CN112813074A - Method for specifically regulating and controlling rice CYP78A11 gene expression and plant transformation vector - Google Patents

Abstract

The invention discloses a method for specifically regulating and controlling the expression of a rice CYP78A11 gene and a plant transformation vector, wherein the method regulates and controls the expression of the CYP78A11 gene by an enhancer and a promoter together, thereby improving the yield of crops; the nucleotide sequence of the enhancer comprises SEQ ID NO.4 or SEQ ID NO. 5; the promoter is a CYP78A11 gene self promoter or a promoter with similar functions. The invention provides a brand-new CYP78A11 gene expression regulation method and application thereof in corn, wherein the plant height and the growth potential of the corn are improved by properly over-expressing the CYP78A11 gene in the corn, so that the biomass of the corn is improved by 5-50%, the yield of the corn is increased by 5-30% finally, and a new thought and method are provided for improving the yield of the corn.

Description

Method for specifically regulating and controlling rice CYP78A11 gene expression and plant transformation vector

(I) technical field

The invention relates to a method for specifically regulating and controlling the expression of a rice CYP78A11 gene, which can be applied to improve the yield of corn, in particular to a method for improving the yield of corn by utilizing the specifically regulated and controlled expression of the CYP78A11 gene, which can reasonably regulate and control the yield-related traits of the corn, such as plant height, grain weight, growth potential and the like, so as to achieve the purpose of increasing the yield.

(II) background of the invention

The regulation of gene expression is very important to the growth and development of plants and to agronomic traits. The expression quantity, the expression time and the expression position of the gene can influence the growth and development of plants. People can select a proper expression regulation method of a certain functional gene through research, and the purposes of improving plant characters and increasing yield are achieved. For example, researchers have selected 5 promoters to be selected from 16 cotton fiber or ovule-associated promoters, selected the promoter FBP7 by constructing a plant expression vector and performing later-stage character analysis, and have improved cotton yield by more than 15% by using FBP7 to mediate expression of IAA synthetase in the ovule of cotton (Zhang M et al, (2011) Nat Biotechnol 29, 453-one 458).

Plant gene expression regulation is a very complex process, and the expression regulation is performed in many ways, including transcriptional regulation, post-transcriptional regulation, and translational regulation. The promoter is a DNA sequence located at the 5' upstream of the structural gene, can activate RNA polymerase, enables the RNA polymerase to be accurately combined with template DNA, has the specificity of transcription initiation, and is an important element for regulating and controlling gene expression. A review by Potenza et al details and summarizes the studies on the promoters (Potenza et al (2004) In Vitro Cell Dev Biol-Plant,40: 1-22). The promoter mainly comprises a constitutive expression promoter, a tissue specific expression promoter, a promoter capable of inducing expression and the like. Constitutive promoters, for example, The CaMV 35S promoter of cauliflower mosaic virus (Terada and Shimamoto, (1990) Molecular & General Genetics,220: 389-; tissue-specific promoters, for example, the STM-specific promoter (U.S. Pat. No.5880330), the ARSK1 root-specific expression promoter (United States Patent Application 20040067506), the AP1 floral meristem promoter (Sasaki, Katsutomo, et al (2011) Plant biotechnology,28: 181-188); inducible promoters, such as the RBCS1 promoter and Arabidopsis rd29A promoter (Zhang Chun Xiao et al (2004.) progress in the study of plant Gene promoters, proceedings of genetics, 031(012), 1455-. Enhancers are another important element in regulating gene expression. Enhancers are small regions of DNA that bind to proteins, and following binding, gene transcription is enhanced. However, the enhancement effect of different enhancers varies greatly, from several fold to several thousand fold.

CYP78A is a subfamily of The P450 family, and has important functions (Bak et al, (2011) The Arabidopsis Book/American Society of Plant Biologists,9: e 0144). Among the plant CYP78A genes, the CYP78A gene CYP78a1 gene in corn was relatively early in functional studies. The gene is expressed predominantly in developing inflorescences and may be involved in fatty acid metabolism, but the specific function of CYP78A1 in maize is not known since there is no corresponding mutant (Imaishi et al (2000) Biosci Biotech Bioch,64: 1696-1701). Kazumaru et al have shown that CYP78A gene CYP78A11 in rice has the function of regulating growth and development of leaf primordium and length of vegetative growth period of rice (Miyoshi, K.et al (2004) Proc. Natl.Acad.Sci.USA,101(3), 875-.

The invention unexpectedly discovers that the corn biomass can be improved, and the corn grain weight and the corn yield can be increased by regulating the expression of the CYP78A11 gene in corn seeds through the combination of an enhancer and the CYP78A11 gene promoter.

Disclosure of the invention

The invention aims to provide a method for specifically regulating and controlling the expression of a rice CYP78A11 gene by utilizing an enhancer and a promoter so as to improve the yield of crops, in particular to improve the plant shape and the growth potential of corn and improve the yield.

The technical scheme adopted by the invention is as follows:

the invention provides a method for specifically regulating and controlling the expression of a rice CYP78A11 gene, which regulates and controls the expression of the CYP78A11 gene by an enhancer and a promoter together, thereby improving the yield of crops; the nucleotide sequence of the enhancer comprises SEQ ID NO.4 or SEQ ID NO. 5; the promoter is a CYP78A11 gene self promoter or a promoter with similar functions, and the nucleotide sequence is preferably shown in SEQ ID NO. 1.

The CYP78A11 gene provided by the invention is derived from rice, but the ordinary skilled in the art can determine whether they have the functions of improving the plant shape and the growth potential of corn and increasing the yield according to the method provided by the invention and the prior art. These genes include coding genes having homology of 95%, 90%, 85%, 80% with the nucleotide sequence shown in SEQ ID NO. 2. The polynucleotide sequence of the CYP78A11 gene of the invention can be cloned from a plant into the corresponding homologous gene by PCR, DNA hybridization, etc., as is common in the art. According to the polynucleotide sequence design primer provided by the invention, a part of or the whole sequence of the homologous gene can be obtained by a PCR method. Once a partial sequence of the gene is obtained, the full-length gene can be cloned by various methods. On the other hand, the polynucleotide provided by the invention is used for preparing a probe, and homologous genes of the probe can be cloned from a DNA library of a plant by a DNA hybridization method.

Further, preferably, the CYP78A11 gene has a nucleotide sequence shown by SEQ ID NO.2 and an amino acid sequence shown by SEQ ID NO. 3.

The region of the promoter of the present invention can be determined by experiment. Typically, the promoter will be a DNA fragment of at least 0.25kb, 0.5kb, 1.0kb, 2.0kb or 3.0kb outside the 5' end of the reading frame encoding the protein. The natural promoter of CYP78A11 or its homologous gene can be used to control the expression of its own gene, or can be functionally linked to homologous genes of other plants to control the expression of these genes in other plants, so as to obtain improved transgenic plants. Meanwhile, after the CYP78A11 gene promoter is artificially modified, the promoter with the similar expression mode of the CYP78A11 gene promoter, of which the nucleotide homology is more than 98%, 95%, 90% and 85%, can also be used for the expression regulation of the CYP78A11 gene.

The enhancer provided by the invention also comprises other enhancers with similar enhancement effect to that shown in SEQ ID NO.4 or SEQ ID NO. 5.

