CN101001867A

CN101001867A - Methods for modifying plant characteristics

Info

Publication number: CN101001867A
Application number: CN 200580019334
Authority: CN
Inventors: 方绮雯; R·I·彭内尔; 吴传银; 陈志红; T·塔塔林诺娃; 张红宇
Original assignee: Ceres Inc
Current assignee: Ceres Inc
Priority date: 2004-04-23
Filing date: 2005-04-22
Publication date: 2007-07-18

Abstract

Isolated polynucleotides, polypeptides, and transgenic plants are described. The transgenic plants can exhibit one or more altered phenotypic characteristics relative to a control plant, including increased height, increased seed weight, increased photosynthetic rates, decreased levels of campestanol, or increased levels of 6-deoxocathasterone.

Description

The method of improvement plant characteristics

The cross reference of related application

The U.S. Provisional Application sequence number 60/565 that the application requires 35U.S.C. § to submit on April 23rd, 2004 for 119 times, 031 and the U.S. Provisional Application sequence number 60/644 submitted on January 18th, 2005,612 right of priority, both full contents all are incorporated among the application.

Technical field

The present invention relates to coded polypeptide, the polynucleotide such as the polypeptide that works in the plain steroid biosynthesizing of rape path comprises the transgenic plant of described polynucleotide and uses the method that described polynucleotide is improved the plant phenotype feature.More specifically, the present invention relates to show the transgenic plant of one or more following metabolites of enhanced level: sucrose, glutaminate/ester (L-glutamic acid), or linolic acid.In addition, the present invention relates to show the plain sterone (6-deoxocathasterone) of 6-deoxidation rape of enhanced level and/or the transgenic plant that reduce the campesterol (campestanol) of level.Described transgenic plant also may show the enhanced growth potential, the size that increases (for example, highly), the seed production of increase, the seed circularity of homogeneous is (for example more, in monocotyledons), the seed weight that every strain plant increases, the seed that every strain plant increases, the speed of growth faster, more efficient photosynthesis, or the drought-resistant property of improvement.

Background of invention

Because the demand to the agricultural and the growth of forestry industry that whole world propagation of population causes has caused making great efforts increase plant biomass and/or size.Although being used to increase a kind of mode of plant size is by plant breeding program, such breeding plan expends time in typically and is labor-intensive.On the other hand, give the external nucleic acid that needs feature and the characteristic of plant is carried out Genetic Control, can be still less time-consuming, and may be applied to various floristics by introducing.

Plant produces a large amount of steroid and sterol, is called the plain steroid (BRs) of rape, and some in them act as tethelin.Existence is more than 40 kinds of known BRs, typically at one or more C-2, and C-6, C-22 and C-23 position have characteristic oxygen part.Brassinolide (BL) is the bioactive form that has most of promotes growth BRs.Arabidopis thaliana CPD and DWF4 are the cytochrome P of the enzymatic step in catalysis BL biosynthesizing path ₄₅₀Albumen; They have 43% homology at amino acid levels.In the BL biosynthetic process, DWF4 catalysis campesterol in the C-22 oxidation forming the plain sterone of 6-deoxidation rape, and CPD catalysis downstream close on step: the plain sterone of 6-deoxidation rape at the hydroxylation of C-23 to produce 6-deoxyteasterone.

Summary of the invention

The application provides isolating polynucleotide, by its encoded polypeptides with comprise its transgenic plant.Transgenic plant can be showed the phenotypic characteristic with respect to the needs of control plant, such as following one or more: the height of increase, the enhanced photosynthetic rate, one or more metabolites of increase level, such as sucrose, glutaminate/ester or linolic acid, with the plain sterone of the 6-deoxidation rape that increases level, the campesterol of reduction level, the enhanced activity (for example, enzymatic activity), the water use efficiency of raising, the seed weight that every strain plant increases, seed and the drought-resistant property of enhanced that every strain plant increases.

The application also provides and is known as the Arabidopis thaliana P of DWF4 ₄₅₀The polypeptide that protein functional is suitable (homologue with directly to homologue) with such homologue and straight to the consistent peptide sequence of homologue.DWF4 plays an important role in the plain steroid of rape synthetic, and it act as plant-growth and promotes hormone.Therefore, except other things, the present invention also provides coding P ₄₅₀The isolating polynucleotide of polypeptide.In some cases, P ₄₅₀Polypeptide can work in the plain steroid biosynthesizing of rape path.For example, some P ₄₅₀Polypeptide can be exercised the enzymatic activity of DWF4, for example, campesterol in the oxidation of C-22 to form the plain sterone of 6-deoxidation rape.

Present disclosure also provides the transgenic plant that comprise polynucleotide described herein and is used to prepare the method for described transgenic plant.The expression of described polypeptide in plant can cause one or more expression effects, such as the plant size that increases (for example, highly, biomass) and/or the speed of growth faster; The seed production that increases; The seed circularity that increases (for example, for monocotyledons, such as rice section, Triticum, and Zea); The metabolite of increase level, such as sucrose, glutaminate, or linolic acid; The campesterol of reduction level; The plain sterone of the 6-deoxidation rape of increase level; The enhanced photosynthetic rate; The water use efficiency that improves; Or the drought-resistant property of improving.In other situation, that polypeptide expression can provide is non-existent usually in the plant (for example, do not exist fully or only do not exist in some tissue) biochemistry or enzymatic activity perhaps can provide such biochemical activity of enhanced level.In some cases, biochemistry or enzymatic functions that described polypeptide expression has existed in can complementary plant perhaps can cause the enzymatic activity (for example, the activity of increase, the activity of reduction, or different activity) that changes.Suppress P ₄₅₀The expression of polypeptide in plant, for example, by antisense, RNAi, or based on the method for ribozyme, the shade tolerance (for example, as downtrod prolongation under dark condition is indicated) that can cause plant to be improved.

Therefore, on the one hand, provide isolating polynucleotide.Isolating polynucleotide can comprise that coding has the aminoacid sequence about 85% listed with SEQ ID NO:2 or the nucleic acid molecule of the polypeptide of multisequencing homology more; For example, comprise the aminoacid sequence that SEQ ID NO:2 lists, or form by the aminoacid sequence that SEQ ID NO:2 lists.Isolating polynucleotide can comprise that coding comprises the polypeptide with the corresponding aminoacid sequence of consensus sequence (SEQ ID NO:4) listed in Fig. 2, for example, and the nucleic acid molecule of the polypeptide of corresponding described consensus sequence.Polypeptide effectively the catalysis campesterol in the oxidation of C-22 to form the plain sterone of 6-deoxidation rape.

Polynucleotide can also comprise effectively the nucleic acid with coding said polypeptide, the controlling elements that connects such as the promotor or the constitutive promoter of wide expression.In some cases, the promotor of wide expression is selected from by p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, the group that YP0144 and YP0190 form.In some cases, constitutive promoter is 35S.

In some cases, encoded polypeptide does not show the aminoacid sequence of listing with SEQ ID NO:1 or SEQ IDNO:3 93% or more sequence homology.In other situation, isolating polynucleotide can comprise coding comprise with Fig. 2 in the nucleic acid of polypeptide of the corresponding aminoacid sequence of consensus sequence (SEQ IDNO:4) listed, wherein said polynucleotide also comprises the wide expression promotor controlling elements that is connected with the nucleic acid of coding said polypeptide effectively.

The application also provides the recombinant vectors that comprises above-mentioned polynucleotide.Recombinant vectors can comprise the controlling elements that is connected to effectively on the described polynucleotide, wherein said polynucleotide comprises that coding has the nucleic acid molecule of the polypeptide of the aminoacid sequence about 85% listed with SEQ ID NO:2 or more sequence homology, comprising that perhaps coding comprises the nucleic acid molecule of the polypeptide of the corresponding aminoacid sequence of listing with Fig. 2 of consensus sequence (SEQ ID NO:4).

On the other hand, transgenic plant are also provided.Transgenic plant can comprise at least a external source polynucleotide, and described at least a external source polynucleotide comprises the nucleic acid of the such polypeptide of coding, that is, (a) aminoacid sequence of listing with SEQ ID NO:2 has an appointment 85% or multisequencing homology more; Perhaps (b) comprises the corresponding aminoacid sequence of listing with Fig. 2 of consensus sequence (SEQ ID NO:4), and condition is that encoded polypeptide does not show the aminoacid sequence of listing with SEQ ID NO:1 or SEQ ID NO:3 and has 93% or more sequence homology.Described polypeptide effectively the catalysis campesterol in the C-22 oxidation, to form the plain sterone of 6-deoxidation rape.In some cases, the external source polynucleotide also comprises the controlling elements on the nucleic acid that is connected to coding said polypeptide effectively.Described controlling elements can be the promotor or the constitutive promoter of wide expression.The promotor of wide expression can be selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.The p326 promotor can cause the expression of described polypeptide in branch and stem apex effectively.Transgenic plant can show the phenotype that changes with respect to control plant.The phenotype of described change can be to be selected from by one or more of the following group of forming: with respect to control plant, the metabolic pattern that changes, the increase of the plain sterone level of 6-deoxidation rape, the minimizing of campesterol level, the photosynthetic rate that increases, the seed production that increases, the seed weight that every strain plant increases and the height that increases.The metabolic pattern that changes can be sucrose, L-glutamic acid or the linolic acid with respect to control plant increase level.Transgenic plant can be monocotyledonss, such as rice section, and Triticum, switchgrass, Secale, Hordeum, jowar belongs to, or Zea.Transgenic plant can be dicotyledonss.

On the other hand, transgenic plant are not Arabidopis thaliana (Arabidopsis thaliana) or tobacco (Nicotiana tabacum) plant.Such transgenic plant can comprise at least a external source polynucleotide, described at least a external source polynucleotide comprises the nucleic acid of the polypeptide that coding is such, that is, the aminoacid sequence of (a) listing with SEQ ID NO:2 has an appointment 85% or multisequencing homology more; Perhaps (b) comprises the corresponding aminoacid sequence of listing with Fig. 2 of consensus sequence (SEQ ID NO:4).Described external source polynucleotide can also comprise the controlling elements on the nucleic acid that connects the rice coding said polypeptide effectively.Described controlling elements can be the promotor or the constitutive promoter of wide expression.The promotor of described wide expression can be selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.The p326 promotor can cause the expression of described polypeptide in branch and stem apex effectively.Transgenic plant can show the phenotype that changes with respect to control plant.The phenotype that changes can be to be selected from by one or more of the following group of forming: with respect to control plant, the metabolic pattern that changes, the increase of the plain sterone level of 6-deoxidation rape, the minimizing of campesterol level, the photosynthetic rate that increases, the seed production that increases, the seed weight that every strain plant increases and the height that increases.The metabolic pattern that changes can be sucrose, L-glutamic acid or the linolic acid with respect to control plant increase level.Transgenic plant can be monocotyledonss, such as rice section, and Triticum, switchgrass, Secale, Hordeum, jowar belongs to, or Zea.Transgenic plant can be dicotyledonss.Polypeptide effectively the catalysis campesterol in the oxidation of C-22, to form the plain sterone of 6-deoxidation rape.

In another embodiment, the transgenic plant that comprise at least a external source polynucleotide are provided, described at least a external source polynucleotide comprises the nucleic acid of the such polypeptide of coding, that is, (a) aminoacid sequence of listing with SEQ ID NO:2 has an appointment 85% or multisequencing homology more; Perhaps (b) comprises the corresponding aminoacid sequence of listing with Fig. 2 of consensus sequence (SEQ ID NO:4), and wherein said transgenic plant show the increase of the plain sterone level of 6-deoxidation rape with respect to control plant.Described external source polynucleotide can also comprise the controlling elements on the nucleic acid that is connected to coding said polypeptide effectively.Controlling elements can be the promotor or the constitutive promoter of wide expression.The promotor of described wide expression can be selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.The p326 promotor can cause the expression of described polypeptide in branch and stem apex effectively.Transgenic plant can show the phenotype that changes with respect to control plant, it is selected from by one or more of the following group of forming: with respect to control plant, the metabolic pattern that changes, the minimizing of campesterol level, the photosynthetic rate that increases, the seed production that increases, the seed weight that every strain plant increases and the height that increases.Transgenic plant can be monocotyledonss, such as rice section, and Triticum, switchgrass, Secale, Hordeum, jowar belongs to, or Zea, or dicotyledons, as above-mentioned.Polypeptide effectively the catalysis campesterol in the C-22 oxidation, to form the plain sterone of 6-deoxidation rape.

On the other hand, the application provides the transgenic plant that comprise at least a external source polynucleotide, described at least a external source polynucleotide comprises the nucleic acid of the such polypeptide of coding, that is, (a) aminoacid sequence of listing with SEQID NO:2 has an appointment 85% or multisequencing homology more; The perhaps consensus sequence (SEQ ID NO:4) listed of (b) corresponding diagram 2, wherein said transgenic plant show the increase of the plain sterone level of 6-deoxidation rape with respect to control plant.Described external source polynucleotide can also comprise the controlling elements on the nucleic acid that is connected to coding said polypeptide effectively.Controlling elements can be the promotor or the constitutive promoter of wide expression.The promotor of described wide expression can be selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.The p326 promotor can cause the expression of described polypeptide in branch and stem apex effectively.Transgenic plant can also show the phenotype that changes with respect to control plant, it is selected from by one or more of the following group of forming: with respect to control plant, the metabolic pattern that changes, the increase of the plain sterone level of 6-deoxidation rape, the photosynthetic rate that increases, the seed production that increases, the seed weight that every strain plant increases and the height that increases.Transgenic plant can be monocotyledonss, such as rice section, and Triticum, switchgrass, Secale, Hordeum, jowar belongs to, or Zea, or dicotyledons.Polypeptide effectively the catalysis campesterol in the C-22 oxidation, to form the plain sterone of 6-deoxidation rape.

In another embodiment, provide the transgenic plant that comprise at least a external source polynucleotide.Described at least a external source polynucleotide can comprise the nucleic acid of the such polypeptide of coding, that is, (a) aminoacid sequence of listing with SEQ ID NO:2 has an appointment 85% or multisequencing homology more; The perhaps consensus sequence (SEQ ID NO:4) listed of (b) corresponding diagram 2, wherein said external source polynucleotide can also comprise the controlling elements of the wide expression on the nucleic acid that is connected to coding said polypeptide effectively.The promotor of wide expression can be selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.Transgenic plant can show the phenotype that changes with respect to control plant, it is selected from by one or more of the following group of forming: with respect to control plant, the metabolic pattern that changes, the minimizing of campesterol level, the increase of the plain sterone level of 6-deoxidation rape, the photosynthetic rate of increase, the seed production of increase, seed weight that every strain plant increases and the height that increases.The metabolic pattern that changes can be sucrose, L-glutamic acid or the linolic acid with respect to control plant increase level.Transgenic plant can be monocotyledonss, such as rice section, and Triticum, switchgrass, Secale, Hordeum, jowar belongs to, or Zea.Transgenic plant can be dicotyledonss.Polypeptide effectively the catalysis campesterol in the C-22 oxidation, to form the plain sterone of 6-deoxidation rape.

On the other hand, provide the transgenic plant that comprise at least a external source polynucleotide.Described at least a external source polynucleotide can comprise the nucleic acid of the such polypeptide of coding, that is, (a) aminoacid sequence of listing with SEQID NO:2 has an appointment 85% or multisequencing homology more; The perhaps consensus sequence (SEQ ID NO:4) listed of (b) corresponding diagram 2, wherein said transgenic plant show the photosynthetic rate of increase with respect to control plant.Described external source polynucleotide can also comprise the controlling elements on the nucleic acid that is connected to coding said polypeptide effectively.Controlling elements can be the promotor or the constitutive promoter of wide expression.The promotor of wide expression can be selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.The promotor of wide expression can be p326, and it can cause the expression of described polypeptide in branch and stem apex effectively.Transgenic plant can also show the phenotype that changes with respect to control plant, it is selected from by one or more of the following group of forming: with respect to control plant, the metabolic pattern that changes, the minimizing of campesterol level, the increase of the plain sterone level of 6-deoxidation rape, the seed production that increases, the seed weight that every strain plant increases and the height that increases.The metabolism distribution that changes can be sucrose, L-glutamic acid or the linolic acid with respect to control plant increase level.Transgenic plant can be monocotyledonss, such as rice section, and Triticum, switchgrass, Secale, Hordeum, jowar belongs to, or Zea, maybe can be dicotyledons.Polypeptide effectively the catalysis campesterol in the C-22 oxidation, to form the plain sterone of 6-deoxidation rape.

The method that is used to produce transgenic plant also is provided.The method that is used to produce transgenic plant can comprise (a) with any polynucleotide introduced plant cell described herein, to produce the plant transformed cell; (b) produce transgenic plant from described plant transformed cell.The seed of any transgenic plant described herein also is provided.

On the other hand, provide isolated polypeptide.Isolated polypeptide can (a) have aminoacid sequence about 85% or the more sequence homology of listing with SEQ ID NO:2; Perhaps (b) comprises the corresponding aminoacid sequence of listing with Fig. 2 of consensus sequence (SEQ ID NO:4), and condition is that encoded polypeptide does not show aminoacid sequence 93% or the more sequence homology of listing with SEQ ID NO:1 or SEQ ID NO:3.

Present disclosure also provides the method for one or more phenotypic characteristics that change plant.For example, be provided for increasing the method for the level of one or more metabolites that are selected from the group of forming by sucrose, glutaminate/ester and linolic acid in the plant.Described method comprises:

(a) with previous described any polynucleotide introduced plant cell, to produce the plant transformed cell; (b) from described plant transformed cells produce transgenic plant, wherein said transgenic plant show the increase level of one or more metabolites.

In another embodiment, a kind ofly be used for increasing the method for level that plant is selected from one or more metabolites of the group of being made up of sucrose, glutaminate/ester and linolic acid and comprise: (a) in order to produce the plant transformed cell, in vegetable cell, introduce the isolating polynucleotide of the nucleic acid molecule comprise that coding is following: 1) have the aminoacid sequence about 85% listed with SEQ ID NO:2 or the polypeptide of more sequence homology, or 2) comprise the polypeptide of the corresponding aminoacid sequence of listing with Fig. 2 of consensus sequence (SEQ ID NO:4); (b) produce transgenic plant from described plant transformed cell, wherein said transgenic plant show the increase level of one or more metabolites.

The method that is used for increasing the plain sterone level of plant 6-deoxidation rape has also been described.Described method comprises a) in order to produce the plant transformed cell, be incorporated herein described isolating polynucleotide to vegetable cell, for example, it comprises the following nucleic acid molecule of coding: 1) have the aminoacid sequence about 85% listed with SEQ ID NO:2 or the polypeptide of more sequence homology, or 2) comprise the polypeptide of the corresponding aminoacid sequence of listing with Fig. 2 of consensus sequence (SEQ ID NO:4); With

(b) produce transgenic plant from described plant transformed cell, wherein said transgenic plant show the plain sterone of 6-deoxidation rape of increase level.

On the other hand, provide the method that is used for reducing plant rape oil sterol levels.Described method comprises: a) in order to produce the plant transformed cell, be incorporated herein described isolating polynucleotide to vegetable cell, for example, it comprises the following nucleic acid molecule of coding: 1) have the polypeptide of aminoacid sequence of listing with SEQ IDNO:2 about 85% or more sequence homology, or 2) comprise the polypeptide of the corresponding aminoacid sequence of listing with Fig. 2 of consensus sequence (SEQ ID NO:4); With

(b) produce transgenic plant from described plant transformed cell, wherein said transgenic plant show the campesterol of minimizing level.

On the other hand, the method that is used to strengthen the photosynthesis of plants rate is provided, it comprises that (a) is in order to produce the plant transformed cell, be incorporated herein described isolating polynucleotide to vegetable cell, for example, it comprises the following nucleic acid molecule of coding: 1) have the aminoacid sequence about 85% listed with SEQ ID NO:2 or the polypeptide of more sequence homology, or 2) comprise the polypeptide of the corresponding aminoacid sequence of listing with Fig. 2 of consensus sequence (SEQ ID NO:4); With

(b) produce transgenic plant from described plant transformed cell, wherein said transgenic plant show the enhanced photosynthetic rate.

Unless otherwise defined, all technology used herein and scientific terminology have with the present invention under the field in the common identical meaning of understanding of those of ordinary skill.Although can be used for practice or check the present invention to those methods similar or of equal value described herein and material, describe appropriate means and material hereinafter.In addition, described material, method and example only are exemplary, are not to be intended to restriction.Mentioned all publications, patent application, patent and other reference of this paper all incorporated this paper into by reference.In the situation of conflict, this detailed description comprises definition, will control.

List in the details of one or more embodiments of the present invention accompanying drawing hereinafter and the detailed description.Further feature of the present invention, purpose and advantage will be from describing and accompanying drawing, and Accessory Right is apparent in requiring.

The accompanying drawing summary

Fig. 1 lists the aminoacid sequence (SEQ ID NO:1) of Arabidopis thaliana (Arabidopsis) DWF4 and the aminoacid sequence of two peptide species suitable with Arabidopis thaliana DWF4 function: from the SEQID NO:2 of corn with from the SEQ ID NO:3 of paddy rice.About SEQ ID NO:2, Ceres clone ID is the inside title of described sequence.About SEQ ID NO:3, " gi " corresponding title in public database NCBI then is the Short Description of BAC clone sign and protein sequence.

