CN102492718A - Application of genes of auxin influx carrier AUX1/LAX family in breeding of corns and broomcorns - Google Patents
Application of genes of auxin influx carrier AUX1/LAX family in breeding of corns and broomcorns Download PDFInfo
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Abstract
The invention discloses an application of genes of an auxin influx carrier AUX1/LAX family in breeding of corns and broomcorns, which is characterized in that diffremtn members of the genes of the AUX1/LAX family are cloned from different higher plants and are respectively connected with different promoters to built fusion genes, recombination genes are transferred into cells of the corns and the broomcorns by the transgenic technology, so that transgenic plants and later generations thereof are obtained, engineering plants with characters changed are selected for creating new germplasm with application prospect in breeding of the corns and the broomcorns.
Description
Technical field
The invention belongs to the biotechnology breeding field of farm crop; Relate to a kind of application that changes the scheme of corn and Chinese sorghum proterties through gene fusion construct and transgenic; Specifically, relate to the application of growth hormone input carrier AUX1/LAX family gene in corn and Chinese sorghum breeding.
Background technology
The development of plant genetic engineering depends on the application with difference in functionality gene.Growth hormone plays an important role in growth and development of plants, and the expression of, transhipment synthetic through growth hormone, metabolism and relevant signaling genes is modified with and possibly produces material impact to growth and development of plant.
Growth hormone is in the tender part of plant children or divide in the vigorous tissue syntheticly, arrives site of action through its distinctive mode of transport, in a plurality of processes of growth and development of plants, plays an important role.Growth hormone difference distribution within plant tissue influences a plurality of growth courses, and varying environment signal and endogenous signal can influence the growth hormone changes in distribution through synthetic the transhipment with iuntercellular in the part of growth hormone.IAA is the principal mode of growth hormone in most plants.
Lower concentration growth hormone promotes cell elongation and division in many systems, and the growth hormone of high density then suppresses the division and the elongation of cell.The growth hormone signal has promoted the expression of phytokinin arrestin, and phytokinin has been induced growth hormone signal suppressing albumen.Growth hormone has and the most significantly promotes adventive root and lateral root to take place and the growth effect in plant hormone; Participation root hair is educated, primary root is grown, lateral-root primordia forms and the gravitropism reaction of root; Ethene is through metabolism play a role (Pitts et al, 1998 of regulation and control growth hormone; Rahman et al, 2002).The decision that interaction regulation and control radicle forms and lateral root takes place of growth hormone and phytokinin.Growth hormone is created local peak concentration in the active transhipment of iuntercellular, thereby regulates and control the initial sum process of many growth incidents.Auxin concentration gradient in the plant tissue comprise growth hormone in cell with intercellular uneven distribution.The auxin concentration of cell can be transported a plurality of aspect adjusted in synthetic, passivation, activation, decomposition and intercellular polarity.And these different aspects be adjusted in different plant species, different development stage all plays an important role.
In the plant root tip vegetative point, the polarity of growth hormone transportation forms what is called " growth hormone accumulation peak " with local synthetic having facilitated at quiescent center (being stem cell organization center) position, for the foundation of quiescent center with kept crucial effects.(Dubrovsky etc. take place in the lateral root organ; 2008) shown the growth hormone peak concentration and grown that programming adjustment and organ are initial to have a corresponding relation on space-time, heredity or medicine disturb the difference distribution of growth hormone and the growth incident that response process has hindered appearance when the growth hormone gradient exists.In addition, as if the growth hormone difference distribution is enough to start growth course.The exquisiteness test that the growth hormone trace is applied to shoot apical meristem shows the local growth (Reinhardt et al., 2003) that auxin concentration is enough to start and accomplish leaf or flower that increases.Growth hormone can be modified its development program in the accumulation of given cell, i.e. the time of growth hormone accumulation with determining positions the development program time and the position of adapting.The archusia synthetic that random stimulus causes in pericyclic cell increases, and has improved the plain concentration of local growth, causes that the initial lateral root of these competent cells takes place.
There are two kinds of different growth hormone transport pathway in plant materials: polarity carrier mediated in phloem transportation and the cortex is transported.In phloem haulage system, the transportation of growth hormone and the transportation of other nutritive substance do not have fundamental difference; The polarity transportation then is the peculiar one type of active transport from the cell to the cell of growth hormone, depends on localized carrier of film and energy expenditure (Lomax etc., 1995), and travelling speed is also slower than vascular system travelling speed, is generally 5-20mm/h.Highly input, the output carrier proteins of regulation and control, polarity orientation are given the credit in the polarity transportation of growth hormone in tissue to a great extent.The polarity transportation forms with the organ top growth hormone in the plant body be the concentration gradient at center, and the auxin concentration of keeping in the different tissues is poor, realizes the regulation and control of development of plants.And exist positive regeeration between growth hormone signal and the transhipment of growth hormone polarity and regulate (Vieten etc., 2005).The albumen of the plain polarity transportation of mediating growth has AUX1/LAX family, PIN-formed family, ABCB family protein etc.