The method for specifically regulating and controlling the expression of the rice CYP78A11 gene comprises the steps of forming functional connection between the CYP78A11 gene nucleotide sequence and an enhancer, a promoter and a terminator, and constructing a CYP78A11 gene expression cassette; the CYP78A11 gene expression cassette is introduced into the plant of crop (preferably corn) to obtain expression, improve the plant shape and growth potential of crop and increase yield.

The terminator controlling the expression of the gene may be a natural terminator providing the gene, or may be a terminator of another gene of the same plant or another plant gene. Commonly used terminators include Octopine synthase terminator and nopaline synthase terminator derived from Agrobacterium and the like. References include: guerineau et al (1991) mol.Gen.Genet.,262: 141-144; proudfoot (1991) Cell,64: 671-674; sanfacon et al (1991) Genes Dev.,5: 141-149; mogen et al (1990) Plant Cell,2: 1261-; munroe et al (1990) Gene,91: 151-158; ballas et al (1989) Nucleic Acids Res.,17: 7891-7903; and Joshi et al (1987) Nucleic Acids Res.,15: 9627-. Preferably, the nucleotide sequence of the terminator is shown as SEQ ID NO. 6.

The invention also provides a plant transformation vector for CYP78A11 gene expression, which is prepared by transferring a CYP78A11 gene expression frame into a binary vector, wherein the binary vector can be obtained by modifying a pCambia1300 vector (the NCBI database serial number is AF234296), and specifically, the binary vector of pCambia1300-pZmUBI-g10evo is obtained by artificially synthesizing a nucleotide sequence (shown as SEQ ID NO: 7) containing a pZmUBI promoter and a g10evo gene and connecting the nucleotide sequence into the pCambia1300 vector after enzyme digestion by a restriction endonuclease connection method.

The plant transformation vector comprises a CYP78A11 gene expression frame and a screening marker gene expression frame. The selection marker gene can be used for selecting transformed cells, and commonly used genes include antibiotic resistance genes such as neomycin phosphotransferase II (NEO) and Hygromycin Phosphotransferase (HPT), herbicide resistance genes such as glyphosate resistance gene EPSPS, and the like. Other selectable marker genes may also be used as selectable genes for transformation in accordance with the present invention.

The CYP78A11 gene expression cassette of the invention is composed of a CYP78A11 gene promoter, a CYP78A11 gene nucleotide sequence, a CYP78A11 gene terminator and a DNA fragment with enhancer effect; the CYP78A11 gene promoter nucleotide sequence is shown in SEQ ID NO.1, the CYP78A11 gene nucleotide sequence is shown in SEQ ID NO.2, the CYP78A11 gene terminator sequence is shown in SEQ ID NO.6, and the DNA fragment with the enhancer effect is shown in SEQ ID NO.4 or SEQ ID NO. 5.

The expression frame of the screening marker gene is constructed by a promoter, an EPSPS leader peptide, the screening marker gene and a terminator. The promoter of the screening marker gene is a promoter of the corn pUBI, and the nucleotide sequence of the promoter is shown as 1bp-2026bp in SEQ ID NO. 7; the nucleotide sequence of the EPSPS leader peptide is shown as 2027bp-2248bp in SEQ ID NO. 7; the screening marker gene is g10evo, and the nucleotide sequence of the screening marker gene is shown as 2249bp-3536bp in SEQ ID NO. 7; the terminator is CaMV 35S terminator of cauliflower mosaic virus, and the nucleotide sequence is shown as 2120bp-2310bp in pCambia1300 vector (NCBI database sequence number: AF 234296).

The polynucleotide sequence encoding the CYP78a11 protein of the present invention may have a variety of different variations including, but not limited to: 1) different polynucleotide sequences are obtained due to different codons encoding the same amino acid, and the sequences encode protein polypeptides with the same activity; 2) genetic polymorphisms of biological origin (Genetic Polymorphism), i.e. the diversity between different individuals or populations of the same plant; 3) variations in polynucleotide sequence are introduced by manual manipulation. The artificially introduced variation may be random variation or targeted variation. One of ordinary skill in the art can generate point mutations, insertion or deletion variations, etc., by molecular biological methods. The introduction of a variation in polynucleotide sequence by manual manipulation also includes obtaining a hybrid Gene still having a normal function by the method of Gene Shuffling, etc. For example, U.S. Pat. No. 2002/0058249; stemmer (1994) proc.natl.Acad.Sci.USA,91: 10747-10751; stemmer (1994) Nature,370: 389-391; crameri et al, (1997) Nature Biotech, 15:436- > 438; moore et al, (1997) J.mol.biol.,272: 336-; zhang et al (1997) Proc.Natl.Acad.Sci.USA,94: 4504-; crameri et al (1998) Nature,391: 288-; and U.S. Pat. No.5, 605,793and 5,837, 458.

The invention further provides an identification method of the obtained trans-CYP 78A11 gene corn, which comprises observing plant phenotype, spraying herbicide, molecular biology method and the like, and of course, the method can be used together in a plurality of methods in practical application. The trans-CYP 78A11 corn plant provided by the invention and a control plant have obvious phenotypic difference and can be distinguished by naked eyes. The transgenic CYP78A11 maize plant exhibits an increased plant height, an increased vigor, a broadened leaf, an increased biomass, an increased yield per plant, or a combination of two or more of these phenotypic changes. If the marker gene and the target gene are closely linked, the target gene can be identified by spraying herbicide. For example, by constructing the CYP78A11 gene on a vector using a glyphosate resistant gene (EPSPS) as a selection marker, glyphosate resistant plants are likely to be plants in which the CYP78A1 or homologous genes thereof are overexpressed. The transgenic CYP78A11 maize plant can also be identified by molecular biology methods, which mainly comprise Southern hybridization, PCR and other technologies to detect the DNA of a target gene; detecting the mRNA level of the target gene by using fluorescent quantitative PCR or quantitative PCR and other technologies; western hybridization, enzyme-linked immunosorbent assay (ELISA) and other technologies, and the protein level of the target gene is detected.

The CYP78A11 gene expression regulation and control method provided by the invention has other realization methods, for example, the position of the gene in a genome is found, and then a regulation and control sequence of gene expression, such as CaMV 35S enhancer, is inserted into a site capable of enhancing the gene expression through a DNA fixed-point insertion technology. One enhancer often affects the expression of nearby genes, and some can increase the expression of genes that are 20kb or more apart. The technique of DNA site-directed insertion of plants is well established at present (Townsend, Jeffrey A., et al. (2009) Nature,459:442- & lt445.; Jiang, W., et al. (2013) & Nat.Biotechnol.,31: 233- & lt239; Cong, L.et al. (2013) Science,339: 819- & lt823). Therefore, by using a site-directed insertion method, it is possible to increase the expression of the CYP78A gene by inserting an enhancer near the gene.

The gene provided by the invention can be introduced into plants to obtain transgenic high-yield plants, and the plants include but are not limited to corn, wheat, barley, sorghum, rice, sugarcane, soybean, carrot, potato, cotton, sunflower, rape, oak, turfgrass and pasture grass.

The skilled person can now use the existing techniques to introduce the polynucleotides provided by the present invention into plants for expression. A more commonly used method is the gene gun method (Klein et al,1987, Nature (London),327: 70-73; U.S. Pat. No.4,945,050) or the Agrobacterium-mediated method ((De Blaere et al,1987, meth.enzymol.,143: 277). The present invention is not limited to this method.

