Molecular Characteristics Of Genes Involved In Sorbitol Metabolism In Apple

Sorbitol is the primary photosynthetic product, and major translocated and storaged form of photosynthate in many species of Rosaceae. Those plant development and fruit quality depend on sorbitol. In addition, sorbitol is an efficient osmolyte. Its accumulation can improve plant tolerance of salinity, drought, low temperature, fireblight disease and oxidative stress. Sorbitol can also form complex with boron to facilitate phloem boron transport. In metabolism of sorbitol, NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH), sorbitol transporter (SOT), NAD-sorbitol dehydrogenase (SDH) play key roles. Up to now, though sorbitol and its metabolic enzymes have been investigated, information about molecular characteristics of those enzymes is still lacking. In present study, cloning, real-time PCR, sub-cellular localization and transformation were used to illustrate the molecular characteristics of enzymes involved in sorbitol metabolism. The main results were as follows:1. The full-length cDNA sequence of S6PDH gene was cloned from apple. It was 1080 bp long, encoding 314 amino acid residues. Based on the ORF, two genomic DNA sequences of S6PDH gene were also isolated, one is 3595bp, and the other is 3402bp. These two gDNA sequences had six extrons and five introns. The difference between them was the length of last three introns.Expression pattern of S6PDH during leaf development were detected by real-time RT-PCR. S6PDH mRNA level was low in young leaves, increased markedly with leaf growth, reached maximum in mature leaves, and then reduced in old leaves, paralleling with the enzyme activity during leaf development. Under salt or drought stress conditions, S6PDH mRNA level was increased prominently with the prolongation of stress treatment.Therefore, the expression of S6PDH seemed closely related with the transition from sink to source function of the leaves, was also suggested to be regulated at the transcriptional level.The binary plasmid vector pBI-S6PDH including the S6PDH coding region was introduced into kiwifruit by agrobacterium-mediated method. The kanamycin resistant plantlets were identified by PCR and southern blot. Five plantlets were obtained for future research.2. Subcellular localization analysis with GFP using gun-mediated bombardment method revealed that S6PDH protein was located in cytoplasm of onion epidermal cell. Immunocytochemical study showed that after incubated by antibody of S6PDH and labeled by goat anti-rabbit immunoglobulin, colloidal gold mainly sedimentated in cytoplast and chloroplast, which indicated that S6PDH mainly located in cytoplast and chloroplast in apple leaves.3. The 2396bp promoter of S6PDH gene was cloned. It included many cis-acting elements and showed transcriptional activation identified by transformed tobacco GUS histochemical assay. The result of 5` deletion derivates transformed into tobacco indicates that the regions of -2122~-1719bp and -1066~0bp may play an important role in regulating gene transcription.4. Three cDNAs encoding different isoforms of NAD-SDH from apple were isolated. The cDNA of MdSDH7, MdSDH8 and MdSDH9 were 1418bp, 1446bp and 1388bp, encoding 371aa, 368aa and 368aa, respectively. gDNA of MdSDH7 was 2176bp including 6 exons and 5 introns, gDNA of MdSDH8 and MdSDH9 were 1239bp and 1246bp respectively and both include 2 exons and 1 intron.Expression pattern of SDHs was detected by real-time PCR. MdSDH7 expression level was higher at the late developmental stage of apple fruit,MdSDH8 expression level was higher at both middle and late developmental stages,whereas MdSDH7 expression level was higher at the middle developmental stage. The activity of NAD-SDH was high in young fruit (from 0 to 15 DAF). It dropped significantly as fruit developed, but increased slightly in 92DAF, and then dropped again. In general, the activity of NAD-SDH changed in a similar way as that of the relative growth rate of the fruit.Expression of MdSDH8 and MdSDH9 were high in source leaves whereas that of MdSDH7 was high in sink leaves. The activity of NAD-SDH was high in young leaf, dropped as leaf became mature, and then increased again in senescent leaf.In young fruit,the expression level of MdSDH7, MdSDH8 and MdSDH9 were all higher in seed than that in flesh or pericarp. In mature fruit,the expression level of MdSDH7 was higher in seed and pericarp, whereas that of MdSDH8 and MdSDH9 was higher in seed and flesh, respectively. The changes in NAD-SDH activity were proportionate to those in RGR, Thus, it is considered that SDH activity was associated with sink strength. The fluctuation pattern of SDH activity did not correspond to that of the transcripts, these results may be due to interaction of SDH isogenes.5. Four cDNAs encoding different isoforms of SOT from apple were isolated. The cDNA of MdSOT7, MdSOT8, MdSOT9 and MdSOT10 were 1681bp, 1755bp, 1797bp and 1849bp, encoding 538aa, 526aa, 491aa and 535aa, respectively. gDNA of the four genes were 2616bp, 2230bp, 2397bp and 3013bp, respectively, and all of them include 3 exons and 2 introns which differ in length and position. Expression pattern of SOTs were detected by real-time RT-PCR during leaf and fruit development. The expression level of MdSOT7 was consistent with leaf development. The expression level of MdSOT8, MdSOT9 and MdSOT10 was higher in mature leaf. As apple fruit developed, MdSOT7 expression level was higher in the early and late developmental stages,MdSOT8 expression level was higher in the middle stage, MdSOT9 expression level remained unchanged, and MdSOT10 was higher in the early developmental stage.