CN104651358B - Separated tissue-specific promoter and its application from corn - Google Patents

Abstract

The invention discloses separated tissue-specific promoter and its application from corn, belong to separation and the application field of plant tissue specificity promoter.The present invention is separated from corn obtains the tissue-specific promoter that nucleotides sequence is classified as SEQ ID NO.1, and the present invention is further truncated acquisition SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, still has the function of the sequence of tissue-specific promoter shown in SEQ ID NO.5 and SEQ ID NO.6.The invention also discloses recombinant plant expression vectors and recombinant host cell containing the tissue-specific promoter.The present invention further discloses the tissue-specific promoters to improve crop seed quality, Crop Improvement character and the application for cultivating genetically modified plants new varieties etc..

Description

Tissue-specific promoter isolated from maize and its application

technical field

The invention relates to a promoter isolated from plants, in particular to a tissue-specific promoter isolated from corn (Zea mays), and the present invention also relates to a recombinant plant expression vector and a host cell containing the tissue-specific promoter, the present invention The invention further relates to their application in improving crop seed quality, improving crop traits, cultivating new plant varieties, etc., and belongs to the field of separation and application of plant tissue-specific promoters.

Background technique

The promoter is a DNA sequence that RNA polymerase specifically recognizes and binds to, and is an important cis-acting element, generally located in the upstream region of the 5' end of the structural gene. Gene regulation in higher plants is mainly carried out at the transcription level. The promoter controls the initiation time and expression level of gene expression, and also plays a decisive role in the type of RNA polymerase used. Therefore, the promoter is the key to understanding gene expression patterns and transcription regulation. The key mechanism is central to the regulation of plant gene transcription.

According to the transcription mode and function of promoters, they can be divided into three categories: constitutive promoters, tissue or organ-specific promoters and inducible promoters. At present, most of the promoters used in plant genetic engineering are constitutive promoters, which enable the high-level expression of exogenous target genes in various plant tissues and parts, but constitutive promoters also have many disadvantages, for example, they cannot be efficiently expressed in time and space. Regulate the expression of target genes, excessive consumption of intracellular material and energy (Gittins JR, Pellny TK, Hiles ER, Rosa C, Biricolti S, et al. (2000) Transgene expression driven by heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase small-subunitgene promoters in the vegetable tissues of apple(Malus pumila mill.).Planta210:232-240.); a large number of heterologous proteins or metabolites accumulate in plants, breaking the metabolic balance of plants, which is not conducive to plant growth (Robinson DJ (1996) Environmental risk assessment of releases of transgenic plants containing virus-derived inserts.Transgenic research 5:359-362.); easy to cause gene silencing or co-suppression (Kumpatla SP, Chandrasekharan MB, Iyer LM, Guofu L, Hall TC ( 1998) Genome intruder scanning and modulation systems and transgene silencing. Trends in Plant Science 3:97-104; Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJ (2000) Transcriptional silencing and promotermethylation triggered by double-stranded RNA. EMBO J 19:5194-5201.). Therefore, scientists are constantly looking for more effective tissue or organ-specific promoters to replace constitutive promoters in order to more precisely regulate the expression of foreign genes.

The gene transcription process under the regulation of tissue or organ-specific promoters generally only occurs in some specific tissues or organs. Tissue or organ-specific expression promoters can regulate the expression of exogenous genes more economically and effectively, and play a specific role in specific required parts, which can not only increase the expression abundance of exogenous genes, but also minimize biological energy consumption , so as not to affect the normal growth of the plant.

Corn (Zea mays) is the largest food crop in China and an important genetically modified crop. Maize tissue-specific promoters can be divided into root, stem, leaf, flower, embryo, endosperm, fruit, xylem, green tissue and other tissue or organ-specific promoters. Each type of tissue-specific promoter has some special characteristics. functional element. The tissue-specific promoter is isolated from maize, and the tissue-specific promoter can be used to drive the specific high-efficiency expression of the target gene in the plant tissue, which can be used for molecular improvement of maize or production of new maize varieties with special uses, etc. Is of great significance.

Contents of the invention

One of the objects of the present invention is to provide a tissue-specific promoter isolated from maize (Zea mays).

The second object of the present invention is to provide a recombinant expression vector containing the above-mentioned tissue-specific promoter and a host cell containing the recombinant expression vector.

The third object of the present invention is to apply the tissue-specific promoter and the recombinant expression vector containing the tissue-specific promoter to construct transgenic plants, improve crop seed traits, and breed new plant varieties with excellent traits.

To achieve the above object, the present invention firstly provides a tissue-specific promoter isolated from corn (Zea mays), the polynucleotide sequence of which is shown in (a), (b), (c) or (d) :

(a), the polynucleotide sequence shown in SEQ ID NO.1; or

(b), a polynucleotide capable of hybridizing to the complementary sequence of SEQ ID NO.1 under stringent hybridization conditions, the polynucleotide still has the function or activity of a tissue-specific promoter; or

(c) A polynucleotide sequence with at least 60% homology to the polynucleotide sequence of SEQ ID NO.1, and the polynucleotide has the function or activity of a tissue-specific promoter; preferably, the polynucleotide sequence with SEQ ID NO.1 The polynucleotide sequence of ID NO.1 has at least 80% homology to the polynucleotide sequence, and the polynucleotide has the function or activity of a tissue-specific promoter; more preferably, the A polynucleotide sequence with at least 90% homology to the polynucleotide sequence, and the polynucleotide has the function or activity of a tissue-specific promoter; most preferably, it is at least the same as the polynucleotide sequence of SEQ ID NO.1 A polynucleotide sequence with more than 90% homology, and the polynucleotide has the function or activity of a tissue-specific promoter; or

(d) A polynucleotide variant obtained by deleting, substituting or inserting one or more bases on the basis of SEQ ID NO.1, the polynucleotide variant comprising the sequence shown in SEQ ID NO.2, And the polynucleotide variant still has the function or activity of a tissue-specific promoter.

