CN101173257B - Gene for regulating sensibility of plants to auxin and uses thereof - Google Patents

Abstract

The invention discloses the use of phospholipase D zeta 2 protein in regulating the sensitivity of plants to auxin or the response to low phosphorus stress. The invention also discloses a method for preparing plants with high sensitivity to auxin or high responsiveness to low phosphorus stress. The present invention associates phospholipase D zeta2 protein with plant auxin sensitivity and low phosphorus stress response for the first time, and can prepare transgenic plants with high sensitivity to auxin or high response to low phosphorus stress, thereby greatly reducing field stress. The application amount of auxin increases the absorption of low phosphorus by plants and greatly reduces agricultural costs.

Description

Genes Regulating Sensitivity of Plants to Auxin and Their Applications

technical field

The invention belongs to the field of plant genetic engineering and relates to the application of a phospholipid hydrolase in plants, in particular to the application of PLDzeta2 in regulating the response of plants to plant hormone auxin and the response of plants to low phosphorus stress.

Background technique

Phospholipids are a class of lipids composed of phosphoric acid that widely exist in the body, mainly including glycerophospholipids and sphingomyelins. The phospholipids commonly referred to mainly refer to glycerophospholipids, which have many types and fast turnover in the body. They are both hydrophilic and hydrophobic. A class of facultative molecules that are not only the basic components of cell membranes, but also can be used as signal substances to respond to external environmental stimuli. Common phospholipid molecules in plants include phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholesterol, phosphatidylinositol-4,5-diphosphate, etc. These phospholipids are not only important components of the plant cell inner membrane system , and also plays an important role in the signal transduction of plant cells.

Phospholipase D (PLD) is a kind of phospholipid hydrolase ubiquitous in bacteria, fungi, plants and animals, which can hydrolyze specific ester bonds in phospholipid molecules to generate phosphatidic acid (PA) and free head group molecules. In plants, PLD and its product PA are involved in many important cellular processes such as stress, vesicle transport, and cytoskeletal protein reorganization. There are 12 genes encoding PLD in the Arabidopsis genome. Different PLD genes are involved in plant seed germination, seedling growth, main root elongation and root hair morphogenesis, pollen tube germination and top growth, leaf senescence and fruit ripening. , and play an important regulatory role in the response of plants to the plant hormone abscisic acid (ABA), drought, water loss, low temperature, low phosphorus and other adversity stresses.

In previous studies, the inventors used the method of PCR screening library to screen the Arabidopsis hypocotyl cDNA phage library, and cloned a cDNA sequence of a PLD coding gene. Sequencing proved that the sequence was the cDNA sequence of PLDzeta2, encoding The product is PLDzeta2 protein. The cDNA sequence has been submitted to the EMBL gene bank with accession number AM182458.

However, the function of the PLDzeta2 gene is still poorly understood in this field, so it is necessary to further study the function of the PLDzeta2 gene in order to apply it to plant cultivation.

Contents of the invention

The object of the present invention is to provide a kind of phospholipase D zeta2 (PLDzeta2) and its application.

In the first aspect of the present invention, the use of phospholipase D zeta 2 protein is provided for regulating the sensitivity of plants to auxin, or regulating the response of plants to low phosphorus stress.

In another preferred embodiment of the present invention, the phospholipase D zeta 2 protein can be used to improve the sensitivity of plants to auxin; or, the phospholipase D zeta 2 protein can be used to promote the plant's response to low phosphorus stress. response.

In another preferred embodiment of the present invention, the plants are selected from the group consisting of Brassica plants of the family Brassicaceae, Gramineae plants, Solanaceae plants, and the like.

In another preferred example of the present invention, the Brassica plants of the genus Brassica include (but are not limited to): Arabidopsis thaliana, rapeseed, Chinese cabbage;

Described gramineous plants include (but not limited to): rice, wheat; or

The plants of the family Solanaceae include (but are not limited to): potatoes, tomatoes.

In another preferred embodiment of the present invention, the phospholipase D zeta 2 protein has the amino acid sequence shown in SEQ ID NO:2. Alternatively, it is an amino acid sequence having higher homology with the amino acid sequence shown in SEQ ID NO: 2 (such as homology > 60%; preferably, homology > 80%; more preferably, homology > 90% or higher).

In a second aspect of the present invention, there is provided a method for increasing the sensitivity of plants to auxin, said method comprising:

When auxin concentration ≥ 10nM (preferably, be 10-1000000nM, more preferably, be 10-10000nM; Most preferred 10-1000nM), improve the expression of phospholipase D zeta 2 albumen in the plant, thereby improve plant growth hormone sensitivity; or

When auxin concentration≤1nM (preferably, be 0.00001-1nM, more preferably, be 0.0001-1nM), reduce the expression of phospholipase D zeta 2 protein in the plant, thereby improve the sensitivity of plant to auxin.

In a third aspect of the present invention, there is provided a method for preparing plants (such as transgenic plants) with high sensitivity to auxin when the auxin concentration is ≥ 10 nM, the method comprising:

(1) transfer the coding gene of phospholipase D zeta 2 protein into plant cells, tissues or organs, and obtain the plant cell, tissue and organ transformed into the coding gene of phospholipase D zeta 2 protein; and

(2) regenerate the plant cells, tissues or organs obtained in step (1) into which the gene encoding the phospholipase D zeta 2 protein is transferred and select transgenic plants.

