Cloning And Functional Analysis Of AsA Biosynthesis Related Genes And Their Promoter Activities From Kiwifruit

Ascorbic acid (AsA) is an important antioxidant in animals and plants, which playsimportant roles in eliminating reactive oxygen species (ROS) while allowing for crucialsignaling to regulate plant growth and defenses and an essential enzyme cofactor in manymetabolism processes. AsA is clearly an essential dietary component for humans, with aprotective role proposed for many disorders through increasing resistance of immune system.Ascorbic acid (AsA) is a vital compound in plants with a range of functions as a growthregulation factor in regulating flowering time, or as a stress response factor, which is involvedin many stress-induced oxidative processes, e.g., responses to water loss, pathogens andoxidizing agents, high temperature, photo-oxidative stress and ozone. Up to now,GDP-mannose-3’,5’-epimerase (GME), GDP-L-galactose phosphotylase (GGP), L-galactosephosphatase (GPP) may play key roles in ASA biosynthesis and regulation. In present study,gene cloning, real-time PCR, and promoter characteristics were used to illustrate themolecular characteristics of enzymes involved in ASA biosynthesis under different treatments.To gain a better understanding of the relationship between AsA concentrations andtranscriptional levels of the above genes, and to investigate the regulatory mechanisms fortheir expression in kiwifruit under stress conditions, we performed a systematic investigationof AsA, mRNA expression, and promoter activities in response to various treatments. For this,we evaluated three kiwifruit species (A. eriantha, A. deliciosa cv.‘qinmei’ and A.rufa) thatproduce different amounts of AsA. Furthermore, to evaluate the relationship between levels ofAsA in fruits development and GGP transcripts, we systematically investigated the regulatorymechanisms for GGP expression in the three kiwifruit species. The main results were asfollows:1. The full-length cDNA sequence of GME gene was cloned from A. deliciosa‘qinmei’and A.rufa, named AdGME (GenBank Accession NO. GU339037) and ArGME (GenBankAccession NO. JN132110). They were both1143-bp long, both encoded a polypeptide of376amino acids. Using the MegAlign program of MEGA, we determined that these amino acidsequences shared99.7%identity. RT-qPCR analyses in two species demonstrated that ABA, dark or light did not seem to alter GME expression, but GA3resulted in a decrease in thetranscript levels of GME. However, application of SA, wounding, cold or heat, GMEtranscript exhibited an increase in both species.2. The promoters of GME genes were cloned by Genomic DNA-walking method. Theywere both1225-bp long, named ParGMEå'ŒPadGME. We comparatively analysed thepromoter sequences of ParGME (GenBank Accession NO. JQ693500) and PadGME(GenBank Accession NO. JQ693499). Sequence alignment revealed that they shared99.16%similarity. They showed transcriptional activation identified by transformed tobacco GUShistochemical assay. The promoter activities of GME genes were not significantly inducedwhen the transformed tobacco exposing to light or wounding. While the promoter activities ofGME genes were significantly induced when treated by SA or heat.3. The full-length cDNA sequence of AdGPP gene was cloned from A. deliciosa. Theexpression patterns of AdGPP gene were studied when kiwifruit plants were exposing todifferent conditions. RT-qRCR analyses demonstrated: during the first12h of darkness,AdGPP expression gradually but continually declined. After48h of dark treatment, plantswere then exposed to light, and AdGPP expression quickly recovered within1h. Following apeak at Hour2, then AdGPP expression began to decrease to the original level. Treatmentwith SA caused only a slight increase in expression over the entire period. ABA or hypoxicconditions were associated with high AdGPP expression at first, followed by a gradualdecline in transcript levels.4. The AdGPP promoter was isolated from A. deliciosa genomic DNA by GenomicDNA-walking method. After sequencing, we cloned a1395-bp fragment (GenBank AccessionNo. JX122767). The AdGPP promoter showed transcriptional activation identified bytransformed tobacco GUS histochemical assay. The promoter activities of AdGPP were notsignificantly induced when the transformed tobacco exposing to light or wounding. While thepromoter activities of AdGPP gene were significantly induced when treated by SA or heat.5. The full-length cDNA sequence of GGP genes were cloned from A.eriantha andA.rufa, named AeGGP (GenBank Accession No. KC146049) and ArGGP (GenBankAccession No. KC146048). Some correlation between relative levels of GGP mRNA andAsA concentrations in three species during fruit development. Transcripts were the mostabundant in A. eriantha, and the lowest in A. rufa. RT-qPCR results with the kiwifruit leavesrevealed that expression of the GGP genes was induced by light, MeJA, heat, hypoxicconditions; and was very sensitive to light conditions.6. The promoters of GGP genes were cloned from three kiwifruit species by GenomicDNA-walking method. GUS activity of the entire AeGGP promoter was the highest and that of ArGGP promoter was the lowest under normal growing conditions. When compared thethree promoters, we found that ArGGP promoter has a nearly300-bp insertion. The result of5`deletion derivates transformed into tobacco indicates that some negative cis-elements mayexist in the ArGGP promoter. The promoter activities of GGP genes were significantlyinduced when application of ABA or MeJA. Light, heat or hypoxic conditions can also inducethe promoter activities of GGP genes. According to the results of5`deletion derivates ofAeGGP or AdGGP promoter, we deduced that G-box is important for the expression of GGPin kiwifruit under different types of illumination and ABRE or CGTCA-motif may play animportant role in regulating GGP promoter activities after application of ABA or MeJArespectively.