A polygalacturonase-inhibiting protein (pgip) gene from Malus domestica cv Granny Smith apple plants was cloned by degenerate oligo-primed polymerase chain reaction (PCR) and Inverse PCR. An alignment of the pear and bean PGIP sequences was used to design degenerate PCR primers in highly conserved regions. Degenerate PCR allowed the amplification of a 351bp internal fragment of the pgip gene, termed ipgip. The DNA sequence of ipgip was used to design Inverse PCR primers. Inverse PCR enabled cloning of the remainder of the gene, from which a composite pgip gene sequence was constructed. A new set of PCR primers were designed to the 5' and 3' ends of the gene, which allowed amplification of the full-length gene from apple genomic DNA. This method has broad application to isolation of homologues of any gene for which some sequence information is known.

Introduction

Diseases resulting from pre- and post-harvest fungal infections have an enormous deleterious impact on world agriculture (Vasil, 1998). The recent rapid advances in agricultural biotechnology suggest that genetic engineering for increased quantitative resistance will make an increasing contribution to future disease control. Genes identified as encoding anti-microbial proteins can now be rapidly and precisely introduced into elite germplasm, creating novel pathogen resistant lines (Shah, 1997). An important part of the strategies designed to engineer increased resistance of plants to fungal diseases is the discovery and characterisation of plant antifungal proteins and the isolation of their encoding genes.

The polygalacturonase-inhibiting proteins (PGIPs) are key members of a class of anti-fungal proteins that potently inhibit the activity of cell wall-degrading fungal enzymes (De Lorenzo and Cervone, 1997). PGIPs inhibit the catalytic activity of fungal endopolygalacturonases, thereby favouring the accumulation of oligogalacturonides that are capable of triggering other defence responses.

Conventional PCR allows the amplification of sequences within known boundaries. Several methods have been developed for the amplification of DNA sequences that flank regions of known sequences. These include TGW-PCR (targeted gene walking PCR) (Parker et al., 1991) and Inverse PCR (Triglia et al., 1988; Ochman et al., 1988; Silver and Keerikatte, 1989). Inverse PCR allows the amplification of sequences that lie outside the boundaries of known sequences by inverting the unknown sequence. This is done by self-ligating digested DNA and opening the circular DNA molecules at a different site.

This paper describes the cloning of an apple pgip gene from Malus domestica cv Granny Smith by employing two PCR-based methods.

Results and Discussion

Degenerate oligo-primed PCR

The internal region of the pgip gene (ipgip) was cloned by degenerate oligo-primed PCR. Conserved regions between the pear (Stotz et al., 1993) and bean (Toubart et al., 1992) PGIPs which may represent important functional domains within the protein, were used to design degenerate primers for PCR. The primers were designed to amplify a 351bp product when used in a PCR reaction. The expected size product of 351bp was obtained for PCR reactions containing either a cloned bean pgip gene (plasmid pLD1), bean or pear genomic DNA as template (Fig. 1A, lanes 2, 3 and 4, respectively). A fragment of the same size, presumably within the pgip gene, was amplified in the PCR reaction containing apple genomic DNA as template (Fig. 1A, lane 5). This indicated the presence of pgip in the apple genome. The PCR product was cloned into a vector and the sequence of the insert (351bp product) in the resultant plasmid, pIPGIP (4.2kb), was determined by automated sequencing.

Inverse PCR

The remaining regions of the gene were cloned using Inverse PCR. For Inverse PCR cloning, the genomic DNA has to be cut with restriction enzymes that flank the region of interest. In this study two restriction enzymes were used, namely BglII and NsiI. BglII was used to clone the front or 5' end of the gene, whereas NsiI was used to clone the back or 3' end of the gene. For Inverse PCR reactions, primers were designed using sequence data of the ipgip region. The templates for the PCR reactions were prepared by digesting the DNA with either BglII or NsiI, followed by self-ligation and linearisation of the circular molecules at a different restriction site.