In another preferred embodiment of the present invention, the method includes the steps of:

(s1) providing an Agrobacterium carrying an expression vector, the expression vector containing the gene encoding the phospholipase D zeta 2 protein;

(s2) plant cells, tissues or organs are contacted with the Agrobacterium in step (s1), so that the coding gene of the phospholipase Dzeta 2 protein is transferred into the plant cells and integrated into the chromosomes of the plant cells;

(s3) select plant cells, tissues, organs that have been transferred to the gene encoding the phospholipase D zeta 2 protein; and

(s4) regenerating the plant cells, tissues and organs in step (s3) and selecting transgenic plants.

In another preferred embodiment of the present invention, the concentration of auxin is 10-1000000nM (more preferably, 10-10000nM; most preferably, 10-1000nM).

In another preferred example of the present invention, the expression vector is selected from (but not limited to): p35S-1301, p35S-1302.

In the fourth aspect of the present invention, there is provided a method for preparing a plant with high sensitivity to auxin or a plant with high responsiveness to low phosphorus stress when the auxin concentration is ≤ 1 nM, the method comprising:

(1) Agrobacterium carrying an antisense expression vector (or a carrier that can finally reduce gene expression) is provided, and the antisense expression vector contains the coding gene (or gene fragment) of the phospholipase D zeta 2 protein initiated in the reverse direction ;

(2) Plant cells, tissues or organs are contacted with the Agrobacterium in step (1), so that the coding gene of the phospholipase D zeta 2 protein that is started in the reverse direction contained in the antisense vector is transferred into plant cells, and Integrate into the chromosomes of plant cells;

(3) select the plant cells, tissues, and organs that have been transferred to the gene encoding the phospholipase D zeta 2 protein that is started in the reverse direction contained in the antisense vector; and

(4) Regenerating the plant cells, tissues and organs in step (3) and selecting transgenic plants.

10. The method according to claim 9, wherein the concentration of auxin is 0.00001-1 nM (more preferably, 0.0001-1 nM).

In another preference, the "gene encoding the phospholipase D zeta 2 protein initiated in the reverse direction" refers to the position of the start codon at the 5' end of the gene when the gene is cloned into the vector. The position of the corresponding promoter is far away, while the position of the 3' end terminator is close to the position of the promoter of the vector (that is, it is opposite to the direction of conventional gene cloning into the vector).

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Fig. 1 is a graph comparing the sensitivity changes of wild-type plants and PLDzeta2 deletion mutant atpldζ2 to auxin (IAA). WT is wild-type Arabidopsis thaliana, and atpdζ2 is a mutant line with deletion of PLDzeta2 gene. atpldζ2 has reduced sensitivity to auxin compared to wild type, i.e., is less sensitive to auxin than wild type.

Figure 2 is a graph showing the comparison of auxin sensitivity between wild-type plants and PLDzeta2 overexpression and deletion transgenic lines. WT is wild-type Arabidopsis, pO-PLDζ2-LO11 and pO-PLDζ2-LO19 are two transgenic lines with up-regulated expression of PLDzeta2 gene, pO-PLDζ2-LA3 and pO-PLDζ2-LA11 are two transgenic lines with down-regulated expression of PLDzeta2 gene Transgenic strains. pO-PLDζ2-LA3 and pO-PLDζ2-LA11 decreased the sensitivity to auxin compared with the wild type. Contrary to the auxin response of transgenic plants with down-regulated PLDzeta2 gene expression, the up-regulated transgenic lines pO- PLDζ2-LO11 and pO-PLDζ2-LO19 had increased sensitivity to auxin compared with wild-type plants. Among them, Control represents the wild-type control plant, that is, WT.

Figure 3 is a map of p35S-1301/AtPLDζ2 sense overexpression binary vector.

Figure 4 is a map of p35S-1301/antisense AtPLDζ2 deletion expression binary vector.

Detailed ways

After long-term research, the inventor first associates the phospholipase D zeta2 (PLDzeta2 for short, or PLDζ2) gene with the auxin sensitivity of plants, and finds that the overexpression or low expression (including non-expression) of the PLDzeta2 gene can regulate the plant's response to the auxin. Auxin sensitivity; and, the PLDzeta2 gene is also involved in the plant's response to low phosphorus stress, and the low expression (including no expression) of PLDzeta2 promotes the plant's adaptation to low phosphorus stress. The present invention has been accomplished based on this.

As used in the present invention, the "plants with high sensitivity to auxin" or "plants with increased sensitivity to auxin" refer to a plant (such as a transgenic plant) that can grow under appropriate growth conditions. The sensitivity to the hormone is 10% or higher (more preferably 20% or higher) higher than that of wild-type plants under the same conditions. For example, in the case of 1 nM IAA treatment, the transgenic plants were more than 15% more sensitive to IAA than the control plants (see Table 1 and Table 2).

As used in the present invention, the "plant with low sensitivity to auxin" or "plant with reduced sensitivity to auxin" refers to a plant (such as a transgenic plant) that is sensitive to auxin under appropriate growth conditions. The sensitivity is 10% or less (more preferably 20% or less) lower than that of wild-type plants under the same conditions.

