CN101379190A

CN101379190A - Crisp caudex 2 gene family and correlation method and purpose

Info

Publication number: CN101379190A
Application number: CNA200680052191XA
Authority: CN
Inventors: K·S·杜加
Original assignee: Pioneer Hi Bred International Inc
Current assignee: Pioneer Hi Bred International Inc
Priority date: 2006-02-03
Filing date: 2006-02-03
Publication date: 2009-03-04
Also published as: EP1987149A1; CA2635964A1; BRPI0621197A2; AU2006337212A1; WO2007089244A1

Abstract

This invention relates to isolated polynucleotides encoding BRITTLE STALK 2-like (Bk2L) family polypeptides. The invention also relates to the construction of a chimeric gene encoding all or a portion of a Bk2L polypeptide, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the Bk2L polypeptide in a transformed host cell.

Description

Crisp caudex 2 gene family and methods involving and purposes

invention field

Invention field relates to molecular biology of plants, especially crisp stem 2-sample (BRITTLESTALK2-Like) gene, crisp stem 2-sample polypeptide, and its purposes.

Background of invention

Plant primary growth is mainly driven by the increase of cell, and the latter realizes the irreversible generation of turgescence in cell by primary cell wall.Although need cell fission to produce new cell, the growth being caused by the expansion of these cells is not only derived from their division.Being embedded into cellulose micro-fibers in hemicellulose matrix and the xylogen in cell walls, is the main determining factor (people such as Appenzeller, Cellulose 11:287-299 (2004)) of tensile strength.Cell expands along the axle perpendicular to primitive fiber direction conventionally.For example, radial the depositing of primitive fiber helps cell along the expansion of the longitudinal axis.

The difference of secondary wall and primary wall is, its rich cellulose and xylogen, and its deposition starts from the end that cell expands.The synthetic regulation and control of primary cell wall have been applied to change growth velocity and the size (highly) of plant, and the regulation and control of secondary wall can be for improving Biomass accumulation and tissue intensity people such as (, Cellulose 11:287-299 (2004)) Appenzeller.

Mierocrystalline cellulose is the main wall composition of maturation plant cell normally, forms nutritive issue.By forming interchain and intrachain hydrogen bond, minimize the energy the cellulose family crystalline texture causing and it is become take density as one of basis mechanically the strongest known organic molecule.Then naturally, Mierocrystalline cellulose is the main determining factor of structure organization intensity.

Plant physical strength is one of most important agronomy attribute.Separated and characterized stem strength deficient plants mutant.First the physical properties based on stalk, the crisp stalk of barley (bc) mutant has been described, compare with wild-type plant, it has that 80% of cellulose amount reduces and 2 times of minimizings of breaking tenacity people such as (, Plant Physiol.97:509-514 (1991)) Kokubo.The crisp stalk 1 of rice (bc1) mutant shows the minimizing (people such as Qian, Chi.Sci.Bull.46:2082-2085 (2001)) of cell wall thickness and content of cellulose.The people such as Li have described the discriminating of the crisp stem 1 of rice (BC1), and it is the gene (The Plant Cell 15 (9): 2020-2031 (2003)) of coding COBRA-sample albumen.Their discovery shows, BC1 works in the biosynthesizing that regulates secondary cell wall, so that the main physical strength of rice plant to be provided.

The stem of the crisp stem 2 of corn (bk2) mutant shows the significantly reduced physical strength of wild-type copy (Langham, MNL 14:21-22 (1940)) than them.Corn bk2 mutant has stem and leaf that be highly brittle and easily fracture.The main chemical compositions that mutant stem lacks is Mierocrystalline cellulose.Therefore, stem physical strength seems mainly to depend on cellulosic amount in the stem of unit length under fringe.

In addition, the gene of coding cellulose synthase catalytic subunit (CesA) has been thought and has been related to Cell wall synthesis, and extended familys in plant represent.After complete genome order-checking, in mouse ear mustard, identified 10 kinds of genes, by EST, checked order and from corn, isolate 12 kinds of genes (U.S. Patent number 6,803,498 and 6,930,225).Be reported that 3 kinds of CesA genes from each mouse ear mustard and corn can produce secondary wall, and remaining obvious generation primary wall (people such as Taylor, Proc.Natl.Acad.Sci.U.S.A.100:1450-1455 (2003)).From the sudden change in 3 kinds of CesA genes of mouse ear mustard, cause xylem that stem-sample stalk withers and the physical strength of reduction.When relevant CesA transgenation from rice, stalk becomes fragile, and this is indicating the effect of these genes in secondary wall formation.In each case, the physical strength of reduction is relevant with the content of cellulose of minimizing.

Generally speaking, the sudden change that participates in the CesA gene of primary wall formation can cause serious phenotypic alternation, and the sudden change of secondary wall-formation gene can not resemble them, do not affect physical strength and change exophenotype (people such as Appenzeller, Cellulose 11:287-299 (2004)) largely.

Because not enough stem strength is the subject matter of corn breeding, thereby be desirable to provide the plant quality of standing property or composition and the method for silage quality that cellulose concentration in manipulation cell wall changes axis intensity and/or raising.

Summary of the invention

The present invention includes:

In one embodiment, comprise separated polynucleotide, the nucleotide sequence that it comprises (a) coding polypeptide relevant with stem physical strength, wherein based on Clustal V comparison method, compare with SEQID NO:16 or 18, the aminoacid sequence of described polypeptide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or the arbitrary integer between 80% and 100%, or (b) complementary sequence of this nucleotide sequence, wherein said complementary sequence and nucleotide sequence are comprised of the Nucleotide of similar number, and be 100% complementation.

In another embodiment, the method that comprises change (preferably increasing) axis physical strength, it comprises (a) and import recombinant DNA construction body in reproducible vegetable cell, to generate the vegetable cell transforming, described recombinant DNA construction body is included in the promotor that has function in plant, this promotor is operably connected to (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:4, 6, 8, 10, 12, 14, 16, with 18 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity, or (b) above-mentioned (a) (i) total length complementary sequence of described polynucleotide, (b) from the vegetable cell regeneration of transgenic plant of described conversion, wherein said transgenic plant comprise described recombinant DNA construction body in its genome, and wherein compare with the control plant that does not comprise described recombinant DNA construction body, described transgenic plant show the change (preferably increasing) of stem physical strength.The method can also comprise, and (c) obtains the progeny plants that is derived from described transgenic plant, and wherein said progeny plants comprises this recombinant DNA construction body in its genome.

In another embodiment, comprise the method for evaluating axis physical strength, it comprises (a) and import recombinant DNA construction body in reproducible vegetable cell, to generate the vegetable cell transforming, described recombinant DNA construction body is included in the promotor that has function in plant, this promotor is operably connected to (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ IDNO:4, 6, 8, 10, 12, 14, 16, with 18 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity, or (b) above-mentioned (a) (i) total length complementary sequence of described polynucleotide, (b) from the vegetable cell regeneration of transgenic plant of described conversion, (c) evaluate the stem physical strength of described transgenic plant.The method can also comprise, and (d) obtains the progeny plants that is derived from described transgenic plant; (e) evaluate the stem physical strength of described progeny plants.

In another embodiment, comprise the method for evaluating axis physical strength, it comprises (a) and import recombinant DNA construction body in reproducible vegetable cell, to generate the vegetable cell transforming, described recombinant DNA construction body is included in the promotor that has function in plant, this promotor is operably connected to (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ IDNO:4, 6, 8, 10, 12, 14, 16, with 18 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity, or (b) above-mentioned (a) (i) total length complementary sequence of described polynucleotide, (b) from the vegetable cell regeneration of transgenic plant of described conversion, (c) obtain the progeny plants that is derived from described transgenic plant, (d) evaluate the stem physical strength of described progeny plants.

The present invention also comprises:

In one embodiment, comprise a kind of plant, in its genome, comprise: (a) the first recombinant DNA construction body, it comprises at least one promotor that has function in plant, this promotor is operably connected at least one polynucleotide that are selected from the first separation of lower group: (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:2,4,6,8,10,12,14,16, and 18 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQID NO:1,3,5,7,9,11,13,15, and 17 relatively, there is at least 60% sequence identity; (iii) above-mentioned (a) (i) or (a) total length complementary sequence of the polynucleotide of (ii); (b) the second recombinant DNA construction body, it comprises at least one promotor that has function in plant, and this promotor is operably connected at least one polynucleotide that are selected from the second separation of lower group: (iv) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:20,22,24,26,28,30,32,34,36,38,40, with 42 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity; (v) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:21,23,25,27,29,31,33,35,37,39, and 41 relatively, there is at least 60% sequence identity; (vi) above-mentioned (b) (iv) or (b) total length complementary sequence of the polynucleotide of (v).

In another embodiment, comprise a kind of plant, in its genome, comprise at least one and regulate sequence, this adjusting sequence is operably connected to (a), and at least one is selected from the separated polynucleotide of lower group: (i) polynucleotide, and it is encoded based on Clustal V comparison method, with SEQID NO:2,4,6,8,10,12,14,16, with 18 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:1,3,5,7,9,11,13,15, and 17 relatively, there is at least 60% sequence identity; (iii) above-mentioned (a) (i) or (a) total length complementary sequence of the polynucleotide of (ii); (b) at least one is selected from the separated polynucleotide of lower group: (i) polynucleotide, and it is encoded based on Clustal V comparison method, with SEQ ID NO:20,22,24,26,28,30,32,34,36,38,40, with 42 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:21,23,25,27,29,31,33,35,37,39, and 41 relatively, there is at least 60% sequence identity; (iii) above-mentioned (b) (i) or (b) total length complementary sequence of the polynucleotide of (ii), wherein compare with not comprising described at least one control plant that is operably connected to the adjusting sequence on described (a) and (b), described plant shows the cell walls content of cellulose of increase or the growth velocity of increase.

In another embodiment, comprise a kind of plant, in its genome, comprise inhibition DNA construct, this construct is included in the promotor that has function in plant, this promotor is operably connected to all or part of of (a) following nucleotide sequence: (i) nucleotide sequence, its coding is based on Clustal V comparison method, with SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, with 18 comparisons, aminoacid sequence has at least 50% sequence identity, or from 51% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (ii) the total length complementary sequence of above-mentioned (a) nucleotide sequence (i), or (b) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity, or from 51% to the arbitrary integer sequence identity that is up to (and comprising) 100%, and wherein said target genes encoding is selected from Bk2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, the polypeptide of Bk2L8 and Bk2L9, wherein compare with the control plant that does not comprise described inhibition DNA construct, described plant shows the stem physical strength of reduction.

In another embodiment, comprise a kind of plant, in its genome, comprise inhibition DNA construct, this construct is included in the promotor that has function in plant, this promotor is operably connected to all or part of of (a) following nucleotide sequence: (i) nucleotide sequence, its coding is based on Clustal V comparison method, with SEQ ID NO:6 comparison, aminoacid sequence has at least 50% sequence identity or from 51% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (ii) the total length complementary sequence of above-mentioned (a) nucleotide sequence (i); Or (b) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity or from 51% to the arbitrary integer sequence identity that is up to (and comprising) 100%, and wherein said target genes encoding Bk2L3 polypeptide, wherein compare the organ size that described plant shows the plant height of reduction and/or reduces with the control plant that does not comprise described inhibition DNA construct.

In another embodiment, comprise a kind of plant, in its genome, comprise inhibition DNA construct, this construct is included in the promotor that has function in plant, this promotor is operably connected to all or part of of (a) following nucleotide sequence: (i) nucleotide sequence, its coding is based on Clustal V comparison method, with SEQ ID NO:10 comparison, aminoacid sequence has at least 50% sequence identity or from 51% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (ii) the total length complementary sequence of above-mentioned (a) nucleotide sequence (i); Or (b) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity or from 51% to the arbitrary integer sequence identity that is up to (and comprising) 100%, and wherein said target genes encoding Bk2L5 polypeptide, wherein compare with the control plant that does not comprise described inhibition DNA construct, described plant shows male sterile.

accompanying drawing and sequence table summary

From forming following detailed description, accompanying drawing and the sequence table of the application's a part, can understand more fully the present invention.

Figure 1A-1F has shown SEQ ID NO:2,4,6,8,10, and described in 12,14,16 and 18, the Clustal V of the aminoacid sequence of Bk2 and Bk2-sample albumen comparison, is wherein used default parameter.

The chart that Fig. 2 shows has contrasted the identity per-cent (with the divergence per-cent in second triangle) between 9 aminoacid sequences shown in Figure 1A-1F, wherein uses Clustal V comparison method.

Fig. 3 has shown the Solexa MPSS of gene Bk2 ^tMgene expression analysis.

Fig. 4 has shown the associated of expression pattern between Bk2 gene and CesA gene family member.

Fig. 5 A-5B has shown the MPSS from Solexa ^tMresearch obtain from the association between all different B k2 of corn and the expression level of CesA gene.

Fig. 6 has shown Bk2L albumen from corn, from the BC1L albumen of rice with from the germline of the COBL albumen (NCBI registration number is in bracket) of mouse ear mustard, has analyzed.Numeral along branch is the bootstrap value that 5,000 of heuristic searchings repeat to obtain.Only shown the bootstrap value that obtains the monosystem group that the >50% time supports.The amino acid difference of branch length and deduction is proportional.

SEQ ID NO:1 is 1784 base pair nucleotide sequences, it contains the open reading-frame (ORF) (ORF) (Nucleotide 89-1438) from crisp stem 2 (Bk2) gene of corn, 5 ' (the Nucleotide 1-88) in the latter Gai ORF district and the extra untranslated region (UTR) of 3 ' (Nucleotide 1439-1784) side joint.

SEQ ID NO:2 is the aminoacid sequence that is derived from the derivation of the crisp stem 2 of corn (Bk2) polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:1.

SEQ ID NO:3 is 3152 base pair nucleotide sequences, it contains the ORF (Nucleotide 586-2586) from crisp stem 2-sample 1 (Bk2L1) gene of corn, 5 ' (the Nucleotide 1-585) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 2587-3152) side joint district.

SEQ ID NO:4 is the aminoacid sequence that is derived from the derivation of the crisp stem 2-of corn sample 1 (Bk2L1) polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:3.

SEQ ID NO:5 is 2094 base pair nucleotide sequences, it contains the ORF (Nucleotide 281-1624) from crisp stem 2-sample 3 (Bk2L3) gene of corn, 5 ' (the Nucleotide 1-280) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 1625-2094) side joint district.

SEQ ID NO:6 is the aminoacid sequence that is derived from the derivation of the crisp stem 2-of corn sample 3 (Bk2L3) polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:5.

SEQ ID NO:7 is 2102 base pair nucleotide sequences, it contains the ORF (Nucleotide 326-1672) from crisp stem 2-sample 4 (Bk2L4) gene of corn, 5 ' (the Nucleotide 1-325) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 1673-2102) side joint district.

SEQ ID NO:8 is the aminoacid sequence that is derived from the derivation of the crisp stem 2-of corn sample 4 (Bk2L4) polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:7.

SEQ ID NO:9 is 2422 base pair nucleotide sequences, it contains the ORF (Nucleotide 216-2249) from crisp stem 2-sample 5 (Bk2L5) gene of corn, 5 ' (the Nucleotide 1-215) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 2250-2422) side joint district.

SEQ ID NO:10 is the aminoacid sequence that is derived from the derivation of the crisp stem 2-of the corn sample 5 (Bk2L5) of the ORF of nucleotide sequence described in SEQ ID NO:9.

SEQ ID NO:11 is 1845 base pair nucleotide sequences, it contains the ORF (Nucleotide 184-1563) from crisp stem 2-sample 6 (Bk2L6) gene of corn, 5 ' (the Nucleotide 1-183) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 1564-1845) side joint district.

SEQ ID NO:12 is the aminoacid sequence that is derived from the derivation of the crisp stem 2-of corn sample 6 (Bk2L6) polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:11.

SEQ ID NO:13 is 1644 base pair nucleotide sequences, it contains the ORF (Nucleotide 85-1425) from crisp stem 2-sample 7 (Bk2L7) gene of corn, 5 ' (the Nucleotide 1-84) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 1426-1644) side joint district.

SEQ ID NO:14 is the aminoacid sequence that is derived from the derivation of the crisp stem 2-of corn sample 7 (Bk2L7) polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:13.

SEQ ID NO:15 is 2108 base pair nucleotide sequences, it contains the ORF (Nucleotide 144-2105) from crisp stem 2-sample 8 (Bk2L8) gene of corn, 5 ' (the Nucleotide 1-143) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 2106-2108) side joint district.

SEQ ID NO:16 is the aminoacid sequence that is derived from the derivation of the crisp stem 2-of corn sample 8 (Bk2L8) polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:15.

