| The potato(Solarium tuberosum L.) is the fourth food crop with multiple uses in the world, of which potato chips and fries are fashionable. To provide a uniform flow of tubers to fresh market and processing from harvest, cold storage at about 4℃is usually adopted, which could reduce the excessive loss of moisture, development of rots and sprout growth. Cold storage, however, causes a large accumulation of reducing sugar(RS) that reacts with the amino acids under high temperature and resulted in undesirable dark brown color of the products. The starch-sugar conversion involves multiple metabolic pathways with multi-regulating and-feedback regulating factors. Therefore, mechanism of"cold-sweetening"is a basic issue for improving potato post-harvest quality. Only the invertase and reversible sucrose synthase reactions catalyze known paths of cleavage of sucrose in vivo. Invertases(Inv) include acid invertases which can be subgrouped as vacuolar and cell wall invertases, and neutral(or alkaline) invertases. Previous report indicated a strong positive correlation between acid invertase activity and the ratio of RS and sucrose, which was accounted for the conversion of starch-sugar. The present research focus on the cloning of complete CDS(coding sequence) of endogenous acid invertase gene, regulation of its expression through RNAi and antisense strategy, and their influence on RS content. The main results obtained are as following:1. The complete CDS of invertase gene was obtained from a tetraploid potato clone JH, which was considered resistant to cold-sweetening in previous work, by RT-PCR with specific primers derived from the complete reported CDS(Genebank accession No. L29099). The result of sequencing showed that the over-length of the CDS was 1920 bp encoding 639 amino acids with 95% homology to the cDNA of acid invertases in tomato(S70040), and was 98%, 96% and 96% similar to that of the vacuolar invertases in potato(X70368.1), tomato(Z12025) and Lycopersicon pimpinellifolium(Z12026). The protein prediction analysis showed a transmembrane region(34-56 in amino acids) and an N-term signal sequence(1-49). Along with the sequence there were two conservative sites, NDPNG(120-124) and WECVD(299-303), and several potential glycosylation sites, suggesting that the cloned cDNA was protentially vacuolar invertase gene.2. Performed with PCR amplification and digestion, the cloned cDNA yielded three fragments which represents 5'-end(397 bp), the conservative portion(625 bp) and 3'-end(955 bp). The above fragments and together with the full-length CDS were individually cloned into pBI121 vector controlled by the constitutive Cauliflower Mosaic Virus(CaMV) 35S promoter and pBIC vector controlled by patatin class I promoter(CIPP) in antisense orientation. Analysis of stored transgenic tubers revealed that, controlled by 35S promoter, when stored at low temperature(4℃), expression of antisense acid invertase repressed the endogenous invertase activity of the four transgenic lines(ES4, ES6, ES9 and ES19) that were stored for 6 weeks by 33%, 29%, 41% and 47% and by 10%, 13%, 13% and 12% for 10 weeks period. These variations led to significant RS content decrease by 26%, 23%, 23% and 19% for 6 weeks and by 34%, 29%, 34% and 32% for 10 weeks. When storage temperature increased to 20℃, the average decrease of endogenous acid invertase activity was over 60% for 6 weeks storage, while no remarkable alteration in RS content during this period. However, more than 30% reduction in RS content and non-detectable variation in acid invertase activity was observed for 10 weeks storage. Comparison between transgenic tubers harboring 35S and CIPP promoters showed that, stored at 4℃, antisense cDNA driven by CIPP promoter repressed the endogenous invertase activity by 53% for 6 weeks storage and by 21% for 10 weeks period, which turned down the RS content by 35% in tubers stored for 6 weeks and by 67% for 10 weeks, although little variation were observed between the two promoters at 20℃. No significant difference was detected on the acid Inv activity and RS content among transgenic tubers expressing antisense cDNA varied in fragment size indicating a similar efficiency of inhibition. However, the tuber-specific promoter, CIPP, performed a higher efficiency of inhibition than did the constitutive 35S promoter.3. RNAi vector inserted with portion of inv cDNA was also introduced into potato plants. Successfully expression of RNAi vector in potato plants aroused a range of variations in transgenic lines by an average of 59.3% reduction in acid invertase activity, in which the best repression was found in Line Ni-4 by 78% decrease in invertase activity. Comparatively, the average of 58.4% reduction was found on invertase activity in well inhibited antisense transgenic lines, in spite of the occurrence of 84% decrease(Line ES4-3). The little difference suggested a powerful potential of RNAi technology.4. Analysis of expression of antisense mRNA showed a large accumulation of antisense mRNA in both transgenic plants and tubers driven by 35S promoter whereas only in transgenic tubers controlled by CIPP promoter, indicating again the tissue-specific expression of CIPP. However, the accumulation of mRNA of endogenous acid invertase in RNAi transgenic plant was obviously decreased compared with the control plant. Since the reduction of endogenous acid inv mRNA were observed both in antisense and RNAi transgenic plants, the regulation of Invertase in cold-sweetening of tubers was probably focused on transcriptional levels.5. Further analysis of selected transgenic plants indicated that, under low temperature storage, the strongest suppression was found in tubers of the transformant ES4-3 which accounted for 28% of enzyme activity of the control. This reduction in Inv activity led to a 68.7% decrease in the accumulation of reducing sugar, further resulting in a 92.4% and 7.2% increase in the amount of sucrose and starch content respectively compared with wild-type tubers. Anther available transgenic lines EC4-5 and EC19-5 achieved remarkable reduction in Inv activity, reaching 66.6% and 77.1% of the control respectively, while the former revealed a relative little decrease(8.69%) in RS content contrast to the latter(69.5%), resulting a moderate elevation of 7.6% and 12.2% in sucrose. In general, low level of RS accumulation at low temperature was resulted from less sucrose conversion without obvious stimulation of starch synthesis, indicating that cold-induced accumulation of reducing sugar was mainly derived from sucrose cleavage rather than starch degradation.6. Chips were prepared from good repressed transgenic tubers and untransformed control tubers and processing variety'Atlantic' stored at low temperature(4℃). Comparison of chip color index showed a decrease trend in transgenic chips in contrast to the control tuber and a large variation between two production seasons.
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