In the present invention, the 181bp promoter deletion fragment (the fragment between -181 and +1) shown in SEQ ID NO.2 obtained by gradually deleting the partial sequence from the polynucleotide sequence shown in SEQ ID NO.1 from the 5' end .

In the present invention, the polynucleotide shown in SEQ ID NO.1 or SEQ ID NO.2 is operably connected with the GUS gene to construct a recombinant plant expression vector respectively; corn is used as the acceptor material, and the transient expression is transformed by gene gun The system is used to verify the function of the promoter, and the test results show that the GUS gene driven by SEQ ID NO.1 or SEQ ID NO.2 is only expressed specifically in the embryo of corn seeds, and is expressed in the endosperm, roots, stems, leaves, etc. of corn seeds. There is no GUS expression activity in the tissue site; the test results confirm that the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 isolated from maize in the present invention is a tissue-specific promoter.

The present invention further gradually deletes the full-length promoter shown in SEQ ID NO.1 from the 5' end to obtain four truncated promoter fragments P _85502-4 (SEQ ID NO.3), P _85502-0 (SEQ ID NO.4), P _85502-2 (SEQ ID NO.5), P _85502-6 (SEQ ID NO.6); the four truncated sequences were respectively connected to the expression vector with reporter gene to construct The stable expression vectors p85502-4G3, p85502-0G3, p85502-2G3 and p85502-6G3 were obtained; the stable expression vector p85502-0G3 and the stable expression vector p85502-2G3 were respectively transformed into maize materials by the method mediated by Agrobacterium, and the stable expression The vector p85502-4G3 and the stable expression vector p85502-6G3 were respectively transformed into Arabidopsis thaliana according to the Agrobacterium-mediated method, and stably transformed transgenic plants were obtained; the results of functional verification showed that the promoter deletion fragment P _85502-0 still had The function of initiating reporter gene expression in tissue, the promoter deletion fragment P _85502-0 is an embryo tissue-specific promoter with reduced promoter activity under normal physiological conditions, but exhibits inducible expression in leaf sheath tissue under mechanical injury induction characteristics. From the staining results of various tissues of transgenic Arabidopsis thaliana, it can be seen that the shorter P ₈₅₅₀₂ deletion fragment still has promoter activity, but P _85502-4 still maintains the characteristic of inducible expression compared with P _85502-6 .

The result of driving the expression of the reporter gene GUS in transgenic maize/Arabidopsis thaliana shows that these four promoter deletion fragments still have promoter activity, but their activation strength and tissue specificity have some changes. Therefore, the present invention provides a deletion fragment of the tissue-specific promoter shown in SEQ ID NO.2, whose polynucleotide sequence is shown in (a), (b), (c) or (d):

(a), the polynucleotide sequence shown in SEQ ID NO.2; or

(b), a polynucleotide capable of hybridizing to the complementary sequence of SEQ ID NO.2 under stringent hybridization conditions, and the polynucleotide still has the function or activity of a promoter; or

(c) A polynucleotide sequence having at least 60% homology with the polynucleotide sequence of SEQ ID NO.2, and the polynucleotide has the function or activity of a promoter; preferably, it is identical to the sequence of SEQ ID NO. The polynucleotide sequence of 2 has at least 80% homology polynucleotide sequence, and the polynucleotide has the function or activity of a promoter; more preferably, it has at least 90% homology with the polynucleotide sequence of SEQ ID NO.2 A polynucleotide sequence with more than % homology, and the polynucleotide has the function or activity of a promoter; most preferably, a polynucleotide sequence with at least 95% homology with the polynucleotide sequence of SEQ ID NO.2 acid sequence, and the polynucleotide has the function or activity of a promoter; or

(d) A polynucleotide variant obtained by deleting, substituting or inserting one or more bases on the basis of SEQ ID NO.2, the polynucleotide variant contains the 47th- The 6bp base at position 53, and the polynucleotide variant still has the function or activity of a promoter.

The present invention provides the promoter deletion fragment shown in SEQ ID NO.3, its polynucleotide sequence is shown in (a), (b), (c) or (d):

(a), the polynucleotide sequence shown in SEQ ID NO.3; or

(b), a polynucleotide capable of hybridizing to the complementary sequence of SEQ ID NO.3 under stringent hybridization conditions, and the polynucleotide still has the function or activity of a promoter; or

(c), a polynucleotide sequence having at least 60% homology with the polynucleotide sequence of SEQ ID NO.3, and the polynucleotide has the function or activity of a promoter; preferably, with the sequence of SEQ ID NO. The polynucleotide sequence of 3 has at least 80% homology polynucleotide sequence, and the polynucleotide has the function or activity of a promoter; more preferably, it has at least 90% homology with the polynucleotide sequence of SEQ ID NO.3 A polynucleotide sequence with more than % homology, and the polynucleotide has the function or activity of a promoter; most preferably, a polynucleotide sequence with at least 95% homology with the polynucleotide sequence of SEQ ID NO.3 acid sequence, and the polynucleotide has the function or activity of a promoter; or

(d) A polynucleotide variant obtained by deleting, substituting or inserting one or more bases on the basis of SEQ ID NO.3, and the polynucleotide variant still has the function or activity of a promoter.