Growth hormone gets into the dual mode completion that cell can mediate through the growth hormone input carrier (auxin influx carrier) of polar contribution on passive diffusion and the plasma membrane.Flow into cell by the growth hormone of input carrier mediation and can make the contrary concentration gradient transportation of growth hormone, can prevent also that growth hormone is passive to diffuse in the adjacent cells, thus guaranteed in different cells and the tissue auxin concentration with distribute suitable.AUX1/LAX family participates in the transhipment of growth hormone polarity, the plain cell that flows into of their mediating growths.Arabidopis thaliana AUX1 is the growth hormone input carrier of first discovery, its sudden change causes that growth hormone polarity transportation is obstructed, root to the susceptibility of growth hormone descend, root gravitropism forfeiture and phyllotaxy arrange unordered.4 growth hormone input carriers of arabidopsis gene group coding AUX1 (auxin resistant 1), LAX1, LAX2 and LAX3 (Kramer, 2004).AUX1 is the input carrier of first discovery.The protein with permease character that AUX1 is made up of 485 amino-acid residues infers that it has 11 to stride diaphragm area (Marchant etc., 2002).The similarity of AUX1 and other 3 LAX aminoacid sequences is 73%～82%.They all have the function of growth hormone input carrier.Paddy rice contains 4 possible growth hormone and flows into carrier, called after Os auxin transporter protein 1, Os auxin transporter protein 2, Os auxin transporter protein 3, Os auxin transporter protein 4.The ZmA UX1 gene of from corn, cloning is compared with Arabidopis thaliana AUX1; Similarity at aminoacid sequence is 81%; It is reported and in the tip of a root (tip of a root 1cm segment) of primary root, crown root, seminal root and the lateral root of sprouting 12 days corn seedlings, express the abundance height, and almost detect less than this genetic expression than upper curtate at root system.In situ hybridization shows that ZmAUX1 expresses abundance high (.Hochholdinger etc., 2000) in epidermic cell, pericycle and the endodermis cell of the tip of a root.The applicant passes through systematic comparison and the analysis to the growth hormone input carrier of corn, paddy rice, Arabidopis thaliana; The similarity of finding Os auxin transporter 3 in ZmAUX1 and Arabidopis thaliana auxintransporter 2 and 3, the paddy rice is high; And more approaching on amino acid composition, peptide chain length and iso-electric point, molecular weight, answer called after Zm auxin transporter protein 3.ZmAUX1 still is called in narration in this specification sheets for ease.
The degeneration-resistant border characteristic of plant and the receptivity of inorganic nutrients are most of directly related with Root morphology and physiological property.Adjusting through auxin concentration and polar contribution is expected to realize: 1) improve crop to fertilizer utilization ratio, save production cost and reduce environmental pollution; 2) improve the utilization ratio of root system, increase the dry crop yield moisture; 3) improve crop and resist saliferous ability, increase crop yield in the salt marsh arable land; 3) create high yield, high-quality crop varieties resistant to lodging.
Summary of the invention
To present present Research; Produce the auxin concentration gradient and change and cause transgenic corns and the Chinese sorghum that plant root proterties, plant height and lodging resistance change through changeing the AUX1/LAX family gene thereby the purpose of this invention is to provide one, the latter is used for the genetic improvement of corn, Chinese sorghum.
AUX1/LAX family gene of the present invention comprises that from different higher plants, clone and the close or identical protein gene of peptide sequence Arabidopis thaliana UX1/LAX family; Also comprise synthetic or the polymerized nucleoside acid sequence of recombinating, the encoding egg white gene that promptly sequence is close or identical with function or its RNAi structure by different sequences.
Gene of the present invention is used and is comprised with complete sum Partial cDNA gene form, complete sum portion gene group gene form and their RNAi structure formation.
According to the invention is with clone AUX1/LAX family gene member's from higher plant such as Arabidopis thaliana, paddy rice, corn total length or the partial sequence gene fusion construct in the middle interstitial granules of recombinating respectively; Then fusion gene is inserted in the plant expression vector; Adopt transgenic technology that recombination is imported vegetable cell, obtain transfer-gen plant; Through detecting transgene expression and plant being carried out macroscopical identification, therefrom filter out transfer-gen plant and offspring thereof that objective trait obviously changes, create the new germ plasm and the new variety that in crop breeding, have application prospect.
Wherein, the said full length sequence that is used for the AUX1/LAX family gene of gene fusion construct can be the cDNA sequence, also can be genome sequence, also consistent the or close dna fragmentation with AUX1/LAX family gene sequence of synthetic.Described AUX1/LAX family gene is from higher plant; Corn sibling species or subspecies like Arabidopis thaliana, paddy rice, common cultivation corn and Zea; Can be from Chinese sorghum and arabian cron, the also consistent or close dna fragmentation with AUX1/LAX family gene sequence of synthetic.