Fig. 2 is the common sequence (SEQ ID NO:4) of the polypeptide suitable with Arabidopis thaliana DWF4 function.Described common sequence comprises having the active P of DWF4 ₄₅₀Polypeptide for example, has 22 α-hydroxylase activity.Described common sequence shows which amino acid to occur in each position.Described common sequence contains lowercase and capitalization.Capitalization is represented the amino acid abbreviations of a letter of standard, and the kind of lowercase represented amino acid: " t " refers to p1 amino acid, and it is specially L-Ala, glycine, Serine and Threonine; " p " refers to polare Aminosaeren, and it is specially l-asparagine and glutamine; " n " refers to the amino acid of negative charge, and it is specially aspartic acid and L-glutamic acid; "+" the electric charge residue of making a comment or criticism, it is specially Methionin, arginine, and Histidine; " r " refers to aromatic residues, and it is specially phenylalanine, tyrosine, and tryptophane; " a " refers to aliphatic residue, and it is specially Isoleucine, Xie Ansuan, leucine, and methionine(Met).Admissible amino acid breach usefulness in the consensus sequence "＜" sign flag.For example,＜1〉represent that equaling 1 amino acid whose breach allows;＜2-4〉represent that 2-4 amino acid whose breach allows.

Fig. 3 lists DWF4 antisense sequences (DWF4a/s; SEQ IDNO:5); Referring to embodiment 3.

Fig. 4 demonstrate Arabidopis thaliana DWF4 and the comparison of rice (Oryza Sativa) DWF4 on amino acid levels.These two sequences have 69% homology to each other.The film grappling, proline(Pro) enrichment, O ₂-combination, the steroid combination, the position of Unknown Function and heme-binding domain is represented by underscore 1-6 respectively.Identical amino acid is sealed with the lead shade.

The demonstrate nucleotide sequence of many promotors used herein of Fig. 5.

Fig. 6 shows between transgenic plant described herein and the contrast at the chart of different light strength ratios than photosynthetic rate (PS).PS leads the CO at 380ppm ₂Concentration, 25 ℃ of temperature and 0,20,50,100,200,500,1000,1500,2000 μ mol m-2s-1 measure on complete boot leaf.The PS of each luminous point leads the mean value of representing 3 strain plants.Lines are represented standard deviation.

Fig. 7 code displaying Arabidopis thaliana described herein, the polymerized nucleoside acid sequence of Zea mays (Zea mays) and rice DWF4 polypeptide.

Fig. 8 shows many DWF4 polypeptide directly to homologue, and for example C-22 α-hydroxylase is directly to the peptide sequence of homologue.

Detailed Description Of The Invention

Constitutive promoter: at great majority, but unnecessary is whole, under environmental condition and developmental condition or the Cell Differentiation, conduct mentioned in this article " constitutive promoter " but promoter promote the transcribing of detection level of the sequence that in all plant tissues, effectively connects. The example of constitutive promoter comprises cauliflower mosaic virus (CaMV) 35S transcription initiation zone, the sub-p13879 of arabidopsis thaliana promoter and p32449, be derived from 1 of Agrobacterium tumefaciems (Agrobacterium tumefaciens) T-DNA ' or 2 ' promoter, other transcription initiation zone of many plant genes is such as corn ubiquitin-1 promoter.

The promoter of wide expression: when it many, but need not to be all, when promoting to transcribe in the plant tissue, it is as used herein " wide expression " that promoter can be called. For example, the sequence that the promoter of wide expression can promote effectively to connect is at stem, branch, and stem apex (shoot apex), and transcribing in the blade, but can be faintly or do not promote fully at tissue be such as transcribing in the breeding tissue of flower and the seed of growing. In some cases, the promoter that effectively is connected to the wide expression on the sequence can be to exceed at least 2 times (for example, at least 3,5 than the transcriptional level in the seed of root tissue or growth, 10, or 20 times) level promotes transcribing in plants shoots. In other situation, the promoter of wide expression can be to exceed level promotion the transcribing in plants shoots of at least 2 times (for example, at least 3,5,10, or 20 times) than the transcriptional level in the breeding tissue of flower. Consider above-mentionedly, do not think that CaMV 35 S promoters are promoters of wide expression. The promoter example that is used for wide expression of the present invention comprises p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.

Domain: domain is to be used for dactylotype or the label that characteristic is described protein family and protein fragments. Such dactylotype or label can comprise conservative: (1) primary sequence; And/or (2) secondary structure; And/or (3) three-dimensional conformation. Usually, domain can be relevant with protein family or motif. Typically, these families and/or motif are with external and/or activity in vivo is relevant. Domain can be any length, comprises the whole of protein sequence. Cytochrome P₄₅₀Domain, relevant family and motif, and describe hereinafter with the detailed description of the related activity of polypeptide of the present invention. As described herein, has the polypeptide that shows with one or more specified structures territory of the sequence homology of another polypeptide particular percentile, at least a biochemical activity that is shown by another polypeptide can be shown, perhaps plant phenotype can be affected in a similar manner.

Endogenous: term " endogenous " refers in the context of the invention into the natural part of cell or from organic any polynucleotide, polypeptide or the protein sequence of described cytothesis.

External source: " external source " refers to introduce by any mode except property hybridization the polynucleotide of host cell or organism. Typically, the external source polynucleotide stably is incorporated in the genome of host cell or organism. The example of the method for polynucleotide introduced plant and plant cell can be described hereinafter, and it comprises agriculture bacillus mediated conversion, biological projectile method, electroporation, in planta technology, etc. , contain the plant of exogenous nucleic acid herein, can be called T for elementary genetically modified plants₁Plant is called T for the first generation₂Plant, and for second and the suceeding generation plant be called T₃，T ₄, etc. T₂The offspring is T₁The autogamous result of plant. T₃The offspring is T₂The autogamous result of plant. Should be appreciated that the external source polynucleotide can be introduced into ancester cell, rather than cell or the plant of research. For example, the genetically modified plants that contain all exogenous nucleic acids can be the offsprings of hybridizing between the plant of stable conversion and the non-transgenic plant. Think that such offspring contains described external source polynucleotide. Therefore, BC1, BC2, and BC3 plant and F1, F2, and the F3 plant can contain the external source polynucleotide similarly.

The polypeptide that those have at least a common characteristic described in this phrase of polypeptide that function is suitable. Such feature comprises sequence similarity or homology, biochemical activity, and the transcriptional profile similitude, and phenotype is active. Typically, the albumen that function is suitable is shared some sequence similarities or homology. In this definition, think that homologue or straight homologues are that function is suitable. In addition, the albumen that function is suitable can be shared at least a biochemical activity. For example, the polypeptide that function is suitable can be " biochemical suitable ", for example, can act on the identical reactant to provide identical product. Biochemical quite thing can or can not show identical dynamics, with the affinity of reactant, or the turnaround time that produces described product, but owing to produced identical end product, can think that still function is suitable.

The polypeptide that another kind of function is suitable is " phenotype suitable (phenotypic comparables) ", and the physical features that its impact is identical is such as the metabolic pattern of the plant size that increases or height or change. Even the physical features that the polypeptide impact is identical, but degree is different, can think that still polypeptide is that phenotype is suitable. For example, the feature (for example, causing the height that increases) that the impact of suitable polypeptide is identical, the quantitative assay that is wherein caused by one of suitable polypeptide be another about 20% or more; For example, about 20-30%; About 30-40%; About 40-50%; About 50-60%; About 60-70%; About 70-80%; About 80-90%; Or about 90-100%. Therefore, although a kind of albumen can be the suitable thing of phenotype with plant height increase by 10% and another kind of plant with plant height increase by 15%, two peptide species.

Gene: when being used for the context of the invention, term " gene " comprises all regulation and control and the coded sequence that is adjacent to associate with the single hereditary individuality with Genetic Function. Gene can comprise the non-coding sequence of regulating Genetic Function, and described Genetic Function includes, but are not limited to, the effect of specific polyadenous glycosidation, transcriptional control, DNA conformation, the chromatin conformation, the methylated degree of base and position and those of binding site of controlling the albumen of all these. The gene that comprises " extron " (coded sequence), it can be interrupted encoding proteins by " introne " (non-coding sequence). The Genetic Function of gene can only need rna expression or protein production, or can only need the combination of albumen and/or nucleic acid, the expression that need not to be correlated with. In some cases, the gene that closes on each other can be shared sequence by this way, that is, a gene is with overlapping another gene. Gene can find in the genome of organism, artificial chromosome, plasmid, carrier etc., or finds as separate entities independently.

Heterologous sequence: " heterologous sequence " is those ineffective connections and/or is not approximating those sequences in essence. For example, think that the promoter from corn is allos for the arabidopsis coding region sequence. And, think that the promoter of the gene that comes the own coding corn growth factor is allos for the sequence of the described growth factor receptors of coding corn. The controlling element sequence, such as the terminal terminator sequence of UTRs or 3 ' that does not originate in essence the initial identical gene of described coded sequence, being considered to for described coded sequence is allos. Effectively connect in essence and element adjacent one another are for not being allos each other. On the other hand, these identical elements connect with remaining valid, if but other padding sequence placed will to become between them be allos. Therefore, the promoter of the corn gene of express amino acid transport protein and coded sequence are not allos each other, but effectively promoter and the coded sequence of the corn gene of connection are allos in novel mode.

Homology: in the present invention, " homology " refer to, for example, with genes of interest, nucleic acid, polynucleotide, or polypeptide share to a certain degree sequence similarity or the gene of homology, nucleic acid, polynucleotide, or polypeptide. This similitude can be only in the fragment of described sequence, and can representative structure territory (for example, structure or functional domain). Unnecessary with the biochemical activity of source entity or phenotype effect is identical or similar, although they can be identical or similar.

Inducible promoter: in the context of the present invention, " inducible promoter " refer under certain conditions, such as light, and chemical concentration, the condition in the protein concentration, organism, cell or organelle, etc., the promoter of being regulated and control. The representative instance of the inducible promoter that can use with polynucleotide of the present invention is PARSK1, promoter from the arabidopsis gene of encoding serine-threonine kinase enzyme, and described promoter is subject to dehydration, the inducing of abscisic acid and sodium chloride (Wang and Goodman, Plant 18:37 (1995)). Can comprise oxygen free condition, the temperature of rising, or the existence of light by the example that inducible promoter affects the environmental condition of transcribing.

Effectively connect: " effectively connecting " control element to coded sequence is attached on the described coded sequence by this way, so as with the condition of control element compatibility under obtain the expression of described coded sequence. Described control element needn't be adjacent with coded sequence. Therefore, for example, but the interference of not translating the sequence of transcribing be may reside between promoter and the coded sequence, so that described promoter " connects " effectively to described coded sequence.

Straight homologues: in the present invention, " straight homologues " refers to encode and exercises similar biochemical activity or affect in a similar manner the gene outcome of phenotype or the second nucleic acid of polypeptide as the product of the first nucleic acid. Described straight homologues typically also will have with the first nucleic acid sequence similarity or homology to a certain degree. Therefore, directly can reveal the polypeptide that sequence similarity is to a certain degree arranged with the polypeptide of the first nucleic acid coding by coding schedule to homologous nucleic acid. Described sequence similarity can be at one or more functions and/or structure domain or along the total length of the polypeptide of the coded sequence of described nucleic acid and/or their correspondences and find.

Sequence homology percentage: usually, term " homology " refers to the accurately nucleotides of corresponding two kinds of polynucleotides respectively or polypeptide-p-nucleotides or amino acid-p-amino acid. Two or more sequences (polynucleotide or polypeptide) can compare by " the sequence homology percentage " of determining them. With in this article the time, term " sequence homology percentage " refers to the degree of any inquiry sequence that provides and the homology between the subject nucleotide sequence. For any inquiry nucleic acid or amino acid sequence, for example, C22-α hydroxylase can followingly be determined with respect to the homology percentage of theme nucleic acid or amino acid sequence.

Appliance computer program ClustalW, it allows the comparison of nucleic acid or protein sequence to carry out along their total length (overall contrast), will address inquires to nucleic acid or amino acid sequence and one or more theme nucleic acid or amino acid sequence and compare. ClustalW calculates the optimum Match between inquiry and one or more subject nucleotide sequences, and compares them, in order to can determine homology, and similitude and otherness. The breach of one or more residues can be inserted and address inquires to sequence, among subject nucleotide sequence or both, so that sequence alignment is maximized. For the fast in pairs comparison of nucleotide sequence, use the demonstration parameter: the size of word: 2; Window size: 4; Get separating method: percentage; Top diagonal number: 4; With breach point penalty (penalty): 5. For the multiple ratio of nucleotide sequence pair, use following parameter: the open point penalty of breach: 10.0; Breach extends point penalty: 5.0; Change with weight: be. For the fast in pairs comparison of protein sequence, use following parameter: the size of word: 1; Window size: 5; Get separating method: percentage; Top diagonal number: 5; Breach point penalty: 3. For the multiple ratio of protein sequence pair, use following parameter: weight matrix: blosum; The open point penalty of breach: 10.0; Breach extends point penalty: 0.05; Hydrophily breach: open; Hydrophily residue: GPSNDQERK; Residue specificity breach point penalty: open.

Output is the sequence contrast, the relation between its reflection sequence. For example, ClustalW can be at Baylor College of Medicine (BCM) Search Launcher network address＜http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html〉or at European Bioinformatics Institute network address＜http://www.ebi.ac.uk/clustalw operation.

In order determine to address inquires to the homology percentage between sequence and the subject nucleotide sequence, ClustalW is with the base of shorter sequence or amino acid number base or the amino acid number divided by coupling, and takes advantage of described result with 100. Output is that described subject nucleotide sequence is about the homology percentage of described inquiry sequence. For example, if inquiry sequence and subject nucleotide sequence respectively are that 500 base-pairs are long, and have (or identical) base of 200 couplings, so described subject nucleotide sequence will have the sequence homology for described inquiry sequence 40%. If the sequence of two kinds of comparisons is different length, so with the coupling number divided by shorter sequence in two kinds of sequence lengths. For example, if between 400 inquiry polypeptide and 500 amino acid whose theme polypeptide 100 amino acid couplings are arranged, so described theme polypeptide will have with the homology of addressing inquires to polypeptide 25%. If be less than 150 bases or 50 amino acid on the shorter sequence length, so with the number of coupling divided by 150 (for nucleic acid bases) or 50 (for amino acid), and multiply by 100, to obtain homology percentage.

In some embodiments, the amino acid sequence of suitable theme polypeptide with the sequence homology of amino acid sequence (for example, SEQ ID NOS:2 and 3) greater than 40% of the polypeptide of addressing inquires to (for example,＞40% has,＞50%,＞60%,＞70%,＞75%,＞80%,＞85%,＞86%,＞87%,＞88%,＞89%,＞90%,＞91%,＞92%,＞93%,＞94%,＞95%,＞96%,＞97%,＞98%, sequence homology perhaps＞99%). In some embodiments, the nucleotide sequence of suitable theme nucleotides with the nucleotide sequence of addressing inquires to nucleic acid greater than 70% sequence homology (for example,＞75% has,＞80%,＞85%,＞90%,＞9 1%,＞92%,＞93%,＞94%,＞95%,＞96%,＞97%,＞98%, sequence homology perhaps＞99%).

Should be noted that the homology percent value can be rounded up to immediate 1/10th. For example, with 78.11,78.12,78.13, and 78.14 be rounded up to 78.1, and with 78.15,78.16,78.17,78.18, and 78.19 are rounded up to 78.2. Be to be further noted that always integer of length value.

Plant promoter: " plant promoter " is the promoter of can initial in plant cell (promotion) transcribing. Such promoter needs not to be plant-derived. For example, be derived from the promoter of plant virus, such as the CaMV35S promoter, or be derived from the promoter of Agrobacterium tumefaciems, such as the T-DNA promoter, can be plant promoter. The representative instance of plant-derived plant promoter is corn ubiquitin-1 (ubi-1) promoter. Other plant promoter also is for known to a person of ordinary skill in the art.

Promoter: when being used in this paper, term " promoter " refers to be positioned at the sequence of the upstream of genetic transcription starting point and determines subregion, and it participates in identification and the combination of RNA polymerase and other albumen, regulates with initial sum and transcribes. Basic promoter is the essential minmal sequence of the assembling needed transcription complex of transcription initiation. Basic promoter generally includes " TATA box " element, and it is usually located between the 15th and 35 nucleotides of transcription initiation site upstream. Sometimes, basic promoter also comprises " CCAAT box " element (typically CCAAT sequence) and/or GGGCG sequence, and it is usually located at transcriptional start point upstream the 40th and 200 nucleotides, preferably between 60-120 nucleotides.

Regulating and controlling sequence: the application that term " regulating and controlling sequence " and " control element " can exchange, and refer to that impact transcribes or translation initiation and speed and any nucleotide sequence of the stability of described translation or polypeptide product and/or motility. Regulating and controlling sequence includes, but not limited to promoter, the promoter control element, and protein binding sequence, 5 ' and 3 ' UTRs, transcription initiation site, terminator sequence, the poly-adenosine sequence, the particular sequence in the introne, coded sequence, etc.

Preciseness: when being used for this paper, " preciseness " is probe length, probe compositions (G+C content), and salinity, organic solvent concentration, and the function of hybridization temperature or wash conditions. Preciseness is typically by parameter T_mWith with T_mDifferent temperature and compare T_mIt is the temperature when the complementary molecule that has 50% in the hybridization is hybridized. Height preciseness condition provides T_m-5 ℃ to T_mThose of-10 ℃ condition. Middle or medium preciseness condition provides T_m-20 ℃ to T_mThose of-29 ℃ condition. Low preciseness is submitted to and is provided T_m-40 ℃ to T_mThose of-48 ℃ condition. Hybridization conditions and T_m(℃) relation table be shown in the math equation:

T _m＝81.5-16.6(log ₁₀[Na ⁺])+0.41(％G+C)-(600/N)(1)

Wherein N is the length of probe. This equation is very useful at 14-70 nucleotides probe identical with target sequence for length. Following T for the DNA-DNA heterozygote_mEquation be effective to 50 to greater than the probe in 500 nucleotides scopes, and be used for comprising the condition of organic solvent (formamide).

T _m=81.5+16.6log{[Na+]/(1+0.7[Na+]) }+0.41 (%G+C)-500/L 0.63 (% formamide) (2)

Wherein L is the length of heterozygote middle probe.(P.Tijessen, " Hybridization with NucleicAcid Probes " in Laboratory Techniques in Biochemistry and MolecularBiology, P.C.vand der Vliet, ed., c.1993 by Elsevier, Amsterdam.) T of equation (2) _mBe subjected to the influence of heterozygote character; For DNA-RNA heterozygote, T _mBe higher than calculated value 10-15 ℃, for RNA-RNA heterozygote, T _mExceed 20-25 ℃.Since when using long probe, the every minimizing 1% of homology, T _mReduce about 1 ℃ (Bonner etc., J.Mol.Biol.81:123 (1973)), support homologous genes or relevant family member to detect so the preciseness condition can be adjusted to.

Suppose balance, and therefore, under probe surplus and competent time conditions, most preferably carry out,, draw equation (2) to obtain balance according to hybridization of the present invention.Need reach balance time can shorten such as the polymer of dextran sulfate or other high volume by comprise hybridization promotor in hybridization buffer.

Can be in the hybridization process, or after hybridization takes place, control preciseness by the salt and the temperature condition that change used washing soln.When being used to calculate the preciseness of washing soln, above the formula of Xian Shiing is equal available.Preferred washing soln preciseness is in the above-described scope; High preciseness is to be lower than T _m5-8 ℃, middle or medium preciseness is to be lower than T _m26-29 ℃ and low preciseness are to be lower than T _m45-48 ℃.

Basically do not have: when at least 85 weight % were A among whole A+B in the composition, the composition that contains A was " not having basically " B.Preferably, A comprise in the composition whole A+B at least about 90 weight %, more preferably at least about 95 weight % or even 99 weight %.For example, can think that plant gene or dna sequence dna do not have other plant gene or dna sequence dna basically.

Translation initiation site: in the context of the present invention, " translation initiation site " ATG normally more generally was first ATG during cDNA transcribed.Yet single cDNA can have a plurality of translation initiation sites.

Transcription initiation site: in the present invention, " transcription initiation site " is used for describing the point of transcription initiation.This point typically is positioned at about 25 Nucleotide places, TFIID binding site downstream, such as the TATA box.Transcribing can be initial in intragenic one or more sites, and individual gene can have a plurality of transcription initiation sites, and some in them can be that specificity is used for transcribing at concrete cell type or tissue.

Untranslated region (UTR): " UTR " but be the nucleotide base of transcribing any adjacent series of not translating.These zones of not translating can be relevant with concrete function, such as the stability that increases mRNA courier.The example of UTRs includes, but not limited to the poly-adenosine signal, terminator sequence, the sequence between transcriptional start point and first exon (5 ' UTR), and the sequence (3 ' UTR) that is positioned at last exon and mRNA end.

Carrier: carrier means any genetic elements that the polymerized nucleoside acid sequence can be transported to target cell, such as plasmid, and phage, transposon, cosmid, karyomit(e), virus, etc.Usually, when when suitable controlling elements associates mutually, carrier can duplicate.Therefore, described term comprises clone and expression vector, and virus vector and in conjunction with carrier.

Nucleic acid or polynucleotide are when using in this article, and term " nucleic acid " or " polynucleotide " exchange to be used, and is meant RNA and DNA, comprise cDNA, genomic dna (for example synthesizes, chemosynthesis) DNA and contain the DNA (or RNA) of nucleic acid analog.Polynucleotide can have any three-dimensional structure, and can be in justice or antisense orientation are arranged.The limiting examples of polynucleotide comprises gene, gene fragment, exon, intron, messenger RNA(mRNA) (mRNA), transfer RNA, ribosome-RNA(rRNA), rrna, cDNA, the reorganization polynucleotide, branch's polynucleotide, plasmid, carrier, the DNA of isolating any sequence, the RNA of isolating any sequence, nucleic acid probe, and primer.