The present invention also provides the promoter deletion fragment shown in SEQ ID NO.4, its polynucleotide sequence is shown in (a), (b), (c) or (d):

(a), the polynucleotide sequence shown in SEQ ID NO.4; or

(b), a polynucleotide capable of hybridizing to the complementary sequence of SEQ ID NO.4 under stringent hybridization conditions, and the polynucleotide still has the function or activity of a promoter; or

(c), a polynucleotide sequence having at least 60% homology with the polynucleotide sequence of SEQ ID NO.4, and the polynucleotide has the function or activity of a promoter; preferably, with the sequence of SEQ ID NO. The polynucleotide sequence of 4 has at least 80% homology polynucleotide sequence, and the polynucleotide has the function or activity of a promoter; more preferably, it has at least 90% homology with the polynucleotide sequence of SEQ ID NO.4 A polynucleotide sequence with more than % homology, and the polynucleotide has the function or activity of a promoter; most preferably, a polynucleotide sequence with at least 95% homology with the polynucleotide sequence of SEQ ID NO.4 acid sequence, and the polynucleotide has the function or activity of a promoter; or

(d) A polynucleotide variant obtained by deleting, substituting or inserting one or more bases on the basis of SEQ ID NO.4, and the polynucleotide variant still has the function or activity of a promoter.

The present invention further provides the promoter deletion fragment shown in SEQ ID NO.5, its polynucleotide sequence is shown in (a), (b), (c) or (d):

(a), the polynucleotide sequence shown in SEQ ID NO.5; or

(b), a polynucleotide capable of hybridizing to the complementary sequence of SEQ ID NO.5 under stringent hybridization conditions, and the polynucleotide still has the function or activity of a promoter; or

(c) A polynucleotide sequence having at least 60% homology with the polynucleotide sequence of SEQ ID NO.5, and the polynucleotide has the function or activity of a promoter; preferably, with the sequence of SEQ ID NO.5. The polynucleotide sequence of 5 has at least 80% homology polynucleotide sequence, and the polynucleotide has the function or activity of a promoter; more preferably, it has at least 90% homology with the polynucleotide sequence of SEQ ID NO.5 A polynucleotide sequence with more than % homology, and the polynucleotide has the function or activity of a promoter; most preferably, a polynucleotide with at least 95% or more homology with the polynucleotide sequence of SEQ ID NO.5 acid sequence, and the polynucleotide has the function or activity of a promoter; or

(d) A polynucleotide variant obtained by deleting, substituting or inserting one or more bases on the basis of SEQ ID NO.5, and the polynucleotide variant still has the function or activity of a promoter.

The present invention provides the promoter deletion fragment shown in SEQ ID NO.6, its polynucleotide sequence is shown in (a), (b), (c) or (d):

(a), the polynucleotide sequence shown in SEQ ID NO.6; or

(b), a polynucleotide capable of hybridizing to the complementary sequence of SEQ ID NO.6 under stringent hybridization conditions, and the polynucleotide still has the function or activity of a promoter; or

(c) A polynucleotide sequence having at least 60% homology with the polynucleotide sequence of SEQ ID NO.6, and the polynucleotide has the function or activity of a promoter; preferably, with the sequence of SEQ ID NO. The polynucleotide sequence of 6 has at least 80% homology polynucleotide sequence, and the polynucleotide has the function or activity of a promoter; more preferably, it has at least 90% homology with the polynucleotide sequence of SEQ ID NO.6 A polynucleotide sequence with more than % homology, and the polynucleotide has the function or activity of a promoter; most preferably, a polynucleotide sequence with at least 95% homology with the polynucleotide sequence of SEQ ID NO.6 acid sequence, and the polynucleotide has the function or activity of a promoter; or

(d) A polynucleotide variant obtained by deleting, substituting or inserting one or more bases on the basis of SEQ ID NO.6, and the polynucleotide variant still has the function or activity of a promoter.

The "replacement" mentioned in the present invention refers to the replacement of other bases with different bases; the "deletion" refers to the lack of one or more bases; the "insertion" refers to nucleotides The change in the natural molecule is due to the addition of one or more bases.

The present invention further provides a recombinant plant expression vector containing the tissue-specific promoter and a host cell containing the recombinant plant expression vector.

The tissue-specific promoter described in the present invention is operably linked with the heterologous DNA sequence to be transcribed to obtain a recombinant plant expression vector that specifically expresses the heterologous DNA sequence in the crop seed embryo.

The recombinant plant expression vector may also contain a selectable marker gene.

In addition, the tissue-specific promoters of the invention can be operably linked to marker sequences to determine the activity of marker sequences, which typically include genes that confer antibiotic resistance or herbicide resistance, such as: tetracycline resistance gene, hygromycin resistance gene, glufosinate or glufosinate resistance gene, etc.

Any plant transformation method can be used to introduce the recombinant plant expression vector constructed in the present invention into the cells, tissues or organs of the target plant to obtain a transformant; then the transformant can be regenerated by a plant tissue culture method to obtain a complete plant and its clone or its progeny; the transformation method includes: Agrobacterium-mediated transformation, protoplast transformation, Ti plasmid, Ri plasmid, plant virus vector, microinjection, electroporation, particle bombardment, etc.; the target plant includes Monocotyledonous plants, dicotyledonous plants; preferably, the target plants are grasses, for example, corn, rice, barley, wheat, sorghum and other crops.