The transformation methods and procedures vary from plant to plant. The more widely used method is generally to introduce immature embryos, mature embryos, undifferentiated callus or protoplasts of a plant by Agrobacterium or a gene gun. Then screening culture is carried out by using a corresponding screening culture medium. Then differentiation is carried out to obtain transformed buds, and the transgenic seedlings which can be planted can be obtained through the culture of a rooting culture medium. Further, herbicide-resistant transgenic plants can be screened by spraying herbicides, for example, nicosulfuron can kill non-transgenic rice.

Compared with the prior art, the invention has the following beneficial effects: the invention provides a brand-new CYP78A11 gene specific expression regulation method and application thereof in corn, wherein the plant height and the growth potential of the corn are improved by properly over-expressing the CYP78A11 gene in the corn, so that the biomass of the corn is improved by 5-50%, the corn yield is increased by 5-30% finally, and a new thought and method are provided for improving the corn yield.

(IV) description of the drawings

FIG. 1: the T-DNA structure of the plant transformation vector 1300-pZmUbi-g10evo-p35S-CYP78A11 is shown schematically. p35S is the 35S promoter of artificially synthesized CaMV, CYP78A11 is a DNA fragment containing the coding region of CYP78A11 gene and a terminator, pUBI is the promoter of the artificially synthesized maize UBI-1 gene, g10evo is the 35S terminator of the artificially synthesized EPSPS synthetase gene and CaMV on the pCambia1300 vector.

FIG. 2: the T-DNA structure of the plant transformation vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11 is shown schematically. pCYP78A11 is the promoter of the rice CYP78A11 gene itself obtained by PCR, CYP78A11 is a DNA fragment containing the coding region of CYP78A11 gene and a terminator, pUBI is the promoter of the artificially synthesized maize UBI-1 gene, g10evo is the artificially synthesized EPSPS synthetase gene and 35S terminator of CaMV on the pCambia1300 vector.

FIG. 3: the T-DNA structure of the plant transformation vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-p35S or 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-E2 is shown schematically. pCYP78A11 is the promoter of the rice CYP78A11 gene itself obtained by PCR, CYP78A11 is a DNA fragment containing the coding region and terminator of CYP78A11 gene, pUBI is the promoter of the artificially synthesized maize UBI-1 gene, g10evo is the artificially synthesized EPSPS synthetase gene and 35S terminator of CaMV on pCambia1300 vector, En is the artificially synthesized enhancer.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the molecular biological and biochemical methods used in the following examples of the invention are all known techniques. The details of the Molecular Cloning published by the company John Wiley and Sons, Inc. of Ausubel, Molecular Protocols in Molecular Biology, and Cold Spring Harbor Laboratory Press (2001), Sambrook et al, A Laboratory Manual,3rd ED., are described in detail.

Example 1 construction of Rice CYP78A11 Gene overexpression vector

1. Rice CYP78A11 gene fragment

The rice CYP78A11 gene coding region (the nucleotide sequence is shown as SEQ ID NO.2, the amino acid sequence is shown as SEQ ID NO. 3) and the terminator DNA fragment (the nucleotide sequence is shown as SEQ ID NO. 6) are obtained by PCR. The 2 primers for PCR were: CYP78A11-F1 (5'-GGATCCAACAATGGCAATGGCCACCGCCAC-3') and CYP78A11-R1 (5'-GGTACCCATCTCACAAGCTCACACGGC-3').

Taking the genome of the Xishui 134 of the Zhejiang main-cultivated rice variety as a template, cloning a DNA fragment of about 2.2kb obtained by PCR into a pMD-18-T-Vector (TaKaRa) by using primers CYP78A11-F1 and CYP78A11-R1, and determining a sequence to obtain the DNA fragment of 5 '-SEQ ID NO.2-SEQ ID NO. 6-3', wherein the DNA fragment not only encodes the DNA sequence (the nucleotide sequence is shown in SEQ ID NO. 2) of the amino acid of the CYP78A11 rice, but also comprises an intron and a terminator, and the nucleotide sequence of the terminator is shown in SEQ ID NO. 6. The reaction system of PCR is: 5x PrimeStar^TM Buffer(Mg²⁺plus) (from TaKaRa), 10. mu.l; dNTP mix (2.5 mM each), 4. mu.l; primer CYP78A11-F1 (10. mu.M), 1. mu.l; primer CYP78A11-R1 (10. mu.M), 1. mu.l; 100 ng of template DNA; PrimeStar^TMHS DNA Polymerase (2.5U/. mu.l), 0.5. mu.l; sterilized distilled water, added to a final volume of 50. mu.l. The PCR conditions were: pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 30sec, annealing at 62 ℃ for 30sec, extension at 72 ℃ for 140sec, 32 cycles; extension at 72 ℃ for 10 min.

2. Promoter fragment of rice CYP78A11 gene

The promoter fragment pCYP78A11 of the rice CYP78A11 gene was obtained by PCR. The 2 primers for PCR were: pCYP78A11-F1 (5'-GGTACCGATTAGATAAAGCCACTTCCAC-3') and pCYP78A11-R1 (5'-GGATCCAGGTGTTTGTGGTTTTGCTTGTG-3'). KpnI and BamHI restriction sites are respectively designed in the primers. A DNA fragment (namely a promoter fragment pCYP78A11 of a CYP78A11 gene) of about 1.9kb obtained by PCR (polymerase chain reaction) is cloned into a pMD-18-T-Vector (TaKaRa) by taking a genome CYP78A11 gene of a Xishui 134 rice variety mainly planted in Zhejiang as a template, and the sequence is determined to obtain a promoter sequence shown as SEQ ID NO. 1. The reaction system of PCR is: 5x PrimeStar^TM Buffer(Mg²⁺plus) (from TaKaRa), 10. mu.l; dNTP mix (2.5 mM each), 4. mu.l; primer pCYP78A11-F1 (10. mu.M), 1. mu.l; primer pCYP78A11-R1 (10. mu.M), 1. mu.l; 100 ng of template DNA; PrimeStar^TMHS DNA Polymerase (2.5U/. mu.l), 0.5. mu.l; sterilized distilled water, added to a final volume of 50. mu.l. Of PCRThe conditions are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 30sec, annealing at 62 ℃ for 30sec, extension at 72 ℃ for 2min, 32 cycles; extension at 72 ℃ for 10 min.

3. Artificial synthetic enhancer E2

The p35S promoter containing enhancer is artificially synthesized, the sequence is shown as SEQ ID NO.4, and the enhancer E2 is artificially synthesized, the sequence is shown as SEQ ID NO. 5.

4. Binary vector

In order to obtain a binary vector containing a glyphosate-tolerant gene g10evo as a screening marker, a DNA sequence pUBI-g10evo containing a corn UBI-1 promoter pUBI, a rice EPSPS leader peptide sequence and a g10evo gene is artificially synthesized, and is shown as SEQ ID NO. 7. The binary vector pCambia1300 is digested by KpnI and XhoI, and the digested vector is recovered; carrying out double enzyme digestion on the synthesized fragment pUBI-g10evo by utilizing KpnI and XhoI, and respectively recovering the fragments after enzyme digestion; the 2 recovered fragments were ligated to obtain binary vector 1300-pZmUbi-g10 evo.

