change total carbon source over to be to screen containing in the seminose subculture medium of 10g, 26～28 ℃ of dark culturing 10～20 days; Afterwards well-grown callus is transferred to and broke up in the division culture medium for 6～8 weeks, cultivate 12hd ^-1, illuminance 1000～2000Lux, culture temperature is 26～28 ℃; Treat to be transferred to when seedling grows to 6～8cm short root cultivation in the root media, obtain resistant plant; Described height oozes medium component: MS+2,4-D 2.0mgL ^-1+ 0.2molL ^-1N.F,USP MANNITOL+0.2molL ^-1Sorbyl alcohol; Described succeeding transfer culture based component is: MS+2,4-D 2.0mgL ^-1The described seminose subculture medium that contains refers to add seminose and sucrose in subculture medium, and the seminose span is 2gL ^-1～8gL ^-1, the sucrose span is 8gL ^-1～2gL ^-1, seminose and sucrose summation are 10gL ^-1Described differentiation culture based component is: MS+6-BA 1.0mgL ^-1+ NAA 0.1mgL ^-1+ KT 2.0mgL ^-1+ seminose 3～4gL ^-1+ sucrose 7～6gL ^-1+ agar 7gL ^-1PH5.8; The composition of described root media is: 1/2MS+NAA 3.0mgL ^-1+ 6-BA 0.1mgL ^-1+ sucrose 40gL ^-1PH5.8;

(2) rapid detection of transfer-gen plant: adopt the dichlorophenol sulfonphthalein method, tissue to be measured is inoculated in the MS liquid nutrient medium that contains seminose and dichlorophenol sulfonphthalein, under dark condition, behind 20～35 ℃ of cultivation 1～5d, observe the substratum colour-change, substratum color yellowing or orange, then the pmi gene successfully imports; In the described MS liquid nutrient medium that contains seminose and dichlorophenol sulfonphthalein, mannose content is 2～5gL ^-1, dichlorophenol sulfonphthalein content is 30～60mgL ^-1