As used in the present invention, the "plant with high responsiveness to low phosphorus stress" refers to a plant (such as a transgenic plant) that can adapt to low phosphorus stress better and faster, such as wild-type Arabidopsis plants in low phosphorus (1μM pi) treatment, a large number of root hairs can be produced in about 8 days, and a large number of root hairs can be produced in 2 days in the PLDzeta2-deficient mutant. The "response to low phosphorus stress" refers to the physiological activities of plants that are different from normal metabolism when phosphorus nutrients are deficient, which is the self-regulation of plants adapting to the deficiency of phosphorus nutrients. The responses mainly include increasing the number of lateral roots and root hairs, Promote the hydrolysis of phospholipid molecules and increase the content of inorganic phosphorus in the body.

Phospholipase D zeta2

Phospholipase D zeta2 (PLDzeta2) is a PLD gene widely present in plants (such as Arabidopsis, rice, cabbage, rape, wheat, etc.), and PLDzeta2 is highly conserved in various plants and has a very high protein content. homology.

In the present invention, the PLDzeta2 protein used may be naturally occurring, for example, it may be isolated or purified from plants. In addition, the PLDzeta2 protein can also be artificially prepared, for example, the recombinant PLDzeta2 protein can be produced according to conventional genetic engineering recombination techniques. Preferably, the present invention can use recombinant PLDzeta2 protein.

Any suitable PLDzeta2 protein may be used in the present invention. The PLDzeta2 protein includes full-length PLDzeta2 protein or its biologically active fragments. Preferably, the amino acid sequence of the PLDzeta2 protein can be substantially identical to the sequence shown in SEQ ID NO: 2.

The amino acid sequence of PLDzeta2 protein formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. The PLDzeta2 protein or its biologically active fragments include a part of conservative amino acid replacement sequence, and the amino acid replacement sequence does not affect its activity or retains part of its activity. Appropriate substitution of amino acids is a well-known technique in the art, which can be readily performed and ensures that the biological activity of the resulting molecule is not altered. These techniques allow those skilled in the art to recognize that, in general, changes to single amino acids in non-essential regions of a polypeptide do not substantially alter biological activity. See Watson et al. Molecular Biology of The Gene, Fourth Edition, 1987, The Benjamin/Cummings Pub. Co. P224.

Any biologically active fragment of PLDzeta2 protein can be used in the present invention. Here, the biologically active fragment of the PLDzeta2 protein refers to a polypeptide that can still maintain all or part of the functions of the full-length PLDzeta2 protein. Usually, the biologically active fragment maintains at least 50% of the activity of the full-length PLDzeta2 protein. Under more preferred conditions, the active fragment can maintain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length PLDzeta2 protein.

Modified or improved PLDzeta2 proteins can also be used in the present invention, eg, PLDzeta2 proteins that have been modified or improved to enhance their half-life, effectiveness, metabolism, and/or potency of the protein can be used. The modified or improved PLDzeta2 protein may be a conjugate of a PLDzeta2 protein, or it may comprise substituted or artificial amino acids. The modified or improved PLDzeta2 protein or gene may be different from the naturally occurring PLDzeta2 protein or gene, but it can also regulate the sensitivity of the plant to auxin or the response to low phosphorus stress, and will not bring other adverse effects or toxicity. That is to say, any variation that does not affect the biological activity of the PLDzeta2 protein or the biological function of the gene can be used in the present invention.

Any high homology with the PLDzeta2 protein (for example, the homology with the PLDzeta2 protein is 70% or higher, preferably, the homology is 80% or higher, more preferably, the homology is 90% or higher) and having the same function as the PLDzeta2 protein are also included in the present invention.

As an example of the present invention, there is provided a PLDzeta2 protein (also known as AtPLDzeta2 or AtPLDζ2) obtained from Arabidopsis thaliana (Arabidopsis thaliana), the coding gene of the described PLDzeta2 protein is 3370bp in full length, comprising 5'UTR of 53bp and The 176bp 3'UTR encodes a polypeptide of 1046AA, the gene is shown in SEQ ID NO: 1, and the encoded protein is shown in SEQ ID NO: 2.

Uses of PLDzeta2

The present invention provides the use of PLDzeta2 protein, which is used to regulate the sensitivity of plants to auxin or the response of plants to low phosphorus stress; or to screen for regulating the sensitivity of plants to auxin or the response of plants to low phosphorus stress Substance (that is, the substance regulates the sensitivity of plants to auxin or the response to low phosphorus stress by regulating the expression of PLDzeta2 protein).

Those skilled in the art all understand that the regulation of auxin for plant growth is dual, and the growth of plants can be promoted under the lower situation of auxin concentration (such as ≤ 0.1nM), and the higher (such as ≥ 10nM) of auxin concentration can promote the growth of plants. ) can inhibit the growth of plants. For plants such as Arabidopsis thaliana, the critical concentration for judging whether the auxin concentration promotes or inhibits the growth of the plant is about 1-10nM, that is, when the auxin concentration is higher than 10nM, the growth of the plant is inhibited, and the growth of the plant is inhibited. When the concentration of the hormone is lower than 1nM, the growth of plants can be promoted. And, for various auxins or different species, the critical concentration is different but close (ie about 1-10 nM).