5. Construction of vector 1300-pZmUbi-g10evo-p35S-CYP78A11

Carrying out double enzyme digestion on the vector 1300-pZmUbi-g10evo by utilizing HindIII and KpnI, and recovering the vector; the artificially synthesized p35S promoter fragment (shown as SEQ ID NO: 4) was subjected to double digestion with HindIII and BamHI, and the promoter fragment was recovered; carrying out double enzyme digestion on the rice CYP78A11 gene obtained by PCR and a terminator fragment (shown as ' 5' -SEQ ID NO.2-SEQ ID NO.6-3 ') by using BamHI and KpnI, and recovering the fragment; the 3 enzyme-cleaved fragments are connected in three segments to obtain a final vector 1300-pZmUbi-g10evo-p35S-CYP78A11, and the structural schematic diagram of the vector T-DNA is shown in figure 1.

6. Construction of vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11

Carrying out single enzyme digestion on the vector 1300-pZmUbi-g10evo by utilizing KpnI, carrying out dephosphorylation (self-ligation prevention) on the enzyme digestion vector by utilizing FastAP, and then recovering the enzyme digestion vector to recover the vector; carrying out double enzyme digestion on a promoter fragment (shown as SEQ ID NO: 1) of the rice CYP78A11 gene obtained by PCR by utilizing KpnI and BamHI, and recovering the promoter fragment; carrying out double enzyme digestion on the rice CYP78A11 gene obtained by PCR and a terminator fragment (shown as ' 5' -SEQ ID NO.2-SEQ ID NO.6-3 ') by using BamHI and KpnI, and recovering the fragment; the 3 enzyme-cleaved fragments are connected in three segments to obtain a final vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11, and the structural schematic diagram of the vector T-DNA is shown in figure 1.

7. Construction of vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-p35S

Carrying out single enzyme digestion on the vector 1300-pZmUbi-g10evo by SacI, carrying out dephosphorylation on the enzyme digestion vector by FastAP to prevent self-ligation, and then recovering the enzyme digestion vector; the artificially synthesized p35S promoter (shown as SEQ ID NO: 4) was digested with SacI, the digested fragments were recovered, the vector and fragments were ligated, and the transition vector with a vector structure such as 1300-p35S-pZmUbi-g10evo was selected. Carrying out single enzyme digestion on the transition vector 1300-p35S-pZmUbi-g10evo by utilizing KpnI, carrying out dephosphorylation (self-ligation prevention) on the enzyme digestion vector by using FastAP, and then recovering the enzyme digestion vector and recovering the vector; carrying out single enzyme digestion on the vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11 by using KpnI, and recovering a pCYP78A11-CYP78A11 fragment containing a CYP78A11 promoter, a coding region and a terminator; the 2 fragments are connected to obtain a final vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-p35S, and the structural diagram of the vector T-DNA is shown in figure 3.

8. Construction of vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-E2

Carrying out single enzyme digestion on the vector 1300-pZmUbi-g10evo by SacI, carrying out dephosphorylation on the enzyme digestion vector by FastAP to prevent self-ligation, and then recovering the enzyme digestion vector; artificially synthesized E2 (shown as SEQ ID NO: 5) is subjected to enzyme digestion by SacI, enzyme digestion fragments are recovered, the vector and the fragments are connected, and a transition vector with a vector structure of 1300-E2-pZmUbi-g10evo is screened. Carrying out single enzyme digestion on the transition vector 1300-E2-pZmUbi-g10evo by utilizing KpnI, carrying out dephosphorylation (self-ligation prevention) on the enzyme digestion vector by using FastAP, and then recovering the enzyme digestion vector and recovering the vector; carrying out single enzyme digestion on the vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11 by using KpnI, and recovering a pCYP78A11-CYP78A11 fragment containing a CYP78A11 promoter, a coding region and a terminator; the 2 fragments are connected to obtain a final vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-E2, and the structural schematic diagram of the vector T-DNA is shown in figure 3.

Finally, the above 4T-DNA plasmids were individually transferred to Agrobacterium LB4404 by electroporation, and positive clones were selected by YEP solid medium containing 15. mu.g/mL tetracycline and 50. mu.g/mL kanamycin and maintained for the subsequent plant transformation.

Example 2 transformation of maize

Methods for transforming maize have become more established, for example, the method for transforming maize with Agrobacterium has been described by Frame et al (Frame et al, (2002) Plant Physiol,129: 13-22). Respectively taking a vector 1300-pZmUbi-g10evo-p35S-CYP78A11,

1300-pZmUbi-g10evo-pCYP78A11-CYP78A11, 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-p35S and 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-E2 (i.e. Agrobacterium containing T-DNA vector) are scribed, picked and inoculated to prepare Agrobacterium for transformation.

Taking Hi-II corn ears 8-10 days after pollination. All immature embryos (1.0-1.5 mm in size) were collected. Agrobacterium containing the T-DNA vector was co-cultured with immature embryos for 2-3 days (22 ℃). Transfer immature embryos to callus induction medium (medium containing 200mg/L Timentin for Agrobacterium killing, reference (Frame et al, (2002) Plant Physiol,129:13-22)) and dark culture at 28 ℃ for 10-14 days. All calli were transferred to selection medium with a final concentration of 2mM glyphosate (Frame et al, (2002) Plant Physiol,129:13-22) and incubated in the dark at 28 ℃ for 2-3 weeks.

All tissues were transferred to fresh selection medium containing 2mM glyphosate at final concentration and incubated at 28 ℃ for 2-3 weeks in the dark. Then, all screened viable embryonic tissues were transferred to regeneration medium (Frame et al, (2002) Plant Physiol,129:13-22), and cultured in the dark at 28 ℃ for 10-14 days, one strain per dish. Transferring the embryonic tissue to a fresh regeneration medium, and culturing for 10-14 days at 26 ℃ by illumination. All fully developed plants were transferred to rooting medium (Frame et al, (2002) Plant Physiol,129:13-22), cultured under light at 26 ℃ until roots were fully developed, and then transplanted to greenhouse for single Plant culture, and the herbicide resistance of transgenic maize was tested. Spraying a 41% glyphosate aqueous solution diluted by 300 times and used for Monsanto, wherein after 7 days, leaves are yellow, and withered leaves are negative; the plants are positive plants which grow in the same way as the plants which grow in the.

Example 3, vector 1300-pZmUbi-g10evo-p35S-CYP78A11 transgenic maize had significantly increased plant height and leaf width compared to the non-transgenic control, but there was no significant increase in individual plant yield.

In order to study the effect of overexpression of the rice CYP78A11 gene on the growth and development of maize, a vector 1300-pZmUbi-g10evo-p35S-CYP78A11 which mediates the expression of the CYP78A11 gene by a constitutive promoter p35S was constructed by the method of example 1, and 34 transgenic maize lines (named TS) of the vector were obtained by the method of example 2. T0 generation plants were transplanted into the greenhouse and the vigor and plant height of the mature transgenic maize-containing plants and non-transgenic control plants were analyzed in comparison. 21 of the 34 CYP78A1 over-expressed plants obtained by the method have significantly enhanced growth potential compared with a control, significantly increased plant height compared with the control and significantly widened leaves. The plant height, leaf width and individual plant yield of the maturity of two typical lines are shown in Table 1.