Isolated nucleic acid molecule can produce by standard technique.For example, polymerase chain reaction (PCR) technology can be used for obtaining the isolating nucleic acid that contains nucleotide sequence described herein.PCR is meant the method or the technology of enzymatic amplification purpose nucleic acid therein.Typically application purpose zone end or sequence information in addition design oligonucleotide primer, and its sequence with the reverse strand of the template that will be amplified is consistent.PCR can be used for comprising the sequence from complete genome DNA or whole-cell rna from DNA and the concrete sequence of RNA amplification.Primer typically is 14-40 Nucleotide on length, but can (for example, length is 10,15,20,25,27,34 from 10 Nucleotide to a hundreds of Nucleotide on length, 40,45,50,52,60,65,70,75,82,90,102,150,200,250 Nucleotide).For example, comprehensive round pcr exists PCR Primer:A Laboratory Manual,Dieffenbach, C. and Dveksler, G. edits, and Cold Spring Harbor Laboratory Press describes in 1995.When application RNA originates as template, can use ThermoScript II and synthesize complementary DNA (cDNA) chain.Ligase chain reaction (LCR), strand displacement amplification, self-sustained sequence replication or also can be used for obtaining isolating nucleic acid based on the amplification of nucleotide sequence.For example, referring to, Lewis, 1992, Genetic Engineering News, 12:1; Guatelli etc., 1990, Proc.Natl.Acad.Sci.USA, 87:1874-1878; And Weiss, 1991, Science, 254:1292.

The isolating nucleic acid of the present invention can also be chemosynthesis, as single nucleic acid molecule (for example, use the phosphoramidite technology, it is synthetic to use automated DNA in 3 '-5 ' direction) or as a series of oligonucleotides.For example, can synthesize one or more pairs of long oligonucleotides (for example,＞100 Nucleotide) of the sequence that contains needs, each is to containing short complementary fragment (for example, about 15 Nucleotide), during to annealing, forms duplex with the described oligonucleotide of box lunch.Archaeal dna polymerase is used for extending oligonucleotide, and each oligonucleotide is connected in the carrier then to forming one, double-stranded nucleic acid molecule.

The isolating nucleic acid of the present invention can also be by mutagenic obtained.For example, the application standard technology comprises oligonucleotide directed mutagenesis and site-directed mutagenesis by PCR, reference nucleic acid sequence can be suddenlyd change.Referring to, Short Protocols in Molecular Biology,The 8th chapter, Green PublishingAssociates and John Wiley ﹠amp; Sons, Ausubel, editors such as F.M., 1992.

Nucleic acid analog can be modified on base portion, sugar moieties or phosphoric acid skeleton, to improve, for example, the stability of nucleic acid, crossability or solvability.To the modification of skeleton comprise uncharged connection (for example, methyl orthophosphoric acid, phosphotriester, phosphamide (phosphamidates), carboxylamine, etc.) with charged the connection (for example, thiophosphatephosphorothioate, phosphorodithioate, etc.).Modification to skeleton can also connect by binding peptide, for example, causes the PNA-type to connect.Comprise in the modification of base portion making deoxythymidine be modified into deoxyuridine, with Deoxyribose cytidine be modified into 5-methyl-2 '-Deoxyribose cytidine or 5-bromo-2 '-Deoxyribose cytidine.The modification of sugar moieties comprise 2 ' hydroxyl of modifying ribose form 2 '-O-methyl or 2 '-O-allyl group sugar.Can modify phosphoric acid ribodesose skeleton, to produce morpholino nucleic acid, wherein each base portion connects hexa-atomic morpholine ring, or produces peptide nucleic acid(PNA), and wherein deoxidation phosphoric acid skeleton is replaced by pseudopeptide backbone, and keeps 4 bases. Referring toSummerton and Weller, Antisense Nucleic Acid Drug Dev.(1997) 7 (3): 187-195; With Hyrup etc. (1996) Bioorgan.Med.Chem.4 (1): 5-23.In addition, for example, deoxidation phosphoric acid skeleton can be by thiophosphatephosphorothioate or phosphorodithioate skeleton, phosphamide, or the alkyl phosphate skeleton replaces.Nucleic acid can be (that is, justice or antisense strand being arranged) double-stranded or strand.

When being used for this paper, " isolating ", when referring to nucleic acid or polynucleotide, be meant from genome, other nucleic acid that for example exists in the Plant Genome or the nucleic acid or the polynucleotide of Polynucleotide molecular separation, it comprises that common side is connected in the nucleic acid or the polynucleotide of the one or both sides of described genomic nucleic acid or polynucleotide.When being used in this paper, term " isolating " about nucleic acid or oligonucleotide also comprises the sequence that any non-natural exists, because the sequence that such non-natural exists is not found in natural, and do not have the sequence of direct neighbor in naturally occurring genome.

For example, isolating nucleic acid or polynucleotide can be dna moleculars, and condition is to find usually directly to be removed with one of described dna molecular side nucleotide sequence even or not exist in naturally occurring genome.Therefore, isolating nucleic acid as the isolating molecule that does not rely on other sequence (for example includes, but not limited to, the nucleic acid of chemosynthesis, or handle cDNA or the genomic DNA fragment that produces by PCR or restriction endonuclease) and the dna molecular that exists, and the plasmid, virus (for example, the retrovirus that are attached to carrier, self-replicating, slow virus, adenovirus, or simplexvirus) on DNA, or prokaryotic organism or Eukaryotic genomic dna.In addition, isolating nucleic acid can comprise engineering nucleic acid, such as the dna molecular as the part of heterozygosis or integrative nucleic acid.For example,, or contain and become hundred nucleic acid that exist in other nucleic acid up to a million on the gel slice of genomic dna restrictive diges-tion liquid, do not think isolating nucleic acid at cDNA library or genomic library.

Polypeptide term " polypeptide " is used with its most wide in range meaning, refers to the compound of two or more subunit amino acid, amino acid analogue or other peptide mimics.Described subunit can connect by peptide bond or other key, for example, ester, ether, etc.Term " amino acid " is meant natural and/or non-natural or synthetic amino acid, comprises the D/L optical isomer.The albumen, analogue, mutant and the fragment thereof that comprise total length by this definition.

As for polypeptide, " isolating " means described to a certain extent polypeptide and separates in the cellular constituent of natural discovery usually with it.Isolated polypeptide can produce single master tape on non-reduced poly acrylamide gel.In some cases, polypeptide is " purifying ".When being used in this paper, term " purifying " preferably refers to, for example, in mixture with respect to all polypeptide, exist at least about 75 weight % or more (for example, at least 80%, 85%, 90%, 95%, the polypeptide of same type 97%, 98%, 99%, or 100%).For example, isolated polypeptide can by chemosynthesis, or be passed through recombinant production acquisition in host cell or transgenic plant by extracting from natural origin.

For the recombinant production polypeptide, the nucleotide sequence that contains the nucleotide sequence of the desired polypeptides of encoding can be connected in the expression vector, and be used for transform bacteria, eucaryon or plant host cell (for example, insect, yeast, Mammals, or vegetable cell).In bacterial system, can use intestinal bacteria (Escherichia coli) bacterial strain, such as BL-21.Suitable intestinal bacteria (E.coli) carrier comprises the pGEX serial carrier that can produce the fusion rotein with glutathione S-transferase (GST).Depend on used carrier, the typically index growth of the intestinal bacteria of conversion then, stimulated with isopropylthiogalactoside (IPTG) before results.Usually, the fusion rotein of expression is soluble, and by being adsorbed onto on the glutathione agarose pearl, then wash-out in the presence of free glutathione, easily purifying from lysing cell.The pGEX carrier design becomes to comprise zymoplasm or Xa factor protease cracking site, so that clone's target gene product can partly discharge from GST.Alternatively, 6X His-mark can be used for assisting and separate.

In eukaryotic host cell, many expression systems based on virus can be used for express polypeptide.For example, can with the coding polypeptide of the present invention nucleic acid clone to baculovirus vector, such as pBlueBac (Invitrogen, Carlsbad, CA) in, and be used for then with wrap common transfection insect cell from Autographa californica, such as Spodoptera frugiperda (Sf9) cell by the wild-type DNA of nuclear polyhedral virus (AcMNPV) more.The recombinant virus that produces polypeptide of the present invention can be differentiated by standard method.

By the expression vector that application has suitable controlling elements and selective marker, can produce the mammal cell line of stably express polypeptide.For example, pcDNA3 carrier for expression of eukaryon (Invitrogen, Carlsbad, CA) be suitable at cell, such as express polypeptide in Chinese hamster ovary (CHO) cell, COS-1 cell, human embryo kidney (HEK) 293 cells, NIH3T3 cell, BHK21 cell, mdck cell, ST cell, PK15 cell or the human vascular endothelial (HUVEC).In some instances, the pcDNA3 carrier can be used for express polypeptide in the BHK21 cell, and wherein said carrier comprises CMV promotor and G418 antibiotics resistance gene.According to the introduction of expression vector, for example, can select stable clone by antibiotics resistance to G418, kantlex or Totomycin.Alternatively, the sequence of amplification can be connected on the mammalian expression vector, such as pcDNA3 (Invitrogen, San Diego, CA), and then using wheat embryo extract or rabbit reticulocyte lysate in in-vitro transcription and translation.

In other situation, can use recombinant nucleic acid construct transformed plant cells, with express polypeptide, as previous and described in the following embodiments.Then, can use the known technology of personnel that this area has ordinary skill, extract and the described polypeptide of purifying.

Polynucleotide and polypeptide

Because the transgenic plant that comprise them can show the phenotypic characteristic of change with respect to control plant, so polynucleotide described herein is interesting.For example, transgenic plant can show the metabolic pattern of change.The metabolic pattern that changes can comprise sucrose, L-glutamic acid or the linolic acid of improvement level, as discussed below.In some cases, transgenic plant can show the plain sterone of 6-deoxidation rape of increase level, and/or reduce the campesterol of level.In some cases, the transgenic plant of expressing such polypeptide can show the photosynthetic rate of raising.

The phenotypic characteristic of these changes can be used for developing or the optimizing plant prod.For example, polynucleotide of the present invention and/or polypeptide can be used for increasing sucrose in the plant, L-glutamic acid or linoleic level; Or increase the level of the plain sterone of 6-deoxidation rape in the plant; Or reduce the level of campesterol in the plant; Or raising photosynthesis of plants rate.In some cases, improve, for example, increase the plain sterone of 6-deoxidation rape of level and the campesterol of minimizing level more than a kind of phenotypic characteristic.Therefore, transgenic plant can have the growth potential of raising, have the biomass of increase, highly, seed production, seed weight or the seed circularity that improves.Therefore, described polynucleotide or polypeptide are effective to prepare the transgenic plant that have special applications in agricultural and forestry industry.

Especially, provide isolating P ₄₅₀Polynucleotide and peptide sequence comprise polynucleotide sequence variants, homologue, directly to homologue, syzygy and fragment and and P ₄₅₀The polypeptide that the polypeptide function is suitable.Isolating P ₄₅₀Polynucleotide and polypeptide can be the homologues of Arabidopis thaliana Dwf4 polynucleotide or DWF4 polypeptide and/or directly to homologue.Isolating P ₄₅₀Polynucleotide and polypeptide can be the homologues of coding 22-α hydroxylase or 22-α hydroxylase polypeptide and/or directly to homologue.Therefore, this paper has described isolating Dwf4 polynucleotide or DWF4 peptide sequence, and it comprises the DWF4 polypeptide suitable with Arabidopis thaliana DWF4 function.DWF4 is a cytochrome P ₄₅₀Polypeptide, except other activity, its catalysis campesterol in the C-22 hydroxylation to produce the plain sterone of 6-deoxidation rape.Therefore, in some cases, peptide sequence can show biochemical activity or influence plant phenotype in the mode similar to the DWF4 polypeptide, and representative and Arabidopis thaliana DWF4 albumen biochemistry or the suitable polypeptide of phenotypic function.

Polynucleotide of the present invention comprises the Codocyte pigment P ₄₅₀The nucleic acid of polypeptide.SEQ ID NOs:1-3 lists 3 kinds of P from Arabidopis thaliana, corn and paddy rice respectively ₄₅₀Proteic polynucleotide and peptide sequence.Arabidopis thaliana and paddy rice sequence are the DWF4 polypeptide of coding 22-α hydroxylase enzyme.By with Semen Maydis polypeptide sequence library at many polypeptide databases, comprise P ₄₅₀, plant and privately owned database carry out sequence relatively, and express the phenotypic characteristic of the transgenic plant of described Semen Maydis polypeptide (SEQ ID NO:2) by assessment, identify that Semen Maydis polypeptide (SEQ ID NO:2) is that DWF4 is directly to homologue with respect to the transgenic plant of expressing Arabidopis thaliana DWF4 polypeptide (SEQ IDNO:1) or paddy rice DWF4 polypeptide (SEQ IDNO:3).Referring to following embodiment and Fig. 4, Fig. 4 has listed the comparison between Arabidopis thaliana DWF4 polypeptide and paddy rice DWF4 polypeptide.

Also considered the homologue of described polynucleotide (and encoded polypeptides), directly to homologue, fragment, syzygy, complement or reverse complement.As indicated above, the homologue of polypeptide and directly can be called the suitable polypeptide of function to homologue.The polypeptide homologue that function is suitable shows the sequence homology that the Semen Maydis polypeptide sequence of listing with SEQID NO:2 has special level.For example, isolating polynucleotide can comprise that aminoacid sequence that coding is listed with SEQ ID NO:2 has an appointment 85% or multisequencing homology more, and is for example about 86,87,90,92,95,96,97,98,99, or the nucleic acid of the polypeptide of 100% sequence homology.The albumen that function is suitable can be Arabidopis thaliana DWF4 directly to homologue.The albumen that function is suitable can be have C-22 α-hydroxylase activity polypeptide directly to homologue.Fig. 8 lists the many straight peptide sequence to homologue of Arabidopis thaliana DWF4.

In some cases, isolating polynucleotide can comprise the nucleic acid of the polypeptide that coding is such, that is, described polypeptide comprises the corresponding aminoacid sequence of consensus sequence (SEQ ID NO:4) of the DWF4 polypeptide of listing with Fig. 2.For example, isolating polynucleotide can comprise the nucleic acid of the corresponding polypeptide of consensus sequence (SEQ ID NO:4) of the DWF4 polypeptide that coding and Fig. 2 list.In some cases, described polynucleotide also comprises the promotor of the wide expression on the nucleic acid that effectively is connected to coding said polypeptide.Can use the promotor of any wide expression, include, but not limited to this paper further describes those.

In some cases, isolating polynucleotide can comprise the nucleic acid of the polypeptide that coding is such, promptly, described polypeptide comprises the corresponding aminoacid sequence of consensus sequence (SEQ ID NO:4) of the DWF4 polypeptide of listing with Fig. 2, condition is that encoded polypeptide does not show the aminoacid sequence of listing with SEQ ID NO:1 or SEQ ID NO:3 and has 93% or more (for example, 94%, 94.5%, 95%, 95.5%, 96%, 97%, 98%, or 99%) sequence homology.Can use the polypeptide that comprises with the corresponding aminoacid sequence of consensus sequence, for example, prepare transgenic plant with one or more following phenotypic characteristics: improvement (for example, improve) photosynthetic rate, the plain sterone of the 6-deoxidation rape of increase level, the campesterol of minimizing level, the metabolic pattern of improvement, for example, the sucrose of increase level, L-glutamic acid or linolic acid, the seed production of raising, the seed circularity of raising, the plant height that increases, etc.

In some cases, polypeptide described herein can be the albumen suitable with Arabidopis thaliana DWF4 function directly to homologue, this exercises the biochemical activity of at least a DWF4 or influences plant phenotype in the mode similar to DWF4 by polypeptide and determines.Therefore, polypeptide can the catalysis reaction similar to DWF4, or influences plant phenotype in the mode similar to DWF4.For example, known Arabidopis thaliana DWF4 catalysis campesterol in the C-22 oxidation to form the plain sterone of 6-deoxidation rape.DWF4 also catalysis 6-oxo campesterol hydroxylation to produce the plain sterone (cathasterone) of rape.Polypeptide of the present invention can also be finished a step or two steps of these enzymatic steps.

In some cases, the polypeptide that function is suitable shows at least 60% Arabidopis thaliana DWF4 albumen biochemical activity, for example, and at least 70%, 80%, 90%, or 95% biochemical activity.The method of assessment biochemical activity is known to those of ordinary skill in the art, and comprises that enzymatic detects (for example, assessment V _Max, K _m, K _Cat, K _iDeng), the radioactive tracer picked-up detects, or the like.Especially, for the substrate and the product level of the enzymatic step of being given, can use the known analytical technology of those of ordinary skill in the art and (for example, GC-MS) assess.For example, the chemical intermediate level in the BL approach can be assessed in the transgenic plant of the polynucleotide that comprises the polypeptide described herein of encoding.With respect to the level in the control plant, can comparison level.The level of chemical intermediate can be in each period of growing in the transgenic plant, for example, at seedling or mature period, or use various tissues (for example, seed, blade, branch, stem, flower, or the like) assess.The increase of the plain sterone level of the minimizing of campesterol level and/or 6-deoxidation rape can indicate polypeptide be Arabidopis thaliana DWF4 directly to homologue.

Recombinant vectors and host cell

The present invention also provides recombinant vectors and the host cell that comprises above-mentioned any isolating polynucleotide.As hereinafter explanation more fully, various recombinant vectorss are that those of ordinary skill in the art is known.Recombinant vectors can comprise having transcribing and/or the translational control sequence of any needs, such as promotor, and the sequence of the present invention of the terminal terminator sequence of UTRs and 3 '.Carrier can also comprise replication orgin, scaffold attached region (SARs), and mark, homologous sequence, intron, etc.Described carrier can also comprise the marker gene of giving the selectable phenotype of vegetable cell.Described mark is encoding human agent for killing resistance, particularly antibiotics resistance typically, such as resistance to kantlex, G418, bleomycin, Totomycin, or the resistance of Herbicid resistant such as chlorine sulphur grand (chlorosulfuron) or phosphinotricin.

Typically, the carrier of reorganization will comprise polynucleotide and effectively be connected to controlling elements on the polynucleotide, so that for example, in host cell, the polypeptid coding sequence in the polynucleotide can be transcribed and translate.Controlling elements can be a promotor, and some in them are known to those skilled in the art.For example, plant promoter can comprise, such as the promotor that instructs described gene to transcribe in all or some tissue of regenerated plant, for example, constitutive promoter is such as the promotor of 35S or wide expression, such as p326.In such circumstances, promotor is connected on the nucleic acid of coding desired polypeptides effectively.Alternatively, plant promoter can instruct sequence of the present invention transcribing in particular organization (tissue-specific promoter), perhaps is in addition under accurate more environment control (inducible promoter).As previously described, various plant promoters comprise composing type, tissue-specific, the wide expression type and inducible promoter, are known to those skilled in the art.Can also be included in the poly-adenosine zone of 3 of coding region ' end.Described poly-adenosine zone can be derived from natural gene, is derived from various other plant genes, or is derived from T-DNA.

The promotor that can comprise in some cases, wide expression.Can application examples as, the promotor of wide expression, such as p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.In such circumstances, the polynucleotide that is connected to effectively on the promotor of wide expression can be above-mentioned any polynucleotide, for example, those of nucleic acid that comprise the total DWF4 aminoacid sequence that coding SEQID NOs:1-3 or Fig. 2 list, or comprise the polynucleotide of the nucleotide sequence of the such polypeptide of coding, that is, described polypeptide shows with SEQ ID NOs:1-3 at least about 85% (for example, at least about 86%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100%) sequence homology.Using constitutive promoter, in the situation such as 35S, polynucleotide can comprise that aminoacid sequence that coding is listed with SEQ ID NO:2 has 85% or multisequencing homology (for example, about 86,87 more, 90,92,95,96,97,98,99, or 100% sequence homology) nucleic acid, the nucleic acid of the polypeptide of the corresponding aminoacid sequence of consensus sequence (SEQ ID NO:4) that maybe can comprising encodes has the DWF4 polypeptide of listing with Fig. 2, condition is that encoded polypeptide does not show the aminoacid sequence of listing with SEQ ID NO:1 or SEQ ID NO:3 and has 93% or more (for example, 94,94.5,95,95.5,96,97,98,99 or 100%) sequence homology.

Recombinant vectors can be used for transforming various vegetable cells with the preparation transgenic plant.The technology that is used to transform the higher plant kind of broad variety known in the art.Typically, preparation comprises the recombinant DNA construction body of polymerized nucleoside acid sequence of the present invention, and described polymerized nucleoside acid sequence is inserted in the carrier that is suitable for transformed plant cells.Construct can application standard recombinant DNA technology .1989 such as () Sambrook preparation.Carrier framework can be any typical carrier in this area, such as plasmid, and virus, artificial chromosome, BACs, YACs and PACs.

Transgenic plant

The present invention also provides the transgenic plant that comprise external source polynucleotide described herein or recombinant vectors.By many known methods, comprise electroporation, microinjection and biological projectile method can be incorporated into previous described any polynucleotide or recombinant vectors in the genome of various plant hosts.Alternatively, described polynucleotide or carrier can be connected with suitable T-DNA side joint zone, and are incorporated in the conventional agrobacterium tumefaciens host carrier.Agrobacterium tumefaciens mediated transformation technology like this comprises the application of releasing (disarming) and binary vector, is known in the art.Other transgenosis and transformation technology comprise by calcium or PEG protoplast transformation, the absorption of the naked DNA of electroporation mediation; The electroporation of plant tissue, and microparticle bombardment.

The ectopic expression of sequence of the present invention can be used " knocking in " method and realize.Here, first composition, " activation subsystem (activator line) " is the transgenic plant that comprise transcription activating that effectively is connected on the promotor.Second composition comprises the cDNA sequence of the needs in the targeted integration sequence/zone that effectively is connected to transcription activating.Second composition being transformed into " activation subsystem " or being used to transform host plant is that it is hybridized by conventional cultural method with " activation subsystem " to produce " target ".In every kind of situation, the result is identical.That is to say that described promotor promotes the proteic generation of transcription activating, it is in conjunction with the expression of targeted integration zone with the cDNA of promotion needs then.

