Studies On The Molecular And Physiological Characteristics And Function Of Fructokinase In Citrus

Citrus is one of the most important fruits in the world. The cultivated area of citrus in China is maximum in the world. After entry of China into WTO, our domestic market faces the competition from abroad. Therefore, the breeding and production of high quality citrus fruit is urgent. Sugars play an essential role in flavor characteristics of the citrus fruit and are also a commercial measure of fresh fruit quality. Sugars are not only the substance that affect sweetness of fruit, but also from the material basis for synthesis of organic acid, carotenoids, and other nutrient and aromatic ingredients of citrus fruit. Studying the physiological and molecular mechanism of sugar transporting and accumulating process in tissues of developing citrus fruit will supply an important basis for scientific regulating citrus quality by genetic tools.In order to clarify the mechanism of sugar accumulation in citrus fruit, we cloned two fructokinase genes from satsuma mandarin (Citrus unshiu Marc.) and analyzed the different expression in various organs. Based on these results, we isolated the full-length cDNA and genomic DNA of one fructokinase gene. To elucidate the function of fructokinase on sugar accumulation in citrus fruit, the relationship of fructokinase with development of satsuma mandarin fruit and sugar accumulation was studied. On the side, an antisense and an intron-spliced RNAi expression vectors were constructed and preliminary study on transformation was carried out. The results were summarized below.(1). RNA isolation is one of the most important techniques in the studies of molecular biology. The cell composition in fruit trees is more complex so it's hard to isolate high quality RNA from those tissues. Therefore, technique improving and isolating high quality RNA become the key points in the studies of molecular biology in fruit trees. Four total RNA extraction methods including modified TRIzol, CTAB, SDS/phenol, and single-step method were applied to isolate total RNA from various citrus tissues. The quality of RNA isolated by the four modified methods was improved markedly after our efforts and the RNA was fit for the latter studies.(2). Two Citrus unshiu Marc, fructokinase genes were isolated from genomic DNA using degenerate primers: Cufrkl (GenBank: AY 118083) 1213 bp in length, encoded a deduced protein of 205 amino acids, including 4 exons and 3 introns; Cufrk2 (GenBank: AF521003) 793 bp in length, encoded 204 amino acids, including 2 exons and 1 intron. Both amino acid sequences possessed sugar-binding domains and were 64~78% identical with previously characterized fructokinase genes of other plants. The amino acid sequences encoded by Cufrkl and Cufrk2 were 68% identical.(3). Northern blot showed that the transcripts of Cufrkl and Cufrk2 were distinct to a certain extent. Both transcripts could be detected in young leaves and in fruit at early developmental stages (from 8th May to 3rd Jun). Transcripts of Cufrkl could be detected in petals at a very low level but those of Cufrk2 were at a high level. Only transcripts of Cufrkl were detected in fruit on 22nd Oct. Transcripts of both genes were undetectable in stems and in the peel from ripe fruit.(4). One complete cDNA of fructokinase gene from citrus (Citrus unshiu Marc.) were isolated byusing of PCR, RT-PCR and rapid amplification of cDNA ends (RACE), named CuFRKl (GenBank: AY561840). The full-length cDNA of CuFRKl was 1459 bp in length and contained a complete ORF from 168 to 1220 bp, encoding 350 amino acids with two sugar-binding domains and three ATP-binding domains. The deduced amino acids of CuFRKl were about 62% to 78% identical to the previously characterized fructokinase genes from other plants.(5). The full-length genomic DNA of CuFRKl was isolated from satsuma mandarin leaves genomic DNA. The sequences were 3169 bp in length containing 7 exons and 6 introns. The 7 exons 1161 bp in length included a complete ORF. The size of introns ranged from 106 to 623 nucleotides. As in most plant introns, the content of A+T w...