Usually, according to the application in agriculture, it is selected to prepare plants with overexpression of PLDzeta2 gene (such as transgenic plants) or plants with low expression of PLDzeta2 gene.

As a preferred method, the PLDzeta2 protein can be used to increase the sensitivity of plants to auxin, such as enabling plants to grow rapidly under the condition of applying very little auxin (such as ≤1nM), thereby reducing the cost of plant cultivation The purpose; also such as making the plant localized under the condition of applying a high dose (such as ≥10; more preferably ≥100nM) of auxin (according to different needs, auxin can be applied at a certain stage of plant growth) Or all parts (the sensitivity of different organs of the plant to auxin is different) growth inhibition, so that the plant dwarfs or other physiological mechanisms (such as promoting rooting, preventing fruit shedding, or prolonging the flowering period of plants, etc.), to meet the requirements of agricultural applications need. As another preferred mode, the PLDzeta2 protein can also be used to improve the plant's response to low phosphorus stress, and promote the plant's absorption of low phosphorus, thereby achieving the purpose of reducing plant cultivation costs.

Since the transport mechanism or response mechanism of plants to various auxins is similar, the PLDzeta2 can not only regulate the sensitivity of plants to IAA, but also can regulate the sensitivity of plants to various auxins. The growth Factors include, but are not limited to, indole acetic acid (IAA), indole butyric acid (IBA), naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and the like.

In order to demonstrate the above-mentioned purposes of PLDzeta2, the present inventor cloned the PLDzeta2 gene, constructed a sense expression vector with the full length of the PLDzeta2 gene, and constructed an antisense expression vector with the N-terminal DNA fragment of the non-conserved region, and then combined the sense and antisense expression vectors of the PLDzeta2 gene The sense expression vector was transformed into Agrobacterium, and Arabidopsis was transformed by Agrobacterium-mediated method, and the transgenic strain of Arabidopsis was established. Through the analysis of PLDzeta2 mutants, sense and antisense expression transgenic plants, it was found that PLDzeta2 regulates the response of plants to auxin by regulating the cycle of auxin in the cell membrane and in the cell in plant cells, and PLDzeta2 also regulates the plant response to auxin. response to low phosphorus stress.

In addition, the present inventors discovered and obtained the T-DNA insertion mutant strain of PLDzeta2 by searching the Arabidopsis T-DNA insertion mutant library of the Salk Institute (http://signal.salk.edu/tabout.html) , further experiments proved that the strain is a mutant of PLDzeta2 deletion expression; at the same time, through the comparative study of PLDzeta2 overexpression and deletion expression transgenic lines and wild-type plants, it was found that PLDzeta2 regulates the response of plants to auxin, and is involved in the Plant response to low phosphorus stress.

Using PLDzeta2 to regulate the sensitivity of plants to auxin can make transgenic plants very sensitive to exogenously applied auxin; at the same time, the loss of expression of this gene can increase the response of plants to low phosphorus stress. This finding is not only of theoretical significance, At the same time, it also shows the application prospect of PLDzeta2 gene in modern agriculture.

Using the PLDzeta2 gene to regulate the sensitivity of plants to auxin can make overexpressed or underexpressed transgenic plants very sensitive to exogenously applied auxin, and the low or missing expression of the gene can promote the response of plants to low phosphorus stress .

Those skilled in the art know that auxin promotes plant growth at low concentrations (eg ≤ about 1 nM) and inhibits plant growth at high concentrations (eg ≥ about 10 nM). Therefore, usually according to the application in agriculture, it is selected to prepare plants with overexpression of PLDzeta2 gene (such as transgenic plants) or to prepare plants with low expression of PLDzeta2 gene.

When auxin is used to promote the growth and development of plants, by preparing plants with low expression of PLDzeta2 gene, the plants can show high sensitivity to auxin when the auxin concentration is low (such as ≤ about 1nM) (i.e. Compared with the wild type, it is more sensitive to auxin), so it can thrive under the condition of applying a small amount of auxin, and achieve the purpose of reducing the cost of applying auxin.

On the other hand, when auxin is used to slow down or inhibit the growth of plants, by cultivating plants with high expression of PLDzeta2 gene, the plants show high sensitivity to auxin when the auxin concentration is high (such as ≥ about 10nM) (ie more sensitive to auxin compared to wild type) so that growth inhibition, dwarfing or other physiological mechanisms can occur upon application of appropriate amounts of auxin. For example, when cultivating potatoes, by preparing plants with high sensitivity to auxin, adding a high concentration of auxin at an appropriate stage of plant cultivation can inhibit the overgrowth of the aerial part and promote the expansion of potato tubers, thereby achieving the purpose of increasing yield .

In addition, the present inventors also found that the product phosphatidic acid produced by the hydrolysis of phospholipids by PLDzeta2 also has the function of regulating the sensitivity of plants to auxin.

Method of modulating plant sensitivity to auxin or response to low phosphorus stress

The present invention also provides a method for regulating plant sensitivity to auxin, said method comprising: making said plant excessively express PLDzeta2 protein, or low expression PLDzeta2 protein (including making phospholipase Dzeta 2 protein non-expression or low expression ).

