Study On The Role Of StSN2 In Potato Dormancy

Potato（Solanum tuberosum L.）is an important staple crops,which has the characteristics of wide adaptability,high yield and rich nutrition value.It is of great significance to ensure national food security,promote poverty alleviation in poor areas and the strategy of Rural Revitalization.Potato tubers of different varieties have dormancy period of 30-150 days at room temperature,and sprout after dormancy period.Untimely dormancy release and germination will lead to the reduction of commercial tubers,the decline of nutritional quality,the accumulation of toxic substance solanine alkali,affect the vitality of seed potato,diet,nutrition and health,processing and sales,and seriously restrict the development of potato industry.In the early stage,St SN2,the key gene to maintain dormancy,was screened and identified through transcriptome and proteomic studies of potato tuber dormancy and germination.By detecting the transcription level of St SN2 in varieties at different dormancy stages,it was found that the expression of St SN2 was the highest when the tuber was mature,and decreased gradually with the release of tuber dormancy.In order to explore the function of St SN2 in maintaining potato tuber dormancy,transgenic materials with different expression levels were constructed by transgenic technology.Through the analysis of dormancy characters and physiological indexes,it was clear that St SN2 had the function of maintaining tuber dormancy.The function of St SN2 to maintain potato tuber dormancy was elucidated from the physiological and molecular level by using proteomic technology,exploring the action path of St SN2 in regulating tuber dormancy,combined with q RT-PCR,COIP-MS and enzyme activity detection.The main results are as follows:（1）St SN2 gene was cloned from potato‘Chuan Yu 10’（C10）.Its Open Reading Frame was 315 bp and encoded 104 amino acids.Bioinformatics analysis showed that the relative molecular weight of the protein encoded by St SN2 was 11.04 KD,and it had a conserved region containing 12 cysteine residues,which belonged to hydrophilic protein.Phylogenetic analysis showed that St SN2 was closely related to SN2 gene of tomato,capsicum and tobacco.Tissue expression analysis showed that St SN2 was expressed in root,stem,leaf and other tissues.The expression of St SN2 in tuber was significantly higher than that in other tissues,especially in tuber dormancy.（2）42 P_35scamv-St SN2 overexpression lines,35 antisense lines,45 tuber specific P_patatin-St SN2 overexpression lines and 60 antisense lines were prepared by Agrobacterium mediated method.Phenotypic analysis showed that changing the expression level of St SN2gene significantly affected the dormancy period of tuber.Compared with C10 tubers,the dormancy period of overexpressed tubers was prolonged by 3-4 weeks,and that of antisense tubers was shortened by 2-3 weeks.However,the effects of St SN2 on plant height,main stem number and stem diameter were not significant.（3）The transgenic tubers stored for 45 days were used as materials（C10 as control）for proteomic analysis.A total of 46924 effective mass spectrograms,25216 peptides and4709 proteins were identified,381 proteins with FC（Chang fold）≥1.2 times,236 with FC≥1.3 times,61 with FC≥1.5 times,and 6 with 2.0 times.Subcellular location prediction showed that the differential proteins were mainly located in cytoplasm,nucleus and chloroplast.KEGG pathway analysis found that the differential proteins were mainly enriched in starch and sucrose metabolism,plant hormone signal transduction,phenylalanine biosynthesis and other pathways.（4）The expression of St SN2 responded to the dormancy regulatory hormones ABA and BR,and the response to ABA was the most significant.Proteomic analysis showed that St SN2 up regulated the expression of positive regulatory proteins PYL,AAO,VIP1,and down regulated the negative regulatory protein PP2C,and q RT-PCR results showed that the corresponding gene level of these proteins also showed the same change trend.Further found that St SN2 induced the expression of positive regulatory factors ABI3 and ABI5 downstream of ABA,indicating that St SN2 enhanced ABA signal.Co IP-MS results showed that St SN2 interacted with Sn RK2.4（positive regulator of ABA signal）,and St SN2promoted the expression of Sn RK2.4.（5）Proteomic analysis showed that St SN2 regulated the metabolic pathway of starch and sucrose,and promoted the expression ofβ-amylase,pyruvate kinase,malate dehydrogenase and other proteins,which was consistent with the results of q RT-PCR.Enzyme activity analysis showed that St SN2 inhibited the expression of sucrose synthase,glucose-1-phosphoadenylate transferase,pyruvate kinase and other proteins.The content of starch,sucrose and glucose was further analyzed.The results showed that the level of sucrose and glucose in St SN2 overexpressed tubers decreased,the starch increased,while the antisense expression tubers showed the opposite St SN2 inhibited the activities of sucrose synthetase,amylase and pyruvate kinase,and promoted the activities of sucrose phosphate synthetase（SPS）.It was proved that St SN2 could maintain potato dormancy by inhibiting the pathway of sugar decomposition and utilization.To sum up,St SN2 regulates tuber dormancy mainly by enhancing ABA signal and inhibiting carbohydrate decomposition.The results of this paper will enrich the functional research of St SN2 and lay a foundation for further research on the mechanism of potato tuber dormancy regulation network.