Table 1:

line of	Plant height (cm)	Blade width (cm)	Yield per plant (g)
				CK	214.6±8.7	15.1±3.9	132±2.3
TS5	267.4±9.9	23.7±5.5	135±2.8
				TS7	272.1±9.5	25.1±6.2	133±2.9

CK is a non-transgenic control maize plant, a transgenic recipient maize; TS is a T-DNA transformed plant of the vector 1300-pZmUbi-g10evo-p35S-CYP78A11, wherein the number (5, 7) behind the TS is a random number for different lines so as to distinguish different transformation events.

As can be seen from table 1, transgenic maize plants had significantly increased plant height and leaf width compared to the non-transgenic control, with a maximum increase in plant height of 26.8% and a maximum increase in leaf width of 66%, but there was no significant difference in individual plant yield. Our analysis probably resulted from the fact that CYP78a11 was overexpressed in too great a magnitude and inappropriately in time, space, and amount due to the constitutive promoter we used, resulting in a significant increase in biomass, but not in increased maize yield. Therefore, we considered whether corn could be improved by regulating the expression level of CYP78a11 gene by other means of gene expression regulation.

Example 4 the vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11 transgenic maize has no significant difference in plant height, leaf width and single plant yield from non-transgenic controls.

Because the constitutive promoter p35S is used for mediating the expression of the rice CYP78A11 gene in the corn to obviously improve the biomass and leaf width of the corn, but the yield of the corn is not obviously improved, in order to optimize the expression regulation mode of the CYP78A11 gene, an expression vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11 which utilizes the CYP78A11 gene self promoter to mediate the over-expression of the CYP78A11 gene is constructed by the method of the embodiment 1, and 45 transgenic corn strains (named as TC) of the vector are obtained by the method of the embodiment 2. T0 generation plants were transplanted into the greenhouse and the vigor and plant height of the mature transgenic maize-containing plants and non-transgenic control plants were analyzed in comparison. We obtained 45 CYP78a11 overexpressing plants with no significant difference in vigor, plant height, leaf width and individual yield compared to non-transgenic controls. The plant height, leaf width and individual plant yield of the maturity of two typical lines are shown in Table 2.

Table 2:

line of	Plant height (cm)	Blade width (cm)	Yield per plant (g)
				CK	214.6±8.7	15.1±3.9	132±2.3
TC12	216.1±9.1	16.4±4.3	131±2.5
				TC23	219.7±9.2	15.7±3.8	135±2.8

CK is a non-transgenic control maize plant, a transgenic recipient maize; TC is T-DNA transformed plant of vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11, wherein the number (12, 23) following the TS is randomly numbered for different lines to facilitate differentiation of different transformation events.

As can be seen from Table 2, there were no significant differences in plant height, leaf width and individual yield of the transgenic maize plants compared to the non-transgenic controls. Our analysis is probably due to the fact that the promoter pCYP78A11 of CYP78A11 itself is used, and the overexpression amplitude is too small to cause the change of the characters such as biomass, plant height and single plant yield of the transgenic maize plants. Therefore, we considered whether corn could be improved by controlling the expression level of CYP78a11 gene in a more suitable manner of gene expression regulation.

Example 5 vectors 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-p35S and 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-E2 transgenic maize gave significantly increased individual yields compared to controls.

Since the constitutive promoter p35S or the tissue-specific promoter pCYP78A11 of CYP78A11 per se can not be used for mediating the expression of the rice CYP78A11 gene in corn to remarkably improve the yield of a single plant of a transgenic corn plant, in order to optimize the expression regulation mode of the CYP78A11 gene, 1300-pZUbi-g 10evo-pCYP78A11-CYP78A11-p35S and 1300-pZUbi-g 10evo-pCYP78A11-CYP78A 11-CYP78A11-E2 which jointly mediate the overexpression of the CYP78A11 gene by using the promoter and an enhancer of the CYP78A11 gene are constructed by the method of example 1, and 31 transgenic corn lines (named TE1) and 37 (named TE2) of the vectors are respectively obtained by the method of example 2. T0 generation plants were transplanted into the greenhouse and the vigor and plant height of the mature transgenic maize-containing plants and non-transgenic control plants were analyzed in comparison. Of the 31 TE1 plants we obtained, 18 plants had significant differences in growth vigor, plant height, leaf width and individual yield compared to non-transgenic controls; of the 37 TE2 plants obtained, 22 plants had significant differences in vigor, plant height, leaf width and individual yield compared to the non-transgenic control. The plant height, leaf width and individual plant yield of four typical lines in the mature period are shown in Table 3.

Table 3:

line of	Plant height (cm)	Blade width (cm)	Yield per plant (g)
				CK	214.6±8.7	15.1±3.9	132±2.3
TE1-9	234.1±9.0	19.3±4.1	143±2.9
				TE1-18	242.1±9.2	18.6±4.2	141±2.5
TE2-22	239.4±8.9	19.2±4.3	148±2.9
				TE2-31	234.9±9.1	19.5±4.5	141±2.7

CK is a non-transgenic control maize plant, a transgenic recipient maize; TE1 is a T-DNA transformed plant of a vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-p35S, wherein the number (9, 18) behind TE1 is a random number for different lines so as to distinguish different transformation events; TE2 was a T-DNA transformed plant with the vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-E2, where the numbers (22, 31) following TE2 were randomly numbered for different lines to facilitate differentiation of different transformation events.

As can be seen from Table 3, the transgenic maize plants all have significant differences in plant height, leaf width and individual plant yield compared to the non-transgenic controls. Compared with a control group, the transgenic corn with the vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-p35S has the advantages that the plant height is increased by 12.8 percent at most, the leaf width is increased by 27.8 percent at most, and the yield of a single plant is increased by 8.3 percent at most; compared with a control group, the transgenic corn with the vector 1300-pZmUbi-g10evo-pCYP78A11-CYP78A11-E2 has the advantages that the plant height is increased by 9.1 percent at most, the leaf width is increased by 29.1 percent at most, and the yield of a single plant is increased by 12.1 percent at most. Therefore, the method for regulating and controlling the CYP78A11 gene expression by combining the promoter and the enhancer can be used for improving the corn yield-related traits and further improving the yield of a corn single plant.

Sequence listing

<110> Zhejiang university

<120> method for specifically regulating and controlling rice CYP78A11 gene expression and plant transformation vector

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1936

<212> DNA

<213> Unknown (Unknown)

