| Potato(Solanum tuberosum L) is the fourth important crop, and plays crucial roles in food security and economic development in the world. With increase living standards, the consumption of potato processing products has been elevated. French fries and chips dominate the processing market among other products. In order to prevent loss from sprouting, water loss and disease infection in processing industries, potato tubers are often stored at low temperature. However, low temperature (<10℃) storage often leads to reducing sugars (RS) accumulation converted from starch in stored tubers, which is known as the cold-induced sweetening (CIS). Reducing sugars can interact with amino acids and causes a nonenzymatic Maillard reaction at high frying temperature to generate carcinogen acrylamide and distorted color, seriously affects the quality of the products. Previous research on the mechanism of potato CIS mainly focuses on the key enzyme genes related to the carbohydrate metabolism. However, the activity regulation of these enzymes and the main pathways of potato CIS remain unknown.A total of 188 genes were identified to be differentially expressed in CIS-resistant wild type species S. berthaultii upon cold stimulation. Based on the expression pattern of these genes, it was hypothesized that starch degradation, sucrose decomposition and glycolysis pathways play important roles during potato CIS. In our research, the transcripts of part of the ESTs in potato genotypes distinct in resistance to CIS were investigated. We obtained ESTs had higher expression in the CIS-resistant genotypes tubers stored at low temperature. Through cloning and function complementation of the ESTs significantly activated in the CIS-resistant genotypes, the roles of the genes and their potential regulatory pathways in potato CIS were explicated. The main results obtained are as following.1. The expression analysis of the genes related to potato CISThree CIS-resistant potato genotypes and three CIS-sensitive genotypes were employed, their tubers stored at 4℃ and 20℃ for 0 d,5 d,15 d,30 d,45 d,60 d, were sampled to analyze the expression patterns of 43 selected ESTs. The results showed that there were 8 ESTs expressed in CIS-resistant tubers only, including 4 ESTs C20-3-A14^ C20-1-G06、C4-1-I07 and C20-2-N12 with assigned functions, and 4 ESTs C20-3-E15、 C20-5-M08、C20-1-F10 and C20-2-B24 with unknown function. Interestingly, ESTs C20-3-E15 and C20-5-M08 were highly activated only in CIS-resistant tubers stored at low temperature, speculating their potential roles in potato CIS worth further investigation.2. Cloning and the structure analysis of C20-3-E15 and C20-5-M08Using the method of rapid-amplification of cDNA ends (RACE), and sequence matched in the Potato Genome Sequence Consortium database, full length cDNAs were cloned from the cDNA of S. berthaultii tubers by ESTs C20-3-E15 and C20-5-M08. The full length cDNA of C20-3-E15 is of 799 bp and consists of a 621 bp open reading frame (ORF) encoding a deduced protein of 207 amino acids. The gDNA sequence of the gene contains 4 exons and 3 introns. The promoter sequence of the gene contains the elements related to carbohydrates metabolism, amylases, cold stress and anaerobic respiration. The deduced amino acid sequence analysis revealed that the protein has "Tryp_alpha_amyI" domain, similar to amylase inhibitors, so the gene was denoted SbAI (S. berthaultii Amylase Inhibitor). The full length of C20-5-M08 is 796 bp and consists of a 504 bp ORF which encoding a deduced protein of 168 amino acids, a 39 bp 5’-untranslated region and a 253bp 3’-untranslated region. The gDNA sequence of the gene contains 2 exons and one intron, and the promoter sequence contains the elements related to amylases, cold stress, anaerobic respiration and carbohydrates metabolism. The amino acid sequence analysis revealed that the deduced protein has an IBR (In between Rings) and a RING finger domain, so the gene was denoted SbRFPl (S. berthaultii RING finger grotein 1).3. The function study of potato amylase inhibitor gene SbAIPrimers designed according to a-amylase genes, P-amylase genes, isoamylase genes and starch phosphorylase genes that are related to starch degradation from the Potato Genome Sequence Consortium database and NCBI (nr/nt) database, the expression patterns of these genes in various organs of CIS-resistant genotype AC030-06 and CIS-sensitive genotype E potato 3 (E3) were evaluated to provide a base for function dissection of SbAI and SbRFPl. The results showed that the activity of a-amylase was mainly constituted by Amy23, while the activity of β-amylase was mainly from BAM1 and BAM9. The amylase activity and RS content of the stored tubers indicated that Amy23^ BAM1 and BAM9 may play important roles in CIS and sprouting of potato tubers.The overexpression (OE) and RNAi (RI) vectors of SbAI were constructed and transformed into E3 and AC 142-01, respectively. Compared with wild type tubers, the OE-tubers stored at low temperature were lighter in chip color, lower in RS content, amylase activity and starch degradation rate. However, RI-tubers performed in contrast to the OE-tubers. These results strongly suggest that the SbAI gene is most likely to be involved in regulation of potato CIS. With the method of bimolecular fluorescence complementation (BiFC), it was confirmed that SbAI interacted with Amy23, BAM1 and BAM9 in living tobacco cells, and the SbAI protein inhibited the amylase activity, implying that SbAI reduces the RS content in cold storage potato tubers through regulating the amylase activity.4. The function study of potato SbRFPl geneIn this research, the OE and RI vector of SbRFPl were constructed and transformed into E3 and AC 142-01, respectively. The OE transgenic tubers stored at low temperature showed a lighter chip color and lower RS content compared with the wild type tubers, while the RI-transgenic tubers exhibited darker chip color and higher RS content. These results led to a speculation that the SbRFPl gene may be associated with potato CIS. The regulatory mechanism of SbRFPl in potato CIS was further investigated by analyzing expression of the genes encoding main enzymes involved in carbohydrate metabolic pathway in cold stored transgenic tubers. The results showed that expression of the SbRFPl gene impacted to some extent on the expression of a-amylase gene Amy23, β-amylase gene BAM1 and acid invertase gene StvacINVl. It was further reinforced by analyzing the activity of amylases and invertase. The P-amylase activity, invertase activity and starch degradation obviously inhibited in the OE-transgenic tubers and elevated in the RI-transgenic tubers. A positive linear relationship was established between invertase activity and hexoses sucrose ratio. These results exploited that the function of SbRFP1 gene in potato CIS is possibly accomplished through regulating the activity of β-amylase and acid invertase.In conclusion, SbAI and SbRFPl identified in present research are novel genes for regulation of potato CIS, and play critical roles in potato CIS process through regulating the β-amylase activity. The results provide theoretical and substantial base for looking insight the mechanism of potato CIS. |
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