Wherein, the full length sequence of described ZmAUX1 is from the corn sibling species or the subspecies of common cultivation corn and Zea, also the sequence one of the synthetic close dna fragmentation of making peace.
Wherein, the used promotor of gene fusion construct can be root-specific promoter, also can be stem, fruit ear, tassel specificity promoter, and inducible promoter.
The said fusion gene of the present invention is made up of 3 ' tail district of promoter sequence and target gene encoding sequence, plant gene.Fusion gene can be in vegetable cell effective expression.
The AUX1/LAX family gene that the present invention clones can be separately or union and recombination in planting thing expression vector, change target gene the corn and the Chinese sorghum of excellent genes type over to by the latter again, obtain transgenic corns and Chinese sorghum that economical character changes.
The corn and the Chinese sorghum of the commentaries on classics AUX1/LAX family gene that the present invention produces can utilize separately, also can utilize through obtaining to have 2～3 genetically modified plant concurrently after the hybridization between the transfer-gen plant.
The structure of the discriminating of AUX1/LAX family gene, clone and plant expression vector
Through the examination and the sequence assembly in full genome and cDNA library,, in corn and paddy rice, identify 4 AUX1/LAX family genes respectively in conjunction with utilizing the AtAUX1 gene order to carry out similarity relatively.Employing clones target gene through conventional clone technology from the donor plant, confirm through order-checking.If from the uncompleted species cloning AUX1/LAX of gene order-checking family gene, then seek and clone target gene through the utilization of homologous sequence.In the present invention, the AUX1/LAX family gene that will from corn or paddy rice, clone merges with total length or partial sequence form or with RNAi structure formation and promotor, produces fusion gene (target gene can justice or antisense form link to each other with promotor).The proterties required according to engineering plant with AUX1/LAX gene of selecting of from different higher plants, cloning and selected broad spectrum or tissue specificity or the fusion of inducing specific expression promoter, produces different AUX1/LAX fusion genes.Adopt routine techniques that fusion gene is inserted in the plant expression vector then.Adopt transgenic technology that fusion gene is imported vegetable cell, obtain transfer-gen plant.
With the pCAMBIA1300-Pubi::AUX1/LAX-Tnos-P35S::EPSP plasmid construction is example, briefly sets forth the building process of plant expression vector.The AUX1/LAX gene starts with corn ubiquitin protein promotor Pubi, and the selection markers gene is herbicide glyphosate resistant gene EPSP, and the latter is started by cauliflower mosaic virus 35S RNA promotor P35S.The plant expression vector construction route is: at first; The pT-easy-AUX1/LAX and the intermediate carrier pCDNA3 that are connected with foreign gene cut through restriction enzyme respectively; Reclaim AUX1/LAX fragment and carrier segments, through dephosphorization be connected, the AUX1/LAX gene is inserted among the pCDNA3.Connect correct plasmid and cut, reclaim the fusion gene fragment that is added with Sac I restriction enzyme site, be inserted into then among the plant expression vector pCUE (pCAMBIA1300-Pubi::MCS-Tnos-35S::EPSP) through Sac I enzyme.Recon is identified the fusion gene direction of insertion through Xho I+Not I or Hind III+BamHI double digestion.The AUX1/LAX gene can positive and negative adopted form be inserted among the plant expression vector pCUE.Extract DNA and purifying from the intestinal bacteria that contain recombinant plasmid, utilize the liquid nitrogen flash freezer method to transform among Agrobacterium LBA4404 and the EHA105.Transform the Agrobacterium bacterium colony that produces and adopt the PCR method to identify, be used for agriculture bacillus mediated corn, the genetic transformation of Chinese sorghum then.
The acquisition of transfer-gen plant and evaluation
Through the genetic transformation conversion method plant expression vector that makes up is imported in corn and the Chinese sorghum excellent genes type, obtain transformed plant.When adopting aseptic seedling stem apex method for transformation, T0-T2 transformant plant is carried out herbicide screening in tri-leaf period.If with the herbicide glyphosate is selective agent, concentration is 1.25～1.5%.At weedicide spray back 12～15d, anti-, the sense degree of statistics plant weedicide.Because the high density Glyphosate 62 IPA Salt causes that the maize leaf jaundice is dead, can plant be divided into I, II, III, IV level according to the dead degree of jaundice, I level resistance is the strongest.With resistance be T1 and the T2 of I level or II level for plantlet of transplant bagging selfing to the land for growing field crops, results filial generation seed carries out PCR simultaneously and detects.Detect through herbicide screening and PCR, select that Herbicid resistant is strong gathers in the crops seed of future generation with PCR male plantlet of transplant to the land for growing field crops, eliminate the herbicide sensitive strain to be.According to Herbicid resistant and PCR detected result, from T3 generation pick out transgenic isozygoty and be (in T1 and/or T2 generation, present Mendelian's segregation ratio, and T3 plant Herbicid resistant does not have separations) be used for that tree characteristics is observed and physiology mensuration.