The isolated tissue-specific promoter of the present invention is widely used in improving the quality of crop seeds, improving plant traits, cultivating new plant varieties and the like.

The tissue-specific promoter of the present invention is operably linked to the target heterologous DNA sequence to be transformed, which can guide or regulate the transcription or expression of the target heterologous gene to be transformed in the embryo of the plant seed, and obtain the expected traits of transgenic plants or new plant varieties; for example, the tissue-specific promoter of the present invention is operably linked to the target heterologous DNA sequence to be transformed (wherein, the heterologous DNA sequence to be transformed is also connected to the 3' The non-coding region is operably connected, and the 3' non-coding region may contain a terminator sequence, an mRNA cutting sequence, etc.) to obtain a recombinant plant expression vector capable of expressing the heterologous DNA sequence of interest in plant seed embryos. The target heterologous DNA sequence to be transformed is not limited, and may be a regulatory gene, an antisense gene of a regulatory gene, or a small RNA capable of interfering with endogenous gene expression; the described heterologous DNA sequence to be transformed may be from a non- A nucleic acid molecule or gene of the target gene species, or a nucleic acid molecule or gene that has been artificially engineered or modified from or exists in the same species.

Generally speaking, the heterologous DNA sequences to be transformed are mostly related genes that improve crop seed quality, increase crop resistance to environmental stress, improve crop traits or metabolism, for example, can be: related genes that improve plant physiology, growth and development , related genes that increase yield, strengthen nutrition, improve resistance to environmental stress (resistance to diseases and insect pests, salt and alkali resistance, drought resistance, high temperature resistance, frost resistance, etc.) and other related genes, these genes either provide beneficial traits for plants, or Improve or improve the quality of crop seeds, promote the development of seed embryos or improve the resistance of crop seeds to diseases and insect pests. It is also possible to construct a recombinant plant expression vector after the related genes that promote the accumulation of trace elements such as iron, zinc, and potassium in crop seeds are operably connected to the tissue-specific promoter of the present invention, and the recombinant plant expression vector is transformed into After in vivo plant tissues or cells, the tissue-specific promoter of the present invention can drive genes related to the accumulation of iron, zinc, potassium and other trace elements in crop seeds to perform specific high-efficiency expression in crop seed embryos, effectively increasing the concentration of iron in crop seeds. , zinc, potassium and other trace elements, and finally achieve the purpose of improving the quality of crop seeds.

The target heterologous DNA sequence to be transformed in the present invention may also include a sequence with RNA activity or a sequence that produces a polypeptide product, for example, it may be an antisense sequence, RNAi sequence, ribozyme sequence, splice body, amino acid coding sequence, etc. sequences and their fragments.

Definition of terms involved in the present invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are now described.

The term "stringent hybridization conditions" means conditions of low ionic strength and high temperature known in the art. Typically, under stringent conditions, a probe hybridizes to its target sequence to a detectably higher degree (eg, at least 2-fold over background) than to other sequences. Stringent hybridization conditions are sequence-dependent, and in different circumstances The conditions will be different, and longer sequences hybridize specifically at higher temperatures. The target sequence that is 100% complementary to the probe can be identified by controlling the stringency of hybridization or washing conditions. For detailed guidance on nucleic acid hybridization, refer to relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nuclear acidassays.1993). More specifically, the stringent conditions are usually selected to be lower than the specified ionic strength for specific sequences The thermal melting point ( _Tm ) at pH is about 5-10°C. _Tm is the temperature at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium (at a specified ionic strength, pH and nucleic acid concentration ) (because the target sequence is present in excess, 50% of the probes are occupied at _T at equilibrium). Stringent conditions can be those in which the salt concentration is below about 1.0 M sodium ion concentration at pH 7.0 to 8.3 , typically about 0.01 to 1.0 M sodium ion concentration (or other salt), and a temperature of at least about 30° C. for short probes (including, but not limited to, 10 to 50 nucleotides) and at least about 30° C. for long probes For needles (including, but not limited to, greater than 50 nucleotides), at least about 60° C. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, normal The signal can be at least twice background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42° C.; or 5×SSC, 1% SDS, incubate at 65° C., wash in 0.2×SSC and wash in 0.1% SDS at 65° C. The washes can be performed for 5, 15, 30, 60, 120 minutes or longer.

The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce a recombinant host cell, e.g., direct uptake, transduction, f pairing or known in the art. other known methods. Exogenous polynucleotides may remain as non-integrating vectors such as plasmids or may integrate into the host genome.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.) . Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the explicitly designated sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted at position 3 with mixed bases and/or deoxyinosine residues (Batzer et al. , Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J.Biol.Chem.260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol Cell.Probes 8: 91-98 (1994)).

The term "promoter" refers to the upstream of the coding sequence of the target gene, which provides the recognition site for RNA polymerase and other factors necessary for correct transcription initiation, and initiates or directs the transcription of the target gene into mRNA.

The term "tissue-specific promoter": a promoter that regulates or drives the specific expression of a gene of interest in a tissue or organ.

The term "heterologous DNA sequence" means that the DNA sequence is of foreign origin to that particular host cell, or if from the same original source but with modifications or alterations to the original sequence.

The term "endogenous gene" is derived from the host's own genes, including DNA or RNA sequences.