SEQ ID NO:17 is 1335 base pair nucleotide sequences, it contains the ORF (Nucleotide 1-1332) from crisp stem 2-sample 9 (Bk2L9) gene of corn, 5 ' (Nucleotide 0) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 1963-1965) side joint district.

SEQ ID NO:18 is the aminoacid sequence that is derived from the derivation of the crisp stem 2-of corn sample 9 (Bk2L9) polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:17.

SEQ ID NO:19 is 3780 base pair nucleotide sequences, it contains from the ORF of the CesA1 gene of corn (Nucleotide 201-3428), 5 ' (the Nucleotide 1-200) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 3429-3780) side joint district.

SEQ ID NO:20 is the aminoacid sequence that is derived from the derivation of the corn C esA1 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:19.

SEQ ID NO:21 is 3725 base pair nucleotide sequences, it contains from the ORF of the CesA2 gene of corn (Nucleotide 179-3403), 5 ' (the Nucleotide 1-178) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 3404-3725) side joint district.

SEQ ID NO:22 is the aminoacid sequence that is derived from the derivation of the corn C esA2 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:21.

SEQ ID NO:23 is 2830 base pair nucleotide sequences, it contains from the ORF of the CesA3 gene of corn (Nucleotide 3-2468), 5 ' (the Nucleotide 1-2) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 2469-2830) side joint district.

SEQ ID NO:24 is the aminoacid sequence that is derived from the derivation of the corn C esA3 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:23.

SEQ ID NO:25 is 3773 base pair nucleotide sequences, it contains from the ORF of the CesA4 gene of corn (Nucleotide 338-3571), 5 ' (the Nucleotide 1-337) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 3572-3773) side joint district.

SEQ ID NO:26 is the aminoacid sequence that is derived from the derivation of the corn C esA4 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:25.

SEQ ID NO:27 is 3704 base pair nucleotide sequences, it contains from the ORF of the CesA5 gene of corn (Nucleotide 272-3502), 5 ' (the Nucleotide 1-271) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 3503-3704) side joint district.

SEQ ID NO:28 is the aminoacid sequence that is derived from the derivation of the corn C esA5 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:27.

SEQ ID NO:29 is 3568 base pair nucleotide sequences, it contains from the ORF of the CesA6 gene of corn (Nucleotide 63-3242), 5 ' (the Nucleotide 1-62) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 3243-3568) side joint district.

SEQ ID NO:30 is the aminoacid sequence that is derived from the derivation of the corn C esA6 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:29.

SEQ ID NO:31 is 3969 base pair nucleotide sequences, it contains from the ORF of the CesA7 gene of corn (Nucleotide 144-3404), 5 ' (the Nucleotide 1-143) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 3405) side joint district.

SEQ ID NO:32 is the aminoacid sequence that is derived from the derivation of the corn C esA7 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:31.

SEQ ID NO:33 is 3813 base pair nucleotide sequences, it contains from the ORF of the CesA8 gene of corn (Nucleotide 215-3499), 5 ' (the Nucleotide 1-214) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 3500-3813) side joint district.

SEQ ID NO:34 is the aminoacid sequence that is derived from the derivation of the corn C esA8 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:33.

SEQ ID NO:35 is 3799 base pair nucleotide sequences, it contains from the ORF of the CesA9 gene of corn (Nucleotide 238-3477), 5 ' (the Nucleotide 1-237) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 3478-3799) side joint district.

SEQ ID NO:36 is the aminoacid sequence that is derived from the derivation of the corn C esA9 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:35.

SEQ ID NO:37 is 3470 base pair nucleotide sequences, it contains from the ORF of the CesA10 gene of corn (Nucleotide 29-3265), 5 ' (the Nucleotide 1-28) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 3266-3470) side joint district.

SEQ ID NO:38 is the aminoacid sequence that is derived from the derivation of the corn C esA10 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:37.

SEQ ID NO:39 is 3231 base pair nucleotide sequences, it contains from the ORF of the CesA11 gene of corn (Nucleotide 21-3044), 5 ' (the Nucleotide 1-20) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 3045-3231) side joint district.

SEQ ID NO:40 is the aminoacid sequence that is derived from the derivation of the corn C esA11 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:39.

SEQ ID NO:41 is 3028 base pair nucleotide sequences, it contains from the ORF of the CesA12 gene of corn (Nucleotide 52-2835), 5 ' (the Nucleotide 1-51) in the latter Gai ORF district and 3 ' the extra UTR of (Nucleotide 2836-3028) side joint district.

SEQ ID NO:42 is the aminoacid sequence that is derived from the derivation of the corn C esA12 polypeptide of the ORF of nucleotide sequence described in SEQ ID NO:41.

The single-letter code that sequence table contains nucleotide sequence character and amino acid whose 3 alphanumeric codes, J.219 the described IUPAC-IUBMB standard of (2): 345-373 (1984) is consistent for their definition and Nucleic Acids Res.13:3021-3030 (1985) incorporated by reference in this article and Biochemical.The sign and the format character that for Nucleotide and amino acid sequence data, use are combined in the rule described in 37C.F.R. § 1.822.Sequence description and appended sequence table meet described in 37C.F.R. § 1.821-1.825 Nucleotide and/or the disclosed rule of aminoacid sequence about patent application.

发明详述

在本申请中引用的所有专利、专利申请和出版物，都特此整体引作参考。

如在本说明书和所附权利要求书中所使用的，数形式“一种”、 “一个”和“该”包括复数对象，除非上下文清楚地作出相反说明。因而，例如，“一种植物”包括多种这样的植物，“一个细胞”包括1个或多个细胞和本领域技术人员已知的其等同方案，依此类推。

在本说明书的上下文中，将使用许多术语。

“转基因的”包括其基因组已经因为存在异源核酸而改变的任意细胞、细胞系、愈伤组织、组织、植物部分或植物，所述异源核酸例如重组DNA构建体，包括最初这样改变的那些转基因植物从最初的转基因植物有性杂交或无性繁殖建立的那些。本文使用的术语“转基因的”不包括通过常规植物育种方法或通过天然存在的事件对基因组(染色体的或染色体外)的改变，所述天然存在的事件例如随机的异体受精，非重组病毒感染，非重组细菌转化，非重组转座，或自发突变。

当应用于植物细胞时，“基因组”不仅包括在核内发现的染色体 DNA，而且包括在细胞的亚细胞组分(例如，线粒体，质体)内发现的细胞器DNA。

“植物”包括完整植物、植物器官、植物组织、种子和植物细胞和它们的后代。植物细胞包括、但不限于，来自种子，悬浮培养物，胚，分生组织区域，愈伤组织，叶，根，芽，配子体，孢子体，花粉，和小孢子的细胞。

″后代″包含植物的任意后续世代。

″转基因植物″包括在基因组内包含异源多核苷酸的植物。优选地，所述异源多核苷酸稳定整合入基因组中，使得该多核苷酸传递连续世代。所述异源多核苷酸可独地或作为重组DNA构建体的一部分整合入基因组中。

″异源″序列是指源自外来物种的序列，或者，如果来自相同物种，通过故意的人工干预组成和/或基因组基因座从它的天然形式显著修饰。

“多核苷酸”，“核酸序列”，“核苷酸序列”，或“核酸片段”可换使用，是链或双链RNA或DNA的聚合物，其任选地含有合成的、非天然的或改变的核苷酸碱基。核苷酸(通常它们的5′-磷酸形式存在)用如下的字母命名来表示:“A”表示腺苷酸或脱氧腺苷酸(分别对于RNA或DNA)，“C”表示胞苷酸或脱氧胞苷酸，“G”表示鸟苷酸或脱氧鸟苷酸，“U”表示尿苷酸，“T”表示脱氧胸苷酸，“R”表示嘌呤 (A或G)，“Y”表示嘧(C或T)，“K”表示G或T，“H”表示A或C或 T，“I”表示肌苷，且“N”表示任意核苷酸。

″多肽″，″肽″，“氨基酸序列”和″蛋白″在本文中可换使用，是指氨基酸残基的聚合物。该术语应用于氨基酸聚合物，其中的一个或多个氨基酸残基是对应的天然存在的氨基酸的人工化类似物，也应用于天然存在的氨基酸聚合物。术语″多肽″，″肽″，“氨基酸序列”，和″ 蛋白″也包括修饰，包括、但不限于，糖基化，脂质结合，硫酸化，谷氨酸残基的γ-羧基化，羟基化和ADP-核糖基化。

" messenger RNA(mRNA) (mRNA) " refers to not have intron and can be translated into by cell the RNA of albumen.

" cDNA " refers to complementary with mRNA template and uses ThermoScript II from the synthetic DNA of mRNA template.CDNA can be strand, maybe can use the Klenow fragment of DNA polymerase i to change into double chain form.

" maturation " albumen refers to translate the polypeptide of post-treatment; That is any propetide, having existed from wherein remove initial translation product or the former polypeptide of peptide.

" precursor " albumen refers to the initial product of mRNA translation; That is, propetide and peptide are former still exists.Propetide and peptide are former be may be, but not limited to,, thin inner cellular localization signal.

" separated " refers to following material, for example nucleic acid molecule and/or albumen, its be substantially devoid of or removed in naturally occurring environment, conventionally follow this material or with the interactional component of this material.Separated polynucleotide can be from they naturally occurring host cell purifying.Can the known conventional nucleic acid purification process of operation technique personnel, obtain separated polynucleotide.This term also comprises the polynucleotide of recombination of polynucleotide and chemosynthesis.

" restructuring " refers to that 2 scripts are artificial combination of sequence section separately, for example, handle separated nucleic acid segment by chemosynthesis or by gene engineering." restructuring " also comprises cell or the carrier of having modified by importing heterologous nucleic acids, or be derived from the cell of the cell of such modification, but by naturally occurring event (for example do not comprise, spontaneous mutation, natural conversion/transduction/swivel base) change to cell or carrier, those that for example produce without deliberate manual intervention.

" recombinant DNA construction body " refers to the combination of the natural nucleic acid fragment that conventionally can not find together.Therefore, recombinant DNA construction body can comprise adjusting sequence and the encoding sequence that is derived from different sources, or is derived from adjusting sequence and the encoding sequence in identical source, but arranges to be different from the mode of natural common discovery.

" adjusting sequence " refers to and is positioned at the upstream (5 ' non-coding sequence), inside of encoding sequence or the nucleotide sequence of downstream (3 ' non-coding sequence), and it can affect the transcribing of relevant encoding sequence, RNA processing or stability or translation.Regulate the sequence can be including, but not limited to, promotor, translation leader, intron, and polyadenylation recognition sequence.

" promotor " refers to the nucleic acid fragment of transcribing that can control another nucleic acid fragment.

" promotor that has function in plant " is the promotor of transcribing that can control in vegetable cell, and no matter whether its origin is from vegetable cell.

" be operably connected " and refer to the association of individual chip amplifying nucleic acid fragment, make the function of a fragment be subject to the adjusting of other fragment.For example, in the time regulating the transcribing of nucleic acid fragment, promotor is operably connected with this nucleic acid fragment.

" expression " refers to the generation of function product.For example, the expression of nucleic acid fragment can refer to that the transcribing of nucleic acid fragment (for example, producing transcribing of mRNA or functional r NA) and/or mRNA are translated as precursor or maturation protein.

" phenotype " is the detectable feature of phalangeal cell or organism.

For example, by nucleic acid fragment (, recombinant DNA construction body) insert in the context in cell, " importing " refer to " transfection " or " conversion " or " transduction ", comprises that nucleic acid fragment is to mixing in eucaryon or prokaryotic cell prokaryocyte, and the genome that wherein said nucleic acid fragment can mix cell (for example, karyomit(e), plasmid, plastid or mitochondrial DNA) in, change into self-replicating, or transient expression (for example, the mRNA of transfection).

" cell of conversion " is the arbitrary cell that has wherein imported nucleic acid fragment (for example, recombinant DNA construction body).

" conversion " used herein refers to stable conversion and instantaneous conversion.

" stable conversion " refers to that nucleic acid fragment is to the importing in host organisms genome, causes the upper stable heredity of heredity.Once stable conversion, enters nucleic acid fragment stable integration in host organisms and any offspring's genome.

" instantaneous conversion " refers to that nucleic acid fragment, to the importing in the nucleus of host organisms or the organoid that contains DNA, causes not having the upper stable hereditary genetic expression of heredity.

" allelotrope " is one of several alternative forms that occupy the gene of the specific gene seat on karyomit(e).The not homoallelic DNA sequence dna of gene is different.When allelotrope that specific gene seat place on the pair of homologous karyomit(e) at diplont exists is identical, this plant is isozygotied at this locus place.If the allelotrope that the specific gene seat place on the pair of homologous karyomit(e) of diplont exists is different, this plant is heterozygosis at this locus place.If it is upper that transgenosis is present in one of pair of homologous karyomit(e) of diplont, this plant narrows at this locus place.

" contig " refers to the nucleotide sequence assembling from 2 or a plurality of component nucleotide sequence, and described component nucleotide sequence has the common or overlap of sequence homology.For example, can contrast and compare the nucleotide sequence of 2 or a plurality of nucleic acid fragments, to differentiate common or overlapping sequence.When common or overlapping sequence is present between 2 or a plurality of nucleic acid fragment, sequence (with their corresponding nucleic acid fragment) can be assembled into the nucleotide sequence of single adjacency.

" codon degeneracy " refers to the divergence of genetic code, and it allows the variation of nucleotide sequence, and do not affect the aminoacid sequence of the polypeptide of coding.Therefore, the present invention relates to any nucleic acid fragment of comprising nucleotide sequence, all or major portion of described nucleotide sequence coded aminoacid sequence described herein.Technician knows particular host cell in " the codon preference " of using Nucleotide codon to show in specifying specific amino acids.Therefore, when nucleic acid fragment improves the expression in host cell, wish designing nucleic acid fragment, the frequency that the preferred codon that makes frequency that its codon is selected approach host cell is selected.

" synthetic nucleic acid fragment " can be used method known to those skilled in the art, from the oligonucleotide fundamental unit assembling of chemosynthesis.Connect and these fundamental units of annealing, to form larger nucleic acid fragment, then they can assemble by enzyme process, to build whole target nucleic acid fragment." chemosynthesis " relevant with nucleic acid fragment refers to, assembled in vitro component Nucleotide.Use the method for fully establishing, can realize the manual chemosynthesis of nucleic acid fragment, or use one of numerous commercially available machines, can carry out the chemosynthesis of automatization.Therefore, the optimization based on nucleotide sequence, can be for best base be because expressing customization nucleic acid fragment, to reflect the codon preference of host cell.If codon is selected those codons of deflection host preference, technician knows the possibility of successful genetic expression.Preferably codon determine can be based on gene investigation, described GENE SOURCES is from the host cell that can obtain sequence information.

Term " amplification " refers to, uses at least one nucleotide sequence as template, build nucleotide sequence a plurality of copies or with a plurality of copies of nucleic acid array complementation.Amplification system comprises polymerase chain reaction (PCR) system, ligase chain reaction (LCR) (LCR) system, amplification (NASBA based on nucleotide sequence, Cangene, Mississauga, Ontario), Q-β replicative enzyme system, amplification system based on transcribing (TAS), and strand displacement amplification (SDA).Referring to, for example, DiagnosticMolecular Microbiology:Principles and Applications, the people such as D.H.Persing, Ed., American Society for Microbiology, Washington, D.C. (1993).The product of amplification is called amplicon.

Term " chromosomal localization " comprises, the chromosomal length that the DNA linear segments comprising with reference to karyomit(e) can be measured.With reference to 2 unique DNA sequence dnas, mark, can define chromosomal localization.

Term " mark " comprises the locus for the unique location on differential staining body on karyomit(e)." polymorphism mark " comprises in a variety of forms the mark that (allelotrope) occurs, makes multi-form mark when being present in a homology centering, allows to transmit each karyomit(e) of this centering.Use one or more marks, can define genotype.

Use many control methodss for detection of homologous sequence, including, but not limited to, LASARGENE bioinformation is calculated the Megalign program (DNASTAR Inc., Madison, WI) of bag, can measure sequence alignment and identity percentage calculation.Except as otherwise noted, use Clustal V comparison method (Higgins and Sharp (1989) CABIOS.5:151-153), utilize default parameter (GAP PENALTY=10, GAP LENGTHPENALTY=10), carry out a plurality of comparisons of sequence provided herein.Use Clustal V method by being KTUPLE=1 to comparison and the default parameter that calculates the identity per-cent of protein sequence, GAP PENALTY=3, WINDOW=5 and DIAGONAL S SAVED=5.For nucleic acid, these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.After aligned sequences, by observe " sequence distance " table on same program, use Clustal V program, can obtain " identity per-cent " and " divergence " value; Except as otherwise noted, with which, calculate and provide in this article and claimed identity per-cent and divergence.

