JP2002537760A - Root-specific promoter - Google Patents

Abstract

(57) [Problem] To provide a promoter that enables the expression of a gene in the root of a plant. SOLUTION: DNA having the sequence shown in FIG. 5 and being a gene promoter region was isolated from the root of corn. Promoters can be used to express foreign genes in the roots of plants. This is particularly useful for expressing insecticidal toxins such as delta-endotoxin of Bacillus thuringiensis to confer resistance to insect attack on the roots of plants by Coleoptera insects.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a gene promoter sequence that controls the expression of a gene in a root tissue of a plant.

[0002] In the improvement of plants by molecular techniques, it is desirable that the expression of the introduced foreign gene be restricted to those tissues whose expression would have obvious effects. Mainly for
There are two reasons. First, limited expression is less harsh on plant metabolism than global (structural) expression. Second, if the expressed protein has no effect on edible parts, it is a good way to express the foreign gene in humans or parts of plants that are not used for edible use in animals. It is. The second reason is
It may be important if the effect of ingesting the expressed protein is not completely known.

A known goal in genetic improvement of cereal plants is to introduce resistance to insect attack. Certain insect species attack green leaf tissue, while others, such as Coleoptera, attack roots. Similarly, there are certain microorganisms that attack underground plant tissue and induce disease, and any genetic mutation that confers resistance to such organisms,
Require expression of resistance-conferring genes in roots.

An object of the present invention is to provide a root-specific gene promoter sequence and a means for isolating the sequence from root DNA.

According to the present invention, there is provided a DNA sequence having the sequence shown in FIG. 5, which defines the promoter of a plant gene expressed in the root.

[0006] The DNA can be isolated from root tissue of a particular target plant species. A preferred specific target plant is Zea mays .

[0007] The present invention also provides a gene construct comprising, as a sequence, the above-described gene promoter of the present invention, a coding region located downstream of and regulated by the promoter, and a 3 'untranslated region containing a polyadenylation signal. I will provide a.

[0008] Preferably, the coding region encodes a protein that is toxic to root attacking organisms, more preferably, the protein is a Bacillus thuri ngiensis insecticidal endotoxin.

Further, according to the present invention, there is provided a plant genome into which the gene construct of the present invention has been inserted by transformation.

[0010] The promoter sequence of the present invention can be isolated from a genomic sequence to which cDNA derived from a gene expressed in a root hybridizes. A genomic library is screened using the cDNA as a probe. Those genomic fragments that hybridize to cDNA probes have not only the structural gene, but also a promoter sequence associated with it. The promoter can then be isolated by cutting the sequence around the translational start site of the structural gene sequence. The sequences of such suitable cDNAs are shown in FIGS. 1 and 2, which were isolated from maize.

[0011] These cDNAs were placed in (1) a plasmid called pMR7 in E. coli DH5α host and (2) a plasmid called pMR7 / 10.1 in E. coli DH5α host, and the National Collection of Industrial and Marine Bacteria (Aber
deen, United Kingdom) on March 15, 1990, under accession number 40267. These deposits were made under the Budapest Treaty on the Deposit of Microorganisms for Patents.

Many genes encoding insecticidal proteins have been described in the literature, particularly the delta-endotoxin gene of Bacillus thuringiensis.

The present invention is described by way of illustration with the following examples.

Example 1 Total RNA was extracted from the root tissue of corn plants grown for 5 days and 14 days. Total RNA was also isolated from corn leaves and immature cobs for use in the comparative studies described below.

The RNA sample was purified by the guanidium thiocyanate / cesium chloride method, and the poly (a) + mRNA was purified by an oligo (dT) column. The corresponding cDNA was synthesized by the oligo dT priming method, and after adding a linker, the cDNA was cloned into the plasmid pUC13.

The results of each of these methods were monitored by label insertion. Clone digests randomly picked from the cDNA library showed a size distribution for the insert between 300 and 1300 base pairs.

[0017] The recombinants were individually transferred to microtiter wells and the library totaled about 7000
Consisted of clones.

