WO2000029594A1 - Root-specific promoter - Google Patents

Abstract

A DNA which has the sequence shown in Figure 5 and which defines a gene promoter region has been isolated from maize roots. The promoter may be used for driving expression of foreign genes in the roots of plants. This is particularly useful for expressing an insecticidal toxin, such as a delta-endotoxin of Bacillus thuringiensis, to impart resistance to insect attack on the roots of plants by Coleopteran insects.

Description

ROOT-SPECIFIC PROMOTER

This invention relates to a gene promoter sequence which directs expression of a gene to the root tissue of plants.

In the genetic improvement of plants by molecular techniques,, -t is desirable that expression of inserted foreign genes be restricted to tissue where that expression will have significant effect. There are two principal reasons for this. First, restricted expression, rather than total (constitutive) is likely to be less demanding on the metabolism of the plant . Secondly, it would be good practice to direct expression of the foreign gene to those parts of the plant which are not used for human or animal food when the expressed protein has no effect on such food parts. This second reason may be important when the effect which ingestion of the expressed protein may have is not be fully known. One widespread target for genetic improvement of crop plants is the introduction of resistance to insect attack. Certain insect species attack green leaf tissue, whereas other, for example Coleoptera, attack the roots. Similarly there are certain disease-inducing microorganisms which attack the below-ground plant tissue and any genetic modification to impart resistance to such organisms will require expression of the resistance-imparting gene in the roots. An object of the present invention is to provide a root-specific gene promoter sequence and means for isolating same of root DNA.

According to the present invention there is provided a DNA sequence, defining a promoter of a root-expressed plant gene, having the sequence set forth in Figure 5 herewith.

The said DNA may be isolated from the root tissue of a particular target plant species of interest. The preferred species is Zea mays.

The invention also provides a gene construct comprising, in sequence, the aforesaid gene promoter of the invention, a coding region located downstream and controlled by the said promoter and a 3' -untranslated region including a polyadenylation signal.

Preferably the coding region encodes a protein which is toxic to root-attacking organisms and more preferably the protein is an insecticidal endotoxin of Bacillus thurinαiensis .

Further according to the invention there is provided a plant genome into which the gene construct of the invention has been inserted by transformation. The promoter sequence of the invention may be isolated from the genomic sequence to which a cDNA derived from a root-expressed gene hybridises. A genomic library is screened using the said cDNA as a probe . Those geaomic fragments which hybridise to the cDNA probe carry not only the structural gene but the promoter sequence associated therewith. The promoter may then be isolated by cleavage of the sequence around the location of the translation start point of the structural gene sequence. The sequences of suitable such cDNAs are shown in Figures 1 and 2 were isolated from maize.

These cDNAs have been deposited (1) in a plasmid designated pMR7 in an E.coli DH5α host and (2) in a plasmid designated pMR7/10.1 in an E.coli DH5α host, at the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, on 15th March 1990, under the Accession Number 40267. These deposits were made under the terms of the Budapest Treaty on the deposit of microorganisms for patent purposes. ~

Many genes specifying insecticidal proteins, particularly delta-endotoxin genes of Bacillus thurinqiensis have been reported in the literature.

The invention will now be described, by way of illustration, by the following Examples. EXAMPLE 1

Total RNA was extracted from root tissue of five-day old and fourteen-day old maize plants.

For use in certain comparative tests which will be described later, total RNA was also isolated from maize leaf and immature cob.

The RNA samples were purified using the guanidinium thiocyanate/caesium chloride method and poly(a)+mRNA purified on an oligo(dT) column. The corresponding cDNAs were synthesised using the oligo dT priming method and the cDNA cloned into plasmid pUC13 aftear linkering. The success of each of^"these stages was monitored by incorporation of a label. Digests of randomly picked clones from the cDNA library showed a size distribution for inserts of between 300 and 1300 base pairs. Recombinants were individually transferred to microtitre wells, in total the library consisted of about 7,000 clones.

Clones representing genes with root enhanced expression were identified by differential screening. Identical filters were prepared from the microtitre plates and hybridised separately with probes prepared by first strand synthesis of root rr-RNA and four week old leaf mRNA. The autoradiographs were superimposed and recombinants showing root enhanced "expression were selected as showing a more intense signal with the root probe than with the leaf probe. Interestingly, none of the selected clones showing differential hybridisation fell into the highly expressed category; all examples of this type showed equally intense signals to both probes.

By this procedure, 235 clones were selected as potentially showing a degree of differential hybridisation after the first screen. This number was reduced to thirteen after further screens . The cDNA inserts of these thirteen clones ranged from 300 to 1100 base pairs as judged by restriction digestion or PCR. The inserts of each of the thirteen candidate inserts were then used in Northern hybridisations to confirm their tissue specificity.

RNAs from the five-day and fourteen day old root tissue and, fθr comparison, from leaf and cob tissue were probed to identify any which were expressed in root tissue but not in leaf or cob.

By these procedures, the clone designated pMR7 showed enhanced expression in both the five and the fourteen day old root and only insignificant expression in leaf and cob.

Figure 3 herewith shows the autoradiograph of a Northern blot probed with pMR7. For comparison purposes, Figure 4 shows the auto- radiograph of a Northern blot probed with pMR12 which was typical of those clones which do not show root enhanced expression. Comparison of Figures 3 and 4 shows that whereas pMR7 hybridised to both five- and fourteen-day old root RNA with little hybridisation to either leaf- or cob RNA, pMR2 gave strong signals on five-day old root RNA, much reduced signal on fourteen day old root but strong signals to both leaf and cob RNA.

