Abstract:

Two pairs of primers, designed from conserved regions of plant vacuolar and cell wall bound acid invertase genes respectively, were used to amplify 741bp (A) and 524 bp (B) fragments from Citrus unshiu genomic DNA by polymerase chain reaction (PCR). The fragments were cloned into pUCm-T, and then sequenced. The deduced amino acid sequence of fragment A is 79%, 79%, 78% identical to genes from Daucus carota (X75353), Solanum tuberosum (X70368) and Lycopersicon esculentum (Z12025)respectively, and was suggested to encode a soluble acid invertase located in vacuole, while B is 78%, 78%, 77% identical to genes from Fragaria ananassa (AF000521), Arabidopsis thaliana (AP001307) and Pisum sativum (X85327) respectively, and was suggested to encode a insoluble acid invertase in cell wall. Lower homology among A, CUAI1, B and CSCWI suggested that A and B were two new members of acid invertase gene family in Citrus, respectively belong to putative CUAI2 and CUCWI. There was no interaction among these members (CUAI1, CUAI2, CUCWI or CSCWI) except for CUCWI and CSCWI in Southern blot analysis. The interaction between CUCWI and CSCWI could be eliminated by using CSCWI's partial sequence which was lower homologous to CUCWI as probe, and weakened by shortening hybridization time, increasing hybridization and washing temperature, washing membrane in lower concentration of SSC.

Key words: Citrus, Acid invertase, Gene family, Cloning, Characterization