A clone corresponding to the gene whose expression in the root was amplified was identified by differential screening. The same filter was prepared from the microtiter plate and hybridized separately with the root mRNA and a probe prepared by first-strand synthesis of leaf mRNA grown for 4 weeks. Overlap the autoradiograph, as showing a stronger signal in the root probe than the leaf probe,
Recombinants showing amplified expression at the root were selected. Interestingly, the selected differentially hybridized clones were not among the high expressing classes, and all samples of this type showed comparable intensity signals for both probes.

According to this procedure, 235 clones were selected after the initial screening as potentially indicative of some degree of differential hybridization.
Further screening reduced this number to 13.

As determined from restriction digestion or PCR, c of these 13 clones
DNA inserts ranged from 300 to 1100 base pairs. Each of the 13 candidate inserts was then used for Northern hybridization to confirm its tissue specificity.

R from 5 or 14 day old root tissues and leaf and cob tissues for comparison
NA was tested to identify those that were expressed in the root but not in the leaves or cobs.

In this procedure, the clone, designated pMR7, showed amplified expression at the roots at 5 and 14 days of age and showed little expression in leaves and cobs.

FIG. 3 shows an autoradiograph of a Northern blot probed with pMR7. For comparison purposes, FIG. 4 shows an autoradiograph of a Northern blot probed with MR12, a typical clone showing no amplified expression in the root. A comparison of FIGS. 3 and 4 shows that pMR7 hybridizes to both 5 and 14 day old root RNAs and hardly hybridizes to both leaf or cob RNAs, whereas pMR2 is 5 day old. 14-day-old root RNA shows a strong signal for the root RNA of
A strong signal was shown for both NAs.

Therefore, pMR7 was selected for further analysis. The insert of pMR7 was 700 base pairs in length and the entire sequence was determined by synthesizing oligonucleotides at approximately 200 base pair intervals, walking the full length, and directly sequencing the plasmid. There is a poly (A) + tail. The insert of pMR7 is shown in FIG.

The corn genomic DNA digest was tested using pMR7 as a probe.
Southern blots showed that the copy number of the corresponding gene was low, that is, only a few hybridizing bands could be detected at the stringency level used.

Screening of the partial Mbo I genomic library identified a number of putative positives from which upstream promoter sequences that govern expression in root tissues can be identified and sequenced.

[0027] The insert of pMR7 was used to screen a second maize seedling root cDNA library cloned into vector 1ZAPII.
From a number of positively hybridized clones, one, namely pMR7 /
10.1 was selected for further analysis. DNA sequencing revealed pMR7 /
10.1 is completely homologous to pMR7 but has been shown to be longer in length, probably representing a clone of the full-length cDNA. The sequence of pMR7 / 10.1 is shown in FIG.

Example 2 A gene-specific probe, which is the entire 3 'untranslated region of the MR7 gene, was radiolabeled,
A commercially available maize genomic library obtained from Clontech (USA) (W22 series) was used to screen. Probes obtained by PCR using cDNA as a template were 350 bp in length and had lower GC content than total cDNA, thereby reducing the possibility of non-specific hybridization.

After three rounds of plaque purification, five clones were selected for further analysis. Each strongly hybridized to oligonucleotide probes designed over the length of the pMR7 cDNA, confirming that they were closely related to the original cDNA. Restriction analysis of purified DNA from these lambda clones shows that four of them (# 7, 11, 14, and 15) are clearly related based on similarity of restriction profiles. Was done. Number 10, another clone,
Had different profiles. Hybridization with a probe specific for the MR7 gene confirmed this relationship. Five or more lambda isolates, each probing digest resulted in a single or few hybridization bands, with the 10th having a different profile than the other four.

For further analysis, number 7 was selected from the four more closely related lambda clones based on the larger insert size estimated by restriction analysis to be approximately 16 kb (the other inserts were The size ranged from 9.0 to 13.5 kb).

To identify genomic fragments containing the MR7 promoter, the insert from lambda clone 7 was subcloned into the pUC18 vector. pMRP1 represents a 10 kb EcoRI fragment subcloned from lambda clone # 7. By partial sequencing using internal primers, this fragment, in contrast to the different but distinct MR7 genes, which were either related, was transformed into the pMR7 / 10.
It was confirmed that the DNA contained DNA related to the DNA of the present invention.