<400> 1

ggtaccgatt agataaagcc acttccacga caggccttct cctgtaacca aatatgaggt 60

cttgcgagac gggttgctct aacctatcac acgtgacccg tcaccacaat atctagtttt 120

atagtagtag gcgtctttct cgaacaacgc ggggtaaacg tttaaaataa ccctataatt 180

atagtccgta cgtatatttt ttgagatccg agcaatgagt aaaagtttta aatacttagc 240

tacatattaa cgttcaccag taaaattaca ccaacatgta atcaaacaac aaagcaaaat 300

gtggtcagat taaatcaggt gagtattatc ggtggcccga gtaacgcaag tagggcgtgg 360

ggagctatcc agacaagcaa acaaacaact atatgggtcc cggcttaaaa catgtagggt 420

aggcaagtgt actctctctg ttctctcgag tagcaatcaa agctaacact tgttctctct 480

taatgttttt tttcttgaca ttaatcctac ctagtatatt ttaaaataaa tgatgagatt 540

ggtttttaac catttgtttt tctcaaaaaa tagtgtatat ataaataata tataaaaatc 600

acttcggtga aaaccattta atagaagaag ctttaagaaa aaacaagtaa acatattttt 660

aaaattttat aataattaat actccctccg ttttttaata gatgacgcca ttgacttttt 720

ctcacatatt tgaccattcg tcttattaga aaaatttaca taattacaat ttattttgtt 780

atgagttgtt ttatcactca tagtacttta agtgtgattt atatcatata tatttgcata 840

aaatttttga ataagacgaa tggtcaaaca tgtgacaaaa agtcaacggc gttatctatt 900

aaaaaacgga ggtagtaatt aattaatcat aaacaaatgt cttatctcgt tttactttca 960

acggagcgga gttaccacac gtccaccccc aaaacgaact ctgcctaaat gtagtactac 1020

ctccattata aaatataagc attttttgtc tagatttaaa gctaaaaatg cttatatttt 1080

tgggacggag ggagtatata gcactatagg cctatttaga tccctagtaa aattttacac 1140

cctgtcacat cgagtgtttg gacacaaaca taaagtatta aatataaaaa aataactaat 1200

tatacagatt gcgtgtaaat tgcgagacaa atcttttaag cctaattaca tcatgatttg 1260

acaatgttgt gctacagtaa acatttacta ataatagatt aattaggctt aataaattta 1320

tctcgcggtt tacaggtgga ttctgtaatt tattttgtta ttagactata tttaatattt 1380

caaatgtgtg cctgtatatc tgatgtggca cgcaaaaact ttacacttct ggatctaaat 1440

acaagtcgaa aaatgatgaa taattatata ccgttgtttt caaaatgtta aatggtatat 1500

tactactccc tccgtactcg taaagtaagt tgttttggac agcgacatgg tctctaaaat 1560

acaactttga cttcttattt ctataaaaat atttaataaa aagtgatata tgtatatttt 1620

tatgaaagta tttttcaaga caaatctatt catataattt tacattttca aattcaacaa 1680

cttgagagtg atttatgatt tatatttgta aagtttgatt taaacattat acgaaacaac 1740

tttctttacg agtagtacgg agggagtaca cgtgacatat ccacattttc atcatccttt 1800

tattttaggc cattctggtc acccctataa ataccacctc cacccaatcc ttcccacccc 1860

acttcatcac aagccaaaca gcccaactcc cgagctcgag ccacacaagc aaaaccacaa 1920

acacctggat ccaaca 1936

<210> 2

<211> 1754

<212> DNA

<213> Unknown (Unknown)

<400> 2

atggcaatgg ccaccgccac cgcctcctcc tgcgtcgacg ccacgtggtg ggcgtacgcc 60

ctcccggcgc tcctcggcgc cgacaccctc tgcgcccacc cggcgctgct cgccggcgcc 120

gtcctcctgg ccttcgccac cgccgcggtg ctcgcctggg ccgcgtcccc cggcgggccg 180

gcgtgggcgc acggccgcgg ccgcctcggc gcgacgccca tcgaggggcc ccgggggctc 240

cccgtgttcg gcagcatctt cgcgctctcc cggggcctcc cgcaccgcgc gctcgacgcg 300

atgtcgcgcg acgcggcggc gccacgggcg agggagctca tggcgttctc cgtcggggag 360

acgccggcgg tggtgtcgtc gtgcccggcg acggcgaggg aggtgctcgc gcacccgtcg 420

ttcgccgacc gcccgctgaa gcgctcggcg cgggagctgc tgttcgcgcg cgccatcggg 480

ttcgccccca gcggcgagta ctggcgcctc ctccgccgca tcgcctccac ccacctcttc 540

tcccctcgcc gcgtcgccgc gcacgagccg gggcgccagg ccgacgccac ggcgatgctg 600

tccgccatgg ccgccgagca gtccgccacc ggcgccgtcg tgctccgccc ccacctccag 660

gccgccgcgc tcaacaacat catgggcagc gtgttcggcc ggcgctacga cgtctcctcc 720

tcctccggcg ccgccgccga cgaggccgag cagctcaaga gcatggtgcg cgaggggttc 780

gagctcctcg gcgcgttcaa ctggtccgac cacctcccat ggctcgccca cctctacgac 840

cccaaccacg tcgcccgccg ctgcgccgcg ctcgtccccc gcgtccaggc gttcgtccgc 900

ggcgtcatcc gcgaccaccg cctccgccgc gactcctcct ccaccgccgc cgacaatgcc 960

gacttcgtcg acgtcctcct ctccctcgag gcccacgaga acctcgccga ggacgacatg 1020

gtcgccgtcc tctgggtaaa aaaaaaaaaa aaaaaacaaa ttctactcaa acatttcaaa 1080

ctcaaatgtt tttttaaaaa tgtttttgtg tattttggca ggagatgata tttcgtggga 1140

cggacacgac ggcgttggtg acggagtggt gcatggcgga ggtggtgagg aacccggcgg 1200

tgcaggcgag gctgagggcg gaggtggacg cggcggtggg cggcgacggg tgtcccagcg 1260

acggcgacgt ggcgcggatg ccgtacctgc aggcggtggt gaaggagacg ctgagggcgc 1320

acccgccggg gccgctgctg agctgggcgc ggctggccac cgccgacgtg gggctcgcca 1380

acggcatggt ggtgccggcg ggcacgacgg cgatggtgaa catgtgggcc atcacccacg 1440

acggcgaggt gtgggccgac ccggaggcgt tcgcgccgga gcggttcatc ccgtcggagg 1500

gcggcgccga cgtcgacgtc cgcggcggcg acctccgcct ggcgccgttc ggcgccgggc 1560

gccgcgtctg ccccggcaag aacctcggcc tcgccaccgt caccctctgg gtcgcccgcc 1620

tcgtccacgc cttcgactgg ttcctccccg acggctcgcc gccggtgtcc ctcgacgagg 1680

tcctcaagct ctccctcgag atgaagaccc ctctcgccgc cgccgccacc ccccgccgcc 1740

gccgcgccgc ctga 1754

<210> 3

<211> 555

<212> PRT

<213> Unknown (Unknown)