Except as otherwise noted, " BLAST " provided herein sequence identity/similarity refers to and uses BLAST 2.0 routine packages, the value of using default parameter people such as (, Nucleic Acids Res.25:3389-3402 (1997)) Altschul to obtain.The software that carries out BLAST analysis can openly obtain, for example, and by NCBI (National CenterforBiotechnology Information).This algorithm comprises, first by differentiating the short word of length W in search sequence, its with database sequence in the word of equal length while comparing, mate or meet some on the occasion of threshold value scoring T, thereby differentiate that high Grading sequence is to (HSPs).T is called neighborhood word score threshold value (people such as Altschul, the same).These initial neighborhood words hit the seed of the retrieval of the longer HSP that contains them as startup discovery.Then along the both direction of each sequence, extend described word and hit, until the comparison of accumulation score can improve.For nucleotide sequence, operation parameter M (the right reward score of residue of coupling; >0 always) and the N (point penalty of mispairing residue; <0 always) calculate the score of accumulation.For aminoacid sequence, with score matrix, calculate the mark of accumulation.When following situation, stop at the extension that in each direction, word hits: the maximum value that the comparison mark of accumulation reaches from it numerical value X that declined; Due to the accumulation of one or more negative score residues comparisons, the score of accumulation reaches zero or lower than zero; Or arrive the ending of any one sequence.The parameter W of BLAST algorithm, T and X have determined susceptibility and the speed of comparison.What BLASTN program (for nucleotide sequence) acquiescence was used is word length (W) 11, desired value (E) 10, cutoff 100, M=5, N=-4, relatively two chains.For aminoacid sequence, BLASTP program acquiescence is used, word length (W) 3, desired value (E) 10, with BLOSUM62 score matrix (referring to Henikoff & Henikoff, Proc.Natl.Acad.Sci.USA 89:10915 (1989)).

As used herein, " from 51% to the arbitrary integer that is up to (and comprising) 100% " refers to 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ".

As used herein, " from 61% to the arbitrary integer that is up to (and comprising) 100% " refers to 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ".

As used herein, " from 81% to the arbitrary integer that is up to (and comprising) 100% " refers to 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ".

As used herein, " 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; or any other integer between 80% to 100% " refer to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

The recombinant DNA of standard used herein and molecule clone technology are well-known in the art, are more completely described in Sambrook, J., Fritsch, E.F. and Maniatis, T.MolecularCloning:A Laboratory Manual; Cold Spring Harbor Laboratory Press:Cold Spring Harbor, 1989 (" Sambrook " hereinafter).

Go to now preferred embodiment:

Preferred embodiment comprises separated polynucleotide and polypeptide, recombinant DNA construction body, and the composition that comprises these recombinant DNA construction bodies (for example plant or seed), and use the method for these recombinant DNA construction bodies.

Preferred separated polynucleotide and polypeptide

The present invention includes polynucleotide and the polypeptide of following preferred separation:

In a preferred embodiment, the nucleotide sequence that separated polynucleotide comprise (a) coding polypeptide relevant with stem physical strength, wherein based on Clustal V comparison method, with SEQID NO:2,4,6,8,10,12,14,16 or 18 relatively, the aminoacid sequence of described polypeptide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer between 80% to 100%; Or (b) complementary sequence of the nucleotide sequence of above-mentioned (a), wherein said complementary sequence and nucleotide sequence are comprised of the Nucleotide of similar number, and are 100% complementation (that is, the total length complementary sequences of nucleotide sequence (a)).Preferably, described polypeptide is relevant with Maize Stem physical strength, and the aminoacid sequence of described polypeptide is compared with SEQ ID NO:16 or 18.

In another preferred embodiment, separated polynucleotide comprise (a) based on Clustal V comparison method, with SEQ ID NO:1,3,5,7,9,11,13,15, and 17 comparisons, there is at least 60% sequence identity or from 61% to the nucleotide sequence that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (b) the total length complementary sequence of the nucleotide sequence of above-mentioned (a).

In another preferred embodiment, the aminoacid sequence that the isolated polypeptide relevant with stem physical strength comprises is based on Clustal V comparison method, with SEQ ID NO:2,4,6,8,10,12,14,16 or 18 relatively, has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer between 80% to 100%.

Several method can be for measuring the stem physical strength of plant.Preferably, can electricity consumption mechanical experiment systematic survey physical strength.The in the situation that of Maize Stem physical strength, in a kind of preferred method, as follows the internode under fringe is carried out to 3 crooked experiments: use Instron, 4411 types (Instron Corporation, 100 Royall Street, Canton, Massachusetts 02021), between anchor point is 200mm across width, thirdly with the speed combination load cell of 200mm/ minute; Making internode rupture required load can be as the tolerance (" 3 crooked experiments " hereinafter) of physical strength.Verified, cellulosic amount height correlation (referring to U.S. Patent Application No. 2004068767 A1, incorporated by reference in this article) in the Maize Stem of breaking tenacity and per unit length between knot.

Compare with the control plant of express polypeptide, in plant this polypeptide lack the reduction that can cause this axis physical strength time, this polypeptide " relevant to stem physical strength ".

Compare with the maize plant that contrasts of express polypeptide, in maize plant this polypeptide lack the reduction that can cause this maize plant stem physical strength time, this polypeptide " relevant to Maize Stem physical strength ".

Should be appreciated that those skilled in the art can understand, the present invention includes more than specific exemplary sequence.Cause specific site produce the amino acid be chemically equal to, but do not affect the variation of nucleic acid fragment of functional property of the polypeptide of coding, be well-known in the art.For example, the codon of amino acid alanine (a kind of hydrophobic amino acid) can be substituted by the codon of the residue that the lower residue (for example glycine) of the another kind of hydrophobicity of coding or hydrophobicity are higher (for example α-amino-isovaleric acid, leucine or Isoleucine).Similarly, also can predict the variation that causes an electronegative residue to be substituted by another residue, for example aspartic acid is substituted by L-glutamic acid, or a positively charged residue is substituted by another variation, for example Methionin is substituted by arginine, to produce the product being equal in function.Also can predict and cause the Nucleotide of the N-end of peptide molecule and the change of C-terminal portions to change, to change the activity of polypeptide.The modification of each proposition is within the scope of the general knowledge of this area, and the mensuration of the bioactive reservation of the product of coding is also within the scope of the general knowledge of this area.

Preferred recombinant DNA construction body and inhibition DNA construct

The present invention also comprises recombinant DNA construction body, it comprises at least one and (is for example operably connected at least one adjusting sequence, preferably, in described plant, have the promotor of function) on polynucleotide, the polynucleotide that wherein said polynucleotide comprise any separation of the present invention.

In a preferred embodiment, recombinant DNA construction body is included in the promotor that has function in plant, this promotor is operably connected to (a) polynucleotide, its coding is based on ClustalV comparison method, with SEQ ID NO:2,4,6,8,10,12,14,16, and 18 comparisons, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (b) the total length complementary sequence of the described polynucleotide of above-mentioned (a).

In another preferred embodiment, recombinant DNA construction body is included in the promotor that has function in plant, this promotor is operably connected to (a) polynucleotide, based on Clustal V comparison method, with SEQ ID NO:1,3,5,7,9,11,13,15, and 17 comparisons, its nucleotide sequence has at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%, or (b) the total length complementary sequence of the described polynucleotide of above-mentioned (a).

The present invention also comprises inhibition DNA construct.

In a preferred embodiment, suppress DNA construct and be included in the promotor that has function in plant, this promotor is operably connected to all or part of of (a) following nucleotide sequence: (i) nucleotide sequence, its coding is based on Clustal V comparison method, with SEQ ID NO:2,4,6,8,10,12,14,16, and 18 comparisons, aminoacid sequence has at least 50% sequence identity or from 51% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (ii) the total length complementary sequence of above-mentioned (a) nucleotide sequence (i); Or (b) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity or from 51% to the arbitrary integer sequence identity that is up to (and comprising) 100%, and wherein said target genes encoding is selected from the polypeptide of Bk2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9.

" inhibition DNA construct " is a kind of recombinant DNA construction body, when transforming or stable integration enters in the genome of plant, causes " silence " of target gene in plant.Described target gene can be that this is plant endogenous or genetically modified." silence " used about target gene herein, typically refers to the inhibition of the mRNA of expression of target gene or the level of albumen/enzyme, and/or the inhibition of enzyme activity level or protein functional.Term " inhibition " comprises reduction, reduces, declines, goes down, suppresses, eliminates and stops." silence " or " gene silencing " do not refer in particular to mechanism, and is inclusive, is not limited to, and antisense, co-suppression, virus suppress, hair clip suppresses, stem-ring suppresses, the scheme based on RNAi, and the scheme based on little RNA.

Suppress DNA construct and can comprise a region that is derived from target gene, and can comprise all or part of nucleotide sequence of the sense strand (or antisense strand) of target gene.According to the scheme that will use, can be consistent with all or part of sense strand (or antisense strand) 100% of target gene or be less than 100% consistent (for example, at least 50% or 51% to 100% arbitrary integer) between consistent in described region.

It is known in the art suppressing DNA construct, once can easily build after selected target target gene, including, but not limited to, co-suppression construct, antisense constructs, virus suppresses construct, hair clip suppresses construct, and stem-ring suppresses construct, double-stranded RNA-production construct, more generally, for example siRNA (short interfering rna) construct and miRNA (microRNA) construct of RNAi (RNA interference) construct and little RNA construct.

" Antisense Suppression " refers to suppress the production of sense-rna transcript of the expression of target protein.

" sense-rna " refers to complementary with all or part of target primary transcript or mRNA and blocks the rna transcription thing (U.S. Patent number 5,107,065) of the expression of separated target nucleic acid fragment.The complementarity of sense-rna can be the arbitrary portion with specific gene transcript, that is, at 5 ' non-coding sequence, 3 ' non-coding sequence, intron, or encoding sequence.

" co-suppression " refers to the production that has adopted rna transcription thing of the expression that can suppress target protein.RNA refers to a kind of rna transcription thing " to have justice ", and it comprises mRNA, and can be in cell or In Vitro Translation become albumen.The crossing of nucleotide sequence at sense orientation with natural mRNA by concentrating on homology expressed, designed in the past the co-suppression construct in plant, the minimizing that it can cause having all RNA of homology with the sequence of cross expressing (referring to the people such as Vaucheret (1998) Plant J.16:651-659; And Gura (2000) Nature 404:804-808).

Another version has been described the application (the open WO 98/36083 of PCT, open on August 20th, 1998) of inhibition of the contiguous mRNA encoding sequence of guidance of plant virus sequence.

The application of " hair clip " structure has been described in nearest work, this structure has been mixed the mRNA encoding sequence of all or part of complementary direction, it causes potential " stem-ring " structure (the open WO 99/53050 of PCT, open on October 21st, 1999) of the RNA of expression.In this case, stem is formed by the polynucleotide corresponding with target gene, and described target gene inserts with sense or antisense direction with respect to promotor, and ring forms by some polynucleotide of target gene, and they do not have complementary sequence in construct.This can increase co-suppression or reticent frequency in the transgenic plant of recovery.The summary suppressing about hair clip, referring to Wesley, the people such as S.V. (2003) Methods in Molecular Biology, Plant Functional Genomics:Methods andProtocols 236:273-286.

Wherein stem forms and encircles by least 30 Nucleotide from the gene that will suppress the construct being formed by random nucleotide sequence, also effectively for suppressing (WO 99/61632, open on December 2nd, 1999).

Poly--T and poly--A sequence have also been described for setting up the purposes (WO 02/00894, open on January 3rd, 2002) of the stem of stem-ring structure.

Another version comprises, promotes the formation of the stem in stem-ring structure with synthetic tumor-necrosis factor glycoproteins.Verified, with transgenic organism prepared by such recombinant dna fragment, have and fall the low-level albumen by forming the nucleotide fragments coding of ring, as described in the open WO 02/00904 of disclosed PCT on January 3rd, 2002.

RNA disturbs process people such as (, Nature 391:806 1998) Fire refer to sequence-specific PTGS in the animal of short interfering rna (siRNA) mediation.The so-called PTGS of corresponding process (PTGS) in plant or RNA are reticent, in fungi also referred to as oppressive (quelling).Think that the process of PTGS is for stoping conservative cytophylaxis mechanism in the evolution of expression of alien gene, conventionally by different floras and door total people such as (, Trends Genet.15:358 1999) Fire.The protection of expressing from alien gene like this; may react on the generation of double-stranded RNA (dsRNA) and evolve; described double-stranded RNA (dsRNA) is derived from virus infection; or be derived from by the cell response of the homology single stranded RNA of specificity break virus geneome RNA, transposon element is to the random integration in host genome.The existence of dsRNA in cell, can, by the mechanism that does not have fully to characterize, trigger RNAi reaction.

The existence of long dsRNA in cell, can stimulate the activity of the rnase iii enzyme that is called the enzyme of dicing (dicer).The enzyme of dicing participates in dsRNA to processing people such as (, Nature 409:363 (2001)) Berstein that is called the dsRNA short-movie section of short interfering rna (siRNA).The length of the short interfering rna being obtained by the enzymic activity of dicing is approximately 21 to approximately 23 Nucleotide normally, and the duplex that comprises approximately 19 base pairs (people such as Elbashir, Gene Dev.15:188 (2001)).The little temporary transient RNA (stRNA) that the enzyme of dicing has also been considered to relate to 21-and 22-Nucleotide is from the precursor RNA excision of the conserved structure that participates in translation and control people such as (, Science293:834 (2001)) Hutvagner.RNAi reaction has also characterized the endonuclease enzyme complex of the reticent mixture (RISC) of so-called RNA-induction, and it can mediate the cutting with the antisense strand of siRNA duplex with the single stranded RNA of sequence complementarity.The cutting of target RNA occurs in the centre (people such as Elbashir, Gene Dev.15:188 (2001)) with the region of the antisense strand complementation of siRNA duplex.In addition, RNA disturbs and also can comprise the little RNA (gene silencing for example, miRNA) mediating, supposition is by regulating the cell mechanism of chromatin Structure, thus stop the transcribing of target-gene sequence (referring to, for example, Allshire, Science 297:1818-1819 (2002); The people such as Volpe, Science 297:1833-1837 (2002); Jenuwein, Science 297:2215-2218 (2002); With the people such as Hall, Science 297:2232-2237 (2002)).Like this, miRNA molecule of the present invention can be for mediated gene silencing, and this is by the interaction with rna transcription thing, or by the interaction with specific gene sequence, and wherein such interaction causes transcribing or the gene silencing of post-transcriptional level.

In multiple systems, studied RNAi.The people such as Fire (Nature 391:806 (1998)) first observe RNAi in caenorhabditis elegant.Wianny and Goetz (NatureCell Biol.2:70 (1999)) have described the RNAi being mediated by dsRNA in mice embryonic.The people such as Hammond (Nature 404:293 (2000)) have described with the RNAi in the drosophila cell of dsRNA transfection.The people such as Elbashir (Nature 411:494 (2001)) have described by importing the RNAi of the duplex induction of synthetic 21-Nucleotide RNA in the mammalian cell cultivating, and described mammalian cell comprises human embryo kidney (HEK) and HeLa cell.

Little RNA plays an important role in controlling gene is expressed.The adjusting of many growth courses (comprise and blooming), is controlled by little RNA.Now may be by using the transgenic constructs that produces little RNA in plant, the variation of the genetic expression of processing plant gene.

Little RNA seems by the base pairing performance function with complementary RNA or DNA target sequence.When in conjunction with RNA, RNA cutting or translation that little RNA can trigger target sequence suppress.When in conjunction with DNA target sequence, think that little RNA can mediate the DNA methylation of target sequence.Regardless of concrete mechanism, the result of these events is inhibition of gene expression.

Think that the sequence between little RNA and their RNA target is complementary, contribute to determine to adopt which mechanism, i.e. RNA cutting or translation suppress.It is believed that and the perfect complementary siRNA of target, by RNA, cut and play a role.Some miRNA has perfectly or approaches perfectly and the complementarity of their target, at least some such miRNA, has confirmed RNA cutting.Other miRNA and their target have several mispairing, and suppress their target on apparently in translation skill.In addition, be not subject to the constraint of the concrete theory of the mechanism of action, universal law is to occur perfection or approach perfect complementarity causing RNA cutting, and when miRNA/ target duplex contains many mispairing, preference translation suppresses.It obviously makes an exception is the microRNA 172 (miR172) in plant.One of target of miR172 is APETALA2 (AP2), although miR172 and AP2 have, approaches perfectly complementarity, seems to cause the translation of AP2 to suppress, rather than RNA cutting.