The term "selectable marker gene": the expression of the gene in a plant cell confers a selective advantage on the cell, the selective advantage possessed by these cells transformed with these selectable marker genes may be due to their growth compared to non-transformed cells The ability to grow in the presence of negative selection agents such as antibiotics or herbicides. Selectable marker genes also refer to combinations of genes whose expression in a plant cell confers a negative as well as a positive selective advantage on that cell.

The term "operably linked" refers to a functional linkage between two or more elements, which may be contiguous or non-contiguous.

The term "transformation": A method of introducing a heterologous DNA sequence into a host cell or organism.

The term "expression": transcription and/or translation of an endogenous or transgene in a plant cell.

The term "coding sequence": a nucleic acid sequence transcribed into RNA.

The term "plant expression vector": one or more DNA vectors used to effect plant transformation; these vectors are often referred to in the art as binary vectors. Binary vectors together with vectors with helper plasmids are the most commonly used for Agrobacterium-mediated transformation. Binary vectors usually include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc.

Description of drawings

Figure 1 Electropherogram of RNA quality detection; R: root; S1, S2, S3: stems of the 4th, 3rd, and 2nd segments; L1, L2, L3: 2nd, 4th, and 6th leaves; SH1, SH2, SH3: 2nd segments , 4, 6 leaf sheaths; SA: ST: stem; stem tip; T: male flower; EN: endosperm; E: embryo; K: kernel.

Figure 2 Schematic diagram of the original validation vector pCAMBIA3301.

Figure 3. Schematic representation of p85502-EZ.

Figure 4. Schematic representation of the pUM3G intermediate vector.

Figure 5 is a schematic diagram of the expression vector p85502G3.

Fig. 6 Transient expression results of the embryo-specific promoter; the development time of the embryo is 20 days.

Fig. 7 The expression of GUS gene in seeds promoted by embryo-specific promoter; 1, 2, 3, 4 and 5 in the figure represent different maize seeds respectively.

Fig. 8 GUS staining images of tissues of T1 generation transgenic maize stably transformed with p85502G3, (a): leaf; (b): root; (c): leaf sheath.

Figure 9 Transient expression results of reporter gene expression driven by promoters of different lengths.

Fig. 10 p85502-4G3 stable expression vector map.

Fig. 11p85502-0G3 stable expression vector map.

Figure 12 The map of p85502-2G3 stable expression vector.

Figure 13 The map of p85502-6G3 stable expression vector.

Figure 14 GUS staining diagrams of each missing fragment of the promoter in T1 generation transgenic maize tissues; (a)-(e): GUS staining diagrams of T1 generation transgenic maize tissues stably transformed with p85502-0G3; (f)-(k ): GUS staining diagram of each tissue of T1 generation transgenic maize stably transformed with p85502-2G3; (l)-(n): GUS staining diagram of each tissue of T1 generation transgenic Arabidopsis stably transformed with p85502-4G3; (o) -(q): GUS staining images of various tissues of T1 generation transgenic Arabidopsis stably transformed with p85502-6G3; (a)/(b)/(f)/(g): longitudinal section of seeds 30 days after pollination, (b)/(g) is negative control; (c)/(i): leaf sheath; (d)/(j): leaf; (e)/(k): root; (h): coloring of graph (i) Partial cross-section enlarged 25 times; (l)/(o): inflorescence; (m)/(p): silique; (n)/(q): leaf.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Cloning and sequence analysis of maize tissue-specific promoter

1. Using gene chip data to screen promoters that drive specific expression genes in maize seeds

Preparation of gene chip materials: take the roots, leaves, stems and interstems of maize B73 at the trumpet stage, mature but unpollinated young ears and filaments, embryos and A total of 14 samples, including endosperm, were set up with 3 replicates for each sample type, and the gene expression profiles of 42 samples were analyzed using a gene chip (Maize GenomeArray from Affymetrix).

Bioinformatics analysis: Using bioinformatics methods to analyze the data of 42 samples, a gene Zm85502 (the promoter sequence of the gene is shown in SEQ ID NO.1) was found to be specific in the middle and late stages of embryonic development. a large number of expressions.

Table 1 Analysis of gene Zm85502 expression in different tissues by gene chip

2. Quantitative RT-PCR screening of promoters driving embryo-specific expression genes

The inbred line B73 was taken as materials: roots, stems, leaves, leaf sheaths, shoot tips at the trumpet stage, and embryos and endosperms 10 days, 15 days, 20 days, and 25 days after pollination. Among them, the three segments of the morphological upper end of the stem were taken, and the leaves and corresponding leaf sheaths were taken from the second, fourth, and sixth leaves and leaf sheaths counted from the morphological lower end, respectively, and three replicates were set for each sample.

RNA extraction and cDNA acquisition: After the tissue samples were ground into powder with a liquid nitrogen pre-cooled mortar, RNA was extracted using the general TRIzol reagent extraction method. The quality of RNA was detected by 1.5% agarose gel electrophoresis, and the electrophoresis picture is shown in FIG. 1 . According to the amount of RNA required by the reverse transcription kit (Promega Company), the DNA was removed first, and then reverse transcribed into cDNA. After reverse transcription was incubated on a PCR machine at 42°C for 1 hour, 25mM EDTA was added to each reaction system to terminate the reaction. The cDNA sample master solution of each tissue was diluted 25 times before use, and each reaction used 5 microliters of the diluted cDNA. Actin was used as the internal reference gene, and three parallel points were made for each reaction between the target gene and the internal reference gene. The verified primers are listed in Table 2.