Any promotor that acts in plant may be used to first composition, such as constitutive promoter, and tissue or organ specific promoters, or the promotor of wide expression, as described earlier such.The sub-polypeptide of suitable transcription activating include, but not limited to encode those of HAP and GAL4.Described binding sequence by sub-albumen identification of selected transcription activating and target is used for second composition.

Can cultivate the transformed plant cells that produces by aforesaid method, have the plant of the phenotype that is transformed with regeneration.Regeneration techniques can depend on the manipulation of plant hormone in the tissue culture growth substratum, and can depend on biocide and/or the weedicide mark of introducing with described purpose polynucleotide.Regeneration can also obtain from plant protoplast, corpus callosum, transplant, organ, pollen, embryo or its part.

Cell transformed, corpus callosum, tissue or plant can be by selecting or screening particular feature or active engineered plant material, for example, by those of marker gene or antibiotics resistance gene coding, and determine and separate.Such screening and system of selection are that those of ordinary skill in the art is known.In addition, can Applied Physics and chemical process identify transformant.Method comprises DNA analysis and pcr amplification (for example, being used for the detection of polynucleotide); Be used to detect the RNA trace of transcribing, S1 Rnase protection, primer extension, or RT-PCR amplification with checking R NA; Be used to detect the enzymatic check of the enzyme or the ribozyme activity of polypeptide and polynucleotide; And gel electrophoresis of protein, western blotting, immunoprecipitation, and enzyme linked immunosorbent detection are to detect polypeptide.Other technology, such as in situ hybridization, enzyme dyeing, and immunostaining also can be used for detecting the existence or the expression of polypeptide and/or polynucleotide.The method of carrying out all technology of mentioning is known.After polynucleotide stably was attached in the transgenic plant, it can be introduced in other plant by sexual hybridization, for example, and by the breeding and propagation technology of standard.

Above-mentioned polynucleotide can be used for transforming many plants and vegetable cell system, comprises monocotyledons and dicotyledons.Polynucleotide and polypeptide will find concrete application in agricultural and field of forestry.Suitably the floristics of group comprises dicotyledons, such as red blue Pittosporum, and alfalfa, Glycine, Coffea, Semen Brassicae campestris (high sinapinic acid and rape) or Helianthus.Monocotyledons also is suitable for, and belongs to such as Zea mays, Triticum, Secale, Hordeum, Avena, Oryza, broomcorn millet, Amaranthaceae, switchgrass or jowar.Vegetable crop or root crop, such as Lactuca, Radix Dauci Sativae, allium, Cauliflower, Pisum, sweet corn, popcorn with corn, tomato genus, potato, Macroptilium (comprising Kidney bean, lima bean, dried beans (dry beans), green soya bean) etc., be suitable for, and fruit crop, belong to (for example, watermelon, muskmelon), peach, pear, Malus, cherry genus, tangerine, lemon, natsudaidai, Prunus, Mangifera, Musa and palm such as grape, Fragaria, Ananas, melon.

Therefore, method described herein can be used to belong to following each purpose dicotyledons: Magniolales, anistree order (Illiciales), Laurales (Laurales), Piperales (Piperales), Aristolochiales (Aristochiales), Nymphaeales (Nymphaeales), Ranales (Ranunculales), Papeverales, Sarraceniaceae, Trochodendrales (Trochodendrales), Hamamelidales (Hamamelidales), Eucommiales (Eucomiales), Leitneriales, Myricales (Myricales), Balanopsidales (Fagales), Casuarinales (Casuarinales), Caryophyllales (Caryophyllales), Batales, knotweed order (Polygonales), Plumbaginales (Plumbaginales), Dilleniales (Dilleniales), Theales (Theales), Malvales (Malvales), Urticales (Urticales), Lecythidales (Lecythidales), Violales (Violales), Salicales (Salicales), Capparales, heather order (Ericales), Diapensiales (Diapensales), persimmon order (Ebenales), Primulales (Primulales), Rosales (Rosales), Fabales, Podostemales (Podostemales), Haloragales, Myrtales (Myrtales), Cornales (Cornales), handkerchief Lip river ladder order (Proteales), Santalales (Santales), Rafflesiales (Rafflesiales), Celastrales (Celastrales), Euphorbiales (Euphorbiales), Rhamnales (Rhamnales), Sapindales (Sapindales), Juglandales (Juglandales), Mang ox seedling order (Geraniales), polygalales (Polygalales), Umbellales, Gentianales (Gentianales), Polemoniales (Polemoniales), Lamiales (Lamiales), Plantaginales (Plantaginales), Scrophulariales (Scrophulariales), bellflower order (Campanulales), Rubiales (Rubiales), Dipsacales (Dipsacales) and aster order (Asterales).Method described herein can also be used to belong to following each purpose monocotyledons: Alismatales, Hydrocharitales (Hydrocharitales), Najadales (Najadales), Triuridales (Triuridales), Commelinales (Commelinales), Eriocaulales (Eriocaulales), Restionales (Restionales), annual bluegrass order (Poales), Juncales (Juncales), Cyperales (Cyperales), Typhales (Typhales), Bromeliales (Bromeliales), Zingiberales, Arecales (Arecales), Cyclanthales (Cyclanthales), pandanales (Pandanales), Arales, Lilliales and blue order (Orchidales) perhaps are used to belong to the plant of Gymnospermae, for example, pinales (Pinales), Ginkgoales (Ginkgoales), Cycadales (Cycadales) and Gnetales (Gnetales).

The present invention has application to the floristics of ten minutes broad range, comprise from following each kind that belongs to: allium (Allium), Alseodaphane (Alseodaphne), Anacardium (Anacardium), Arachis (Arachis), Asparagus (Asparagus), Atropa (Atropa), Avena (Avena), fine jade Phoebe (Beilschmiedia), Btassica (Brassica), both citrus (Citrus), Citrullus (Citrullus), Capsicum (Capsicum), Vinca (Catharanthus), red blue Pittosporum (Carthamus), Cocculus (Cocculus), cocoanut (Cocos), Coffea (Coffea), Croton, Cucumis (Cucumis), Cucurbita (Cucurbita), Daucus, Duguetia, oil palm belongs to (Elaeis), Eschscholtzia (Eschscholzia), Ficus (Ficus), Fragaria (Fragaria), sea papaver (Glaucium), Glycine (Glycine), Gossypium (Gossypium), Helianthus (Helianthus), Heterocallis, Hevea, Hordeum (Hordeum), poison tobacco (Hyoscyamus), Lactuca (Lactuca), glue Calamus (Landolphia), linum (Linum), Litsea (Litsea), lolium (Lolium), lupinus (Lupinus), tomato belongs to (Lycopersicon), Malus (Malus), Manihot, Majorana, Medicago (Medicago), Musa (Musa), Nicotiana (Nicotiana), Olea (Olea), Oryza (Oryza), millet belongs to (Panicum), Pannesetum, papaver (Papaver), elargol Chrysanthemum (Parthenium), Persea (Persea), Phaseolus (Phaseolus), Pinus (Pinus), Pistachia, Pisum (Pisum), pear (Pyrus), Prunus (Prunus), Rhaphanus (Raphanus), Rhizocarya, Ricinus (Ricinus), Secale (Secale), Senecio (Senecio), Chondodendron (Sinomenium), sinapsis alba belongs to (Sinapis), Solanum (Solanum), and jowar belongs to (Sorghum), Stephania (Stephania), Theobroma (Theobroma), Semen Trigonellae belongs to (Trigonella), Triticum (Triticum), Vetch (Vicia), Vinca (Vinca), Vitis (Vitis), Vigna (Vigna) and Zea (Zea).

Being used for implementing suitable group of kind of the present invention comprises and produces alkaloidal plant, for example, from following plant: papaveracease (Papaveraceae), Berberidaceae (Berberidaceae), Lauraceae (Lauraceae), Menispermaceae (Menispermaceae), Euphorbiaceae (Euphorbiaceae), pulse family (Leguminosae), Boraginaceae (Boraginaceae), Apocynaceae (Apocynaceae), asclepiadaceae (Asclepiadaceae), Liliaceae (Liliaceae), Gnetaceae (Gnetaceae), Erythroxylaceae (Erythroxylaceae), convolvulaceae (Convolvulaceae), Ranunculaceae (Ranunculaeceae), Rubiaceae (Rubiaceae), Solanaceae (Solanaceae), and Rutaceae (Rutaceae) family.Papaveracease family, for example, this family contains about 250 kinds and mainly finds species in the north temperate zone in the world, and comprise following plants, such as Eschscholtzia californica (Californiapoppy) and opium poppy (Opium poppy).Available belongs to and (for example comprises papaver (Papaver) in the papaveracease family, Papaver bracteatum, ghost opium poppy (Papaver orientale), Papaversetigerum, and opium poppy (Papaver somniferum)), Sanguinaria, Dendromecon, sea papaver (Glaucium), Herba meconopsis integrifoliae belongs to (Meconopsis), Chelidonium (Chelidonium), and Eschscholzioideae is (for example, Eschscholtzia (Eschscholzia), and Argemone (Argemone) (for example, Argemone hispida Eschscholtzia californica (Eschscholziacalifornia)),, Mexican Pricklepoppy (Argeinone mexicana) and Argemone munita) generic.Being used for implementing of the present invention other produces alkaloidal kind and comprises Croton salutaris, Croton balsamifera, the root of fangji (Sinomenium acutum), root of Taiwan Stephania (Stephania cepharantha), Stephaniazippeliana, Litsease biferea, Alseodaphne perakensis, camphor tree leaf Cocculus trilobus (Cocculuslaurifolius), Duguetia obovata, Rhizocarya racemifera, and Beilschmiediaoreophila.

Another the suitable group that is used for implementing kind of the present invention comprises the plant that produces terpenoid, for example, plant from following dependent of dead military hero: Aesculus (Aesculus), Anamirta, punching Nelumbo (Andrographis), artemisia (Artemisia), Betula (Betula), Bixa (Bixa), Cannabis (Cannabis), Centella (Centella), crowndaisy chrysanthemum belongs to (Chrysanthemum), Tanacetum (Tanacetum), Cinnamomum (Cinnamomum), both citrus (Citrullus), Luffa (Luffa), Coleus (Coleus), turmeric (Curcuma), Cymbopogon (Cymbopogan), Daphne (Daphne), Euphorbia (Euphorbia), Glycine (Glycine), Glycyrrhiza (Glycyrrhiza), Gossypium (Gossypium), Guayule, Hevea, Rabdosia (Isodon), Rabdosia (Rabdosia), Rabdosia (Rabdosia), Mentha (Mentha), Salvia (Salvia), Rosmarinus (Rosmarinus), Quassia (Simarouba), Taxus (Taxus), Thymus vulgaris (Thymus) and Thunder God Calamus (Tripterygium).

Other suitable kind comprises tomato (Lycopersicum esculentum), Nicotiana (Nicotiana spp.) (for example, tobacco (Nicotiana tabacum)), Capsicum (Capsicumspp.) (comprising capsicum (C.annuum)), greyish white guayule (Parthenium argentatumGray), spearmint (Mentha spicata), Mentha pulegium Linn. (M. pulegium), Mentha piperita (M.piperita), thyme (Thymus vulgaris L.), wild marjoram (Origanum vulgare), Rosmarinus officinalis (Rosmarinus officinalis), Herba Melissae officinalis (Melissa officinalis), cocoa tree (Theobroma cacao), lavender (Lavandula augustifolia), medicine Salvia japonica Thunb. (Salviaofficinalis), fruitlet coffee (Coffea arabica), Hevea benthamiana, Peru rubber tree (Hevea guianensus), rubber tree (Hevea brasiliensis), cassava glue (Manihotglaziovii), Manihot dichotoma, Castilla elastica, Indian ficus (Ficus elastica), Funtimia elastica, Landolphia kirkii, Landolphia gentilli, Landolphiaheudelotii, Landolphia owariensis, folium eucalypti rattan (Crytostegia grandiflora), Crytostegia niadagascariansis, Taraxacum megalorhizon, bakelite (Palaquimgutta), Manilkara bidentata and Manilkara zapata.

In some cases, transgenic plant are not Arabidopis thaliana or tobacco plant.In some cases, transgenic plant are not the members of Solanaceae (Solanaceae) family or Cruciferae (Brassicaceae) family.For example, in following situation, transgenic plant can not be Arabidopis thaliana or tobacco plant, that is, when it comprised at least a external source polynucleotide, wherein said at least a external source polynucleotide comprised the nucleic acid of the polypeptide that coding is such:

(a) aminoacid sequence of listing with SEQ ID NO:2 has an appointment 85% or more sequence homology; Or

(b) consensus sequence of listing with Fig. 2 (SEQ ID NO:4) is corresponding.

Transgenic plant can show any biochemical activity of aforementioned polypeptides.For example, transgenic plant can show at least a biochemical activity of Arabidopis thaliana DWF4, for example, and 22 α-hydroxylase activity.The method that is used for assessing biochemical activity is for known to a person of ordinary skill in the art; Referring to, for example, above.

Transgenic plant can be used for producing the plant (for example, comparing with control plant) of the plant phenotype with change.When expressing in plant, for example, at reasonable time or in suitable tissue, polypeptide can influence the phenotype of plant (for example, transgenic plant).The phenotype effect is typically with respect to the control plant of not expressing purpose external source polynucleotide, such as corresponding wild plant, not having transgenosis purpose external source polynucleotide still to have with described purpose transgenic plant is isogenic corresponding plants, perhaps wherein said polypeptide expression is suppressed or does not induce (promptly, when expression is to control following time at inducible promoter) corresponding homogenic plant, and assess.Be lower than 10% and (for example, be lower than 9%, 8%, 7%, 6% when plant shows, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%) during the mRNA of the polypeptide that is shown by the purpose plant of amount or coding said polypeptide, thinks that plant " do not express " polypeptide.Expression can be assessed by the known method of those of ordinary skills, for example, and RT-PCR amplification, RNA trace, S1RNAse protection, primer extension, western blotting; Gel electrophoresis of protein, immunoprecipitation, enzyme linked immunosorbent detection, chip detection, and mass spectrum.Should be noted that if polypeptide is expressed, expression can be in whole plants or assessed in the tissue at needs selectively under the control of organizing property specially or wide expression promotor.Similarly, if polypeptide was expressed in specific time, for example, at the specified time of growing or when inducing, expression can optionally be assessed in the period of needs.

The phenotype effect can be the metabolic pattern that changes with respect to control plant.For example, transgenic plant can show one or more following metabolites of increase level: sucrose, glutaminate/ester (L-glutamic acid), or linolic acid.In some cases, when polypeptide described herein was expressed in transgenic plant, described transgenic plant can show than the plant of not expressing described polypeptide from 10% to about 30% more (for example, about 12 to about 30%; About 15 to about 25%, about 18 to about 25%, or about 10 to about more than 20%) sucrose concentration (for example, in leaf tissue).In some cases, when polypeptide described herein was expressed in transgenic plant, described transgenic plant can show than the plant of not expressing described polypeptide from about 10% to about 65% more (for example, about 10 to about 30%; About 20 to about 45%; About 30 to about 60%; About 40 to about 65%; About 30 to about 55%; About 20 to about more than 30%) aminoglutaric acid concentration (for example, in leaf tissue).In other situation, when polypeptide described herein was expressed in transgenic plant, described transgenic plant can show than the plant of not expressing described polypeptide from about 10% to about 50% more (for example, about 15% to about 35%; About 20% to about 45%; About 30% to about 48%; About 15% to about more than 35%) linolic acid concentration (for example, in leaf tissue).In some cases, with respect to control plant, transgenic plant can not show some other amino acid, carbohydrate, and the increase of lipid acid and organic acid level, for example, some compound that shows in the table 3.

With respect to control plant, the phenotype effect can be the enhanced photosynthetic rate.The method that is used for measuring the floristic photosynthetic rate that provides be for those of ordinary skill in the art known.For example, with respect to control plant, transgenic plant can be in specific temperature, light intensity (for example, photosynthesis photon flux intensity (PPFD)), and humidity, or show the enhanced photosynthetic rate under the gas concentration lwevel.The temperature of assessment photosynthetic rate can be from about 5 ℃ to about 45 ℃, or between any value (for example, about 10 ℃, about 20 ℃, about 25 ℃, about 30 ℃, or about 32 ℃).Humidity value can be from about 5% to about 80% scope, or between any value (for example, about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, or about 70%).Light intensity (PPFD) can be from about 0mol m ^-2s ^-1To about 4000mol m ^-2s ^-1, or between any value (for example, 25,50,100,200,300,400,500,600,700,800,900,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,2000,2200,2500,2750,2900,3000,3200,3400,3600,3800, or 3900mol m ^-2s ^-1).Gas concentration lwevel can be in the scope from about 25ppm to about 1000ppm, or between any value (for example, about 50, about 75, about 100, about 200, about 300, about 360, about 380, about 400, about 500, about 600, about 700, about 760, about800, about 900, or about 950ppm).Can application of temperature, light intensity, any combination of humidity and gas concentration lwevel.For example, in some cases, at the 360ppm gas concentration lwevel, about 50-55% humidity level, about 25 ℃ of temperature, about 1000 to about 2000mol m ^-2s ^-1The PPFDs scope, transgenic plant show the enhanced photosynthetic rate with respect to control plant.

The phenotype effect can be increase or the minimizing in the chemical intermediate level of BL approach.For example, the phenotype effect can be the increase with respect to the plain sterone level of 6-deoxidation rape of control plant.The phenotype effect can be the minimizing with respect to the campesterol level of control plant.Measure the analytical procedure of chemical intermediate, comprise the intermediate of BL approach,, be known in the art, and also describe at this paper such as campesterol and the plain sterone of 6-deoxidation rape.Increasing or reduce can be any amount with respect to control plant, for example, with respect to control plant more than 1.2 times, 1.5 times, 1.8 times, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 50 times, or 100 times increase or minimizing.In some cases, with respect to control plant, the increase of the plain sterone of 6-deoxidation rape can be higher than 2 times, or is higher than 3 times, or is higher than 4 times, or is higher than 5 times, or is higher than 6 times.

With respect to control plant, transgenic plant can also show the growth potential of increase, the size of increase (for example, highly), the seed production that increases, the seed circularity of homogeneous is (for example, in monocotyledons more, such as Oryza), the drought-resistant property of growth velocity, or enhanced faster.For example, when polypeptide described herein was expressed in transgenic plant, described transgenic plant can show higher by (for example, about 10% to about 15% than the plant of not expressing described polypeptide from about 7% to about 20%; About 12% to about 18%; About 8% to about 18%; About 15% to about 20% is higher) height.In other situation, when polypeptide described herein was expressed in transgenic plant, described transgenic plant can show higher by (for example, from about 10% to about 20% than the plant of not expressing described polypeptide from about 10% to about 95%; From about 10% to about 50%; From about 10% to about 70%; From about 20% to about 60%; From about 20% to about 75%; From about 25% to about 85%; From about 30% to about 70%; From about 35% to about 90%; From about 40% to about 60%; From about 40% to about 85%; From about 50% to about 80%; From about 50% to about 90%; Higher from about 70% to about 90%) seed production (number seeds of every strain plant).In some cases, when polypeptide described herein was expressed in transgenic plant, described transgenic plant can show higher by (for example, from about 5% to about 10% than the plant of not expressing described polypeptide from about 5% to about 20%; From about 8% to about 12%; From about 10% to about 15%; Higher from about 8% to about 18%) the seed weight of every strain plant.

Should be noted that the phenotype effect is typically assessed by one or more experimental analysis significance,statisticals.Should be appreciated that, when phenotype relatively with the time spent of doing of assessment polypeptide, with suitable parameter or nonparametric statistics as, Chi-side's check, Si Shi t-check, Mann-Whitney check, or F-checks o'clock thinks that the difference of polypeptide is significance,statistical in p≤0.05.

Other phenotype effect can be assessed by the known method of those of ordinary skills, and the specified time cell length that is included in growth is measured; The mensuration that BL uses; Sterol detects check; The detection of reaction product or by product; Detection for the level of enzymatic step substrate that provides and/or product; Detect with dose response for the enzymatic substrate of inferring.

Method

Polynucleotide described herein and polypeptide can be used for producing the phenotype with change, for example, and the plant of the phenotypic characteristic of change.Therefore, provide the method that is used to change one or more phenotypic characteristics.Phenotypic characteristic can be following one or more with respect to control plant: the metabolic pattern of change; The photosynthetic rate that changes; The plain sterone of the 6-deoxidation rape of increase level; The campesterol of minimizing level; The seed production that increases; The seed weight of the every strain plant that increases; With the height that increases.Method described herein typically can comprise a) in order to produce the plant transformed cell, introduce the isolating polynucleotide of the nucleic acid molecule comprise that coding is following to vegetable cell: 1) have the aminoacid sequence about 85% listed with SEQ ID NO:2 or the polypeptide of more sequence homology, or 2) comprise the polypeptide of the corresponding aminoacid sequence of listing with Fig. 2 of consensus sequence (SEQ ID NO:4); (b) produce transgenic plant from described plant transformed cell.Can use any method, comprise described herein those, assess the phenotypic characteristic of the change of thus obtained plant.In some cases, can be changed more than a kind of phenotypic characteristic, for example, the increase of the plain sterone level of 6-deoxidation rape and the minimizing of campesterol.

