CN102134569A - Maize MAPK gene promoter and application thereof - Google Patents

Abstract

The present invention discloses a corn MAPK gene promoter and its application, wherein the corn MAPK gene promoter is a promoter capable of responding to various environmental stress signals, and its nucleotide sequence is shown in sequence 1 in the sequence list , or, is the DNA sequence of a gene promoter that reaches more than 85% identity with Sequence 1; the promoter is used to construct one or more induced responses including drought, cold, salt, germs, light, heavy metal and trauma The carrier, thus applied to the breeding of transgenic stress-resistant plants.

Description

A kind of promoter of maize MAPK gene and its application

technical field

The invention relates to a promoter of a plant MAPK gene, in particular to a promoter of a corn MAPK gene and application thereof.

Background technique

Corn is one of the most important food crops in the world. Drought is a major environmental factor limiting global maize production. According to conservative estimates, only in the tropics, the loss of maize due to drought is at least 20 million tons per year, and the loss rate is at least 17%. According to conservative estimates, corn accounts for 21.1% of my country's total grain output. For many years, in the national key research plan, the goal of maize breeding has always emphasized high quality, high yield, and multi-resistance, but the actual evaluation is based on maize yield as a hard indicator, which will objectively induce breeding experts to ignore the quality and adaptability of maize breeding. sex. In addition, the drought resistance trait of maize is a quantitative trait controlled by polygenes, and the drought resistance mechanism is very complicated, so the understanding of it is limited, which objectively leads to the difficulty of drought resistance breeding in maize.

For a long time, the identification and expression of drought stress response genes have been hoped at home and abroad as a breakthrough to analyze the drought resistance mechanism of maize. With the development and application of DNA chip or microarray technology, a large number of plant drought stress response genes and various gene expression profiles have been discovered. The expression of these genes has obvious time, space, tissue and condition specificity. Form a complex network regulatory system, but the mechanism of regulation of these responsive gene expression has not yet made a reasonable explanation.

A large number of studies have shown that the temporal, spatial, tissue-specific and conditional specificity of gene expression largely depends on the gene promoter. The composition of the promoters of different genes varies greatly. For example, it is generally believed that the promoter recognized by ribonucleic acid (RNA, Ribonucleic Acid) polymerase II contains a TATA box and or an initiation factor that controls specific transcription initiation. For many years, the TATA box was considered to be the main core element controlling the basic transcription machinery, but the promoters of many other genes lack the TATA box element and contain a promoter, even for the same promoter, due to epigenetic changes within the promoter ( epigenetic alteration), such as methylation or hypermethylation, can lead to transcriptional effects on different genes. In addition, many genes, especially some family genes, have no obvious differences in functional domains, but their transcription processes are very different, such as human and mouse heat shock transcription factors HSF1, HSF2, HSF3 and HSF4, where HSF1 and HSF3 are related to heat HSF2 has no response to classical stressors. Although HSF4 is similar to HSF1 and HSF2, it is expressed in a tissue-specific manner. One of the reasons is that the promoters of these heat shock transcription factors HSFs The heat shock element is different.

In addition, since many traits of maize such as stress resistance are controlled by multiple genes, it is difficult to achieve systemic resistance of the whole maize plant by transforming a single gene. Studies have proved that transforming a small amount of genes is difficult to endow crops with the resistance traits that meet production needs. Internationally, attempts have been made to transform more than two genes at the same time. In addition, due to the limitations of current transgenic technology, attempts to transform more than two genes at the same time The successes are few and far between.

Mitogen-activated protein kinase (MAPK, Mitogen-Activated Protein Kinase) is a class of serine/threonine protein kinase that exists in all eukaryotes, and is an important factor in intracellular signal transduction. 4 MAPK subfamilies. They play a role in signal transmission and cascade amplification through phosphorylation and dephosphorylation of proteins in organisms, and can respond to a variety of environmental stress signals, such as cold and drought, salt and trauma. Several MAPK kinases have been found in plants that can respond to multiple stresses including drought simultaneously, such as MAPK in alfalfa cells, SIMK-like SIPK in tobacco cells, and MAPK and MAPKKK in Arabidopsis. It can be predicted that the combination of the promoter of this type of gene and other genes can change the expression response mode of the gene to the environment or can make the gene specifically respond to a certain environment.