Table 2 Validation primers

Real-time quantitative RT-PCR was used to further verify the results of the gene chip analysis, and to verify the expression specificity and expression intensity of the gene Zm85502 (the promoter sequence of the gene is SEQ ID NO.1) in the embryo. It was found that the results of quantitative RT-PCR were basically consistent with the experimental results of gene chip analysis, and the gene was mainly expressed in embryos.

Table 3 RT-PCR analysis of gene Zm85502 expression in different tissues

3. Cloning of the promoter

Using the 2.0kb sequence shown in SEQ ID NO.1 as the sequence containing the full-length "tissue-specific high expression" promoter to design cloning primers, the designed cloning primers are as follows:

85502f 5-cccgggAGAGCACAGTAACATCAACACT-3;

85502r 5-ctgcagCTCGGCTCCACTACCGCGCGCGTT-3;

Using the genomic DNA of the inbred line B73 as a template, the target promoter clone was obtained by high-fidelity DNA polymerase KOD amplification. The PCR amplification procedure was as follows: 1. 94°C for 4min, 2. 98°C for 10sec, 3. 61°C for 30sec, 4. , 68°C for 2min, 2-4 partial cycles 30 times, and then 68°C for 10min. The PCR cloned fragment was loaded into the cloning vector pEASY-Blunt (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), the sequence was verified to be correct by sequencing, and the new vector was named p85502-EZ.

Experimental example 1 Candidate promoter-driven reporter gene specific expression test in maize embryo

Plant expression vector construction: in order to verify whether the fragment about 2.0kb shown in SEQ ID NO.1 has tissue-specific promoter function, the fragment shown in SEQ ID NO.1 is cloned into plant expression vector pCAMBIA3301 (purchased from Cambia company , http://www.cambia.org) (Figure 2) to verify the function of the promoter. In order to facilitate cloning, in this experiment, the multiple cloning site of the pCAMBIA3301 vector was modified, and it was double-digested with EcoR I and Bgl II, and a synthetic small fragment containing multiple restriction sites was inserted here (the sequence is as follows shown), to replace the original multiple cloning site to form the intermediate vector pUM3G:

5- GAATTC GGTACC CGGG(EcoR Ⅰ/Hind III/Kpn Ⅰ/Sma Ⅰ)ctattgcggtgcaggctgccagagcggcggctgtgacgctgtctttgccggcgccatcaccgccaactccactcttctcgcagaatgatgatagatccaccatggttaacctagacttgtccatcttctggattggccaacttaattaatgtatgaaataaaaggatgcacacatagtgacatgctaatcactataatgtgggcatcaaagttgtgtgttatgtgtaattactagttatctgaataaaagagaaagagatcatccatatttcttatcctaaatgaatgtcacgtgtctttataattctttgatgaaccagatgcatttcattaaccaaatccatatacatataaatattaatcatatataattaatatcaattgggttagcaaaacaaatctag TCTAGA CTGCAG CCATGG TAGATCT(Xba Ⅰ/Pst Ⅰ/Nco Ⅰ/BglⅡ)-3；

p85502-EZ (Fig. 3) was digested with Sma I and Pst I, and the promoter fragment was connected into the pUM3G intermediate vector (Fig. 4) treated with Sma I and Pst I to obtain the expression vector p85502G3 ( Figure 5), the reporter gene is GUS.

Acquisition of transformed materials: Considering that the transcripts of most of the candidate genes reached the highest value about 20 days after corn pollination, the young ears of corn were taken 20 days after pollination, bracts and filaments were removed, and 5% sodium hypochlorite was used to Soak for disinfection and sterilization for 30 minutes, and then wash with sterile water three times. In the ultra-clean workbench, the immature embryos were peeled off with a scalpel under aseptic conditions, and the immature embryos were centrally soaked in liquid MS medium in order to remove the starch on the surface of the embryos and maintain the vitality of the embryos. After taking enough embryos, wash them once with liquid MS medium, and then transfer them to solid hypertonic culture for hypertonic treatment for 4 hours for later use. Place 9-12 young embryos in each dish, and place three embryos in 3-4 rows.

Transformation: References for the production of microprojectiles and bombardment methods of gene guns (Rumei Chen et.al., 2008, Transgenic Res.17(4):633-43), just change the split membrane to the specification of 1100psi. One bombardment per dish, 2-3 parallels for each construction, and the amount of plasmid used was 1 μg/gun. One hour after the bombardment, the immature embryos were transferred to recovery medium for 24 hours at 28°C in the dark.

GUS histochemical staining: transfer the dark-cultured maize transformation material to a sterile 2 ml centrifuge tube (1 gun/tube) in an ultra-clean workbench, add 400 microliters of GUS staining solution to each tube, fasten the cap of the tube, and centrifuge The tubes were placed horizontally in a 37°C thermostat for at least 8 hours. The function of the candidate promoter shown in SEQ ID NO.1 was verified by observing and analyzing the presence and depth of the coloring spots of immature embryos. It can be seen from the test results ( FIG. 6 ), that the GUS reporter gene driven by the promoter shown in SEQ ID NO.1 has high-intensity expression in maize embryos.

Stable transformation of maize to further identify the function of the embryo-specific promoter: the stable transformation vector p85502G3 (Figure 5) was transformed into the maize material Hi II by the method mediated by Agrobacterium, and stably transformed plants were obtained. The maize stable transformation process is as follows:

(1) Take Hi II young ears 10 days after pollination, first prepare 5% sodium hypochlorite solution with sterile water to soak and sterilize the young ears for 15 minutes, and then soak and wash them three times with sterile water.

