| The potato steroidal glycoalkaloids (SGAs) is one of the secondary metabolites in potato (Solanumtuberosum). The two major SGAs, α-chaconine and α-solanine, are accounted for more than90%of thetotal glycoalkaloids in potato tubers. The content of SGAs in potato tubers should not exceed20mg·100g-1 FW, otherwise it will affect palatability and become an health threaten agent. Therefore, introductionof potato varieties with low SGAs content in tubers is one of the primary goals of potato breeding.Accumulation of SGAs in potato tubers was influenced by genotype and environmental factors, suchas temperature and light; especially, the post-harvest storage conditions also plays impact on theaccumulation of SGAs. Light is one of the most important environmental factors that affect thebiosynthesis of the SGAs, however, the impact of different light wavelength and the mechanism oflight-induced biosynthesis of SGAs remain unknown. In this study, we aimed to analyze the impact oflight wavelength and light mediated signal transduction of the SGAs accumulation in potato tubers, toidentify the signal molecules that might influence the accumulation and biosynthesis of SGAs. We clonedthe genes of key enzyme involved in the SGAs biosynthesis, analyzed the cloned gene sequence,constructed expression vector and interference expression vector, then constructed the transgenic tobaccoand potato. The results from this study brought great insight into the metabolic regulation of the SGAsaccumulation in potato tubers; therefore, provided references to take appropriate breeding strategy toreduce the accumulation of SGAs in the potato tubes and to adapt the proper storage to reduce theaccumulation of SGAs in tubers during long term storage. The main findings are as follow:1. The tubers of vary varieties were selected to analyze the impact of light wavelength on thesteroidal glycoalkaloids accumulation in potato tubers. The results showed that the different lightwavelengths had varied effect on SGAs accumulation in potato tubers. Although under the samewavelength, the content of the steroidal glycoalkaloids varies among genotypes. The red light showedsignificant induction capacity on the accumulation of steroidal glycoalkaloids in potato tuber, followed bythe blue light. The data also showed that the dark colored tubers produce more light-induced SGAs.2. The influence of light wavelength on the second messengers—G protein, calmodulin and cAMP inpotato tubers also analyzed. The results showed that the content of those secondary signal moleculesincreased most significantly under the red light. Therefore, the results suggested that the red light was oneof the most important signaling molecules for SGAs biosynthesis and accumulation. It is proposed that thered light might stimulate the acceptor (phyB) and trigger the accumulation of SGAs via the secondmessengers.3. The expressing level of hmgr, pss1, sgt1, sgt2, sgt3and phytochrome (phyA and phyB) wereanalyzed with Real-Time PCR in four cultivars and the wild species—S.chacoense. The results showedthat the response of the genes varies among varieties and tissues, but the genes mediated the biosynthesisof chaconine and solanine are high in all tissues, which indicated that the content of secondary metabolitecould vary significantly when the activities of several metabolizing-related enzyme altered. Thus, tocontrol the activities of key enzyme and rate-limiting enzymes which catalyze the anabolism of steroidal glycoalkaloids, the co-transformation of the tubers specified promoters and the related genes might beneeded.4. The cDNA sequence of the potato sgt3was obtained from the total RNA of zhuangshu3potatotubers by reverse transcription polymerase chain reaction (RT-PCR). The protein function and structure ofthe similar sgt3gene were predicted and analyzed. The blast results showed that the gene sequence shareda high degree of similarity with the sgt3gene in GenBank (accession No: DQ266437) and the homologywas99.54%, the similarity of the amino acid sequence was99%. The full-length of cDNA was1500bp,which contained505amino acids, UDPG glycosyltransferase conserved domain and many importantfunctional sites. The3D structure of protein was predicted by homology comparative modeling inSwiss-Model, the results showed that the3D structure of SGT3was highly similar with that of theglycosyltransferase, so the results suggested that SGT3could be a member of glycosyltransferasesuperfamily and has the function of synthesizing steroidal glycoalkaloid. Sgt3similar gene obtained herewas rhamnosyl transferase.5. The fragements of sgt3: conserved domain sgt3-i1, non-conserved domain sgt3-i2and sgt3-i3contained both conserved and non-conserved domains were cloned into the intermediate vectorpUCCRNAI. the constitutive RNAi vector promoted by CaMV35S promoter carrying two copies ofpartial sequences of sgt3-i2and sgt3-i3in an inverted-repeat orientation (called pCSRI1, pCSRI2,pCSRI3) and super-expression vector pCST3, inverted repeats and their introns were connected toexpression vectors (pCAMBIA2300-Actin1-OCS, pBIC and pCAMBIA3300), constructing the actinpromoter carrying two copies of part sequences of sgt3-i1in an inverted-repeat orientation (calledpCARi1), tissue specific RNAi vector promoted by CIPP promoter carrying two copies of part sequencesof sgt3-i2and sgt3-i3in an inverted-repeat orientation (called pBCRI2, pBCRI3) and super-expressionvector pBCT3, then transfer those vectors into the Agrobacterium tumefaciens (LBA4404).6. The pCARI1/LBA4404, pCSRI2/LBA4404, pCSRI3/LBA4404and pCST3/LBA4404wereintroduced into tobacco T12via Agrobacterium-mediated genetic transformation. A total of18resistantplants were obtained. The PCR analysis indicated that12out of18plants carried the target fragment. Theherbicide resistance test showed that the transgenic tobacco showed some resistance to glufosinate,indicating that target gene was expressed in transgenic tobacco plants.7. The five interference vectors (pCARI1, pCSRI2, pCSRI3, pBCRI2, pBCRI3) and twosuper-expression vectors (pCST3and pBCT3) were constructed and introduced into potato stems andtubers via Agrobacterium-mediated transformation. Eight transgenic plants were obtained.
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