CN101484581A

CN101484581A - Plant artificial chromosome platforms via telomere truncation

Info

Publication number: CN101484581A
Application number: CNA200780025280XA
Authority: CN
Inventors: W·于; J·M·维加; J·伯奇勒
Original assignee: University of Missouri System
Current assignee: University of Missouri System
Priority date: 2006-05-09
Filing date: 2007-05-09
Publication date: 2009-07-15

Abstract

The invention provides engineered plant minichromosomes generated by telomere mediated truncation of native chromosomes. These minichromosomes are faithfully transmitted from one generation to the next and provide an ideal platform for breeding genes into desired plant varieties with out problems, such as linkage drag, associated with standard breeding methods.

Description

By the artificial chromosome platforms of the plant of telomere brachymemma

Background of invention

The application requires the U.S. Provisional Patent Application sequence number (SN) 60/798 of application on May 9th, 2006, the U.S. Provisional Patent Application sequence number (SN) 60/862 of application on October 24th, 830 and 2006,733 right of priority, whole disclosures of described patent application all are attached to herein by reference especially.

1. invention field

The present invention relates to molecular biology and plant genetics field.More precisely, the present invention relates to the artificial minichromosomes platform of plant and production and using method.

2. the description of association area

Chromosomal keeping and hereditary common three the essential elements that need in the biology: replication orgin, kinetochore and telomere, as identifying with the yeast artificial chromosome before.For example, Murray and Szostak (1983) have described the cloning system based on the linear yeast artificial chromosome of external structure.Yet in the many cells eukaryotic system, none can be kept artificial chromosome in the nucleic acid elements that has identified.

A kind of method that produces artificial chromosome is by from the beginning making up, and is about to kinetochore, telomere and selective marker assembles and import (United States Patent (USP) the 7th, 015, No. 372) in the plant again.Yet known to by maiotic effective transmission, karyomit(e) has minimum size restriction (Schubert, 2001).Though the Mammals minichromosomes that from the beginning great majority make up can transmit the report that does not also have this class minichromosomes to transmit in kind of system during mitotic division.Up to now, the artificial chromosome that from the beginning makes up that only transmits by reduction division in the eukaryote is yeast artificial chromosome (Murray and Szostak, 1983; Murray and Szostak, 1986).In addition, studies show that for normal delivery that plant kinetochore size must surpass about 1 megabasse (Kaszas and Birchler, 1998), this size surpassed at present can assembled in vitro size.

By contrast, some have the karyomit(e) of natural centric brachymemma can be by reduction division transmission (Shinohara etc., 2000; Tomizuka etc., 1997,2000; Voet etc., 2001; Shen etc., 1997,2000; Schubert, 2001; Zheng etc., 1999; Kato etc., 2005; McClintock, 1938; Brock and Pryor, 1996; Nasuda etc., 2005).The chromosomal a kind of method of brachymemma that has been applied to mammlian system is brachymemma (telomere mediated truncation) (Farr etc., 1991,1992,1995 of telomere mediation; Barnett etc., 1993; Itzhaki etc., 1992; Heller etc., 1996; Mills etc., 1999; Saffery etc., 2001).Yet, also do not study the method that discloses the brachymemma that is used for the mediation of plant chromosome telomere up to now.

Other method of chromosome deletion has been applied to botanical system.For example, by X ray or gammairradiation Pollen Maydis (McClintock, 1938; Brock and Pryor, 1996) or by B-9 and transposition (Zheng etc., 1999 of duplicating 9S; Kato etc., 2005) change has taken place in the maize chromosome that is produced.Yet the chromosomal change that produces by these methods lacks stable delivery during reduction division or mitotic division, can't carry the site-specific recombination site, can not make heterologous sequence carry out effectively expressing, and perhaps chromosomal change is difficult to produce.Therefore, this area is starved of generation and the using method that is used for plant minichromosomes.

Summary of the invention

The present invention is by providing the plant minichromosomes that can effectively transmit through mitotic division and reduction division, thereby overcome the limitation of this area.Term " minichromosomes (minichromosome) " is meant the engineering karyomit(e) for preparing by disappearance part natural dyeing body.Therefore, minichromosomes is completely different with the artificial chromosome that from the beginning prepares by recombinant DNA technology.In specific embodiments, the invention provides plant minichromosomes as herein described, described plant minichromosomes obtains the brachymemma of initial natural phant karyomit(e) by inserting telomere repeat sequence.Therefore, plant minichromosomes of the present invention can be defined as and comprise at least one by the chromosome arm of brachymemma.In other the embodiment, two arms of plant minichromosomes are all by brachymemma at some.In some respects, plant minichromosomes can effectively transmit during plant mitotic division and reduction division, and described plant is identical with the plant variety of initial karyomit(e) (starting chromosome).Those skilled in the art will appreciate that this class minichromosomes comprises functional kinetochore and replication orgin usually, the two can be for keeping strictly according to the facts.

In one aspect of the invention, plant minichromosomes comprises natural kinetochore.Natural kinetochore is present in the endogenous chromosome, and forms (Jiang etc., 2003) by the kinetochore tumor-necrosis factor glycoproteins.These sequences are effectively separated for karyomit(e) during cell fission (for example mitotic division and reduction division).Therefore, aspect some, plant minichromosomes is defined as and comprises the natural phant centromeric sequence in the present invention.In other embodiments, minichromosomes of the present invention can be defined as and not comprise neocentromere.

Minichromosomes also can comprise engineering telomeric sequence (engineered telomere sequence).Term used herein " engineering telomeric sequence " is meant compared with the different telomeric sequence of the telomeric sequence that is present in the natural dyeing body.For example, the engineering telomeric sequence is positioned at the position that can be different from the telomere that is present in the natural dyeing body with respect to centric position.Specifically, compare with natural telomere, the engineering telomeric sequence on the minichromosomes of the present invention may be more near the kinetochore of minichromosomes.For example, in plant minichromosomes of the present invention, the engineering telomeric sequence can be defined as apart from the about 10kb in kinetochore of natural dyeing body sequence to about 10Mb.In a more particular embodiment, the engineering telomere can be apart from the about 10kb in kinetochore of natural dyeing body sequence to about 5Mb or about 100kb extremely about 1Mb, 2Mb, 3Mb, 4Mb or 5Mb.Plant minichromosomes of the present invention also can comprise two engineering telomeric sequences, and they can be different apart from the centric distance of minichromosomes.

The engineering telomeric sequence can derive from multiple source.For example, the engineering telomeric sequence can derive from plant variety or the mutation identical with natural dyeing body sequence, and perhaps deriving from respect to natural dyeing body sequence is different plant varieties.For example; telomeric sequence can be the telomeric sequence by the pAtT4 clone; described pAtT4 comprises the direct repeat or derivatives thereof (Richards and Ausubel, 1988) of Arabidopis thaliana type (Arabidopsis-type) telomere motif TTTAGGG (SEQ ID NO:1).In this case, each tumor-necrosis factor glycoproteins comprises 430 base pairs of telomeric sequence.The engineering telomere repeat sequence can derive from corn, wheat, oat, paddy rice, Arabidopis thaliana or soybean.Those skilled in the art will appreciate that the engineering telomere can comprise a plurality of telomere repeat sequences.For example, in certain embodiments, engineering telomere of the present invention can comprise between between 2 and 100, between 2 and 50 or the tumor-necrosis factor glycoproteins between 2 and 10, comprises 6 tumor-necrosis factor glycoproteinss.In some embodiments of the present invention, provide the telomeric sequence on the telomere brachymemma carrier (telomeretruncation vector), for example the brachymemma of telomere shown in this paper carrier (for example pWY76, pWY86 and pJV21).In one embodiment of the invention, can adopt telomere brachymemma carrier to produce the minichromosomes of brachymemma.Telomere brachymemma carrier can be included in the minichromosomes, perhaps can therefrom separate.In one embodiment of the invention, telomere brachymemma carrier can lack during producing minichromosomes.For example, transgenosis can be imported into then in the minichromosomes and comprise the genetically modified minichromosomes of one or more interpolations with generation.The minichromosomes that forms constitutes one embodiment of the invention by this method.

An aspect of plant minichromosomes is their size.Minichromosomes optimized encoding minimum purpose as herein described can produce the native gene of harmful effect to the plant phenotype that comprises minichromosomes.Therefore, preferred preparation has lacked most of endogenous chromosome, and keeps enough big or small minichromosomes with stable delivery simultaneously.When natural karyomit(e) was A chromosome, this point may be extremely important.Therefore the B chromosome essential function of not encoding usually unlikely produces harmful phenotype.Yet it is too little so that can not keep getting off strictly according to the facts through mitotic division and reduction division still to observe minichromosomes.Therefore, in certain embodiments of the invention, plant minichromosomes comprises the natural phant chromosome sequence, wherein about 1% to about 99.9% natural dyeing body sequence deletion.In some embodiments, in the minichromosomes about 10%, 20%, 25%, 35%, 50%, 75%, 85%, 90%, 93%, 95%, 97%, 99%, 99.5% or 99.9% or from the natural dyeing body sequence deletion of any scope that wherein can derive from.

In some others of the present invention, plant minichromosomes can limit according to its size.For example, the big I of plant minichromosomes is defined as about 0.1Mb to about 20Mb or 30Mb.In other embodiment, any scope that the plant minichromosomes size can be about 1Mb, 1.5Mb, 2Mb, 2.5Mb, 3Mb, 4Mb, 5Mb, 7.0Mb, 9Mb, 10Mb, 20Mb, 50Mb or 100Mb or wherein can derive from.For example, in some cases, plant minichromosomes size as herein described is between about 1Mb and 10Mb, perhaps between about 5Mb and 10Mb.

Aspect some, plant minichromosomes can be defined as by mitotic division and transmit with 100% frequency of the present invention.More often than not, plant chromosome limits by the frequency that it transmits during reduction division.For example, plant minichromosomes can be by transmit (50% is the ideal transmission) greater than about frequency of 10%, 20%, 25% or 30% during reduction division.Therefore, plant chromosome of the present invention can comprise the A chromosome or the B chromosome of brachymemma, and when minichromosomes transmitted in the plant identical with initial karyomit(e) kind, the frequency that the A chromosome of described brachymemma or B chromosome transmit during reduction division was greater than 35%.

Can use various plants to prepare plant minichromosomes of the present invention.In some embodiments, this plant can be defined as dicotyledons or monocotyledons.For example, plant can be clover, the friction standing grain belongs to (Tripsacum), corn, rape (canola), paddy rice, soybean, tobacco, turfgrass, oat, rye, Arabidopis thaliana or wheat.Aspect some, minichromosomes can be defined as and derive from maize plant of the present invention.

In other embodiments, plant minichromosomes of the present invention can derive from the plant A chromosome.For example, minichromosomes can comprise the natural dyeing body sequence that derives from A chromosome, and for example under the situation of corn minichromosomes, it can comprise and derives from 1,2,3,4,5,6,7,8,9 or No. 10 chromosomal native sequences of corn.In one embodiment, plant minichromosomes of the present invention derives from No. 7 karyomit(e)s of corn.