(2), under aseptic conditions, strip off the immature embryos with a length of about 1.5mm-2.0mm and place them in a liquid infection medium added with acetosyringone (see the paper Molecular Breeding for details on the medium formula, 2001, p. 8 volumes, pp. 323–333).

(3) Scrape and resuspend an appropriate amount of recombinant cloned cells containing the target expression vector cultured on YEB solid medium with corresponding resistance at 28°C for 4 days and resuspend in liquid infection medium with acetosyringone Medium, 28°C constant temperature shaker at low speed to restore culture to OD ₂₆₀ to 0.4-0.6.

(4) Wash the stripped immature embryos twice with a liquid infection medium, discard the cleaning solution, add bacteria cells with an OD ₂₆₀ =0.4-0.6, invert and mix for 20 times, and let stand in the dark for 5 minutes.

(5), suck and discard the bacterial liquid, and wash the impregnated immature embryos twice with the liquid infection medium, together with the second cleaning solution and the immature embryos, pour them into the solid co-culture medium without screening pressure (see the paper for details of the medium formula) Molecular Breeding, 2001, volume 8, page number: 323-333), the immature embryos are evenly distributed on the culture medium, and the smooth surface of the immature embryos is cultivated close to each other, with the curved surface facing upward.

(6) Discard the cleaning solution, and place the culture in a 25° C. incubator in the dark for 3 days. The immature embryos after 3 days of co-cultivation were transferred under aseptic conditions to solid recovery medium without screening pressure, and cultured at 28°C in the dark for 7-10 days.

(7) Transfer the well-grown and sterile immature embryo derivatives of the recovery culture to the selection medium with basta selection pressure for selection and culture at 28° C. in the dark for 1-2 months, and subculture once every 2 weeks.

(8) After the resistant callus with a growth rate significantly higher than that of the general callus appears, after it is propagated to a certain point, a certain amount of resistant callus is transferred to a differentiation medium with multiple hormones (cultivation medium) For the basic formula, see the paper Molecular Breeding, 2001, Volume 8, page number: 323-333) and culture at 28°C in the dark for about 2 weeks to induce the formation of embryoid bodies.

(9) Transfer the embryoid body to a solid rooting medium, and culture it under light conditions at 28°C for about 1 week. After rooting into seedlings, transfer the seedlings to a cylindrical culture tube filled with solid rooting medium, and cultivate them under light at 28°C for about 1 week.

(10), then transfer the test-tube seedlings with 2-3 young leaves to a nutrient bowl with nutrient soil and cultivate them in a light incubator for about 1 week, then transfer them to the greenhouse for further cultivation and finally transplant them into the field.

Figure 7 is the situation that the promoter shown in SEQ ID NO.1 drives GUS gene expression in corn seeds, and it can be found from Figure 7 that the promoter shown in SEQ ID NO.1 can drive the specific high expression of GUS gene in corn embryos ,.

Figure 8 is the GUS staining diagram of the tissues of the full-length promoter P ₈₅₅₀₂ (SEQ ID NO.1) at the stage of T1 transgenic maize with three leaves and one heart; the right half of Figure 8 (a)-(c) is the negative control at the same period Materials, it can be clearly seen that the edge (left and right sides) of the transgenic leaf section is colored, but the natural growth edge (upper and lower sides) of the leaf is not colored, while any part of the control leaf is not colored. It shows that the full-length promoter of P ₈₅₅₀₂₂ (SEQ ID NO.1) is not expressed in vegetative tissue under normal physiological conditions, but exhibits the characteristic of inducible expression in vegetative tissue under the condition of mechanical injury induction.

Test Example 2 Isolation of the shortest promoter and its functional verification test

The promoter shown in SEQ ID NO.1 is gradually deleted from the 5' end to obtain multiple truncated promoter fragments, the lengths of which are 1.4kb, 0.81kb, 0.31kb, 0.18kb and 0.14kb; and then respectively Each truncated fragment was connected into the transient verification vector pCAMBIA3301 (purchased from Cambia Company, http://www.cambia.org), to verify whether each truncated fragment still had the function of a promoter, to determine the shortest expression function promoter sequence.

As can be seen from Figure 9, the truncated sequences of 1.4kb, 0.81kb, 0.31kb and 0.18kb obtained by gradually deleting the promoter shown in SEQ ID NO.1 from the 5' end can all drive the GUS gene in maize seeds. Expressed in the embryo; wherein 0.18kb (the shortest promoter fragment) is a fragment between -181—+1 (SEQ ID NO.2):

-181CTGACGCGCC ACGTCCCGCG CCACCGCCGA GCCGCTT CT-138

-139GTAC TACACCGCAG CCGGCGCAGC GGAATACACG CCAGAAACTA ATCCAGATAG

-81CTAGCCTTTG ATTTCTCACT TCAATCTGAC GAAGTGCACA CCTAGGCAAC AAACTCGAAC

-21GCGCGCGGTAGTGGAGCCGAG+1

-181—+1 is the smallest fragment with function;

-138—+1 is the largest fragment with no function; the difference between the two is 43bp, and this 43bp contains a TATAbox.

The test results confirmed that the truncated promoter fragment shown in SEQ ID NO.2 can drive the high-efficiency expression of the GUS gene in maize embryos, proving that the truncated fragment shown in SEQ ID NO.2 still has a promoter function.

Test Example 3 Functional verification test of truncated promoter for stable transformation of maize

The full-length promoter shown in SEQ ID NO.1 was gradually deleted from the 5' end to obtain four truncated promoter fragments P _85502-4 (SEQ ID NO.3), P _85502-0 (SEQ ID NO. 4), P _85502-2 (SEQ ID NO.5), P _85502-6 (SEQ ID NO.6); referring to the construction method of the stable transformation vector p85502G3 in Test Example 1, the stable expression vector p85502-4G3 was constructed respectively (Fig. 10), p85502-0G3 (Figure 11), p85502-2G3 (Figure 12) and p85502-6G3 (Figure 13);

The stable expression vector p85502-0G3 and the stable expression vector p85502-2G3 were respectively transformed into corn material Hi II according to the method mediated by Agrobacterium in Test Example 1, and stably transformed plants were obtained. The stable transformation process of corn was the same as that in Test Example 1.

The stable expression vector p85502-4G3 and the stable expression vector p85502-6G3 were respectively transformed into Arabidopsis thaliana according to the method mediated by Agrobacterium, and stably transformed transgenic plants were obtained. The method for obtaining the transgenic Arabidopsis is as follows:

1. Prepare the Agrobacterium strain used for transformation, inoculate the Agrobacterium strain used for transformation into liquid YEP medium with Kan and Rif resistance for activation, shake overnight at 28° C. at 200 rpm. Then transfer the activated strains to 200mL liquid YEP medium with Kan and Rif resistance at a volume ratio of 1:400, at 28°C, 200rpm. When the bacterial solution reaches OD600 within 1.5-3.0, centrifuge at 4000g for 10min at 4°C to collect the bacterial cells. Dilute it with 10% sucrose containing 0.02% silwet L-77 (manufactured by GE) to an OD600 of about 0.8--1.0 for later use.

2. Watering: Water the wild-type Arabidopsis seedlings that need to be transformed the day before the transformation.

3. Use scissors to cut off the pollinated siliques on the plant, take some trays, spread absorbent paper on the bottom and soak them with water for later use.

4. Place the plants flat next to a large plate, and place the inflorescences in the plate.

5. Pour in the spare Agrobacterium bacteria solution, put on disposable gloves, gently press the inflorescence into the bacteria solution, and count for 30 seconds.

6. After the transformation is completed, the plants are quickly transferred to the spare tray, and another tray is turned upside down on the top of the tray to shade all the transformed plants for 24 hours.

7. Place the shading-treated plants back under normal light cultivation conditions for cultivation.

8. The above-mentioned conversion steps can be repeated every other week, but the new teachings cannot be cut out. Each build is generally converted 2-3 times.

It can be seen from Figure 13(a) that the promoter deletion fragment P _85502-0 still has the function of initiating reporter gene expression in embryonic tissue, but its activity is significantly lower than that of the full-length promoter P ₈₅₅₀₂ (SEQ ID NO.1). From Figure 13(c), it can be seen that the promoter deletion fragment P _85502-0 maintains the characteristics of inducible expression in leaf sheaths, and Figure 13(d) and (e) illustrate that it has lost the full-length promoter in leaves and roots Properties of inducible expression in vegetative tissues. Based on the above results, it can be seen that the promoter deletion fragment P _85502-0 is an embryo tissue-specific promoter with reduced promoter activity under normal physiological conditions, but it shows inducible expression in leaf sheath tissue under mechanical damage. characteristic.

It can be seen from Fig. 13(f) that the promoter deletion fragment P _85502-2 lost the function of initiating reporter gene expression in embryonic tissue. However, it can be seen from (h)-(k) that P _85502-2 maintains the property of inducing expression in vegetative tissues. Based on the above results, it can be seen that the promoter deletion fragment P _85502-2 has no promoter activity in various tissues under normal physiological conditions, but exhibits inducible expression characteristics in vegetative tissues induced by mechanical damage.

From the staining results of each tissue of transgenic Arabidopsis, it can be seen that the shorter P ₈₅₅₀₂ deletion fragment is still active ((l)/(o)/(m)/(p)), only P _85502-4 and P _{85502- 6} still maintains the characteristic of inducible expression (n)/(q) in comparison.

From the results of the above four deletion fragments driving the expression of the reporter gene GUS in transgenic maize/Arabidopsis thaliana, it can be seen that these four deletion fragments still have promoter activity, but their promoter strength and tissue specificity have some changes.

Claims (6)

1. A tissue-specific promoter isolated from corn (Zea mays), characterized in that its polynucleotide is shown in SEQ ID NO.1. 2. The truncated tissue-specific promoter of the tissue-specific promoter according to claim 1, whose polynucleotide sequence is shown in SEQ ID NO.2. 3. A recombinant plant expression vector containing the tissue-specific promoter according to claim 1 or 2. 4. The application of the tissue-specific promoter according to claim 1 or 2 in regulating or promoting the specific expression of the heterologous gene of interest in the maize seed embryo. 5. The application of the tissue-specific promoter according to claim 1 or 2 in improving crop seed quality, improving plant traits or cultivating new varieties of transgenic plants; the crop or plant is corn. 6. according to the described application of claim 4, it is characterized in that, comprising: after described tissue-specific promoter and target heterologous gene sequence to be transformed are operably connected, transform in maize cell, tissue or organ Transformants are obtained; the transformants are regenerated through tissue culture to obtain complete maize plants or clones.