MicroRNA (miRNA) is that the length having identified in animal and plant is the non-coding RNA (people such as Lagos-Quintana of approximately 24 Nucleotide of about 19-(nt), Science 294:853-858 (2001), the people such as Lagos-Quintana, Curr.Biol.12:735-739 (2002); The people such as Lau, Science 294:858-862 (2001); Lee and Ambros, Science 294:862-864 (2001); The people such as Llave, Plant Cell 14:1605-1619 (2002); The people such as Mourelatos, Gene.Dev.16:720-728 (2002); The people such as Park, Curr.Biol.12:1484-1495 (2002); The people such as Reinhart, Gene.Dev.16:1616-1626 (2002)).The longer precursor transcript that they are about 70-200 Nucleotide from magnitude range processes, and these precursor transcripts have the ability that hairpin structure is stablized in formation.In animal, the enzyme that participates in processing miRNA precursor is called cuts enzyme (Dicer), i.e. a kind of RNA enzyme III-sample albumen (people such as Grishok, Cell 106:23-34 2001; The people such as Hutvagner, Science 293:834-838 (2001); The people such as Ketting, Gene.Dev.15:2654-2659 (2001)).Plant also has the enzyme of cutting-sample enzyme, be DCL1 (called after CARPELFACTORY/SHORTINTEGUMENTS1/SUSPENSOR1 in the past), nearest evidence shows, as cutting enzyme, it participates in processing hair clip precursor, to produce ripe miRNA (people such as Park, Curr.Biol.12:1484-1495 (2002); The people such as Reinhart, Gene.Dev.16:1616-1626 (2002)).In addition, from nearest work, can understand, at least some miRNA hair clip precursor produces as the transcript of longer polyadenylation, several different miRNA and relevant hair clip may reside in (people such as Lagos-Quintana, Science 294:853-858 (2001) in single transcript; The people such as Lee, EMBO J 21:4663-4670 2002).Nearest work also on inspection from cutting the dsRNA product that enzyme processing hair clip produces, select miRNA chain (Schwartz waits people Cell115:199-208 (2003)).Seeming the stability (be G:C and A:U content, and/or mispairing) of 2 ends of dsRNA of processing can affect chain and select, and low stability end is more easily by the expansion of helicase activity.5 ' the end chain at low stability end mixes in RISC mixture, other chain of simultaneously degrading.

MicroRNA seems to be arranged in by combination the complementary sequence of the transcript that these genes produce, and regulates target gene.The in the situation that of lin-4 and let-7, target site is positioned at 3 ' UTR (people such as Lee, the Cell 75:843-854 (1993) of said target mrna; The people such as Wightman, Cell 75:855-862 (1993); The people such as Reinhart, Nature 403:901-906 (2000); The people such as Slack, Mol.Cell 5:659-669 (2000)), between lin-4 and let-7 miRNA and their target site, there are several mispairing.The combination of lin-4 or let-7 miRNA seems to cause the downward of steady-state level of the albumen of said target mrna coding, and does not affect transcript itself (Olsen and Ambros, Dev.Biol.216:671-680 (1999)).On the other hand, nearest evidence shows, miRNA can cause the specific RNA cutting of target transcript in target site in some cases, and this cutting step seems to need 100% complementarity (Hutvagner and Zamore, the Science 297:2056-2060 (2002) between miRNA and target transcript; The people such as Llave, Plant Cell 14:1605-1619 (2002)).Seem likely, miRNA can enter at least 2 approach that target gene regulates: when the complementary <100% of target, albumen is lowered; When target complementarity is 100%, RNA cutting.The microRNA that enters RNA cutting approach is similar to RNA in animal and disturbs the 21-25 Nucleotide short interfering rna (siRNA) that produces in (RNAi) process and PTGS (PTGS) (Hamilton and the Baulcombe (1999) in plant; The people such as Hammond, (2000); The people such as Zamore, (2000); The people such as Elbashir, (2001)), and may mix the reticent mixture (RISC) of inducing with the similar or identical RNA-observing in RNAi.

With the target of information biology identification miRNA, not yet success in animal, this may be due to the following fact, and animal miRNA and their target have low complementarity.On the other hand, information biology scheme has been successfully used to predict target (people such as Llave, the Plant Cell 14:1605-1619 (2002) of Mirnas of plant; The people such as Park, Curr.Biol.12:1484-1495 (2002); The people such as Rhoades, Cell 110:513-520 (2002)), thereby Mirnas of plant seems to have the total complementarity higher than animal miRNA with their target of inferring.The member of most of such prediction target transcript coding involved in plant development moulding of Mirnas of plant or the transcription factor family of cytodifferentiation.

The preferred regulatory element of recombinant DNA construction body and inhibition DNA construct

Many promotors can and suppress in DNA construct for recombinant DNA construction body of the present invention.Result that can be based on hope, selects promotor, can comprise composing type, tissue-specific, induction type or other promotor for expressing at host organisms.

The high-level constitutive expression of the candidate gene under 35S promoter is controlled, can have multiple-effect impact.But, tissue-specific and/or should coerce specific expression and can eliminate undesirable impact, but the ability that strengthens drought tolerance retained.This impact exists mouse ear mustardin observe (people such as Kasuga, Nature Biotechnol.17:287-91 (1999)).Like this, when being driven by different promoters, can test candidate gene effect.

The constitutive promoter being applicable in plant host cell comprises, for example, the core promoter of Rsyn7 promotor, and in WO 99/43838 and U.S. Patent number 6,072,050 disclosed other constitutive promoter; Core CaMV 35S promoter (people such as Odell, Nature 313:810-812 (1985)); Rice Actin muscle (people such as McElroy, Plant Cell 2:163-171 (1990)); Ubiquitin (people such as Christensen, the people such as Plant Mol.Biol.12:619-632 (1989) and Christensen, Plant Mol.Biol.18:675-689 (1992)); PEMU (people such as Last, Theor.Appl.Genet.81:581-588 (1991)); MAS (people such as Velten, EMBOJ.3:2723-2730 (1984)); ALS promotor (U.S. Patent number 5,659,026), etc.Other constitutive promoter comprises, for example, and in U.S. Patent number 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; With 6,177, those that discuss in 611.

When the promotor of selecting for method of the present invention, may wish using-system-specific or grow the promotor regulating.Tissue-promotor specific or that growth regulates is DNA sequence dna, it regulates DNA sequence dna the selective expression in male Inflorescence development, solid or the two the vital vegetable cell/tissue of growth, and the expression of such DNA sequence dna is limited in to the male Inflorescence development of plant or the stage of seed maturity.Any discernible promotor can, in method of the present invention, cause the spatial-temporal expression of hope.

Preferred stem-specific promotor is Herba Medicaginis stem-specific S2A gene (people such as Abrahams, Plant Mol.Biol.27:513-528 (1995)).

Seed or embryo-specific and can comprise for the promotor in the present invention soybean Kunitz trypsin inhibitor (Kti3, Jofuku and Goldberg, Plant Cell 1:1079-1093 (1989)), potato tuber albumen (patatin) (the potato tuber) (people such as Rocha-Sosa, EMBO is (1989) J.8:23-29), convicilin, vicilin, and legumin (pea cotyledon) (Rerie, W.G., wait people Mol.Gen.Genet.259:149-157 (1991), Newbigin, E.J., waits people, Planta 180:461-470 (1990), Higgins, T.J.V, Deng people, Plant.Mol.Biol.11:683-695 (1988)), zein (corn embryosperm) (Schemthaner, J.P., Deng people, EMBO is (1988) J.7:1249-1255), phaseollin (Kidney bean cotyledon) (Segupta-Gopalan, C., Deng people, Proc.Natl.Acad.Sci.U.S.A.82:3320-3324 (1985)), phytohemagglutinin (Kidney bean cotyledon) (Voelker, T. wait people, EMBO is (1987) J.6:3571-3577), B-conglycinin and glycinin (soybean cotyledon) (Chen, Z-L, Deng people, EMBO is (1988) J.7:297-302), gluten (rice endosperm), hordein (barley endosperm) (Marris, C., Deng people, Plant Mol.Biol.10:359-366 (1988)), glutenin and gliadine (wheat endosperm) (Colot, V, Deng people, EMBO is (1987) J.6:3559-3564), with sweet potato storing albumen (sporamin) (sweet potato stem tuber) (Hattori, T., Deng people, Plant Mol.Biol.14:595-604 (1990)).Be operably connected to the promotor of the seed-specific gene of the allos coding region in mosaic gene construct, in transgenic plant, maintain their spatial-temporal expression pattern.Such example comprises, Arabidopis thaliana 2S seed storage protein gene promoter, to express enkephalin peptide in Arabidopis thaliana and colea seed, (the people such as Vanderkerckhove, Bio/Technology 7:L929-932 (1989)), phaseolus vulgaris agglutinin and soya bean β-phaseollin promotor, to express the luciferase (people such as Riggs, Plant Sci.63:47-57 (1989)), with wheat gluten promotor, to express E.C. 2.3.1.28 people such as (, EMBO is (1987) J.6:3559-3564) Colot.

React on the endogenous or for example existence of chemical compound (chemical inducer) of exogenous stimulation, or react on signal environment, hormone, chemistry and/or that grow, inducible promoter is optionally expressed the DNA sequence dna being operably connected.The promotor of induction type or adjusting comprises, for example, by light, heat, coerce, promotor that floods or arid, plant hormone, wound or chemical substance (for example ethanol, jasmonate, Whitfield's ointment, or safener) regulate.

React on the promotor of coercing and comprise following promotor: 1) RD29A promotor (people such as Kasuga, Nature Biotechnol.17:287-291 (1991)); 2) barley promotor, B22E; In developmental Semen Maydis core, the expression of B22E is specific (" the PrimaryStructure of a Novel Barley Gene Differentially Expressed in ImmatureAleurone Layers " .Klemsdae of stalk, S.S. wait people, Mol.Gen.Genet.228 (1/2): 9-16 (1991)); With 3) corn promotor, Zag2 (" Identification and molecularcharacterization of ZAG1; the maize homolog of the Arabidopsis floralhomeotic gene AGAMOUS ", Schmidt, R.J. wait people, Plant Cell 5 (7): 729-737 (1993)).Zag2 transcript can detect in pollination for first 5 days to pollination for 7-8 days, and guides the expression in the carpel of developmental female inflorescence, and to the specific Ciml of the core of developmental Semen Maydis core.Ciml transcript can detect for 4-5 days before pollination to pollination for 6-8 days.Other useful promotor comprises can be derived from any promotor that it expresses gene relevant with developmental female little Hua on maternal.

Promotor can be different from natural gene completely, or comprises the different elements that is derived from the different promoters of finding at nature, or even comprises synthetic DNA section.Skilled person in the art will appreciate that different promotors can instruct in different tissues or cell type or in the different etap or react on the genetic expression of different envrionment conditionss.Also recognize, owing in most of the cases determining the accurate border that regulates sequence not yet completely, the DNA fragmentation of some variant may have identical promoter activity.Cause the promotor that gene is expressed in most cell types to be in most of the cases often called " constitutive promoter ".Constantly find dissimilar new promotor useful in vegetable cell; At Okamuro, J.K., and Goldberg, R.B., in the compilation of Biochemistry of Plant 15:1-82 (1989), can find many examples.

Particularly preferred promotor can comprise: Herba Medicaginis stem-specific S2A gene promoter, RIP2, mLIP15, ZmCOR1, Rab17, CaMV 35S, RD29A, SAM synthetic enzyme, ubiquitin, CaMV 19S, nos, Adh, sucrose synthase, R-allelotrope, or root cells promotor.Other preferred promotor is included in herein disclosed CesA10 in the open 2005/0086712A1 of whole United States Patent (USP) incorporated by reference, CesA11, and any in CesA12 promotor.

Recombinant DNA construction body of the present invention and inhibition DNA construct also can comprise that other regulates sequence, include but not limited to translation leader, intron, and polyadenylation recognition sequence.In another preferred embodiment of the present invention, recombinant DNA construction body of the present invention comprises enhanser or silencer in addition.

Intron sequences can be added on the 5 ' untranslated region or encoding sequence of part encoding sequence, to be increased in the amount of the ripe signal accumulating in cytosol.Verified, in the transcriptional units of plant and animal expression construct, comprise can montage intron, can increase genetic expression at mRNA and protein level and be up to 1000 times.Buchman and Berg, Mol.Cell Biol.8:4395-4405 (1988); The people such as Callis, Gene Dev.1:1183-1200 (1987).In the time of near being placed in 5 ' end of transcriptional units, such intron of genetic expression strengthens conventionally maximum.The use of corn intron A dh1-

S introne

1,2 and 6, Bronze-1 intron is known in the art.Conventionally referring to, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).

If wish expression of polypeptides, conventionally wish in 3 of polynucleotide encoding district '-end comprises polyadenylation district.Polyadenylation district can be derived from natural gene, many other plant genes or T-DNA.3 ' the end sequence adding can be derived from, for example, and nopaline synthase or octopine synthase gene, or be derived from another kind of plant gene, or not too preferably from any other eukaryotic gene.

Translation leader is at the promoter sequence of gene and the DNA sequence dna between encoding sequence.Translation leader is present in the mRNA of the processing completely of translation initiation sequence upstream.Translation leader can affect processing from primary transcript to mRNA, mRNA stability or translation efficiency.The example (Turner, R. and Foster, G.D., Molecular Biotechnology 3:225 (1995)) of translation leader has been described.

Can select plant arbitrarily, for differentiating adjusting sequence and gene for the preparation of recombinant DNA construction body of the present invention and inhibition DNA construct.Be applicable to isolated genes and regulate the example of the plant target of sequence to comprise, but be not limited to clover, apple, apricot, Arabidopis thaliana, choke, rocket salad (arugula), asparagus, junket pears, banana, barley, Kidney bean, beet, blackberry, blueberry, blueberry, green Cauliflower, brussels sprouts, Caulis et Folium Brassicae capitatae, rape, muskmelon, Radix Dauci Sativae, cassava, Semen Ricini, Cauliflower, celery, cherry, witloof, coriander, citrus, money tangerine, trifolium, coconut, coffee, corn, cotton, Cranberries, cucumber, Pseudotsuga menziesii (Mirbel) Franco (Douglas fir), eggplant, witloof, thatch dish (escarole), eucalyptus, fennel, Fructus Fici, garlic, cucurbit, grape, natsudaidai, honeydew, yam bean, Kiwifruit, lettuce, fragrant-flowered garlic, lemon, lime (lime), torch pine, Semen Lini, mango, muskmelon, mushroom, nectarine, nut, oat, oil palm, oilseed rape, gumbo, olive, onion, orange, ornamental plant, palm, pawpaw, Parsley, Holland parsnip, pea, peach, peanut, pears, pepper, persimmon, pine, pineapple, psyllium, plum, pomegranate, white poplar, potato, summer squash, oranges and tangerines, Rediata, radiscchio, little radish, Semen Brassicae campestris, raspberry, rice, rye, jowar, Southern Pine, soybean, spinach, pumpkin, strawberry, beet, sugarcane, Sunflower Receptacle, sweet potato, sweet gum, red tangerine, tea tree, tobacco, tomato, triticale (triticale), turf, turnip, rattan, watermelon, wheat, potato, and cucumber.For differentiating that regulating the particularly preferred plant of sequence is Arabidopis thaliana, corn, wheat, soybean, and cotton.

In another preferred embodiment of the present invention, recombinant DNA construction body of the present invention comprises enhanser in addition.

Preferred composition

A kind of preferred composition of the present invention is plant, comprises recombinant DNA construction body of the present invention (for example those preferred constructs discussed above) arbitrarily in its genome.

Another kind of preferred composition is plant, (for example in its genome, comprise the destruction of at least one gene (it can be genome allos or endogenous) that is selected from Bk2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9, insert, for example transposable element, or series jump).

Another kind of preferred composition is plant, comprises other recombinant DNA construction body discussed below (for example, comprising the nucleotide sequence relevant with SEQ ID NO:20-42 and the construct of aminoacid sequence) in its genome.

Preferred composition also comprises any offspring of plant and any seed obtaining from plant or its offspring.Offspring comprises the offspring who obtains by the self-pollination of plant or outcross.Offspring also comprises hybrid and inbred lines.

Preferably, in the crop of hybrid seed breeding, ripe transgenic plant can selfing, to produce the inbreeding plant of isozygotying.Inbreeding plant can generate the seed of the recombinant DNA construction body that contains new importing.Can cultivate these seeds, to be created on the plant that contains recombinant DNA construction body and show relevant phenotype as herein described in genome, or for the procedure of breeding, to produce hybrid seed, the latter can grow, to generate the plant that contains this recombinant DNA construction body and show relevant phenotype as herein described.Preferably, described seed is corn.

Preferably, described plant is unifacial leaf or dicotyledons, more preferably, and corn or soybean plants, more preferably maize plant, for example corn hybrid plant or corn inbreeding plant.Described plant can be also Sunflower Receptacle, Chinese sorghum, and (canola) drawn in Kano, wheat, clover, cotton, rice, barley or grain.

Preferably, recombinant DNA construction body is stably integrated in Plant Genome arbitrarily.

Particularly preferred embodiment comprises:

1. a kind of plant (preferably corn), in its genome, comprise the recombinant DNA construction body that contains at least one regulatory element, described regulatory element is operably connected to the nucleotide sequence of (a) coding polypeptide relevant with stem physical strength, the aminoacid sequence of wherein said polypeptide is based on Clustal V comparison method, compare with SEQ ID NO:16 or 18, there is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer between 80% to 100%; Or (b) complementary sequence of this nucleotide sequence, wherein said complementary sequence and nucleotide sequence are comprised of the Nucleotide of similar number, and are 100% complementation (that is, the total length complementary sequences of nucleotide sequence (a)).Preferably, described at least one regulatory element is the promotor that has function in plant.

2. a kind of plant (preferably corn), in its genome, comprise (a) the first recombinant DNA construction body, it comprises at least one promotor that has function in plant, this promotor is operably connected at least one polynucleotide that are selected from the first separation of lower group: (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:2,4,6,8,10,12,14,16, with 18 relatively, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:1,3,5,7,9,11,13,15, and 17 relatively, there is at least 60% sequence identity or from 61% to the arbitrary integer that is up to (and comprising) 100% sequence identity; (iii) above-mentioned (a) (i) or (a) total length complementary sequence of the polynucleotide of (ii); (b) the second recombinant DNA construction body, it comprises at least one promotor that has function in plant, this promotor is operably connected at least one polynucleotide that are selected from the second separation of lower group: (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:20,22,24,26,28,30,32,34,36,38,40, with 42 relatively, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:21,23,25,27,29,31,33,35,37,39, and 41 relatively, there is at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (iii) above-mentioned (b) (i) or (b) total length complementary sequence of the polynucleotide of (ii).Preferably, compare with the control plant of described the second recombinant DNA construction body with not comprising described the first recombinant DNA construction body, described plant shows the cell walls content of cellulose of increase and/or the growth velocity of increase.

3. a kind of plant (preferably corn), in its genome, comprise at least one and regulate sequence, described adjusting sequence is operably connected to (a), and at least one is selected from the separated polynucleotide of lower group: (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:2,4,6,8,10,12,14,16, and 18 relatively, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ IDNO:1,3,5,7,9,11,13,15, and 17 relatively, there is at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (iii) above-mentioned (a) (i) or (a) total length complementary sequence of the polynucleotide of (ii); (b) at least one is selected from the separated polynucleotide of lower group: (i) polynucleotide, and it is encoded based on Clustal V comparison method, with SEQ ID NO:20,22,24,26,28,30,32,34,36,38,40, with 42 relatively, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:21,23,25,27,29,31,33,35,37,39, and 41 relatively, there is at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (iii) above-mentioned (b) (i) or (b) total length complementary sequence of the polynucleotide of (ii).Preferably, compare with not comprising described at least one control plant that is operably connected to the adjusting sequence on described (a) and (b), described plant shows the cell walls content of cellulose of increase and/or the growth velocity of increase.

4. a kind of plant (preferably corn), in its genome, comprise at least one and regulate sequence, described adjusting sequence is operably connected to (a), and at least one is selected from the separated polynucleotide of lower group: (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:2 comparison, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:1 comparison, has at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (iii) above-mentioned (a) (i) or (a) total length complementary sequence of the polynucleotide of (ii); (b) at least one is selected from the separated polynucleotide of lower group: (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:38,40, with 42 relatively, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ IDNO:37,39, and 41 relatively, there is at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (iii) above-mentioned (b) (i) or (b) total length complementary sequence of the polynucleotide of (ii).Preferably, compare with not comprising described at least one control plant that is operably connected to the adjusting sequence on described (a) and (b), described plant shows the cell walls content of cellulose of increase.

5. a kind of plant (preferably corn), in its genome, comprise at least one and regulate sequence, described adjusting sequence is operably connected to (a), and at least one is selected from the separated polynucleotide of lower group: (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:6 comparison, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:5 comparison, has at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (iii) above-mentioned (a) (i) or (a) total length complementary sequence of the polynucleotide of (ii); (b) at least one is selected from the separated polynucleotide of lower group: (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:20,32, with 34 relatively, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ IDNO:19,31, and 33 relatively, there is at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (iii) above-mentioned (b) (i) or (b) total length complementary sequence of the polynucleotide of (ii).Preferably, compare with not comprising described at least one control plant that is operably connected to the adjusting sequence on described (a) and (b), described plant shows the growth velocity of increase.

6. a kind of plant (preferably corn), in its genome, comprise at least one and regulate sequence, described adjusting sequence is operably connected at least 2 separated polynucleotide that are selected from lower group: (a) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:2,4,6,8,10,12,14,16, and 18 relatively, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (b) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:1,3,5,7,9,11,13,15, and 17 relatively, there is at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (c) the total length complementary sequence of above-mentioned (a) or polynucleotide (b).

7. a kind of plant (preferably corn), in its genome, comprise inhibition DNA construct, the latter is included in the promotor that has function in plant, this promotor is operably connected to all or part of of (a) following nucleotide sequence: (i) nucleotide sequence, its coding is based on Clustal V comparison method, with SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, with 18 comparisons, aminoacid sequence has at least 50% sequence identity, or from 51% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (ii) the total length complementary sequence of above-mentioned (a) nucleotide sequence (i), or (b) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity or from 51% to the arbitrary integer sequence identity that is up to (and comprising) 100%, and wherein said target genes encoding is selected from the polypeptide of Bk2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9.Preferably, compare with the control plant that does not comprise described inhibition DNA construct, described plant shows the stem physical strength of reduction.Preferably, described inhibition DNA construct comprises co-suppression construct, antisense constructs, and virus suppresses construct, hair clip suppresses construct, and stem-ring suppresses construct, double-stranded RNA-production construct, RNAi construct, or little RNA construct (for example, siRNA construct or miRNA construct).

8. a kind of plant (preferably corn), its genome comprises the destruction that coding is selected from least one gene of BK2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9 polypeptide.Preferably, described destruction causes comparing with the control plant that does not comprise described destruction, and described plant shows the stem physical strength of reduction.Preferably, described destruction comprises an insertion, for example transposable element or series jump.

9. a kind of plant (preferably corn), in its genome, comprise inhibition DNA construct, the latter is included in the promotor that has function in plant, this promotor is operably connected to all or part of of (a) following nucleotide sequence: (i) nucleotide sequence, its coding is based on Clustal V comparison method, with SEQ ID NO:6 comparison, aminoacid sequence has at least 50% sequence identity or from 51% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (ii) the total length complementary sequence of above-mentioned (a) nucleotide sequence (i); Or (b) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity or from 51% to the arbitrary integer sequence identity that is up to (and comprising) 100%, and wherein said target genes encoding Bk2L3 polypeptide.Preferably, compare the organ size that described plant shows the plant height of reduction and/or reduces with the control plant that does not comprise described inhibition DNA construct.Preferably, described inhibition DNA construct comprises co-suppression construct, antisense constructs, and virus suppresses construct, hair clip suppresses construct, and stem-ring suppresses construct, double-stranded RNA-production construct, RNAi construct, or little RNA construct (for example, siRNA construct or miRNA construct).

10. a kind of plant (preferably corn), the destruction of at least one gene that its genome comprises coding Bk2L3 polypeptide.Preferably, described destruction causes comparing with the control plant that does not comprise described destruction, the organ size that described plant shows the plant height of reduction and/or reduces.Preferably, described destruction comprises an insertion, for example transposable element or series jump.

11. a kind of plant (preferably corn), in its genome, comprise inhibition DNA construct, the latter is included in the promotor that has function in plant, this promotor is operably connected to all or part of of (a) following nucleotide sequence: (i) nucleotide sequence, its coding is based on Clustal V comparison method, with SEQ ID NO:10 comparison, aminoacid sequence has at least 50% sequence identity or from 51% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (ii) the total length complementary sequence of above-mentioned (a) nucleotide sequence (i); Or (b) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity or from 51% to the arbitrary integer sequence identity that is up to (and comprising) 100%, and wherein said target genes encoding Bk2L5 polypeptide.Preferably, compare with the control plant that does not comprise described inhibition DNA construct, described plant shows male sterile.Preferably, described inhibition DNA construct comprises co-suppression construct, antisense constructs, and virus suppresses construct, hair clip suppresses construct, and stem-ring suppresses construct, double-stranded RNA-production construct, RNAi construct, or little RNA construct (for example, siRNA construct or miRNA construct).

12. a kind of plant (preferably corn), the destruction of at least one gene that its genome comprises coding BKL5 polypeptide.Preferably, described destruction causes comparing with the control plant that does not comprise described destruction, and described plant shows male sterile.Preferably, described destruction comprises an insertion, for example transposable element or series jump.

13. any offspring of above-mentioned plant, any seed of above-mentioned plant, any seed of the offspring of above-mentioned plant, and from any above-mentioned plant and offspring's cell.

Those of ordinary skills can easily recognize and be applicable to the contrast that contrasts or measure with respect to the plant that comprises recombinant DNA construction body (or suppress DNA construct) in genome or with reference to plant.For example,, as non-restrictive explanation:

● for recombinant DNA construction body (or suppressing DNA construct), be the offspring of hemizygous conversion of plant, this offspring is separated into and comprises or do not comprise the recombinant DNA construction body plant of (or suppressing DNA construct): conventionally, with respect to not comprising the recombinant DNA construction body offspring of (or suppressing DNA construct), measure the offspring who comprises recombinant DNA construction body (or inhibition DNA construct).

● recombinant DNA construction body (or suppress DNA construct), to gradually oozing in inbred lines, for example, gradually oozes in corn, or in a kind, for example, in soybean: conventionally with respect to parent's inbreeding or product germline, measure gradually ooze be.

● 2 hybrid systems, wherein first hybrid system generates from 2 parent's inbred lines, second hybrid system generates from contain recombinant DNA construction body (or suppressing DNA construct) 2 all identical parent's inbred lines except one of parent's inbred lines: conventionally with respect to first hybrid system, measure second hybrid system.

● a kind of plant, in its genome, comprise recombinant DNA construction body (or suppressing DNA construct) (or kind of plant, the destruction that it comprises gene in its genome): can be with respect to do not comprise recombinant DNA construction body (or suppressing DNA construct) in genome, but other side (for example has the genetic background that can compare with described plant, with the plant comparison that comprises this recombinant DNA construction body or inhibition DNA construct or destruction, nuclear genetic material has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) control plant (or with respect to the control plant that does not comprise described destruction), measure described plant.There are many technology based on laboratory that can be used for analyzing, contrasting and characterize plant genetic background; Wherein there is isozyme electrophoresis, restriction fragment length polymorphism (RFLPs), randomly amplified polymorphic DNA (RAPDs), the polymerase chain reaction (AP-PCR) of causing arbitrarily, DNA cloning fingerprinting (DAF), the amplification region of sequence characterization (SCARs), amplified fragment length polymorphism (AFLPs), and repeat (SSRs) also referred to as the simple sequence of micro-satellite.

Recombinant DNA construction body of the present invention, to the importing in plant, can be realized by the technology of any appropriate, includes but not limited to direct DNA picked-up, chemical treatment, electroporation, microinjection, cytogamy, infects, carrier mediated DNA shifts, bombardment, or agriculture bacillus mediated conversion.When hope is integrated in genome by a plurality of or stacking recombinant DNA construction body or separated polynucleotide (for example, to realize the coexpression of the polynucleotide of 2 or a plurality of separation), the polynucleotide of single separation can be imported in parent system, and hybridize by traditional breeding technique, so that the combination of hope or stacking to be provided in progeny plants subsequently.

Set forth below preferred technology, embodiment 3 is the conversions about corn plant cell, and embodiment 8 is the conversions about soybean plant cell.

Main other preferred method that uses agrobacterium tumefaciens to transform dicotyledons and obtain transgenic plant, comprises cotton (U.S. Patent number 5,004,863, U.S. Patent number 5,159,135, U.S. Patent number 5,518,908); Soybean (U.S. Patent number 5,569,834, U.S. Patent number 5,416,011, the people such as McCabe, BiolTechnology 6:923 (1988), the people such as Christou, Plant Physiol.87:671 674 (1988)); Rape (U.S. Patent number 5,463,174); Peanut (people such as Cheng, Plant Cell Rep.15:653 657 (1996), the people such as McKently, Plant Cell Rep.14:699 703 (1995)); Pawpaw; And pea (people such as Grant, PlantCell Rep.15:254 258, (1995)) those disclosed.

Use electroporation, particle bombardment and Agrobacterium-mediated Transformation monocotyledons have also been reported, as preferred method, comprise, for example, the conversion realizing in following plant and plant regeneration: the asparagus (people such as Bytebier, Proc.Natl.Acad.Sci. (USA) 84:5354, (1987)); Barley (Wan and Lemaux, Plant Physiol 104:37 (1994)); Corn (the people such as Rhodes, Science240:204 (1988), the people such as Gordon-Kamm, Plant Cell 2:603 618 (1990), the people such as Fromm, BiolTechnology 8:833 (1990), the people such as Koziel, BiolTechnology 11:194, (1993), the people such as Armstrong, Crop Science 35:550 557 (1995)); Oat (people such as Somers, BiolTechnology 10:1589 (1992)); Orchardgrass (people such as Horn, Plant Cell Rep.7:469 (1988)); Rice (people such as Toriyama, TheorAppl.Genet.205:34, (1986); The people such as Part, Plant Mol.Biol.32:11351148, (1996); The people such as Abedinia, Aust.J.Plant Physiol.24:133 141 (1997); Zhang and Wu, Theor.Appl.Genet.76:835 (1988); The people Plant CellRep.7:379 such as Zhang, (1988); Battraw and Hall, Plant Sci.86:191 202 (1992); The people such as Christou, Bio/Technology 9:957 (1991)); Rye (people such as De la Pena, Nature 325:274 (1987)); Sugarcane (Bower and Birch, Plant is (1992) J.2:409); Tall grass (people such as Wang, BiolTechnology 10:691 (1992)), and wheat (people such as Vasil, Bio/Technology 10:667 (1992); U.S. Patent number 5,631,152).

There are many methods from plant tissue aftergrowth.Concrete renovation process depends on primordial plant tissue and the concrete plant species that will regenerate.

From single plant protoplast transformant or from explant regeneration, growth and the cultivation plant of a plurality of conversions, (Weissbach and Weissbach well-known in the art, In:Methodsfor Plant Molecular Biology, (Eds.), Academic Press, Inc.San Diego, CA, (1988)).This regeneration and process of growth generally include following step: select the cell of conversion, by the common stage of embryonic development, by the plantlet stage of taking root, cultivate the cell of those individuations.The embryo of regeneration of transgenic and seed similarly.After this genetically modified bud of taking root obtaining is planted in suitable plant growth culture medium for example in soil.

Growth or the regeneration of the plant of the nucleic acid fragment external, ectogenic separation that contains the target protein of encoding are well-known in the art.Preferably, the plant self-pollination of regeneration, to provide the transgenic plant of isozygotying.Otherwise, make the plant hybridization of the seed growth that is important from the pollen that obtains of plant of regeneration and agronomy.On the contrary, the plant for the regeneration of pollinating from the pollen of the plant of these important systems.Use method well known to the skilled person, cultivate the transgenic plant of the present invention that contain target polypeptides.

Detecting the mensuration of albumen can be undertaken by SDS-polyacrylamide gel electrophoresis or immunoassay.The level determination of detection substrate or enzyme product can use gas-chromatography or liquid chromatography (for separating of) and ultraviolet or visible spectrophotometry or mass spectroscopy (for detection of) etc. carry out.The mensuration of the level of the mRNA of target enzyme, can realize by RNA blotting or RT-PCR technology.Once aftergrowth, and obtain the progeny plants that transgenosis is isozygotied, will observe the plant with stable phenotype in offspring's similar seed.

Preferred method

The present invention also comprises the method that changes axis physical strength; Evaluate the method for axis physical strength; Evaluate the method for content of cellulose in plant; Change the cell walls content of cellulose of plant and/or the method for growth velocity, the method for giving plants male sterility, and the method for the organ size of reduction plant height and/or plant.Preferably, described plant is monocotyledons or dicotyledons, more preferably corn or soybean plants, more preferably maize plant.Described plant can be also Sunflower Receptacle, jowar, and tongue is drawn in Kano, wheat, clover, cotton, rice, barley or grain.