More preferably, the method is selected from the following two aspects:

(1) when auxin concentration ≥ 10nM (preferably, be 10-1000000nM, more preferably, be 10-10000nM; Most preferred 10-1000nM), improve the expression of phospholipase D zeta 2 protein in the plant, thereby improve plant sensitivity to auxin; or

(2) when auxin concentration≤1nM (preferably, be 0.00001-1nM, more preferably, be 0.0001-1nM), reduce the expression of phospholipase D zeta 2 protein in the plant, thereby improve the sensitivity of plant to auxin .

The present invention also provides a method for improving the responsiveness of plants to low phosphorus stress, the method comprising: reducing the expression of phospholipase D zeta 2 protein in the plant (including making the phospholipase D zeta 2 protein non-expressed or low Express).

After knowing the use of the PLDzeta2 protein, various methods well known to those skilled in the art can be used to promote the expression of the PLDzeta2 protein. For example, an expression unit carrying the PLDzeta2 gene (such as an expression vector or a virus, etc.) can be delivered to the target site through a certain way, and the active PLDzeta2 protein can be expressed. In addition, various methods well known to those skilled in the art can also be used to reduce the expression of PLDzeta2 protein or to make it lack of expression, such as delivering an expression unit (such as an expression vector or virus, etc.) carrying an antisense PLDzeta2 gene to the target site, so that The cells or plant tissues do not express or reduce the expression of PLDzeta2 protein.

As an embodiment of the present invention, the gene encoding the PLDzeta2 protein is cloned into an appropriate vector (such as the binary vector p35S-1301) by conventional methods, and the recombinant vector with the foreign gene is introduced into the In plant cells, tissues or organs expressing the PLDzeta2 protein, and then obtain plants overexpressing the PLDzeta2 gene.

Preferably, the method for preparing the high plant of auxin sensitivity comprises: (1) the coding gene of exogenous PLDzeta2 albumen is transferred into plant cell, tissue or organ, obtains the plant cell, the tissue that is transformed into the coding gene of PLDzeta2 albumen , organs; and (2) regenerating the plant cells, tissues and organs obtained in step (1) into plant plants.

As a preferred example, the method comprises the steps of:

(s1) providing an Agrobacterium carrying an expression vector containing a gene encoding the PLDzeta2 protein;

(s2) contacting the plant cell, tissue or organ with the Agrobacterium in step (s1), so that the gene encoding the PLDzeta2 protein is transferred into the plant cell and integrated into the chromosome of the plant cell;

(s3) selecting transgenic plant cells, tissues, and organs that have been transferred to the gene encoding the PLDzeta2 protein; and

(s4) regenerating the plant cells, tissues and organs in step (s3) and selecting transgenic plants.

As one embodiment, when used to prepare transgenic plants of Arabidopsis thaliana, the inventors used the Agrobacterium carrying the expression vector containing the PLDzeta2 protein to infect the unopened flower buds of Arabidopsis thaliana, after flowering and fruiting of the flower buds , The received seeds contain transgenic seeds, and then directly obtain transgenic plants through resistance screening. When preparing transgenic plants such as rice, the callus of rice is infected with the Agrobacterium carrying the expression vector containing the PLDzeta2 protein, and then the tissue is regenerated into a transgenic plant.

As another preferred example, the method includes:

(1) Agrobacterium carrying an antisense expression vector is provided, and the antisense expression vector contains the coding gene (or gene fragment) of the phospholipase D zeta 2 protein initiated in the reverse direction;

(2) Plant cells, tissues or organs are contacted with the Agrobacterium in step (1), so that the coding gene of the phospholipase D zeta 2 protein that is started in the reverse direction contained in the antisense expression vector is transferred into plant cells, and Integrate into the chromosomes of plant cells;

(3) select the transgenic plant cells, tissues, and organs that have been transferred to the gene encoding the phospholipase D zeta 2 protein encoded by the reverse direction that the antisense expression vector contains; and

(4) regenerating the plant cells, tissues and organs in step (3) and selecting transgenic plants.

After the antisense expression vector is transferred into plant cells, a full-length gene or gene fragment encoded in the reverse direction of PLDzeta2 is obtained by transcription, and the reverse fragment can recognize and pair with the forward mRNA sequence of PLDzeta2 normally transcribed in plants , and then inhibit the normal translation of PLDzeta2 mRNA, eventually resulting in no or low expression of phospholipase D zeta 2 protein in plant cells.

The length of the gene fragment used for the coding gene of the phospholipase D zeta 2 protein contained in the antisense vector is generally 100bp-3kb, preferably 300bp-1.5kb, such as 1kb.

Methods well known to those skilled in the art can be used to construct an expression vector containing the sequence of the gene encoding the PLDzeta2 protein and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as neomycin resistance, green fluorescent protein (GFP), Hyg resistance, Kan resistance, sex.

A vector containing the above-mentioned appropriate gene sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells and the like.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. Transformation of plants can also use methods such as Agrobacterium transformation or biolistic transformation, such as leaf disk method, rice immature embryo transformation method and the like. Transformed plant cells, tissues or organs can be regenerated into plants by conventional methods, so as to obtain transgenic plants.

The obtained transformants can be cultured by conventional methods to express the protein of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant protein in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The main advantages of the present invention are:

For the first time, the phospholipase D zeta2 (PLDzeta2) gene was associated with the auxin sensitivity of plants, and it was found that the PLDzeta2 gene can be used to regulate the sensitivity of plants to auxin. Moreover, this gene can also regulate the response of plants to low phosphorus stress. The gene has wide application value in the field of plant cultivation, and can make plants more sensitive to auxin, thereby greatly reducing the application amount of auxin or phosphorus in the field, and can effectively reduce agricultural costs.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the following documents Methods published in: Carl W. Dieffenbach and Gabriela S. Devksler eds. PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1995. or as recommended by the manufacturer.

Cloning of embodiment 1 PLDzeta2 gene

The method of screening phage library (Alfandari, D., and Darribère, T. (1994). A simple PCR method for screening cDNA libraries. PCR Methods Appl. 4, 46-49) using conventional 96-well plate PCR method obtained Full-length cDNA clone of the PLDzeta2 gene. In the following examples or drawings, the PLDzeta2 gene obtained from Arabidopsis thaliana is also referred to as AtPLDzeta2 or AtPLDζ2.

First, primers were designed at the 3' end of the gene according to the known genome sequence information (i.e. the gene sequence of At3g55940 in GenBank):

AtPLDζ2-1: 5'-TGATCGTTATTCCGCTAC-3' (SEQ ID NO: 3);

AtPLDζ2-2: 5'-AAGGTTCCCTCTTGTCTC-3' (SEQ ID NO: 4).

The primers were used to screen the Arabidopsis thaliana hypocotyl phage cDNA library (purchased from the Arabidopsis thaliana Biological Research Center, ABRC, or refer to http://www.arabidopsis.org/), and the cDNA containing the full-length sequence of AtPLDzeta2 was isolated. cDNA clone pBSK-AtPLDζ2.

The positive clone (BSK-AtPLDζ2 plasmid) obtained by the aforementioned screening was verified by sequencing, and the cDNA sequence of AtPLDζ2 contained therein was shown in SEQ ID NO: 1, including 53 bp of the 5' untranslated region and 176 bp of the 3' untranslated region. A total of 3370bp in length, the cDNA sequence has been submitted to the Genbank gene bank, the accession number is AM182458.

BLAST found that the cDNA sequence was identical to the sequence of the coding region of the PLDzeta2 gene predicted from the Arabidopsis genome, indicating that the gene obtained from the screening library was the coding gene of PLDzeta2.

Example 2 Isolation and identification of T-DNA insertion mutant atpldζ2 of PLDζ2

In order to study the physiological function of the AtPLDζ2 gene, the present inventors also searched the Arabidopsis T-DNA insertion mutant library of the Salk Institute (http://signal.salk.edu/tabout.html). The search results show that the mutant line numbered Salk_119084 is an insertion mutant of AtPLDζ2, and the insertion site is 14bp from the end of the third intron in the corresponding AtPLDζ2 gene (Locus number is At3g55940) to the fourth exon s position.

The mutants obtained from the Salk mutant library were screened for Kan resistance, and then the T-DNA insertion site was detected for the resistant plants. Adopt conventional method to extract DNA from the seedling of this plant, utilize the primer Lba1 (5'TGGTTCACGTAGTGGGCCATCG 3 (SEQ ID NO: 5)') on the T-DNA carrier and gene-specific primer P2 (5'-CTCTAGCAAATGAGAGTCTAG-3' (SEQ ID NO: 6)) carried out PCR amplification to verify the insertion of the T-DNA vector; primers P1 (5'-ATGTCGACGGATAAATTACTAC-3' (SEQ ID NO: 7)) and P2 before and after the T-DNA insertion site were used for PCR amplification. Increased, verified the homozygous insertion of T-DNA.

Plants identified as heterozygous by PCR were collected as a single plant, and the progeny plants were screened for Kan resistance, and the segregation ratio of resistance was calculated. The segregation ratio of plants with Kan resistance and plants without Kan resistance was 3:1, proving that The T-DNA in the mutant has only one inserted copy, so compared with the wild type, the mutant only has the variation of the AtPLDζ2 gene, and other genes are the same as the wild type.

For the homozygous plants of the mutants, total RNA was extracted and reverse-transcribed for RT-PCR. Use primer P3 (ie: AtPLDζ2-1) and primer P4 (ie: AtPLDζ2-2) at the 3' end of the T-DNA insertion site for PCR amplification to verify the mRNA transcription of the PLDzeta2 gene in mutant inbred plants level is suppressed. The results of RT-PCR showed that the expression of the PLDzeta2 gene was completely prevented in the mutant, and the gene was not expressed.

Example 3 Obtaining of Overexpression and Deletion Expression Transgenic Lines of PLDζ2

Using the pBSK-AtPLDζ2 plasmid obtained by screening the cDNA library in Example 1, select the KpnI and BamHI restriction sites at both ends of the cDNA, carry out restriction digestion, and connect to the p35S-1301 binary vector after the same digestion treatment (See Liu W, Xu ZH, Luo D, Xue HW. (2003). Roles of OsCKI1, arice casein kinase I, in root development and plant hormone sensitivity. Plant J.36, 189-202), build into 35S startup The positive-sense binary vector (p35S-1301/AtPLDζ2) driving AtPLDζ2, the map of the positive-sense binary vector is shown in Figure 3, wherein, Over-PLDzeta2 means the positive-sense inserted PLDzeta2 gene.

The non-conserved 1kb fragment at the 5' end of the AtPLDζ2 gene was amplified by PCR, digested with BamHI and SalI, and then ligated into the p35S-1301 binary vector that had been digested with the same restriction enzymes, and constructed to contain the reverse sequence of AtPLDζ2 driven by the 35S promoter. Antisense binary vector (p35S-1301/antisense AtPLDζ2), that is, the position of the start codon at the 5' end of the AtPLDζ2 gene is far from the position of the promoter of the vector, and the position of the stop codon at the 3' end is far from the position of the promoter of the vector The positions are close, and the map of the antisense binary vector is shown in Fig. 4, wherein, A-PLDzeta2 means the antisense inserted PLDzeta2 gene fragment.

The constructed binary vector was transformed into Agrobacterium GV3101 (see Plant Mol Biol 26: 1115-1124, konczc C, Schell J, 1986) by the conventional liquid nitrogen freeze-thaw method, and the conventional Arabidopsis inflorescence axis infection was adopted. Thaliana transformation method Transform Arabidopsis thaliana Col wild-type plants (purchased from Arabidopsis Biological Research Center, ABRC), harvest the seeds of the plants, obtain transgenic plants after Hyg resistance screening, and extract genomic DNA and use PCR method to verify The T-DNA sequence of the binary vector is integrated into the chromosome.

Overexpressed transgenic plants were selected for general 35S-primer (5'-ATGCCATCATTGCGATAAAGG-3'(SEQ ID NO:8)) and AtPLDζ2 5' primer AtPLDζ2-pxA (5'AGCAAATGAGAGTCTAG 3'(SEQ ID NO:9)) Perform PCR amplification to verify that T-DNA is integrated into the chromosome; transgenic plants that lack expression select primer s35S-primer and gene 5, end primer (5'ATGTCGACGGATAAATTACTAC 3'(SEQ ID NO: 10)) to perform PCR amplification to verify T-DNA integrated into the chromosome.

Example 4 Deletion mutant of PLDζ2 atpldζ2 and detection of overexpression and deletion transgenic lines for sensitivity to auxin

The sensitivity of plants to auxin is judged according to the fact that auxin promotes the growth of the main root in the case of low concentration (≤1nM), and inhibits the growth of the main root in the case of ≥10nM. In this embodiment, the inventors use relative root length for analysis, that is, the relative root length is the percentage of the length of the main root in the case of IAA treatment relative to the length of the main root of untreated plants.

Auxin sensitivity test: surface-sterilized ① wild type, ② pldzeta2 deletion mutant (i.e. the T-DNA insertion mutant obtained in Example 2), ③ PLDzeta2 overexpression type (i.e. the overexpression transgenic plant obtained in Example 3) or ④ Arabidopsis seeds of the deletion expression type (that is, the deletion expression transgenic plants obtained in Example 3) were sown on MS medium with different concentrations of IAA (indole acetic acid) (0, 1nM, 10nM, 100nM and 1μM) (1010cm square plate), vernalization at 4°C (in a refrigerator at 4°C, let the seeds of Arabidopsis break dormancy and keep germination consistent) after 2 days of treatment, move them to an artificial climate chamber for germination and growth. After 9 days of vertical cultivation under light, measure the length of the main root respectively, measure at least 30 plants for each treatment, and calculate the average value; repeat 3 times.

The experimental results are shown in Figure 1 and Table 1 and Figure 2 and Table 2. According to Fig. 1 and table 1, under the situation that low-concentration auxin (1nM IAA) is processed, the relative main root length (being 94.97% of untreated contrast plant) does not have obvious change under the situation of IAA processing of wild-type plant, However, the mutant plants were obviously induced by auxin (112.96% of the untreated control plants); in the case of 10nM IAA treatment, the wild-type plants were significantly inhibited by IAA, and their relative root length was 79.31% of the untreated plants , the relative root length of mutant plants was 97.52%, and was not significantly inhibited by auxin; in the case of 100nIAA treatment, wild-type plants were significantly inhibited by IAA, and their relative root length was 61.30% of untreated plants, while The relative root length of mutant plants was 84.83%.

According to Fig. 2 and table 2, under the situation that low concentration auxin (1nM IAA) is processed, the transgenic plant of wild type and two strains of AtPLDζ2 overexpression under the situation of IAA processing relative main root length (respectively is untreated control plant's 97.91%, 94.09% and 97.53%) did not change significantly, but two AtPLDζ2 deletion plants were obviously induced by auxin (respectively 122.02% and 127.13% of the untreated control plants). Transgenic plants overexpressing AtPLDζ2 had increased sensitivity to auxin when a higher concentration of IAA was applied exogenously. When 10nM IAA was applied exogenously, the taproot length of wild-type plants was 82.1% of that of untreated control plants; the taproot lengths of transgenic plants pO-PLDζ2-LO11 and LO19 with overexpression of AtPLDζ2 were 68.9% and 68.9% of untreated control plants, respectively. 55.6%; when exogenously applied 100nM IAA, the main root length of wild-type plants was 60.6% of that of untreated control plants; the main root length of transgenic plants pO-PLDζ2-LO11 and LO19 overexpressed AtPLDζ2 was 48.7% of that of untreated control plants, respectively. % and 33.8%. However, the effect of auxin production in transgenic plants lacking expression was not so significant.

atpldζ2 (i.e., PLDzeta2 deletion mutant) has reduced sensitivity to auxin compared with wild type, and the transgenic lines PO-PLDζ2-LA3 and PO-PLDζ2-LA11 with PLDzeta2 deletion expression have reduced growth sensitivity compared with wild type In contrast, the transgenic lines PO-PLDζ2-LO11 and PO-PLDζ2-LO19 overexpressing PLDzeta2 increased the sensitivity of plants to auxin.

Table 1

0 IAA 1nm IAA 10nm IAA 100nm IAA 1 μm IAA WT 100% 94.97% 79.31% 61.30% 30.17% atpldζ2 100% 112.96% 97.52% 84.83% 42.33%

Table 2

0 IAA 1nm IAA 10nm IAA 100nm IAA 1 μm IAA control 100% 97.91% 82.15% 60.62% 14.50% pO-PLDζ2-LO11 100% 94.09% 68.94% 48.71% 12.56% pO-PLDζ2-LO19 100% 97.53% 55.67% 33.87% 12.03% pO-PLDζ2-LA3 100% 122.02% 97.73% 78.12% 31.74% pO-PLDζ2-LA11 100% 127.13% 98.80% 80.96% 44.30%

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

Claims (9)

1. the purposes of phospholipase D zeta 2 protein, it is characterized in that, be used for regulating the sensitivity of plant to auxin, the aminoacid sequence of wherein said phospholipase D zeta 2 protein is as shown in SEQ ID NO: 2, wherein said The auxin is indole acetic acid. 2. The use according to claim 1, wherein the plant is selected from the group consisting of Brassicaceae Brassica plants, Poaceae plants, or Solanaceae plants. 3. purposes as claimed in claim 2, is characterized in that, described cruciferous Brassica plant comprises: Arabidopsis thaliana, rape, Chinese cabbage; The gramineous plants include: rice, wheat; or The solanaceous plants include: potato and tomato. 4. A method for improving the sensitivity of plants to auxin, said auxin being indole acetic acid, characterized in that said method comprises: When the auxin concentration is ≥10nM, increase the expression of phospholipase D zeta 2 protein in plants, thereby increasing the sensitivity of plants to auxin; or When the auxin concentration is ≤1nM, it reduces the expression of phospholipase D zeta 2 protein in plants, thereby improving the sensitivity of plants to auxin, The amino acid sequence of the phospholipase D zeta 2 protein described therein is shown in SEQ ID NO: 2. 5. A method for preparing plants with high sensitivity to auxin when the auxin concentration ≥ 10nM, the auxin being indole acetic acid, characterized in that the method comprises: (1) transfer the coding gene of phospholipase D zeta 2 protein into plant cells, tissues or organs, and obtain the plant cells, tissues and organs transformed into the coding gene of phospholipase D zeta 2 protein; and (2) Regenerate the plant cells, tissues or organs obtained in step (1) into the gene encoding the phospholipase D zeta 2 protein and select transgenic plants, which are sensitive to auxin when the auxin concentration is greater than or equal to 10nM tall plants, The amino acid sequence of the phospholipase D zeta 2 protein described therein is shown in SEQ ID NO: 2. 6. method as claimed in claim 5, is characterized in that, described method comprises the step: (s1) providing an Agrobacterium carrying an expression vector, the expression vector containing the gene encoding the phospholipase D zeta 2 protein; (s2) plant cells, tissues or organs are contacted with the Agrobacterium in step (s1), so that the coding gene of the phospholipase Dzeta 2 protein is transferred into the plant cells and integrated into the chromosomes of the plant cells; (s3) select plant cells, tissues, organs that have been transferred to the gene encoding the phospholipase D zeta 2 protein; and (s4) Regenerate the plant cells, tissues, and organs in step (s3) and select transgenic plants, that is, plants with high sensitivity to auxin when the auxin concentration is ≥ 10 nM. 7. The method according to claim 5, wherein the concentration of auxin is 10-1000000 nM. 8. A method for preparing plants with high sensitivity to auxin when the auxin concentration is ≤ 1nM, the auxin being indole acetic acid, characterized in that the method comprises: (1) Agrobacterium carrying an antisense expression vector is provided, and the antisense expression vector contains the coding gene of the phospholipase D zeta 2 protein initiated in the reverse direction; (2) Plant cells, tissues or organs are contacted with the Agrobacterium in step (1), so that the coding gene of the phospholipase D zeta 2 protein that is started in the reverse direction contained in the antisense vector is transferred into plant cells, and Integrate into the chromosomes of plant cells; (3) select the plant cells, tissues, and organs that have been transferred to the gene encoding the phospholipase D zeta 2 protein that is started in the reverse direction contained in the antisense vector; and (4) regenerating plant cells, tissues and organs in step (3) and selecting transgenic plants, which are plants with high sensitivity to auxin when the auxin concentration is ≤ 1nM, The amino acid sequence of the phospholipase D zeta 2 protein described therein is shown in SEQ ID NO: 2. 9. The method of claim 8, wherein the concentration of auxin is 0.00001-1 nM.

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