Construction And Functional Characteristics Of Potato Cold-inducible And Tuber-specific Synthetic Promoters And Identification Of St-CBF Transcription Factor

The potato (Solarium tuberosum L.) is the fourth food crop in the world. In addition to its table use, potato tubers are important industrial materials for producing food and starch product. The production of chips and fries have taken an important position in potato precessing industry. To extend its processing period, storing tubers at low temperature is the most efficient and economic way. However, in cold-stored tubers, the accumulation of reducing sugar caused by low temperature results in an unacceptable dark and bitter product because of the browning reaction between reducing sugars and free amino acid groups at high frying temperatures. Now, gene engineering approaches have been important breeding strategies for the improvement of processing quality of potato products (fries and chips) worldwide. However, these approaches require gene expression in tubers at low temperature in order to avoid potential pleiotropic effects on the growth and development of potato plants. To achieve this gene expression manner, promoters with specificity to tubers as well as inducible activity under low temperature are definitely required in potato gene engineering to improve the processing quality of potato products.Based on the cloning of cold-induced promoters, present research constructed synthetic promoters with tuber-specific and cold-responsive patterns, investigated their performance in transgenic potato and their potential applications in potato gene engineering. Moreover, present research performed pilot study to explore putative transcription factors regulating the activities of cold-induced promoters in potato. The main results achieved are as following:1. The cold-inducibility analysis of cold-inducible promoters: By PCR, the cold-inducible promoters of cor15a gene and ci21A gene were amplified from Arabidopsis thaliana (cv. Columbia) genomic DNA and that of Solarium tuberosum (cv. E3) respectively. Promoter sequences analysis showed that cor15a promoter contained the identified low temperature responsive element "LTRE", while ci21A gene promoter did not. Expression vectors, pLB and pCI21A, were constructed by inserting the cor15a promoter and the ci21A promoter into the upstream of GUS gene followed by NOS terminator in pBI121 respectively. Then the pLB and pCI21A were transformed separately into tobacco via Agrobacterium mediated transformation. Transgenic plants were confirmed by PCR and Southern blot. Histochemical staining and fluorescent quantitative analysis of GUS activity in the tested transgenic plants indicated that both the two promoters could be functional in tobacco. The promoter strength and cold-inducibility analysis showed that the expression strength of ci21A promoter was higher than that of corl5a promoter, but the cold-inducibility of cor15a promoter was higher than that of ci21A promoter.2. The functional identification of cor15a promoter in potato: to investigate whether cor15a promoter could be cold-induced in potato, the construct of pLB was introduced into potato "E3" genome via Agrobacterium mediated transfermation of microtubers. The transgenic potato plants were analyzed by PCR and Southern blot. GUS histochemical staining and GUS fluorescent quantitative analysis of the tested transgenic potato plants proved that cor15a promoter could maintain its cold-inducibility in potato. Without cold treatment, no GUS activity could be detected in all of the potato tissues. After cold treatment, various GUS activities were detected in stems, leaves, tubers and stolons, moreover the GUS activities in leaves and tubers were relatively high.3. The functional investigation of the LTRE from cor15a promoter and the TSSR from tuber-specific promoter CIPP: The -297/+70 fragment of cor15a promoter containging LTRE (low temperature responsive element) and the -340/+19 fragment of CIPP containing TSSR (tuber-specific and sucrose responsive sequence) were cloned into the upstream of GUS in pBI121 to generate transformation constructs pL297-121 and pC340-121 respectively, which were subsequently introduced into potato "E3" genome via Agrobacterium mediated transfermation of microtubers. The transgenic potato plants were analyzed by PCR and Southern blot. GUS fluorescent quantitative analysis in transgenic potatoes showed that pL297-121 had cold inducibility in all the tested potato tissues except roots, but its promoter strength was less than that of the full length cor15a promoter. pC340-121 showed higher promoter activity in tubers than in stems, roots and stolons, and no activity was detected in leaves. After cold treatment, the promoter activity of pC340-121 did not show obvious change, indicating the function of TSSR was not affected by low temperature.4. Construction and functional identification of cold-induced and tuber-specific synthetic promoters in potato: By overlapping extension PCR, two chimeric promoters, pCL and pLC, were constructed with different combinations of cor15a promoter fragments, -297/-42 and -297/+70, containing the LTRE (low temperature responsive element) and CIPP promoter fragments, -340/+19 and -340/-28, containing the TSSR (tuber-specific and sucrose-responsive sequence).The TSSR in pCL is closer to the TATA-box than in pLC, in which the LTRE is close to the TATA-box. The two synthetic promoters were cloned into the upstream of GUS gene in pBI121 respectively and then introducede into potao "E3" via Agrobacterium mediated transfermation of microtubers. The transgenic potato plants were obtained by PCR and Southern blot. The cold-inducible and tuber-specific activities of synthetic promoters were investigated by quantitative analysis of GUS activity in transgenic potatoes and the results showed that both the two synthetic promoters could be functional in potato. But pCL with the TSSR closer to the TATA-box showed substantially higher promoter activity than pLC with the LTRE closer to the TATA-box at either normal (20℃) or low temperature (2℃), especially in tubers and stolons. This result suggested that the promoter activity was closely associated with the position of the two functional cis-elements, TSSR and LTRE, to the TATA-box.5. The indentification of St-CBF thanscription factor in potato: An encoding sequence of St-CBF gene was amplified from leaves of potato (E3 and CW2-1) by RT-PCR, using primers designed according to the released EST sequence (accession number CK860811). The DNA sequence of St-CBF is of 600bp in length, encoding a protein of 199 amino acids. The analysis of deduced amino acids sequences of St-CBF showed it was high homologous to other CBF/DREB proteins and contained a typical domain ERF/AP2 of CBF/DREB-type proteins. Moreover, three conserved sequences were also found: the conserved Ser/Thr-rich region located at the N-end, the conserved CBF1/DREB1-type NLS and the conserved DSAW-motif surrounding the ERF/AP2 domain. Tthe St-CBF gene was classed into the CBF1/DREB1 category for the presence of conserved NLS and DSAW-motif, which distinguishing CBF1/DREB1-type proteins from other ERF/AP2 proteins. The encoding sequence of St-CBF was cloned into the expression vector of pGEX-6P-1 and expressed successfully as GST (Glutathione S-Transferase) fusion protein in E. coli. The GST-St-CBF fusion protein could bind specifically to the LTRE sequence from cor15a promoter in vitro, indicating that the St-CBF protein could act as CBF/DREB transcription factors. The Southern blot results showed the St-CBF gene had only one allele in potato genome. In leaves, the expression of St-CBF gene was detected within 15min after potato plants exposed to low temperature (2℃) and remained detectable during low temperature treatment (up to 12h). This result indicted that the St-CBF gene was involved in the gene expression of potato in response to the low temperature stress.