The preferred method that changes (preferably increasing) axis physical strength comprises (a) recombinant DNA construction body is imported to reproducible vegetable cell, to generate the vegetable cell transforming, described recombinant DNA construction body contains at least one regulatory element (preferably, the promotor that has function in plant), described regulatory element is operably connected to the nucleotide sequence of (i) coding polypeptide relevant with stem physical strength, the aminoacid sequence of wherein said polypeptide is based on Clustal V comparison method, with SEQID NO:4, 6, 8, 10, 12, 14, 16 or 18 relatively, have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer between 80% to 100%, or (ii) complementary sequence of this nucleotide sequence, wherein said complementary sequence and nucleotide sequence are comprised of the Nucleotide of similar number, and be 100% complementation, (b) from the vegetable cell regeneration of transgenic plant of described conversion, wherein said transgenic plant comprise described recombinant DNA construction body in its genome, and wherein compare with the control plant that does not comprise described recombinant DNA construction body, described transgenic plant show the change (preferably increasing) of stem physical strength.

A kind of preferred method of evaluating axis physical strength comprises (a) and import recombinant DNA construction body in reproducible vegetable cell, to generate the vegetable cell transforming, described recombinant DNA construction body is included in the promotor that has function in plant, this promotor is operably connected to (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:4, 6, 8, 10, 12, 14, 16, with 18 comparisons, aminoacid sequence has at least 80% sequence identity, or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (ii) above-mentioned (a) (i) total length complementary sequence of described polynucleotide, (b) from the vegetable cell regeneration of transgenic plant of described conversion, (c) evaluate the stem physical strength of described transgenic plant.The method can comprise in addition (d) and obtain the progeny plants that is derived from described transgenic plant; (e) evaluate the stem physical strength of described progeny plants.

The another kind of preferred method of evaluating axis physical strength comprises (a) and import recombinant DNA construction body in reproducible vegetable cell, to generate the vegetable cell transforming, described recombinant DNA construction body is included in the promotor that has function in plant, this promotor is operably connected to (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:4, 6, 8, 10, 12, 14, 16, with 18 comparisons, aminoacid sequence has at least 80% sequence identity, or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (b) above-mentioned (a) (i) total length complementary sequence of described polynucleotide, (b) from the vegetable cell regeneration of transgenic plant of described conversion, (c) obtain the progeny plants that is derived from described transgenic plant, (d) evaluate the stem physical strength of described progeny plants.

A kind of preferred method of evaluating plant cellulose content comprises (a) and import recombinant DNA construction body in reproducible vegetable cell, to generate the vegetable cell transforming, described recombinant DNA construction body comprises the polynucleotide that are operably connected in the promotor that has function in plant, wherein said polynucleotide encoding is based on Clustal V comparison method, with SEQ ID NO:4, 6, 8, 10, 12, 14, 16, with 18 comparisons, aminoacid sequence has at least 80% sequence identity, or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, (b) from the vegetable cell regeneration of transgenic plant of described conversion, (c) evaluate the content of cellulose of described transgenic plant.The method can comprise in addition (d) and obtain the progeny plants that is derived from described transgenic plant; (e) evaluate the content of cellulose of described progeny plants.

The another kind of preferred method of evaluating plant cellulose content comprises (a) and import recombinant DNA construction body in reproducible vegetable cell, to generate the vegetable cell transforming, described recombinant DNA construction body comprises the polynucleotide that are operably connected in the promotor that has function in plant, wherein said polynucleotide encoding is based on Clustal V comparison method, with SEQ ID NO:4, 6, 8, 10, 12, 14, 16, with 18 comparisons, aminoacid sequence has at least 80% sequence identity, or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, (b) from the vegetable cell regeneration of transgenic plant of described conversion, (c) obtain the progeny plants that is derived from described transgenic plant, (d) evaluate the content of cellulose of described progeny plants.

A kind of preferred method of plant that selection has a content of cellulose of change comprises (a) and obtains any plant of the present invention (for example any preferred embodiment discussed above); (b) evaluate the content of cellulose of the plant obtaining in step (a); (c), when content of cellulose is compared change with control plant, select the plant of the evaluation of step (b).Preferably, when content of cellulose increases, even more preferably, when content of cellulose is at least 35%, 40%, 45%, 50%, 55%, or 60% time, and/or when Mierocrystalline cellulose dry matter content be 100mg/cm at least, 200mg/cm, 300mg/cm, 400mg/cm, or during 500mg/cm, select the plant of evaluating.In this paper embodiment 10, set forth the preferred method of measuring content of cellulose.

A kind of preferred method of the cell walls content of cellulose of change (preferably increasing) plant and/or change (preferably improving) growth velocity comprises, in the genome of plant, (be for example integrated into, by the combination of transgenic technology or transgenic technology and traditional breeding method) one or more recombinant DNA construction bodies, thereby at least one is selected from the separated polynucleotide of lower group coexpression (a): (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, with 18 comparisons, aminoacid sequence has at least 80% sequence identity, or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:1,3,5,7,9,11,13,15, and 17 relatively, there is at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%, (iii) above-mentioned (a) (i) or (a) total length complementary sequence of the polynucleotide of (ii), (b) at least one is selected from following separated polynucleotide: (i) polynucleotide, and it is encoded based on Clustal V comparison method, with SEQ IDNO:20,22,24,26,28,30,32,34,36,38,40, with 42 relatively, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:21,23,25,27,29,31,33,35,37,39, and 41 relatively, there is at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%, (iii) above-mentioned (b) (i) or (b) total length complementary sequence of the polynucleotide of (ii).

Preferably, the method that increases plant cell wall content of cellulose comprises, in the genome of plant, be integrated into one or more recombinant DNA construction bodies, thereby at least one is selected from the separated polynucleotide of lower group coexpression (a): (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:2 comparison, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:1 comparison, has at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (iii) above-mentioned (a) (i) or (a) total length complementary sequence of the polynucleotide of (ii); (b) at least one is selected from the separated polynucleotide of lower group: (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:38,40, with 42 relatively, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:37,39, and 41 relatively, there is at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (iii) above-mentioned (b) (i) or (b) total length complementary sequence of the polynucleotide of (ii).

Preferably, the method that improves plant growth rate comprises, in the genome of plant, be integrated into one or more recombinant DNA construction bodies, thereby at least one is selected from the separated polynucleotide of lower group coexpression (a): (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:6 comparison, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:5 comparison, has at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (iii) above-mentioned (a) (i) or (a) total length complementary sequence of the polynucleotide of (ii); (b) at least one is selected from the separated polynucleotide of lower group: (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:20,32, with 34 relatively, aminoacid sequence has at least 80% sequence identity or from 81% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%; (ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:19,31, and 33 relatively, there is at least 60% sequence identity or from 61% to the arbitrary integer sequence identity that is up to (and comprising) 100%; (iii) above-mentioned (b) (i) or (b) total length complementary sequence of the polynucleotide of (ii).

A kind of preferred method of giving plants male sterility comprises: (a) in reproducible vegetable cell, import inhibition DNA construct, the latter is included in the promotor that has function in plant, this promotor is operably connected to all or part of of (i) following nucleotide sequence: (A) nucleotide sequence, its coding is based on Clustal V comparison method, with SEQ ID NO:10 comparison, aminoacid sequence has at least 50% sequence identity, or from 51% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (B) the total length complementary sequence of above-mentioned (i) nucleotide sequence (A), or (ii) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity or from 51% to the arbitrary integer sequence identity that is up to (and comprising) 100%, and wherein said target genes encoding Bk2L5 polypeptide, (b) from the vegetable cell regeneration of transgenic plant of described conversion, wherein said transgenic plant comprise described inhibition DNA construct in its genome, and wherein compare the organ size that described transgenic plant show the plant height of reduction and/or reduce with the control plant that does not comprise described inhibition DNA construct.The method can comprise in addition: (c) obtain the progeny plants that is derived from described transgenic plant, wherein said progeny plants comprises described inhibition DNA construct in its genome.

A kind of preferred method that reduces plant height and/or reduce plant organ size comprises: (a) to importing in reproducible vegetable cell, suppress DNA construct, the latter is included in the promotor that has function in plant, this promotor is operably connected to all or part of of (i) following nucleotide sequence: (A) nucleotide sequence, its coding is based on Clustal V comparison method, with SEQ ID NO:6 comparison, aminoacid sequence has at least 50% sequence identity, or from 51% to the polypeptide that is up to the arbitrary integer sequence identity of (and comprising) 100%, or (B) the total length complementary sequence of above-mentioned (i) nucleotide sequence (A), or (ii) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity or from 51% to the arbitrary integer sequence identity that is up to (and comprising) 100%, and wherein said target genes encoding Bk2L3 polypeptide, (b) from the vegetable cell regeneration of transgenic plant of described conversion, wherein said transgenic plant comprise described inhibition DNA construct in its genome, and wherein compare the organ size that described transgenic plant show the plant height of reduction and/or reduce with the control plant that does not comprise described inhibition DNA construct.The method can comprise in addition: (c) obtain the progeny plants that is derived from described transgenic plant, wherein said progeny plants comprises described inhibition DNA construct in its genome.

Separated nucleic acid and albumen and any embodiment of the present invention can be for the vegetation types of wide scope, monocotyledons especially, and for example kind gramineous, comprises dichromatism chinese sorghum and corn.The nucleic acid of separation of the present invention and albumen also can be for the kinds from following genus: Cucurbita, rose belongs to, Vitis, white walnut, Fragaria, Lotus, Medicago, donkey food Macroptilium, Trifolium, Trigonella, Vigna, both citrus, linum, Geranium, cassava, Daucus, Arabidopis thaliana, Btassica, Rhaphanus, mustard belongs to, Atropa, Capsicum, Datura, Hyoscyamus, tomato belongs to, Nicotiana, Solanum, green winter Solanum, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Brome, Asparagus, antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, lolium, Oryza, Avena, Hordeum, Secale, Triticum, raw, Ce Sinobambusa, Dendrocalamus, with pears Sinobambusa.

embodiment

Further explained in the following embodiments the present invention, wherein umber and percentages, the number of degrees are degree Celsius, except as otherwise noted.Although should be appreciated that these embodiment have indicated the preferred embodiments of the invention, provide as just explanation.From discussion and these embodiment above, those skilled in the art can determine essential characteristic of the present invention, in the situation that not deviating from its spirit and scope, can make multiple changes and improvements of the present invention, make it be applicable to multiple use and condition.Thereby those skilled in the art are from description above, can understand except show herein and describe those of the present invention multiple improvement.Such improvement also within the scope of the appended claims.

embodiment 1

the sign of the corn cDNA of coding Bk2-sample albumen

The crisp stem 2 of corn (bk2) phenotype reported first was in (Langham in 1940, MNL 14:21-22 (1940)), by phenotype map mark umc95 around of 100 centimorgan district and the chr9L between bn17.13 people such as (, MNL 65:52-53 (1991)) Howell.In the past, confirmed clone csc1c.pk005.k4:fis (SEQ ID NO:1) crisp stem 2 polypeptide (SEQ ID NO:2) (the international application no PCT/US2005/035450 that encodes, it requires the U.S. Provisional Application submitted on October 6th, 2004 number 60/615,868 right of priority, its complete content is incorporated by reference in this article).Other 2 members (SEQ ID NO:7 and 8 and SEQ IDNO:13 and 14) of Bk2 gene family are also disclosed.In the disclosure content, these genes are called after Bk2-sample (Bk2L).

The BLASTN or the TBLASTN algorithm that use NCBI (NCBI) to provide, other corn cDNA sequence to several database retrievals in nucleic acid and amino acid levels and the crisp stem 2 of corn (Bk2) sequence (SEQ ID NO:1) homology, described database is including, but not limited to, the inside proprietary database of DuPont (the Local Alignment gopher on basis; The people such as Altschul, J.Mol.Biol.215:403-410 (1993); The people such as Altschul, NucleicAcids Res.25:3389-3402 (1997)) and the Maize genome search sequence (GSS) that can openly obtain and TIGR Maize genome set (TIGR gene index database, The Institute forGenomic Research, Rockville, MD 20850; The people such as Quackenbush, J.NucleicAcids Res.28 (1): 141-145 (2000)).6 newcomers (Bk2L1, Bk2L3, Bk2L5, Bk2L6, Bk2L8 and Bk2L9) of Bk2 gene family have been isolated.Table 1 listed disclosed in this manual except Bk2 self all Bk2-sample albumen.

table 1

crisp stem 2-sample albumen

Figure 1A-1F has shown the Clustal V comparison of the aminoacid sequence of report in table 1, wherein uses default parameter.The chart that Fig. 2 shows has contrasted the identity per-cent (with the divergence per-cent in second triangle) between 9 aminoacid sequences shown in Figure 1A-1F, wherein uses Clustal V comparison method.

By public data storehouse being carried out to BLAST (the Local Alignment gopher on basis of EST; Altschul, S.F., waits people, J.Mol.Biol.215:403-410 (1993)) retrieve, further identify the possible function of the polypeptide of each cDNA coding.To (comprising all nonredundant GenBank CDS translations at BLAST " nr " database, be derived from the sequence of 3-dimension structure Brookhaven albumen database, the up-to-date key plate of SWISS-PROT protein sequence database originally, EMBL, and DDBJ database) in the sequence that comprises carry out similarity retrieval.The BLASTN algorithm that uses NCBI (NCBI) to provide, the similarity of analytical sequence.DNA sequence dna is translated in all open reading-frame (ORF)s, and the BLASTX algorithm (Gish, W. and States, the D.J. that use NCBI to provide, Nature Genetics 3:266-272 (1993)), with " nr " database in all protein sequences contrasts similaritys that can openly obtain of comprising.Be displayed in Table 2 " scoring " result of the aminoacid sequence of the whole Bk2-sample albumen of being encoded by the cDNA clone's who comprises appointment whole cDNA inset.Data in table 2 have also shown SEQ ID NO:2,4,6,8,10, and the calculation result of the identity per-cent of aminoacid sequence described in 12,14,16 and 18 has been pointed out sequence in the general identifier number column of NCBI.

table 2

the BLAST result of the sequence of the polypeptide of coding and Bk2-sample albumen homology

Gene (SEQ ID NO :)	The general identifier numbering of NCBI (registration number)	Scoring (bit)	Identity per-cent
Gene (SEQ ID NO :)		Scoring (bit)	Identity per-cent	Bk2L1 (SEQ ID NO:3)	NCBI GI 34733385 (AAQ81633.1)	1266	100％
Bk2L3 (SEQ ID NO:5)	NCBI GI 30090026 (AAO17706.1)	868	95％	Bk2L1 (SEQ ID NO:3)	NCBI GI 34733385 (AAQ81633.1)	1266	100％
Bk2L3 (SEQ ID NO:5)	NCBI GI 30090026 (AAO17706.1)	868	95％	Bk2L4 (SEQ ID NO:7)	NCBI GI 30090026 (AAO17706.1)	922	97％
Bk2L5 (SEQ ID NO:9)	NCBI GI 52076665 (BAD45565.1)	1079	79％	Bk2L4 (SEQ ID NO:7)	NCBI GI 30090026 (AAO17706.1)	922	97％
Bk2L5 (SEQ ID NO:9)	NCBI GI 52076665 (BAD45565.1)	1079	79％	Bk2L6 (SEQ ID NO:11)	NCBI GI 50939113 (XP_479084.1)	742	81％
Bk2L7 (SEQ ID NO:13)	NCBI GI 50939113 (XP_479085.1)	751	82％	Bk2L6 (SEQ ID NO:11)	NCBI GI 50939113 (XP_479084.1)	742	81％
Bk2L7 (SEQ ID NO:13)	NCBI GI 50939113 (XP_479085.1)	751	82％	Bk2L8 (SEQ ID NO:15)	NCBI GI 34898176 (NP_910434.1)	838	65％
Bk2L9 (SEQ ID NO:17)	NCBI GI 50927043 (XP_473354.1)	597	63％	Bk2L8 (SEQ ID NO:15)	NCBI GI 34898176 (NP_910434.1)	838	65％

Fig. 6 has shown Bk2L albumen from corn, from the BC1L albumen of rice with from the germline of the COBL albumen (NCBI registration number is in bracket) of Arabidopis thaliana, has analyzed.Numeral along branch is that heuristic searching surpasses 5,000 bootstrap values that repeat to obtain.Only shown the bootstrap value that obtains the monosystem group that the >50% time supports.The amino acid difference of branch length and deduction is proportional.

embodiment 2

the gene expression analysis of Bk2-sample albumen

Use the massive parallel signal order-checking (MPSS of Solexa ^tM) technology (referring to table 3) (people such as Brenner, Nat.Biotechnol.18:630-634 (2000); The people such as Brenner, Proc.Natl.Acad.Sci.U.S.A.97:1665-1670 (2000)), checked the tissue specificity of the expression of disclosed Bk2-sample gene family in table 1.MPSS ^tMcomprise the marker of setting up 17 bases from the mRNA sample of reverse transcription.To sign order-checking simultaneously, and distribute to gene or EST.For the abundance of these signs provides the digital value that is standardized as PPM (PPM), the sign then contrasting between different tissues is expressed or sign abundance.Thereby, MPSS ^tMplatform can be for measuring the expression pattern of specific gene, and its expression level in different tissues.Numeral is the mean value in a plurality of libraries of each tissue of listing in secondary series.

table 3

in corn, the PPM of Bk2 gene family expresses

Tissue	Library number	Bk	2	Bk 2L 1	Bk 2L 3	Bk 2L 4	Bk 2L 5	Bk 2L 6	Bk 2L 7	Bk 2L 8	Bk 2L 9
Tissue	Library number	Bk	2	Bk 2L 1	Bk 2L 3	Bk 2L 4	Bk 2L 5	Bk 2L 6	Bk 2L 7	Bk 2L 8	Bk 2L 9	Flower pesticide	3	1	73	49	51	0	0	0	9	1
Fringe	17	0	48	17	30	0	0	0	0	0		Flower pesticide	3	1	73	49	51	0	0	0	9	1
Fringe	17	0	48	17	30	0	0	0	0	0	Embryo	10	0	18	61	18	0	0	0	0	3
Endosperm	26	0	18	48	23	0	4	1	3	0	Embryo	10	0	18	61	18	0	0	0	0	3
Endosperm	26	0	18	48	23	0	4	1	3	0	Shell	1	75	68	490	16	0	0	39	0	0
Benevolence	5	2	86	103	51	0	1	1	15	1	Shell	1	75	68	490	16	0	0	39	0	0
Benevolence	5	2	86	103	51	0	1	1	15	1	Leaf	46	17	20	87	10	0	0	0	32	0
Meristematic tissue	20	2	60	81	19	0	0	0	2	0	Leaf	46	17	20	87	10	0	0	0	32	0
Meristematic tissue	20	2	60	81	19	0	0	0	2	0	Pericarp	6	4	16	290	54	0	0	0	2	0
Pollen	2	0	6	2	13	794	0	0	0	0	Pericarp	6	4	16	290	54	0	0	0	2	0
Pollen	2	0	6	2	13	794	0	0	0	0	Root	43	52	69	263	14	0	0	2	8	0
Seedling	7	8	16	72	21	0	0	0	8	0	Root	43	52	69	263	14	0	0	2	8	0
Seedling	7	8	16	72	21	0	0	0	8	0	Fringe silk	9	0	36	69	47	29	0	2	0	0
Small ear	12	17	86	205	111	0	0	12	0	0	Fringe silk	9	0	36	69	47	29	0	2	0	0
Small ear	12	17	86	205	111	0	0	12	0	0	Stem	15	172	48	474	15	0	0	8	14	0
Male flower fringe	2	4	72	53	62	0	0	16	0	0	Stem	15	172	48	474	15	0	0	8	14	0
Male flower fringe	2	4	72	53	62	0	0	16	0	0	Vascular bundle	2	182	56	117	11	0	0	0	7	0
Wheel	7	152	9	126	33	0	0	0	2	2	Vascular bundle	2	182	56	117	11	0	0	0	7	0

Bk2 (table 3, the 3rd row and Fig. 3) expresses with in separated vascular bundle at shell, leaf, root, stem, still in benevolence, meristematic tissue, pollen or fringe silk tissue, does not express.This expression pattern is consistent with the effect of Bk2 gene in secondary wall formation, because express its institute, all contains in a organized way at least some lignified cells.In Fig. 4 (also referring to Fig. 5 A, the 2nd row), shown the relation conefficient analysis of the expression level of Bk2 and the expression level of 12 corn C esA genes.The expression pattern of Bk2 gene is very similar to the CesA gene of previously disclosed formation secondary wall, be CesA10,11 and 12 expression pattern is (referring to the U.S. Patent number 6,930 of authorizing on August 16th, 2005, Fig. 5 in 225, its complete content is incorporated by reference in this article).More specifically, with respect to any other gene in such, Bk2 and corn C esA10,11, and each in 12 genes demonstrates higher relation conefficient, about >0.8.Because 3 CesA genes are coexpression also, their corresponding albumen may form functional complex together with Bk2 albumen.Table 4 listed known up to now all formation primary walls and the CesA albumen of secondary wall (U.S. Patent number 6,930,225, the same; The U.S. Patent number 6,803,498 that on October 12nd, 2004 authorizes, its complete content is incorporated by reference in this article).Corn C esA10,11, and the directed orthologous gene from Arabidopis thaliana and rice of 12 genes and they has been considered to participate in secondary wall formation (people such as Tanaka, Plant Physiol.133:73-83 (2003); The people such as Taylor, Proc.Natl.Acad.Sci.U.S.A.100:1450-1455 (2003); The people such as Appenzeller, Cellulose11:287-299 (2004)).The coexpression of the CesA gene of Bk2 and formation secondary wall, has supported the effect of Bk2 in corn secondary wall formation.

table 4

form the CesA albumen of primary wall and secondary wall

Demonstrating with another Bk2L gene of the correlated expression of CesA gene is Bk2L3.The CesA gene that before the expression pattern of Bk2L3 is very similar to, the participation primary wall of report forms (people such as Holland, Plant Physiol.123:1313-1323 (2000); Dhugga, Curr.Opin.Plant Biol.4:488-493 (2001); The people such as Appenzeller, Cellulose 11:287-299 (2004)).3 genes, CesA1 specifically, 7 and 8, seem to form functional fiber element synthase mixture, for primary wall, form.The expression of Bk2L3 gene and this 3 CesA gene height correlations, seem to be similar to by 3 CesA albumen and a secondary wall cellulose synthase mixture that Bk2 albumen forms, these 4 albumen can form functional fiber element synthase mixture, for primary wall, form.

Bk2L5 only expresses in pollen.Some expression most probable in fringe silk is derived from the pollen tube of growth therein.Bk2L8 is leaf-preferred seemingly, and Bk2L6 is endosperm-specific.

In Fig. 5 A and 5B, shown from Solexa MPSS ^tMresearch from the association between all different B k2 of corn and the expression level of CesA gene.

embodiment 3

indication embodiment

under the specific strong promoter of stem is controlled, by crossing expression corn Bk2-sample gene, structure build the stem strength of enhancing

Build chimeric transgenosis, in tissue-specific mode, directly cross expression Bk2 gene/polypeptide.The corn cDNA that transgenic constructs comprises coding Bk2L3 and/or Bk2L6 (for example, SEQ IDNO:5 or SEQ ID NO:11), it is operably connected to (people such as Abrahams, Plant Mol.Biol.27:513-528 (1995)) in the promotor from the specific S2A gene of stem of clover.Then the DNA that contains Bk2L3 or Bk2L6 ORF is merged to the S2A promotor on 5 ' end and the pinII terminator on 3 ' end, to generate the expression cassette shown in Fig. 3.Then construct is connected on the selection cassette that contains bar gene that CaMV 35S promoter drives and pinII terminator.Those skilled in the art can understand, can adopt different promotors, 5 ' end sequence and/or 3 ' end sequence to reach comparable expression of results.Use Zhao (U.S. Patent number 5,981, mandate on November 9th, 840,1999; Its content is hereby incorporated by reference) the method for transformation based on Agrobacterium, by transforming immature maize with this expression cassette, generate rotaring gene corn plant.Although for S2A promotor-Bk2L3 (or Bk2L6) expression cassette maize transformation plant has been described method below, those of ordinary skills will appreciate that, the method can be connected to any constructs of the promotor on corn Bk2L3 (or Bk2L6) gene or the maize plant that expression cassette production transforms for using to comprise, for expressing plant.

From corn dividing from immature embryo, make embryonic breeding touch agrobacterium suspension, wherein this bacterium can be transferred to S2A promotor-Bk2L3 (or Bk2L6) expression cassette (explaining) above at least one cell (step 1: infect step) of at least one immature embryo.In this step, immature embryo is immersed in agrobacterium suspension with initial inoculation.By embryo and Agrobacterium co-cultivation for some time (step 2: be total to culturing step).After infecting step, immature embryo is cultivated on solid medium.After this common cultivation stage, carry out optional " static " step.In this static step, culturing embryo under at least one antibiotic existence, known this microbiotic can suppress the growth (step 3: static step) of Agrobacterium when not adding the selective agent of Plant Transformation strain.On the solid medium of selective agent, cultivate immature embryo to remove Agrobacterium thering is microbiotic but do not have, and provide stationary phase for infected cell.After this, containing on the substratum of selective agent the embryo of cultivating through inoculation, in growth, through transformed calli, be collected (step 4: select step).Preferably, cultivate immature embryo having on the solid medium of selective agent, cause the selective growth through transformant.By select cultured calli on substratum at solid, the callus that makes to obtain is regenerated subsequently and is become plant (step 5: regeneration step).

embodiment 4

indication embodiment

under stem high specificity promotor is controlled, by being used in, in promoter region, contain enhanser unit the transgene expression of the corn Bk2-sample gene of part, builds the stem strength strengthening

By allos enhancer element being placed in to the promoter region of natural B k2L3 (or Bk2L6) gene, strengthen the expression of Bk2L3 (or Bk2L6) gene.Build an expression cassette, it comprises for example CaMV35S of the enhancer element that merges with the natural promoter of Bk2L3 (or Bk2L6) and full-length cDNA.Then can, by transforming immature maize with the expression cassette described in embodiment 3, produce rotaring gene corn plant.

Embodiment 5

indication embodiment

by crossing of corn Bk2-sample and CesA gene, express, build the stem strength strengthening

In view of form the gene major effect plant tissue of secondary wall physical strength, do not affect form phenotype, the gene that forms primary wall can affect plant growth rate, thereby their regulation and control can be for improving growth velocity.Confirmed in the past

corn gene CesA

1,7, and 8 in Various Tissues coexpression, show that they may form functional enzyme mixture.Bk2L3 and this 3 CesA gene co-expressings, show that the protein product of all these 4 genes forms a kind of functional enzyme mixture strongly.By different promoters, preferably its in expressing the corn of promoters driven of the gene relevant with cell elongation simultaneously mistake express this 4 genes, these genes, as single polygene construct or as the construct separating of the various combination that contains these genes, can be produced the transgenic plant of the growth velocity with increase.Other Bk2L gene also can be used with 3 above-mentioned CesA assortments of genes arbitrarily, to produce the transgenic plant of the growth velocity with increase.

Embodiment 6

indication embodiment

Except contributing to physical strength, secondary wall accounts for the major part of plant biomass.In view of physical strength can be for reducing plant lodging, quality and quantity biomass are important for many other purposes, comprise alcohol production.Bk2 gene and corn C esA10,11, together with 12 genes, provide an approach that increases cellulose ratios in cell walls.The efficiency of alcohol production is directly related with cellulosic amount in biomass.With Mierocrystalline cellulose instead of woody element, aspect silage digestibility, be also useful.

About forming the gene of primary wall, as described in Example 5, can make Bk2 gene and CesA10,11, and 12 gene co-expressings, but under secondary wall-specific promotor is controlled, to produce, there are the stem strength of raising and the transgenic plant of biomass quality.

Embodiment 7

indication embodiment

build the down-regulation of corn Bk2-sample gene

Owing to forming the CesA gene of primary wall, contribute to cell to expand, their limited down-regulation can be for reducing plant height or organ size.More specifically, the expression of Bk2L3 gene and the CesA gene height correlation that forms primary wall.In view of needing all members of expressive function enzyme complex to improve enzymic activity, only a member's down-regulation may be enough to reduce active.For example the down-regulation of Bk2L3 (and/or Bk2L5, for male sterile), can realize by any technology in co-suppression, RNAi, sense-rna or microRNA, produces short and small transgenic plant.Highly reduce the crop plants that and needs lower for some harvest indexs improve.For example, modern wheat and rice varieties are obviously lower than their older copy.The ability that reduces plant height is mainly the reason of the Green Revolution of every kind of such crop.

embodiment 8

indication embodiment

under stem-specific strong promoter is controlled, recombinant DNA structure in dicotyledons cell build the expression of body

Can be structured in 5 of cDNA fragment ' comprise the expression cassette (people such as Abrahams from the promotor of Herba Medicaginis stem-specific S2A gene, Plant Mol.Biol.27:513-528 (1995)), and for expressing polypeptide of the present invention the soybean transforming.PinII terminator can be placed in 3 of cDNA fragment '.Such construct can be for crossing expression Bk2-sample gene.Will appreciate that, those skilled in the art can adopt different promotors and/or 3 ' end sequence to reach suitable expression of results.

Use suitable Oligonucleolide primers, the polymerase chain reaction of cloning by cDNA (PCR), can prepare the cDNA fragment of this gene.Cloning site can be mixed to oligonucleotide, so that suitable DNA fragmentation direction to be provided when inserting expression vector.Then increase as mentioned above, separated fragment is inserted and carried in the pUC18 carrier of seed expression cassette.

Then can be with the expression vector soybean transformation embryo of the sequence that comprises the polypeptide of the present invention of encoding.For inductor somatic embryo, i.e. cotyledon, is cut into 3-5mm by the seed surface sterilization of soybean varieties A2872, immature long, 26 ℃, in illumination or dark, on suitable nutrient agar, cultivate 6-10 week.Then cut off the somatic embryo that generates secondary embryo, be placed in suitable liquid nutrient medium.Repeat to select as after the somatic embryo of the embryo breeding in early stage spherical stage bunch the suspension that maintains as described below.

At rotary shaker (150rpm), above in 26 ℃, soybean embryo generation suspension culture is remained in 35ml liquid nutrient medium, with luminescent lamp, provide circulation at daytime/night in 16:8 hour.By about 35mg tissue is inoculated in 35ml liquid nutrient medium, culture was carried out to succeeding transfer culture in every two weeks.

The method of then bombarding by particle gun (people (1987) Nature (London) 327:70-73 such as Klein, U.S. Patent number 4,945,050), soybean transformation embryo generation suspension culture.DuPont Biolistic ^tMpDS1000/HE instrument (helium retrofit) can transform for these.

Can be for promoting the selectable marker gene of transformation of soybean, the 35S promoter (Odell comprising from cauliflower mosaic virus, Deng people, (1985) Nature 313:810-812), from the hygromycin phosphotransferase gene of plasmid pJR225 (from intestinal bacteria; Gritz, waits people, (1983) Gene 25:179-188) and the transgenosis that forms from 3 ' region of the nopaline synthase gene of the T-DNA of agrobacterium tumefaciens Ti-plasmids.Can be separated into restriction fragment by comprising phaseollin 5 ' district (this fragment coding polypeptide of the present invention) and the seed expression cassette in phaseollin 3 ' district.Then this fragment can be inserted to unique restriction site of the carrier that carries marker gene.

In 50 μ l60mg/ml, add 1 μ m gold grain suspension (in order): 5 μ l DNA (1 μ g/ μ l), 20 μ l spermidines (0.1M), and 50 μ l CaCl ₂(2.5M).Then particle product is stirred 3 minutes, in Eppendorf centrifuge, rotate 10 seconds, remove supernatant liquor.Then in 400 μ l70% ethanol, wash the coated particle of DNA-1 time, Eddy diffusion is in 40 μ l dehydrated alcohols.Can be by DNA/ particle suspension liquid sonication 3 times, each 1 second.Subsequently by the coated gold grain application of sample of 5 μ l DNA+ to each larger vector dish.

The suspension culture in 2 week age of about 300-400mg is placed in to 60 empty x 15mm culture dish, use transfer pipet by residual liquid from tissue removal.For each transformation experiment, normally bombard about 5-10 dull and stereotyped tissue.Film destroy pressure setting is 1100psi, casing is evacuated to the vacuum tightness of 28 inches of mercury.Tissue is placed on from keeping somewhere sieve (retaining screen) approximately 3.5 inches of places, and bombards 3 times.After bombardment, tissue is divided into 2 half, is put back in liquid, cultivate as mentioned above.

5-7 days after bombardment, is fresh culture by changing liquid cultivation matrix, and 11-12 days after bombardment, is replaced by the fresh culture that contains 50mg/ml Totomycin.This selection substratum upgrades weekly.In 7-8 week after bombardment, observe from the downright bad embryo of unconverted occurs bunch and grow the green tissue through transforming.Take isolated chlorenchyma away, be seeded in each flask, to generate embryo generation suspension culture new, vegetative conversion.Each new strain is used as independently transformation event and is processed.These suspension subsequently can succeeding transfer culture, and is maintained immature embryo bunch, or is regenerated as whole strain plant by maturation and the sprouting of each somatic embryo.

embodiment 9

indication embodiment

under the specific strong promoter of stem is controlled, recombinant DNA construction body in microorganism cells express

The cDNA (SEQ ID NO:1,3,5,7,9,11,13,15 or 17) of coding crisp stem 2-sample polypeptide of the present invention can be inserted to T7 coli expression carrier pBT430.This carrier is derivative people (1987) Gene 56:125-135 such as () Rosenberg of pET-3a, and its uses phage t7 RNA polymerase/T7 promoter systems.By first destroying EcoRI and the HindIII site in pET-3a in their zero position, build plasmid pBT430.The oligonucleotide adapter that contains EcoRI and Hind III site is inserted to the BamHI site of pET-3a.This can set up the pET-3aM with extra unique cloning site, and this site is for inserting expression vector by gene.Then, use the mutagenesis that is positioned oligonucleotide, the NdeI site in translation initiation position is changed into NcoI site.The DNA sequence dna of pET-3aM in this region, 5 '-CATATGG changes into 5 '-CCCATGG in pBT430.