<400> 3

Met Ala Met Ala Thr Ala Thr Ala Ser Ser Cys Val Asp Ala Thr Trp

1 5 10 15

Trp Ala Tyr Ala Leu Pro Ala Leu Leu Gly Ala Asp Thr Leu Cys Ala

20 25 30

His Pro Ala Leu Leu Ala Gly Ala Val Leu Leu Ala Phe Ala Thr Ala

35 40 45

Ala Val Leu Ala Trp Ala Ala Ser Pro Gly Gly Pro Ala Trp Ala His

50 55 60

Gly Arg Gly Arg Leu Gly Ala Thr Pro Ile Glu Gly Pro Arg Gly Leu

65 70 75 80

Pro Val Phe Gly Ser Ile Phe Ala Leu Ser Arg Gly Leu Pro His Arg

85 90 95

Ala Leu Asp Ala Met Ser Arg Asp Ala Ala Ala Pro Arg Ala Arg Glu

100 105 110

Leu Met Ala Phe Ser Val Gly Glu Thr Pro Ala Val Val Ser Ser Cys

115 120 125

Pro Ala Thr Ala Arg Glu Val Leu Ala His Pro Ser Phe Ala Asp Arg

130 135 140

Pro Leu Lys Arg Ser Ala Arg Glu Leu Leu Phe Ala Arg Ala Ile Gly

145 150 155 160

Phe Ala Pro Ser Gly Glu Tyr Trp Arg Leu Leu Arg Arg Ile Ala Ser

165 170 175

Thr His Leu Phe Ser Pro Arg Arg Val Ala Ala His Glu Pro Gly Arg

180 185 190

Gln Ala Asp Ala Thr Ala Met Leu Ser Ala Met Ala Ala Glu Gln Ser

195 200 205

Ala Thr Gly Ala Val Val Leu Arg Pro His Leu Gln Ala Ala Ala Leu

210 215 220

Asn Asn Ile Met Gly Ser Val Phe Gly Arg Arg Tyr Asp Val Ser Ser

225 230 235 240

Ser Ser Gly Ala Ala Ala Asp Glu Ala Glu Gln Leu Lys Ser Met Val

245 250 255

Arg Glu Gly Phe Glu Leu Leu Gly Ala Phe Asn Trp Ser Asp His Leu

260 265 270

Pro Trp Leu Ala His Leu Tyr Asp Pro Asn His Val Ala Arg Arg Cys

275 280 285

Ala Ala Leu Val Pro Arg Val Gln Ala Phe Val Arg Gly Val Ile Arg

290 295 300

Asp His Arg Leu Arg Arg Asp Ser Ser Ser Thr Ala Ala Asp Asn Ala

305 310 315 320

Asp Phe Val Asp Val Leu Leu Ser Leu Glu Ala His Glu Asn Leu Ala

325 330 335

Glu Asp Asp Met Val Ala Val Leu Trp Glu Met Ile Phe Arg Gly Thr

340 345 350

Asp Thr Thr Ala Leu Val Thr Glu Trp Cys Met Ala Glu Val Val Arg

355 360 365

Asn Pro Ala Val Gln Ala Arg Leu Arg Ala Glu Val Asp Ala Ala Val

370 375 380

Gly Gly Asp Gly Cys Pro Ser Asp Gly Asp Val Ala Arg Met Pro Tyr

385 390 395 400

Leu Gln Ala Val Val Lys Glu Thr Leu Arg Ala His Pro Pro Gly Pro

405 410 415

Leu Leu Ser Trp Ala Arg Leu Ala Thr Ala Asp Val Gly Leu Ala Asn

420 425 430

Gly Met Val Val Pro Ala Gly Thr Thr Ala Met Val Asn Met Trp Ala

435 440 445

Ile Thr His Asp Gly Glu Val Trp Ala Asp Pro Glu Ala Phe Ala Pro

450 455 460

Glu Arg Phe Ile Pro Ser Glu Gly Gly Ala Asp Val Asp Val Arg Gly

465 470 475 480

Gly Asp Leu Arg Leu Ala Pro Phe Gly Ala Gly Arg Arg Val Cys Pro

485 490 495

Gly Lys Asn Leu Gly Leu Ala Thr Val Thr Leu Trp Val Ala Arg Leu

500 505 510

Val His Ala Phe Asp Trp Phe Leu Pro Asp Gly Ser Pro Pro Val Ser

515 520 525

Leu Asp Glu Val Leu Lys Leu Ser Leu Glu Met Lys Thr Pro Leu Ala

530 535 540

Ala Ala Ala Thr Pro Arg Arg Arg Arg Ala Ala

545 550 555

<210> 4

<211> 805

<212> DNA

<213> Unknown (Unknown)

<400> 4

aatcttgagc tcatggtgga gcacgacact ctcgtctact ccaagaatat caaagataca 60

gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc 120

ctcggattcc attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt 180

ggcacctaca aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc 240

gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt 300

ccaaccacgt cttcaaagca agtggattga tgtgataaca tggtggagca cgacactctc 360

gtctactcca agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt 420

caacaaaggg taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc 480

atcaaaagga cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga 540

aaggctatcg ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg 600

aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt 660

gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca agaccttcct 720

ctatataagg aagttcattt catttggaga ggacacgctg aaatcaccag tctctctcta 780

caaatctatc tctggatccg agctc 805

<210> 5

<211> 341

<212> DNA

<213> Unknown (Unknown)

<400> 5

gagctccatg gtggagcacg acactctcgt ctactccaag aatatcaaag atacagtctc 60

agaagaccaa agggctattg agacttttca acaaagggta atatcgggaa acctcctcgg 120

attccattgc ccagctatct gtcacttcat caaaaggaca gtagaaaagg aaggtggcac 180

ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt caagatgcct ctgccgacag 240

tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac 300

cacgtcttca aagcaagtgg attgatgtga taacagagct c 341

<210> 6

<211> 429

<212> DNA

<213> Unknown (Unknown)

<400> 6

tctcgccgac cacgtcttac ctacgctaat taagcttcgt tttagtctcg tcttcctcct 60

tgagcttgca agagactagg gtttgatgac gtcatccgat gatcgtgctt agttccaatt 120

ttggcagctg cgtgtgtgga gctaagccac gaacagatat ataattaatc tcatatatat 180

atatgtagat gattaattat gtatgtatgt atgaatgaat gtaattaggg cttgatttgt 240

gtgacgaaca tgtcgcgtcg tcatcatttt gataaatggc tggctgtagc tagctacctc 300

gccccccatt atatttgtac cagttgatgt aacgtgacgt aacgtaatgt aatgtaatgt 360

aatgtaatgt aatgtttgat catggattaa ttaattatta ctaatcatgc cgtgtgagct 420

tgtgagatg 429

<210> 7

<211> 3571

<212> DNA

<213> Unknown (Unknown)

<400> 7

ggtaccgagc tcgcatgcct acagtgcagc gtgacccggt cgtgcccctc tctagagata 60

atgagcattg catgtctaag ttataaaaaa ttaccacata ttttttttgt cacacttgtt 120

tgaagtgcag tttatctatc tttatacata tatttaaact ttactctacg aataatataa 180

tctatagtac tacaataata tcagtgtttt agagaatcat ataaatgaac agttagacat 240

ggtctaaagg acaattgagt attttgacaa caggactcta cagttttatc tttttagtgt 300

gcatgtgttc tccttttttt ttgcaaatag cttcacctat ataatacttc atccatttta 360

ttagtacatc catttagggt ttagggttaa tggtttttat agactaattt ttttagtaca 420

tctattttat tctattttag cctctaaatt aagaaaacta aaactctatt ttagtttttt 480

tatttaataa tttagatata aaatagaata aaataaagtg actaaaaatt aaacaaatac 540

cctttaagaa attaaaaaaa ctaaggaaac atttttcttg tttcgagtag ataatgccag 600

cctgttaaac gccgtcgacg agtctaacgg acaccaacca gcgaaccagc agcgtcgcgt 660

cgggccaagc gaagcagacg gcacggcatc tctgtcgctg cctctggacc cctctcgaga 720

gttccgctcc accgttggac ttgctccgct gtcggcatcc agaaattgcg tggcggagcg 780

gcagacgtga gccggcacgg caggcggcct cctcctcctc tcacggcacg gcagctacgg 840

gggattcctt tcccaccgct ccttcgcttt cccttcctcg cccgccgtaa taaatagaca 900

ccccctccac accctctttc cccaacctcg tgttgttcgg agcgcacaca cacacaacca 960

gatctccccc aaatccaccc gtcggcacct ccgcttcaag gtacgccgct cgtcctcccc 1020

ccccccccct ctctaccttc tctagatcgg cgttccggtc catggttagg gcccggtagt 1080

tctacttctg ttcatgtttg tgttagatcc gtgtttgtgt tagatccgtg ctgctagcgt 1140

tcgtacacgg atgcgacctg tacgtcagac acgttctgat tgctaacttg ccagtgtttc 1200

tctttgggga atcctgggat ggctctagcc gttccgcaga cgggatcgat ttcatgattt 1260

tttttgtttc gttgcatagg gtttggtttg cccttttcct ttatttcaat atatgccgtg 1320

cacttgtttg tcgggtcatc ttttcatgct tttttttgtc ttggttgtga tgatgtggtc 1380

tggttgggcg gtcgttctag atcggagtag aattctgttt caaactacct ggtggattta 1440

ttaattttgg atctgtatgt gtgtgccata catattcata gttacgaatt gaagatgatg 1500

gatggaaata tcgatctagg ataggtatac atgttgatgc gggttttact gatgcatata 1560

cagagatgct ttttgttcgc ttggttgtga tgatgtggtg tggttgggcg gtcgttcatt 1620

cgttctagat cggagtagaa tactgtttca aactacctgg tgtatttatt aattttggaa 1680

ctgtatgtgt gtgtcataca tcttcatagt tacgagttta agatggatgg aaatatcgat 1740

ctaggatagg tatacatgtt gatgtgggtt ttactgatgc atatacatga tggcatatgc 1800

agcatctatt catatgctct aaccttgagt acctatctat tataataaac aagtatgttt 1860

tataattatt ttgatcttga tatacttgga tgatggcata tgcagcagct atatgtggat 1920

ttttttagcc ctgccttcat acgctattta tttgcttggt actgtttctt ttgtcgatgc 1980

tcaccctgtt gtttggtgtt acttctgcag gtcgactcta gaaacaatgg cggcgaccat 2040

ggcgtccaac gctgcggctg cggctgcggt gtccctggac caggccgtgg ctgcgtcggc 2100

agcgttctcg tcgcggaagc agctgcggct gcctgccgca gcgcgcggag ggatgcgggt 2160

gcgggtgcgg gcgcggggtc ggcgggaggc ggtggtggtg gcgtccgcgt cgtcgtcgtc 2220

ggtggcagcg ccggcggcga aggctgagat gtccgacgcc ctgcccgcca ccttcgacgt 2280

gatcgtgcat ccagctcgcg aactccgcgg cgagcttcgc gctcagccat ccaagaacta 2340

caccactcgc tacctcctcg ccgctgccct cgctgagggc gagacccgcg tggtgggcgt 2400

ggctacctct gaggacgccg aggccatgct ccgctgcctc cgcgactggg gcgctggcgt 2460

ggagcttgtg ggcgatgacg ccgtgatccg cggtttcggc gctcgcccac aggccggtgt 2520

gaccctcaac ccaggcaacg ctgccgcggt ggcccgcctc ctcatgggcg tggccgctct 2580

cacctctggc accactttcg tgaccgacta cccggactcc ctcggcaagc gccctcaggg 2640

cgacctcctt gaggccctcg aacgcctcgg tgcctgggtg tcctccaacg acggtcgcct 2700

cccgatctcc gtgtccggcc cagtgcgcgg tggcaccgtg gaggtgtccg ccgagcgctc 2760

ctcccagtac gcctccgccc tcatgttcct cggccctctc ctcccggacg gactcgaact 2820

ccgcctcacc ggcgacatca agtcccacgc tccgctccgc cagacactcg acaccctctc 2880

tgacttcggc gtgcgcgcca ctgcctccga cgacctccgc cgcatctcca tcccgggtgg 2940

ccagaagtac cgcccaggcc gcgtgctcgt gccgggcgac tacccgggct ccgctgccat 3000

cctcaccgcc gctgccctcc tcccaggcga ggtgcgcctc tctaacctcc gcgagcacga 3060

cctccagggc gagaaggagg ccgtgaacgt gctccgcgag atgggcgctg acatcgtgcg 3120

cgagggcgat accctcaccg tgcgcggtgg ccgccctctc cacgccgtga ctcgcgacgg 3180

cgattccttc accgacgccg tgcaagccct caccgccgct gctgccttcg ccgagggcga 3240

caccacctgg gagaacgtgg ccactctccg cctcaaggag tgcgaccgca tctctgacac 3300

ccgcgctgag cttgagcgcc tcggcctccg cgcacgcgag accgccgact ctctctccgt 3360

gactggctct gctcacctcg ctggtggcat caccgccgac ggccacggcg accaccgcat 3420

gatcatgctc ctcaccctcc tcggcctccg cgcagacgct ccactccgca tcaccggcgc 3480

acaccacatc cgcaagtcct accctcagtt cttcgctcac ctcgaagccc tcggcgctcg 3540

cttcgagtac gctgaggcca ccgccctcga g 3571

Claims (9)

1. A method for specifically regulating CYP78A11 gene expression is characterized in that the method regulates the CYP78A11 gene expression by the common specificity of an enhancer and a promoter; the nucleotide sequence of the enhancer comprises SEQ ID NO.4 or SEQ ID NO. 5; the promoter is a CYP78A11 gene self promoter or a promoter with similar functions.

2. The method of claim 1, wherein the promoter nucleotide sequence is set forth in SEQ ID NO 1.

3. The method according to claim 1, wherein the CYP78A11 gene has the nucleotide sequence shown in SEQ ID NO. 2.

4. The method according to any one of claims 1 to 3, wherein the CYP78A11 gene expression is controlled by functionally linking the nucleotide sequence of the CYP78A11 gene with an enhancer, a promoter and a terminator to construct an expression cassette for the CYP78A11 gene; the CYP78A11 gene expression cassette is introduced into a crop plant and expressed.

5. The method of claim 4, wherein the terminator nucleotide sequence is set forth in SEQ ID NO 6.

6. The method of claim 4, wherein said crop comprises corn, wheat, barley, sorghum, rice, sugarcane, soybean, carrot, potato, cotton, sunflower, canola, oak, turf grass, pasture grass.

7. A plant transformation vector for the expression of the CYP78a11 gene according to claim 1, wherein said plant transformation vector is a binary vector comprising an expression cassette for the CYP78a11 gene.

8. The plant transformation vector of claim 7, wherein said binary vector comprises a selectable marker gene expression cassette comprising a promoter, an EPSPS leader peptide, a selectable marker gene, and a terminator.

9. The plant transformation vector of claim 8, wherein said promoter is the maize pUBI promoter, the nucleotide sequence of which is shown as 1bp-2026bp in SEQ ID NO. 7; the nucleotide sequence of the EPSPS leader peptide is shown as 2027bp-2248bp in SEQ ID NO. 7; the screening marker gene is g10evo, and the nucleotide sequence of the screening marker gene is shown as 2249bp-3536bp in SEQ ID NO. 7; the terminator is CaMV 35S terminator of cauliflower mosaic virus, and the nucleotide sequence is shown as 2120bp-2310bp in pCambia1300 vector.

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