Results obtained for the BglII step of the Inverse PCR cloning of the apple pgip gene are shown in Fig. 1B. Using gene-specific Inverse PCR primers, AP-PGIP-INV_L and AP-PGIP-INV_R, the expected size product of 4.2 kb was obtained for the positive control reaction with pIPGIP as template (Fig. 1B, lane 4). A product of 820bp was amplified in the PCR reaction containing apple genomic DNA digested with BglII (Fig. 1B, lane 3). This product was cloned to produce pAppBglII. Sequence analysis revealed that the pAppBglII insert shared an overlap of 98bp and 166bp with the 5' and 3'-ends of the ipgip fragment, respectively (Fig. 2, panel B). Due to the sequence identity shared between the two fragments, the sequences were joined using computer software. On the assumption that the apple pgip gene was the same length as other cloned pgip genes, an additional 168bp downstream of the BglII site needed to be cloned in order to isolate the 3'-end of the gene.

A new set of Inverse PCR primers (AP-PGIP-INV_L-2 and AP-PGIP-INV_R-2) resulted in the amplification of a 560bp product (data not shown). This PCR product was cloned to produce pAppNsiI. Sequence analysis showed that pAppNsiI contained 300bp downstream of the BglII restriction site (Fig. 2, panel C). A stop codon (TAA) was identified 167bp from the BglII site and this represented the 3'-end of the pgip gene. The insert of pAppNsiI shared an overlap of 190bp with the insert of pAppBglII. The DNA sequence of the 5'-end of the pgip gene (pAppBglII and ipgip sequences) was linked with the DNA sequence of the 3'-end of the pgip gene (sequence from pAppNsiI) using computer software to give the complete sequence of a composite apple pgip gene (Fig. 2, panel D). Based on the sequencing data for the composite gene, a new set of PCR primers were designed to the 5' and 3' ends of the composite gene, which allowed amplification of the full-length gene from apple genomic DNA. Sequence analysis of the full-length gene revealed that it was identical to a pgip gene from Golden Delicious apples (Yao et al., 1999; GenBank accession no. U77041) which was published during the course of this work.

The use of degenerate oligo-primed PCR is a powerful method to clone new or uncharacterised genes that are related to a known gene family (Compton , 1990). The two most critical factors in degenerate oligo-primed PCR are the design of the primers as well as the PCR conditions. The primers should be designed to an amino acid region with minimal degeneracy in codon usage (Compton, 1990). PCR conditions must be optimised to give a balance between efficiency and specificity.

Inverse PCR is a convenient and versatile method of cloning unknown sequences upstream or downstream of known sequences (Triglia et al., 1988). Potential disadvantages of Inverse PCR have, however, been described. One of the drawbacks of Inverse PCR is the requirement for two restriction enzyme sites that flank the priming region. The lack of data on restriction sites as well as the size of chromosomal DNA greatly reduces the successful cloning rates. An additional problem with Inverse PCR is the inefficient PCR amplification of closed circular double-stranded DNA. The presence of introns in the target gene must also be considered when carrying out Inverse PCR from genomic DNA. In this study the apple pgip gene did not have introns.

In the study reported in this paper, several different enzymes were used to digest the apple genomic DNA, but subsequent Inverse PCR amplification was only successful for some of the enzymes used. One of the reasons could be that the sites were too far apart so that in the subsequent PCR, the amplification of large products was inefficient. Some DNA is lost at each clean-up step after digestion, ligation and re-digestion, therefore it is difficult to determine the amount of target DNA added to the PCR reaction. The absence of amplification products could therefore be due to insufficient target template. Several PCR methods have subsequently been developed to overcome difficulties experienced with Inverse PCR. One of these methods, called random primed gene walking PCR (Trueba and Johnson, 1996) allows the amplification of specific PCR products. This enables direct sequencing of unknown regions without the need for DNA cloning.

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