Can suitably digest the plasmid DNA that contains cDNA, to discharge the nucleic acid fragment of this albumen of coding.Then can be on 1% low melting-point agarose gel this fragment of purifying.Damping fluid and agarose contain 10ug/ml ethidium bromide, so that DNA fragmentation develops.Then can, according to manufacturer's specification sheets, pass through GELase ^tM(Epicentre Technologies, Madison, WI) digestion, from sepharose purifying fragment, ethanol precipitation, dry, Eddy diffusion is in 20uL water.Use T4 DNA ligase (New England Biolabs (NEB), Beverly, MA), suitable oligonucleotide adapter can be connected in fragment.Use low melting-point agarose as above, can be purified into from unnecessary adapter the fragment of the adapter that contains connection.Digested vector pBT430, with alkaline phosphatase (NEB) dephosphorylation, with phenol/chloroform Deproteinization as above.The carrier pBT430 that then can prepare 16 ℃ of connections and fragment 15 hours, be transformed into DH5 electroreception state cell (GIBCO BRL) subsequently.Can on the agar plate that contains LB substratum and 100ug/mL Ampicillin Trihydrate, select transformant.Then pass through restriction enzyme analysis, the correct direction of the T7 promotor of the transformant of the gene that screening contains the polypeptide of the present invention of encoding.

For high level expression, the plasmid clone that contains cDNA inset (being correct direction with respect to T7 promotor) can be transformed into coli strain BL21 (DE3) (people (1986) J.Mol.Biol.189:113-130 such as Studier).Grown culture in 25 ℃ of LB substratum that containing Ampicillin Trihydrate (100mg/L).In the optical density(OD) of 600nm approximately 1, add IPTG (isopropylthio-beta galactose glycosides, inductor) to the final concentration of 0.4mM, continue at 25 ℃ of incubation 3h.Then by centrifugal cell harvesting, in the 50mM Tris-HCl pH 8.0 that Eddy diffusion contains 0.1mM DTT and 0.2mM phenyl methyl sulfonic acid fluoride in 50uL.Add 1mm granulated glass sphere in a small amount, with microprobe ultrasonic processor by mixture supersound process 3 times, each approximately 5 seconds.Centrifugal mixture, the protein concentration of mensuration supernatant liquor.By SDS-polyacrylamide gel electrophoresis, 1ug albumen that can the separated solvable fraction from culture.Can observe the protein band that moves to the molecular weight of expection on gel.

embodiment 10

the sign of the stem tissue of the wild-type of corn (Bk2) and crisp stem (bk2-ref) mutant

From Maize genome COOP original seed center (USDA/ARS & CropSciences/UIUC, S-123 Turner Hall, 1102 S.Goodwin Avenue, Urbana, IL 61801-4798), obtain the corn original seed with reference to allelotrope (bk2-ref) that contains bk2.After blooming approximately 2 weeks, evaluate the different qualities of 3 strain greenhouse growing plants (each is bk2-ref/bk2-ref and its wild-type sibling Bk2/bk2-ref naturally, and the two is derived from the seed obtaining from identical selfing fringe).Use 4411 type Instron electromechanical experimental installations (Instron Corp., Canton, MA), 3 internodes under fringe (

internode

3,4, and 5, from fringe node numbering) are carried out to 3 crooked experiments.Between anchor point is 20cm across width.Anvil vertically presses to region between the knot of about 3cm on the node of the stem of horizontal positioned with the constant speed of 20cm/min, until it subsides or fractures.Tolerance by the ultimate load of fracture as the intensity of differentiation internode and stem.

Stem portion below fringe node, measures total dry matter.By boil the stem material 2 times 30 minutes of pulverizing with damping fluid (25mMMOPS, pH 7), the third and fourth internode below the fringe node in each strain of 3 strain plants, measures structural dry-matter and content of cellulose in duplicate.By remaining material be suspended in methyl alcohol/chloroform (3/1, v/v) in 1 hour, dry, weigh.By Updegraff method (Updegraff, Anal.Biochem.32:120-124 (1969)), measure crystalline cellulose.In brief, by the stem material grinding be placed in boiling water bath (acetic acid: water: the 8:2:1 mixture of nitric acid) 1 hour, water, then use the crystalline thing of 95% washing with alcohol 3 times, dry in Speedvac subsequently.The following Klason xylogen of measuring: the stem material grinding with 72% (w/w) sulfuric acid incubation 1 hour, the 1:20 diluent washing with 72% sulfuric acid in water 2 times, 65 ℃ of heating 30 minutes, washes with water 1 time, spends the night 80 ℃ of dried residue.Mensuration sugar as described in people such as (, Cellulose 11:287-299 (2004)) Appenzeller forms.

In a word, in stem tissue the reduction of physical strength and cellulose amount reduce and epidermis under and the nonuniform deposition height correlation of secondary cell wall material in all sclerenchyma fibers of dimension pipe.Cellulosic amount is lower, and the wall of mutant is thinner, and the dry matter content of stem that is reflected as per unit length is lower.

table 5

the measurement of stem composition and physical strength

Proterties	Wild-type	bk2-ref
Proterties	Wild-type	bk2-ref	Fringe height (cm)	102.00±8.8	106.33±11.8
Diameter stem (mm)	23.84±0.27	23.40±0.46	Fringe height (cm)	102.00±8.8	106.33±11.8
Diameter stem (mm)	23.84±0.27	23.40±0.46	Stem heavy (g)	89.43±3.39	62.08±8.46
Moisture (%)	79.20±0.21	84.87±1.04	Stem heavy (g)	89.43±3.39	62.08±8.46
Moisture (%)	79.20±0.21	84.87±1.04	Dry-matter (g/cm)	0.68±0.04	0.43±0.07
Destroy displacement (mm)	11.83±0.46	6.51±1.10	Dry-matter (g/cm)	0.68±0.04	0.43±0.07
Destroy displacement (mm)	11.83±0.46	6.51±1.10	Failure load (kg)	23.68±2.25	9.04±2.66
Insoluble dry-matter (%)	51.57±1.00	45.20±1.69	Failure load (kg)	23.68±2.25	9.04±2.66
Insoluble dry-matter (%)	51.57±1.00	45.20±1.69	Mierocrystalline cellulose (%)	33.30±0.56	23.76±0.68
Xylogen (%)	9.07±0.21	10.28±0.63	Mierocrystalline cellulose (%)	33.30±0.56	23.76±0.68
Xylogen (%)	9.07±0.21	10.28±0.63	Remaining cell wall (%)	9.20±1.68	11.16±2.03
Mierocrystalline cellulose (g/cm)	0.24±0.114	0.11±0.019	Remaining cell wall (%)	9.20±1.68	11.16±2.03

Sequence table

Claims

1. the polynucleotide of separation, it comprises:

(a) nucleotide sequence of the coding polypeptide relevant with stem physical strength, the aminoacid sequence of wherein said polypeptide is based on Clustal V comparison method, compare with SEQ ID NO:16 or 18, there is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer between 80% to 100%; Or

(b) complementary sequence of this nucleotide sequence, wherein said complementary sequence and nucleotide sequence are comprised of the Nucleotide of similar number, and are 100% complementations.

2. the recombinant DNA construction body that comprises the polynucleotide of claim 1, described polynucleotide are operably connected in the promotor that has function in plant.

3. change the method for axis physical strength, it comprises:

(a) to the recombinant DNA construction body that imports claim 2 in reproducible vegetable cell, to generate the vegetable cell of conversion; With

(b) from the vegetable cell regeneration of transgenic plant of described conversion, wherein said transgenic plant comprise described recombinant DNA construction body in its genome, and wherein compare with the control plant that does not comprise described recombinant DNA construction body, described transgenic plant show the change of stem physical strength.

4. the method for claim 3, comprises in addition (c) and obtains the progeny plants that is derived from described transgenic plant, and wherein said progeny plants comprises this recombinant DNA construction body in its genome.

5. the method for claim 3, wherein said transgenic plant show the increase of stem physical strength.

8. a kind of plant comprises recombinant DNA construction body claimed in claim 2 in its genome.

9. the separated polynucleotide of claim 1, wherein measure described stem physical strength by 3 crooked experiments.

10. evaluate the method for axis physical strength, it comprises:

(a) in reproducible vegetable cell, import recombinant DNA construction body, to generate the vegetable cell transforming, described recombinant DNA construction body is included in the promotor that has function in plant, this promotor is operably connected to (i) polynucleotide, its coding is based on Clustal V comparison method, with SEQ ID NO:4,6,8,10,12,14,16, and 18 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity, or (b) above-mentioned (a) (i) total length complementary sequence of described polynucleotide;

(b) from the vegetable cell regeneration of transgenic plant of described conversion; With

(c) evaluate the stem physical strength of described transgenic plant.

The method of 11. claims 10, comprises in addition:

(d) obtain the progeny plants that is derived from described transgenic plant; With

(e) evaluate the stem physical strength of described progeny plants.

12. evaluate the method for axis physical strength, and it comprises:

(b) from the vegetable cell regeneration of transgenic plant of described conversion;

(c) obtain the progeny plants that is derived from described transgenic plant; With

(d) evaluate the stem physical strength of described progeny plants.

13. a kind of plant comprise in its genome:

(a) the first recombinant DNA construction body, it comprises at least one promotor that has function in plant, and this promotor is operably connected at least one polynucleotide that are selected from the first separation of lower group:

(i) polynucleotide, it is encoded based on Clustal V comparison method, with SEQ IDNO:2,4,6,8,10,12,14,16, and 18 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity;

(ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQID NO:1,3,5,7,9,11,13,15, and 17 relatively, there is at least 60% sequence identity; With

(iii) (a) (i) or (a) the total length complementary sequence of the polynucleotide of (ii); With

(b) the second recombinant DNA construction body, it comprises at least one promotor that has function in plant, and this promotor is operably connected at least one polynucleotide that are selected from the second separation of lower group:

(iv) polynucleotide, it is encoded based on Clustal V comparison method, with SEQ IDNO:20,22,24,26,28,30,32,34,36,38,40, and 42 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity;

(v) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQID NO:21,23,25,27,29,31,33,35,37,39, and 41 relatively, there is at least 60% sequence identity; With

(vi) above-mentioned (b) (iv) or (b) total length complementary sequence of the polynucleotide of (v),

And wherein compare with the control plant of described the second recombinant DNA construction body with not comprising described the first recombinant DNA construction body, described plant shows the cell walls content of cellulose of increase or the growth velocity of increase.

14. a kind of plant comprise at least one and regulate sequence in its genome, and this adjusting sequence is operably connected to:

(a) at least one is selected from the separated polynucleotide of lower group:

(i) polynucleotide, it is encoded based on Clustal V comparison method, with SEQ ID NO:2,4,6,8,10,12,14,16, and 18 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity;

(ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:1,3,5,7,9,11,13,15, and 17 relatively, there is at least 60% sequence identity; With

(iii) above-mentioned (a) (i) or (a) total length complementary sequence of the polynucleotide of (ii); With

(b) at least one is selected from the separated polynucleotide of lower group:

(i) polynucleotide, it is encoded based on Clustal V comparison method, with SEQ ID NO:20,22,24,26,28,30,32,34,36,38,40, and 42 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity;

(ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ IDNO:21,23,25,27,29,31,33,35,37,39, and 41 relatively, there is at least 60% sequence identity; With

(iii) above-mentioned (b) (i) or (b) total length complementary sequence of the polynucleotide of (ii),

And wherein compare with not comprising described at least one control plant that is operably connected to the adjusting sequence on described (a) and (b), described plant shows the cell walls content of cellulose of increase or the growth velocity of increase.

The plant of 15. claims 14, wherein:

(a) described at least one polynucleotide are selected from:

(i) polynucleotide, it is encoded based on Clustal V comparison method, and with SEQ ID NO:2 comparison, aminoacid sequence has the polypeptide of at least 80% sequence identity;

(ii) polynucleotide, its nucleotide sequence, based on Clustal V comparison method, with SEQ ID NO:1 comparison, has at least 60% sequence identity; With

(b) described at least one separated polynucleotide are selected from:

(i) polynucleotide, it is encoded based on Clustal V comparison method, with SEQ ID NO:38,40, and 42 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity;

(ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ IDNO:37,39, and 41 relatively, there is at least 60% sequence identity; With

And wherein compare with not comprising described at least one control plant that is operably connected to the adjusting sequence on described (a) and (b), described plant shows the cell walls content of cellulose of increase.

The plant of 16. claims 14, wherein:

(a) described at least one polynucleotide are selected from:

(i) polynucleotide, it is encoded based on Clustal V comparison method, and with SEQ ID NO:6 comparison, aminoacid sequence has the polypeptide of at least 80% sequence identity;

(ii) polynucleotide, its nucleotide sequence, based on Clustal V comparison method, with SEQ ID NO:5 comparison, has at least 60% sequence identity; With

(b) described at least one separated polynucleotide are selected from:

(i) polynucleotide, it is encoded based on Clustal V comparison method, with SEQ ID NO:20,32, and 34 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity;

(ii) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ IDNO:19,31, and 33 relatively, there is at least 60% sequence identity; With

And wherein compare with not comprising described at least one control plant that is operably connected to the adjusting sequence on described (a) and (b), described plant shows the growth velocity of increase.

17. a kind of plant comprises at least one and regulates sequence in its genome, described adjusting sequence is operably connected at least 2 separated polynucleotide that are selected from lower group:

(a) polynucleotide, it is encoded based on Clustal V comparison method, with SEQ ID NO:2,4,6,8,10,12,14,16, and 18 comparisons, aminoacid sequence has the polypeptide of at least 80% sequence identity;

(b) polynucleotide, its nucleotide sequence is based on Clustal V comparison method, with SEQ ID NO:1,3,5,7,9,11,13,15, and 17 relatively, there is at least 60% sequence identity; With

(c) the total length complementary sequence of above-mentioned (a) or polynucleotide (b).

18. a kind of plant comprise in its genome:

Suppress DNA construct, this construct is included in the promotor that has function in plant, and this promotor is operably connected to:

(a) following nucleotide sequence is all or part of: (i) nucleotide sequence, and it is encoded based on ClustalV comparison method, with SEQ ID NO:2,4,6,8,10,12,14,16, and 18 comparisons, aminoacid sequence has the polypeptide of at least 50% sequence identity, or (ii) the total length complementary sequence of above-mentioned (a) nucleotide sequence (i); Or

(b) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity, and wherein said target genes encoding is selected from the polypeptide of Bk2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9

And wherein compare with the control plant that does not comprise described inhibition DNA construct, described plant shows the stem physical strength of reduction.

The plant of 19. claims 18, wherein said inhibition DNA construct comprises co-suppression construct, antisense constructs, virus suppresses construct, and hair clip suppresses construct, and stem-ring suppresses construct, double-stranded RNA-production construct, RNAi construct, or little RNA construct.

20. a kind of plant comprise in its genome:

(a) following nucleotide sequence is all or part of: (i) nucleotide sequence, its coding is based on ClustalV comparison method, with SEQ ID NO:6 comparison, aminoacid sequence has the polypeptide of at least 50% sequence identity, or (ii) the total length complementary sequence of above-mentioned (a) nucleotide sequence (i); Or

(b) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity, and wherein said target genes encoding Bk2L3 polypeptide

And wherein compare the organ size that described plant shows the plant height of reduction and/or reduces with the control plant that does not comprise described inhibition DNA construct.

21. a kind of plant comprise in its genome:

(a) following nucleotide sequence is all or part of: (i) nucleotide sequence, its coding is based on ClustalV comparison method, with SEQ ID NO:10 comparison, aminoacid sequence has the polypeptide of at least 50% sequence identity, or (ii) the total length complementary sequence of above-mentioned (a) nucleotide sequence (i); Or

(b) be derived from all or part of sense strand of target gene or the region of antisense strand, the nucleotide sequence in described region is based on Clustal V comparison method, with all or part of sense strand or the antisense strand comparison that obtain described region, there is at least 50% sequence identity, and wherein said target genes encoding Bk2L5 polypeptide

And wherein compare with the control plant that does not comprise described inhibition DNA construct, described plant shows male sterile.