Using the restriction site identified at the 5 'end of the pMR7 cDNA, lambda clone 7
The internal upstream region of the MR7 gene was identified and subsequently isolated based on hybridization to a specific oligonucleotide probe designed for cDNA upstream of the site. ATG translation start point (ATG is 3'Nc
4.2 kb of NcoI, which corresponds to the gene region immediately upstream of the
The fragment was subcloned into pUC18 (pMRP2).

A 1.9 kb XbaI fragment was also identified from within the pMRP2 insert that corresponds to the region expected to contain an active gene promoter.

The 4.2 kb region of pMRP2 was sequenced. FIG. 5 shows the arrangement. Short sequences with homology to "sequence motifs" of many promoters described in the literature are observed.

The primer extension technique was used to identify the start of transcription within the primer region. A point that could be the transcription start point was identified 25 nucleotides downstream of the A + T region, thought to correspond to the "TATA" box of the MR7 promoter.

To confirm the activity of the putative promoter region, both a 4.2 kb NcoI fragment and a 1.9 kb XbaI fragment were cloned into a “promoter analysis construct” for easy analysis of β-glucuronidase (GUS). They were fused to the gene. In the former (pMRP3), a correct ATG-mediated fusion of the NcoI site was made. In the latter (pMRP4), the fusion is transcriptional and the resulting expression construct also
An "amplified" maize AdhI intron I sequence was included in the transcribed region.

[0037] Plasmid DNA of both pMRP3 and pMRP4 was used in transient expression experiments in corn protoplasts from several sources, including root, leaf and endosperm tissues. In each case, the GUS expression from the construct was classified as "highly expressed", higher than the control plasmid in which GUS expression was driven by a "standard" promoter such as 35S or corn Adh. High levels of GUS expression from these two constructs were also demonstrated by bombardment of root, leaf and coleoptile tissue of corn seedlings.

[0038]

[Sequence list]

[Brief description of the drawings]

FIG. 1A. Insertion sequence of pMR7.

FIG. 1B. Insertion sequence of pMR7, continued from FIG. 1A.

FIG. 2A: Sequence of pMR7 / 10.1.

FIG. 2B: Sequence of pMR7 / 10.1, continuation of FIG. 2A.

FIG. 2C: Sequence of pMR7 / 10.1, continuation of FIG. 2B.

FIG. 3 is an autoradiograph of a Northern blot tested for pMR7.

FIG. 4 is an autoradiograph of a Northern blot tested for MR12.

FIG. 5A: 4.2 kb region of pMRP2 (root gene promoter).

FIG. 5B: 4.2 kb region of pMRP2 (root gene promoter), FIG. 5A
Continued.

FIG. 5C: 4.2 kb region of pMRP2 (root gene promoter), FIG. 5B
Continued.

FIG. 5D: 4.2 kb region of pMRP2 (root gene promoter), FIG. 5C
Continued.

FIG. 5E: 4.2 kb region of pMRP2 (root gene promoter), FIG. 5D
Continued.

FIG. 5F: 4.2 kb region of pMRP2 (root gene promoter), FIG. 5E
Continued.

[Procedure amendment]

[Submission date] July 14, 2000 (2000.7.14)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Contents of correction]

[Claims]

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Claims (7)

[Claims] 1. A DNA sequence having the sequence according to FIG. 5, which defines the promoter of a plant gene expressed in the root. 2. The DNA of claim 1, wherein said sequence is isolated from root tissue of a particular target plant species. 3. The DNA according to claim 2, wherein the target plant species is Zea mays . 4. A gene construct comprising the gene promoter according to claim 1, a coding region located downstream of the promoter and controlled by the promoter, and a 3 ′ untranslated region containing a polyadenylation signal as a sequence. . 5. The genetic construct of claim 4, wherein the coding region encodes a protein that is toxic to root attacking organisms. 6. A protein is insecticidal endotoxins of Bacillus Ju ringgit ene cis (Bacillus thuringie nsis), the gene sequence of claim 5. 7. A plant genome into which the gene construct according to any one of claims 4 to 6 has been inserted by transformation.