T3 for the seed germination of the positive strain of PCR system 4 days, is got spire (comprising bud scale) and young root and carries out Real-time RT-PCR detection.Detect through transfer-gen plant Real-time RT-PCR, filter out target gene expression intensity and the adjoining tree transgenic homozygous lines that there were significant differences.If with corn inbred line neat 319 is acceptor, is respectively conversion carrier with target gene justice plasmid pCAMBIA1300-Pubi::AUX1/LAX (+)-Tnos-P35S::EPSP and target gene antisense plasmid pCAMBIA1300-Pubi::AUX1/LAX (-)-Tnos-P35S::EPSP and carries out genetic transformation and offspring's screening and evaluation.In the T3 of the independent transformant that 18 target gene justice plasmids produce for strain was, the expression abundance of the ZmAUX1 of spire was 5 times of root approximately.In different transgenic lines, the ZmAUX1 gene expression abundance and neat 319 the raising that has in various degree of comparing, the ZmAUX1 expression amount of most strains system is 1.67～2.3 times of neat 319 plant of contrast.And in antisense gene strain system, the expression abundance of ZmAUX1 has the decline of different amplitudes, and the ZmAUX1 expression amount is about 60% of neat 319 plant of contrast in most strains are.
Change the phenotype analytical of the positive and negative adopted gene corn plant of ZmAUX1
In transgenic does not contrast strain system and the seed of different transgenic lines is sowed at the big case of uncovered that size is consistent, vermiculite content equates.The 1/2MS nutritive medium is watered in 3 leaf phase final singlings every other day.Transfer-gen plant and adjoining tree over-ground part form and growth velocity and adjoining tree difference are little.Corn grows to 6 leaves during the phase; With tap water flush away vermiculite, isolate root system gently, the adopted gene plant well developed root system of becoming a full member; Show that seminal root length and lateral root number increase than adjoining tree; It is consistent with the morphologic observation result that living weight is measured the result, and the root/shoot ratio comparison is according to increasing, and weight of root system is 120～150% of an adjoining tree.Antisense gene plant root system is close or slightly reduce with contrast, but the part strain is the upperground part biomass certain increase is arranged.
T3 is cultured to the V6 phase for transfer-gen plant and adjoining tree in sufficient phosphorus nutrition liquid, measures the living weight of their root length, radical order, cauline leaf and root system respectively.In sufficient phosphorus nutrition liquid transfer justice ZmAUX1 strain is that corn has flourishing root system, compares with the antisense plant with adjoining tree, though crown root number and seed radical no significant difference have more lateral root.The lateral root sum comparison of the Yi Jiyin strain of becoming a full member system increases by 55%～101% according to plant, and system is different because of strain.In 4 antisense gene strain systems, the lateral root number that is plant except that a strain is less than the adjoining tree, and all the other are more or less the same with adjoining tree on radical order and composition.The mensuration root length draws, and the length of primary root, seminal root and the lateral root of the Yi Jiyin strain of becoming a full member system is all compared the remarkable increase according to plant and antisense strain system, and the long comparison of total root is according to increasing by 44%～96%; And the root length overall of 4 antisense gene strain systems is 95.9～115% of contrast strain systems, and is promptly less with contrast difference.
T3 is cultured to the V6 phase for transfer-gen plant and adjoining tree in scarce phosphorus nutrition liquid, measures the living weight of root length, radical order, cauline leaf and the root system of transgenic and adjoining tree respectively.Under low-phosphorous culture condition, the radical order of all strain systems and root length all have bigger increase than in sufficient phosphorus nutrition liquid, cultivating, and wherein the length of seminal root increases particularly evident.Relatively the Root morphology parameter of transfer-gen plant and adjoining tree is found; Has comparison according to more flourishing root system at scarce phosphorus nutrition liquid transfer justice gene plant; Though the difference with adjoining tree and antisense gene plant on crown root number and seed radical is not remarkable; But the adopted gene plant of becoming a full member has more lateral root, and give birth on crown root, primary root, seminal root the comparison of lateral root number average according to and antisense gene plant many.The lateral root sum of adopted gene plant of becoming a full member is 112～152% of an adjoining tree.In 4 antisense gene strain systems, the lateral root number that is except that a strain is less than the adjoining tree, and all the other are more or less the same with adjoining tree on radical order and composition.On root length, the length of primary root, seminal root and the lateral root of the Yi Jiyin strain of becoming a full member system is all compared according to plant and antisense plant has increase, and total root length is 193～221% of contrast.4 antisense strains are that total root length then is about 83% of contrast strain system.
V6 phase corn living weight is measured the result and is shown that the adopted ZmAUX1 gene of becoming a full member has promoted the plant root growth, has increased the plant root/shoot ratio under the different phosphorus supply levels, and this is consistent with result under the vermiculite culture condition.Handled 8 days in scarce phosphorus nutrition liquid when plant, though obviously influenced the allocation proportion of glucide to rhizome, influenced root/shoot ratio, promptly low-phosphorus stress is handled and has been promoted root system development, and the root growth that slowed down is little to the influence of plant living weight.
Detect the growth hormone polarity transportation Expression of Related Genes level that seed germination produced the spire (comprising bud scale) and the root of seedling in 4 days; Discovery is at ground, the underground part of transgenic line and adjoining tree; The expression amount of crossing growth hormone outflow carrier PIN1 family gene in the strain system of expressing ZmAUX1 all increases; This possibly be that growth hormone flows into the adaptations that increase causes cell to produce; Through plain the transporting keeping the auxin concentration of cell normal level of accelerating growth, thereby keep the intravital auxin concentration gradient of plant.And the localized several PIN-like expression of gene amounts of cell inner membrance do not have notable difference.The changing conditions of these genes shows that auxin concentration and signal have the effect of feedback regulation to growth hormone polarity transhipment Expression of Related Genes, can influence genetically modified effect to a certain extent.But the growth hormone inflow and outflow that has increased certainly will cause the variation of growth hormone allocation proportion between " storehouse " and " source ", and these all can growing and the reaction of low-phosphorus stress is affected root system.
From the transgenic line of well developed root system, select the good strain system of economical character to be used for the rearing new variety of crops such as corn, Chinese sorghum.
Embodiment
Embodiment 1: change the corn inbred line that Os auxin transporterprotein 3 genes are created well developed root system
Change Os auxin transporter protein 3 genes be intended to be this gene at the corn root specifically expressing, therefore, need with the different expression promoter gene fusion construct of Gent.Barley phosphorus transporter body 1 gene is specific expressed and induced by low-phosphorus stress in root; Therefore; Adopt PCR method with its promotor part (843bp at the ATG codon 5 ' upper reaches among the GenBank accession number AF543197; Non-total length) from the barley genome, amplifies called after Prsp.After order-checking is just tested, it is recombinated among the intermediate carrier pCDNA3.Utilize Hind III and Xbal I restriction enzyme site that this promotor orientation is recombinated among the plant expression vector pCPE then, obtain plasmid pCAMBIA-Prsp::MCS-Tnos-P35S::EPSP (pRPE).This carrier contains the herbicide glyphosate resistant gene EPSP that is started by P35S, and promptly the selection markers gene contains the slotting box of goal gene that Prsp starts.The plant expression vector pRPE that builds is used for the structure of the plant expression vector of Os auxin transporterprotein3 gene.
The foundation of receptor system is material with key self-mating system used in China's agriculture prodn, like the selfed seed of Zheng 58, prosperous 7-2, DH4866 etc.Isolated culture induces stem apex to produce the sprout tuber that grows thickly, and is that acceptor carries out genetic transformation with the sprout tuber that grows thickly.Used substratum has:
Seed germination substratum: KNO
31900mg/l, NH4NO
31650mg/l, CaCl
22H
2O 440mg/l, MgSO
47H
2O 370mg/l, KH
2PO
4H
2O 170mg/l, FeSO
47H
2O 27.8mg/l, ZnSO
47H
2O 10mg/l, MnSO
44H
2O 22.3mg/l, H
3BO
310mg/l, KI 0.83mg/l, Na
2MoO
42H
2O 0.5mg/l, CuSO
45H
2O0.025mg/l, CoCl
26H
2O 0.025mg/l, vitamin 10.0mg/l, pyridoxine hydrochloride 1.0mg/l, nicotinic acid 1.0mg/l, glycocoll 2.0mg/l, inositol 100.0mg/l, vitamin H 0.05mg/l, casein hydrolysate 500mg/l, sucrose 30g/l, agar powder 7g/l, pH 5.8～6.0.Be used for seed germination.Liquid nutrient medium then removes agar powder.
The A substratum: the seed germination substratum adds 6-BA 4.5～9.0 μ mol/l and 2, and 4-D 1.0～3.0 μ mol/l are used to induce isolated culture bud point to produce grow thickly the bud tissue block and the bud tissue block succeeding transfer culture of growing thickly.
B substratum: additional 6-BA 4.5 μ mol/l of seed germination substratum and IBA (indolebutyric acid) 1.8 μ mol/l, the bud tissue block that is used to grow thickly differentiation seedling.
Become the seedling substratum: additional 6-BA 2.25 μ mol/l of seed germination substratum and IBA 3.6 μ mol/l, the budlet that is used to grow thickly develops into seedling.
Root media: the seed germination substratum adds IBA 2.8～3.6 μ mol/l, is used for the little seedling rooting of unrooted.
Minimum medium and plant growth regulating substance pressure sterilizing; Microbiotic and weedicide isoreactivity composition filtration sterilization add behind medium sterilization.
Seed sterilization and sprouting: corn seed with 0.1% mercury chloride immersion 10--15 minute, washs 3--5 time with sterilized water with 70% alcohol immersion 10 minutes more then.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of (30---40 milliliter/250 milliliter triangular flask) sterilized water in the bottle, is placed under the dark condition (23---30 ℃) 1--2 days after sealing.(showing money or valuables one carries unintentionally) back seed of sprouting is placed on the minimum medium and continues to sprout down in dark condition.
Stem tip culture with grow thickly that the bud tissue block is induced, subculture, differentiation: when the plumule elongation of germinated seeds ends 3---5 centimetre; Peel off coleoptile and spire; Cut the epicotyl and the stem apex that are about 5 millimeter; Be inoculated into (24--27 ℃) cultivation under dark on the A substratum, and in time cut the plumular axis of elongation and peel off spire.Cultivate after 6--10 days, stem apex begins the irregular growth of expanding, and several wartys or digitation on the meristematic tissue that expands, occur.After 20 days, begin to form indefinite bud and embryoid on the surface of warty or digitation.General per 4 all succeeding transfer culture once.In succeeding transfer culture, budlet is on the high side if the bud tissue block of growing thickly is grown thickly, and 2,4-D concentration is got 3.0 μ mol/l; Bud tissue block callusization is heavier if grow thickly, though a large amount of meristematic cell groups is arranged, indefinite bud seldom appears in the surface, can be with 2, and 4-D concentration is reduced to 1.0 μ mol/l, continues to cultivate then to produce a large amount of wartys or digitation again.The bud tissue block of growing thickly on the A substratum, minority has the generation of adventive root.Exist equally with spire, adventive root also influences the generation of expanding growth and embryoid and the budlet that grows thickly of tissue block, need remove early.The bud tissue block of growing thickly was transferred on the B substratum after 2--3 days, and color and luster is flavescence gradually, and quality is more pliable and tougher, and microspike appears in the surface after 5--6 days.ESEM is observed visible each phase embryoid and indefinite bud down.Embryoid and indefinite bud are grown rapidly, form the budlet that grows thickly on the tissue block surface.
With the bud tissue block of growing thickly is the conversion and the plant regeneration of acceptor
(every liter of substratum contains: tryptone 10g at additional antibiotic LB substratum will to have the agrobacterium tumefaciens (like AGL0 and LBA4404) of binary vector (the Mini--Ti plasmid has selective agent resistant gene and Prsp::Os auxin transporter protein 3 genes); Yeast extract 5g; NaCl 10g, pH 7.0, pressure sterilizing) in 28 ℃ down concussion cultivate; Concussion speed be 110rpm (rev/min), make bacterium be in logarithmic phase.Under 3000rpm centrifugal 10 minutes then, abandon supernatant.Thalline is with liquid seeds germination medium (be that the seed germination medium component reduces by half, the remove agar powder) washing of 1/2 concentration, centrifugal again collection.(acetosyringone, As) the liquid inducing clumping bud substratum of 1/2 concentration of 100 μ mol/l suspends, and dilutes 5--20 and doubly is used for transforming with adding Syringylethanone with thalline again.
Getting the bud tissue block of growing thickly of cultivating 13--20 days behind the subculture is transgene receptor.Transform with agrobacterium-mediated transformation, transform the back material and recover darkling to cultivate.Grow thickly budlet or the bud tissue block of growing thickly of agroinfection were cultivated 7--12 days bacteria growing inhibiting in dark on the substratum that is added with cephamycin (Cefotaxime) 250mg/l or Pyocianil (Carb) 500mg/l.Recover to cultivate growing thickly budlet or the bud tissue block step sizing 3-4 generation on the substratum that is added with selective agent Glyphosate 62 IPA Salt 0.04% of growing thickly after back or the antibacterial cultivation, obtain transgenic cell and budlet.The exhausted big bud tissue block of growing thickly of counting is dead gradually in screening and culturing.Remove 2 with what the tissue block of survival was transferred to no selective agent, recover to cultivate generation budlet in back on the A substratum of 4-D.
Budlet is placed on into irradiation growth down on the seedling substratum, light intensity 2000-3000lx, illumination 14-15 hour/day.Seedling length changes in the root media during to 3-4 sheet leaf takes root.Cultivated about 15 days, about 40% seedling produces new root.For the seedling of not taking root, its base portion of cut wound is transferred on the new root media and is cultivated, and most of plant produce root system after 10 days.Take root behind the seedling flush away substratum, being transplanted to the vermiculite is to grow in the flowerpot of cultivation medium.Plant grows under natural lighting, day warm 22--28 ℃, at temperature 15--21 ℃ of night, waters the inorganic salt composition of the seed germination substratum of 1/2 concentration every other day.Grow about 2 weeks, nursery transplant produces flourishing root system, is colonizated in the field then.
The resistance of transfer-gen plant detects and selects and utilizes
The blade of getting the transplant survival plant carries out molecular Biological Detection and confirms transfer-gen plant.Then with transfer-gen plant (T0) bagging self-fertility.To broadcast in the greenhouse or have the field of safeguards from the T1 seed of different T0 plant, and get blade after emerging and carry out transgenic PCR and detect.Selected plant is carried out Southern blotting checking, and adopt RT-PCR technical inspection transgene expression intensity.The plant that T1 generation filters out continues the bagging selfing, and its filial generation is proceeded molecular biology identification, measured plant height and each panel length, observation root system and other main economical character and preferential plant selfing and reserve seed for planting.
T3 is cultured to the V6 phase for changeing Os auxin transporter protein 3 gene plants with adjoining tree in the nutritive medium of different phosphorus supply levels, measures root length, the radical order of transgenic and adjoining tree respectively, measure the living weight of cauline leaf and root system.When plant grew in low-phosphorous nutritive medium, the radical of adjoining tree fell now, was 88% under the sufficient phosphorus cultivation.Relatively each strain system finds that to the reaction of low-phosphorus stress the susceptibility that transfer-gen plant all shows low-phosphorus stress reduces, and the plant that grows in lateral root number, total radical and the sufficient phosphorus nutrient solution is more or less the same or is higher relatively.This phenomenon also exists in the variation of plant living weight.Gathering continuous triple-substituted observation statistical information draws; When introducing corn Os auxintransporter protein 3 genes to the key self-mating system of corn; Variation has taken place in plant forms especially root traits; Cross and express Os auxin transporter protein 3 and increased the lateral root number, promoted the elongation of primary root, but economical characters such as plant height are not had obvious influence.
Embodiment 2: the plant type of changeing ZmAUX1 gene alteration Chinese sorghum
The purpose of commentaries on classics ZmAUX1 gene is suitably to reduce the Chinese sorghum plant height or draws back plant top internode and promote root system development; Therefore; At first need the coding region of ZmAUX1 gene or the promotor of its RNAi structure and specifically expressing to be connected to form fusion gene; Utilize suitable carrier that fusion gene is imported the Chinese sorghum cell again, from the transfer-gen plant offspring, select to have the material of objective trait.
1. according to paddy rice sucrose synthase-1 (RSs-1) promoter sequence (the Plant Molecular Biology 19:881-885 of report such as Ming-Bo Wang; 1992.) the design primer; With high quality paddy rice genomic dna is template; Adopt PCR method to amplify paddy rice sucrose synthase-1 promotor PRSs-1, this promotor is about 1.7kb, and promotor gene is at the plant phloem specifically expressing.The promoter fragment that clones is connected with opening code-reading frame and the 3 ' tail region of ZmAUX1 after confirming through checking order, and constructs fusion gene PRSs-1::ZmAUX1.Again the latter is recombinated among the plant medial expression vector pCDNA.This carrier contains the bar gene that is started by P35S, i.e. selection markers gene.The plant expression vector pCDNA-PRSs-1::ZmAUX1 that builds changes agrobacterium tumefaciens over to and is used for the Chinese sorghum genetic transformation.
2. the Chinese sorghum aseptic seedling obtains
The seed of good self-mating system of Chinese sorghum or kind with 70% alcohol immersion 4 minutes, soaked 8 minutes with 0.1% mercury chloride again, then with sterilized water washing 3-5 time.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into small amount of aseptic water in the bottle in dark condition (25-28 ℃) 1-2 days down.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium and sprout down in dark condition.When treating that the plumule elongation ends 3 centimetres of left and right sides, peel off coleoptile and 2-3 sheet spire, emergent stem pointed tip growing tip.
3. Agrobacterium is cultivated and activation
(the Mini-Ti plasmid is pCDNA-PRSs-1::ZmAUX1 will to have binary vector; Have herbicide resistance gene bar and PRSs-1::ZmAUX1 fusion gene) agrobacterium tumefaciens (AGL1 or LBA4404) in additional antibiotic LB substratum 28 ℃ down concussion cultivate; Concussion speed is 110r/min, makes bacterium be in logarithmic phase.Under 3000r/min centrifugal 10 minutes then, abandon supernatant.Thalline is with the washing of 1/2MS liquid nutrient medium, centrifugal collection.Thalline is suspended with the 1/2MS liquid nutrient medium that adds 100 μ mol/l Syringylethanones, dilution 5-20 doubly is used for transforming again.
4. the Chinese sorghum aseptic seedling transforms
(1) bacterium liquid is poured in the petridish of 4.5 cm diameters, the inclination petridish is immersed in the bacterium liquid, 0.5 * 10 the aseptic seedling of exposing the stem apex growing tip
5The Pa normal atmosphere was handled 8-12 minute down.
(2) the bud point after the dip-dye blots with aseptic filter paper gently, and germinated seeds is placed on the modified MS medium and in dark, cultivated 2-3 days, and culture temperature is 22-24 ℃.Then aseptic seedling is placed under the scattered light and cultivated 2 days.
(3) aseptic seedling after the illumination cultivation is transplanted in the flowerpot that is covered with upper strata vermiculite lower floor loam, and covers the plant top with vermiculite.Let plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 15-21 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.
5. transformed plant screening and field planting
After transformed plant grows 3 leaves; Spraying herbicide
(Hoechst Schering AgrEvo GmbH; Contain weedicide glufosinate ammonium) aqueous solution, concentration is that 9.6ml-12.8ml
falls drop with plant and is advisable.Unconverted adjoining tree stops growing after back 4 days in sprinkling, begins death after 9 days.Transformed plant is after sprinkling, and some individual variations are similar with adjoining tree, and other individual continuing grow, and change not obvious.When the plant of waiting to survive grew to 5 leaves, to the field, the bagging selfing was set seeds with its field planting.
6. transfer-gen plant progeny analysis
T1 handles with 12.8ml
aqueous solution to 3 leaf phases for plant length, the individual ratio of observation statistics resistance and susceptibility; Adopt round pcr to detect foreign gene, and the segregation ratio of statistics foreign gene in progeny plant.The survival plantlet of transplant arrives land for growing field crops, bagging selfing.T2 except that the bagging selfing is set seeds, adopts round pcr to detect foreign gene for plant, and carries out Southern blotting checking, adopts RT-PCR technical inspection transgene expression intensity.Measure the variation of length with the blade size of the different internodes of plant for selected transgenic line, measure 3-7 leaf phase seedling growth velocity, root/shoot ratio and biomass of individual tree, observe the variation of root system variation and degeneration-resistant proterties, and be contrast with transfer-gen plant not.Select that plant height and panel length change and adventive root increases transgenic line carries out the Chinese sorghum yield traits under field cultivating condition observation and comparison, select anti-fall the strain system of high yield to get into that biological safety is tested and the Chinese sorghum breeding is tested.
Claims (10)
1. the application of growth hormone input carrier AUX1/LAX family gene in corn or Chinese sorghum breeding; It is characterized in that; From higher plant, clone the AUX1/LAX family gene; Be used for corn or Chinese sorghum transgenic with the different promoters fusion, produce engineering plant and offspring thereof that growth hormone concentration gradient and tree characteristics change.
2. the application of growth hormone input carrier AUX1/LAX family gene in corn or Chinese sorghum breeding according to claim 1 is characterized in that, said AUX1/LAX family gene is cloned in Arabidopis thaliana, paddy rice, corn or barley.
3. the application of growth hormone input carrier AUX1/LAX family gene in corn or Chinese sorghum breeding according to claim 1; It is characterized in that; Clone the AUX1/LAX family gene through being connected to form fusion gene, utilize promotor to control genetically modified expression specificity and expression intensity with different promoters.
4. the application of growth hormone input carrier AUX1/LAX family gene in corn or Chinese sorghum breeding according to claim 1; It is characterized in that; Fusion gene is used for corn, Chinese sorghum transgenic; Different AUX1/LAX genes can separately or be united and changed recipient plant over to, the transfer-gen plant that generation auxin concentration gradient changes.
5. the application of growth hormone input carrier AUX1/LAX family gene in corn or Chinese sorghum breeding according to claim 1; It is characterized in that; Transfer-gen plant and offspring thereof are through screening and mensuration, and the strain system that selects inheritance stability and the obvious change of objective trait is used for the genetic improvement of corn, Chinese sorghum.
6. like claim 1 and 2 application of said growth hormone input carrier AUX1/LAX family gene in corn or Chinese sorghum breeding, it is characterized in that the AUX1/LAX family member comes from from common cultivation corn, corn sibling species or subspecies, paddy rice or Arabidopis thaliana.
7. like claim 1 and 6 application of said growth hormone input carrier AUX1/LAX family gene in corn or Chinese sorghum breeding; It is characterized in that; Described AUX1/LAX family gene is complete or Partial cDNA gene form, complete or portion gene group gene form, or their RNAi structure.
8. like claim 1 or 3 application of said growth hormone input carrier AUX1/LAX family gene in corn or Chinese sorghum breeding, it is characterized in that, the promotor of selecting for use be broad spectrum, root-specific, stem is specific, fruit ear is specific or induction type.
9. like claim 1 or 8 application of said growth hormone input carrier AUX1/LAX family gene in corn or Chinese sorghum breeding; It is characterized in that; From paddy rice, clone Os auxin transporter protein 3 genes, they are merged with the different expression promoter Prsp of Gent respectively, change corn in the plant expression vector of recombinating; Obtain the transgenic line of well developed root system, anti-low-phosphorous characteristic, be used for corn breeding.
10. like claim 1 or 8 application of said growth hormone input carrier AUX1/LAX family gene in corn or Chinese sorghum breeding; It is characterized in that; From corn, clone the ZmAUX1 gene, the promotor PRSs-1 of it and vascular-specific expression is merged, change in the Chinese sorghum improved seeds; Obtain the transgenic strain of well developed root system, plant height reduction, be used for the Chinese sorghum breeding.
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| CN104650203B (en) * | 2015-02-09 | 2019-06-25 | 山东农业大学 | A kind of ZmAUX1 albumen and its encoding gene and application |
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