The research on the promoter of maize gene has made some progress at home and abroad. On January 7, 2009, there were 198 plant promoters recorded in The Eukaryotic Promoter Database, of which 21 were maize promoters. The 21 maize promoters that have been reported include 11 prolamin gene promoters, 2 photogatherin gene promoters, 1 sucrose synthase gene promoter, 1 uridine diphosphate (UDP, Uridine DiphosPhate) - Glucose-amyloglucosyltransferase gene promoter, 3 anthocyanin synthesis-related gene promoters, 2 alcohol dehydrogenase gene promoters and 1 heat shock protein memory promoter.

At present, the research on the promoter of plant MAPK gene in response to drought stress has not been paid much attention at home and abroad, and there is no research report on the promoter of MAPK gene. In the early research, our laboratory performed large-scale sequencing analysis on the EST of maize expression genes under drought stress, produced a cDNA microarray of 11855 genes, and used this microarray to analyze the genes of maize seedlings under drought stress on a large scale MAPK gene was found to be significantly up-regulated under drought stress (data not shown). Therefore, the cloning of the MAPK gene promoter provides a theoretical basis for further elucidating the gene expression network regulation and drought resistance mechanism of maize under drought stress, and also has potential application value in the construction of expression vectors in the research of drought resistance breeding of transgenic maize.

references:

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Contents of the invention

The technical problem to be solved by the present invention is to provide a maize MAPK gene promoter and its application, which can respond to various environmental stress signals, so as to construct a gene expression vector that induces responses to various environmental stress signals.

In order to solve the above technical problems, the invention provides a corn MAPK gene promoter is one of the following nucleotide sequences:

(1) the DNA sequence of sequence 1 in the sequence listing;

(2) A DNA sequence having more than 85% identity with the DNA sequence defined in Sequence 1.

Preferably, the gene promoter contains DNA sequence elements responsive to various environmental stress signals.

Preferably, the DNA sequence elements responsive to the multiple environmental stress signals include:

1 copper-responsive element;

4 light-responsive elements;

1 dehydration/light-responsive element;

2 salt or pathogen-responsive elements (salt/pathogen-responsive element);

2 TATA boxes (TATAAAT or TAATA) associated with the transcription rate;

1 pathogen-responsive element;

1 methyl jasmonic acid (MeJA-responsive element) response element;

1 wound-responsive element;

1 CAAT box (CAAT box) with a function similar to TATA box.

In order to solve the above technical problems, the present invention provides the application of the aforementioned maize MAPK gene promoter in the breeding of transgenic stress-resistant plants.

Preferably, the gene promoter is used to construct a vector that induces response to one or more of drought, cold, salt, pathogen, light, heavy metal and wound, so as to be applied to the breeding of the transgenic stress-resistant plant.

The present invention provides the promoter of the MAPK gene derived from corn that can be induced by water shortage, and the promoter contains drought (or water shortage), abscisic acid (ABA, ABScisic Acid), K ⁺ influx, cold, salt, pathogenic bacteria , light, sugar, heavy metal and wound-responsive DNA sequence elements, and thus may be induced in maize under stress conditions of drought (or lack of water), ABA, K ⁺ influx, cold, salt, pathogens, light, sugar, heavy metal and wound stress Initiate gene expression. The promoter has almost no homology with the previously reported maize gene promoter, so it is a newly discovered maize gene promoter in the present invention, and it is of great significance in stress-resistant breeding of transgenic plants.

Description of drawings

Fig. 1 is the flow chart of the method embodiment that the present invention utilizes chromosome walking to clone the corn MAPK gene promoter;

Figure 2 is a chromosomal DNA fragment amplified upstream of the MAPK gene by chromosome walking PCR;

Fig. 3 is the DNA sequence of the maize MAPK gene promoter (Pro36N) cloned by the present invention and its predictive containing response element;

Fig. 4 is the method for detecting the expression construction of corn MAPK gene promoter;

Fig. 5 is the histochemical staining detection of the amplified maize MAPK gene promoter (Pro36N) promoting glucuronidase (GUS, glucuronidase) gene expression.

Detailed ways

The promoter provided by the invention is derived from the gene of maize mitogen-activated protein kinase (MAPK, Mitogen-Activated Protein Kinase), contains the DNA sequence element of drought, cold, salt, germ, light, heavy metal and wound response, can be in plant The gene expression can be induced and activated under the stress conditions of drought, cold, salt, pathogen, light, heavy metal and trauma, so it is of great significance in stress-resistant breeding of transgenic plants. The promoter has almost no homology with the reported gene promoters, so it is a newly discovered promoter of plant genes.

The technical solutions of the present invention will be described in detail below in conjunction with the accompanying drawings and preferred embodiments. The following examples are only used to illustrate and explain the present invention, but not to limit the technical solution of the present invention.

Materials used in embodiments of the invention include:

The expressed sequence tags (ESTs) of the MAPK gene were obtained from the research project of the maize EST sequencing project in our laboratory and submitted to the GenBank of the National Center for Biotechnology Information (NCBI) (GenBank, index number EC857736).

Escherichia coli (Escherichia coli) strain DH5α was preserved in our laboratory.

Restriction endonuclease, T4 ligase and PCR amplified DNA cloning vector pGEM-T easy Vector were purchased from Promega.

X-gluc (imported aliquots) and gel DNA recovery kit were purchased from Hengyin Biological Company.

DNA Marker, common Taq enzyme, etc. were purchased from Fermentas Company.

Gel recovery kit and PCR product purification kit were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

The Agrobacterium tumefacien strain LBA4404, the plant expression vector pCambia1301, and the auxiliary plasmid pRK2073 used as an auxiliary plasmid DNA transfer between bacteria in the combination of the three parents were collected and preserved in our laboratory.

The Chromosome Walking Kit Genome Walking Kit was purchased from TAKARA Company; the chromosome location of the EST of the maize MAPK gene used the BLASTN-HTG program in the database of the Plant Genomics website (PlantGDB, whose website is http://www.plantgdb.org/) .

The DNA sequencing of the MAPK gene promoter was entrusted to Shanghai Sangon Bioengineering Technology Service Co., Ltd. The prediction of gene transcription start sites was performed using the NNPP version 2.2 program (http://www.fruitfly.org/seq_tools/promoter.html). The conventional cis-acting regulatory elements of plant gene promoters are predicted according to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and PLACE (PlantCis-acting Regulatory DNA Elements, http://www.dna. affrc.go.jp/PLACE/) database provided by the method.

The cultivation of plants adopts hydroponics, and the culture medium is Hoagland's solution (Hoagland and Arnon1938).

Agrobacterium and Escherichia coli medium are common LA (10 grams of peptone, 5 grams of yeast powder, 5 grams of NaCl, 15 grams of agar powder, 1000 milliliters of water, pH7.0) and LB (LA without adding agar powder).

The three specific primers used in the first round of chromosome walking PCR amplification are:

SP1: 5'-ACGTACTCCGTCATCATGTCGCTCTC-3',

SP2: 5'-ACGTTGGCGGAGTGGATGTACTTGAG-3',

SP3: 5'-TTGGACCGGATGATGTGTGCAG-3'.

The 3 specific primers used in the second round of walking PCR amplification are:

Pro-SP1: 5'-GCGCTGTGCAGGATCAAATTAAGAG-3',

Pro-SP2: 5'-GTCCAACCAATCAATCCATGCCCATC-3',

Pro-SP3: 5'-TGCAGTTCGCGAGAACGTACCAGAC-3'.

The method of Jefferson (1987) was used for the histochemical detection of gene expression.

Example 1

As shown in Figure 1, for the method embodiment flow process of cloning corn MAPK gene promoter of the present invention, comprise the steps:

10: locating the EST sequence of the maize MAPK gene on the maize chromosome;

According to the free BLASTN-HTG program analysis in the PlantGDB (http://www.plantgdb.org/) database, the EST sequence of the maize MAPK gene was mapped to the chromosome 9 of maize.

20: Walk the positioned chromosome;

Chromosome walking PCR amplification adopts TAIL-PCR (Liu et al., 1995) amplification strategy, and the DNA fragments amplified each time are shown in Figure 2, and cloned into the pGEM-T easy Vector plasmid vector under the action of T4 ligase Perform sequencing.

30: connecting the PCR product generated by chromosome walking with the plant gene expression vector containing the reporter gene;

Under the action of T4 ligase, the plasmid vector cloned into pGEM-T easy Vector was sequenced to obtain the DNA sequence of the maize MAPK gene promoter (numbered as Pro36N).

40: Detect the expression of the reporter gene, and identify the DNA fragment of the promoter of the maize MAPK gene.

The above identification includes the prediction of the DNA sequence of the cloned maize MAPK gene promoter and the sequence of the response elements contained therein.

According to the method process shown in Figure 1, a DNA fragment (as shown in Figure 2) amplified by sequence-specific PCR was obtained, i.e. the DNA fragment of the maize MAPK gene promoter, which was determined to be 387bp in length through sequencing, as shown in Figure 3 . Neural Network Promoter Prediction (NNPP version 2.2) was used to predict the transcription start site of MAPK gene, and the method provided in PlantCARE and PLACE database was used to predict and analyze, and it was found that the DNA segment contained the following corresponding DNA Sequence element:

1 copper-responsive element;

4 light-responsive elements;

1 dehydration/light-responsive element;

2 salt or pathogen-responsive elements (salt/pathogen-responsive element);

2 TATA boxes (TATAAAT or TAATA) associated with the transcription rate;

1 pathogen-responsive element;

1 methyl jasmonic acid (MeJA-responsive element) response element;

1 wound-responsive element;

1 CAAT box (CAAT box) with a function similar to TATA box.

The DNA sequence of the maize MAPK gene promoter is shown in Sequence 1.

Example 2

Construction of β-glucuronidase (GUS, β-Glucuronidase) gene expression vector with maize MAPK gene promoter (Pro36N) and detection of GUS gene expression

The pseudo-promoter DNA fragment cloned on the pGEM-T easy Vector plasmid vector was digested with SalI/NcoI and recovered. pCAmbia1301 was digested with SalI/NcoI to remove the 35S promoter DN before the GUS gene. Under the action of ligase, the pseudo-promoter DNA fragment digested by Sal I/Nco I was ligated with pCAmbia1301 to construct a new recombinant plasmid. In the same way, a recombinant plasmid with a 672 bp non-promoter DNA of peanut was constructed as a negative control plasmid, as shown in FIG. 4 .

The constructed recombinant plasmid was introduced into Agrobacterium LBA4404 cell through triparental combination (between Escherichia coli DH5α containing the auxiliary plasmid pRK2073, Escherichia coli DH5α containing the recombinant plasmid, and Agrobacterium LBA4404 cell). Agrobacterium LBA4404 cultured to OD ₆₀₀ =0.6 carrying the recombinant plasmid was injected into the veins of maize leaves at the three-leaf stage using a disposable syringe (with a 4 ^# needle). 1/5 Hoagland nutrient solution at 26°C, 12h light/24h, cultured for 2 days, followed by Jefferson (1987) method for histochemical staining. The specific method of histochemical staining is: soak in X-gluc staining solution (100mM (pH 7.0) phosphate buffer; 0.1% (V/V) Triton-X 100; 10mM EDTA; 0.5mg/ml X-Gluc; 1% (V/V) dimethyl sulfoxide), 37 ° C overnight, 70% ethanol decolorization. The results of tissue staining are shown in Figure 5.

The constructed recombinant plasmid was introduced into Agrobacterium LBA4404 cell through triparental combination (between Escherichia coli DH5α containing the auxiliary plasmid pRK2073, Escherichia coli DH5α containing the recombinant plasmid, and Agrobacterium LBA4404 cell). Agrobacterium LBA4404 cultured to OD ₆₀₀ =0.6 carrying the recombinant plasmid was injected into the veins of maize leaves at the three-leaf stage using a disposable syringe (with a 4 ^# needle). 1/5 Hoagland nutrient solution at 26°C, 12h light/24h, cultured for 2 days, followed by Jefferson (1987) method for histochemical staining.

The specific method of histochemical staining is: soak in X-gluc staining solution (100mM (pH 7.0) phosphate buffer; 0.1% (V/V) Triton-X 100; 10mM EDTA; 0.5mg/ml X-Gluc; 1% (V/V) dimethyl sulfoxide), 37 ° C overnight, 70% ethanol decolorization. The results of tissue staining are shown in Figure 5.

in:

A: negative control. A 672bp non-promoter DNA from peanut was used to replace the upstream CaMV35S promoter of the GUS gene on pCambia1301 for plasmid construction.

B: Original pCambia1301 positive control.

C: Maize MAPK gene promoter (Pro36N) amplified by chromosome walking PCR. The amplified Pro36N promoter (including the start codon, with a total length of 387bp) replaced the construction of the upstream CaMV35S promoter of the GUS gene on pCambia1301 for plant expression.

The detection method adopts the method of Agrobacterium-mediated transient expression in infected maize leaves combined with histochemical staining. The results of histochemical staining, see Fig. 5C, indicated that Pro36N can initiate the expression of the reporter gene gus, which is a true promoter.

The purpose of this example is to verify that the maize MAPK gene promoter Pro36N can initiate the expression of the reporter gene gus, and is a real promoter.

gene sequence list

Sequence 1: promoter DNA sequence of maize MAPK gene

1 TGCAGTTCGCGAGAACGTACCAGACGAGCCCTACCCGCCGTCGGATCCG 49

50 AACAGCTTCCCTTCCATCAGATTCAGAGCGCCGTCCCGCTCACTTTCTC 98

99 GCTACCTTCCTCAGCCGGAGTCGTGGGCAGAGCAGAGCGATCGAGGCCTG 148

149 CTTCAGCAGTTCAGCTGCAGCCGCCGCCCGCCCGTCCCCAGTCCCAC 196

197 CTTCTTTTATAAATCCCACGCCTGCGCTTCCTCTTTTGACGAATCGGTC 245

246 GCGAGAGGAGAAACATAGGAGTCAATTAATTTGCCTGTTCTGCTTGCTG 294

295 CTGACCTTGTGAGTGAGCCGCCGGTCTTTCCCCTTCCTCGCCGAGGGGAA 343

344 ATAATAAGGCAGCCACAGCCAGGGCCGCCCCGAGGAAGAAGATG 387

Claims (5)

1. a corn MAPK gene promoter is one of following nucleotide sequences:

(1) dna sequence dna of sequence 1 in the sequence table;

(2) dna sequence dna with described sequence 1 qualification has conforming dna sequence dna more than 85%.

2. according to the described gene promoter of claim 1, it is characterized in that, contain the dna sequence dna element of multiple environment-stress signal response.

3. according to the described gene promoter of claim 2, it is characterized in that the dna sequence dna element of described multiple environment-stress signal response comprises:

1 copper response element (copper-responsive element);

4 light response elements (light-responsive element);

1 lack of water/light response element (dehydration/light-responsive element);

2 salt or pathogenic bacteria bacterium answer element (salt/pathogen-responsive element);

2 TATA boxes relevant (TATAAAT or TAATA) with transcription rate;

1 pathogenic bacteria bacterium answers element (pathogen-responsive element);

1 methyl jasmonic (MeJA-responsive element) response element;

1 wound response element (wound-responsive element);

1 has the CAAT box (CAAT box) that is similar to the effect of TATA box.

4. the application of the described corn MAPK of claim 1 gene promoter aspect the breeding of transgenosis adversity resistant plant.

5. according to the described application of claim 4, it is characterized in that, described gene promoter is used for making up the carrier of one or more evoked responses that contain arid, cold, salt, germ, light, heavy metal and wound, thereby is applied to the breeding of described transgenosis adversity resistant plant.

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