The DWF4 expression of polypeptides level that changes

Overexpression

As described previously, can be with polynucleotide described herein, recombinant vectors, host cell and transgenic plant are processed into the overexpression that produces desired polypeptides.The overexpression of polypeptide can be used for changing plant with respect to control plant, for example, do not express the phenotypic characteristic of the control plant of described polypeptide, such as increasing plant height, change metabolic pattern, increase the level of the plain sterone of 6-deoxidation rape, reduce the level of campesterol, increase photosynthetic rate, improve seed production, or the like.In addition, polypeptide can make up overexpression with the overexpression of another kind of polypeptide, for example, and the another kind of P that participates in the BL biosynthetic pathway ₄₅₀Polypeptide is such as CPD.Such co expression of polypeptide can produce additional or collaborative effect to biochemical activity (for example, enzymatic activity) or the phenotype (for example, highly) of plant.Can also use fusion polypeptide, and typically comprise described herein in reading frame with another kind of polypeptide, such as participating in the biosynthetic polypeptide of BL (for example, the polypeptide that CPD) merges.

The inhibition of expressing

Alternatively, polynucleotide described herein and recombinant vectors can be used for preventing or suppressing endogenous P ₄₅₀Albumen, such as DWF4, the expression in the purpose floristics.There are many methods can be used for the expression of suppressor gene in plant.Antisense technology is a kind of known method.In this method, the nucleic acid fragment of native gene is cloned and is connected on the promotor effectively, so that the antisense strand of RNA is transcribed.Then, recombinant vectors is transformed in the rice plant, as indicated above such, and the antisense strand of generation RNA.Described nucleic acid fragment needs not to be the complete sequence of wanting repressed native gene, but typically identical with at least a portion of wanting repressed native gene basically.Usually, higher homology can be used for compensating the application of shorter sequence.Typically, the sequence of at least 30 Nucleotide of application (for example, at least 40,50,80,100,200,500 Nucleotide or more).

Catalytic RNA molecule or ribozyme also can be used for suppressing to express.Ribozyme can be designed to match with any target RNA-specific basically, and, functionally make described target spot RNA inactivation thus at specific position cracking phosphodiester backbone.Comprise that in ribozyme ribozyme sequence gives their RNA lytic activities, increase their inhibition activity thus.The method of design and application target spot RNA specific ribozyme is known to those skilled in the art.Referring to, usually, WO 02/46449 and its reference of quoting.

Can also use the method for disturbing (RNAi) based on RNA.It is the cell mechanism of a kind of regulate gene expression and virus replication that RNA disturbs.This mechanism is mediated by double-chain small disturbance RNA molecule (siRNA).All inherent mRNA that cell contains the sequence identical with siRNA by destruction reply introduce described cell external double-stranded RNA (for example, siRNA).

It is believed that RNAi comprises initial sum effector step.In initial step, the dsRNA of input is digested the siRNA s (siRNAs) of 21-23 Nucleotide, and it also is called " guide RNA s ".As enzyme Dicer, a member of dsRNA specific ribonucleic acid enzyme RNase III family progressively during cracking dsRNA (for example, directly introduce or by transgenosis or virus), produces described siRNAs in the progressive mode of ATP dependent form.Successive cracking incident is degraded into the duplex (siRNAs) of 19-21bp with RNA, and each has 2-Nucleotide 3 ' overhang.In the effector step, described siRNA duplex bind nucleic acid enzyme complex is with the reticent mixture of formation RNA-inductive, or RISC.It is that activation RISC is necessary that the ATP dependent form of SiRNA duplex is launched.RISC is by base pairing interaction target homeodomain transcription thing for activity, and from 3 of siRNA ' is terminal about 12 Nucleotide is fallen in the mRNA cracking.

The method of the siRNAs of design and preparation target target mRNA is known to those skilled in the art; Referring to, for example, WO 99/32619 and WO 01/75164.In a kind of method of design, begin to carry out AA dinucleotides sequence scanning from target transcript AUG initiator codon.19 Nucleotide of every kind of AA sequence and 3 ' close on are recorded as potential siRNA target spot site.Can select 2 kinds or 4 kinds of sequences then, partly based on following standard:

1) siRNAs with 30-50%GC content more is added with activity than the siRNAs that those have higher G/C content;

2) because 4-6 Nucleotide many (T) act as the termination signal of RNA pol III, so, in target sequence, should avoid more than the fragment of 4 T ' s or A ' s when designing will be from the sequence of RNA pol III promoter expression the time;

3) because some zones of mRNA can be made of or combination the modulin height, it can be effective to selecting siRNA target spot site along the different positions of described full length gene; And

4) it can be effective to comparison potential target spot site and suitable genome database (people, mouse, rat, etc.), and get rid of and consider to have any target sequence more than the homology of 16-17 adjacent base pair with other non-purpose encoding sequence.

In some embodiments, the RNA interfering construct comprises the sequence that is transcribed into the double-stranded RNA with stem-ring structure.A chain of double-stranded RNA stem portion comprises with the similar or identical sequence of the adopted encoding sequence of having of desired polypeptides, and that is to about 2,500 Nucleotide from about 10 Nucleotide on length.Can be with the length that the similar or identical sequence of adopted encoding sequence is arranged from 10 Nucleotide to 500 Nucleotide, from 15 Nucleotide or 300 Nucleotide, from 20 Nucleotide to 100 Nucleotide, or from 25 Nucleotide to 100 Nucleotide.Another chain of double-stranded RNA stem portion comprises the antisense sequences of desired polypeptides, and can have than described and have the length of adopted sequence correspondence shorter, identical, or longer length.The loop section of double-stranded RNA can be from 10 Nucleotide to 5,000 Nucleotide, for example, and from 15 Nucleotide to 1,000 Nucleotide, from 20 Nucleotide to 500 Nucleotide, or from 25 Nucleotide to 200 Nucleotide.The loop section of RNA can comprise intron. Referring to, for example, WO 99/53050.

Then, can applied chemistry synthesize, in-vitro transcription, siRNA expression vector and PCR expression cassette prepare designed siRNA.

Goods

The present invention also provides goods.Goods can comprise transgenic plant described herein, for example, and the transgenic plant in container, sack, jar or cultivation vessel.Goods can comprise or many seeds of transgenic plant described herein.Typically, the seed mixture of homogeneous is nursed one's health and the wrapping material of packing into by manner known in the art basically, to form goods.One bag of seed so preferably has the packaging label of following sack, for example, ties up to mark or label on the wrapping material, is printed on the label on the wrapping material, or is inserted in the label in the bag.Packaging label can indicate the plant that grows up to from described seed and be suitable for producing the specified polypeptide of selecting in advance.Packaging label can also indicate wherein contained seed in conjunction with providing needed phenotypic characteristic, the transgenosis of as discussed above those.

Embodiment

Embodiment 1-p326:DWF4 expresses the analysis to plant-growth and growth in paddy rice

With Ti-plasmids, the UASHap1 (Hap1 upstream activating sequence) that it contains specified p326 upstream and is connected to the promotor of Hap1 encoding sequence effectively and is connected to green fluorescent protein (GFP) encoding sequence effectively is incorporated among the rice breeding Kitaake by the corpus callosum of using Agrobacterium and can transform.The expression of Hap1 encoding sequence causes the accumulation of Hap1 albumen and GFP.GFP can detected expression be limited in observing expression still less in the stem and blade of rice plants in root tissue.Do not detect expression in the seed before root meristem, flower or rudiment.To be called CRS-BIN1A7 from this transformation event deutero-strain system.

Also introduce Ti-plasmids in Kitaake, it contains the UAS of 5 copies _Hap1Upstream and being connected to effectively on the genome encoding sequence of Arabidopis thaliana 22-α hydroxylase (DWF4).Described gDNA is from environmental WS.The activation of UAS (passing through Hap1) causes the accumulation with the DWF4 transcript of transcribing of DWF4 gDNA.

To be derived from independently

transgenic line

17 and 36 and be called 17-3, the T2 UAS:DWF4 strain system CRS-BIN1A7 plant pollination of 36-5 and 36-6, with generation F1 for seed.With 17-3,3 groups of F1 of 36-5 and 36-6 offspring are called R150 for plant, R149 and R147.By the existence of fluoroscopic examination F1, and detect the existence of UAS:DWF4 by PCR for plant Hap1:GFP.The existence of DWF4 gDNA transcript confirms by RT-PCR.To be called R150P5, R150P7, the F1 of R149P2 and R149P5 for the F2 of plant for seed germination, and with negative control growth side by side in 5 jars, as shown in table 1.

Table 1.

F1 is for abbreviation	p326：Hap1	UAS：DWF4	Logarithm
F1 is for abbreviation	p326：Hap1	UAS：DWF4	Logarithm	?R150P5 ?R150P2	Exist	Exist	1
				?R150P5 ?R150P2	Exist	Exist	1
				?R150P7 ?R150P6	Exist	Exist	2
				?R150P7 ?R150P6	Exist	Exist	2
				?R149P2 ?R149P6	Exist	Exist	3
				?R149P2 ?R149P6	Exist	Exist	3
				?R147P5 ?R147P2	Exist	Exist	4

The pairing of table 1. phenotype plant.Every pair contains F1 that a strain contains p326:Hap1 and UAS:DWF4 and contains the control plant of p326:Hap1 or UAS:DWF4, growth in same jar for a plant and a strain.

Blade length, width of blade, panel length, the thickness of stem, paniculiform thickness, the branch number, the mensuration of number seeds and seed weight is used for assessing the effect of enhanced DWF4 activity to growth, growth and output.The mensuration of blade shows, the F1 that contains p326:Hap1 and UAS:DWF4 and enhanced DWF4 for plant in, sheath and blade comparison are long by about 15% according to the blade of plant, and therefore the length between first segment increased significantly.The width of blade can also be reduced to a certain extent, although the variation of data makes any difference not remarkable.The compound action that these DWF4 inductive change is in height approximately to increase by 15%, and makes blade grow thickly to the plant top a little to the effect of panel length.On the blade texture, there is not obvious variation.Yet, containing p326:Hap1 and UAS:DWF4 and showing in the active plant of enhanced DWF4, blade angle has increased.When independent growth, the F1 of the DWF4 transcript that shows enhanced level for plant in, the length of sheath and blade has also increased, and the blade of these plants is darker green.

Contain p326:Hap1 and UAS:DWF4 and show number (tillering for about 11) that the active F1 of enhanced DWF4 tillers on for plant with only contain p326:Hap1 or only contain the tillering number of control plant of UAS:DWF4 identical.Yet, demonstrate stable difference about the mensuration of some parameters of seed production.Contain p326:Hap1 and UAS:DWF4 and show the active F1 of enhanced DWF4 for plant in, each tiller and the F2 of every strain plant for the seed weight of number seeds and every strain plant, all increased 20-30%.And the weight of seed has increased: from the F2 that contains p326:Hap1 and UAS:DWF4 and show the active plant of enhanced DWF4 for the seed comparison according to plant heavily about 10%.

The 95th day (when results) carried out after being determined at of tillering number and number seeds was transplanted to soil.For the mensuration of seed weight, seed was collected after being transplanted to soil on the 95th day, and 37 ℃ of dryings 14 days.All plants carry out about 11 and tiller: tillering number does not change with enhanced DWF4 is active.Yet the number seeds of the number seeds that each is tillered and every strain plant has increased 20-30%, and the weight of every seed increases about 10%.The combination of these effects is that the increase (output) of every strain plant seed weight reaches about 30%.At each seed of tillering, there is the difference greater than 1 standard deviation in the seed of every strain plant in the seed weight of the seed weight of every seed and every strain plant.The expression of described digital proof dicotyledons 22-α hydroxylase in monocotyledons causes heavier seed.

To weigh for seed to 100 F2 that select at random from each of 4 pairs of identical plants, and from the heaviest to the lightest displaying seed weight.Described data show, being increased in minimum about 30% the seed of seed weight is the most outstanding.Have 3 to (2-4 to) 4 centerings, with respect to the control plant of growth in same jar, in the lightest about 30% the seed from the plant that contains the DWF4 that improves the standard, the seed weight increase reaches about 40%.At remaining in about 70%, seed weight increase amount still less.Have 1 to (the 1st pair) 4 centerings, seed weight has all increased in the seed of all kinds equably.These results show that when expressed on growth property ground in monocotyledons, 22-α hydroxylase was given the seed circularity of homogeneous more.

The width of stem foot and the width of panicle base have also been measured.With respect to only containing p326:Hap1 or only containing the control plant of UAS:DWF4, in the plant that contains p326:Hap1 and UAS:DWF4, it is about 30% that the width increase of stem reaches, and paniculiform width increase reaches about 20%.Therefore, the expression of DWF4 in paddy rice causes the increase of plant weight and seed production, and the increase on the thickness of axle is enough to support the plant weight and the seed production that increase.Visual observations shows that described plant is not inclined to lodging.

These data show that the vegetalitas of 22-α hydroxylase in monocotyledons is expressed, and when not having the expression of hydroxylase in the seed of inflorescence and growth, provides the seed production of increase.

Embodiment 2-p326:DWF4 expresses the analysis to metabolism in paddy rice

The UAS:DWF4 strain is DWF4-BIN1B16-2, and 17-3 and 27-3 are derived from independently transgenic line 16,17 and 27 respectively, use the CRS-BIN1A7 plant pollination, to produce F1 for seed.3 groups of F1 are called R148 for plant, and R150 and R151 by the existence of fluoroscopic examination Hap1:GFP, and detect the existence of UAS:DWF4 by PCR.Determine the existence of DWF4gDNA transcript by RT-PCR.The R148P5 that calls oneself in the future, the F1 of R150P7 and R151P1 for the F2 of plant for seed germination, and growth in darkness 3 days, and detect the existence of p326:Hap1 and UAS:DWF4 then.With 5 couples of GFP (+)/DWF4 (+) and GFP (+)/DWF4 (-) chorista, every pair from same strain F1 for plant, and come in 5 jars and grow, as shown in the table 2.

Table 2.

T2 is for abbreviation	Hap1：GFP	UAS：DWF4	Logarithm
T2 is for abbreviation	Hap1：GFP	UAS：DWF4	Logarithm	R148P5-13 R148P5-12	Exist	Not existence	1
				R148P5-13 R148P5-12	Exist	Not existence	1
				R148P5-14 R148P5-15	Exist	Not existence	2
				R148P5-14 R148P5-15	Exist	Not existence	2

?R150P7-14 ?R150P7-19	Exist	Not existence	3
?R150P7-14 ?R150P7-19	Exist	Not existence	3
?R150P7-18 ?R150P7-20	Exist	Not existence	4
?R150P7-18 ?R150P7-20	Exist	Not existence	4
?R151P1-41 ?R151P1-40	Exist	Do not exist	5

The pairing of table 2. plant.GFP (+)/DWF4 (+) and GFP (+)/DWF4 (-) F2 grows side by side for chorista.Hap1:GFP represents that with UAS:DWF4 2-becomes two elements of sub-system.

F2 grows in the greenhouse for plant.Begin to bloom about 12 days of back and in another boot leaf 5 minutes (between 5:00pm and the 5:05pm) collect the boot leaf of every strain plant, freezing on dry ice, and-80 ℃ of preservations.For chemical analysis, with these blade freeze-drying, with methyl alcohol and dichloromethane extraction, and in the gas chromatography-mass spectrum of deriving (GC-MS) layering before in polarity and nonpolar phase.Extraction is carried out 2 times and 3 times, is used for the repeat samples that GC-MS analyzes with generation.The amount that is used for the freeze-drying leaf tissue of each extraction is displayed in Table 3.

Table 3.

The T2 abbreviation	?1	?2	?3
The T2 abbreviation	?1	?2	?3	R148P5?13	?28.9	?29.1	?30.1
R148P5?12	?29.8	?29.7	?32.0	R148P5?13	?28.9	?29.1	?30.1
R148P5?12	?29.8	?29.7	?32.0
R148P5?14	?29.9	?28.6	?-
R148P5?14	?29.9	?28.6	?-	R148P5?15	?29.5	?29.2	?22.7
				R148P5?15	?29.5	?29.2	?22.7
				R150P7?14	?29.3	?30.0	?29.6
R150P7?19	?28.6	?30.2	?30.0	R150P7?14	?29.3	?30.0	?29.6
R150P7?19	?28.6	?30.2	?30.0
R150P7?18	?31.2	?29.6	?30.4

?R150P7?20	?29.7	?30.0	?27.4
?R150P7?20	?29.7	?30.0	?27.4
?R151P1?41	?28.9	?27.4	?28.5
?R151P1?41	?28.9	?27.4	?28.5	?R151P1?40	?29.5	?19.2	?-

Table 3. is used for the sample that MxP analyzes.The T2 abbreviation is meant each sheet of 10 boot leaves that are used for the GC-MS analysis.Hurdle ' 1 ', ' 2 ' and ' 3 ' show to be used for the amount of the freeze-drying leaf tissue of extraction at every turn, the mg of unit.For two boot leaves (R148P5 14 and R151P1 40), has only the tissue of enough twice extractions.

Data gathering and processing comprise the visible inspection of stratographic and compare, comprise principle component analysis (principal component analysis, PCA) and graduation classification analysis (hierarchicalclustering analysis, multivariate analysis HCA), and experiment/check analysis.Described data gathering and processing are used for determining the metabolite ratio.Analyzed 82 kinds of compounds, comprised amino acid, carbohydrate, lipid acid and organic acid.The compound of being analyzed is displayed in Table 4.

Table 4.

The L-L-Ala	Fumaric acid	Xylitol/arabitol
The L-L-Ala	Fumaric acid	Xylitol/arabitol	Glycine	Succsinic acid	N.F,USP MANNITOL
The L-Xie Ansuan	Citromalic acid	Inositol	Glycine	Succsinic acid	N.F,USP MANNITOL
The L-Xie Ansuan	Citromalic acid	Inositol	The L-leucine	Oxysuccinic acid	Maltose alcohol
The L-Isoleucine	2 hydroxybenzoic acid	Undecanoic acid	The L-leucine	Oxysuccinic acid	Maltose alcohol
The L-Isoleucine	2 hydroxybenzoic acid	Undecanoic acid	The L-Serine	Ribonic acid-g-lactone 1	Sad ME (C8:0)
The L-proline(Pro)	α-ketogluconic acid	Sad ME (C10:0)	The L-Serine	Ribonic acid-g-lactone 1	Sad ME (C8:0)
The L-proline(Pro)	α-ketogluconic acid	Sad ME (C10:0)	The L-Threonine	Quininic acid	Lauric acid ME (C12:0)
Homoserine	Shikimic acid	Tetradecanoic acid ME (C14:0)	The L-Threonine	Quininic acid	Lauric acid ME (C12:0)
Homoserine	Shikimic acid	Tetradecanoic acid ME (C14:0)	Trans-the 4-L-oxyproline	Citric acid	Palmitinic acid ME (C16:0)
The L-aspartic acid	Isocitric acid	Stearic acid ME (C18:0)	Trans-the 4-L-oxyproline	Citric acid	Palmitinic acid ME (C16:0)
The L-aspartic acid	Isocitric acid	Stearic acid ME (C18:0)	The L-methionine(Met)	3-phoshoglyceric acid	Oleic acid ME (C18:1)
The L-halfcystine	Glyconic acid	Linolic acid ME (C18:2)	The L-methionine(Met)	3-phoshoglyceric acid	Oleic acid ME (C18:1)
The L-halfcystine	Glyconic acid	Linolic acid ME (C18:2)	L-L-glutamic acid	Wood sugar/pectinose	Linolic acid ME (C18:3)
L-glutaminate	Fucose 1	Mountain Yu acid ME (C22:0)	L-L-glutamic acid	Wood sugar/pectinose	Linolic acid ME (C18:3)

The L-phenylalanine	Fructose	1	Lignoceric acid ME (C24:0)
The L-phenylalanine	Fructose	1	Lignoceric acid ME (C24:0)	Altheine	N.F,USP MANNITOL	The 1-tetradecanol
The L-ornithine	Semi-lactosi 1	Cetyl alcohol		Altheine	N.F,USP MANNITOL	The 1-tetradecanol
The L-ornithine	Semi-lactosi 1	Cetyl alcohol	L-Methionin	Glucose	1	The 1-Stearyl alcohol
The L-Histidine	Sucrose	The 1-V-1326	L-Methionin	Glucose	1	The 1-Stearyl alcohol
The L-Histidine	Sucrose	The 1-V-1326	The L-tryptophane	Maltose	1	The 1-policosanol
DL-lactic acid	Trehalose	The 1-triacontanol	The L-tryptophane	Maltose	1	The 1-policosanol
DL-lactic acid	Trehalose	The 1-triacontanol	Oxyacetic acid	Isomaltose	1	Squalene
Pyruvic acid	Raffinose	Cholesterol	Oxyacetic acid	Isomaltose	1	Squalene
Pyruvic acid	Raffinose	Cholesterol	Oxalic acid	Gycerol	Stigmasterol
Phosphoric acid	Tetrahydroxybutane	Sitosterol	Oxalic acid	Gycerol	Stigmasterol
Phosphoric acid	Tetrahydroxybutane	Sitosterol	R-Glyceric acid	Ribitol ISTD	Campesterol
Phenylformic acid			R-Glyceric acid	Ribitol ISTD	Campesterol

The compound that table 4. is analyzed by GC-MS (MxP).For lipid acid, described ratio is meant carbon atom number and unsaturated link(age) number.

The result of metabolism pattern analysis shows, in 5 strains contain the plant of p326:Hap1 and UAS:DWF4 (GFP (+)/DWF4 (+) plant), 2 strains are arranged, with respect to the contrast that only contains p326:Hap1 (GFP (+)/DWF4 (-) plant), in 5 strains contain the plant of p326:Hap1 and UAS:DWF4 (GFP (+)/DWF4 (+) plant) 2 strains are arranged, the level of its free sucrose has increased about 20%.Sucrose concentration in all the other 3 strain plants also is higher than the concentration in the contrast, although described increase is not a significance,statistical.

Described result shows that also with respect to contrast, in the boot leaf of all 5 strain F2 for plant, L-glutamic acid and linoleic concentration have increased.Being increased between about 15% and about 65% of L-glutamic acid.Linoleic being increased between about 18% and about 40%.Other amino acid, carbohydrate, other lipid acid and organic acid concentration are significantly not different with respect to contrast.