Thus pMR7 has been selected for further analysis. The insert of pMR7 is 700 base pairs in length and has been fully sequenced by walking through its length by synthesising oligonucleotides at approximately 200 base pair intervals and performing direct plasmid sequencing. There is a poly (A) + tail. The sequence of the pMR7 insert is given as Figure 1 herewith.

Maize genomic DNA digests have been probed using pMR7 as a probe. Southern blots have indicated that the corresponding gene is of low copy number, that is, only a small number of hybridising bands are detectable at the level of stringency used.

From the screen of a partial Mbol genomic library a number of-putative positives have been identified and from these the^" upstream promoter sequence which directs expression to root tissue can be isolated and sequenced.

The pMR7 insert was used to screen a second maize seedling root cDNA library constructed in the cloning vector 1ZAP II. From a number of positively hybridising clones, one, pMR7/10.1, was selected for further analysis . DNA sequencing indicated that pMR7/10.1 was completely homologous with pMR7 but was of longer length, perhaps representing the full length cDNA clone. The sequence of pMR7/10.1 is given as Figure 2 herewith. EXAMPLE 2 A 'gene-specific' probe, representing the entire 3' untranslated region of the MR7 gene, was radioactively labelled and used to screen a commercial corn genomic library obtained from Clontech, USA (line W22) . The probe, obtained by PCR using the cDNA as a template, was 350bp in length and of lower G+C content than the entire cDNA, thereby reducing the chances of non-specific hybridisation.

Five clones were selected for further analysis after three rounds of plaque purification. Each hybridised strongly to oligonucleotide probes designed throughout the length of the pMR7 cDNA, confirming that they were closely related to the original cDNA. Restriction analysis of purified DNA obtained from these lambda clones indicated that 4 of them (numbers 7, 11, 14 and 15) were clearly related on the basis of similarity of restriction profiles. The other clone, number 10 had a different profile. Hybridisation of the MR7 gene-specific probe confirmed this relationship. Single or few hybridisation bands resulted from probing digests of each of the 5 lambda isolates, number 10 having a different profile than the other four. Of the four more closely related lambda clones, number 7 was chosen for further analysis on the basis of its larger insert size of approximately 16kb, estimated from restriction analysis (the other inserts ranging in size from 9.0 to 13.5kb) .

In order to identify a genomic fragment containing the MR7 promoter, the insert from lambda clone 7 was subcloned into pUC18 vectors. pMRPl represents a-lϋkb EcoRI fragment subcloned from lambda clone number 7.^" Partial sequencing with an internal primer confirmed that this fragment contained DNA related to that of pMR7/l0.1 cDNA, as opposed to any related but distinguishably different classes of the MR7 gene.

Utilising restriction sites identified at the 5' end of the pMR7 cDNA, the upstream region of the MR7 gene contained within lambda clone 7 was identified and subsequently isolated on the basis of hybridisation to specific oligonucleotide probes designed against sequence in the cDNA upstream of the aforementioned sites. A 4.2kb Ncol fragment was subcloned into pUClδ (pMRP2) which represents the region of the gene immediatly upstream of the ATG translation startpoint (the ATG being a part of the 3' Ncol restriction site).

From within the insert of pMRP2 , a 1.9kb Xbal fragment was also identified which represented a region expected to'contain an active gene promoter. The entire 4.2kb region of pMRP2 was sequenced. The sequence is given in Figure 5 herewith. Short sequences sharing homology with a number of promoter 'sequence motifs' described in the literature ca be recognised. The technique of primer extension was utilised to identify the transcription start point within the promoter region. A possible transcription start point was identified 25 nucleotides downstream of the A+T region thought to represent the 'TATA' box of the MR7 promoter.

To confirm the activity of the putative promoter regions, both the 4.2kb Ncol fragment and the 1.9kb Xbal fragment were cloned into a 'promoter assay construct', in which they were fused to a the easily a"ssayable B-glucuronidase (GUS) gene. In the former case (pMRP3), there was precise fusion through the ATG of the Ncol site. In the latter case (pMRP4) , the fusion was a transcriptional one, the resulting expression construct also containing the 'enhancing' maize Adhl Intron I sequence within the transcribed region.

Plasmid DNA of both pMRP3 and pMRP4 were used in transient expression experiments in maize protoplasts derived from several sources, including root, leaf and endosperm tissue. In each case, expression of GUS from the constructs was classifed as 'high', being greater than control plasmids in which GUS expression was driven by 'standard' promoters such as 35S and maize Adh. High level GUS expression from these two constructs was also demonstrated by bombardment of root, leaf and coleoptile tissues of maize seedlings.

Claims

1. A DNA sequence defining a promoter of a root-expressed plant gene having the sequence set forth in Figure 5 herewith.

2. A DNA as claimed in claim 1, in which the said sequence is isolated from the root tissue of a particular target plant species of interest.

3. A DNA as claimed in claim 2, in which the target plant species is Zea mays.

4. A gene construct comprising, in sequence, the gene promoter as claimed in claim 1. a coding region located downstream and controlled by the said promoter and a 3' -untranslated region including a polyadenylation signal.

5. A gene construct as claimed in claim 4, in which the coding region encodes a protein which is toxic to root-attacking organisms.

6. A gene sequence as claimed in claim 5, in which the protein is an insecticidal endotoxin of Bacillus thurineiensis.

7. A plant genome into which the gene construct claimed in any of claims 4 to 6 has been inserted by transformation .