CN102066566A

CN102066566A - Bollworm insect resistance management in transgenic plants

Info

Publication number: CN102066566A
Application number: CN2009801221653A
Authority: CN
Inventors: C·S·赫尔南德茨; A·范弗里埃特; J·范里; J·F·曼扎纳罗
Original assignee: Bayer CropScience NV
Current assignee: Bayer CropScience NV
Priority date: 2008-06-13
Filing date: 2009-04-16
Publication date: 2011-05-18
Also published as: BRPI0914615A2; CA2727637A1; EP2300618A1; WO2009149787A1

Abstract

This invention relates to the use of a combination of different proteins insecticidal to Helicoverpa zeaor Helicoverpa armigeran an insect resistance management process, wherein such proteins are: a) a Cry2A protein such as Cry2Aa, Cry2Ab, or Cry2Ae and b) a Cry1A, Cry1F or VIP3A protein, particularly wherein such proteins binds saturably to the insect midgut membrane of Helicoverpa zeaor Helicoverpa armigera, as well as plants and seeds expressing such combination of proteins, which are used to delay or prevent the development of resistance in populations of such insect species.

Description

Bollworm insect-resistant management in the transgenic plant

Invention field

The present invention relates to the control in plant pest management field, particularly insect.The present invention relates in the insect-resistant management plan, use transgenic plant cells and plant, the genome of wherein said cell and plant (or more typically, forerunner's (predecessor) vegetable cell or plant) be provided at least two genes, the different proteins that each genes encoding has pesticidal to the real noctuid (Helicoverpa zea) of paddy or boll fruit armyworm (Helicoverpa armigera), wherein these protein saturable ground are in conjunction with the brush border membrane of this type of insect species, described protein is: a) Cry2A albumen and b) Cry1A, Cry1F or VIP3A albumen, for example VIP3A, Cry1Ac, Cry1Ab or Cry1A.105 albumen.In one embodiment, this type of plant is used to delay or prevents that the resistance to farm crop from appearring in bollworm (cotton bollworm) colony.

In addition, the invention provides simultaneously or use Cry2A albumen and VIP3A, Cry1A or Cry1F albumen in succession or express this type of Cry2A albumen and VIP3A, Cry1A or the proteic plant of Cry1F, delaying or to prevent bollworm, particularly real noctuid of paddy or boll fruit armyworm, resistance occur.

This type of plant transformed has advantage than with single insecticidal protein gene plant transformed or with Cry1F-and Cry1A-encoding gene plant transformed, is especially delaying or is preventing that bollworm colony from occurring at aspect the resistance of the expressed insecticidal protein of this type of plant.

The invention still further relates to the method that is used to produce transgenic plant, particularly corn, cotton, rice, soybean, Chinese sorghum, tomato, Sunflower Receptacle and sugarcane, described transgenic plant comprise at least two different insect Cry albumen extremely, and these albumen show the uncontested combination of binding site in the IBB in real noctuid of paddy or the boll fruit armyworm larva.In plant, express coding Cry2A albumen and VIP3A, Cry1F or the proteic mosaic gene of Cry1A albumen, particularly VIP3Aa, Cry1Ab or Cry1Ac simultaneously, in that to prevent or delay that insecticidal protein that bollworm colony expresses in to this type of plant occurs aspect the resistance particularly useful.

The invention further relates to and be used to prevent or delay the real noctuid of paddy or boll fruit armyworm colony that it comprises providing also have this type of plant of expressing the proteic gene of Cry2A expressing the method that resistance appears in VIP3 or Cry1A and/or the proteic transgenic plant of Cry1F.Because Cry2A albumen and Cry1A or VIP3 or Cry1F albumen are uncontested to the specific binding site in the middle IBB of real noctuid of paddy or boll fruit armyworm larva, so these combinations can be used to obtain to resist the long-term provide protection of described larva.

The invention still further relates in the real noctuid of paddy or boll fruit armyworm insect species colony to containing the method for real noctuid of control paddy in the area that the proteic plant of VIP3, Cry1F and/or Cry1A produces resistance or boll fruit armyworm insect, described method comprises step: sowing in described area, plantation or growth contain the seed or the plant of the proteic gene of at least one coding Cry2A.In one embodiment of the invention, described plant also can comprise the another kind of gene of not sharing the insecticidal protein of binding site in real noctuid of paddy or boll fruit armyworm with Cry2A, VIP3, Cry1F or Cry1A albumen of (except that the proteic gene of coding Cry2A) coding.

Background of invention

Insect pest is produced whole world farm crop and is being suffered enormous economic loss, and the peasant can face every year because insect pest causes the threat of production loss.Insect-resistant genetically engineered in the farm crop has become an attractive method of the reduction cost relevant with the chemical prevention practice with crop management.Based on express the insecticidal protein that is derived from Gram-positive soil bacteria bacillus thuringiensis (Bacillus thuringiensis) (being abbreviated as " Bt " in this article) in plant, since nineteen ninety-six, first-generation insect-resistant is put on market by farm crop.

With insect resistance is appearred in some synthetic insecticide fast and compares, although used a lot of year, do not report yet so far insect to mix insecticidal protein for example the proteic plant of bacillus thuringiensis resistance appears.This may be owing to the insect-resistant supervisory routine that is used for these type of transgenic plant, for example express the high dosage level at the protein of major objective insect and use the sanctuary (refuge area) (natural sanctuary or structure sanctuary) that contains the plant that has or not this type of insecticidal protein.

The method of expressing bacillus thuringiensis or other insecticidal protein gene in order to give the anti-insect-resistant of plant in plant is well-known in the art, and the novel method of agricultural insect control is provided, and this method also is safe, attractive and cost-efficient aspect environment simultaneously.This method continue successfully an important determinative whether (or when) insect the resistance to the insecticidal protein of expressing in the transgenic plant can appear.Compare with foliar application (foliar application) (insecticidal protein degraded fast usually afterwards), transgenic plant will apply lasting selective pressure to insect.Select to know the test and find out that the selective pressure that continues can produce the insecticidal protein proteic adaptability of bacillus thuringiensis Cry for example in insect from the laboratory.

Real noctuid of paddy and boll fruit armyworm are respectively most important many hosts property lepidopterous insects pest species in the new old world.These insects have the history that quite apace insecticide development resistance, and they are not so good as other important lepidopterous insects insect to the susceptibility of a lot of Bt deutero-insecticidal proteins usually.Therefore, these insect species are that for example the most possible material standed for of resistance appears in Bt cotton or Bt maize plant to the Bt plant.

The most popular protein that imports in the plant for the control lepidopterous insects comprises Cry1A, Cry1F and VIP3A albumen.Cry1F albumen and Cry1Ac competing phase midgut binding site together in real noctuid of paddy and boll fruit armyworm, are proposed in conjunction with test based on competition.And, do not find that evidence shows that Cry1F has any site that is not shared (Hernandez and Ferre, 2005) in these insect species.Therefore, these two kinds of albumen combinations in same plant are not the methods that is applicable to the resistance management of real noctuid of paddy or boll fruit armyworm insect.Have been found that Cry1Fa has only low-affinity to the Cry1Ac binding site, this low-affinity might reflect the proteic hypotoxicity of Cry1F in viewed these insect species (people such as Liao, 2002).

So universally recognized opinion is arranged: the mode of action of Cry2 toxin is unique, and the Cry toxin that is different from other 3 structural domains, this be because their non-specific ground and/or not saturable ground combine (people such as English, 1994 with the binding site of limited number not; People such as Lee, 2006).Since the publication of people such as English (1994), Cry2A described in this publication is proteic should to be reaffirmed significantly in conjunction with feature, and several authors mention that also being used to of describing in the document prepares Cry2A albumen and carry out method (for example, people such as Luo, 2007) in conjunction with test.In addition, EPA biopesticide data single 006487 (2002) has been stated: Cry2Ab albumen, and Cry2 albumen usually, produce highly effective ionic channel with compensation with self or with the combining of a large amount of nonspecific binding sites.( www.epa.gov/opp00001/biopesticides/ingredients/factsheets/factsheet_006487.htm)。In addition, people (2000b) such as people (1994) such as English and Karim have reported in the real noctuid of paddy Cry1A and Cry2A albumen is competed at least in part or shared common binding site.In addition, statement among the non-control state of the USDA-APHIS application 06-298-01p (2006): the shared a lot of total binding sites of Cry1A and Cry2A albumen ( Www.aphis.usda.gov/brs/aphisdocs/06 29801p.pdf).

Still groundless direct saturability test confirms the proteic saturable bonded report of Cry2A, uses the binding site (being BBMV) of fixed concentration in this determination test, adds the cumulative labelled protein of concentration to described binding site.With the report of this area with find different, the inventor finally proves that at this paper the Cry2A toxin can be in special and saturable mode in conjunction with the acceptor in the sensitive insect, and the Cry2A toxin is not shared (or competition) binding site with the Cry1A toxin in bollworm (real noctuid of paddy and boll fruit armyworm).Presents has comprised and has been presented in the test of direct saturability Cry2A albumen saturable ground in conjunction with the reported first of the midgut brush border membrane of sensitive insect, and comprises the first report of the binding competition between the different Cry2A albumen of analysis.

Summary of the invention

This paper is provided in the transgenic plant the real noctuid of control paddy or boll fruit armyworm and invades and harasses and obtain the real noctuid of paddy or the boll fruit armyworm insect method of accumulation more slowly to the resistance development of described plant simultaneously, and it is included in and expresses Cry2Ae albumen and the b that a) described insect species is had insecticidal in the described plant) described insect species is had Cry1A, Cry1F or a proteic combination of VIP3A of insecticidal.

This paper also is provided for preventing in real noctuid of insect species paddy or boll fruit armyworm colony or delays at the insect-resistant development of the transgenic plant of expressing insecticidal protein controlling the method for described insect pest, and it is included in to express in the described plant has the Cry2Ae albumen of insecticidal and Cry1A, Cry1F or the proteic combination of VIP3A that the real noctuid of paddy or boll fruit armyworm is had insecticidal to the real noctuid of paddy or boll fruit armyworm.

In one embodiment of the invention, provide in the real noctuid of insect species paddy or boll fruit armyworm colony to expressing the method that the proteic plant of VIP3A, Cry1A or Cry1F produces real noctuid of control paddy in the area of resistance or boll fruit armyworm, it is included in the described area sowing or plantation and expresses and at least a real noctuid of paddy or boll fruit armyworm are had the step of the proteic plant of Cry2Ae of insecticidal.

This paper also provides, to expressing the method that the proteic plant of Cry2Ae produces real noctuid of control paddy in the area of resistance or boll fruit armyworm, it is included in the described area sowing or plantation and expresses real noctuid of paddy or boll fruit armyworm are had Cry1F, the VIP3 of insecticidal or the step of the proteic plant of Cry1A in the real noctuid of paddy or boll fruit armyworm colony.

Also provide according to the present invention, be used to obtain to comprise the method for plant of the mosaic gene of at least two kinds of different insecticidal proteins of coding, wherein as measure in conjunction with experiment in the competition of the brush border membrane vesicle that uses real noctuid of paddy or boll fruit armyworm insect larvae, described protein does not have shared binding site in the larva of real noctuid of paddy or boll fruit armyworm species, said method comprising the steps of: but obtain to comprise coding real noctuid of paddy or boll fruit armyworm are had the proteic expression of plants mosaic gene of Cry2Ae of insecticidal and coding has insecticidal to real noctuid of paddy or boll fruit armyworm Cry1A, but the plant of the proteic expression of plants mosaic gene of VIP3 or Cry1F.This paper further provides such method, wherein said plant obtains in the following way: but transform plant with the mosaic gene of described coding Cry2Ae and Cry1A, VIP3 or the proteic expression of plants of Cry1F, and acquisition comprises seed and the progeny plants of the described plant of described mosaic gene; Maybe will comprise the mother plant hybridization of mother plant and the mosaic gene that comprises described coding Cry1A, VIP3 or Cry1F of the mosaic gene of described coding Cry2Ae, and obtain to comprise the progeny plants and the seed of described mosaic gene; Or comprising coding but real noctuid or boll fruit armyworm have and transform coding in the plant of mosaic gene of the proteic expression of plants of Cry2Ae of insecticidal but real noctuid of paddy or boll fruit armyworm are had Cry1A, the VIP3 of insecticidal or the mosaic gene of proteic second expression of plants of Cry1F to paddy, and obtain to comprise the progeny plants and the seed of these at least two mosaic genes.

In another embodiment of the invention, the method that is used to obtain express the plant of at least two kinds of different insecticidal proteins is provided, wherein as can measure in conjunction with experiment in the competition of the brush border membrane vesicle that uses real noctuid of paddy or boll fruit armyworm larva, described protein does not have shared midgut binding site in the larva of real noctuid of species paddy or boll fruit armyworm, and wherein said protein is: the Cry2Ae albumen and the b that a) the real noctuid of paddy or boll fruit armyworm are had insecticidal) the real noctuid of paddy or boll fruit armyworm are had a Cry1A of insecticidal, VIP3 or Cry1F albumen, the VIP3 or the Cry1A albumen that particularly the real noctuid of paddy or boll fruit armyworm are had insecticidal.

This paper also provides sowing; the method of plantation or growing plant; wherein said plant is protected and can be to bollworm resisting; described plant comprises the mosaic gene of at least two kinds of different insecticidal proteins of expression; wherein as measure in conjunction with experiment in the competition of the brush border membrane vesicle that uses real noctuid of paddy or boll fruit armyworm larva; described protein does not have shared binding site in the larva of real noctuid of species paddy or boll fruit armyworm, said method comprising the steps of: sowing; plantation or growth comprise coding real noctuid of paddy or boll fruit armyworm are had the proteic mosaic gene of Cry2Ae of insecticidal and coding has insecticidal to real noctuid of paddy or boll fruit armyworm Cry1A; VIP3 or Cry1F albumen; the plant that preferably the real noctuid of paddy or boll fruit armyworm is had the VIP3 or the proteic mosaic gene of Cry1A of insecticidal.

This paper also provides: the purposes of at least two kinds of different insecticidal proteins in transgenic plant, be used for preventing or delaying the insect-resistant development of real noctuid of paddy or boll fruit armyworm colony, wherein as passing through competition in conjunction with measuring, described protein does not have shared binding site in the insect midgut of described insect species, described application is included in the described transgenic plant to express to have the Cry2Ae albumen of insecticidal and the real noctuid of paddy or boll fruit armyworm are had Cry1F, VIP3 or the Cry1A albumen of insecticidal the real noctuid of paddy or boll fruit armyworm; With coding real noctuid of paddy or boll fruit armyworm had the proteic mosaic gene of Cry2Ae of insecticidal and coding has insecticidal to real noctuid of paddy or boll fruit armyworm Cry1F, the proteic mosaic gene of VIP3 or Cry1A, particularly encode real noctuid of paddy or boll fruit armyworm are had the proteic mosaic gene of Cry2Ae of insecticidal and coding has insecticidal to real noctuid of paddy or boll fruit armyworm VIP3 or the proteic mosaic gene of Cry1A, purposes, be used for preventing or delay the insect-resistant development of the real noctuid of insect species paddy or boll fruit armyworm colony, to control described insect pest the transgenic plant of expressing insecticidal protein.

In the embodiment of this paper, provide real noctuid of paddy or boll fruit armyworm are had the Cry2Ae albumen of insecticidal and described species insect is had Cry1A, the VIP3 of insecticidal or the purposes of the proteic combination of Cry1F, be used to prevent or delay the resistance development that described species insect kills the transgenic plant of insect toxins to the expression heterology, particularly when described purposes realizes by express described protein combination in plant.

This paper also provides: comprise the application of the proteic plant of Cry2Ae in following area that the real noctuid of paddy or boll fruit armyworm is had insecticidal, colony in the described insect species in described area has produced resistance to comprising the proteic plant of Cry1F, VIP3 and/or Cry1A, and wherein said application can be included in the described area sowing, plantation or growth and comprise the proteic plant of Cry2Ae that the real noctuid of paddy or boll fruit armyworm is had insecticidal; With comprise Cry1F, VIP3 and/or the application of the proteic plant of Cry1A in following area that the real noctuid of paddy or boll fruit armyworm is had insecticidal, colony in the described insect species in described area has produced resistance to comprising the proteic plant of Cry2Ae, and wherein said application can be included in the described area sowing, plantation or growth and comprise Cry1F, VIP3 and/or the proteic plant of Cry1A that the real noctuid of paddy or boll fruit armyworm is had insecticidal.

This paper also provides: coding has the proteic mosaic gene of Cry2Ae of insecticidal and coding has insecticidal to real noctuid of paddy or boll fruit armyworm Cry1A to real noctuid of paddy or boll fruit armyworm, the proteic mosaic gene of VIP3 or Cry1F, particularly encode and the real noctuid of paddy or boll fruit armyworm are had the proteic mosaic gene of Cry2Ae of insecticidal and coding paddy reality noctuid or boll fruit armyworm are had the application in the method for the plant that is used for obtaining expressing at least two kinds of different insecticidal proteins of the Cry1A of insecticidal or the proteic mosaic gene of VIP3, wherein as what can measure in competition combination experiment (the brush border membrane vesicle of insect larvae as described in for example passing through to use), described insecticidal protein does not have shared binding site in the larva of species paddy reality noctuid or boll fruit armyworm.

In one embodiment of the invention, provide coding that real noctuid of paddy or boll fruit armyworm are had the purposes of the proteic mosaic gene of Cry2Ae of insecticidal, be used to obtain to comprise the plant of at least two kinds of different insecticidal proteins, wherein as measuring in conjunction with experiment in the competition of for example brush border membrane vesicle by insect larvae as described in using, described insecticidal protein does not have shared midgut binding site in the larva of real noctuid of species paddy or boll fruit armyworm, wherein said Cry2Ae mosaic gene is present in and also comprises coding has insecticidal to real noctuid of paddy or boll fruit armyworm Cry1A, in the plant of VIP3 or the proteic mosaic gene of Cry1F.

In one embodiment, above-mentioned application comprises step: by transforming the plant that plant obtains to comprise these type of different insecticidal proteins with the described Cry2Ae of coding with Cry1A, VIP3 or the proteic mosaic gene of Cry1F, with the seed and the progeny plants that obtain by plant that will comprise the proteic mosaic gene of the described Cry2Ae of coding and the plant hybridization that comprises coding described Cry1A, VIP3 or the proteic mosaic gene of Cry1F to comprise the plant of these type of different insecticidal proteins and obtain to comprise the described plant of described mosaic gene.