Embodiment 3-DWF4 antisense

Experimentize, wherein the CaMV35S promotor is connected on the Arabidopis thaliana DWF4 antisense polynucleotide effectively.Described construct is introduced Arabidopis thaliana, and find with respect to the corresponding plant that lacks described construct, thus obtained transgenic plant show to prolong under shady and cool condition and are suppressed.

Embodiment 4-uses 35S promoter, expresses corn 22-α hydroxylase in the Kitaake paddy rice

Preparation has the construct that effectively is connected to the nucleic acid of coding SEQ ID NO:2 on the CaMV35S promotor, and is introduced into the Kitaake rice plants.With respect to the control plant that lacks described construct, T2 shows the height of increase for plant.

Embodiment 5-uses p326 or 35S promoter, expresses Indica Rice 22-α hydroxylase in Arabidopis thaliana

Evaluation is from the cDNA clone (SEQID NO:16) of Indica rice, and the aminoacid sequence of its aminoacid sequence and Arabidopis thaliana DWF4 has 69% homology.This clone is called OsDWF4.Use that p326 and 35S promoter express in Arabidopis thaliana that OsDWF4 causes and when p326:DWF4 and 35S:DWF4 observed those similar a series of phenotypes during expression in Arabidopis thaliana respectively.These phenotypes comprise the prolongation of hypocotyl, petiole and inflorescence.In addition, OsDWF4 is introduced in the Arabidopis thaliana semidwarf.The semidwarf phenotype of these plants is because the expression of DWF4 antisense sequences (DWF4a/s) causes.Described semidwarf phenotype has been proofreaied and correct in the expression of OsDWF4 in such plant, and this shows that DWF4 and OsDWF4 are functional directly to homologue.

The evaluation of DWF4 homologue

The BLAST Analysis and Identification of Arabidopis thaliana DWF4 sequence (Genbank AF044216) goes out Japonica paddy rice clone, Genbank AC 104473 (locus id AAN60994).Amino acid levels Japonica clone and Arabidopis thaliana DWF4 (Genbank AF044216) clone 69% homology is arranged.Find that Indica paddy rice SEQ ID NO:16 contains in the position 539 C-T with respect to Japonica paddy rice clone GenbankAC104473 and replaces.The replacement of position 539 causes the leucine of position 180 in the Indica sequence.

Arabidopis thaliana DWF4 aminoacid sequence and Indica paddy rice aminoacid sequence have in 3 primary structure territories (structural domain A, structural domain B and heme-binding domain)＞85% homology, but in film grappling territory 21% homology is arranged, as shown in the table 5.

Table 5.

Whole protein		Grasp	Hinge	A	B	C	Protoheme
Whole protein								Identical	Similar
69.00％	80.00％							Identical	Similar	21.00％	71.40％	94.10％	92.30％	76.90％	88.20％

Table 5. amino acid sequence homology.Described numeral is described the sequence homology between DWF4 and the OsDWF4.Structural domain A is O ₂-in conjunction with the territory.Structural domain B is that steroid is in conjunction with the territory.Domain C has unknown function.Structural domain A provides important function by C and heme-binding domain for DWF4 and is high conservative.Grasp and land are more very not conservative.

Transform and transgenic plant strain system

The DWF4 encoding sequence is connected in p326 promotor and the introducing Ti-plasmids carrier effectively.(SEQ ID NO:16) is connected on p326 and the 35S promoter effectively with the OsDWF4 sequence, and each construct is introduced in the Ti-plasmids carrier.P326 gives strong, the wide expression of the most cells that spreads all over except apical meristem and flower cell and tissue, at root lower a little expression is arranged.35S promoter is given constitutive expression basically.The Ti carrier contains the selectable mark of Basta  resistance.

Use the flower infiltration, construct is introduced Arabidopis thaliana WS plant.SR01370 strain system contains p326:DWF4 and SR01390 and SR01392 strain system and contains p35S:OsDWF4.Contain Herbicid resistant selective marker and the genetically modified T2 of DWF4 for plant by weedicide being coated onto on the blade and identifying respectively by PCR.The T2 that contains single T-DNA insertion identifies by the research segregation ratio for chorista.

Table 6.

Strain is a title	Construct
Strain is a title	Construct	?SR01370	?p326：DWF4
?SR01334	?p326：OsDWF4	?SR01370	?p326：DWF4
?SR01334	?p326：OsDWF4	?SR01390	?p35S：OsDWF4
?SR01392	?p35S：OsDWF4	?SR01390	?p35S：OsDWF4
?SR01392	?p35S：OsDWF4	?SR01219	?p35S：DWF4a/s
?SR01557	P35S:DWF4a/s and	?SR01219	?p35S：DWF4a/s

	?p326：OsDWF4
	?p326：OsDWF4	?SR01159	?YP0009：DWF4
?SR01130	?YP0104：DWF4	?SR01159	?YP0009：DWF4
?SR01130	?YP0104：DWF4	?SR01187	?YP0126：DWF4
?BinD?1-11	?p13879：DWF4	?SR01187	?YP0126：DWF4
?BinD?1-11	?p13879：DWF4	?SR?1029-6	?P13879：ANT

Phenotype analytical

SR01370, the phenotype of SR01334 and SR01390/SR01392 plant can be observed for vision at T1.For the strain system that demonstrates possible phenotypic difference, long by measuring root on the 4th day in rudiment (DAG) back, with measure hypocotyl length, rosette diameter, plant height, silique length and weight DAG the 13rd day, every strain T1 female parent at least 18 strain T2 plants are analyzed.T2 does not have the wild-type that transforms and other T1 strain system of containing other uncorrelated cDNAs with comparing for wild chorista.By using Leica TCS SP2 scan laser Laser Scanning Confocal Microscope observation of cell size, and the chlorophyll autofluorescence that sends from top 15cm fluorescence stalk cell of imaging.

The T1 phenotype

DAG the 34th day, be that 3 strain 6SR01370 strains system shows the petiole of prolongation and curling a little blade with respect to other T1 that contains other cDNAs for strain.These phenotypes are features of DWF4gDNA phenotype, and this shows that the p326:DWF4 transgenosis also influences the brassinolide level.DAG the 20th and 40 days, similar phenotype was tangible in 18 strains of 10 strains in the SR01334 strain system and 20 strain SR01390 strains system independently in 10 strains respectively.

The T2 phenotype

P326 and 35S express in the Arabidopis thaliana rice shoot consumingly.When the same phase of growing is compared with unconverted wild-type rice shoot, DWF4 and OsDWF4 transgenosis cause the hypocotyl that prolongs in for rice shoot at T2.DAG the 13rd day, every strain is that the mensuration of hypocotyl length in the 10 strain plants shows, T2 is for SR01370-2 and SR01370-5, SR01334-2 and SR01334-4, with the SR01390-7/SR01392-5 hypocotyl be unconverted wild-type hypocotylar twice at the most and t-check show for all strains to be that variable is significant on 0.05 level.More not remarkable to hypocotylar effect comparison SR01334-2 and SR01334-4 to SR01370-2 and SR01370-5, this shows that allos OsDWF4 transgenosis has stronger effect to the hypocotyl growth than Arabidopis thaliana DWF4 gene itself.

For in the measurement of growing late period, every strain T1 generation 18 strain T2 plants are grown in the greenhouse, and chorista is carried out gene type assay by using PCR.DAG the 21st day, contain p326:DWF4 (SR01370-1 and SR01370-2) at all, p326:OsDWF4 (SR01334-2 and SR01334-4), and the T2 of 35S:OsDWF4 (SR01390-7/SR01392-5) is for the blade of observing the petiole of prolongation and curling a little in the strain system.Measurement shows, behind the bolting 4 days, on diameter, contains every kind of genetically modified T2 and goes out＞7% for plant than wild-type chorista is bigger.Si Shi t-check analysis shows that for some strain systems, variation is significant (P on 0.05 level _1334-2=0.0467, P _1334-4=0.063, P _1370-1=0.221, P _1370-2=0.0022, P _1390-7=0.064, P _1392-5=0.0053).Although T2 is the population mixture that contains homozygote and heterozygote for colony, the diameter that the DWF4 transgenic plant increase can make a distinction from non-transgenic plant.

T2 is also higher than wild-type for plant, as shown in FIG. 7.Measurement shows, DAG the 30th day, T2 was for SR01370-1 and SR01370-2, SR01334-2 and SR01334-4 and SR01390-7/SR01392-5 than wild-type chorista higher～9% (Fig. 7 A).Si Shi t-check analysis shows that for some strain system, variation is significant (P on 0.05 level _1334-4=0.0008, P _1370-2=0.001, P _1390-7=0.0414, P _1392-5=0.00027).Consider that these measurements are that population mixture from homozygote and heterozygote carries out, being increased in of plant height may be more significant in the homozygote.The T2 that contains DWF4 or OsDWF4 shows for the confocal microscopy of the stem of plant, from the cell of the cortex of shoot apex 15cm than wild-type contrast bigger (data not shown).

In Arabidopis thaliana silique tissue, endogenous DWF4 transcript and brassinolide itself are accumulated to high level.35S and p326 promotor also cause strongly expressed in the wall of silique, therefore check the silique of transgenic plant, whether there are some adjections in the silique growth to observe.DAG the 33rd day, be that 10 strain T2 are collected in the silique of the 5th joint of elementary inflorescence substrate for each strain of plant from every strain, and measure and weigh.SR0137Q-1 and SR01370-2, SR01334-2 and SR01334-2, and the silique of SR01390-5 and SR01392-5 plant is different from the wild-type chorista those on weight.Yet the silique of silique wall wild-type contrast that contains the genetically modified plant of each DWF4 is nearly～20% longer and narrower significantly, and the both is at the 5th joint of elementary inflorescence and in elementary and other place secondary inflorescence.Described result shows, p326:DWF4, and p326:OsDWF4 and the 35S:OsDWF4 expression in Arabidopis thaliana causes the prolongation of silique.

The gene complementation of DWF4a/s

Determine 1.035 kb DWF4 antisense sequences (SEQ IDNO:5), it is corresponding to grappling and the twisting district of DWF4 cDNA, rather than structural domain A, structural domain B, domain C or heme-binding domain.This sequence is made up of 67% of DWF4 cDNA.On nucleotide level, described 1.035kb sequence has the sequence homology with OsDWF4 39.6%, and through the unlikely antisense performance function as OsDWF4 of design.In Ti-plasmids, the DWF4 sequence is connected on the 35S promoter effectively with antisense orientation (35S:DWF4a/s).

The Ti-plasmids that will contain 35S:DWF4a/s is introduced Arabidopis thaliana, produces SR01219 plant strain system.Referring to table 6.T2 is that SR01219-24 carries out gene type assay by PCR for strain, and shows and contain single T-DNA.T1 and T2 show the length that reduces for the SR01219 plant, but are not short lifes.

Gene complementation is undertaken by OsDWF4 is introduced in the DWF4a/s background.The T3 that single T-DNA is inserted transforms with p326:OsDWF4 once more for 35S:DWF4a/s plant homozygote (SR01219-24-11), produces SR01557 strain system.Use the existence of PCR confirmation p326:OsDWF4, it uses the PCR primer that the sequence of p326 sequence and OsDWF4 cDNA sequence is crossed in amplification.

Screening shows the hypocotylar T1 of prolongation for the SR01557 rice shoot under white light, long day (LD) condition.Such rice shoot, and some more common short hypocotyl rice shoots are transferred in the jar, are used for gene type assay and phenotype analytical.When these plants reached bow structure (rosette) stage (DAG the 23rd day), they showed prolongation and blade that curl a little, perhaps are wild-type in appearance.These data show that the semidwarf phenotype relevant with the SR01219-24-11 plant proofreaied and correct by OsDWF4, and the hypocotyl hinge handle that prolongs is the result of the OsDWF4 that improves the standard.

DAG the 10th day, use RT-PCR and check endogenous DWF4 transcript to close the expression of external source OsDWF4 transcript in the SR01557 plant.For RT-PCR,, collect RNA from bion DAG the 32nd day.For qRT-PCR, collect RNA from 200 strain rice shoots DAG the 10th day.Contain DWF4, the plasmid of CPD and OsDWF4 sequence is as the contrast of these experiments.

RT-PCR shows that when comparing with unconverted wild-type transcript, all 5 strain T1 are for DWF4a/s, and the OsDWF4 plant contains low-level DWF4 transcript.QRT-PCR shows that these endogenous DWF4 transcript levels reduce＞50%, shows that described 1.035 kb DWF4a/s partly reduce endogenous DWF4 level effectively.RT-PCR shows, SR01557-4, and SR01557-7 and SR01557-12 plant are with high level expression OsDWF4, and SR01557-2 and SR01557-3 do not have.T1 causes the OsDWF4 phenotype for SR01557-4 and SR01557-7 plant, and T1 does not have for SR01557-2 and SR01557-3 plant, and this shows that the OsDWF4 transcript is responsible for gauged phenotype.PCR result also shows the primer of the DWF4 that is used for increasing do not increase CPD or OsDWF4, and the primer of the OsDWF4 that is used for increasing do not increase DWF4 and CPD, and this shows that described PCR primer is special for their transcripts separately.

These results show that Arabidopis thaliana DWF4 encoding sequence shows 22 α-hydroxylase activity in paddy rice, and can work in the plain steroid biosynthesizing of rape, for example, form the plain sterone of 6-deoxidation rape by catalysis from the substrate campesterol.Described result shows that also the paddy rice encoding sequence shows 22 α-hydroxylase activity in Arabidopis thaliana, and can work in the plain steroid biosynthesizing of rape, for example, and by using the formation of campesterol as substrate and the plain sterone of catalysis 6-deoxidation rape.Consider that together these results show that dicotyledons 22 α-the hydroxylase activity polypeptide can be used, and vice versa in monocotyledons.At last, these results show that also these hydroxylase encoding sequences show sufficient enzymatic activity, and with the plain sterone of the high-caliber 6-deoxidation rape of generation in plant, and they are functional directly to homologue.

Embodiment 6-assesses YP0009, YP0104 and YP0126 promotor

Transform and transgenic plant strain system

When as the HAP1 syzygy, be called YP0009, the promotor of YP0104 and YP0126 stimulates the mainly expression in root, stem and leaf of UAS:GFP respectively.With YP0009, YP0104 and YP0126 promotor are connected on the DWF4gDNA in Ti-plasmids carrier (CRS-BIN1A) effectively.

Use the flower infiltration, construct is introduced the environmental Ws plant of Arabidopis thaliana.YP0009:DWF4 is contained in SR01159 strain system, and YP0104:DWF4 contains in SR01130 strain system and YP0126:DWF4 is contained in SR01187 strain system.Referring to table 6.Contain the T2 that single T-DNA inserts and obtain identifying, and be used for T2 and represent type analysis for chorista.Corresponding is that homozygous T3 also obtains identifying for plant for single insertion, and as qRT-PCR (for the promotor that sufficient tissue is provided) and phenotype analytical.

Phenotype analytical

Write down the T1 representative type of inferring, and the every T1-2 of each construct is carried out phenotype analytical for incident 18 strain T2 for plant.For showing the clearly strain system of phenotype, also every T2 generation 10 strain T3 are carried out phenotype analytical for plant.The wild-type chorista is with comparing.Bow structure size, plant height, branch number, gas life organize dry weight and seed weight to be used for assessing the genetically modified effect of DWF4.

The expression of DWF4 in stem

In Arabidopis thaliana, the YP0104 promotor is mainly expressed at the epidermis and the cortex of stem.Detect the existence of YP0104:DWF4 in SR1130 T2 for strain is by PCR, and the plant of test positive is carried out phenotype analytical.Every strain SR1130 strain system, find in the clearly evidence-two strain SR1130 strain system (SR1130-1-3 and 1130-5-6) of the plant height that reduces from T1 to T3 generation every strain all than wild-type chorista shorter～10%, and Si Shi t-check analysis shows, described variation is that significant (T2 is for plant, P on 0.05 level _1130-1-3=0.026, P _1130-5-6=0.038; T3 is for plant, P _1130-1-3=0.038, P _1130-5-6=0.0018).

Contain the T2 of YP0104:DWF4 and T3 and show also that for the inspection of plant with respect to the contrast of wild-type chorista, the density of silique has increased on the elementary inflorescence.When 4 strain T3 determine for the silique number at the tip 16cm place of the elementary inflorescence of plant, find its with respect to SR1136-1-3 contrast increased～11%, with respect to the SR11236-5-6 contrast increased～3%; The t-check analysis shows that in this variation some are significant (P on 0.05 level _1130-1-3=0.011, P _1130-5-6=0.38).As if branch biomass and seed production not influenced by the DWF4 transgenosis.And the YP0104 promotor has activity in the epidermis of stem and cortex, it in leaf or seed, do not have can the measurement level expression.

Expression in the DWF4 counterfoil

In Arabidopis thaliana, the YP0009 promotor is mainly expressed in the cortex of root and center pillar.Check the existence of YP0009:DWF4 in SR1159 T2 for strain is by PCR, and the plant of test positive is carried out phenotype analytical.Although the YP0009 promotor has activity in root, use YP0009 and express DWF4 gDNA does not cause any kind of in root or branch visible phenotypic.

The expression of DWF4 in branch

In Arabidopis thaliana, the YP0126 promotor is mainly expressed in the epidermis of branch and leaf and mesophyll.Check the existence of YP0126:DWF4 in SR1187 T2 for strain is by PCR, and leaf tissue is carried out qRT-PCR to confirm existing of DWF4 transcript; Plant to test positive carries out phenotype analytical.Although the YP0126 promotor has activity in blade, use YP0126 and express DWF4 gDNA less than do not cause the visible phenotype at branch or in root.

The constitutive expression trend of DWF4 produces higher plant.Yet these results show that the YP0104:DWF4 transgenosis causes shorter plant, and shows, express DWF4 by using YP0104 in the epidermis of stem and cortex, and it may reduce the height of stem.

Compare with YP0104, in root, express DWF4, or YP0126 expresses at that time in blade, do not observe phenotype when using YP0009.Therefore can be preferably in plant the promotor of extensive or constitutive expression.

Embodiment 7-assessment promotor YP0216

Transform and transgenic plant strain system

The YP0216 introducing is contained in the Ti-plasmids (CRS-BIN1A) of DWF4 gDNA.When the syzygy of set of applications Hap1, described promotor stimulates UAS:GFP mainly to express at the shoot apex of stem.

Use the flower infiltration, construct is introduced the environmental Ws plant of Arabidopis thaliana.Obtain 10 individual transformation events, form the strain system that is called SR0977.Containing the T2 that single T-DNA inserts by the chorista Analysis and Identification for strain is, and identifies for these insertions it is that homozygous T2 and T3 are for plant and be used for phenotype analytical.

Phenotype analytical

Will be from the plant (SR0977-2 and SR0977-3) of two transformation events, each incident 10 strain T3 is used for qRT-PCR and all phenotype analytical research for plant.The wild-type chorista is grown in contrast for the plant next door at T2 and T3.The measurement of plant height, branch number, branch dry weight and total seed weight is used for assessing the genetically modified effect of DWF4.

In Arabidopis thaliana, the YP0216 promotor is expressed in the epidermis of stem shoot apex and cortex consumingly.Check the existence of YP01216 in SR0977 T2 for strain is by PCR, and check the existence of T3 homozygote transcript by qRT-PCR; T3 to test positive carries out phenotype analytical for plant.When comparing with the wild-type chorista, T3 does not show visible morphology phenotype for plant.

Plant height and branch amount purpose are measured (using T3 for plant), show with the measurement (using T2 and T3) of branch and seed weight for plant, not there are differences between the contrast of transgenic line and wild-type, perhaps any significant difference is twice transgenic event feature one of only.For example, branch weight and total seed weight data show, SR0977-2-10 plant and the difference between their isolating wild-types are significant (for branch weight in 0.05 level the t-check analysis, P=0.03, for seed weight, P=0.001), still in the SR0977-3-5 plant, there is not such difference.Therefore, the variation evidence in SR0977-2 colony although be significant, is not the common characteristic that contains the genetically modified plant of YP0216:DWF4.The measurement of lobe numbers shows between the contrast of transgenic line and wild-type chorista and not there are differences.RT-PCR from the RNA of 1cm place, stem summit shows that the YP0216:DWF4 transgenosis has obtained expression.

The co expression of embodiment 8-DWF4 and ANT

The constitutive expression of ANT in Arabidopis thaliana increased the size of plant.ANT coding AP2-structural domain transcription factor.The constitutive expression of DWF4 in Arabidopis thaliana also increased the size of plant.Two kinds of polynucleotides are expressed in single plant, make up the effect of expressing the plant size with assessment DWF4 and ANT.

Transform and transgenic plant strain system

In all experiments, constitutive promoter 35S and p13879 are used for controlling gene express.Use p13879 expression ANT and cause bigger plant, may continue the splitted cycle by prolonging the cell that forms from meristem, and under the homozygote condition, cause sterile.

DWF4 gDNA is called 7098806 ANT cDNA clone and is called 5 ' ANT disappearance and 35S or the p13879 combined utilization of ANT Δ N3 (ANT/ Δ).Use CRS-BIN1A with in DWF4 and the ANT sequence introduced plant.

Express the F1 of two kinds of genes for plant with carrying the genetically modified parent's hybridization of DWF4 and ANT with generation.Single T-DNA is contained in p13879:DWF4 strain system (BinD 1-11), shown in hereinafter, and is used as T3 for homozygote.P13879:ANT strain system (SR 1029-6) also contains single T-DNA and is used as heterozygote.With single p13879:ANT plant as female with as male this single p13879:DWF4 plant hybridization.

