Molecular characterisation of genes expressed during fruit development in kiwifruit

Abstract

A cDNA library was constructed from kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var deliciosa cv Hayward) using fruit harvested 8-10 days after anthesis (DAA). The library was differentially screened for cDNA clones corresponding to genes that were preferentially expressed in fruit 8-10 DAA compared to leaf. Twenty-one clones were isolated representing six genes and five of these, designated pKIWI50l-505, were further characterised. The gene for pKIWI503 had low levels of expression in young fruit and was induced late in fruit development and during fruit ripening. The genes for the other four cDNA clones were highly expressed in young fruit (8-10 DAA) with low levels of expression in the later stages of fruit development. The genes for pKIWI5O1 and 504 have a second lower peak of expression at 44 DAA which is approximately 40% of the level at 8-10 DAA. The gene for pKIWI505 is highly expressed in both root and fruit 8-10 DAA. The nucleotide sequences of the differential clones were determined and compared with sequences in the databases. pKIWI504 has strong homology to metallothionein-like proteins and is homologous to l5 of the 21 differential cDNAs isolated from the library. pKIWI503 has homology to a subgroup of plant metallothionein-like proteins, and pKIWI505 is a partial clone with homology to the β-subunit of the mitochondrial ATP synthase. The genes for pKIWI501 and 502 encode novel proteins. The cDNA library was screened with a tomato clone for 3-hydroxy-3-methylglutary1-coenzyme A reductase (HMGR). In tomato HMGR has been shown to be expressed early in fruit development. A single kiwifruit clone was isolated (H1) and partial nucleotide sequence was determined. The deduced amino acid sequence showed a high level of homology with HMGR sequences from potato and rubber. However, expression of the gene for H1 was detected in kiwifruit roots but not in fruit. The copy number of the genes corresponding to pKIWI501-505 was estimated from Southern hybridisations with DNA extracted from a diploid line of A. chinensis, a close relative and probable progenitor of A. deliciosa. pKIWI504 corresponded to a single copy gene, pKIWI501, 503 and 505 to genes with low numbers of copies (two to seven), and pKIWI502 to a member of a large gene family. pKIWI501 and 503 were selected for further characterisation based on the expression pattern during fruit development and the low copy number of the corresponding genes. A genomic library was constructed from A. chinensis DNA and genomic clones corresponding to pKIWI503 (λ3) and 501 (λ2B) were isolated. Partial nucleotide sequence was determined for λ3. A single nucleotide difference was observed in the 204 bp of overlapping sequence between the cDNA clone (pKIWI503) and the genomic clone. Attempts to clone the λ3 upstream promoter region were unsuccessful. Nucleotide sequence was determined for 3.2 kb of λ2B, including the entire coding region of the gene and a 1.53 kb region of 5' untranslated sequence. Fourteen nucleotide differences (nine in the coding region) were identified between the genomic clone and pKIWI501. The 1.53 kb 5'flanking region of λ2B was isolated and fused to the gusA reporter gene. The construct was introduced into Arabidopsis, tomato and A. chinensis using Agrobacterium-mediated transformation. The activity of the gusA gene was analysed in the Arabidopsis and tomato transgenic plants using histochemical and fluorometric assays. The 1.53 kb promoter region was able to direct expression of the reporter gene in the two heterologous species. The gusA gene activity was preferentially expressed in the vasculature of the plant and was detected in the leaves, roots, tomato fruit and Arabidopsis siliques. The highest level of activity was detected in the roots. Preliminary analysis of the transgenic tomato plants revealed expression in the fruit throughout the different stages of fruit development. However, there was temporal control of expression during the development of the siliques in Arabidopsis, with a peak of expression observed when siliques were approximately half their final size. This temporal control is similar to the expression of the original gene in kiwifruit development, but with a delay in the timing of peak expression.