In other embodiment, plant minichromosomes of the present invention comprises the natural dyeing body sequence of plant B chromosome.Use for some, the B chromosome of telomere brachymemma can provide benefit, because they comprise dispensable gene, expects that these dispensable genes can not disturb plant phenotype or can not disturb chromosomal genetically modified expression.Telomere method for truncating as herein described causes disappearance natural dyeing body sequence from B chromosome, particularly away from centric district.Lack control unseparated B chromosome district by the telomere brachymemma, thus based on the minichromosomes of B chromosome with normal separation.In fact, can utilize the plant B chromosome of any kind to prepare plant minichromosomes according to methods described herein.For example, plant minichromosomes can comprise the natural B chromosome sequence, and these B chromosome sequences derive from corn, rye or Chinese sorghum, perhaps derives from any other plant variety with B chromosome, perhaps the self-contained any plant variety (Jones and Rees, 1982) that has imported B chromosome.In some embodiments, chromosomal brachymemma is carried out in comprising the vegetable cell of a plurality of B chromosomes, for example comprises the cell of at least 2,3,4,5,6,7,8,9,10,11 or more a plurality of B chromosome copy.

Of the present invention aspect some, what can preferably lack B chromosome contains the minichromosomes plant, and minichromosomes does not separate because the existence of complete B chromosome may cause the B chromosome deutero-.Therefore, in some embodiments, the invention provides by importing complete B chromosome or part B chromosome and handle the method for the content of the plant minichromosomes that comprises the B chromosome sequence.For example, can regulate the content of plant minichromosomes by importing the unseparated B chromosome sequence in regulation and control B kinetochore.

Plant minichromosomes of the present invention can comprise selective marker, and this selective marker provides growth vigor for the vegetable cell that carries mark under the particular growth condition, thereby can identify plant, vegetable cell or the plant part that comprises minichromosomes.Selective marker can work in bacterial cell.Minichromosomes also can comprise provides " feminine gender " of microbiotic, weedicide or other insecticide sensitivity selective marker, thereby can select the cell of the plant, vegetable cell or any other target organism that comprise specific minichromosomes.In addition, in some aspects, plant minichromosomes can comprise male fertile and recover gene, for example Rf3 gene.

Plant minichromosomes also can comprise the site-specific recombination sequence.For example, minichromosomes of the present invention can comprise lox or FRT site.Site-specific is reassembled as the gene shift-in and shifts out artificial chromosome (plant minichromosomes for example of the present invention) easy method is provided.Can also this mode provide random mutagenesis.The single-minded reorganization of practical site is well-known in the art with the method that mediated gene in the body shifts.Can the single-minded reorganization of practical site add on the minichromosomes gene is sequential, so that be provided for the gene of a complete biochemical route or a cover agronomy good character.Aspect some, the telomeric sequence that is inserted into initial plant chromosome comprises the site-specific recombination sequence of the present invention.Aspect this, can produce the plant minichromosomes that comprises the site-specific recombination sequence fast.

In some other embodiment, plant minichromosomes can comprise the gene of minichromosomes copy number in the control cell.An example of this gene is the element of the above-mentioned unseparated B chromosome of mediation.One or more structure genes also can be included in the minichromosomes.Especially expection is usefully with can to insert gene in the minichromosomes as many and keep function again simultaneously and transmit the structure gene of artificial chromosome strictly according to the facts.This can comprise 1,2,3,4,5,6,7,8,9 or more a plurality of structure gene.

In another embodiment, the invention provides the method that is used for expressing one or more foreign genes at the cell of plant, vegetable cell or any other target organism.Foreign gene can derive from any biology, comprises plant, animal and bacterium.In one embodiment, foreign gene is given and is specified the improved economical character of plant.For example, transgenosis can be given insect-resistance (insectresistance), herbicide tolerant, carbohydrate metabolism change, fatty acid metabolism change, disease resistance and nuisance resistance proterties such as (pest resistance).The present invention also comprises and can use minichromosomes to shift a plurality of foreign genes simultaneously to comprising complete biochemistry or regulating in the plant of approach.In another embodiment, the present invention includes the plant minichromosomes that can be used as dna cloning vector.This carrier can be used for plant and animal order-checking plan.Under some particular cases, transgenosis can import initial karyomit(e) with telomere repeat sequence, comprises genetically modified plant minichromosomes thereby produce.

In another embodiment, provide the vegetable cell that comprises plant minichromosomes.The kind of this vegetable cell can be identical with the kind of initial plant chromosome, yet in some cases, can be different varieties.Therefore, plant or the plant seed that comprises plant minichromosomes also constitutes integral part of the present invention.In some aspects, the plant that comprises minichromosomes is a maize plant.

Generally speaking, method and composition of the present invention comprises the karyomit(e) brachymemma (thing) of telomere mediation.For example, the carrier that comprises the plant telomere is imported in the vegetable cell.This class carrier can import in the vegetable cell by method well-known in the art, for example the conversion of DNA bombardment or edaphic bacillus (Agrobacterium) mediation.For example, in certain embodiments, can use agrobacterium-mediated conversion and produce by A chromosome deutero-plant minichromosomes.On the contrary, in some cases, can use the DNA bombardment and produce by B chromosome deutero-plant minichromosomes.After on the one or more positions that telomeric sequence are incorporated into plant chromosome, can be in integration points with the karyomit(e) brachymemma.Therefore, method of the present invention can comprise uses the heterologous nucleic acids that comprises at least two telomere repeat sequences to transform initial plant chromosome, and makes initial plant chromosome brachymemma so that produce plant minichromosomes.Can integrate chromosomal one or two arm of brachymemma by telomere.Then, can the gained plant minichromosomes be screened to determine its size and source by for example southern blotting technique method or FISH.

In other methods of the present invention, the heterologous nucleic acid sequence that initial plant dyeing body and function comprises telomere repeat sequence and additional sequences (for example site-specific recombination sequence, transgenosis or other selected sequence) transforms.Perhaps, initial plant chromosome can transform to produce plant minichromosomes with the heterologous nucleic acid sequence that comprises telomere repeat sequence.Subsequently, plant minichromosomes can transform with second heterologous nucleic acid sequence that comprises additional sequences (for example transgenosis).For example, additional sequences can comprise FRT or lox site.In some cases, additional sequences also can comprise the gene of giving insect-resistance, herbicide tolerant, carbohydrate metabolism change, fatty acid metabolism change, disease resistance, male fertile recovery or nuisance resistance.Therefore, in certain embodiments, extra gene can be that male fertile recovers gene, for example Rf3 gene.This aspect possibility particularly advantageous of the present invention can be delivered to daughter cell very efficiently because recover the plant minichromosomes of male fertile.

As for any other method described herein or composition, the embodiment that all can adopt this method of the present invention and/or Composition Aspects to discuss.Therefore, the embodiment of relevant a kind of method or composition can be applicable to other method and composition of the present invention equally.

Do not add before the term used herein and comprise its plural form when number is modified.As this paper appended claims, when " comprising " coupling, do not add before the term when number is modified and to refer to one or more with term.

The term that uses in claims " or " be used to refer to " and/or ", unless offer some clarification on only refer to the two one of, perhaps the two repels mutually, though the present disclosure support only refer to the two one of and " and/or " definition." other " used herein can refer at least two kinds or more than.

In the application's full text, term " about " is used to refer to the inherent variability that comprises instrumental error, measures method that numerical value adopts, or the deviation that exists in the research object.

Other purpose of the present invention, feature and advantage will be conspicuous from following detailed description.Yet, should be appreciated that, the detailed Description Of The Invention and the specific embodiment of the expression preferred embodiment of the invention just provide by the mode of example, because describe in detail from the present invention, various changes in the spirit and scope of the invention and modification will be conspicuous to those skilled in the art.

The accompanying drawing summary

The following drawings constitutes the part of this specification sheets, and is used to further specify some aspect of the present invention.Can understand the present invention better with reference to the one or more in these accompanying drawings and in conjunction with specific embodiments details described herein.

Fig. 1: the example of karyomit(e) brachymemma construct.Expression construct pWY76 and pWY86 and contrast construct pWY96 among the figure.The implication of letter sign is as follows among the figure: LB, T-DNA left margin; RB, the T-DNA right margin; Tvsp, the terminator of soybean nutritional storage protein gene; Bar, the bialaphos resistant gene is as selectable marker gene; TEV, tobacco plaque virus 5 ' non-translational region; P35S, the 2X35S promotor of cauliflower mosaic virus (CaMV); Tnos, the Nos terminator of Agrobacterium tumefaciens (Agrobacterium tumefaciens); Tmas, the Mas terminator of Agrobacterium tumefaciens; Pnos, the Nos promotor of Agrobacterium tumefaciens; Pmasl ', the Mas promotor of Agrobacterium tumefaciens; Lox and FRT, the site-specific recombination site; HPT, the hygromycin B resistant gene; GFP, green fluorescence protein gene; DsRed, red fluorescent protein; FLP, the site-specific recombinase gene; Telomere is by the telomere unit (Richards and Ausubel, 1988) of the isolating pAtT4 of Arabidopis thaliana (Arabidopsisthaliana).Arrow is represented the direction of transcribing, perhaps expression karyomit(e) orientation under the situation of telomere repeat sequence.

The structure of Fig. 2: pJV21.Four funtion parts also mark with box indicating.LB, left margin; RB, right margin; Lox, the lox66 site; Cre, the Cre recombinase gene; Bar, the bialaphos resistant gene; 35S, the CaMV 35S promoter; Ubi, corn ubiquitin promotor; N3 ', the terminator sequence of nopaline synthase gene; Telo, 400bp Arabidopis thaliana telomere repeat sequence; NotI and AscI, restriction enzyme sites.

Detailed Description Of The Invention

A purpose of plant breeding is to select needed proterties, for example disease resistance, insect resistace, growth rate, nutritional need and output. Regrettably, in many plant varieties, different mutation are best for geographic region and climatic province. Yet, required proterties or gene are cultivated into each mutation spend possibly time several years, because must select any specific trait, keep again the peculiar advantage of specific mutation simultaneously. The change of some proterties or the accumulation of proterties also may need a plurality of transgenosis, and this just makes and gradually oozes complicated. Therefore, giving the desirable gene of agronomy merit can be provided by different chromosome, thereby may be not and other gene linkage, and is easy to select in progeny plant. Yet, until now, also do not have the record of plant minichromosomes that can encoding gene.

The present invention can be used for mixing the limitation that required genetically modified plant minichromosomes has overcome this area by providing. In some cases, minichromosomes can gradually infiltrate in any specific mutation of plant because it not can with any background gene linkage of encoding on other chromosome. These minichromosomes also can comprise the site-specific recombination site, and this is so that the quick shift-in or shift out any botanical variety that comprises minichromosomes of gene. Therefore, the expression platform of these new plant minichromosomes by providing heredity upward to separate, thus reduced the problem relevant with chain towing (linkage drag).

I. recombination system

In certain embodiments of the invention, plant minichromosomes comprises at least one site-specific recombinase site. In several biology, identify site-specific integration enzyme recombinase system, included but not limited to Cre/lox system (Abremski etc., 1983 of bacteriophage P1; United States Patent (USP) the 4th, 959, No. 317; United States Patent (USP) the 5th, 658, No. 772), FLP/FRT system (Golic and the Lindquist of yeast, 1989), the Pin recombinase (Enomoto etc. of Escherichia coli (E.coli), 1983), the Gin/gix recombinase (Maeser etc. of bacteriophage μ, 1991), R/RS system (Onouchi etc., 1991 of Lu Shi Zygosaccharomyces (Zygosaccharomyces rouxii) pSR1 plasmid; Araki etc., 1992) and the R recombinase (Onouchi etc., 1995) of Lu Shi Zygosaccharomyces. Studies show that, all these systems all in plant, work (O ' Gorman etc., 1991; Maeser etc., 1991; Onouchi etc., 1991; Dale and Ow, 1991). It is generally acknowledged that site-directed integration system such as Cre/lox or FLP/FRT need the cyclic DNA intermediate. With regard to these systems, Cre/lox and FLP/FRT are used widely.

The FLP/FRT system is original (Golic and Lindquist, 1989 of saccharomyces cerevisiae (Saccharomyces cerevisiae); Relevant summary is referring to Futcher, and 1988). In the yeast, recombinase (FLP) is present in the 2 μ plasmids, and identification is as the inverted repeats (FRT) of 599 base-pairs (bp) of target site. Minimum function sequence units in the 599bp repetitive sequence only comprises 34bp; By 1 separated 2 13bp inverted repeats of asymmetric 8bp spacer region, although usually there is the nonessential repetitive sequence (Sauer, 1994) of the 3rd 13bp sequence. Both sides cause DNA generation inversion between the FRT target site usually in abutting connection with the DNA rearrangement of the FLP mediation of the FRT sequence of inverted repeats. In this case, two FRT sites all are retained. The FLP recombinase also can be identified the FRT target site that forward repeats. Both sides cause the DNA between the FRT target site cut in abutting connection with the DNA rearrangement of the FLP mediation in the FRT site that forward repeats usually. In this case, the DNA of cutting-out discharges with the annular form that comprises a FRT site, and the 2nd FRT site still is retained on the template DNA molecule. The FLP recombinase also can mediate the restructuring between the FRT site on the different dna moleculars, and for example, the FLP recombinase can mediate the restructuring between the FRT site on the coloured differently body. Sadowski (1995) points out, is reversible in nature by being binned in of FLP/FRT catalysis.

DNA exchange by FLP/FRT catalysis can be carried out external, because studies show that the restructuring (Meyer-Leon etc., 1984) between the pure FLP recombinase-mediated FRT site. Yeast FLP/FRT combination also has been used for instructing the site-specific restructuring, at Escherichia coli (Cox, 1983) and in fruit bat (Drosophila) genome, sequence (Golic and Lindquist, 1989 of both sides in abutting connection with the FRT site of both downcutting and increase; Golic, 1994). Adopted FLP/FRT to instruct site-specific nature cutting part transgenosis, described transgenosis derives from the corn of homologous recombination and the DNA of rice protoplast (referring to No. the 5th, 527,695, United States Patent (USP) for example). In the corn of stable conversion, utilized the site-directed cutting of FLP/FRT to insert the both sides of Maize genome in abutting connection with the sequence (U.S. Patent number 5,929,301 and 6,175,058) in FRT site. The chromosomal foci selectivity targeted bacteria of foreign DNA and mammal chromosome also can be subjected to FLP/FRT to affect (Huang etc., 1991; O ' Gorman etc., 1991), shown that in non-Yeast genome this is reversible (Huang etc., 1997) by FLP insertion FRT site. Can fully change the FRT site so that generation is recombinated but is irreversible (United States Patent (USP) the 6th, 187, No. 994), perhaps more be conducive to forward reaction (Senecoff etc., 1988) with respect to backward reaction.

Second recombination system that fully characterizes is Cre/lox (Abremski etc., 1983 of bacteriophage P1; Relevant summary is referring to Craig, and 1988; Sauer, 1994; Ow, 1996). The Cre recombinase (causing reCombination (abridging with the letter representation that underscore is arranged in English " causing restructuring ") identification lox (use by abbreviationlocys of crossing over( x) (exchange locus (x)) underlined letter represents) target sequence, the site-specific restructuring between mediation compatibility lox site. (Compatible site) can comprise or not comprise identical sequence in the compatibility site. The length in Lox site is 34 base-pairs, comprises by separated 2 the 13 bp inverted repeats of other spacer region nucleotides of 8bp. The Lox sequence comprises loxB, loxL and loxR site (Hoess etc., 1982) of the loxP (Albert etc., 1995) of bacteriophage P1 and the nucleotide sequence that separates from Escherichia coli. The function variant in the loxP site of having reported includes but not limited to lox66, lox71 and lox72 (Albert etc., 1995; Langer etc., 2002). Can also produce the Lox sequence by multiple synthetic technology known in the art. For generation of the example of the synthetic technology in functional lox site referring to (1982) such as (1981) such as Ogilvie and Ito.

The lox site is a kind of asymmetric nucleotide sequence, and therefore, the lox site on the same dna molecular may have identical or opposite direction each other. Restructuring between the lox site of equidirectional causes the DNA section between two lox sites to lack. In this case, produce connection between the initial dna molecular end of gained, the lox site is retained. The DNA section of disappearance forms the ring-shaped DNA molecule that still contains single lox site. Restructuring on the same dna molecular between rightabout lox site causes the nucleotide sequence generation inversion of DNA section between two lox sites. In addition, being close to the DNA section that is positioned at two lox sites on the different dna moleculars may occur to exchange each other. All these recombination event all are by the product catalysis of Cre code area, and are reversible. Yet, can fully change the lox site, be (Albert etc., 1995 of anti-reverse recombining reaction so that recombination event occurs; Araki etc., 1997; PCT publication No. WO 01/11058), perhaps so that two sites are recombinase " inconsistent " restructuring substrate (Hoess etc., 1986; Trinh and Morrison, 2000; Lee and Saito, 1988; EP 1 035 208). Can also by getting rid of the recombinase source, for example by breeding or the specific promoter of regulating of use, prevent back reaction.

The Cre recombinase also can be realized site-directed integration. For example, adopt the Cre recombinase that the lacZ reporter is incorporated in Chinese hamster ovary (CHO) cellular genome, a lox site is on the lacZ targeting vector, and a lox site is positioned at first cho genomic dna (Fukushige and Sauer, 1992). Show that the Cre recombinase mediates the restructuring between the lox site in following biology: yeast (Sauer, 1987) and plant, for example tobacco and arabidopsis are (referring to No. the 5th, 658,772, United States Patent (USP) for example; Medberry etc., 1995; Albert etc., 1995) and mammalian cell, for example mouse (Sauer and Henderson, 1988; Fukushige and Sauer, 1992; Sauer, 1998). Also having research to report utilizes the Cre/lox recombination system that large BAC (bacterium minichromosomes) fragment site-specific nature is incorporated into (Choi etc., 2000) in plant and the fungal gene group. It is generally acknowledged, in order to realize being incorporated in the individual gene group lox site site-directed, must will comprise the ring-shaped DNA molecule transfered cell in single lox site. Therefore, method of the present invention can make the dna molecular that lacks auxiliary sequencel realize site-directed integration, and the existence of described auxiliary sequencel is normally in order to copy in bacterial host cell and to keep ring molecule. The people (2000) such as the people such as Wallace (2000) and Day have discussed respectively practical site directional integration conduct preselected method that repeats the site of express transgenic in genome in embryonic stem cell or tobacco.

Utilize a plurality of lox site, restructuring can be initiated and realize to the Cre recombinase, and described site includes but not limited to the variant in loxP and a plurality of wild type loxP site, such as lox66 (Albert etc., 1995). The DNA exchange of being instructed by the lox site occurs in the 8bp spacer region, and the 13bp inverted repeats in two related lox sites is exchanged. For example, site-directed restructuring, the second single lox site restructuring on the single lox site on one of them dna molecular and the second dna molecular produces each sequence in abutting connection with the lox site of DNA both sides of wherein integrating. If the single lox site on the related different molecular is identical, then resulting also identical in abutting connection with two lox sites of inserting DNA. Yet if two of starting molecule single lox sites are not identical on the 13bp inverted repeats, resulting two lox sites in abutting connection with inserting DNA are different from initial lox site. For example, if the first single lox66 site participates in site-directed integration with the second single lox71 site, then resulting lox site in abutting connection with inserting the DNA both sides comprises loxP and lox72 site sequence (Albert etc., 1995).

On two recombinant molecules, adopt DNA both sides that the site-directed integration in identical lox or FRT site causes inserting in abutting connection with identical recombination site, this is a kind of reaction that easily reverses by recombinase. In order to prevent the disappearance of insetion sequence, usually need to get rid of the enzyme source of recombinase, for example, by separating or by under the control that recombinase gene is placed inducible promoter and get rid of the source of inducing. Perhaps, those skilled in the art can adopt the site-specific recombination sequence of such design, so that behind integrating remark, gained site and back reaction are incompatible or slow down and to recombinate.

Those skilled in the art will appreciate that can be by any method known in the art, and (for example lox or FRT) provides integrase, for example Cre or FLP recombinase to one or more target sites. For example, can and be deposited in the host cell expression of the suitable regulating and controlling sequence in the plasmid that keeps separately and instantaneous supply recombinase by gene. Recombinase gene and suitable regulating and controlling sequence can be inserted in the biological genome, and in host cell stably express. Perhaps, can use sexual hybridization or breeding contains the recombinase importing in the cell in one or more target lox or FRT site; In this case, contain recombinase gene biology (for example plant) can with the plant hybridization that contains lox or FRT target site, the filial generation that derives from this hybridization may contain recombinase and one or more target site. In some cases, the mRNA of the required recombinase of coding can be imported host cell with coding and recombinase protein is provided. In other cases, the recombinase protein importing that separates can be comprised in the host cell of target recombination site. In any situation of these situations, the promoter that instructs recombinase to express can be but be not limited to the mode of composing type or induction type. What those skilled in the art it is also understood that is, the gene of recombinase gene (for example Cre or FLP) can be separated from bacteriophage P1 or saccharomyces cerevisiae respectively, and be directly used in new host system, the expression of perhaps selecting for codon in the transformed host can make the gene order optimization. Those skilled in the art will appreciate that available suitable recombinase is identified naturally occurring and synthetic target site in a similar manner and mediation is recombinated.

The gene substitution of recombinase-mediated or the example of gene excision utilize adjacency with two target sites of the sequence of replaced or excision usually. For example, the people such as Odell (United States Patent (USP) the 5th, 658, No. 772) disclose and have used two loxP sites and Cre recombinase to produce the specific gene displacement in tobacco. Also use the Cre/lox system in genetically modified plants, to activate and remove transgenosis (PCT publication No. WO 01/40492) in the induction type mode. The people such as Baszczynski (United States Patent (USP) the 6th, 187, No. 994) disclose and have used a plurality of not identical FRT sites and FLP recombinase to change to produce several genes in corn. The people such as Baszczynski (United States Patent (USP) the 6th, 262, No. 341) also disclose with having two target sites specific chimeric Cre/FLP recombinases and have realized the restructuring of a side in abutting connection with lox sequence, opposite side in abutting connection with the dna sequence dna of FRT sequence. In the various situations of these situations, the integration of sequence or excision have produced the exogenous dna fragment as the part reorganization scheme. Yet site-directed integration utilizes a target site in the acceptor gene group only. The invention provides the mediated targeted integration of Cre of ring molecule nonreplicative, external generation, that easily transform, this ring molecule contains and will import the first single lox site in the second single lox site of target gene group before entering.

II. be used for gene of the present invention

Those skilled in the art will appreciate that minichromosomes of the present invention can be used for expressing several genes. In some preferred embodiments, these genes are given good proterties on the plant agronomy that comprises minichromosomes of the present invention. The below provides some to be used for non-limiting gene example of the present invention.

A. Herbicid resistant

Known multiple herbicide resistance gene, these genes can be used for the present invention. An example is the gene of giving the Herbicid resistants such as imidazolone (imidazalinone) or sulfonylureas, and described herbicide suppresses growing point or separate living tissue. The exemplary gene of this class encoding mutant type ALS and AHAS enzyme is referring to such as (1990) such as (1988), the Gleen etc. (1992) such as Lee and Miki.

Can use the resistant gene of glyphosate (respectively by saltant 5-enol pyruvic acid-3-phosphoric acid shikimic acid synthase (5-enolpyruvl-3 phosphoshikimate synthase; EPSP) and the aroA gene resistance of giving) and the resistant gene of other phosphine compound; careless ammonium phosphine (phosphinothricin acetyltransferase (phosphinothricin acetyl transferase, PAT) gene and streptomyces hygroscopicus (Streptomyces hygroscopicus) phosphinothricin acetyltransferase (bar) gene) for example. Referring to such as No. the 4th, 940,835, the people's such as Shah United States Patent (USP), it discloses the nucleotide sequence of EPSPS form that can conferring glyphosate resistance. Can obtain by ATCC preserving number 39256 dna molecular of encoding mutant type aroA genes, disclose the nucleotide sequence of this mutated genes in No. the 4th, 769,061, the United States Patent (USP) of Comai. The people's such as the people's such as Kumada European Patent Application No. 0333033 and Goodman United States Patent (USP) discloses the nucleotide sequence of the glutamine synthetase gene of conferring herbicide (for example L-phosphinothricin) resistance for the 4th, 975, No. 374. The nucleotide sequence of phosphinothricin acetyltransferase gene is referring to No. the 0242246th, the people's (1989) such as the people such as Leemans, DeGreef European application, and it has put down in writing the generation of the genetically modified plants of the chimeric bar gene activity of expressing coding phosphinothricin acetyltransferase. The exemplary gene of giving phenoxy propionic acid and cyclohexane diketone (such as Sethoxydin and haloxyfop) resistance is Acct-S1, Accl-S2 and the Acct-S3 gene that the people such as Marshall (1992) disclose.

The gene of giving the Herbicid resistant of inhibited photosynthesis also is known, for example triazine (psbA and gs+ gene) and benzonitrile (nitrilase gene). The people such as Przibilla (1991) have described the Plasmid Transformation chlamydomonas (Chlamydomonas) with encoding mutant type psbA gene. The United States Patent (USP) of Stalker discloses the nucleotide sequence of nitrilase gene for the 4th, 810, No. 648, can obtain to contain by ATCC preserving number 53435,67441 and 67442 dna molecular of these genes. The clone of glutathione S-transferase coding DNA and expression are referring to (1992) such as Hayes.

B. the recovery of male sterility and male fertile

The example of giving the restorer of male sterile gene and male fertile is known in the art, comprise the disclosed gene of following patent and restorer: United States Patent (USP) the 3rd, 861, No. 709, United States Patent (USP) the 3rd, 710, No. 511, United States Patent (USP) the 4th, 654, No. 465, United States Patent (USP) the 4th, 727, No. 219, United States Patent (USP) the 5th, 530, No. 191, United States Patent (USP) the 5th, 625, No. 132 and United States Patent (USP) the 5th, 689, No. 041, the disclosure of described patent all is attached to herein by reference especially.

Male sterility gene can increase the efficient that hybridizes with it, because they have been got rid of in specifying hybridization to the needs of castrating as maternal milpa physics.

If when needing to adopt male sterility system of the present invention and corn plant, it may be useful utilizing equally one or more male fertile restorer genes. For example, if use cytoplasmic male sterilty (CMS), 3 inbred strais of seed demand then hybridize: (1) has the cytoplasm male sterility line of CMS kytoplasm; (2) have the fertilizability inbred strais of normal kytoplasm, this is to be isogenic (" maintainer ") with CMS for karyogene; (3) the different fertilizability inbred strais with normal kytoplasm, it carries restoring gene (" recovery " is). CMS system is by breeding with the maintainer pollination, and its all filial generations all are male sterility, because the CMS kytoplasm derives from female parent. With the hybrid of restorer hybridization in, so these male sterile plants can be effectively as maternal, partly carry out physics and castrate and need not arrenotoky to female parent.

The male fertile recovery is that the existence of gene causes producing the complete F that can educate ₁The hybrid generation.If do not have the recovery gene in the male parent, then obtain the male sterile hybrid.When using maize plant nutritive issue (for example being used for silage), this class hybrid is useful, but in most of the cases, seed still is considered to the crop part of most worthy, so the essential fertility of recovering these crop hybrids.Therefore, one aspect of the present invention relates to hybrid maize plant CH389090, and this maize plant comprises the genetic loci that can recover male fertile in other male sterile plants.Can be applicable to the male infertility gene of plant of the present invention and the example of corresponding recovery system is known by the field of plant breeding technician, can be referring to for example United States Patent (USP) the 5th, 530, No. 191, United States Patent (USP) the 5th, 689, No. 041, United States Patent (USP) the 5th, 741, No. 684 and United States Patent (USP) the 5th, 684, No. 242, the disclosure of described patent all is attached to herein by reference especially.For example, recovery is that gene can be Rf3 gene (Duvick, 1965; Chase and Gabay-Laughnan 2004; Gabay-Laughnan etc., 2004) or be used for gene (Mariani etc., 1990 of Barnase/Barstar recovery system; Mariani etc., 1992).

C. disease resistance

Plant defense is interacted by specificity between corresponding nontoxic (Avr) gene product in (R) product of the disease resistence gene in the plant and the pathogenic agent usually and activates.Department of botany can transform to be transformed into the plant of anti-special pathogen strain with clone's resistant gene.Referring to (1994) such as for example Jones (clone of the tomato Cf-9 gene of yellow branch spore (Cladosporium fulvum) resistance); (1994) (Arabidopis thaliana RSP2 genes of pseudomonas syringae resistance) such as Martin etc. (1993) the tomato Pto gene of mutation (Pseudomonas syringae pv.) resistance (pseudomonas syringae cause a disease) and Mindrinos.

Virus attack albumen or also can be used for the virus disease resistance by the compound toxin of its deutero-.For example, in the transformed plant cells, virus capsid protein is accumulated and has been given the virus infection that the virus that produces this coat protein gene and correlated virus are caused and/or the resistance of disease development, referring to (1990) such as Beachy.The resistance of coat protein mediation is endowed and transforms plant with antagonism alfalfa mosaic virus, cucumber mosaic virus, annulus orae, potato virus X, marmor upsilon, tobacco plaque virus, Tobacco rattle virus and tobacco mosaic virus (TMV).

Also can use special viral antibody.Referring to (1993) such as for example Tavladoraki, they point out that the transgenic plant of expressing recombinant antibody gene are protected and avoid virus infection.

For example, people (1992) such as Logemann discloses the transgenic plant of expressing barley rrna inactivation gene has increased the mycosis resistance.

D. glutinous property cereal starch

Glutinous property feature is the example of recessive character.In this example, must grow and selfing by first generation (BC1) filial generation that obtain of backcrossing.Then the selfed seed that derives from the BC1 plant is tested and carry the recessive gene of glutinous property proterties with definite BC1 plant.In other recessive character, need carry out the check of extra filial generation, for example make extra filial generation (for example BC1S1) growth, with the recessive gene of determining that plant is carried.

E. insect-resistance

An example of insect-resistance gene comprises bacillus thuringiensis (Bacillusthuringiensis) albumen, its derivative or its synthetic pattern polypeptide.Referring to (1986) such as for example Geiser, they disclose the clone and the nucleotide sequence of Bt delta-endotoxin genes.In addition, the dna molecular of coding delta-endotoxin genes can be by for example ATCC preserving number 40098,67136,31995 and 31998, available from American type culture collection (American Type CultureCollection, Manassas, Virginia).Another example is a lectin.Referring to (1994) such as for example Van Damme, they disclose some nucleotide sequences of kafir lily (Clivia miniata) mannose binding lectin gene.Also can adopt vitamin-binding protein, for example avidin.Referring to PCT application US93/06487, the content of described application is attached to herein by reference.This application has instructed avidin and avidin homologue as the purposes at the larvacide of insect.

And another insect-resistance gene is an enzyme inhibitor, for example proteolytic enzyme (protease) or proteolytic ferment (proteinase) inhibition or amylase inhibitor.Referring to (1993) (nucleotide sequences of streptomyces nitrosporeus (Streptomyces nitrosporeus) α-Dian Fenmei inhibition) such as (1993) such as (1987) such as for example Abe (nucleotide sequence of rice cysteine protease inhibitor), Huub (the cDNA nucleotide sequence of encoding nicotiana protease inhibitor I) and Sumitani.Also can adopt insect specificity hormone or pheromone.Referring to the disclosure of people (1990) such as for example Hammock about clone's juvenile hormone esterase (a kind of passivation of neotonin) of baculovirus expression.

Other example that also has comprises insect specific antibody or its deutero-immunotoxin and arrested development albumen.Referring to (1994) such as Taylor, they point out to make enzyme deactivation by producing single chain antibody fragments in the transgene tobacco.

F. improved fatty acid metabolism, phytinic acid metabolism and carbohydrate metabolism

Can adopt and give the gene that improves fatty acid metabolism.For example, can adopt stearyl ACP delta 8 desaturase genes (referring to (1992) such as Knutzon).Various fatty acid desaturases (gene) have also been disclosed, yeast saccharomyces cerevisiae OLE1 gene for example, its coded delta 9 fatty acid desaturases, this is a kind of enzyme (McDonough etc., 1992) that is formed single unsaturated Zoomeric acid (16:1) and oleic acid (18:1) lipid acid by palmityl (16:0) coenzyme A or stearyl (18:0) coenzyme A; The gene (Fox etc. 1993) of coding castor-oil plant stearyl acyl carrier protein Δ-9 desaturase; Cyanobacteria cytoalgae (Synechocystis) is responsible for linolic acid (18:2) is changed into the Δ 6-desaturase and the Δ 12-desaturase (Reddy etc. 1993) of gamma-linolenic acid (18:3 γ); Derive from the gene (Arondel etc. 1992) of coding ω-3 desaturase of Arabidopis thaliana); Plant Δ 9-desaturase (PCT application publication number WO91/13972) and soybean and rape (Brassica) Δ 15 desaturases (European patent application published EP 0616644).

Also can transform in the plant, thereby improve the phytinic acid metabolism by importing the phytinic acid enzyme coding gene to improve the decomposition of phytinic acid, more free phosphoric acids being added to.For example, the disclosure of the nucleotide sequence of relevant aspergillus niger (Aspergillus niger) phytase gene can be referring to (1993) such as Van Hartingsveldt.For example, in corn, this can finish by clone and then the importing DNA relevant with single allele, and described single allele is that to cause with low-level phytinic acid be the reason (referring to (1990) such as Raboy) of the maize mutant type of feature.

Knownly can be used to change glycometabolic several genes.For example, the plant available code changes the gene transformation of the enzyme of starch branching pattern.Referring to (1988) such as Shiroza (nucleotide sequence of Streptococcus mutans (Streptococcus mutans) fructosyl transferase gene), Steinmetz etc. (1985) (nucleotide sequence of subtilis (Bacillus subtilis) levansucrase (levansucrase) gene), Pen etc. (1992) (expressing the generation of the transgenic plant of Bacillus licheniformis (Bacilluslicheniformis) α-Dian Fenmei), Elliot etc. (1993) (nucleotide sequence of tomato conversion enzyme gene), (1993) (maize endosperm starch branching enzyme II) such as Sergaard etc. (1993) (the site-directed mutagenesis of barley alpha-amylase gene) and Fisher.Derive from corn and the Z10 gene of the 10kD zein storage protein of encoding also can be used to change in the cell 10kD zein with respect to other components contents (Kirihara etc., 1988).

III. plant and uses thereof

The invention provides the transgenic plant that comprise plant minichromosomes of the present invention, comprising but be not limited to clover, corn, rape, paddy rice, soybean, tobacco, turfgrass, oat, rye and wheat.The seed that also comprises plant, described seed comprises minichromosomes of the present invention.

For example, in certain embodiments, comprise the gene that the seed of minichromosomes of the present invention can be expressed the conferring herbicide tolerance.A useful example of herbicide tolerant gene provides glyphosate (N-((phosphonomethyl)) glycine) resistance, comprises the isopropyl amine salt form of this class weedicide.

When enforcement is of the present invention, can be used to improve the required proterties of controlling plant diseases with acquisition with other disease control proterties combination of plant.The disease control proterties of using the distinct mode of action is combined, can be provided than having the single more consistent and more persistent protection of plant that prevents and treats proterties, reduce because the probability of resistance takes place in the field for transgenic plant.

The invention still further relates to commodity that contain one or more minichromosomes of the present invention and the commodity of producing by recombinant plant that contains one or more minichromosomes or seed.The commodity that contain one or more sequences of the present invention often include but not limited to pulverize cereal, oil product, crushing cereal, whole grain cereal or plant seed, perhaps comprise the recombinant plant that contains one or more sequences of the present invention or any any food that pulverizes cereal, oil product, crushing cereal, whole grain cereal of seed.In the included product of one or more commodity or this paper, detect one or more sequences of the present invention and be actually the evidence that commodity or product comprise minichromosomes of the present invention.

Another aspect of the invention relates to the tissue culture of the plant that comprises minichromosomes.Term used herein " tissue culture " is meant the composition of the isolated cell that comprises identical or different type or constitutes the aggregate of this class cell of plant part.The type of exemplary tissue culture is protoplastis, callus and is complete vegetable cell in plant or plant part, for example embryo, pollen, flower, kind benevolence, fringe, cob, leaf, bract (husk), handle, root, the tip of a root, flower pesticide, corn are grown palpus, or the like.In a preferred embodiment, tissue culture comprises embryo, protoplastis, meristematic cell, pollen, leaf or the flower pesticide that obtains from these plant part prematurity tissues.The preparation of plant tissue cultures and maintenance method are (described each patent all are attached to herein by reference for United States Patent (USP) the 5th, 538, No. the 5th, 550,318, No. 880 and United States Patent (USP)) well-known in the art.For instance, the tissue culture that comprises organ (for example male flower fringe or flower pesticide) has been used to produce aftergrowth (United States Patent (USP) the 5th, 445, No. the 5th, 322,789, No. 961 and United States Patent (USP); The disclosure of described patent all is attached to herein by reference).

A kind of corn tissue culture type is male flower fringe/anther culture thing.The male flower fringe contains flower pesticide, flower pesticide and then encase sporule.Microspore development becomes pollen.For flower pesticide/microspores culture thing, if the male flower fringe is the plant integral part, then preferably pick out in the stage when sporule is monokaryon, that is to say to include only 1 rather than 2 or 3 nuclears.The method of determining the correct stage is well known to those skilled in the art, and comprises Kapp mycin (mitramycin) fluorescence colour, Trypan Blue (preferably) and aceto carmine pressed disc method.Having observed mid-term in mononuclear microspore stage is the etap that the method for the disclosed subsequently final generation plant of this paper is made maximum reaction.

Generally can under about any low temperature below 25 ℃, carry out pre-treatment although contain the plant organ (for example male flower fringe) of sporule, preferred 4-25 ℃ scope, preferred 8-14 ℃ scope especially.Although other temperature also produces the embryoid and the plant that regenerates, compared with temperature pre-treatment outside the preferable range, subzero treatment produces optimum response speed (response rate).Speed of response is counted regenerated plant numerical table with the embryoid number that excites in the culture or every sporule and is shown.The illustrative methods of microspores culture is referring to No. the 5th, 445,961, No. the 5th, 322,789, United States Patent (USP) for example and United States Patent (USP), and the disclosure of described patent is attached to herein by reference especially.

In addition, in certain embodiments of the invention, initial plant or vegetable cell transform with nucleic acid (for example telomere brachymemma carrier).Method with the nucleic acid transformed plant cell is well known to those skilled in the art.For example, this method can adopt agrobacterium-mediated conversion or the dna particle bombardment that this paper enumerates.Developed the ad hoc approach that some is used for conversion of high molecular weight nucleic acid.For example, BIBAC system (Hamilton, 1997 of Hamilton and colleague's design thereof; Hamilton etc., 1996; The patent No. 5,733,744), be used for big T-DNA insertion fragment and be converted into Plant Genome.BIBAC combines the T-DNA forwarding function of Ti-plasmids and keeps the ability of the big dna fragmentation of bacterial artificial chromosome (BAC).As for BAC, it has the replication orgin of the intestinal bacteria F plasmid of single copy, is used for remaining on intestinal bacteria with plasmid is stable.It also has the replication orgin and the T-DNA border that is used for the T-DNA conversion of the edaphic bacillus Ri plasmid of single copy.A similar system, the transformed artificial chromosome (TAC) with coliphage P1 starting point also has been used for the conversion (Liu etc., 1999) of big DNA.

IV. comprise the breeding of the plant of minichromosomes

As indicated above, can use initial natural phant karyomit(e) to produce plant minichromosomes according to the present invention.In some embodiments, be used for natural dyeing body of the present invention and can be maize chromosome, for example corn B chromosome or corn A chromosome.For example, can use the initial karyomit(e) of any corn listed in the table 1 (Zea mays) karyomit(e) as preparation minichromosomes of the present invention.

The size of table 1 expression corn A chromosome, unit is that megabasse is to (Mb) or centimorgan (cM).Unit is that the big I of cM is measured by two kinds of diverse ways that people such as Sharopova (2002) introduce.

Table 1: corn A chromosome size

What it is also understood that is in some cases, and minichromosomes comprises the site-specific recombination site.Therefore, all available can the carrier that transgenosis imports minichromosomes conversion of any botanical variety that comprises minichromosomes by the site-specific reorganization.In a single day plant minichromosomes produces, and then can easily gradually be seeped in the required genetic background by plant breeding technology well-known in the art.

Therefore, the invention provides the breeding method of the plant that is used to comprise minichromosomes.According to this method, can be with first mother plant and the hybridization of second mother plant, wherein first plant and second plant comprise minichromosomes, and perhaps wherein at least a plant comprises minichromosomes.For example, the maize plant (summer corn (Zea mays L.)) that comprises minichromosomes can be hybridized by nature or mechanical skill.

Flowering plant just produces open-pollinated when wind or insect are propagated out pollen from plant.Yet, can import the type of pollen into or carry out the machinery pollination by control by artificial pollination.In one embodiment, hybridization comprises following steps:

(a) sow the seed of first mother plant and second mother plant in the approaching mode of pollinating, the seed of seed that is preferably the first inbreeding plant and the second different inbreeding plants;

(b) with the cultivating seeds of first mother plant and second mother plant or be grown to serve as flowering plant;

(c) with the hero of having spent of first mother plant or second mother plant, prevent that pollen from producing,, castrate mother plant thereby provide perhaps as maternal male sterile plants thereby promptly flower is handled;

(d) allow between first mother plant and second mother plant, natural cross pollination to take place;

(e) results are castrated the seed that mother plant produced; And if desired,

(f) seeds cultivation with results becomes plant, preferred hybrid plant.

Usually by with alternate row, sub-district or with any other mother plant of cropping pattern plantation easily, plant mother plant in the approaching mode of pollinating each other.If the sexually matured asynchronism(-nization) of mother plant then may need the ripe slower plant of plantation earlier, thereby guarantee between maternal receivability pollen period, can obtain the pollen of male parent.Two parents' of cultivation plant, and allow to grow into when blooming.Be preferably in this growth phase, according to the cultivation personnel with due regard to, plant generally handled with fertilizer and/or other agricultural chemicals.

When blooming, be male parent, then from extracing the product pollen organ of these mother plants to avoid self-pollination as all plants of pistillate parent if comprise the plant of minichromosomes.This can carry out by hand, but also can carry out with machine if needed.Perhaps, when maternal plant comprises when giving male sterile cytogene or nuclear gene, then may not need to extract the organ that produces pollen.In addition, can use chemical gametocide to make the male flower of female plant sterile.In this case, can handle without chemical agent, perhaps can comprise the gene that causes chemical agent castration effect resistance as the mother plant of staminiferous plant.Castrate the agent influence and participate in process or the cell that growth, maturation or pollen discharge.The plant of castrating the agent processing through this class is male sterile, but keeps female fertility usually.The application of chemical gametocide is referring to No. the 4th, 936,904, United States Patent (USP) for example, and its disclosure all is attached to herein by reference.In addition, PCT publication No. WO 98/44140 discloses that (Monsanto, St.Louis MO) are used, to produce the male sterile maize plant with glyphosate resistance maize plant and ROUNDUP weedicide.

After the castration, make the plant continued growth usually again, owing to natural cross pollination takes place in wind action, this is common in Gramineae (for example corn) pollination.Because pistillate parent is castrated, all pollen of paternal plant all can be used for pollination, have extractd the part of blooming that has pollen because be used as all plants of female plant before from hybridization.Certainly, during this crossover process, parent's mutation is so grown, promptly isolate, thereby the anything unexpected pollution of external source pollen is reduced to minimum, prevent that perhaps the anything unexpected of external source pollen from polluting with other field plant.These isolation technologies all have in one's pocket those skilled in the art.

Two mother plant continued growths that can make hybridization can destroy male row up to maturation after the end of perhaps blooming.The seed of only gathering in the crops pistillate parent is to obtain new F ₁Cenospecies.Then, can be in the season of growth subsequently, with the new F that is produced ₁The hybrid seed sowing is in the commercialization field, or the continuation breeding system is so that develop new inbred lines.

Perhaps, in another embodiment of the invention, one or both comprised plant minichromosomes of first mother plant and second mother plant.Therefore, any plant that comprises minichromosomes of the present invention all constitutes integral part of the present invention.Hybridization used herein can refer to selfing, backcrosses, with other mutation or same intervarieted cross, with population hybridization etc.Therefore, adopt the plant comprise minichromosomes all within the scope of the present invention as all maize plants that the parent produced.

Embodiment

Following embodiment is used to further specify all respects of the present invention.One skilled in the art will appreciate that on behalf of the present inventor, disclosed technology operate good technical and/or composition in the embodiment of this invention in following examples, therefore can be regarded as constituting the optimal way of its enforcement.Yet, those skilled in the art will appreciate that according to present disclosure, in disclosed specific embodiments, carry out multiple modification, still can obtain identical or similar results and can not depart from the spirit and scope of the present invention.

Embodiment 1

The universal method of brachymemma telomere

The chromosomal brachymemma of telomere mediation is by realizing the telomere repeat sequence of 6 copies by transforming in the plasmid importing endogenous plant karyomit(e).At this in particular cases, the Arabidopis thaliana telomere of being cloned before by Richards and Ausubel (1988) has prepared telomere repeat sequence.Corn HiII (Armstrong and Green, 1985) immature embryo has carried out transforming (Frame etc., 2002) according to agrobacterium-mediated conversion scheme with the standard binary vector.Between agrobacterium-mediated transition phase, T-DNA protects with edaphic bacillus VirD2 and VirE albumen, and is incorporated in the Plant Genome by unusual recombination mechanism.A small amount of disappearance may take place, and great majority occur in battery limit (BL), the left side, occur in battery limit (BL), the right sometimes.Yet these disappearances can not destroyed telomeric sequence.The existence of telomere repeat sequence makes when integrating the karyomit(e) brachymemma.Confirm the generation of the relevant karyomit(e) brachymemma of telomere, and in mammalian cell, be used to produce minichromosomes (Farr etc., 1991,1992,1995; Barnett etc., 1993; Itzhaki etc., 1992; Heller etc., 1996; Mills etc., 1999; Saffery etc., 2001).Adopt the karyomit(e) brachymemma construct of two telomere mediations, confirmed that the telomere of the Arabidopis thaliana in the same way unit of 6 copies can make the karyomit(e) brachymemma in corn.

The double base construct that has made up two kinds of karyomit(e) brachymemmas is used for agrobacterium-mediated gene transformation (pWY76 and the pWY86 of corn HiII immature embryo; Referring to Fig. 1).For pWY76 and pWY86, make 93 and 83 transgenic lines recoveries respectively.Use the transgenosis that fluorescence in situ hybridization (FISH) method (Kato etc., 2004) has detected T0 transgenic plant Metaphase Chromosomes.Observe totally 123 transgenosis integration sites for pWY76, observe 107 sites, have 54 and 55 integration sites to be positioned at end of chromosome respectively for pWY86.Taken place therein also to observe pollen abortion in the transgenic lines of karyomit(e) brachymemma.In addition, by the karyotyping probe mixture, in some transgenic lines, detect the disappearance of far-end FISH mark.For example, in a pWY76 transgenic lines, No. 3 the short arm of a chromosome are by brachymemma.Southern blotting technique method analysis revealed hybridization histogram is unintelligible, and this is the characteristic feature (Farr etc., 1991) that heterologous gene newly is linked into telomere.These studies show that, the karyomit(e) brachymemma has taken place in these transgenic lines.In order to compare, also simultaneously the construct of other two kinds of no telomere tumor-necrosis factor glycoproteinss is transformed with pWY76 and pWY86 transgenosis; Yet, do not observe chromosomal brachymemma.These results show that telomere repeat sequence is the reason of karyomit(e) brachymemma.

By the FISH screening, in a pWY86 system, observe A chromosome deutero-minichromosomes.Find that this minichromosomes originates from karyomit(e) No. 7, its long-armed major part disappearance.Be in the tetraploid system of no B chromosome, to observe this minichromosomes at first.Because it occurs in the polyploid, the defective that is caused by chromosomal brachymemma is compensated by the gametophytic homologue of its diploid.In fact, in the diploid of the chromosome deficiency system, do not observe viewed 50% pollen abortion usually therein, after hybridizing with the transformant of recovering by diplont, the filial generation that contains this minichromosomes is restored, and this makes ploidy level be reduced to triploid.Ploidy level be further reduced to diploid and add HiII and the Mo17 background under minichromosomes.Studies show that further this minichromosomes can not cause any unusual phenotype.When hybridizing with diplont, this minichromosomes is with 38.5% (10/26) ratio transmission as staminiferous plant.The nucleic acid construct that is used for the brachymemma of plant telomere

Adopt binary vector pTF101.1 to make up two brachymemma constructs and no telomere contrast (Paz etc., 2004), be used to produce minichromosomes (referring to Fig. 1 construct collection of illustrative plates).Component on these constructs is arranged as follows in order: pWY76:LB-Tvsp-Bar-2XP35S-lox66-GFP-Tnos-Pmas-HPT-Tmas-6x telomere-RB, pWY86:LB-Tvsp-Bar-2XP35S-lox71-DsRed-Tnos-Pnos-FRT-FLP-T nos-6x telomere-RB and pWY96:LB-Tvsp-Bar-2XP35S-lox71-DsRed-Tnos-Pnos-FRT-FLP-T nos-Pmas-HPT-Tmas-RB.

Comprise promoterless lox66-GFP or lox71-DsRed fusion gene among pWY76 and the pWY86 respectively, allow other gene specificity to be incorporated on the lox site.In addition, the Pnos-FRT-FLP-Tnos box that comprises on the pWY86 allows to carry out the site-specific reorganization to carry out further gene integration on the FRT site.

Embodiment 2

The telomere brachymemma of B chromosome

For the chromosomal brachymemma by the telomere mediation produces little B chromosome, (Black Mexican Sweet, the B chromosome of maize cell BMS) import corn HiII parent A system, and make it to put aside to multiple copied will to derive from the black sweet corn of Mexico.Transform (biolistic transformation) (Frame etc. by particle gun, 2000), relevant chromosomal brachymemma construct of two telomeres and pACH25 construct (Christensen and Quail, 1996) are mixed HiII hybrid immature embryo or the HiII parent A immature embryo that has a plurality of B chromosomes with target.Can select transgenic event in the substratum that has replenished bialaphos effectively by the bar gene of the corn ubiquitin promoters driven of pACH25 construct.In a single day the telomeric sequence of B chromosome and A chromosome construct truncate is incorporated on the karyomit(e), just can produce minichromosomes from the B chromosome and the A chromosome of brachymemma.By FISH, filter out minichromosomes in the tip of a root Metaphase Chromosome by regenerated herbicide resistant plants (T0) acquisition.Adopt pACH25 plasmid and pWY96 construct (referring to the embodiment 1) probe as the FISH screening, this has the sequence (vide infra) of nearly all pWY76 and pWY86 except that telomeric sequence.The pWY96 probe can effectively be hybridized with pWY76 and pWY86 transgenosis.Use the B repetitive probe to identify the brachymemma (Alfenito and Birchler, 1993) of B chromosome.48 minichromosomes have been identified; Wherein 41 from B chromosome, and 7 from A chromosome.All minichromosomes that derive from B chromosome are transferable, its great majority at T1 for recovery.Carry out A chromosome and double to handle (Kato and Birchler, 2006) to preserve minichromosomes on the A of 3 brachymemmas minichromosomes, be kept at diplontic words else if, they will be lost in reduction division.

Embodiment 3

Express the Cre gene with the BIBAC carrier system by agrobacterium-mediated gene transformation

Comprise the Herbicid resistant marker gene for having, transform, adopted BIBAC as the corn of a large amount of genetic material of the 30kb pastoris genomic dna of fluorescence in situ hybridization (FISH) mark and 35S-lox-Cre recombinant expression cassettes ^TMCarrier system.Say the following BIBAC gene transformation plasmid pJV21 that made up simply.PCH20 carrier (Hamilton, 1997) has been carried out improving so that BamHI to SwaI fragment deletion.By on the SwaI site, adding the BamHI joint, with BamHI digestion and connection again, rebuild the BamHI site subsequently.The gained plasmid is called pJV06.Connect by flush end, the 3.4kb HindIII/SalI fragment cloning that contains the 35S-loxP-Cre expression casette that derives from pED97 is prepared pJV08 to the BamHI site.Then, the 400bp telomeric sequence and the ubi-bar expression casette that will derive from pAHC25 (Christensen etc., 1992) is assembled to pBLUESCRIPT ^TM(Strategene, LaJolla, California) in, then by flush end connect be cloned into pJV08 the SrfI site with preparation pJV15, wherein telomeric sequence is placed in the left margin of binary vector.For these research, the ubi-bar gene is used as selective marker.Telomeric sequence TTTAGGG (SEQ ID NO:1) points to left margin.By the flush end connection another 400bp telomeric sequence is cloned into the direct repeat of PacI site with the 800bp telomeric sequence on the preparation LB.The clone has been carried out order-checking to confirm the direction of telomere repeat sequence.Then, will derive from BIBAC1 (Hamilton, 1997; Hamilton etc., 1996) 30kb cerevisiae dna is cloned into the NotI site with preparation pJV21 (Fig. 2).(Carlsbad California), carries out electroporation to competent cell, and construct is transferred among the edaphic bacillus bacterial strain LBA4404 for Gibco, Invitrogen Co. according to manufacturer's specification sheets.In this construct, at the clone 35S-lox66-Cre of contiguous right margin place expression cassette, so that the Cre/lox site specific recombination system is inserted genome by gene transformation.

Genetic transformation

Agrobacterium-mediated gene transformation carries out having with conversion the immature embryo of the HiII plant of 0-12 B chromosome with the pJV21 construct.75 transform system and are restored.Transgenosis is hybridized by DNA and FISH is confirmed (Kato etc., 2004).In 60 systems that with DNA hybridization are feature, 45 cordings have single copy transgenosis.63 in 75 transgenic lines have single transgenosis, are confirmed by FISH.Use bar gene and Cre gene probe respectively,, the transgenosis integrity is analyzed by hybridizing at the left end in T-DNA district and the southern blotting technique of right-hand member.In 75 systems that analyzed, to observe 52 transgenic lines on the lefts circle and have identical copy number with right margin, this has just shown that transgenosis is complete in these cases.

Transgenosis is incorporated into the telomere district

Compare with having the transgenic event that separates band, the DNA hybridization histogram of a transgenic event J11-9 is unintelligible.Unsharp hybridization histogram is by natural telomere activity or inserts the telomere characteristics that new telomere produced during the chromosomal brachymemma of telomere mediation, because Telomere terminal transferase has added the telomere motif TTTAGGG of different numbers in different cells; (SEQ ID NO:1) (Farr etc., 1991; Richards and Ausubel, 1988).In order to prove this genetically modified terminal character, use cutting just in time a plurality of restriction enzymes of the transgenosis distal area before the 800bp telomere repeat sequence digests the J11-9 genomic dna, and (Yu etc. 2006b) use by method ³²The bar gene probe of P mark carries out DNA hybridization.Displacement has taken place according to the distance between 3 restriction enzyme cutting sites (XbaI, XhoI and DraI) and the fringes of chromosome end in unintelligible hybridization histogram.For example, the enzyme DraI formation scope of most proximal end surpasses the maximum sized unintelligible histogram of 6kb, and a unintelligible histogram that XbaI formation scope is 3.2-4.6kb of distal-most end, the unintelligible figure that XhoI forms is between 5-6kb.By contrast, the cutting on the HindIII site of nearer end has formed the separation band.If the HindIII site then can form the separation band at bar gene probe far-end.This HindIII site is present in the natural telomere probably, because do not observe this site at our sequence data of pJV21 construct.Therefore, genetically modified terminal position may be the result of integration incident in the telomere rather than the result that from the beginning telomere forms among the J11-9.In addition, in the J11-9 plant, do not observe growth failure, gametophyte is sterile or transmit and to weaken, this does not just show not losing of the function genetic material that caused by this transgenosis, this phenomenon usually in having the transgenic plant of chromosomal brachymemma, observe (Yu etc., 2006b).

Also shown this genetically modified terminal location by cytological analysis.Make and hybridize that this probe mixes (Kato etc., 2004) mutually with the karyotyping mixture from the karyomit(e) of the J11-9 tip of a root and the 30kb dna probe of fluorescein-dUTP mark.With this mixture produce the HiII transformation receptor caryogram (Yu etc., 2006a).Identify the end of this transgenosis at the 3L chromosome arm.

Genetically modified position and distribution

In order to make transgenosis position and distribution profile, expand fish analysis to all transgenic lines.By FISH, in each karyotype (table 2), observe transgenosis, and integration site is present in or near telomere, kinetochore, euchromatic region, heterochromatin knob (knob) and NOR district.Except that 8S and 10S, all chromosome arms are all included.Transgenosis is randomly dispersed on 10 A chromosomes, and wherein the number on the macrochromosome is more, and the number on the microchromosome is less, as expection.Yet, on any B chromosome, all do not observe transgenosis, there are nearly 12 B chromosomes (" B ") (average 3.9) in the plant although transform.

Table 2: transgenic event with by the corn pJV21 construct of agrobacterium-mediated conversion

The Cre/lox site specific recombination system

With the conversion of BIBAC construct 84 transgenosiss are inserted the corn gene group at random.These transgenosiss are complete mostly, shown in DNA hybridization.Detect 91 transgenosis sites (table 2) by DNA hybridization 75 transgenic lines with the Cre gene probe.In order to confirm genetically modified expression of these Cre and function, report construct pHK52 (Srivastava etc., 1999) is delivered in the immature embryo of transgenic plant filial generation through particle gun.The pHK52 construct contains β-glucuronidase (GUS) gene of the antisense orientation that is under the corn ubiquitin promotor.Antisense gus gene both sides are in abutting connection with the inverted repeats in loxP site.The antisense gus gene is reversible to be transferred to the justice orientation, and expresses in the presence of the Cre recombinase, and these enzyme catalysis both sides are in abutting connection with the inversion of the genetic elements of lox site inverted repeats.Bombardment embryo to all transgenic lines has carried out instantaneous GUS mensuration.This studies confirm that the Cre gene has function (table 2) in 68 systems.For J11-9 and HiII is outbreeding, isolates genetically modified embryo colony.In 30 of 50 embryos altogether (ratio is about 1:1), its GUS expresses and is activated.

Embodiment 4

The telomere brachymemma that B chromosome transforms by particle bombardment

Because B chromosome has and makes its many character that become preferred minichromosomes, therefore carried out further research and can effectively form by B chromosome deutero-plant minichromosomes determining whether.In these researchs, telomere brachymemma construct pWY76 and pWY86 (Fig. 1) have been adopted.In addition, by preparing plasmid pWY86-bar with disappearance 35S-bar expression casette with PmlI/AvrII digestion Y86 and self connection.Christensen and Quail (1996) have just described the plasmid pAHC25 that is used to study before.

Say simply, in the super Jing Tai of laminar flow hood (flow hood), under aseptic condition, dissect the immature embryo between the 1.2-2.0mm.According to described methods of people (2000) such as people such as Songstad (1996) and Frame, carried out the gene transformation of the particle gun mediation of corn immature embryo.With the immature embryo placed face down at the callus inducing medium that contains N6 salt and VITAMIN (pH 5.8) (Chu etc., 1975) in, culture medium supplemented 2.0mg/L 2,4-D, 100.0mg/L inositol, 2.8g/L proline(Pro), 30.0g/L sucrose, 100.0mg/L casein hydrolysate, 3.0g/LGELRITE, 25 μ M Silver Nitrates.Embryo was induced under 28 ℃ 2-4 days, transfer to then in the infiltration substratum (pH 5.8) that contains N6 salt and VITAMIN, culture medium supplemented 2.0mg/L2,4-D, 100.0mg/L inositol, 0.7g/L proline(Pro), 30.0g/L sucrose, 100.0mg/L casein hydrolysate, 36.4g/L sorbyl alcohol, 36.4g/L N.F,USP MANNITOL, 3.0g/L GELRITE, 25 μ M Silver Nitrates carry out 4 hours osmotic treated before bombardment.

Plasmid prepares box (miniprep kit) (Qiagen, Valencia, California) preparation in a small amount with Qiagen.(BIO-RAD, Hercules California) coat the mixture of 1 μ g telomere brachymemma plasmid (pWY76, pWY86 or pWY86-bar) and 0.25 μ gpAHC25 with 3 milligrams 0.6 μ gold particle.Use following parameter, (BIO-RAD) bombards with the PDS1000/He particle gun: 650psi rupture disk (rupture disk) pressure, 6cm range, 6mm slit (gap), 1.2cm from larger vector (macro-carrier) to barrier plate (stoppingplate), 28Torr vacuum when breaking.

Wrap with PARAFILM through the embryo of bombardment in the infiltration substratum, after 28 ℃ cultivation was spent the night down, be transferred in the callus inducing medium next day.

The fish analysis of transgenic plant minichromosomes

By above-mentioned FISH, filter out the minichromosomes of T0 transgenic plant.The probe of pWY96 and pAHC25, is hybridized with transgenosis with texas Red (Texas red) dCTP mark (Kato etc., 2004) by nick translation.B tumor-necrosis factor glycoproteins (Alfenito and Birchler 1993) is with Alexa Fluor-488 dCTP mark, and mixes the B chromosome that the conversion that mediates by particle gun with screening transforms respectively with pWY96 or pAHC25 transgenosis probe.CentC (Ananiev etc., 1998) and NOR (Stein etc., 1998) probe Alexa Fluor-488 dCTP (Invitrogen) mark; CRM (Zhong etc., 2000) uses the Cy5-dCTP mark.The size that minichromosomes is compared with normal A type karyomit(e) or B-type chromosome according to them is identified.Carried out the FISH of meiotic cell according to described methods of people (2006) such as people such as Gao (1999) and Yu.B tumor-necrosis factor glycoproteins texas Red dCTP mark, knob sequence (14) is used the AlexaFluor-488dCTP mark.

281 transgenic events (67 have pWY76, and 87 have pWY86, and 127 have pWY86-bar) of from the bialaphos resistant calli, regenerating.FISH shows that 41 incidents have transgenosis on B chromosome, and it comprises that 24 have the genetically modified minichromosomes of far-end and 20 and have inner genetically modified complete B chromosome.In addition, identify 5 A-B and 1 B-A transposition and 7 fragments that derive from A chromosome.At last, also identify the B chromosome derivative of the brachymemma of 10 no transgenosis signals.Be retained on the ace when the chromosomal brachymemma of telomere mediation appears at such positioning instant transgenosis, and when during mitotic division, losing, these incidents take place probably.If these minichromosomes do not obtain the extra genetically modified support of same incident, just then they may be lost during the selection of conversion process.Therefore, these minichromosomes quantitatively are less than and have genetically modified minichromosomes.

The measurement of minichromosomes size

Size for the B kinetochore tumor-necrosis factor glycoproteins of measuring 86B23, the B tumor-necrosis factor glycoproteins is marked as 10 Metaphase Chromosome image input Fujifilm Image Gauge V3.3 program (Fuji of green green channel (green channel), Tokyo, Japan).Centric amount of minichromosomes and the normal centric amount of B chromosome are measured, calculated each cell minichromosomes kinetochore and the normal centric ratio of B.Calculate average ratio and standard deviation.In order to measure the karyomit(e) size of 86B23, pachytene chromosome with 4 ', 6-diamidino-2-phenylindone (DAPI) dyeing, with Zeiss fluorescent microscope (Carl Zeiss, Inc., Oberkochen, Germany) photographic images, and input Fujifilm Image Gauge program (Fuji).Measure the length of minichromosomes and normal B chromosome, calculated their ratio.Other minichromosomes and the normal B chromosome of same cell are compared, according to the size of its outward appearance from other minichromosomes of estimation directly perceived.The big or small comparative result of the B chromosome of some brachymemmas sees Table 3.

The transmission test of minichromosomes

The result of these researchs shows, the transmission of minichromosomes have nothing in common with each other (table 3) during the reduction division.The transmission of unit price R2 (34.8% from self-pollination) can be suitable with the isochromosome 8S8S (37.5%) (Yu etc., 2006) that reports before.The Type B minichromosomes does not wait (table 3) from 12% (86-74) to 39% (76-15a) by the transmission of male parent.The transmission of minichromosomes is subjected to influence of various factors, for example karyomit(e) size limit value (Schubert, 2001; Spence etc., 2006).Known B kinetochore size and structure can influence the transmission (Kaszas and Birchler 1998) of B chromosome derivative in addition.Most of B chromosome deutero-minichromosomes do not separate in the presence of normal B chromosome.Long-armed distal area of B chromosome and kinetochore are to cause the unseparated reason of B chromosome (Carlson 1978).For example, in the presence of normal B chromosome, male parent passes to filial generation (table 3) with 2 copy minichromosomes.This character of Type B minichromosomes provides mechanism, to produce a series of minichromosomes that target gene is expressed that are used to increase.

Table 3:B minichromosomes size and transmission ratio

Incident	76-15a	86-74	86B23	86B155	R2
Incident	76-15a	86-74	86B23	86B155	R2	The karyomit(e) size	1/2B	1/5B	1/20B	3/4B	NM
The transgenosis location	Far-end	Far-end	Far-end	Far-end	Far-end	The karyomit(e) size	1/2B	1/5B	1/20B	3/4B	NM
The transgenosis location	Far-end	Far-end	Far-end	Far-end	Far-end	Hybridization	As male parent	As male parent	As male parent	Selfing	Selfing
Transmit	14/36	3/25	7/29	9/18	15/46	Hybridization	As male parent	As male parent	As male parent	Selfing	Selfing
Transmit	14/36	3/25	7/29	9/18	15/46	Transmit ^*	30/36	6/25	8/29	16/18	16/46

NM represents not measure. ^*Number with filial generation number/filial generation of testing of minichromosomes. ^*Minichromosomes transmits the subalgebra of number/tested.

The GUS of the histological chemistry assay method of the transgenosis individuality of B chromosome and derivative

As super several B chromosomes of other biology, the B chromosome of corn also is an inert, without any the active gene that records out (Jones, 1995).Yet, it be unclear that B chromosome shortage gene activity and cause by lacking gene, still suppress to cause by the genetic transcription due to its heterochromatin character because of B chromosome.The conversion of corn B chromosome is used to determine the inhibition that whether has transgene expression.In this research, write down 17 and on B chromosome, only had genetically modified transgenic event.This just shows that they may be subjected to support from the transgenosis bar genetic expression of B chromosome, although might on A chromosome, there be transgenosis, because of too little so that can not detect by the FISH method.Analyze by the antagonism callus, having measured in these incidents 9 has gus gene to express.Other incident lack GUS express may be in conversion process gus gene reticent or lack complete gus gene expression cassette and cause.Yet at least 6 incidents, it is (table 4) that is caused by the transgenosis on B chromosome or the derivative that gus gene is expressed.In these cases, the GUS that has measured the main root of being grown by isolating filial generation expresses.For example, in the filial generation of 76-15a, 76-15b and 86-74 and the outbreeding of acceptor strain plant, have genetically modified all filial generations (being respectively 14,11 and 3 strain individualities) and all express GUS on the derivative of B chromosome or brachymemma, none expresses GUS but do not have genetically modified filial generation (being respectively 22,19 and 22 strain individualities) at B chromosome or derivative.In the 86B23 of the self-pollination with minimum minichromosomes filial generation, having on minichromosomes has 23 strains to express GUS in the genetically modified 25 strain individualities, and none shows that GUS expresses in the 26 strain individualities of minichromosomes but do not have.Lack on B chromosome and the derivative and do not have the GUS expression in the genetically modified filial generation, confirmed that GUS expresses from the transgenosis on B chromosome or the derivative, does not have not detected A chromosome incident.Therefore, the transgenosis of inserting B chromosome is expressed on the site of being tested at least.These sites seem along the B chromosome kinetochore up to the long-armed terminal stochastic distribution of B chromosome.

Transgene expression in the table 4:B minichromosomes

Incident	76-15a	76-15b	86-74	86B23	86B155	76-10	86-14
Incident	76-15a	76-15b	86-74	86B23	86B155	76-10	86-14	The karyomit(e) size	1/2B	1B	1/5B	1/20B	3/4B	1B	1B
The transgenosis location	Far-end	Inner	Far-end	Far-end	Far-end	Far-end	The kinetochore	The karyomit(e) size	1/2B	1B	1/5B	1/20B	3/4B	1B	1B
The transgenosis location	Far-end	Inner	Far-end	Far-end	Far-end	Far-end	The kinetochore	GUS+	14	11	3	23	7	2	16
Add up to	14	11	3	25	9	12	23	GUS+	14	11	3	23	7	2	16
Add up to	14	11	3	25	9	12	23	％	100.0	100.0	100.0	92.0	77.8	16.7	69.6

According to (1987) such as Jefferson, (Sigma, St.Louis MO) have carried out histological chemistry's mensuration that gus gene is expressed by the GUS staining kit.The root of cutting of callus or long 2-5mm is directly put into the GUS dyeing solution that 50 μ l are contained in 96 hole PCR plates.Plate is wrapped with PARAFILM, cultivated 1 hour down at 37 ℃.

Reorganization between J11-9 and the R2

For the purposes of minichromosomes as the artificial chromosome platform is described, measured the Cre catalytic exchange in lox site on the R2 minichromosomes.The transfer-gen plant J11-9 that will have far-end transgenosis 35S-lox66-Cre expression cassette is hybridized with R2 as maternal, and R2 contains promoterless lox71-DsRed (Clontech, Mountain View, California) gene.Lox66 and lox71 are the lox recombination sites of sudden change, recombinate by the aforesaid mode that is beneficial to forward reaction most in this site.Two genetically modified successfully reorganization have caused the exchange of two transgenosis distal areas, and the genetic material of J11-9 is added on the R2 minichromosomes.Can monitor reorganization by reversible reaction, this reaction will not have promotor DsRed gene and places 35S promoter control down, thereby produce discernible red fluorescent protein.The fluorescent and stereo microscope that employing has the texas Red spectral filter screens recombination event in the main root of germination seedling.According to fluorometric analysis, identify 4 strain plant the 44 strain F1 filial generations altogether from heterozygosis J11-9 and heterozygosis R2 hybridization.About 8.7% filial generation (about 4 strain plant) should have the two kinds of transgenosiss (table 4) based on R2 and J11-9 transmission.

In order to determine whether red fluorescence is the result of site-specific reorganization, from each strain of the 4 strain plant of expressing red fluorescence, isolate genomic dna, and be used for the recombinant products of two expections of pcr amplification as template.Amplified production is checked order, and the reorganization under the various situations on the lox site is confirmed.The configuration and the expectancy model in order-checking district are complementary.Therefore, confirmed that this minichromosomes is suitable for being used for genetic manipulation by site specific recombination system, and can be as the prototype of minichromosomes type artificial chromosome (AC).

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Sequence table

＜110〉Univ Missouri (THE CURATORS OF THE UNIVERSITY OF MISSOURI)

BIRCHLER，JAMES

VEGA，JUAN?M.

YU，WEICHANG

＜120〉the artificial chromosome platforms of plant by the telomere brachymemma

<130>UVMO：042US

＜140〉the unknown

<141>2007-05-09

<150>60/862,733

<151>2006-10-24

<150>60/798,830

<151>2006-05-09

<160>1

<170>PatentIn?version?3.3

<210>1

<211>7

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaiiana)

<400>1

Claims

1. plant minichromosomes that one or two arm produced by the initial plant chromosome of brachymemma, wherein said minichromosomes comprises the natural phant kinetochore, and stable delivery in the mitotic division of the plant that comprises minichromosomes and reduction division, described plant is identical with the plant variety that therefrom obtains initial plant chromosome.

2. the plant minichromosomes of claim 1, wherein about 25% to about 99.9% initial plant chromosome is by brachymemma.

3. the plant minichromosomes of claim 1, wherein said minichromosomes comprises the engineering telomeric sequence.

4. the plant minichromosomes of claim 3, wherein said engineering telomeric sequence comprises about 2-100 telomere repeat sequence.

5. the plant minichromosomes of claim 3, wherein said engineering telomeric sequence comprises about 6 telomere repeat sequences.

6. the plant minichromosomes of claim 3, wherein said engineering telomeric sequence derives from Arabidopis thaliana.

7. the plant minichromosomes of claim 1, two arms of wherein said initial plant chromosome are all by brachymemma.

8. the plant minichromosomes of claim 1, the size of wherein said minichromosomes is about 1Mb about 100Mb extremely.

9. the plant minichromosomes of claim 1, wherein said initial plant chromosome is an A chromosome.

10. the plant minichromosomes of claim 1, wherein said initial plant chromosome is a B chromosome.

11. the plant minichromosomes of claim 1, wherein said initial plant chromosome derives from dicotyledons.

12. the plant minichromosomes of claim 11, wherein said initial plant chromosome derives from monocotyledons.

13. the plant minichromosomes of claim 12, wherein said initial plant chromosome derives from corn.

14. the plant minichromosomes of claim 1, it also comprises the site-specific recombination site.

15. the plant minichromosomes of claim 15, wherein said site-specific recombination site are FRT or lox site.

16. the plant minichromosomes of claim 1, it also comprises transgenosis.

Be selected from following proterties 17. the plant minichromosomes of claim 16, wherein said transgenosis are given: insect-resistance, herbicide tolerant, carbohydrate metabolism change, fatty acid metabolism change, disease resistance, male fertile recover and the nuisance resistance.

18. a vegetable cell, described vegetable cell comprises the plant minichromosomes of claim 1.

19. a kind of plant, described plant comprises the plant minichromosomes of claim 1.

20. the plant of claim 18, wherein said plant is a corn.

21. a seed, described seed comprises the plant minichromosomes of claim 1.

22. a method that produces plant minichromosomes, this method may further comprise the steps:

(a) transform initial plant chromosome with the heterologous nucleic acids that comprises at least two telomere repeat sequences; With

(b) make initial plant chromosome generation brachymemma to produce plant minichromosomes.

23. also comprising with the site-specific recombination site, the method for claim 22, this method transform initial plant chromosome.

24. the method for claim 22, wherein said site-specific recombination site are FRT or lox site.

25. the method for claim 22, wherein said telomere repeat sequence derives from Arabidopis thaliana.

26. the method for claim 22, wherein said heterologous nucleic acids comprise about 6 telomere repeat sequences.

27. the method for claim 22, wherein said initial plant chromosome transforms by agrobacterium-mediated conversion.

28. the method for claim 22, wherein said initial plant chromosome transforms by the DNA bombardment.

29. the method for claim 22, two arms of wherein said initial plant chromosome are all by brachymemma.

30. the method for claim 22, wherein said initial plant chromosome is an A chromosome.

31. the method for claim 22, wherein said initial plant chromosome is a B chromosome.

32. also comprising with selected encoding sequence, the method for claim 22, this method transform initial plant chromosome.

33. the method for claim 22, wherein said selected encoding sequence are given and be selected from following proterties: insect-resistance, herbicide tolerant, carbohydrate metabolism change, fatty acid metabolism change, disease resistance, male fertile recover and the nuisance resistance.

34. the method for claim 22, wherein said initial plant chromosome is included in the vegetable cell.

35. the method for claim 34, wherein said vegetable cell comprise at least 2 B chromosome copies.

36. the vegetable cell of claim 18, wherein said plant minichromosomes comprises transgenosis.

37. the method for claim 22, wherein said initial plant A chromosome transforms by agrobacterium-mediated conversion.

38. the method for claim 22, wherein said initial plant B chromosome transforms by micropellet bombardment.

39. the plant minichromosomes of claim 17, the wherein said encoding sequence of giving the male fertile recovery is the Rf3 gene.

40. the method for claim 33, the wherein said encoding sequence of giving the male fertile recovery is the Rf3 gene.