Use the F1 that contains DWF4 and ANT transgenosis that PCR identifies that 5 strains obtain by hybridization for plant, and other 5 strains only contain the genetically modified plant of DWF4.In addition, this hybridizes with the same p13879:ANT as female unconverted wild-type plant as hero.PCR shows that 5 strains only contain the genetically modified F1 of ANT for plant be other 5 strain (wild-type compatriot) of wild-type.Altogether these 20 strains F1 is used for all phenotype tests for plant.

Transform once more

The plant that will contain 35S:ANT Δ and kalamycin resistance NPTII gene uses the T-DNA construct of the Herbicid resistant pat gene that contains p13879:DWF4 and improvement to transform once more.The T1 that selects Herbicid resistant is for plant, and application PCR reconfirms the genetically modified existence of ANT.To be Herbicid resistant and be used for all phenotype for NPTIIPCR male T1 for plant and measure.

Phenotype analytical

Plant-growth is to ripe.With bow structure size, plant height, gas life organize dry weight, branch number, number seeds, the measurement of closing seed weight is used for assessing DWF4 and the genetically modified effect of ANT.

Positive and T1 that oppositely select for p13879:DWF4 shows the mixing of phenotype for plant for plant with for 35S:ANT PCR for p13879:ANT and p13879:DWF4 PCR male F1.Some have the petiole and the blade of prolongation, the single transformant of similar 35S and p13879:DWF4, and other have big bow structure and round leaf, the single transformant of similar p13879:ANT.The F1 that contains p13879:DWF4 and p13879:ANT is in height similar to the control plant of only expressing DWF4 for plant; Yet it is sterile that 2 strains are arranged in the 5 strain plants.

These observations show that F1 has the feature of single transformant of DWF4 and the single transformant of ANT for plant.Yet, between F1 is for plant, have many differences.For example, the branch number changes from 22-57, may be owing to the difference penetrance of DWF4 phenotype, and it is relevant with the branch that increases.As another example, seed weight changes from 0-303mg, may be owing to the difference penetrance of ANT phenotype, and it is with sterile relevant.These results show, have the biomass that reduces with respect to the ANT contrast.Having similar variable similar phenotype also observes in for plant at the T1 that contains p13879:DWF4 and p35S:ANT Δ.

In a word, F1 generation and T1 show the composition of ANT and DWF4 phenotype for plant, and this causes variable phenotype is the mixing of ANT and DWF4 feature rather than their summation.

Embodiment 9-assessment promotor p13879 and p32449

In Arabidopis thaliana, use the transgenic plant that wherein p13879 and p32449 promotor are used for expressing DWF4cDNA, the effect of research brassinolide in stress tolerance.Arabidopis thaliana (Ws) the strain system of containing one of two kinds of DWF4 constructs with for twice of every kind of construct independently transformation event one be used from all experiments.These constructs are p13879:DWF4 (BinD) and p32449:DWF4 (CC2).Promotor p13879 and p32449 express in root and branch, especially express in epidermis, cortex and branch meristematic tissue.All T2 contain single T-DNA for strain system (BinD 1-11-1, BinD 3-9-1, CC2 4-2-3 and CC2 7-2-1) and insert.T2 is carried out progeny testing for plant, identifying T2, and be used in all experiments for homozygote.Do not contain wild-type T2 that T-DNA inserts for chorista with comparing.In order to confirm that described homozygote contains correct transgenosis, use PCR all 4 kinds of strain systems are carried out gene type assay.

Growth conditions and measurement

Planting seed in the jar of 5 * 7-inch, and is being controlled at 22 ℃, is growing in the growth room of 16h illumination/8h dark and 70% relative humidity.Plant is watered weekly 2 times, so that total amount is weekly a 2.5L water.For thermal treatment, the plant in 14 day age to be transferred to be arranged on 36 ℃, 16h illumination/8h dark in the growth room of 70% relative humidity, continued for 3 weeks.For deepfreeze, the plant in 7 day age to be transferred to be arranged on 8 ℃, 16h illumination/8h dark in the growth room of 70% relative humidity, continued for 8 weeks.For drought stress, stopped to water in the 14th day from rice shoot, at 22 ℃, the growth conditions of 16h illumination/8h dark and 70% relative humidity is 2 weeks of growth down.

All p13879:DWF4 and p32449:DWF4 rice shoot all show the narrow of the prolongation of petiole and blade, and these are features of the DWF4 of elevated levels.

Thermal treatment: when comparing, contain the genetically modified T2 of DWF4 and show heat stress enhanced susceptibility for plant with the wild-type chorista.Under 36 ℃ and 70% relative humidity after 18 days, all bleached fully for plant and dead from all T2 of two kinds of p32449:DWF4 strains systems.From the plant of two kinds of p13879:DWF4 strains system nearly half bleach fully and dead.After 21 days, all p13879:DWF4 plants are all dead under these conditions.All wild-type plants keep green and survival.These results show that the p32449:DWF4 plant is more responsive more to heat stress than p13879:DWF4 plant.

Deepfreeze: when comparing, contain the genetically modified T2 of DWF4 and do not show for plant cold is coerced the susceptibility that changes with the wild-type chorista.Under 8 ℃ and 70% relative humidity after 7 weeks, from two kinds independently p32449:DWF4 strain system and two kinds independently p13879 strain all plants and the wild-type chorista that are is as broad as long.

Arid is handled: when comparing, contain the genetically modified T2 of DWF4 and do not show the susceptibility that drought stress is changed for plant with the wild-type chorista.Do not having water and after 2 weeks of growth under 70% relative humidity, from two kinds independently p32449:DWF4 strain system and two kinds independently p13879 strain all plants and the wild-type chorista that are is as broad as long.

In addition, when using isolating bow structure, in water reduction rate, not there are differences.Water reduction rate is that plant is measured the sensitivity of the reaction of arid.

Described result shows that with respect to wild-type, p13879:DWF4 and p32449:DWF4 T2 tie up to not change on the resistance of cold or drought stress for strain, and more responsive more to heat stress than wild-type chorista.As if p32449:DWF4 have bigger effect than p13879:DWF4.Difference between these two kinds of strain systems can be explained by the expression pattern of two kinds of different promoters.Although p13879 and p32449 express in many tissues and organ, the transcribing to induce under hot shock condition of the sequence that connects from p32449-reaches 8 times, and the transcribing of sequence that connects from p13879-do not have thermal induction.Therefore, possibly, be the result of the thermal induction of the sequence that connects of p13879-to the bigger susceptibility of heat stress.

The analysis of BL biosynthetic pathway chemistry intermediate in the embodiment 10-transgenic plant

Use gas chromatography-mass spectrum (GC-MS), measure the level of chemical intermediate in the p326:DWF4 rice shoot BL biosynthetic pathway, and compare with the level in the unconverted wild-type contrast rice shoot.By having strengthened in catalytic p326:DWF4 of being reflected at of DWF4 and the p326:ZmDWF4 rice shoot, this provides number seeds, seed circularity and seed production phenotype is the strong evidence of the direct result of the BL level that increases.

Material and method

P326:DWF4 directly merges strain system

With DWF4 gDNA (SEQ ID NO:14) be called ZmDWF4 (SEQ ID NO:15) homology corn cDNA each is connected to respectively on p326 promotor and the OCS terminator effectively, obtain construct CR24 and CR26.Use agriculture bacillus mediated conversion CR24 and CR26 are transformed in the paddy rice, and obtain homozygote strain system again.The T4 that is CR24-3-6 and CR26-1-6 with 100 strains sterilizes with 20% sodium hypochlorite solution for seed, and with sterile distilled water rinse 4 times, and on the MS substratum of 1/2 concentration of replenishing 1.5% sucrose, germinate.Collect the rice shoot and the unconverted wild-type contrast rice shoot in 17 day age, and in liquid nitrogen, pulverize immediately.With powdered samples freeze-drying 5 days, and transport to RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama 351-0198, the plant function laboratory of Japan (Plant FunctionsLab) is used for gas chromatography-mass spectrum (GC-MS) analysis.The fresh weight of every kind of rice shoot sample and dry weight show in following table 7.

Table 7.

Strain system	Fresh weight	Dry weight
Strain system	Fresh weight	Dry weight	Wild-type	23.2166	?3.0359
CR24-3-6	24.2097	?3.1753	Wild-type	23.2166	?3.0359
CR24-3-6	24.2097	?3.1753	CR26-1-6	28.231	?4.1924

Table 7. is used for the weight of 100 rice shoots of BL analysis.The numeral unit of display is the weight of gram.

The analysis of chemical intermediate in the BL biosynthetic pathway

In order to analyze plain steroid (BR) intermediate of endogenous rape in the BL biosynthetic pathway, with freeze dried sample 250mL methyl alcohol: CHC13 (4: 1v/v) extraction is 2 times, and according to Fujioka etc. (2003) such as (2002) and He by GC-MS with BR intermediate purifying and mensuration.

The result

The freeze dried T3 that will isozygoty for Arabidopis thaliana DWF4 gDNA or for homologous corn cDNA (ZmDWF4) is for rice seedling, and the Shozo Fujioka that delivers at RIKEN from the Equivalent of unconverted wild-type contrast.Described GC-MS data show in following table 8.

Table 8.

Compound	?WT	?CR24-3-6	?CR26-1-6
Compound	?WT	?CR24-3-6	?CR26-1-6	?24MC	?5850	?5140	?6900
?CR	?99500	?84200	?92800	?24MC	?5850	?5140	?6900
?CR	?99500	?84200	?92800	?CN	?2190	?1720	?1340
6-oxo CN	?55.4	?53.6	?67.5	?CN	?2190	?1720	?1340
6-oxo CN	?55.4	?53.6	?67.5
6-deoxidation CT	?0.58	?1.3	?3.36
6-deoxidation CT	?0.58	?1.3	?3.36	6-deoxidation TE	?0.25	?0.18	?0.45
6-deoxidation 3DT	?2.44	?2.16	?5.44	6-deoxidation TE	?0.25	?0.18	?0.45
6-deoxidation 3DT	?2.44	?2.16	?5.44	6-deoxidation TY	?7.61	?69.66	?18.4
6-deoxidation CS	?1.14	?1.04	?1.98	6-deoxidation TY	?7.61	?69.66	?18.4
6-deoxidation CS	?1.14	?1.04	?1.98
?CT	?nd	?nd	?nd
?CT	?nd	?nd	?nd	?TE	?0.05	?0.05	?0.09
?TY	?0.68	?0.66	?1.23	?TE	?0.05	?0.05	?0.09
?TY	?0.68	?0.66	?1.23	?CS	?0.25	?0.27	?0.39
?BL	?nd	?nd	?nd	?CS	?0.25	?0.27	?0.39

Table 8. wild-type, the GC-MS result of CR24-3-6 and CR26-1-6 rice shoot.Described numeral is represented the level of BR approach intermediate with the ng/g fresh weight.CN (campesterol) and 6-deoxidation CT (the plain sterone of 6-deoxidation rape) are respectively substrate and the product of DWF4.

When these GC-MS data were drawn with respect to known BL biosynthetic pathway, with respect to the wild-type contrast, the minimizing of campesterol level directly passed up to the increase of the plain sterone level of 6-deoxidation rape.These results show, are increased by the DWF4 transgenosis in the conversion of C-6 oxidative pathway campesterol in latter stage to the plain sterone of 6-deoxidation rape.

For containing the CR24-3-6 plant that effectively is connected to the p326 on the Arabidopis thaliana DWF4 gDNA, there is the increase greater than 2 times in the plain sterone level of 6-deoxidation rape.At 6-deoxidation tea sterone and 3-dehydrogenation-6-deoxidation tea sterone level small minimizing being arranged also and then, is the increase of 6-deoxidation typhasterol (6-deoxotyphasterol) in described approach.First rate-limiting step of DWF4 catalysis BL biosynthetic pathway, and second of CPD catalysis, therefore described result's proof is in the p326:DWF4 plant, and CPD becomes rate-limiting step.Possibly, between the BR intermediate of CPD downstream, exist rapidly to transform, so that in these subsequent stages, fail to see clearly difference.Yet, can be to comprise feedback and other regulation and control ring, (Hong etc., 2003 as proposed for other BR intermediate level in the BL-deficient mutants; Tanabe etc., 2005).

For the CR26-1-6 plant that contains the p326 that effectively is connected on the ZmDWF4 cDNA, on the plain sterone level of 6-deoxidation rape, have～6 times increase, and increase (with respect to about 2 times of wild-type) is arranged on all downstream BR intermediate concentration.This means in the conversion of C-6 oxidative pathway campesterol in latter stage to the plain sterone of 6-deoxidation rape has also been increased by the ZmDWF4 transgenosis.In any p326:DWF4 or p326:ZmDWF4 plant, do not observe in the early stage variation of C-6 oxidative pathway.

Described result provides direct evidence, and a) allos DWF4 polypeptide can pass through to strengthen single rate-limiting step, in of the conversion of paddy rice C-6 oxidative pathway campesterol in latter stage to the plain sterone of 6-deoxidation rape, and the BL biosynthetic pathway in the stimulating plant.The result of the plain sterone of campesterol and 6-deoxidation rape shows that the BL level has increased in the p326:DWF4 rice plants.

General introduction and discussion

The direct GC-MS of chemical intermediate measures and shows that the T3 that contains the p326 promotor that effectively is connected on the DWF4 gDNA is for rice plants in the BL biosynthetic pathway, and contain and comprise corn DWF4 directly to other rice plants of the construct of homologue, more effective in the conversion of the plain sterone of 6-deoxidation rape at campesterol.This is by the catalytic step of DWF4 in the BL biosynthetic pathway.

The effect of DWF4 is to increase the photosynthesis ability, for example, and as the CO that catches by Rice Flag ₂Measurement like that (vide infra).Promotor p326 can promote to express in source tissue, and the mistake expression of DWF4 gene in these tissues can cause campesterol to plain more efficient conversion of sterone of 6-deoxidation rape and the accumulation of BL in these tissues.BL is not far by transhipment in plant, and the BL level that increases a little local action to stimulate CO ₂Catch conversion with sucrose.This sucrose can be loaded into phloem and be transported to seed, strengthens the circularity of seed and may provide the stimulus of extra Buddhist monk's the unknown to tiller to produce more (data not shown) and more seed.The increase of BL also can act on the blade cell wall, and makes them more can expand under turgor pressure.

Described CR24-3-6 and CR26-1-6 plant show the increase of the plain sterone level of 6-deoxidation rape～2 times to～6 times, and reach in downstream intermediate level～2 times increase.

The assessment of embodiment 11-transgenosis photosynthetic efficiency

General introduction

The T3 that isozygotys that LiCor is used for carrying out carrying direct fusion constructs p326-DWF4 (CR24) measures for the photosynthesis (PS) of the boot leaf on the plant.At two kinds of CO ₂Concentration, 380ppm and 760ppm, transgenic plant PS lead in a kind of strain is remarkable higher than wild-type plant.The PS of another kind of strain system lead tend to respect to contrast higher, but be not that statistics is significantly higher.

Material and method

Contain the direct combination of plant of p326:DWF4

DWF4 gDNA is connected on p326 promotor and the OCS terminator effectively, as among the embodiment 10, obtaining construct CR24.Use agriculture bacillus mediated conversion CR24 is transformed in the paddy rice, and produce homozygous lines.With representative strains is that each the T3 that isozygotys of CR24-3-6 and CR24-5-6 is grown in the greenhouse side by side for plant and unconverted wild-type.In order to adapt to, before LiCor measures, the plant in flowering period is moved on in the Conviron growth room.Growth conditions chamber in Conviron is 28 ℃ of 16h illumination with 25 ℃ of 8h dark.3 strain plants of every kind of transgenic line and contrast are measured with the LiCor instrument.

LiCor measures

Use Li-Cor 6400 (Li-Cor Inc.Lincoln, Nebraska) portable photosynthesizer is determined the photosynthesis gaseous interchange, described Li-Cor 6400 portable photosynthesis system equipment 2/3cm vane room and fixed led light sources (6400-02B) are used the red and blue LEDs of a row.At 2 kinds of different CO ₂Concentration (380 μ l l ^-1With 760 μ l l ^-1) and from 0-2000 μ mol m ^-2s ^-19 kinds of different optical density(OD) of scope are determined the optical response curve of every kind of sample.Mid point in flowering period on boot leaf is measured.Estimate by the length (3cm) that the width with blade multiply by described chamber in indoor blade area, and this variable is used for the calculating of photosynthetic rate.Leaf temperature is controlled at 25 ℃, and the humidity of chamber remains between the 50-55%, and velocity of flow remains on 500 μ mol s ^-1Blade is at 1500 μ mol m ^-2s ^-1Radiation intensity under in the chamber, carry out balance.When speed reaches steady state, follow-up photosynthetic rate is taken the logarithm minimum latency 120s and maximum latency 200s for the optical density(OD) that each provides.

The result

As shown in FIG. 6, at 500 μ mol m ^-2s ^-1-2000 μ mol m ^-2s ^-1Optical density(OD) and 380 ppmCO ₂, transgenic line CR24-3-6 has than the higher PS of non-transgenic contrast and leads.At 2000 μ mol m ^-2s ^-1, for control plant, as if PS leads and has descended, and does not have for the CR24-3-6 plant.At 500-2000 μ mol m ^-2s ^-1With 380ppm CO ₂, it is higher that the PS of CR24-5-6 leads the speed of also comparing photograph all the time.

Also measured CO at 760ppm ₂The PS of concentration leads.At identical optical density(OD) and 760ppmCO ₂, strain is that the PS that the PS of CR24-3-6 and CR24-5-6 leads also all the time than the non-transgenic contrast leads higher (data not shown).These data clearly illustrate that the plant of overexpression C-22 α hydroxylase polypeptide may have the enhanced photosynthetic rate in blade.Seem possibly CO ₂This small part that is enhanced to of fixed is responsible for above-mentioned plant size and seed production phenotype.

The GC-MS that embodiment 12-p326:DWF4 expresses in paddy rice analyzes

Material and material

Promotor and encoding sequence

Corpus callosum by using Agrobacterium and can transforming will contain Hap1 upstream p326 promotor, and UAS _Hap1And the Ti-plasmids (CRS-BIN1A) of the sequence of coding GFP is introduced among the rice cultivating kind Kitaake.The stimulation of promotor causes the accumulation of Hap1 albumen and GFP.The detectable expression of GFP is limited in rice plants root, stem and the leaf.Can not detect expression in the seed before branch meristem, flower and rudiment.The DWF4 gDNA upstream UAS that will contain 5 copies _Hap1Ti-plasmids (DWF4-BIN1B) also introduce Kitaake.Described gDNA is from environmental WS (SEQIDNO:14).The activation of UAS (passing through Hap1) causes the accumulation with the DWF4 transcript of transcribing of DWF4 gDNA.

T2 is for the hybridization of plant, gene type assay and pairing

The UAS:DWF4 strain is DWF4-BIN1B 16-2, and 17-3 and 27-3 are derived from independently transgenic line 16,17 and 27 respectively, is offspring's pollination of CRS-BIN1A 7 with the p326:Hap1 strain, to produce F1 for seed.Be called R148 respectively, 3 groups of F1 of R150 and R151 are for the existence of plant by fluoroscopic examination Hap1:GFP, and detect the existence of UAS:DWF4 by PCR, and the existence of DWF4 gDNA transcript confirms by RT-PCR.Will be from F1 for plant R148P5, the F2 of R150P7 and R151P1 also detects the existence of p326:Hap1 and UAS:DWF4 then for seed germination and growth in darkness 3 days.With 5 couples of GFP (+)/DWF4 (+) and GFP (+)/DWF4 (-) chorista, each, is grown in the jar for plant side by side from identical F1, as shown in the table 9.

Table 9.

The T2 abbreviation	Hap1：GFP	?UAS：DWF4	Right
The T2 abbreviation	Hap1：GFP	?UAS：DWF4	Right	R148P5-13 R148P5-12	Exist	Not existence	1
				R148P5-13 R148P5-12	Exist	Not existence	1
				R148P5-14 R148P5-15	Exist	Not existence	2
				R148P5-14 R148P5-15	Exist	Not existence	2
				R150P7-14 R150P7-19	Exist	Not existence	3
				R150P7-14 R150P7-19	Exist	Not existence	3
				R150P7-18 R150P7-20	Exist	Not existence	4
				R150P7-18 R150P7-20	Exist	Not existence	4
				R151P1-41 R151P1-40	Exist	Do not exist	5

The pairing of table 9. plant.GFP (+)/DWF4 (+) and GFP (+)/DWF4 (-) F2 grows side by side for chorista.Hap1:GFP represents that with UAS:DWF4 2-becomes two elements of sub-system.

The plant that contains the direct syzygy of p326:DWF4

DWF4 gDNA is connected on p326 promotor and the OCS terminator effectively, as among the embodiment 10, obtaining construct CR24.Use agriculture bacillus mediated conversion CR24 is transformed in the paddy rice, and produce homozygous lines.Be that 10 strains of each strain of CR24-3-6 and the CR24-5-6 T3 that isozygotys is grown in the greenhouse side by side for the logical unconverted wild-type of 5 strains of plant with representative strains.

The extraction of boot leaf and chemical analysis

All plants all grow in the greenhouse.Beginning to bloom～occur collecting from the boot leaf of every strain plant in 5 minutes (between 5:00pm and 5:05pm) after 12 days and at other boot leaf.Blooming back 15 days and back 15 days of pollination, also in other cotyledon 5 minutes and between 5:00pm and the 5:05pm, collect the seed of growing.All being organized on the dry ice is freezing, and is kept at-80 ℃.For chemical analysis, with these blade freeze-drying, with methyl alcohol and dichloromethane extraction, and before the GC-MS that derives layering in polarity and nonpolar phase.Extraction is carried out 2 times and 3 times, is used for the repeat samples that GC-MS analyzes with generation.The amount that is used for the freeze-drying leaf tissue of each extraction is displayed in Table 10.