The present invention also provides such application: with not containing Cry2, Cry1 or the proteic plant seeding of VIP3, plantation or the growth sanctuary (refuge area) that the real noctuid of paddy or boll fruit armyworm is had insecticidal, for example by the identical growing area that comprises Cry2Ae as herein described, VIP3 and the proteic plant of Cry1 or near sowing, plantation or this type of plant that grows.

This paper also provides above-mentioned application or method, and wherein plant is to express Cry2Ae, VIP3, Cry1F or Cry1A albumen to the high dosage of real noctuid of paddy or boll fruit armyworm.

This paper also provides: growth, sowing or plantation expression Cry albumen or the proteic plant of VIP3 are with the method for control boll fruit armyworm or the real noctuid insect of paddy, it comprises step: plant in this growing area or near this growing area, sowing or growth are less than 20%, less than 15% or less than the sprinkling of 10% growing area the structure sanctuary of sterilant, or less than the structure sanctuary of not spraying sterilant of 5% growing area, or in growing area, do not plant, sowing or growth sanctuary, wherein said sanctuary is positioned at identical growing area, perhaps be positioned at 2 miles of growing area, in 1 mile or in 0.5 mile, and contain and do not comprise this Cry or the proteic plant of VIP3, wherein said expression Cry albumen or the proteic expression of plants of VIP3 have the Cry2Ae albumen of insecticidal to described insect species and described insect species are had the Cry1A of insecticidal, Cry1F or VIP3A albumen, particularly Cry2Ae and Cry1Ab or Cry1Ac or VIP3A albumen, preferred Cry2Ae and Cry1Ab and the proteic combination of VIP3.This paper also provides plant, the growing area of corn or vegetable lamb (field) particularly, it comprises less than 20%, less than 15%, less than 10% or less than 5% structure sanctuary or do not comprise structure sanctuary, wherein said growing area kind is implanted with the Cry1A that expression has the Cry2Ae or the Cry2Ab albumen of insecticidal to boll fruit armyworm or the real noctuid insect of paddy and one of described insect species had insecticidal, Cry1F or VIP3A albumen, particularly Cry2Ae and Cry1Ab, Cry1Ac or VIP3A albumen, the plant of preferred Cry2Ae and Cry1Ab and the proteic combination of VIP3.

In one embodiment of the invention, also provide specifically and saturable ground in conjunction with the purposes of at least 2 kinds of insecticidal proteins of the binding site in the real exigua larvae midgut of paddy, be used to delay or prevent the resistance development of this insect species the plant of expressing insecticidal protein, be the Cry2A albumen that this insect species is had insecticidal wherein at one of protein described in the described plant, Cry2Ab albumen for example, and another kind of protein is the Cry1A that this insect species is had insecticidal, Cry1F or VIP3 albumen, wherein in the saturability test, determine described saturable combination, the binding site (being BBMV) of fixed concentration is used in described test, and adds the cumulative labelled protein of concentration to described binding site.Especially, in this purposes, Cry1A albumen is selected from down group: Cry1Ac, Cry1Ab, Cry1A.105 or Cry1Ac or Cry1Ab hybrid protein, for example the cry1A coding region encoded protein matter of being mentioned by any this paper.There are not competition in this Cry2Ab and Cry1A albumen to their (saturable and special) binding site in the midgut of the real noctuid insect larvae of paddy, as measuring in conjunction with in testing in the BBMV competition.

Cry2Ae albumen used herein is meant: kill insect Cry2Ae albumen, for example the total length Cry2Ae albumen of the SEQ ID No.2 of WO2002/057664 (Cry2Ae1, SEQ ID No.1); Cry2Ae toxic fragment or comprise the protein (described in WO2002/057664) of Cry2Ae toxic fragment, for example Cry2Ae protein fragments and chloroplast transit peptides or the real noctuid of paddy or boll fruit armyworm are had the fusion rotein of another peptide sequence of insecticidal; Or comprise following aminoacid sequence the real noctuid of paddy or boll fruit armyworm are had the protein of insecticidal, the aminoacid sequence of described aminoacid sequence and this paper SEQ ID No.1 or have at least 95,97 or 99% sequence identity with the SEQ IDNo.2 of WO 2002/057664 is particularly in the part corresponding to minimum toxic fragment; Or by the Cry2Ae coding region part encoded protein matter that is included in the Cry2Ae mosaic gene among the cotton event EE-GH6 (in the PCT patent application of the right of priority that requires European Patent Application No. 07075460 or 07075485 (unexposed), describing), the any protein that particularly comprises arbitrary minimum toxic fragment of described Cry2Ae albumen, or the real noctuid of paddy or boll fruit armyworm kept toxic arbitrary variant with the different described Cry2Ae albumen of 1-5 amino acid.

Cry2Ab albumen used herein is meant: people such as Crickmore (1998) or Www.1ifesci.susx.ac.uk/home/Neil_Crickmore/Bt/In the real noctuid of paddy or boll fruit armyworm are had any Cry2Ab albumen of insecticidal, for example total length Cry2Ab albumen, Cry2Ab toxic fragment, Cry2Ab2 albumen (this paper is SEQ ID No.2) or comprise the albumen of Cry2Ab toxic fragment, for example Cry2Ab2 protein fragments and chloroplast transit peptides or the real noctuid of paddy or boll fruit armyworm are kept the fusion rotein of toxic another peptide sequence; Or comprise following aminoacid sequence the real noctuid of paddy or boll fruit armyworm are had the protein of insecticidal, described aminoacid sequence and this paper SEQID No.2 or with the aminoacid sequence of NCBI login CAA39075 (people such as Dankocsik, 1990) has at least 95,97 or 99% sequence identity, particularly in part corresponding to minimum toxic fragment; Or by the Cry2Ab2 coding region part encoded protein matter that is included in the Cry2Ab mosaic gene in the cotton event 15985 (in the non-control state of USDA-APHIS application 00-342-01p, describing), by the Cry2Ab2 coding region part encoded protein matter that is included in the Cry2Ab mosaic gene among the corn event MON89034 (in the non-control state of USDA-APHIS application 06-298-01p, describing), the any albumen that particularly comprises arbitrary minimum toxic fragment of described Cry2Ab albumen, perhaps the real noctuid of paddy or boll fruit armyworm are kept toxicity and have arbitrary variant of the different described Cry2Ab albumen of 1-5 amino acid.

Cry1F albumen used herein comprises: comprise any protein that the real noctuid of paddy or boll fruit armyworm is kept the minimum toxic fragment of toxic Cry1F Argine Monohydrochloride sequence, for example the albumen of NCBI accession number AAA22347 (the SEQ ID No.10 of US 2005049410) or Cry1Fa1 albumen (SEQ ID No.3).This definition also comprises the variant of the aminoacid sequence of SEQ ID No.3 or NCBI accession number AAA22347, for example has the aminoacid sequence of at least 90% sequence identity (as utilizing the GAP program (Madison of GCGWisconsin software package with SEQ ID No.3 or with the Cry1F albumen of NCBI accession number AAA22347, Wisconsin, USA, 10.2 version), use comparison in pairs to determine), particularly in part, have this identity corresponding to minimum toxic fragment.Cry1F albumen used herein comprises the (WO2005/103266 by Cry1F cotton event 281-24-236, referring to the non-control state of USDA APHIS application 03-036-01p) in or corn event TC1507 or TC-2675 (US 7,288,643, WO 2004/099447, the non-control state of USDAAPHIS application 00-136-01p and 03-181-01p) in the protein of Cry1F genes encoding, the any protein that particularly comprises arbitrary minimum toxic fragment of described Cry1F albumen, or the real noctuid of paddy or boll fruit armyworm are had arbitrary variant of the different described Cry1F albumen of the toxic 1-5 of a having amino acid.

In one embodiment of the invention, VIP3 albumen is the protein that the real noctuid of paddy or boll fruit armyworm larva is had insecticidal, it is any VIP3 albumen of listing among the people such as Crickmore (2008), or comprises any protein of arbitrary minimum toxic fragment of these protein.Used VIP3 albumen can be the VIP3A albumen that the real noctuid of paddy or boll fruit armyworm is had insecticidal, for example the VIP3Aa1 albumen of SEQ ID No.4, the VIP3Af1 albumen of SEQ ID No.5, VIP3Aa19 as herein described (NCBI accession number ABG20428, EPA experimental applications permission handbook (EPA experimental use permit factsheet) 006499 (2007), and comprise any albumen that it kills insect fragment or functional domain this paper SEQ ID No.6) or VIP3Aa20 albumen (this paper SEQ ID No.7); And with NCBI accession number AAC37036 (people such as Estruch, 1996) or the VIP3Aa1 albumen of SEQ ID No.4 (particularly minimum toxic fragment) or have any albumen at least 70% sequence identity (as make the GAP program of utilizing GCG Wisconsin software package, use comparison in pairs to measure), that the real noctuid of paddy or boll fruit armyworm had insecticidal with the VIP3Af1 albumen (this paper SEQ ID No.5, the SEQ ID No.4 among the WO03/080656) (particularly minimum toxic fragment) of NCBI accession number CAI43275 with it with it; And be selected from the following VIP3A albumen that the real noctuid of paddy or boll fruit armyworm is had insecticidal: VIP3Ab, VIP3Ac, VIP3Ad, VIP3Ae, VIP3Af, VIP3Ag or VIP3Ah, particularly VIP3Af1, VIP3Ad1 or VIP3Ae1 albumen (are respectively NCBI accession number CAI43275 (ISP3a, SEQ ID No.4 among the WO 03/080656), CAI43276 (ISP3b, SEQ ID No.6 among the WO 03/080656) and CAI43277 (ISP3C, and kill insect fragment, hybrid or variant SEQ ID No.2 among the WO 03/080656)).In one embodiment, VIP3 albumen is to import (the plant that for example comprises incident COT102 in the vegetable lamb, in WO 2004/039986 or the non-control state of USDA APHIS application 03-155-01p, describe) VIP3Aa19 albumen (NCBI accession number ABG20428, SEQ IDNo.6), or (event mir 162 for example in the importing maize plant, the non-control state of USDA APHIS application 07-253-01p) VIP3Aa20 albumen (NCBI accession number ABG20429, SEQ ID NO:2 among the WO2007/142840, this paper is SEQ ID No.7), or the VIP3A albumen that in cotton event COT202 or COT203 (seeing WO 2005/054479 and WO 2005/054480 respectively), produces, or have 1-5 amino acid difference and real noctuid of paddy or boll fruit armyworm are kept the proteic variant of toxic any above-mentioned VIP3.

Cry1A albumen used herein is meant Cry1Ac1 (SEQ ID No.8), Cry1A.105 (SEQID No.9) or Cry1Ab1 (SEQ ID No.10) albumen, and comprise comprising any albumen that the real noctuid of paddy or boll fruit armyworm is kept the minimum toxic fragment of toxic Cry1Ac, Cry1A.105 or Cry1Ab Argine Monohydrochloride sequence, for example comprise SEQ ID No.8 or NCBI accession number AAA22331 (Cry1Ac; People such as Adang, 1985) protein in, SEQ ID No.10 or NCBI accession number AAA22330 (people such as Wabiko, 1986 (Cry1Ab)) protein in, or by corn event MON89034 (the non-control state of USDA APHIS application 06-298-01p, the Cry1A.105 albumen of this paper SEQ ID No.9 of the Cry1A transgenes encoding WO2007/140256, the SEQ ID NO:2 or 4 among the WO 2007/027777), or by cotton event COT67B (USDA APHIS does not remove control state application 07-108-01p, WO 2006/128573) in any albumen of the proteic minimum toxic fragment of Cry1Ab of cry1Ab coding region coding.This definition also comprises NCBI accession number AAA22331 (Cry1Ac1), NCBI accession number AAA22330 (Cry1Ab, people such as Wabiko, 1986) aminoacid sequence in, or the variant of the proteic aminoacid sequence of Cry1A.105 described in the non-control state of the USDA APHIS application 06-298-01p, for example with this Cry1Ac, Cry1A.105 or Cry1Ab albumen, SEQ ID Nos.8 particularly, 9 or 10 albumen, more especially in part corresponding to minimum toxic fragment, has at least 90% amino acid sequence identity (as utilizing the GAP program (Madison of GCG Wisconsin software package, Wisconsin, USA, 10.2 editions), use the pairing comparison to determine), protein with the proteic minimum toxic fragment of Cry1A.

Cry1A albumen comprises in this article by US 6,114, the Cry1Ab albumen of 608 SEQ ID NO:3 coding, particularly (US 6 by corn event MON810,713,259, USDA APHIS does not remove control state (non-deregulated status) application 96-017-01p and replenishes) in the Cry1Ab albumen of cry1Ab coding region coding, (USDA APHIS does not remove control state application 95-195-01p by corn event Bt11, US patent 6,114, the Cry1Ab albumen of the cry1Ab coding region coding 608), (US 7 by cotton event 3006-210-23,179,965, WO 2005/103266, USDA APHIS does not remove control state application 03-036-02p) in the Cry1Ac albumen of transgenes encoding, (USDA APHIS does not remove control state application 07-108-01p by cotton event COT67B, WO 2006/128573) in the cry1Ab coding region, be included in the Cry1Ab coding region among the described cotton event EE-GH5 of PCT patent application PCT/EP2008/002667 (unexposed), United States Patent (USP) 7,049, the Cry1Ab albumen of the Cry1Ab coding region coding of 491 SEQ ID No.2, by corn event MON89034 (the non-control state of USDA APHIS application 06-298-01p, WO 2007/140256, the Cry1A.105 albumen of the Cry1A transgenes encoding the SEQ ID NO:2 or 4 among the WO 2007/027777), by cotton event 15985 or cotton event 531, the Cry1Ac sample albumen of the hybrid cry1Ac coding region coding in 757 or 1076 (the non-control state of USDA APHIS application 94-308-01p, the chimeric Cry1Ac albumen of the cry1A cotton event coding of WO 2002/100163), by being set forth in WO 2006/128568 respectively, WO 2006/128569, WO 2006/128570, the cotton event T342-142 of WO 2006/128571 or WO 2006/128572,1143-14A, 1143-51B, the cry1Ab albumen of the cry1Ab coding region coding among CE44-69D or the CE46-02A is (promptly by WO 2006/128568, WO 2006/128569, the DNA of SEQ ID No.7 among WO 2006/128571 or the WO2006/128572 or by the albumen of the dna encoding of the SEQ IDNo.5 among the WO 2006/128570), or comprise the protein of arbitrary minimum toxic fragment of above-mentioned Cry1A albumen, or it is different but keep arbitrary variant to the toxic above-mentioned Cry1A albumen of real noctuid of paddy or boll fruit armyworm to have 1-5 amino acid.

This paper also provides and comprises at least 2 genetically modified plants or seed, each transgenosis different protein that the real noctuid of paddy or boll fruit armyworm had insecticidal of respectively encoding wherein, described protein saturable ground and specifically in conjunction with the binding site in this type of insect midgut, wherein said albumen same binding site of competing phase not in this type of insect, and wherein said protein is i) Cry2A albumen and ii) Cry1A, Cry1F or VIP3 albumen.In one embodiment, described plant comprises the following proteic transgenosis of coding: i) Cry2Aa, Cry2Ab or Cry2Ae and ii) Cry1Ab, Cry1Ac, CryIFa or VIP3A, particularly Cry2Ae albumen and Cry1Ab and/or VIP3A albumen.In another embodiment, described plant or seed are to comprise coding Cry1A, proteic mosaic gene of Cry1F or VIP3 and coding Cry2A albumen, the particularly corn of the proteic mosaic gene of Cry2Ae or vegetable lamb or seed, wherein said plant or seed comprise the transformation event that is selected from down group: corn event MON89034, corn event mir 162, comprise the proteic genetically modified corn event of coding Cry2Ae, corn event TC1507, corn event Bt11, corn event MON810, cotton event EE-GH6, cotton event COT102, cotton event COT202, cotton event COT203, cotton event T342-142, cotton event 1143-14A, cotton event 1143-51B, cotton event CE44-69D, cotton event CE46-02A, cotton event COT67B, cotton event 15985, cotton event 3006-210-23, cotton event 531, cotton event EE-GH5, cotton event 281-24-236, all incidents such as this paper further define.

This paper also provides and comprises at least 3 genetically modified plants, what wherein each transgenes encoding was different has the protein of insecticidal to the real noctuid of paddy or boll fruit armyworm, described protein saturable and specifically in conjunction with the binding site in this type of insect midgut, wherein said albumen same binding site of competing phase not in this type of insect, and wherein said plant comprises coding Cry1A or the proteic mosaic gene of Cry1F, the coding proteic mosaic gene of Cry2A and the proteic mosaic gene of coding VIP3A, and wherein these incidents are selected from as mentioned described group of paragraph.

In one embodiment of the invention, in purposes of the present invention, method or plant, Cry2Ae, Cry2Ab, VIP3, Cry1F or Cry1A mosaic gene are included in the mosaic gene among above-mentioned corn or cotton event arbitrary.According to the present invention, also comprise: term Cry2Ae wherein is by term Cry2Aa or Cry2Ab alternate any purposes as herein described, method, plant or seed; And relate to Cry2Ae, Cry2Aa or the proteic any such use of Cry2Ab, method, plant or seed, wherein the combining of midgut BBMV of real noctuid of this Cry2A albumen and paddy or boll fruit armyworm is specific and saturable, particularly when direct saturability in conjunction with test in definite saturable in conjunction with the time; Preferred such purposes, method, plant or seed, wherein in standard competition combination test as herein described, in boll fruit armyworm or the real noctuid of paddy, all there is not biology to compete significantly between any described Cry2A albumen and Cry1A, Cry1F or the proteic specificity combination of VIP3.

In above-mentioned plant of the present invention, seed, purposes or method, preferred plant, for example for different mosaic genes being piled up by hybridization or is combined in the same plant, be the plant that comprises any one above-mentioned corn event or any one above-mentioned cotton event, with and comprise the offspring or the filial generation of coding described Cry2A and described VIP3 and/or the proteic mosaic gene of Cry1.

Plant used herein or seed comprise the plant or the seed that can significantly be subjected to any plant species of bollworm destructive, but particularly including corn, cotton, rice, soybean, Chinese sorghum, tomato, Sunflower Receptacle and sugarcane.

This paper also provides: be used to make expression the real noctuid of paddy or boll fruit armyworm to be had the method that the plantation of proteinic plant of insecticidal or commercialization obtain to decontrol or obtain supervision department's approval; Or be used to reduce and contain the method for structure sanctuary that the real noctuid of paddy or boll fruit armyworm is had any proteinic plant of insecticidal that do not produce; Or be used to plant the method for growing area with structure sanctuary, said method comprising the steps of: quote, submit to or rely on insect test binding data, Cry1A, Cry1F or the proteic binding site of VIP3 are not competed, data for example disclosed herein or the class likelihood data of reporting in conjunction with insect midgut film and the described Cry2A albumen of described insect in wherein said data presentation Cry2A protein-specific and saturable ground in other file in described insect.In one embodiment, Cry2A albumen is Cry2Aa, Cry2Ab or Cry2Ae albumen, and Cry1A albumen is Cry1Ac, Cry1Ab or Cry1A-105 albumen, and VIP3 albumen is VIP3Aa albumen.

This paper also provides: plant the growing area (field) that is implanted with the plant that contains insecticidal protein; the described plant of wherein said protein protection avoids the infringement of boll fruit armyworm or the real noctuid insect of paddy; wherein said growing area has less than 20% structure sanctuary or less than 5% structure sanctuary or there is not structure sanctuary in described growing area, and wherein said expression of plants a) has the Cry2Ae albumen and a b of insecticidal to described insect species) described insect species is had Cry1A, Cry1F or a proteic combination of VIP3A of insecticidal.Described plant optimization is corn or vegetable lamb.

The present invention also comprises aforesaid method or plant, wherein except that Cry or VIP3 albumen, the Bt toxin strengthens albumen also expresses in described plant, it is protein or its fragment that wherein said Bt toxin strengthens albumen, promptly, the part of Bt toxoreceptor in the insect preferably comprises or corresponding to the part of binding domains, for example proteic fragment of cadherin sample.These Bt toxin strengthen albumen with one or more Bt kill insect toxins for example Cry albumen fed to targeted insect.These Bt toxin strengthen albumen can strengthen the Bt insecticidal protein to the source insect species of this receptor and to the toxin activity of other insect species.In one embodiment, described Bt toxin strengthens the part that albumen is the midgut epithelial cells Bt toxoreceptor of real noctuid of paddy or boll fruit armyworm insect.

Detailed Description Of The Invention

Owing to for example Bt Cry or the success of the proteic plant of VIP3 and the increase of this type of plant number of the insecticidal protein that comprises importing, resistance management now was than the past even even more important.

Although the different insecticidal proteins that are derived from Bt or other bacterium for example Cry or the proteic insecticidal spectrum of VIP may be different, main path of its performance toxic action is a common.The Bt source insecticidal protein of the transgenic plant that are useful on (at least a targeted insect, having studied its mechanism of action) (for example Cry1 and VIP3 toxin), in the insect intestines, activated by proteolysis, and interact with the midgut epithelium of sensitive species, cause epithelial cracking.In Cry albumen and the proteic toxic action approach of VIP, it is key property (people such as Hofmann, 1988 that the specificity of the acceptor site on the brush border membrane of toxin and these cells combines; People such as Lee, 2003).Binding site is commonly called acceptor, because this combination is saturable and has high-affinity.

When two kinds of different insecticidal proteins were shared receptor binding site in insect, they can not be provided for the good combination of insect-resistant administrative purposes.In fact, to insecticidal protein for example Bt Cry albumen produce the most possible mechanism of resistance---and unique main mechanism of finding in the insect-resistant of the anti-Bt sprays that occurs in the field so far---be the modification of receptors bind.The similar albumen of aminoacid sequence height is shared acceptor site (for example Cry1Ab and Cry1Ac albumen) usually.But, even two different albumen with very different aminoacid sequences also may be in insect species with high-affinity in conjunction with common binding site (for example, Cry1Ab and Cry1F albumen are in diamond-back moth (Plutella xylostella)).And, found that two kinds of albumen that do not have shared binding site in an insect species can (for example enjoy the common binding site in another insect species, people such as Fiuza (1996) find that Cry1Ac and Cry1Ba albumen shares binding site in striped rice borer (Chilo suppressalis), and people such as Ballester (1999) find they in diamond-back moth in conjunction with different binding sites).

The present invention relates in real noctuid of paddy or boll fruit armyworm Cry1F, VIP3 or Cry1A acceptor are not shown the Cry2A albumen of competition, this makes and the most meaningfully make up at least Cry2Ae, Cry2Aa or Cry2Ab albumen and VIP3, Cry1F or Cry1A albumen (preferably Cry2Ae albumen and Cry1Ab, Cry1Ac, Cry1A.105 or VIP3A albumen) at least in identical plant, to prevent or to delay the insect-resistant development of real noctuid of paddy or boll fruit armyworm.This method advantageously should be a part that is used for the global policies of insect-resistant management, and described strategy if needed or necessary, comprises structure sanctuary and with the high dosage marking protein at targeted insect.

The mentioned binding site of this paper only refers to the real noctuid of paddy or boll fruit armyworm are had the specific binding site of the albumen (for example Cry2Ae, Cry2Ab, VIP3A, Cry1Ac or Cry1Ab albumen) of insecticidal.These are binding sites of binding proteins specific matter, promptly, for these binding sites, tagged ligand (for example Cry2Ae or VIP3A albumen) can be by excessive unlabelled cognate ligand (being respectively Cry2Ae or VIP3A albumen) displacement (or competition) with combining of its binding site.Term binding site or acceptor are used interchangeably at this paper, and are equal to.In one embodiment, as directly measuring in the saturability test, be saturable with combining of this type of specific binding site." directly saturability test " used herein is meant such test, wherein hatches the acceptor (being BBMV in this case) of fixed amount with the tagged ligand of cumulative amount.Under saturable bonded situation, (Y-axis is in conjunction with % when binding data is mapped, X-axis is the concentration of tagged ligand), platform (plateau)---or at least from linear departing from---will be tangible, and under saturable bonded situation not, to obviously there be platform---or from linear departing from, but along with tagged ligand concentration increases, keep increasing linearly in conjunction with %.Platform is the maximum combined that can obtain under experiment condition, occupies because all available specific binding sites all have been labeled part.

When in order to delay or to reduce the insect-resistant development of targeted insect species and in plant during the different insecticidal protein of combination, importantly experimental in the targeted insect species (promptly by implementing in conjunction with test) checks that institute proposes whether shared binding site in the midgut at targeted insect of the different insecticidal proteins that make up.In the present invention, when between two kinds of different insecticidal proteins there is competition in single binding site, (mean, when the binding data of testing from the competition combination is mapped, two kinds of albumen reach same platform in the bottom of competition curve), from the insect-resistant management view, these protein are not useful combination in plant.As used herein, when two kinds of different insecticidal proteins during in conjunction with two different binding sites, from the insect-resistant management view, these protein are useful.As used herein, for with different binding site bonded albumen, a kind of albumen is not considered to biology significantly (perhaps to the competition of the proteic binding site of another kind, in other words, be considered to the non-significant competition of biology), when if this competition only takes place under the very high density of this heterology competitor (for example, if, by binding data mapping (in conjunction with % to unmarked ligand concentration) is measured, unlabelled this heterology competitor of 100nM (or more) is only replaced and is seldom measured bonded tagged ligand (for example about 25% or the specificity of tagged ligand still less in conjunction with)).If albumin X only with low-affinity (for example, if, by binding data mapping (in conjunction with % to unmarked ligand concentration) is measured, unlabelled this heterology competitor of 100nM (or more) is only replaced and is seldom measured bonded tagged ligand (for example about 25% or the specificity of tagged ligand still less in conjunction with)) binding site of bonding mark protein Y, but the evidence that in equity (reciprocal) the combination test of applying marking albumin X, does not have any different binding sites, then therefore two kinds of albumen are unsuitable for combination and are used for the resistance administrative purposes effectively in conjunction with identical binding site.

In the present invention, because (combination) feature of metaprotein can be different from non-denatured protein, measure Cry or the proteic combination of VIP3 so use sex change BBMV albumen by ligand blot, be not considered to be present in the midgut or the BBMV prepared product in the reliable measure (it can use radiolabeled or biotinylated proteinic BBMV to measure in conjunction with test, therefore in this determination test in conjunction with being proteic) of actual specific binding site at non-sex change BBMV.

Be used for testing method and the technology shared of a pair of different insecticidal protein, be well known in the art (referring to for example, people such as Van Rie, 1989, people such as Ferr é, 1991) the binding site of insect larvae.At first, determine a pair of insecticidal protein that targeted insect (being real noctuid of paddy or boll fruit armyworm herein) is all had insecticidal activity.By real noctuid of paddy or boll fruit armyworm midgut, use currently known methods (referring to for example, people such as Wolfersberger 1987), preparation brush border membrane vesicle (BBMV), and analyze purified labelled protein (for example Cry2Ae, Cry2Ab, VIP3 or Cry1 albumen) the specificity combination of BBMV therewith.Carry out the test of homology competition assay to determine whether that combination is specific (at this, excessive unlabelled same protein is as the competitor of tagged ligand), and carry out allos competition assay test to determine in these BBMV the whether same binding site of competing phase (at this, excessive unlabelled different albumen are as the competitor of tagged ligand) of another kind of protein.

In the test of this type of allos competition assay, when applying marking albumin X and unmarked protein Y were found competition as competitor, also applying marking protein Y and unmarked albumin X carried out equity experiment as competitor, do not have competition with affirmation.In the test of homology competition assay, if the combination of the remarkable part of labelled protein is by unmarked albumen (being the homology competitor) competition (or displacement), this combination is specific so---the bound fraction of not replaced or competing by cognate ligand is considered to non-specific binding.Can or pass through radio-labeling by well-known biotin labeling, fluorescent mark technology for example by using Na ¹²⁵I (using currently known methods, for example chloramine-T method) carries out mark to being used for albumen of the present invention (for example Cry2Ae, Cry2Ab, VIP3 or Cry1 albumen).

According to the present invention, " nucleotide sequence " is meant the DNA of strand or double chain form or RNA molecule, any protein DNA of the present invention or RNA, the particularly DNA of being used for of optimized encoding." isolated nucleic acid sequences " used herein is meant the nucleotide sequence that no longer is present in its natural surroundings that separates oneself, for example nucleotide sequence in other host bacterium or plant nucleolus genome.

As used herein, " allos " albumen, for example when mentioning in plant when using the allos insecticidal protein, be meant the natural protein that is not present in this organism (for example plant), particularly by the protein that imports the transgenes encoding in the Plant Genome, wherein this protein source is from bacterioprotein.

According to the present invention, term " protein " or " polypeptide " are used in reference to interchangeably, the molecule of forming by amino acid chain, and do not relate to any concrete binding mode, size, three-dimensional structure or source.Therefore, be used for proteinic fragment of the present invention or part still is called " protein " at this paper." isolating protein " used herein is meant, no longer is present in the protein in its natural surroundings.Proteinic natural surroundings be meant when its nucleotides sequence of coding be listed in its natural surroundings (promptly nucleotide sequence separate from environment in) express and can find this proteinic environment during translation.For example, isolating protein can be present in external or other host bacterium in or in the vegetable cell, or it can be secreted from other host bacterium or from vegetable cell.

" insecticidal protein " used herein should be understood that to refer to, have insect active extremely, particularly to the real noctuid of paddy or boll fruit armyworm larva have insect active extremely, whole protein or its part.This can be the chimeric protein of naturally occurring protein or the part that comprises different insecticidal proteins or hybrid protein (for example, by utilizing gene reorganization, mixing is from the proteic structural domain of difference, or mix different proteic parts), maybe can be to have the aminoacid sequence of bacterioprotein basically but the adorned variant of some amino acid.In this, insecticidal protein can be VIP or the Cry albumen that is derived from Bt or other bacterial isolates, or by mutator gene or recombination (can reorganize by gene, for example Cry or the proteic gene of VIP obtain by coding Bt insecticidal protein for mutagenesis etc.) encoded protein matter.

" parent toxin " used herein is interpreted as referring to, the initial translation product of the full-length gene of coding insecticidal protein before any fracture of generation in midgut.Usually, the VIP3 parent toxin has the molecular weight of about 88kD, and Cry1F or Cry1A parent toxin have the molecular weight of about 130-140kD, and the Cry2A parent toxin has the molecular weight of about 60-70kD.

" toxin " used herein or " minimum toxic fragment " is interpreted as insecticidal protein for example Cry2A, VIP3 or Cry1F or the proteic part of Cry1A, it can obtain and still have insect active extremely by tryptic digestion or by proteolysis in intestinal juice in (targeted insect, for example real noctuid of paddy or boll fruit armyworm).Usually, on the SDS-PAGE gel, VIP3 or Cry1 toxin have the molecular weight of about 60-65kD, and the Cry2A toxin has the molecular weight of about 50-58kD.

" VIP3 albumen " used herein or " VIP3 " are meant the protein that the real noctuid of paddy or boll fruit armyworm larva is had insecticidal, and it is in VIP name website in people such as Crickmore (2008, see Table 3) Www.lifesci.susx.ac.uk/home/Neil Crickmore/Bt/VIP.htmlOn any VIP3 albumen of listing, or comprise any protein of arbitrary minimum toxic fragment of these protein.In one embodiment, this is the VIP3A albumen that the real noctuid of paddy or boll fruit armyworm is had insecticidal, for example VIP3Aa1 (NCBI accession number AAC37036), VIP3Af1 (NCBI accession number CAI43275), VIP3Aa19 (NCBI accession number ABG20428) or VIP3Aa20 albumen (NCBI accession number ABG20429), with and any insect fragment of killing; Or on amino acid sequence level with VIP3Aa1 albumen or the NCBI accession number CAI43275 (ISP3A of NCBI accession number AAC37036, the SEQ ID No.4 of WO 03/080656) VIP3Af1 albumen, particularly with their minimum toxic fragment, has at least 70%, particularly at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity (as utilizing the GAP program (Madison of GCG Wisconsin software package, Wisconsin, USA, 10.2 editions), use comparison in pairs to measure) protein.Parameter was used for aminoacid sequence relatively below the GAP program was used: ' blosum62 ' rating matrix, ' room generation point penalty ' (or ' room weight ') of 8 and ' room extension point penalty ' (or ' length weight ') of 2.In one embodiment, VIP3 albumen used herein is for example people (1996 such as Estruch of VIP3A albumen, NCBI accession number AAC37036) described VIP3Aa1 albumen, with be selected from down group the real noctuid of paddy or boll fruit armyworm are had the VIP3A albumen of insecticidal: VIP3Ab, VIP3Ac, VIP3Ad, VIP3Ae, VIP3Af, VIP3Ag or VIP3Ah, VIP3Af1 particularly, VIP3Ad1 or VIP3Ae1 albumen (are respectively NCBI accession number CAI43275 (ISP3a, the SEQ ID No.4 of WO 03/080656), CAI43276 (ISP3b, SEQ ID No.6 among the WO 03/080656) and CAI43277 (ISP3C, the SEQ ID No.2 of WO 03/080656)) and kill the insect fragment.Certainly, except that naturally occurring VIP3 albumen with comprise it and kill the segmental albumen of insect, this paper also comprises by the real noctuid of paddy or boll fruit armyworm being kept hybrid protein or the chimeric protein that the VIP3 albumen that kills insect active is made, the described chimeric VIP3AcAa albumen of people (2007) such as Fang for example, different with some amino acid but keep the great majority of parent's molecule or all real noctuids of paddy or toxic VIP3 protein mutant of boll fruit armyworm or equivalent; For example, have, preferred 5-10, particularly less than 5 interpolation, real noctuid of the amino acid (preferably in the part corresponding to minimum toxic fragment) of displacement or disappearance and proteinic paddy or boll fruit armyworm kill insect active does not have the obviously VIP3 protein variant of change, for example import VIP3Aa19 albumen (the NCBI accession number ABG20428 in the vegetable lamb (for example plant that comprises incident COT102 described in the non-control state of WO2004/039986 or the USDAAPHIS application 03-155-01p), EPA experimental applications permission handbook 006499 (2007)), or import maize plant (event mir 162 for example, the non-control state of USDAAPHIS application 07-253-01p) the VIP3Aa20 albumen in (NCBI accession number ABG20429, the SEQ ID NO:2 among the WO 2007/142840), or the VIP3A albumen that in cotton event COT202 or COT203 (being respectively WO 2005/054479 and WO 2005/054480), produces, or comprise the protein of arbitrary minimum toxic fragment of these VIP3 albumen, or it is different and the real noctuid of paddy or boll fruit armyworm are kept arbitrary variant of toxic these VIP3 albumen to have 1-5 amino acid.

In addition, in one embodiment of the invention, in VIP3 albumen of the present invention, any natural (bacterium) secreting signal peptide of inferring can lack or can be by Met amino acid or Met-Ala dipeptides or by for example chloroplast transit peptides displacement of appropriate signal peptide.Can use the computer based analysis, utilize program for example signal peptide search utility (SignalP V1.1 or 2.0), be used for the matrix of prokaryotic organism gram-positive microorganism and less than 0.5 threshold score, especially 0.25 or littler threshold score, the signal peptide that detection is inferred (Von Heijne, Gunnar, 1986 and people such as Nielsen, 1996).

" Cry1F albumen " used herein or " Cry1F " comprise and comprise any protein that the real noctuid of paddy or boll fruit armyworm is kept the minimum toxic fragment of the proteic aminoacid sequence of toxic Cry1F, for example protein of NCBI accession number AAA22347 (the SEQ ID No.10 of US 2005049410) or Cry1Fa albumen.This also comprise at least 2 of comprising in the proteic minimum toxic fragment of Cry1F or at least one structural domain, preferred 3 structural domains, hybrid protein or chimeric protein, for example be derived from the hybrid protein of the Cry1F among the WO 1999/024581.This definition also comprises the variant of the aminoacid sequence of NCBI accession number AAA22347, the aminoacid sequence that has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the Cry1F albumen of NCBI accession number AAA22347 (the SEQ ID No.10 of US2005049410) for example, wherein utilize the GAP program (Madison of GCG Wisconsin software package, Wisconsin, USA, 10.2 version), use comparison in pairs to measure sequence identity, particularly this identity is at the part corresponding to minimum toxic fragment.Parameter was used for aminoacid sequence relatively below the GAP program was used: ' blosum62 ' rating matrix, ' room generation point penalty ' (or ' room weight ') of 8 and ' room extension point penalty ' (or ' length weight ') of 2.Preferably, this definition comprise have, preferred 5-10, particularly this proteinic paddy reality noctuid of not obvious reduction or boll fruit armyworm kill the protein of insect active less than the amino acid of 5 interpolation, displacement or disappearance, for example have one or more conservative amino acid metathetical Cry1F albumen in order to clone purpose.Cry1F albumen used herein comprises the (WO2005/103266 by Cry1F cotton event 281-24-236, referring to the non-control state of USDA APHIS application 03-036-01p) in, or corn event TC1507 or TC-2675 (US 7,288,643, WO 2004/099447, the non-control state of USDA APHIS application 00-136-01p and 03-181-01p) in the protein of Cry1F genes encoding, particularly comprise the protein of arbitrary minimum toxic fragment of these Cry1F albumen, or it is different and the real noctuid of paddy or boll fruit armyworm are kept arbitrary variant of toxic these Cry1F albumen to have 1-5 amino acid.

" Cry2Ae " used herein albumen is meant insect Cry2Ae albumen extremely, for example total length Cry2Ae albumen, the Cry2Ae toxic fragment of the SEQ ID No.2 of WO2002/057664 or comprise the protein (described in WO 2002/057664) of Cry2Ae toxic fragment, for example Cry2Ae protein fragments and chloroplast transit peptides or the real noctuid of paddy or boll fruit armyworm are had the fusion rotein of another peptide sequence of insecticidal; Or comprise following aminoacid sequence the real noctuid of paddy or boll fruit armyworm are had the protein of insecticidal, the aminoacid sequence of the SEQ ID No.2 of described aminoacid sequence and WO 2002/057664, particularly in part corresponding to minimum toxic fragment, have at least 95,97 or 99% sequence identity; Or by the protein that is included in the Cry2Ae genes encoding among the cotton event EE-GH6 (seeing the PCT patent application of the right of priority of requirement European Patent Application No. 07075460 or 07075485 (unexposed)) or comprise these Cry2Ae albumen arbitrary minimum toxic fragment protein or to have 1-5 amino acid different and the real noctuid of paddy or boll fruit armyworm are kept arbitrary variant of toxic these Cry2Ae albumen.

" Cry2Ab " used herein albumen be meant people such as Crickmore (1998,2008) or Www.lifesci.susx.ac.uk/home/Neil_Crickmore/Bt/In the real noctuid of paddy or boll fruit armyworm are had any Cry2Ab albumen of insecticidal, for example total length Cry2Ab albumen, Cry2Ab toxic fragment or comprise the protein of Cry2Ab toxic fragment, for example Cry2Ab2 protein fragments and chloroplast transit peptides or the real noctuid of paddy or boll fruit armyworm are kept the fusion rotein of toxic another peptide sequence; Or comprise following aminoacid sequence the real noctuid of paddy or boll fruit armyworm are had the protein of insecticidal, described aminoacid sequence and NCBI accession number CAA39075 (people such as Dankocsik, 1990) coding region, particularly in part corresponding to minimum toxic fragment, have at least 95,97 or 99% sequence identity; Or by the protein that is included in the Cry2Ab2 genes encoding in the cotton event 15985 (seeing the non-control state of USDAAPHIS application 00-342-01p), by the protein that is included in the Cry2Ab2 genes encoding among the corn event MON89034 (seeing the non-control state of USDAAPHIS application 06-298-01p), or comprise described Cry2Ab albumen arbitrary minimum toxic fragment any albumen or to have 1-5 amino acid different and the real noctuid of paddy or boll fruit armyworm are kept arbitrary variant of toxic described Cry2Ab albumen.

" Cry1A " used herein albumen is meant Cry1Ac, Cry1A.105 or Cry1Ab albumen, and comprise and comprise any protein that the real noctuid of paddy or boll fruit armyworm is kept the minimum toxic fragment of toxic Cry1Ac, Cry1A.105 or Cry1Ab Argine Monohydrochloride sequence, for example albumen (Cry1Ac of NCBI accession number AAA22331; People such as Adang, 1985), the albumen of NCBI accession number AAA22330 (people such as Wabiko, 1986 (Cry1Ab) or by the Cry1A.105 albumen of the Cry1A transgenes encoding among the corn event MON89034 (the SEQ ID NO:2 or 4 among the non-control state of USDAAPHIS application 06-298-01p, WO 2007/140256, the WO 2007/027777) or by the proteic minimum toxic fragment of Cry1Ab of the coding of the cry1Ab coding region among the cotton event COT67B (USDA APHIS does not remove control state application 07-108-01p, WO2006/128573).This comprise comprise Cry1A albumen for example at least 2 in the minimum toxic fragment of Cry1Ab or Cry1Ac or at least one structural domain, preferred 3 structural domains, hybrid protein or chimeric protein, for example the active chimeric or heterozygosis Cry1A albumen that increases of bollworm is (as US 6,962,705 or US 7, described in 070,982).This definition also comprises NCBI accession number AAA22331 (Cry1Ac1), NCBI accession number AAA22330 (Cry1Ab, people such as Wabiko, 1986) aminoacid sequence in, or the variant of the proteic aminoacid sequence of Cry1A.105 described in the non-control state of the USDA APHIS application 06-298-01p, for example on amino acid sequence level with described Cry1Ac, Cry1A.105 or Cry1Ab albumen (particularly in the part corresponding to minimum toxic fragment) have at least 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity is (as utilizing the GAP program (Madison of GCG Wisconsin software package, Wisconsin, USA, 10.2 editions), use comparison in pairs to measure), protein with the proteic minimum toxic fragment of Cry1A.Parameter was used for aminoacid sequence relatively below the GAP program was used: ' blosum62 ' rating matrix, ' room generation point penalty ' (or ' room weight ') of 8 and ' room extension point penalty ' (or ' length weight ') of 2.Preferably, this definition comprise have, preferred 5-10, particularly this proteinic paddy reality noctuid of not obvious change or boll fruit armyworm kill the protein of insect active less than the amino acid of 5 interpolation, displacement or disappearance, for example (in order for example to clone purpose) have one or more conservative amino acid metathetical Cry1A albumen.

Being used for the proteic example of Cry1A of the present invention comprises by US 6,114, the Cry1Ab albumen of 608 SEQ IDNO:3 coding, particularly (US 6 by corn event MON810,713,259, USDA APHIS does not remove control state application 96-017-01p and replenishes) in the Cry1Ab albumen of cry1Ab coding region coding, (USDA APHIS does not remove control state application 95-195-01p by corn event Bt11, US patent 6,114, the Cry1Ab albumen of the cry1Ab coding region coding 608), (US 7 by cotton event 3006-210-23,179,965, WO 2005/103266, USDAAPHIS does not remove control state application 03-036-02p) in the Cry1Ac albumen of transgenes encoding, (USDA APHIS does not remove control state application 07-108-01p by cotton event COT67B, WO2006/128573) the cry1Ab coding region in, be included in the Cry1Ab coding region among the cotton event EE-GH5 (describing among PCT patent application PCT/EP2008/002667 (not announcing)), US patent 7, the Cry1Ab albumen of the Cry1Ab coding region coding of 049,491 SEQ ID No.2, by corn event MON89034 (the non-control state of USDA APHIS application 06-298-01p, WO2007/140256, the Cry1A.105 albumen of the Cry1A transgenes encoding the SEQ ID NO:2 or 4 among the WO 2007/027777), by cotton event 15985 or cotton event 531,757 or 1076 (the non-control state of USDA APHIS application 94-308-01p, the Cry1Ac sample albumen of the heterozygosis cry1Ac coding region coding the chimeric Cry1Ac albumen of the cry1A cotton event coding of WO 2002/100163), by cotton event T342-142,1143-14A, 1143-51B, CE44-69D or CE46-02A (see WO 2006/128568 respectively, WO 2006/128569, WO 2006/128570, WO 2006/128571 or WO 2006/128572) in the cry1Ab albumen of cry1Ab coding region coding (promptly by WO 2006/128568, WO 2006/128569, the DNA of SEQ ID No.7 among WO 2006/128571 or the WO2006/128572 or by the protein of the dna encoding of the SEQID No.5 among the WO 2006/128570).In one embodiment of the invention, use from above-listed Cry1Ab or Cry1A.105 albumen comprise the protein of its minimum toxic fragment or comprise these Cry1A albumen arbitrary minimum toxic fragment protein or to have 1-5 amino acid different and to arbitrary variant of real noctuid of paddy or toxic these Cry1A albumen of boll fruit armyworm reservation.

In the present invention, find: the Cry2Ae binding site identical with the Cry2Ab protein competition in boll fruit armyworm with Cry2Aa albumen, and this binding site is different from the binding site of (promptly not sharing) Cry1Ac in real noctuid of paddy and boll fruit armyworm.In addition, Cry1Ac does not compete to the combination of Cry2Ab yet in these insect species.And, be reported in Cry1F and Cry1Ac and Cry1Ac and Cry1Ab shared binding site (for example, Hernandez and Ferre, 2005, people such as Karim, 2000b in real noctuid of paddy or the boll fruit armyworm already; People such as Estela, 2004).Therefore, in real noctuid of paddy or boll fruit armyworm Cry1Ab, Cry1F and Cry1Ac in conjunction with the binding site that is different from the binding site of Cry2Ae or Cry2Aa.In addition, VIP3A albumen and Cry2Ab have been reported in conjunction with different binding sites (people such as Lee, 2006).Because Cry2Aa and Cry2Ae and Cry2Ab share the common binding site, thus in paddy reality noctuid and boll fruit armyworm Cry2Ae or the Cry2Aa albumen binding site different with the VIP3A protein binding.Although compare with the Cry1A that is tested, VIP3A or Cry2A albumen, usually Cry1F albumen has lower activity to these insect species, but they belong to most popular Cry1 albumen in plant, and because they and Cry2A albumen do not have shared binding site, so if plant can provide enough high-caliber Cry1F protein expression natch, they also can be used for the insect-resistant management so.Number of C ry1F source protein has higher intrinsic activity to real noctuid of paddy or boll fruit armyworm, and these are preferred Cry1F albumen among the present invention.When selecting between Cry1F and the Cry1A albumen when in given plant species, making up with Cry2A albumen, for example Cry1Ab or Cry1A.105 albumen have higher intrinsic toxicity for real noctuid of paddy and boll fruit armyworm insect species to consider Cry1A albumen, for delaying or preventing that for the resistance development of real noctuid of paddy or boll fruit armyworm, Cry1A albumen will be better choice.

Bt Cry albumen for example Cry1F, Cry2A and Cry1A albumen in its natural host cell (bacillus thuringiensis (Bacillus thuringiensis)) with the parent toxin formal representation, they change into the toxin form by proteolysis in the insect intestines.Cry1F used herein, Cry2A or Cry1A albumen are meant total length parent toxin or toxin, or anyly have an extremely intermediate forms of insect active.In one embodiment, Cry1F albumen comprises the amino acid position 29 that the comprises NCBI accession number AAA22347 protein to the aminoacid sequence of amino acid position 604, and Cry1A albumen comprises and comprises NCBI accession number AAA22331 (Cry1Ac1; People such as Adang, the aminoacid sequence of amino acid position 29 to 607 1985), comprise NCBI accession number AAA22330 (people such as Cry1Ab, Wabiko, 1986) amino acid position 29 to the aminoacid sequence of amino acid position 607 or comprise the protein of the amino acid position 29 of figure IV-1 among the non-control state of the USDAAPHIS application 06-298-01p to the aminoacid sequence of amino acid position 607.Cry2A albumen used herein comprises and comprises US patent 7, the protein of the aminoacid sequence of the amino acid position 50 to 625 of 265,269 SEQ ID No.2, comprise among the non-control state of the USDAAPHIS application 06-298-01p figure IV-2 amino acid position 81 to 746 aminoacid sequence protein or comprise the protein of the amino acid position 50 of NCBI accession number CAA39075 to the aminoacid sequence of amino acid position 626.

" Cry1 " used herein albumen is meant Cry1F or Cry1A albumen as defined above." Cry2A " used herein albumen not only refers to Cry2Ae or Cry2Ab albumen as defined above, and can refer to that any among the people (2008) such as Crickmore has the Cry2A albumen of insecticidal, for example Cry2Aa albumen to the real noctuid of paddy or boll fruit armyworm.VIP3 used herein or cry1 " gene " or " DNA " are meant code book invention VIP3 or the proteic DNA of Cry1.Gene can all or part ofly be naturally occurring, artificial (modification) or synthetic.

Term used herein " incident " (event) is meant: the specific position specificity in Plant Genome is integrated one or more transgenosiss, and it can be considered to comprise the part of the DNA of the sequence inserted and flank plant sequence.Incident can enter in a lot other plants of same species or in the plant of different plant species by hybridization, thereby allows by breeding technique (comprising for example embryo rescue of technology) and comprise the plant hybridization of incident.

" comprising " used herein should be interpreted as referring to exist described feature, integer, step or the component of mentioning, but do not get rid of the existence or the interpolation of one or more further features, integer, step or component or its group.Therefore, term used herein " the DNA/ protein that comprises sequence or regional X " is meant that this DNA or protein include or contain sequence or regional X at least, can comprise other Nucleotide or aminoacid sequence at 5 ' (or N-terminal) and/or 3 ' (or C-terminal) end thus, for example transit peptides (nucleotide sequence) and/or 5 ' or 3 ' leader sequence.

Coding VIP3 used herein or Cry are proteic, and " mosaic gene " is meant the DNA (or coding region) of such coding VIP3 or Cry, its 5 ' and/or 3 ' regulating and controlling sequence, at least 5 ' regulating and controlling sequence or promotor, be different from this VIP3 or the proteic natural host cell of Cry the natural bacteria 5 that drives this VIP3 or Cry protein expression ' and/or 3 ' regulating and controlling sequence, but the VIP3 or the cry DNA that can be operatively connected with the promotor of expression of plants for example, described thus mosaic gene can be expressed in containing its plant.Mosaic gene need not all express in free or each cell plant, for example, use wound-induced type promotor, expression can be by insect gnaw or injure induce, perhaps express and to be confined to mainly by insect those plant parts of attacking of the real noctuid of paddy or boll fruit armyworm larva for example, particularly those are to grower or the most valuable part of peasant, the leaf of the leaf of for example Ye Hesui of maize plant, or vegetable lamb and cotton boll or soybean plants and beanpod.Therefore, expressing the proteic plant of VIP3 used herein, Cry2A, Cry1F or Cry1A is meant, but the plant that comprises necessary this type of proteic expression of plants mosaic gene of coding, this albumen is expressed in related tissue or at relevant time bar thus, and it need not to express in all plant tissues or need not to express in all time.

Be purpose of the present invention, be expressed as two associated nucleotides of per-cent or " the sequence identity " of aminoacid sequence and be meant that the number of position that has identical residue in these two sequences of the best comparison is divided by the positional number that is compared (* 100).Regard room (that is, in comparison,, in a sequence, have residue and in another sequence, do not have residue) as position with different residues in this position.For calculating according to the sequence identity between two sequences of the present invention, can utilize and use Needleman and Wunsch algorithm (1970) and by the Wisconsin Package, 10.2 version, GeneticsComputer Group (GCG), 575Science Drive, Madison, Wisconsin 53711, the GAP program that USA provides.Used GAP parameter is that the room generates point penalty=50 (Nucleotide)/8 (amino acid), point penalty=3 (Nucleotide)/2 (amino acid) and rating matrix " nwsgapdna " (Nucleotide) or " blosum62 " (amino acid) are extended in the room.

GAP uses Needleman and Wunsch overall comparison algorithm to carry out the comparison of two sequences on the sequence total length, makes the matching number maximum and makes room number minimum.Default parameters is that the room generates point penalty=50 (Nucleotide)/8 (albumen) and point penalty=3 (Nucleotide)/2 (albumen) are extended in the room.For Nucleotide, the acquiescence rating matrix of use is " nwsgapdna ", and for protein, the acquiescence rating matrix is " blosum62 " (Henikoff ﹠amp; Henikoff, 1992).

VIP3 or Cry DNA comprise in this article: those coding VIP3 or the proteic DNA of Cry or its variant or heterozygotes, wherein said variant or heterozygote to the real noctuid of paddy or boll fruit armyworm has insecticidal and under stringent hybridization condition with codified VIP3 or the proteic DNA hybridization of Cry." stringent hybridization condition " used herein is meant following condition especially: fixing relevant DNA on filter membrane, and 42 ℃, in 50% methane amide, 5%SSPE, 2x Denhardt reagent and 0.1%SDS prehybridization filter membrane 1 to 2 hour, perhaps 68 ℃, in 6x SSC, 2x Denhardt reagent and 0.1%SDS prehybridization filter membrane 1 to 2 hour.Then sex change (digoxigenin-or radiation-) label probe is directly added in the prehybridization solution, hatched 16 to 24 hours at above-mentioned proper temperature.After hatching, room temperature, in 2x SSC, 0.1%SDS washing filter membrane 30 minutes, then in 68 ℃, 0.5x SSC and 0.1%SDS, carry out each 2 washing of 30 minutes.By making filter membrane be exposed to X-ray film (Kodak XAR-2 or equivalent) 24 to 48 hours with intensifying screen, finish the radioautograph imaging at-70 ℃.[20x SSC=3M NaCl and 0.3M Trisodium Citrate; 100x Denhart reagent=2% (w/v) bovine serum albumin, 2% (w/v) FicollTM and 2% (w/v) polyvinylpyrrolidone; The SDS=sodium lauryl sulphate; 20x SSPE=3.6M NaCl, 0.2M sodium phosphate and 0.02MEDTA pH7.7].Certainly, can use the condition and the parameter that are equal in the method, still keep the stringent hybridization condition of expectation simultaneously.

Proteinic " kill insect active " used herein refers to,---preferably realizes---ability that this protein can kill insects when this protein of feeding insect by for example expressing this protein in the plant at recombinant host.Should be understood that if protein is at least one etap of insect, preferably larval stage has the ability of kill insects this protein has insect active extremely so.

As used herein, insect species colony is meant the plant of expressing insecticidal protein (this plant controlled in the past or killed described insect colony) " having produced resistance " or " having become resistance ", the production loss level of this plant of being caused by identical insect species when introducing this plant is first compared, and detects the multiple and the remarkable unacceptable production loss that are caused by this insect colony in this plant.Must verify this, in fact need more the insecticidal protein of a large amount to control or kill to check member that plant is in fact producing insecticidal protein (being that they are not non-transgenic plants) and this insect colony.In other words, insect colony no longer produces insect manipulated variable (as defined herein) or no longer this insect species colony is had insecticidal the plant that it produces resistance.Therefore, herein, " insect-resistant development " causes detecting the plant damage of increase.In one embodiment, if can on plant, finish their life cycle and continue the infringement plant from the insect of insect species colony, rather than because the insecticidal protein that produces and cause their growth and feed habit to be suppressed (in an extreme form of insect-resistant in this plant, under attack of insect, this plant can be subjected to the infringement as the conventional non-transgenic plant with identical genetic background), then the insect-resistant of this insect species colony is observed easily.In one embodiment, can use in conjunction with test real noctuid of paddy or boll fruit armyworm BBMV to analyze combining of Cry or VIP3 albumen and resistant insects, modify owing to binding site to confirm this resistance in (standard) competition.

This paper also comprises aforesaid method, purposes, plant or vegetable cell, wherein except Cry or VIP3 albumen, the Bt toxin strengthens albumen also expresses in described plant, it is protein or its fragment that wherein said Bt toxin strengthens albumen, promptly, the part of Bt toxoreceptor in the insect preferably comprises or corresponding to the part of binding domains, for example proteic fragment of cadherin sample.This proteinoid is recorded in the disclosed US patent application 20090018075.These Bt toxin can be strengthened albumen with one or more Bt kill insect toxins for example Cry albumen feed to targeted insect.These Bt toxin strengthen albumen can strengthen the source insect species of Bt insecticidal protein opposing this receptor and the toxin activity of resisting other insect species.In one embodiment, described Bt toxin strengthens the part that albumen is the midgut epithelial cells Bt toxoreceptor of real noctuid of paddy or boll fruit armyworm insect.

" paddy real noctuid " used herein (H.zea) is meant real noctuid (the Helicoverpa zea of paddy, Boddie), a kind of important lepidoptera pest is also referred to as (America) bollworm, corn earworm (corn earworm) or tomato fruitworm (tomato fruitworm), Chinese sorghum ear worm (sorghum headworm) or common vetch worm (vetchworm).This insect is a corn, important insect in cotton and the tomato, and attack for example following plant: choke (artichoke), asparagus, Caulis et Folium Brassicae capitatae, netted melon (cantaloupe), kale, cowpea, cucumber, eggplant, lettuce, lima bean (lima bean), muskmelon, gumbo, pea, pepper, potato, pumpkin, snap beans (snap bean), spinach, summer squash (squash), sweet potato (sweet potato), watermelon, alfalfa, clover (clover), flax, oat, grain (millet), rice, Chinese sorghum, soybean, sugarcane, Sunflower Receptacle, tobacco, common vetch (vetch) and wheat.

" boll fruit armyworm " used herein (H.armigera) is meant boll fruit armyworm (Helicoberpa armigera, Hiibner), a kind of important lepidoptera pest, it is also referred to as (Africa) bollworm, tomato grub (tomato grubworm), tomato aphid (tobacco budworm), corn earworm (corn earworm), old world bollworm or scarce bordered straw, and it is one of the insect with the most hosts property of strong transport property.This insect is an insect important in corn and the cotton, but it also attacks plant, for example tobacco, Sunflower Receptacle, Semen Lini, soybean, clover (Lucerne), pea for example Caulis et Folium Brassicae capitatae, eggplant (aubergine), capsicum, tomato and cucumber of pigeonpea or garbanzo, red pepper (chili), gumbo, carnation, Flos Pelargonii and other ornamental plant or flowers crop, fruit, vegetables for example.

" bollworm " (the cotton bollworm/bollworm) that uses with general meaning is meant real noctuid of paddy and/or boll fruit armyworm herein.

Proteinic " insect manipulated variable " used herein is meant that insect (for example insect larvae) infringement that causes that is enough to the plant being food is restricted to the protein content of commercial acceptable level on this plant, for example by kill insects or by suppressing insect growth, reproductivity or growth, insect is diminished to the infringement of plant and make plant biomass not be subjected to remarkable disadvantageous effect.

In one embodiment of the invention, VIP3 of the present invention and/or Cry albumen are expressed being used for plant of the present invention with high dosage.When mentioning when being used for plant of the present invention, statement used herein " high dosage " is meant, the concentration of insecticidal protein in plant (is determined as the per-cent of total soluble protein matter by ELISA, after wherein in standard extraction damping fluid, extracting soluble protein, use Bradford to analyze (Bio-Rad, Richmond, CA; Bradford, 1976) measure this total soluble protein matter), described concentration can kill at least 95% for the insect in the following etap of targeted insect, insect in this etap is compared significantly lower to the susceptibility of insecticidal protein and the 1 instar larvae phase of this insect (first larval stage), preferred low at least 25 times (can in standard insecticidal protein biological assay test, analyze), and expect that therefore this concentration can guarantee the control fully to the targeted insect species.

" structure sanctuary (structured refuge) " used herein is meant, a growing area of grower or peasant's plantation Bt plant or the part in the field, this part is not planted the Bt plant, do not contain the genetically modified plant of Bt (relatively, around the weeds of peasant's growing area or other non-Bt plant as natural sanctuary) but planted.

Being used for estimating and develop at least two kinds kills insect genes and is used for preventing to be found in disclosed European patent application EP 408403 at the general procedure of the resistance development of the transgenic plant of expressing these genes at targeted insect.The definition that is used for the receptor binding assay field can see Www.unmc.edu/ Pharmacology/receptortutorial/definitions/definitions.ht m

According to the present invention, studied the combining of midgut epithelial cells brush border membrane of Cry albumen and real noctuid of paddy or boll fruit armyworm insect larvae.Brush border membrane is the proteic main target spot of VIP3 or Cry, and film vesicle, preferred source is from insect midgut brush border membrane (for the brush border membrane vesicle, this paper is called BBMV), can according to methods known in the art for example people (1987) such as Wolfersberger obtain.

The present invention relates in transgenic plant combination and express at least two kinds of insecticidal protein genes to delay or to prevent resistance development in real noctuid of targeted insect paddy or the boll fruit armyworm colony.Described gene is inserted in the vegetable cell genome, preferably is inserted in its cell nucleus gene group, and the feasible gene that is inserted is positioned at the downstream of promotor and is operably connected with promotor, and wherein said promotor can instruct gene to express in vegetable cell.

In one embodiment of the invention, the plant that the real noctuid of paddy or boll fruit armyworm is had durable resistance is provided, and described plant comprises Cry2A albumen that coding has an insecticidal to the real noctuid of paddy or boll fruit armyworm, and for example Cry2Ab or the proteic mosaic gene of Cry2Ae and coding have Cry1A, the VIP3 of insecticidal and/or Cry1F albumen, the preferred proteic mosaic gene of Cry1Ab, VIP3A or Cry1A.105 as defined above to the real noctuid of paddy or boll fruit armyworm.

This paper also provides: real noctuid of control paddy or boll fruit armyworm are invaded and harassed and are obtained the real noctuid of paddy or the boll fruit armyworm insect method of accumulation more slowly to the resistance development of described plant simultaneously in transgenic plant, and it is included in and expresses Cry2Ae albumen and the b that a) described insect species is had insecticidal in the described plant) described insect species is had Cry1A, Cry1F or a proteic combination of VIP3A of insecticidal; Be used for preventing or delay the real noctuid of insect species paddy or boll fruit armyworm colony at the insect-resistant development of the transgenic plant of expressing insecticidal protein controlling the method for described insect evil, it is included in and expresses Cry2Ae albumen that the real noctuid of paddy or boll fruit armyworm are had an insecticidal and Cry1A, the Cry1F or the proteic combination of VIP3A that the real noctuid of paddy or boll fruit armyworm are had insecticidal in the described plant.

In one embodiment of the invention, provide in the real noctuid of paddy or boll fruit armyworm insect colony to expressing the method that the proteic plant of VIP3A, Cry1F and/or Cry1A produces the described insect species of control in the area of resistance, it is included in the described area sowing or plantation and expresses at least real noctuid of paddy or boll fruit armyworm are had the step of the proteic plant of Cry2Ae of insecticidal.This paper also provides, to expressing the method that the proteic plant of Cry2Ae produces the described insect of control in the area of resistance, it is included in the described area sowing or plantation and expresses real noctuid of paddy or boll fruit armyworm are had Cry1F, the VIP3 of insecticidal or the step of the proteic plant of Cry1A in the real noctuid of paddy or boll fruit armyworm colony.

Also provide according to the present invention, be used to obtain to comprise the method for plant of the mosaic gene of at least two kinds of different insecticidal proteins of coding, wherein as measure in conjunction with test in the competition of the brush border membrane vesicle that uses real noctuid of species paddy or boll fruit armyworm insect larvae, described protein does not have shared binding site in described insect larvae, said method comprising the steps of: but obtain to comprise coding real noctuid of paddy or boll fruit armyworm are had the proteic expression of plants mosaic gene of Cry2Ae of insecticidal and coding has insecticidal to real noctuid of paddy or boll fruit armyworm Cry1A, but the plant of the proteic expression of plants mosaic gene of VIP3 or Cry1F.

Also provide: sowing herein; the method of plantation or growing plant; described plant is protected and avoid bollworm infringement; it comprises the mosaic gene of at least two kinds of different insecticidal proteins of expression; wherein as measure in conjunction with experiment in the competition of using real noctuid of paddy or boll fruit armyworm larva brush border membrane vesicle; described albumen does not have shared binding site in the larva of described insect species, said method comprising the steps of: sowing; plantation or growth comprise coding real noctuid of paddy or boll fruit armyworm are had the proteic mosaic gene of Cry2Ae of insecticidal and coding has insecticidal to real noctuid of paddy or boll fruit armyworm Cry1A; VIP3 or Cry1F albumen; the plant that preferably the real noctuid of paddy or boll fruit armyworm is had the VIP3 or the proteic mosaic gene of Cry1A of insecticidal.

This paper also provides: at least two kinds of different insecticidal proteins are used for preventing or delaying the purposes of the insect-resistant development of real noctuid of paddy or boll fruit armyworm colony in transgenic plant, wherein as passing through competition in conjunction with measuring, described albumen does not have shared binding site in the insect midgut of described insect species, described application comprises: express in described transgenic plant the real noctuid of paddy or boll fruit armyworm are had the Cry2Ae albumen of insecticidal and the real noctuid of paddy or boll fruit armyworm had Cry1F, VIP3 or the Cry1A albumen of insecticidal; With coding real noctuid of paddy or boll fruit armyworm had the proteic mosaic gene of Cry2Ae of insecticidal and coding has insecticidal to real noctuid of paddy or boll fruit armyworm Cry1F, the proteic mosaic gene of VIP3 or Cry1A, proteic mosaic gene of Cry2Ae and the coding that the real noctuid of paddy or boll fruit armyworm are had an insecticidal of particularly encoding has the purposes of the VIP3 or the proteic mosaic gene of Cry1A of insecticidal to real noctuid of paddy or boll fruit armyworm, be used for preventing or delay the real noctuid of insect species paddy or boll fruit armyworm colony to the insect-resistant development of the transgenic plant of expressing insecticidal protein to control described insect evil.

In the embodiment of this paper, provide: real noctuid of paddy or boll fruit armyworm are had the Cry2Ae albumen of insecticidal and described species insect is had Cry1A, the VIP3 of insecticidal or the purposes of the proteic combination of Cry1F, be used to prevent or delay the resistance development that described species insect kills the transgenic plant of insect toxins to the expression heterology, particularly when described application is undertaken by express described protein combination in plant.

This paper also provides: comprise the proteic plant of Cry2Ae that the real noctuid of paddy or boll fruit armyworm are had an insecticidal in the colony of described insect species to comprising the application in the area that the proteic plant of Cry1F, VIP3 and/or Cry1A produces resistance, wherein said application can be included in sowing in the described area, plantation or growth and comprise the proteic plant of Cry2Ae that the real noctuid of paddy or boll fruit armyworm is had insecticidal; With comprise to the real noctuid of paddy or boll fruit armyworm have Cry1F, the VIP3 of insecticidal and/or the proteic plant of Cry1A in the colony of described insect species to comprising the application in the area that the proteic plant of Cry2Ae produces resistance, wherein said application can be included in sowing in the described area, plantation or growth and comprise Cry1F, VIP3 and/or the proteic plant of Cry1A that the real noctuid of paddy or boll fruit armyworm is had insecticidal.

The present invention also provides such application, wherein sow, plant or grow to have not comprise real noctuid of paddy or boll fruit armyworm are had Cry2, the Cry1 of insecticidal or the sanctuary of the proteic plant of VIP3, for example by with this plant seeding, plant or be grown in the identical growing area that comprises Cry2Ae as herein described, VIP3 and the proteic plant of Cry1 or near.

This paper also provides such use or method, and wherein plant is to express Cry2Ae, VIP3, Cry1F or Cry1A albumen at the high dosage of real noctuid of paddy or boll fruit armyworm, and wherein said high dosage as defined herein.

This paper also provides: growth, sowing or plantation expression Cry albumen or the proteic plant of VIP3 are with the method for control boll fruit armyworm or the real noctuid insect of paddy, it comprises step: plant in this growing area or near this growing area, sowing or growth less than the sprinkling of 20% growing area the structure sanctuary of sterilant, or less than the structure sanctuary of not spraying sterilant of 5% growing area, or in growing area, do not plant, sowing or growth sanctuary, wherein said structure sanctuary is positioned at identical growing area, perhaps be positioned at 2 miles of growing area, in 1 mile or in 0.5 mile, and contain and do not comprise this Cry or the proteic plant of VIP3, wherein said expression Cry albumen or the proteic expression of plants of VIP3 have the Cry2Ae albumen of insecticidal to described insect species and described insect species are had the Cry1A of insecticidal, Cry1F or VIP3A albumen, particularly Cry2Ae and Cry1Ab or Cry1Ac or VIP3A albumen, preferred Cry2Ae and Cry1Ab and the proteic combination of VIP3.

In one embodiment of the invention, also provide specifically and saturable ground in conjunction with the purposes of at least 2 kinds of insecticidal proteins of the binding site in the real exigua larvae midgut of paddy, be used to delay or prevent the resistance development of this insect species the plant of expressing insecticidal protein, be the Cry2A albumen that this insect species is had insecticidal wherein at one of protein described in the described plant, Cry2Ab albumen for example, and another kind of protein is the Cry1A that this insect species is had insecticidal, Cry1F or VIP3 albumen, wherein in the saturability test, determine described saturable combination, the binding site (being BBMV) of fixed concentration is used in described test, and adds the cumulative labelled protein of concentration to described binding site.Especially, in this purposes, Cry1A albumen is selected from down group: Cry1Ac, Cry1Ab, Cry1A.105 or Cry1Ac or Cry1Ab hybrid protein, for example the cry1A coding region encoded protein matter of being mentioned by any this paper.There are not the competition to their (saturable and special) binding site in this Cry2Ab and Cry1A albumen in the midgut of the real noctuid insect larvae of paddy, as measuring in conjunction with in testing in the BBMV competition.

This paper also provides and comprises at least 2 genetically modified plants or seed, each transgenosis different protein that the real noctuid of paddy or boll fruit armyworm had insecticidal of respectively encoding wherein, described protein saturable ground and specifically in conjunction with the binding site in this type of insect midgut, wherein said albumen same binding site of competing phase not in this type of insect, and wherein said protein is i) Cry2A albumen and ii) Cry1A, Cry1F or VIP3 albumen.In one embodiment, described plant comprises the following proteic transgenosis of coding: i) Cry2Aa, Cry2Ab or Cry2Ae and ii) Cry1Ab, Cry1Ac, Cry1Fa or VIP3A, particularly Cry2Ae albumen and Cry1Ab and/or VIP3A albumen.In another embodiment, described plant or seed are to comprise coding Cry1A, proteic mosaic gene of Cry1F or VIP3 and coding Cry2A albumen, the particularly corn of the proteic mosaic gene of Cry2Ae or vegetable lamb or seed, wherein said plant or seed comprise the transformation event that is selected from down group: corn event MON89034, corn event mir 162, comprise the proteic genetically modified corn event of coding Cry2Ae, corn event TC1507, corn event Bt11, corn event MON810, cotton event EE-GH6, cotton event COT102, cotton event COT202, cotton event COT203, cotton event T342-142, cotton event 1143-14A, cotton event 1143-51B, cotton event CE44-69D, cotton event CE46-02A, cotton event COT67B, cotton event 15985, cotton event 3006-210-23, cotton event 531, cotton event EE-GH5, cotton event 281-24-236, all incidents such as this paper further define.

In one embodiment of the invention, in purposes of the present invention, method or plant, Cry2Ae, Cry2Ab, VIP3, Cry1F or Cry1A mosaic gene are included in the mosaic gene among above-mentioned specific corn or cotton event arbitrary.According to the present invention, also comprise: term Cry2Ae wherein is by term Cry2Aa or Cry2Ab alternate any purposes as herein described, method, plant or seed; And relate to Cry2Ae, Cry2Aa or the proteic any such use of Cry2Ab, method, plant or seed, wherein the combining of midgut BBMV of real noctuid of this Cry2A albumen and paddy or boll fruit armyworm is specific and saturable, particularly when direct saturability in conjunction with test in definite saturable in conjunction with the time; Preferred such purposes, method, plant or seed, wherein in standard competition combination test as herein described, in boll fruit armyworm or the real noctuid of paddy, all there is not biology to compete significantly between any described Cry2A albumen and Cry1A, Cry1F or the proteic specificity combination of VIP3.

This paper also provides: be used to make expression the real noctuid of paddy or boll fruit armyworm to be had the method that the plantation of proteinic plant of insecticidal or commercialization obtain to decontrol or obtain supervision department's approval; Or be used to reduce and contain the method for structure sanctuary that the real noctuid of paddy or boll fruit armyworm is had any proteinic plant of insecticidal that do not produce; Or be used to plant the method for growing area with structure sanctuary, said method comprising the steps of: quote, submit to or rely on insect test binding data, Cry1A, Cry1F or the proteic binding site of VIP3 are not competed, data for example disclosed herein or the class likelihood data of reporting in conjunction with insect midgut film and the described Cry2A albumen of described insect in wherein said data presentation Cry2A protein-specific and saturable ground in other file in described insect.In one embodiment, Cry2A albumen is Cry2Aa, Cry2Ab or Cry2Ae albumen, and Cry1A albumen is Cry1Ac, Cry1Ab or Cry1A.105 albumen, and VIP3 albumen is VIP3Aa albumen.

Kill the insect significant part in order in intestinal bacteria, other Bt bacterial strain and plant, to express coding Cry2A, VIP3 or the proteic full length DNA sequence of Cry1 or its, can import suitable restriction site at the flank of this dna sequence dna.This can use well-known method to carry out (people such as Stanssens, 1989 by site-directed mutagenesis; People such as White, 1989).Express in order in plant, to obtain enhanced, can announce WO 91/16432 and WO 93/09218 and announcement EP 0 385 962, EP 0 359 472 and US 5 according to PCT, 689,052, gene of the present invention or the codon that kills insect effective gene part used modify to form equivalence, that modify or artificial gene or Gene Partial, maybe this gene or Gene Partial can be inserted in the genome of plastid, plastosome or chloroplast(id), and use suitable promotor (for example to express at this place, people such as Mc Bride, 1995; US patent 5,693,507, WO 2004/053133).

Because the degeneracy of genetic codon, some amino acid code can be replaced into other amino acid code and not change proteinic aminoacid sequence, and, some amino acid can be equal to amino-acid substitution by other, and significantly do not change, preferably do not change, the proteinic insect active that kills does not change the proteinic insect active that kills in the mode of negative sense at least.For example, as long as the proteinic insect active that kills of not obvious reduction, the conservative amino acid displacement in alkalescence (for example Arg, His, Lys), acid (for example Asp, Glu), nonpolar (for example Ala, Val, Gly, Leu, Ile, Met) or polarity (for example Ser, Thr, Cys, Asn, Gln) classification falls within the scope of the present invention.In addition, as long as the proteinic insect active that kills of not obvious reduction, the non-conservation amino-acid substitution also falls within the scope of the present invention.The variant of dna sequence dna of the present invention or equivalent comprise with being used for the proteic natural gene of Cry2A of the present invention, VIP3, Cry1F or Cry1A to be compared and has different codon and use, but coding has identical protein DNA sequence of killing the insect active and the aminoacid sequence of substantially the same (preferably identical).Utilize obtainable codon use table, use as most preferred codon in the plant gene by codon being used adaptability revision, the codon that particularly originates in the gene of purpose plant genus or species uses (Bennetzen ﹠amp; Hall, 1982; People such as Itakura, 1977), can carry out codon optimized (for example making it more be adapted in corn, cotton, rice, soybean, Chinese sorghum, tomato, Sunflower Receptacle or sugarcane, express) to dna sequence dna.The codon use table that is used for various plant species is open by people (2000) such as for example Ikemura (1993) and Nakamura.

In order for example to obtain the enhanced expression in corn, sugarcane or the rice, also intron, preferred unifacial leaf intron can be added in the mosaic gene in monocotyledons.For example, show intron to the 5 ' control region that inserts corn Adh1 gene, can strengthen expression in corn people such as (, 1987) Callis.Similarly, can use US 5,859, the HSP70 intron of describing in 347 is expressed to strengthen.Can be to translate neutral mode, insert and/or change by intron fixed point and (for example codon is used the most preferably codon use that is adjusted to plant (preferred concrete related objective plant species/genus) (people such as Murray by in codon uses, importing, 1989) and the coded aminoacid sequence of not obvious change (preferably not changing)), further change the insecticidal protein gene or it kills the dna sequence dna of insect part, be present in possible inhibition dna sequence dna in this Gene Partial with modification.

In one embodiment of the invention, make bollworm to VIP3, Cry2A, Cry1F or Cry1A albumen sensitivity (real noctuid of paddy or boll fruit armyworm) and insect manipulated variable, preferably kill these proteinic combinations that insect measures and contact, for example can by in the target plant of these mythimna separatas, express these protein or by transforming plant so that these plants and offspring thereof comprise these proteic mosaic genes of coding, realize described contact.In one embodiment, the target plant of these mythimna separatas is corn, cotton, rice, soybean, Chinese sorghum, tomato, Sunflower Receptacle or sugarcane plants, particularly in the North America, Sino-U.S. and state, South America country.Term plant used herein comprises the part of complete stool plant and plant, for example leaf, stem, flower or seed.In one embodiment, in this type of target plant combination of the present invention at least 3 kinds in real noctuid of paddy or boll fruit armyworm in conjunction with the different proteins of different binding sites, described combination is carried out in the following way: to the target plant provide coding these proteic must mosaic gene, described albumen is for example Cry or the proteic any following combination of VIP3: Cry2Ab, Cry1Ab and VIP3A albumen; Cry2Ab, Cry1Ac and VIP3A albumen; Cry2Ab, Cry1F and VIP3A albumen; Cry2Ab, Cry1A.105 and VIP3A albumen; Cry2Ae, Cry1Ab and VIP3A albumen; Cry2Ae, Cry1A.105 and VIP3A albumen; Cry2Ae, Cry1Ac and VIP3A albumen; Or Cry2Ae, Cry1F and VIP3A albumen.

Can with coding Cry2A, VIP3, Cry1F or Cry1A proteic kill the insect significant part kill the insect effective gene, preferred mosaic gene stably be inserted in the cell nucleus gene group of single vegetable cell in a usual manner, and can use plant transformed cell thus to have the conversion plant of insect-resistant with generation in a usual manner.In this, can use the T-DNA carrier transformed plant cells of insect effective gene extremely that comprises in the agrobacterium tumefaciens (Agrobacteriumtumefaciens), after this for example can use and announce the method described in WO 84/02913 and the people (1991) such as disclosed European patent application EP 0 242 246 and Gould, from plant transformed cell regeneration plant transformed at EP 0 116 718, EP 0 270 822, PCT.The structure that is used for the T-DNA carrier of agriculture bacillus mediated Plant Transformation is well-known in the art.The T-DNA carrier can be the binary vector described in EP 0 120 561 and EP 0 120 515, perhaps can be incorporated into common integrative vector in the Agrobacterium Ti-plasmid by homologous recombination described in EP 0 116 718.Preferred T-DNA carrier all comprise between the T-DNA border sequence or be positioned at least right border sequence the left side, with kill the promotor that the insect effective gene can be operatively connected.Border sequence is described among the people such as Gielen (1984).Certainly, can use other bearer type, utilize following method to come transformed plant cells: for example direct gene shifts (for example describing) in EP 0223247, the conversion of pollen-mediated (for example in EP 0270356 and WO 85/01856, describing), protoplast transformation is (for example at US 4,684, describe in 611), the virus-mediated conversion of plant RNA is (for example at EP 0 067 553 and US 4,407, describe in 956), liposome-mediated conversion is (for example at US 4,536, describe in 475) and other method, it is that (for example, US 6 that being used to of for example describing recently transforms some corn plants, 140,553; People such as Fromm, 1990; People such as Gordon-Kamm, 1990) and rice strain system (people such as Shimamoto, 1989; People such as Datta 1990) method and the general method (PCT publication WO92/09696) that is used for transforming monocots.Transform the method described in the especially preferred PCT patent disclosure WO 00/71733 for cotton.Transform for rice, with reference to the method described in WO 92/09696, WO 94/00977 and the WO 95/06722.

Cry2A and VIP3, it is the most useful in the plant of bollworm target (or infringement) that the proteic combination of Cry1F or Cry1A is expressed in, and described plant comprises corn (feed corn and sweet corn), cotton, tomato, choke, asparagus, eggplant (aubergiens), Caulis et Folium Brassicae capitatae, netted melon, kale, cowpea, cucumber, eggplant (eggplant), lettuce, lima bean, muskmelon, gumbo, pea, pepper, potato, pumpkin, snap beans, spinach, summer squash, sweet potato, watermelon, alfalfa, clover, flax, oat, grain, rice, Chinese sorghum, soybean, sugarcane, Sunflower Receptacle, tobacco, common vetch, wheat, tobacco, Semen Lini, pea is pigeonpea or garbanzo for example, red pepper, gumbo, and carnation, Flos Pelargonii and other ornamental plant or flowers crop, or fruit crop; Preferred corn, cotton, rice, soybean, Sunflower Receptacle, tomato or sugarcane plants.Therefore, preferably in these plants any, be used in combination Cry2A albumen and VIP3, Cry1F or Cry1A albumen to delay or to prevent the resistance development of bollworm according to the present invention.Term used herein " corn " is meant Zea mays (Zeamays)." cotton " used herein is meant Gossypium species (Gossypium spp.), particularly upland cotton (G.hirsutum) and sea island cotton (G.barbadense).Term " rice " is meant Oryza species (Oryza spp.), particularly rice (O.sativa)." soybean " is meant Glycine species (Glycine spp), particularly soybean (G.max).Sugarcane is used in reference to saccharum (Saccharum) plant at this paper, and a kind of tall and big perennial gramineous grass originates in the temperate zone to tropical zone, can be used for extracting sugar.Sunflower Receptacle used herein is meant Sunflower Receptacle (Helianthus annuus).

Plant transformed can be used in the conventional plant breeding scheme, has more conversion plants of same characteristic features or partly is incorporated in other kind of identical or relative species will kill the insect effective gene with generation.Comprise as the insect effective gene extremely of stablizing the genome inset available from the seed that transforms plant.Can cultivate to produce Cry2A, VIP3 or Cry1 toxin or the proteinic insect significant part that kills the cell that transforms plant in a usual manner, it can be reclaimed with in the conventional insecticidal mixtures that is used in anti-lepidopterous insects then.

To kill the insect effective gene and insert in the vegetable cell genome, and can instruct this Gene Partial in the downstream of vegetable cell expression promoter (but expression of plants promotor) (promptly 3 ') so that the gene that inserts is arranged in, and under the control of this promotor.This preferably by in the vegetable cell genome, particularly in nucleus or plastid (for example chloroplast(id)) genome, insert mosaic gene and finish.

But can be used for expression of plants promotor of the present invention includes but not limited to: cauliflower mosaic virus (CaMV) isolate CM 1841 (people such as Gardner, 1981), CabbB-S (people such as Franck, 1980) and the strong composing type 35S promoter (" 35S promoter ") of CabbB-JI (Hull and Howell, 1987); By the described 35S promoter of people such as Odell (1985), from the promotor of ubiquitin family (for example, people such as Christensen 1992, the corn ubiquitin promoter of EP 0 342 926, also referring to people such as Cornejo, 1993), gos2 promotor (people such as de Pater, 1992), emu promotor (people such as Last, 1990), Arabidopsis (Arabidopsis) actin promoter is for example by the described promotor of people such as An (1996), the rice actin promoter is for example by described promotor of people such as Zhang (1991) and US 5, promotor described in 641,876; (WO 97/48819 for the cassava vein mosaic virus promotor, people such as Verdaguer (1998)), from pPLEX series startup (WO 96/06932, particularly S7 promotor) of underground trifolium stunt virus (Subterranean Clover Stunt Virus), alcoholdehydrogenase promotor pAdh1S (GenBank accession number X04049, X00581) and drive 1 of T-DNA ' respectively and TR1 ' the promotor of 2 ' genetic expression (people such as Velten, 1984) and TR2 ' promotor (being respectively " TR1 ' promotor " and " TR2 ' promotor ") for example.Perhaps, can utilize non-composing type but one or more tissues or the organ (for example leaf and/or root) of plant had specific promotor, the Gene Partial of inserting is only expressed in the cell of this particular organization or organ thus.For example, can partly place under the control of photoinduction type promotor by killing the insect effective gene, in the leaf of plant (for example corn, cotton, rice, soybean), optionally express and kill the insect effective gene, described photoinduction type promotor can be for example plant itself or another kind of plant, the ribulose-1,5-bisphosphate of pea for example, the promotor of 5-bisphosphate carboxylase small ylidene gene is seen US 5, disclosing in 254,799.For example, can select promotor to make gene of the present invention only express in those tissues that the target insect pest is gnawed or cell, so that compare with the plant of not expressing this gene, the feed of susceptibility target insect causes the insect damage of minimizing to this host plant.But another kind of alternatives is to use the promotor of abduction delivering, for example by the described MPI promotor of people such as Cordera (1994), it is induced by wound (for example wound that is caused by the feed of insect), or chemical-induced type promotor, for example by Aoyama and the described induced by dexamethasone type of Chua (1997) promotor, or temperature-induced type promotor, US5 for example, heat-shocked promotor described in 447,858, or can be by other external stimulus inductive promotor.

Can be inserted in the Plant Genome killing the insect effective gene, make the gene that inserts be positioned at the upstream of 3 suitable ' terminal transcriptional regulatory signal (being transcript formation and polyadenylation signal) (promptly 5 ').This preferably finishes by insert mosaic gene in the vegetable cell genome.The type that polyadenylation and transcript form signal is not crucial, can comprise CaMV 35S gene, nopaline synthase gene (people such as Depicker, 1982), octopine synthase gene (people such as Gielen, 1984) or T-DNA gene 7 (Velten and Schell, 1985) those, its in transformed plant cells as 3 '-the non-translation DNA sequence works.

Be used for that the selection of the marker gene of mosaic gene of the present invention neither be crucial, and can use the conventional dna sequence dna of following protein of any coding or polypeptide, described protein or polypeptide make the vegetable cell of expressing this dna sequence dna easily distinguish (EP0344029) with the vegetable cell of not expressing this dna sequence dna.This marker gene can be under the control of himself promotor, and has the 3 ' non-translation DNA sequence (as above open) of himself, and condition is near the locus of this marker gene gene of being in its discriminating the locus.Marker gene can for example be: herbicide resistance gene is sfr or sfrv gene (EPA 87400141) for example; The encode gene of modified weedicide target enzyme, the target enzyme of described modification is lower than natural (non-modified) target enzyme to the avidity of this weedicide, for example as modified 5-EPSP (U.S. Patent number 4,535,060 of glyphosate target spot; EP 0218571) or as the modified glutamine synthetase (EP 0240972) of glutamine synthetase inhibitor target spot; Or antibiotics resistance gene, for example (PCT announces WO 84/02913 to the neo gene; EP 0193259).

Can abide by different ordinary methods and express, see the summary among the EP 408403 (incorporating this paper into as a reference) to obtain the combination of at least two insecticidal protein genes in transgenic plant.These methods comprise: transform single-gene and hybridize these plants, make the plant hybridization that imported different required genes respectively, transform second gene again in the plant that has transformed a gene, use different plasmid cotransformation plants, be used at least two gene transformation on the transfering DNA so that these genes are inserted on the identical locus, use the translation fusion gene to be used for transforming (referring to for example, people such as Ho (2006)) etc. in different plants.

The transgenic plant of gained can be used for further plant breeding scheme.Can be with institute's plant transformed selfing to obtain the plant of isozygotying for inserted gene.If this plant is an inbred lines, this plant of isozygotying can be used for directly producing seed or is used to the kind that hybridizes as parent system so.Also can use the conventional plant breeding method, this gene recombination be gone in other inbred lines of free pollination colony or identical plant.

Owing to confirm at present convincingly, Cry2A albumen specifically and saturable ground in conjunction with the insect midgut of sensitive target insect pest, so can be by the means known in the art separation by Cry2A protein-specific bonded acceptor molecule.Therefore, the present invention also comprise be used for separation of C ry2A protein receptor, in particular as the method for protein of Cry2Ab acceptor, and this type of separated receptor protein multiple use that can have.This type of separated acceptor molecule can be used for shaker test, to find the not protein of isoacceptor of combination, acceptor is had the bonded protein of raising etc. with discovery.In addition, the dna sequence dna of this type of Cry2A acceptor can provide the screening implement of usefulness, is used for screening the variation (can indicate this proteinic variation that causes resistance development) of this type of DNA.Can use for example PCR of standard DNA testing tool, in the insect that collect in the field, carry out this screening apace.

Sequence table:

SEQ ID No.1:Cry2Ae1 albumen

SEQ ID No.2:Cry2Ab2 albumen

SEQ ID No.3:Cry1Fa1 albumen

SEQ ID No.4:VIP3Aa1 albumen

SEQ ID No.5:VIP3Af1 albumen

SEQ ID No.6:VIP3Aa19 albumen

SEQ ID No.7:VIP3Aa20 albumen

SEQ ID No.8:Cry1Ac1 albumen

SEQ ID No.9:Cry1A.105 albumen

SEQ ID No.10:Cry1Ab1 albumen

The following examples example the present invention, but be not intended to the present invention or its protection domain are construed as limiting.

Except having the explanation in addition in an embodiment, according to Sambrook and Russell (2001) " molecular cloning: laboratory manual " (Molecular Cloning:A Laboratory Manual), the third edition, Cold Spring Harbor Laboratory Press, NY; People such as Ausubel (1994) " modern molecular biology technique, modern technologies " (Current Protocols in Molecular Biology, Current Protocols), the 1st volume of USA and the 2nd volume; Brown (1998) " molecular biology of plants Labfax " (Molecular Biology LabFax), the 2nd edition, all recombinant DNA technologies are carried out in the standard method of describing among the volume I of Academic Press (UK) and the volume II.Being used for the standard material of molecular biology of plants work and method is described among " molecular biology of plants Labfax " (Plant Molecular Biology Labfax (1993)) (by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK combined publication) that R.D.D.Croy shows.The standard material and the method that are used for the polymerase chain reaction are found in Dieffenbach and Dveksler (1995) " PCR primer: laboratory manual " (PCR Primer:A Laboratory Manual), people (2000) PCR-Basics:From Background to Bench such as Cold Spring Harbor Laboratory Press and McPherson, first version, Springer Verlag is among the Germany.

Embodiment

Embodiment 1

1.1. materials and methods

The activation of toxin purifying and toxin

In 28.5 ℃ shaking continuously and additional air under, will be from Bacillus Genetic Stock Collection (Columbus, OH) the bacillus thuringiensis bacterial strain HD73 of expression Cry1Ac growth 48 hours in CCY substratum people such as (, 1981) Stewart.Sedimentary insoluble part with 1M NaCl, 10mM EDTA washed twice, is washed 1 time with 10mM KCl.The Cry1Ac crystal is dissolved in freshly prepd carbonate buffer solution (50mM Na2CO3/NaHCO3,10mM DTT; PH 10.5) in, shaking with 150rpm down in incubated at room 2.5 hours.By in 4 ℃ with 25000 * g centrifugal 10 minutes, discard undissolved residue.By using trypsin SigmaT-8642 in 37 ℃) (w: trypsinase w): protein rate was hatched 2 hours, the Cry1Ac parent toxin of active dissolution with 1: 10.After centrifugal 10 minutes,, filter with 25000 * g in 4 ℃, in MonoQ 5/5 post, use then supernatant liquor dialysis in buffer A (20mM Tris-HCl, pH 8.65) (GE Healthcare UK) carries out the anionresin purifying in the chromatography system.The continuous gradient wash-out activatory Cry1Ac toxin of the buffer B of use to 60% (pH 8.65 for 20mM Tris-HCl, 1M NaCl).

Except at NEE damping fluid (50mM Na ₂CO ₃, 5mM EDTA, 10mM EGTA; PH12.1) outside dissolving in, ground as Cry1Ac is to (Columbus, the bacterial strain ECE126 of expression Cry2Aa OH) grows and activates from Bacillus Genetic Stock Collection.

Grew 47 hours in the C2 substratum that contains 6 μ g/ml paraxin people such as (, 1988) Donovan in 28 ℃ of reorganization bacillus thuringiensis bacterial strain BtIPS78/11 that shake down, will express Cry2Ab2 at 80rpm.Adding phosphate buffered saline (PBS) (the PBS) (8mMNa of 250mM NaCl ₂HPO ₄, 2mM KH ₂PO ₄, 150mM NaCl; PH 7.4) in carry out two washing steps after, cell precipitation is dissolved in the NEE damping fluid.Add take from freshly prepd 25mg trypsinase/ml trypsinase to the 0.3mg/ml ultimate density, mixture is hatched 75 minutes in 37 ℃, centrifugal.With ammonium sulfate precipitation Cry2Ab toxin soiutions, with the gained resolution of precipitate in TEE damping fluid (50mMTris-HCl, 5mM EDTA, 10mM EGTA; PH 8.6) in.

Containing in 28 ℃ of reorganization bacillus thuringiensis berliner mutation 1715cry-bacterial strains that shake down, will contain the plasmid pGA32 that expresses Cry2Ae at 80rpm in the C2 substratum of 20 μ g/ml erythromycin and growing 144 hours.After in PBS+250mM NaCl, carrying out two washing steps, cell precipitation is dissolved in ealkaline buffer (5mM EDTA, pH 12.0 for 0.1M CAPS, 10mM EGTA), hatched 1 hour in 37 ℃.From freshly prepd 7.5mg trypsinase/ml, add trypsin with 3: 1 ratios, w/w).Mixture is in 37 ℃ of overnight incubation, centrifugal.With PBS dialysis Cry2Ae toxin soiutions.

The biological assay test

Tested every kind of proteic each 7 different concns of activatory Cry, used 16 newborn boll fruit armyworm larvas at each concentration.The sample diluting liquid of 50 μ l volumes is applied to the surface that is assigned to the artificial diet in many hollow plates.Put into a larva in each hole.Relative humidity 65 ± 5% and 16:8 (illumination: under photoperiod dark), hatch plate in 25 ± 2 ℃.7 days postevaluation mortality ratio.(LeOra Software, Berkeley CA) analyzes toxicity data to use POLO-PC probable value routine analyzer.

Midgut separates and the BBMV preparation

With boll fruit armyworm (the ANGR bacterial strain, CSIRO Entomology, Australia) and the real noctuid of paddy (USDA-ARS, last instar larvae MS) is dissected, midgut is preserved in-80 ℃ when needs till.By differential magnesium precipitate method people such as (, 1987) Wolfersberger preparation BBMV, be frozen in the liquid nitrogen ,-80 ℃ of storages.Use bovine serum albumin to measure protein concn in the BBMV prepared product by Bradford method (1976) as standard.

The radio-labeling of Cry toxin

Use the chloramine-T method that Cry1Ac and Cry2Ab toxin are carried out mark.Under having the 18mM chloramine-T (among the PBS) of 1/3v with Na ¹²⁵(PerkinElmer, Boston MA) add in the 25 microgram Cry toxin I (0.5mCi).After hatching 45 seconds, the inclined to one side Potassium hydrogen sulfite of 23mM by adding 1/4v is (in H ₂Among the O) termination reaction.At last, add the 1M NaI of 1/4v, with mixture be loaded into the buffering fluid column (20mM Tris-HCl, 150mM NaCl, 0.1%BSA) the PD10 desalting column crossed of balance (GE HealthCare, UK) on.By SDS-PAGE, further xerogel is exposed to X-ray film, analyze elutriated fraction, with the proteic purity of check mark.Radioactivity in the toxin band that discloses according to the radioactivity of wash-out in the toxin that drops into, the protein peak with by SDS-PAGE (Fig. 1) is calculated the specific activity of mark toxin with respect to the radioactive per-cent in the minor band.Through estimating that the specific activity of 125I-Cry1Ac and 125I-Cry2Ab toxin is respectively 3mCi/mg and 2.4mCi/mg.

¹²⁵The combination test of the Cry1Ac of I-mark and Cry2Ab

Before the use, with BBMV with 16000 * g centrifugal 10 minutes, resuspending was in binding buffer liquid (8mMNa ₂HPO ₄, 2mM KH ₂PO ₄, 150mM NaCl; PH 7.4; 0.1% bovine serum albumin).By in 25 ℃ in binding buffer liquid with cumulative amount ¹²⁵I-Cry2Ab is hatched 20 micrograms 1 hour from the BBMV of boll fruit armyworm, carries out saturation experiments.After hatching,, will precipitate with 500 microlitre cold junctions and close the damping fluid washed twice sample centrifugal 10 minutes with 16000 * g.In LKB 1282Compugamma CS gamma counter, measure the radioactivity that is retained in the precipitation.By excessive unmarked Cry2Ab (1 micromole) is added in the reaction, measure non-specific binding.By non-specific binding is deducted, calculate the specificity combination from total binding.

In order to measure the optimum concn that BBMV is used for competitive assay, in final volume is the binding buffer liquid of 0.1ml, the Cry1Ac of 0.4nM and 1.2nM mark and the BBMV of Cry2Ab and cumulative amount were hatched 1 hour in 25 ℃.Use excessive unmarked toxin to calculate non-specific binding.

Under 25 ℃ of unmarked toxin that have a cumulative amount, with 20 microgram BBMV and 1nM ¹²⁵I-Cry2Ab or 5 microgram BBMV and 0.6nM ¹²⁵I-Cry1Ac was hatched 1 hour, and experiment is at war with.For quantitative assay, in gamma counter, measure fraction in conjunction with the mark toxin of BBMV.Use LIGAND program (Munson and Rodbard, 1980), the concentration in calculations incorporated site and the constant that dissociates.For qualitative test, will be deposited in the sample loading buffer (Laemmli, 1970) and boil 10 minutes, in SDS-PAGE, run glue then.Expose after 1 week, detect the mark toxin that is retained in the precipitation by radioautography.

1.2. result

Activatory Cry albumen is to the toxicity of boll fruit armyworm

Tested the toxicity of Cry1Ac and Cry2Ab prepared product.Activatory Cry1Ac and Cry2Ab albumen have toxicity to the boll fruit armyworm larva, LC ₅₀Value is shown in Table 2.And activatory Cry2Ae albumen shows that boll fruit armyworm is had significantly insect active extremely.

¹²⁵I-Cry2Ab combines with the specificity of boll fruit armyworm BBMV

As first method, by hatch the BBMV from boll fruit armyworm with radiolabeled Cry2Ab, the specificity of test Cry2Ab is in conjunction with (Fig. 1).Although in ¹²⁵The pollutent that has other mark in the I-Cry2Ab prepared product, but only corresponding to the band of Cry2Ab in conjunction with BBMV.Excessive unmarked Cry2Ab sharply reduces ¹²⁵The combination of I-Cry2Ab shows that most of this combinations are specific.By contrast, excessive unmarked Cry1Ac does not reduce the combination of mark Cry2Ab, shows Cry1Ac nonrecognition Cry2Ab binding site.

¹²⁵The bonded of I-Cry2Ab and boll fruit armyworm BBMV is saturated

Hatch boll fruit armyworm BBMV by the mark Cry2Ab with cumulative concentration, the combination that has confirmed Cry2Ab is saturable.(under the 0.2mg BBMV albumen/ml), curve is about 5nM in the condition of using ¹²⁵Beginning departs from linear in I-Cry2Ab place reaches maximum value (Fig. 2) when about 20nM.

Use the competitive assay of boll fruit armyworm BBMV

For finding that BBMV is used to compete the optimum concn in conjunction with experiment, hatch fixed concentration with the boll fruit armyworm BBMV of cumulative concentration ¹²⁵I-Cry2Ab.The same as expecting, deduct non-specific binding after, observe the increase of specificity bonded, corresponding to the increase (Fig. 3 A) of binding site.Select the BBMV of 0.2mg/ml concentration to be at war with in conjunction with experiment.With ¹²⁵I-Cry1Ac has carried out similar experiment, shows that the best BBMV concentration that is used for competitive assay is 0.05mg/ml (data not shown).

Observed with unmarked Cry2Ab albumen in the homology competition experiments ¹²⁵I-Cry2Ab replaces confirmation, and the combination of Cry2Ab in this insect is specificity and saturable (Fig. 3 B).Under our experiment condition, also observe some non-specific binding.Use Cry2Aa and Cry2Ae to show in conjunction with test as the competition of allos competitor, these Cry albumen at an easy rate with ¹²⁵I-Cry2Ab competes (Fig. 3 B).By contrast, unlabelled Cry1Ac can not compete in the concentration range of being tested ¹²⁵The combination of I-Cry2Ab (has caused than the Cry1Ac that is shown in the concentration greater concn among Fig. 3 B ¹²⁵The precipitation of I-Cry2Ab).These results show that the Cry2Ab binding site is shared by other two kinds of Cry2 toxin, but is not shared by Cry1Ac.

Use Cry1Ac, Cry2Ab, Cry2Aa and Cry2Ae as competitor, also carried out ¹²⁵The competition experiments of I-Cry1Ac (Fig. 3 C).None Cry2A albumen with ¹²⁵I-Cry1Ac competes combination, confirms that there is different binding sites in Cry1Ac with these Cry2A albumen.

According to the homology competition curve, calculated the incorporating parametric of Cry2Ab and Cry1Ac, dissociation constant (Kd) and binding site concentration (Rt) (table 1).In both cases, homology competition data fitting unit point model equation.As be shown in table 1, and Cry2Ab has the specific binding site of slightly Duoing than Cry1Ac in boll fruit armyworm, and still for their binding site separately, the avidity of Cry2Ab is lower than the avidity of Cry1Ac.

The radio-labeling of Cry2Ab and Cry1Ac has carried out twice independently, and with freshly prepd second radiolabeled Cry1Ac and Cry2Ab prepared product, repeats all experiments mentioned above.The result who obtains with this second group radiolabeled toxin is similar to those above-mentioned results.

Using ¹²⁵In the competition experiments of I-Cry2Ab,, in boll fruit armyworm BBMV, do not see competition to the Cry2Ab binding site for VIP3Aa and Cry1Fa albumen yet.

Cry2Ab combines with the specificity of the real noctuid BBMV of paddy

Be the binding site model that confirms from the experiment of boll fruit armyworm, to obtain, also in the real noctuid of paddy, carried out in conjunction with test.In this insect, when with the BBMV of cumulative amount with ¹²⁵When I-Cry2Ab is hatched, also observed the specificity combination.When using identical BBMV concentration range, the per-cent of bonded toxin is lower than the per-cent (comparison diagram 4A and 3A) of bonded toxin in the boll fruit armyworm in the real noctuid of paddy.

Use ¹²⁵I-Cry2Ab carries out homology and allos competition experiments in the real noctuid of paddy.As in the boll fruit armyworm, unmarked Cry2Ab competition ¹²⁵The binding site of I-Cry2Ab (Fig. 4 B).Homology competition shows the saturable combination of Cry2Ab in this insect indirectly, because curve has reflected the binding site of limited quantity in the concentration range of unmarked competitor.The allos competition experiments show Cry1Ac not with ¹²⁵The I-Cry2Ab competition.

¹²⁵The experiment of I-Cry1Ac shows that Cry2Ab does not compete the binding site of Cry1Ac (Fig. 4 C), has proved that thus also there is different binding sites with Cry1Ac in Cry2Ab in the real noctuid of paddy.

Can directly on the BBMV of the real noctuid of paddy, obtain to combine in the saturability test with the proteic saturable of viewed similar Cry2Ab on boll fruit armyworm BBMV.And in the real noctuid of paddy, Cry2Aa, Cry2Ab and Cry2Ae albumen are also in conjunction with coreceptor.In addition, between any one albumen of Cry2Ab and Cry1Ab, Cry1Ac, Cry1Fa or VIP3Aa albumen, there is not competition to binding site yet.

1.3. interpretation of result

The saturated combination experiment of carrying out with Cry2Ab shows, has the specific receptors of limited quantity in the film of sensitive insect midgut epithelial cells.The similar saturation testing of carrying out with mark Cry1Ac discloses, and under the BBMV of same concentrations, uses than lack four times toxin in the Cry2Ab test, has realized saturated (data not shown) of binding site.Our result be different from the saturated previous result of Cry2Aa in conjunction with test, previous result's conclusion is that the Cry2Aa combination of proteins is saturable (people such as English, 1994, people such as Jurat-Fuentes, 2003 not; EPA biological pesticide handbook 006487 (2002)).For observed more or less linear curve in those Cry2Aa saturation experiments, a lot of possible explanations are arranged.At first, these authors have used the Cry2Aa parent toxin rather than the activatory toxin of mark.And, in a research, carry out mark, and and hang in the air the circulation (people such as English, 1994) that under used condition Cry2Aa can tolerate sex change and renaturation with sex change Cry2Aa albumen.Secondly, under the insoluble pH value of Cry2A parent toxin, use damping fluid people such as (, 2001) Staples.In saturation experiments, the trend of low solubility and Cry2A protein aggregation people such as (, 2001) Perlak may cause radioactive linear the increasing of the recovery that caused by precipitation.The 3rd, the proteic concentration range of Cry2Aa that is used for saturation experiments may be not enough to saturated binding site.Used scope is identical with the scope that is used for the Cry1Ac toxin, but because Cry2A is proteic than low-affinity, may need the Cry2Aa albumen of higher concentration to show the saturated of binding site.The 4th, used BBMV concentration may be not enough to distinguish the specificity combination.If binding site concentration is enough high, the high-level non-specific binding of Cry2A albumen (with vesicle component and/or bottle or strainer) may be covered specificity and combines so.

Using ¹²⁵In some research that the I-Cry2A toxin carries out, BBMV binding ability test (being the mark toxin of fixed concentration and the BBMV of cumulative amount) is expressed as ¹²⁵I-Cry2A saturation testing (being the mark toxin of fixed BBMV concentration and cumulative amount) (Karim and Dean, 2000, people such as Karim, 2000b, people such as Luo, 2007).When the concentration of binding site in the experiment was changing always, the saturability of binding site can not be determined.Therefore, in these researchs, based on type of error experiment with the Cry2A combining classification be saturable people such as (, 2007) Luo or not saturable (Karim and Dean, 2000, people such as Karim, 2000b).This research is based on first part of report of saturable bonded that correct saturation experiments is described the Cry2 toxin.

In this research, the homology competition experiments of Cry2Ab confirms in the real noctuid of boll fruit armyworm and paddy, has the binding site of limited quantity.In fact, if Cry2A, will have basically unlimited quantity so in conjunction with being saturable not can be used for the bonded binding site, and unmarked toxin and mark toxin will almost be impossible for the competition in these sites.In this experiment, the Cry2Ab of certain percentage is in conjunction with seemingly nonspecific.But very possiblely be that the great majority that classify as in the radioactivity of non-specific binding are actually the radioactivity that comes from sedimentary mark Cry2Ab.

The analysis that incorporating parametric from the homology competition experiments is carried out draws, and in two Helicoverpa species that this paper analyzed, the value of the dissociation constant of Cry2Ab toxin (Kd) is than high about 35 times of the value of Cry1Ac, and this binding affinity that shows Cry2Ab is lower.Yet, with regard to the concentration of binding site, the difference between two toxin much lower (about 3 times).Cry2Ab that calculates by competition experiments and the kd value similar to the value that calculates by the saturation experiments in the boll fruit armyworm (data not shown) of Cry1Ac.People such as Mandal (2007) propose, and the low receptors bind avidity of the Cry2A toxin that some author reported is owing to the existence in extra N-terminal zone in this toxin.

As far as our knowledge goes, this is about the first part of report in conjunction with competition experiments between the Cry2A albumen.The allos competitive assay of mark Cry2Ab and unmarked Cry2Aa and Cry2Ae toxin shows that these albumen are shared the common binding site.

Generally speaking, our result clearly illustrates that, Cry2A albumen with high-affinity saturable ground in conjunction with the specificity site among boll fruit armyworm and the real noctuid BBMV of paddy.In addition, Cry2Aa, Cry2Ab and Cry2Ae share this high affinity combined sites, but do not share with Cry1Ac, VIP3 or Cry1F.In addition, for Cry1Aa, Cry1Ab and Cry1Ac, be reported at least one common high affinity combined sites in all insect species of being tested.Confirm that Cry2A albumen has saturable high affinity combined sites and this binding site is different from the proteic binding site of Cry1A, insect-resistant management is had great importance: it shifts the resistance between Cry1A and the Cry2A albumen, and why so rare (Cross-resistance) has provided the biological chemistry explanation, and provides reliable support with the resistance operating strategy of target boll fruit armyworm or paddy reality noctuid for combination cry1A and cry2A gene in same farm crop.

Embodiment 2

Several method can expect be used for transgenic plant for example corn or vegetable lamb obtain at least two insecticidal protein genes for example the combination of Cry2Ae and Cry1Ab gene express.

First method wherein transforms the plant that has transformed first mosaic gene, to introduce second gene based in succession step of converting again.This transforms two different selectable marker genes of preferred use in succession, for example kalamycin resistance gene and give the phosphinothricin acetyl transferase gene to the resistance of careless ammonium phosphine weedicide (for example, well-known pat or bar gene).The application of these two kinds of selective markers has been described in people (1987) such as De Block.

Second method is based on encode on different plasmids two mosaic genes of different insecticidal proteins of cotransformation in single step.Can be by utilizing the selective marker that is connected with gene separately, the integration of two genes of selection.

In addition, can be in transformation event independently, two insecticidal protein genes are transferred to separately individually in the cell nucleus gene group of different plants, can it be combined in the single plant by hybridization subsequently, and can use the dna marker technology to select to comprise these heterogeneic plants.

(WO 2007/142840 will to comprise the MIR162 incident, the non-control state of USDA APHIS application 07-253-01p) maize plant is hybridized with the maize plant of the mosaic gene of the coding region that comprises the SEQ ID No.9 that contains WO 2002/057664, produces expression VIP3A and Cry2Ae insect and controls proteic maize plant.Perhaps, the maize plant hybridization of the maize plant that will comprise the maize plant of incident Bt11 (the non-control state of USDA APHIS application 95-195-01p) or comprise incident MON810 (USDA APHIS petition 96-017-01p) and the mosaic gene of the coding region of containing the SEQ ID No.9 that comprises WO 2002/057664 produces and expresses Cry1Ab and the Cry2Ae insect is controlled proteic maize plant.

The vegetable lamb and the vegetable lamb hybridization that comprises the incident EE-GH5 described in PCT patent application PCT/EP2008/002667 that will comprise the incident EE-GH6 described in the PCT patent application (unexposed) of the right of priority that requires European Patent Application No. 07075460 or 07075485, to obtain to express Cry2A and the proteic vegetable lamb of Cry1A, this plant has the inherent insect-resistant management to real noctuid of paddy and boll fruit armyworm.

In addition, to comprise the vegetable lamb of COT102 incident of USDA APHIS application 03-155-01p (WO 2004/039986) and above-mentioned 2 gene vegetable lambs or with the mosaic gene that contains the SEQ ID coding region N.7 that comprises WO2002/057664 with comprise US patent 7,049, the vegetable lamb hybridization of the mosaic gene of the Cry1Ab coding region of 491 SEQ ID No.2 is to obtain to express VIP3A, Cry1Ab and the proteic vegetable lamb of Cry2Ae.

Can estimate the coexpression of at least two insecticidal protein genes in single transformant by the insect toxicity test with by biochemical method known in the art.Specific probe allows the quantitative analysis of transcript level; Allow quantitative analysis corresponding gene product in the ELISA test with the monoclonal antibody of corresponding gene product cross reaction; Specificity DNA probing needle allows to characterize genetically modified genome conformity in the transformant.

Certainly; except the aforesaid combination of the Cry2A, the VIP3 that are used for bollworm insect-resistant management and Cry1 gene; these plants also can comprise other transgenosis; for example give other lepidopterous insects species of antagonism or antagonism from other insect purpose insect species gene of the provide protection of Coleoptera (Coleopteran) or Homoptera (Homopteran) insect species for example, or the gene of conferring herbicide tolerance etc.

This paper mentions or all patents, patent application and the publication quoted or public's open (comprising internet publication and the application of non-control state) are being incorporated herein by reference with it in full with clearly instructing on the inconsistent degree of this specification sheets.Quoting of any file of this paper do not mean that all this document forms the part of general knowledge known in this field.

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Table 1.Incorporating parametric in the real noctuid of boll fruit armyworm and paddy

^aK _dThe value representation equilibrium dissociation constant, it calculates according to the homology competition experiments, and represents with nanomolar concentration.

^bR _tValue representation binding site concentration, and represent with picomole/milligram BBMV albumen.

^cValue is two multiple mean values.

^dValue is at least four multiple mean values.

E mean value ± SEM.

Table 2.Cry1Ac and Cry2Ab activated protein are to the toxicity (measuring after 7 days) of boll fruit armyworm newborn larvae.

^aFiducial limit is in 95% level.

Table 3.The tabulation of VIP3 albumen

(www.lifesci.susx.ac.uk/home/Neil_Crickmore/Bt/vip.html)

Description of drawings

Fig. 1. combination is from the radioautograph of the 125I-Cry2Ab of the BBMV of boll fruit armyworm

Lack or exist under the excessive competitor, ¹²⁵I-Cry2Ab and BBMV are hatched, then will be through centrifugal association reaction mixture and precipitation carry out SDS-PAGE, X-ray film was exposed for 1 week.Swimming lane 1: ¹²⁵The I-Cry2Ab toxin; Swimming lane 2: hatch lacking under the competitor with BBMV ¹²⁵I-Cry2Ab; Swimming lane 3: homology competition (excessive unmarked Cry2Ab); Swimming lane 4: with the allos competition of Cry1Ac.

Fig. 2 .125I-Cry2Ab is saturated to the specificity bonded of boll fruit armyworm BBMV

Cumulative amount ¹²⁵The BBMV of I-Cry2Ab and fixed amount (20 microgram vesicle albumen) was hatched 1 hour.By centrifugal termination association reaction, measure the radioactivity that is retained in the precipitation.Calculate non-specific binding by hatching, and from total binding, deduct with excessive unmarked Cry2Ab.

Fig. 3. ¹²⁵I-Cry2Ab combines with boll fruit armyworm BBMV's

(A) ¹²⁵I-Cry2Ab combines with the specificity of the BBMV of cumulative concentration.Calculate non-specific binding existing under the excessive unmarked Cry2Ab.Data point is equivalent to use two batches independently ¹²⁵5 multiple mean values of I-Cry2Ab.(B) use ¹²⁵The competitive assay of I-Cry2Ab.(C) use ¹²⁵The competitive assay of I-Cry1Ac.In competition experiments, each data point is represented at least two independent multiple mean values.

Fig. 4. ¹²⁵I-Cry2Ab combines with the real noctuid BBMV's of paddy

(A) ¹²⁵I-Cry2Ab combines with the specificity of the BBMV of cumulative concentration.Calculate non-specific binding existing under the excessive unmarked Cry2Ab.(B) use ¹²⁵The competitive assay of I-Cry2Ab.(C) use ¹²⁵The competitive assay of I-Cry1Ac.Each data point is represented at least two independent multiple mean values.

Claims

1. be used for preventing or delay the real noctuid of insect species paddy or boll fruit armyworm colony to the insect-resistant development of the transgenic plant of expressing insecticidal protein controlling the method for described insect evil, it is included in combination in the described plant and expresses the real noctuid of paddy or boll fruit armyworm are had Cry2Ae, the Cry2Aa of insecticidal or Cry2Ab albumen and VIP3, the Cry1F or the Cry1A albumen that the real noctuid of paddy or boll fruit armyworm are had insecticidal.

In the colony of real noctuid of paddy or boll fruit armyworm insect to comprising the method for real noctuid of control paddy in the area that the proteic plant of VIP3, Cry1F or Cry1A produces resistance or boll fruit armyworm, it comprises step: sowing or plantation comprise Cry2Aa, Cry2Ab or the proteic plant of Cry2Ae that the real noctuid of paddy or boll fruit armyworm is had insecticidal in described area.

In the colony of real noctuid of paddy or boll fruit armyworm insect to comprising the method for real noctuid of control paddy in the area that the proteic plant of Cry2Aa, Cry2Ab or Cry2Ae produces resistance or boll fruit armyworm, it comprises step: sowing or plantation comprise VIP3, Cry1F or the proteic plant of Cry1A that the real noctuid of paddy or boll fruit armyworm is had insecticidal in described area.

4. be used to obtain comprise the method for the plant of at least two kinds of different insecticidal proteins, wherein as measure in conjunction with test in the competition of the brush border membrane vesicle that uses real noctuid of paddy or boll fruit armyworm insect larvae, described protein saturable and specifically in conjunction with the binding site in real noctuid of species paddy or the boll fruit armyworm larva, and described protein is not shared binding site, said method comprising the steps of: obtain to comprise coding has insecticidal to real noctuid of paddy or boll fruit armyworm Cry2Aa, but proteic expression of plants mosaic gene of Cry2Ab or Cry2Ae and coding have the VIP3 of insecticidal to real noctuid of paddy or boll fruit armyworm, but the plant of the proteic expression of plants mosaic gene of Cry1A or Cry1F.

5. the method for claim 4, wherein said plant obtains in the following way: the seed and the progeny plants that comprise the described plant of described mosaic gene with proteic mosaic gene conversion plant of encode described Cry2Aa, Cry2Ab or Cry2Ae albumen and described VIP3, Cry1A or Cry1F and acquisition; The mother plant hybridization and obtaining of mother plant and the mosaic gene that comprises coding VIP3, Cry1A or Cry1F that maybe will comprise the mosaic gene of coding Cry2Ae, Cry2Aa or Cry2Ab comprises the progeny plants and the seed of the mosaic gene of described combination.

6. sowing; the method of plantation or growing plant; the protected infringement of avoiding real noctuid of paddy or boll fruit armyworm of described plant by expressing at least two kinds of different insecticidal proteins; wherein as measure in conjunction with experiment in the competition of using the brush border membrane vesicle; described protein saturable and specifically in conjunction with the binding site in the larva midgut of described insect species; and described protein is not shared binding site, said method comprising the steps of: sowing; plantation or growth comprise coding has insecticidal to real noctuid of paddy or boll fruit armyworm Cry2Ab; proteic mosaic gene of Cry2Aa or Cry2Ae and coding have the VIP3 of insecticidal to real noctuid of paddy or boll fruit armyworm; the plant of Cry1A or the proteic mosaic gene of Cry1F.

7. each method in the claim 2～6 wherein comprises the described plant that kills the proteic mosaic gene of insect VIP3 of encoding and is selected from down group: comprises coding VIP3Aa1, VIP3Aa19, VIP3Aa20 or VIP3Af1 albumen, or has the plant of mosaic gene of the insecticidal protein of at least 70% sequence identity with any described VIP3A albumen, the maize plant that comprises the MIR162 incident of the application of USDAAPHIS described in the WO 2007/142840 07-253-01p, the vegetable lamb that comprises the COT102 incident of the APHIS of USDA described in WO2004/039986 application 03-155-01p, the vegetable lamb that comprises the COT202 incident described in the WO 2005/054479, with the vegetable lamb that comprises the COT203 incident described in the WO 2005/054480.

8. each method in the claim 2～7, the described plant that wherein comprises the Cry1A gene is selected from down group: comprise coding Cry1Ac, Cry1Ab or Cry1A.105 albumen, or has the plant of mosaic gene of the insecticidal protein of at least 90% sequence identity with any described Cry1A albumen, the maize plant that comprises the MON810 incident of the application of USDAAPHIS described in the US patent 6713259 96-017-01p, comprise US patent 6,114, the maize plant of the Bt11 incident of the APHIS of USDA described in 608 application 95-195-01p, the vegetable lamb that comprises the COT67B incident of the application of USDAAPHIS described in the WO 2006/128573 07-108-01p, the vegetable lamb that comprises the 3006-210-23 incident of the application of USDAAPHIS described in WO2005/103266 03-036-02p, comprise the Cry1A gene event described in the WO 2002/100163, promptly, the vegetable lamb of the incident 531 of USDAAPHIS application 94-308-01p, comprise the incident T342-142 described in the WO2006/128568-128572,1143-14A, 1143-51B, the vegetable lamb of any one incident among CE44-69D or the CE46-02A, the vegetable lamb that comprises the incident EE-GH5 described in PCT patent application PCT/EP2008/002667, with comprise the incident that contains the Cry1A gene described in the WO2007/140256, that is the maize plant of the MON89034 incident of USDA APHIS application 06-298-01p.

9. each method in the claim 2～8, the described plant that wherein comprises the Cry1F gene is selected from down group: comprise coding Cry1Fa albumen or have the plant of mosaic gene of the insecticidal protein of at least 90% sequence identity with described Cry1Fa albumen, the maize plant that comprises the TC1507 incident of USDA APHIS application 00-136-01p (WO/2004/099447), the maize plant that comprises the TC-2675 incident of USDAAPHIS application 03-181-01p, the vegetable lamb that comprises the 281-24-236 incident (incident that contains the Cry1F gene of WO 2005/103266) of USDA APHIS application 03-036-01p.

10. each method in the claim 2～9, the described plant that wherein comprises the Cry2Ae gene is selected from down group: comprise coding Cry2Ae albumen or with described Cry2Ae albumen have at least 95% sequence identity insecticidal protein mosaic gene plant, comprise the SEQ ID No.7 that contains WO 2002/057664 or 9 encoding sequence mosaic gene plant, comprise the vegetable lamb of the incident EE-GH6 that describes in the PCT patent application of right of priority of requirement European Patent Application No. 07075460.

11. each method in the claim 2～10, the described plant that wherein comprises the Cry2Ab gene is selected from down group: comprise coding Cry2Ab albumen or with described Cry2Ab albumen have at least 95% sequence identity insecticidal protein mosaic gene plant, comprise the Cry2Ab incident 15985 described in the non-control state of the USDA APHIS application 00-342-01p vegetable lamb, comprise the maize plant of the Cry2Ab event MON 89034 described in the non-control state of the USDAAPHIS application 06-298-01p.

12. each method in the claim 2～6, wherein said chimeric Cry or VIP gene comprise Cry2Ae, Cry2Ab, VIP3A, Cry1A or Cry1F coding region, be selected from any cotton or any one Cry2Ae, Cry2Ab in the corn event, VIP3A, Cry1A or the Cry1F coding region that are contained in described in the claim 8～12, or wherein said Cry2Ab, Cry2Ae, VIP3, Cry1A or Cry1F mosaic gene are included in any one Cry2Ae, VIP3, Cry1F or Cry1A mosaic gene in any one described cotton or the corn event.

13. each method in the claim 1～12, wherein said plant is selected from down group: corn, cotton, rice, soybean, tomato, Sunflower Receptacle and sugarcane.

14. each method in the claim 1～13, wherein said method comprise that also plantation has the sanctuary of the plant that does not comprise coding Cry or the proteic mosaic gene of VIP.

15. each method in the claim 1～14, wherein said plant provide Cry2, Cry1 or VIP3 albumen at the high dosage of real noctuid of paddy or boll fruit armyworm.

16. each method in the claim 1～15, wherein said Cry2A albumen is Cry2Ab albumen, and described combination is a saturable and specific.

17. comprise Cry2A and Cry1 or genetically modified plant of VIP3 or seed at least, wherein each transgenes encoding is different, the real noctuid of paddy or boll fruit armyworm had insecticidal, saturable and specifically in conjunction with the protein of the binding site in the described insect midgut, wherein said protein in this insect not competing phase with binding site, and wherein said Cry2A albumen is the protein that comprises Cry2Aa or the proteic minimum toxic fragment of Cry2Ae, and described Cry1 or VIP3 albumen are to comprise Cry1Ab, Cry1Ac, the albumen of Cry1Fa or the proteic minimum toxic fragment of VIP3A.

18. the plant of claim 17 or seed, it is to comprise at least 2 corn or vegetable lamb or seeds that are selected from down the combination of the transformation event of organizing: corn event MON89034, corn event mir 162, comprise the proteic genetically modified corn event of coding Cry2Ae, corn event TC1507, corn event Bt11, corn event MON810, cotton event EE-GH6, cotton event COT102, cotton event COT202, cotton event COT203, cotton event T342-142, cotton event 1143-14A, cotton event 1143-51B, cotton event CE44-69D, cotton event CE46-02A, cotton event COT67B, cotton event 15985, cotton event 3006-210-23, cotton event 531, cotton event EE-GH5 and cotton event 281-24-236.

19. the plant of claim 18 or seed, wherein said corn or vegetable lamb or seed comprise at least 3 combinations that are selected from down the transformation event of group: corn event MON89034, corn event mir 162, contain the proteinic genetically modified corn event that coding comprises the proteic minimum toxic fragment of Cry2Ae, corn event TC1507, corn event Bt11, corn event MON810, cotton event EE-GH6, cotton event COT102, cotton event COT202, cotton event COT203, cotton event T342-142, cotton event 1143-14A, cotton event 1143-51B, cotton event CE44-69D, cotton event CE46-02A, cotton event COT67B, cotton event 15985, cotton event 3006-210-23, cotton event 531, cotton event EE-GH5 and cotton event 281-24-236.

20. be used to obtain to plant or the method for supervision department's approval that the real noctuid of paddy or boll fruit armyworm is had the proteinic plant of insecticidal is expressed in commercialization, it may further comprise the steps: quote, submit to or rely on to show that Cry2A albumen do not compete the insect test binding data of Cry1A, Cry1F or the proteic binding site of VIP3 in this insect.

Contain and do not produce the method for structure sanctuary that the real noctuid of paddy or boll fruit armyworm is had any proteinic plant of insecticidal 21. be used for reducing growing area, this method may further comprise the steps: quote, submit to or rely on to show that Cry2A albumen do not compete the insect test binding data of Cry1A, Cry1F or the proteic binding site of VIP3 in this insect.

22. the method for claim 20 or 21, it is further comprising the steps of: quote, submit to or rely on to show the direct saturability test of Cry2A albumen saturable ground in conjunction with the binding site in real noctuid of paddy or the boll fruit armyworm midgut.

23. each method in the claim 20～22, wherein said Cry2A albumen is the protein that comprises the proteic minimum toxic fragment of Cry2Ae, and wherein said Cry1A, Cry1F or VIP3 albumen are defined any this protein in the claim 7～9.

24. the growing area of the insect resistant transgenic plant of real noctuid of control paddy or boll fruit armyworm insect, wherein said growing area has less than 20%, less than 15%, less than 10% or less than 5% structure sanctuary, or do not have a structure sanctuary, wherein said combinations of plant is expressed to have the Cry2Ae of insecticidal or Cry2Ab albumen and boll fruit armyworm or the real noctuid insect of paddy is had the Cry1A of insecticidal boll fruit armyworm or the real noctuid insect of paddy, Cry1F or VIP3A albumen, the Cry2Ae and the Cry1Ab that particularly the real noctuid insect of boll fruit armyworm or paddy are had insecticidal, Cry1Ac or VIP3A albumen preferably have the Cry2Ae of insecticidal to the real noctuid insect of boll fruit armyworm or paddy, Cry1Ab and VIP3 albumen.