Data gathering and processing comprise the visible inspection of stratographic and compare, and comprise the multivariate analysis of principle component analysis (PCA) and graduation classification analysis (HCA), and experiment/check analysis, and described data gathering and processing are used for determining the metabolite ratio.Analyzed 82 kinds of compounds, comprised amino acid, carbohydrate, lipid acid and organic acid, as mentioned shown in.

Table 10.

The T2 abbreviation	?1	?2	?3
The T2 abbreviation	?1	?2	?3	?R148P5?13	?28.9	?29.1	?30.1
?R148P5?12	?29.8	?29.7	?32.0	?R148P5?13	?28.9	?29.1	?30.1
?R148P5?12	?29.8	?29.7	?32.0
?R148P5?14	?29.9	?28.6	?-
?R148P5?14	?29.9	?28.6	?-	?R148P5?15	?29.5	?29.2	?22.7
				?R148P5?15	?29.5	?29.2	?22.7
				?R150P7?14	?29.3	?30.0	?29.6
?R150P7?19	?28.6	?30.2	?30.0	?R150P7?14	?29.3	?30.0	?29.6
?R150P7?19	?28.6	?30.2	?30.0
?R150P7?18	?31.2	?29.6	?30.4

Table 10. is used for the sample that MxP analyzes.T2 is meant each sheet of 10 boot leaves that are used for the GC-MS analysis for abbreviation.Hurdle ' 1 ', ' 2 ' and ' 3 ' show to be used for the amount of the freeze-drying leaf tissue of extraction at every turn, the mg of unit.For two boot leaves (R148P5 14 and R151P1 40), has only the tissue of enough twice extractions.

The result

GC-MS is used for analyzing 82 kinds of compounds, comprises amino acid, carbohydrate, lipid acid and organic acid, described compound is present in the boot leaf and the boot leaf and seed of 2 group of 10 strain T3 for rice plants of 5 couples of F2 for rice plants, and each plant comprises active DWF4 transgenosis.MxP result shows, with respect to the chorista control plant (being respectively R148P5 14 and R151P1 41) that only comprises p326:Hap1 (GFP (+)/DWF4 (ten) plant), the 5 strain F2 that come self-contained p326:Hap1 and UAS:DWF4 (GFP (+)/DWF4 (+) plant) are for having the blade of 2 strains to contain in the plant (R148P5 15 and R151P1 40)～increase of 20 free sucrose concentrations.The boot leaf increase a little that also shows sucrose level from residue 3 strains (R148P5 12, R150P7 19 and R150P7 20).Although there is not significance,statistical, sucrose concentration tends to higher a little rather than lower in these remaining plants of all 3 strains.

All 5 strain GFP (+)/DWF4 (+) F2 shows than GFP (+)/DWF4 (-) higher levels of L-glutamic acid of contrast and linolic acid (C18:2) for plant.Being increased in of L-glutamic acid～15% and～65% between, for linolic acid, it is increased in～18% and～40% between.

Other 79 kinds of compound concentrations of analyzing in 5 GFP (+)/DWF4 (+) boot leaf are tended to higher a little and lower a little than GFP (+)/DWF4 (-) contrast.These comprise glucose and fructose, and the two makes sucrose, and glutaminate, and it is synthetic by L-glutamic acid.

With respect to the contrast of unconverted wild-type, on sucrose or linolic acid level, do not show statistics for the metabolic pattern of the boot leaf of plant as the T3 of the p326:DWF4gDNA that directly merges and increase significantly from containing; A kind of T3 is higher for strain system (CR24-3-6) statistics on L-glutamic acid.

With respect to the contrast of unconverted wild-type, on sucrose, L-glutamic acid or linolic acid level, do not show significant difference on the statistics in the metabolic pattern of the seed that contains the growth that other T3 as the direct DWF4gDNA of fusion constructs collected after for plant pollination in 15 days.

On 79 kinds of other compound levels, there is not tendency.The main component analysis revealed, the L-Threonine, the L-Xie Ansuan, the L-phenylalanine, glycerol, L-leucine and L-Methionin (composition 1) contributed all changes in the seed compound～34%.

General introduction and discussion

By using Rice Flag, carried out the MxP analysis of a series of F2 for plant.MxP data from GFP (+)/DWF4 (+) and GFP (+)/DWF4 (-) plant comparison shows that, with respect to the contrast that only contains p326:Hap1, in the plant that contains p326:Hap1 and UAS:DWF4, there be L-glutamic acid, linolic acid and the possible sucrose level that increases.

Because the present invention describes like this, the various improvement that can be used to implement material of the present invention and method for those of ordinary skill in the art will be conspicuous.Should think that such improvement is in by in the defined scope of the present invention of following claim.

Claims

1. isolating polynucleotide, it comprises that coding has the nucleic acid molecule of following polypeptide: (a) aminoacid sequence of listing with SEQ ID NO:2 has an appointment 85% or more sequence homology.

2. isolating polynucleotide, it comprises the nucleic acid molecule of coding and the polypeptide of the corresponding aminoacid sequence of consensus sequence (SEQID NO:4) listed in Fig. 2, condition is that encoded polypeptide does not show the aminoacid sequence of listing with SEQ ID NO:1 or SEQ ID NO:3 and has 93% or more sequence homology.

3. isolating polynucleotide, it comprises the nucleic acid molecule of the polypeptide of the corresponding aminoacid sequence of consensus sequence (SEQID NO:4) of encoding and listing in Fig. 2, wherein said polynucleotide also comprises the wide expression type promotor controlling elements on the described nucleic acid that effectively is connected to coding said polypeptide.

4. claim 1 or 2 isolating polynucleotide, wherein said polypeptide effectively the catalysis campesterol in the C-22 oxidation to form the plain sterone of 6-deoxidation rape.

5. the isolating polynucleotide of claim 1, wherein said polypeptide comprises SEQ ID NO:2.

6. the isolating polynucleotide of claim 1, wherein said polypeptide has the sequence that SEQ ID NO:2 lists.

7. claim 1 or 2 isolating polynucleotide, wherein said polypeptide also comprises the controlling elements on the described nucleic acid that effectively is connected to coding said polypeptide.

8. the isolating polynucleotide of claim 7, wherein said controlling elements is wide expression type promotor or constitutive promoter.

9. the isolating polynucleotide of claim 8, wherein said wide expression type promotor is to be selected from by p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, the group that YP0144 and YP0190 form.

10. the isolating polynucleotide of claim 9, wherein said wide expression type promotor is p326.

11. the isolating polynucleotide of claim 8, wherein said constitutive promoter is 35S.

12. a recombinant vectors, it comprises the polynucleotide of (i) claim 1 or claim 2;

(ii) effectively be connected to the controlling elements on the described polynucleotide.

13. a recombinant vectors, it comprises the polynucleotide of claim 3.

14. a host cell, it comprises the recombinant vectors of claim 12.

15. a host cell, it comprises the recombinant vectors of claim 13.

16. transgenic plant, it comprises at least a external source polynucleotide, and described at least a external source polynucleotide comprises the nucleic acid of the following polypeptide of encoding:

(a) aminoacid sequence of listing with SEQ ID NO:2 has an appointment 85% or multisequencing homology more; Perhaps

(b) consensus sequence of listing with Fig. 2 (SEQ ID NO:4) is corresponding, and condition is that encoded polypeptide does not show the aminoacid sequence of listing with SEQ ID NO:1 or SEQ ID NO:3 and has 93% or more sequence homology.

17. the transgenic plant of claim 16, wherein said polypeptide comprise the aminoacid sequence that SEQ ID NO:2 lists.

18. the transgenic plant of claim 16, wherein said polypeptide have the sequence of listing among the SEQ ID NO:2.

19. the transgenic plant of claim 16, wherein said external source polynucleotide also comprises the controlling elements on the described nucleic acid that effectively is connected to coding said polypeptide.

20. the transgenic plant of claim 18, wherein said controlling elements are wide expression type promotor or constitutive promoter.

21. the transgenic plant of claim 20, wherein said wide expression row promotor is to be selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.

22. the transgenic plant of claim 21, wherein said wide expression type promotor is p326.

23. the transgenic plant of claim 21, wherein said p326 promotor causes the expression of described polypeptide at branch and stem apex.

24. the transgenic plant of claim 20, wherein said transgenic plant show the phenotype of change with respect to control plant.

25. the transgenic plant of claim 24, the phenotype of wherein said change is to be selected from by one or more of the following group of forming: with respect to described control plant, the metabolic pattern that changes, the increase of the plain sterone level of 6-deoxidation rape, the minimizing of campesterol level, the increase of photosynthetic rate, the seed production of increase, every strain plant seed weight that increases and the height that increases.

26. the transgenic plant of claim 25, the metabolic pattern of wherein said change are sucrose, glutaminate/ester or the linolic acid with respect to control plant increase level.

27. the transgenic plant of claim 16, wherein said transgenic plant are Chinese cabbage plant (Brassica plant).

28. the transgenic plant of claim 16, wherein said transgenic plant are monocotyledonss.

29. the transgenic plant of claim 28, wherein said commentaries on classics monocotyledons are Oryza, Triticum, switchgrass, Secale, Hordeum, jowar genus or Zea.

30. the transgenic plant of claim 16, wherein said transgenic plant are dicotyledonss.

31. the transgenic plant of claim 16, wherein said polypeptide effectively the catalysis campesterol in the C-22 oxidation to form the plain sterone of 6-deoxidation rape.

32. transgenic plant, condition are described plants is not Arabidopis thaliana or tobacco plant, it comprises at least a external source polynucleotide, and described at least a external source polynucleotide comprises the nucleic acid of the following polypeptide of encoding:

(b) consensus sequence of listing with Fig. 2 (SEQ ID NO:4) is corresponding.

33. the transgenic plant of claim 32, wherein said polypeptide comprise the aminoacid sequence that SEQ ID NO:2 lists.

34. the transgenic plant of claim 32, wherein said polypeptide have the sequence that SEQ ID NO:2 lists.

35. the transgenic plant of claim 32, wherein said external source polynucleotide also comprises the controlling elements on the described nucleic acid that effectively is connected to coding said polypeptide.

36. the transgenic plant of claim 35, wherein said controlling elements are wide expression type promotor or constitutive promoter.

37. the transgenic plant of claim 36, wherein said wide expression type promotor is to be selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.

38. the transgenic plant of claim 37, wherein said wide expression type promotor is p326.

39. the transgenic plant of claim 36, wherein said p326 promotor causes the expression of described polypeptide at branch and stem apex.

40. the transgenic plant of claim 35, wherein said transgenic plant show the phenotype of change with respect to control plant.

41. the transgenic plant of claim 40, the phenotype of wherein said change is to be selected from by one or more of the following group of forming: with respect to described control plant, the metabolic pattern that changes, the increase of the plain sterone level of 6-deoxidation rape, the minimizing of campesterol level, the photosynthetic rate of increase, the seed production of increase, every strain plant seed weight that increases and the height that increases.

42. the transgenic plant of claim 41, the metabolic pattern of wherein said change are sucrose, glutaminate/ester or the linolic acid with respect to described control plant increase level.

43. the transgenic plant of claim 32, wherein said transgenic plant are monocotyledonss.

44. the transgenic plant of claim 43, wherein said monocotyledons are Oryza, Triticum, switchgrass, Secale, Hordeum, jowar genus or Zea.

45. the transgenic plant of claim 32, wherein said transgenic plant are dicotyledonss.

46. the transgenic plant of claim 32, wherein said polypeptide effectively the catalysis campesterol in the oxidation of C-22, to form the plain sterone of 6-deoxidation rape.

47. transgenic plant that comprise at least a external source polynucleotide, described at least a external source polynucleotide comprises the nucleic acid of the following polypeptide of encoding:

(b) consensus sequence of listing with Fig. 2 (SEQ ID NO:4) is corresponding, and wherein said transgenic plant show the increase of the plain sterone level of 6-deoxidation rape with respect to control plant.

48. the transgenic plant of claim 47, wherein said polypeptide comprise the aminoacid sequence that SEQ ID NO:2 lists.

49. the transgenic plant of claim 47, wherein said polypeptide have the sequence that SEQ ID NO:2 lists.

50. the transgenic plant of claim 47, wherein said external source polynucleotide also comprises the controlling elements on the described nucleic acid that effectively is connected to coding said polypeptide.

51. the transgenic plant of claim 50, wherein said controlling elements are wide expression type promotor or constitutive promoter.

52. the transgenic plant of claim 51, wherein said wide expression type promotor is selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.

53. the transgenic plant of claim 52, wherein said wide expression type promotor is p326.

54. the transgenic plant of claim 53, wherein said p326 promotor causes the expression of described polypeptide at branch and stem apex.

55. the transgenic plant of claim 47, wherein said transgenic plant also show the phenotype that is selected from by one or more changes of the following group of forming with respect to control plant: with respect to control plant, the metabolic pattern that changes, the minimizing of campesterol level, the photosynthetic rate that increases, the seed production that increases, every strain plant seed weight of increase and the height that increases.

56. the transgenic plant of claim 55, the metabolic pattern of wherein said change are sucrose, glutaminate/ester or the linolic acid with respect to described control plant increase level.

57. the transgenic plant of claim 47, wherein said transgenic plant are monocotyledonss.

58. the transgenic plant of claim 57, wherein said monocotyledons are Oryza, Triticum, switchgrass, Secale, Hordeum, jowar genus or Zea.

59. the transgenic plant of claim 47, wherein said transgenic plant are dicotyledonss.

60. the transgenic plant of claim 47, wherein said polypeptide effectively the catalysis campesterol in the C-22 oxidation to form the plain sterone of 6-deoxidation rape.

61. transgenic plant that comprise at least a external source polynucleotide, described at least a external source polynucleotide comprises the nucleic acid of the following polypeptide of encoding:

(a) aminoacid sequence of listing with SEQID NO:2 has an appointment 85% or multisequencing homology more; Perhaps

(b) consensus sequence of listing with Fig. 2 (SEQ ID NO:4) is corresponding, and wherein said transgenic plant show the minimizing of campesterol level with respect to control plant.

62. the transgenic plant of claim 61, wherein said polypeptide comprise the aminoacid sequence that SEQ ID NO:2 lists.

63. the transgenic plant of claim 61, wherein said polypeptide have the sequence that SEQ ID NO:2 lists.

64. the transgenic plant of claim 61, wherein said external source polynucleotide also comprises the controlling elements on the described nucleic acid that effectively is connected to coding said polypeptide.

65. the transgenic plant of claim 64, wherein said controlling elements are wide expression type promotor or constitutive promoter.

66. the transgenic plant of claim 65, wherein said wide expression type promotor is selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.

67. the transgenic plant of claim 66, wherein said wide expression type promotor is p326.

68. the transgenic plant of claim 67, wherein said p326 promotor causes the expression of described polypeptide at branch and stem apex.

69. the transgenic plant of claim 61, wherein said transgenic plant also show the phenotype that is selected from by one or more changes of the following group of forming with respect to control plant: with respect to control plant, the metabolic pattern that changes, the increase of the plain sterone level of 6-deoxidation rape, the photosynthetic rate that increases, the seed production that increases, every strain plant seed weight of increase and the height that increases.

70. the transgenic plant of claim 69, the metabolic pattern of wherein said change are sucrose, glutaminate/ester or the linolic acid with respect to described control plant increase level.

71. the transgenic plant of claim 61, wherein said transgenic plant are monocotyledonss.

72. the transgenic plant of claim 71, wherein said monocotyledons are Oryza, Triticum, switchgrass, Secale, Hordeum, jowar genus or Zea.

73. the transgenic plant of claim 61, wherein said transgenic plant are dicotyledonss.

74. the transgenic plant of claim 61, wherein said polypeptide effectively the catalysis campesterol in the C-22 oxidation to form the plain sterone of 6-deoxidation rape.

75. transgenic plant that comprise at least a external source polynucleotide, described at least a external source polynucleotide comprises the nucleic acid of the following polypeptide of encoding:

(b) consensus sequence of listing with Fig. 2 (SEQ ID NO:4) is corresponding, and wherein said allogenic polypeptide also comprises the wide expression type controlling elements on the described nucleic acid that effectively is connected to coding said polypeptide.

76. the transgenic plant of claim 75, wherein said polypeptide comprise the aminoacid sequence that SEQ ID NO:2 lists.

77. the transgenic plant of claim 75, wherein said polypeptide have the sequence that SEQ ID NO:2 lists.

78. the transgenic plant of claim 75, wherein said wide expression type promotor is selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.

79. the transgenic plant of claim 78, wherein said wide expression type promotor is p326.

80. the transgenic plant of claim 79, wherein said p326 promotor causes the expression of described polypeptide at branch and stem apex.

81. the transgenic plant of claim 75, wherein said transgenic plant also show the phenotype that is selected from by one or more changes of the following group of forming with respect to control plant: with respect to control plant, the metabolic pattern that changes, the minimizing of campesterol level, the increase of the plain sterone level of 6-deoxidation rape, the photosynthetic rate of increase, the seed production of increase, every strain plant seed weight that increases and the height that increases.

82. the transgenic plant of claim 81, the metabolic pattern of wherein said change are sucrose, glutaminate/ester or the linolic acid with respect to described control plant increase level.

83. the transgenic plant of claim 75, wherein said transgenic plant are monocotyledonss.

84. the transgenic plant of claim 83, wherein said monocotyledons are Oryza, Triticum, switchgrass, Secale, Hordeum, jowar genus or Zea.

85. the transgenic plant of claim 75, wherein said transgenic plant are dicotyledonss.

86. the transgenic plant of claim 75, wherein said polypeptide effectively the catalysis campesterol in the C-22 oxidation to form the plain sterone of 6-deoxidation rape.

87. transgenic plant that comprise at least a external source polynucleotide, described at least a external source polynucleotide comprises the nucleic acid of the following polypeptide of encoding:

(b) consensus sequence of listing with Fig. 2 (SEQ ID NO:4) is corresponding, and wherein said transgenic plant show the photosynthetic rate of increase with respect to control plant.

88. the transgenic plant of claim 87, wherein said polypeptide comprise the aminoacid sequence that SEQ ID NO:2 lists.

89. the transgenic plant of claim 87, wherein said polypeptide have the sequence that SEQ ID NO:2 lists.

90. the transgenic plant of claim 87, wherein said external source polynucleotide also comprises the controlling elements on the described nucleic acid that effectively is connected to coding said polypeptide.

91. the transgenic plant of claim 90, wherein said controlling elements are wide expression type promotor or constitutive promoter.

92. the transgenic plant of claim 91, wherein said wide expression type promotor is selected from the group of being made up of following: p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190.

93. the transgenic plant of claim 92, wherein said wide expression type promotor is p326.

94. the transgenic plant of claim 93, wherein said p326 promotor causes the expression of described polypeptide at branch and stem apex.

95. the transgenic plant of claim 87, wherein said transgenic plant also show the phenotype that is selected from by one or more changes of the following group of forming with respect to control plant: with respect to control plant, the metabolic pattern that changes, the minimizing of campesterol level, the increase of the plain sterone level of 6-deoxidation rape, the seed production that increases, every strain plant seed weight of increase and the height that increases.

96. the transgenic plant of claim 95, the metabolic pattern of wherein said change are sucrose, glutaminate/ester or the linolic acid with respect to described control plant increase level.

97. the transgenic plant of claim 87, wherein said transgenic plant are monocotyledonss.

98. the transgenic plant of claim 97, wherein said monocotyledons are Oryza, Triticum, switchgrass, Secale, Hordeum, jowar genus or Zea.

99. the transgenic plant of claim 87, wherein said transgenic plant are dicotyledonss.

100. the transgenic plant of claim 87, wherein said polypeptide effectively the catalysis campesterol in the C-22 oxidation to form the plain sterone of 6-deoxidation rape.

101. a method that is used to produce transgenic plant, it comprises:

(a) with claim 1, claim 2, or the polynucleotide introduced plant cell of claim 3 are to produce the plant transformed cell; With

(b) from described plant transformed cells produce transgenic plant.

102. according to claim 16,32,47,61,75, or the seed of 87 transgenic plant.

103. isolated polypeptide:

(a) have the aminoacid sequence about 85% listed with SEQ ID NO:2 or multisequencing homology more; Perhaps

104. a method that is used for increasing plant the level of one or more metabolites that are selected from the group of being made up of sucrose, glutaminate/ester and linolic acid, described method comprises:

(b) from described plant transformed cells produce transgenic plant, wherein said transgenic plant show described one or more metabolites of the level of increase.

105. a method that is used for increasing plant the level of one or more metabolites that are selected from the group of being made up of sucrose, glutaminate/ester and linolic acid, described method comprises:

(a) in order to produce the plant transformed cell, in vegetable cell, introduce the isolating polynucleotide of the nucleic acid comprise the following polypeptide of encode: 1) and the aminoacid sequence listed of SEQ ID NO:2 have an appointment 85% or the polypeptide of multisequencing homology more, or 2) comprise the polypeptide of the corresponding aminoacid sequence of listing with Fig. 2 of consensus sequence (SEQ ID NO:4); With

106. a method that is used for increasing the plain sterone level of plant 6-deoxidation rape, described method comprises:

(b) from described plant transformed cells produce transgenic plant, wherein said transgenic plant show the plain sterone of 6-deoxidation rape of the level of increase.

107. a method that is used for increasing the plain sterone level of plant 6-deoxidation rape, described method comprises:

108. a method that is used for reducing plant rape oil sterol levels, described method comprises:

(b) from described plant transformed cells produce transgenic plant, wherein said transgenic plant show the campesterol of the level of minimizing.

109. a method that is used for reducing plant rape oil sterol levels, described method comprises:

110. a method that is used to increase the photosynthesis of plant rate, described method comprises:

(b) from described plant transformed cells produce transgenic plant, wherein said transgenic plant show the photosynthetic rate of increase.

111. a method that is used to increase the photosynthesis of plant rate, described method comprises: