Proteomic-based Study To Explore Molecular Mechanism Of Yam Tuber (Dioscorea Opposita Thunb.) Expansion

As the material storage organ of Dioscorea opposita Thunb.,tubers mainly accumulate substances such as starch.The swelling of yam tubers determines the material basis for their growth and development,which is a highly complex physiological process.In addition,the expansion of yam tubers is of great significance for improving their yield and economic benefits.However,the molecular mechanism of yam tuber enlargement is currently unclear.In order to further reveal the molecular regulation mechanism of yam tuber expansion,this study used‘Dahechangyu’yam as experimental materials to determine the morphological,physiological and biochemical indicators of the tubers at seven development stages.On this basis,we further used Label-free quantitative（LFQ）technology to sequence them in proteomics,analyze the dynamic metabolic pathway of the tuber protein level during the expansion period,and screen important proteins associated with the process of tuber expansion,Real time fluorescence quantitative PCR（qRT-PCR）was used to verify the identification results of proteomics.The results of this study will provide new ideas for understanding the mechanism of yam tuber enlargement,and help to further elucidate the protein regulatory mechanism of yam tuber development and enlargement.At the same time,some candidate genes have important reference value for further research and breeding of yam tuber enlargement in the future.The main findings are as follows:（1）As the yam tubers expand,their mass,length,and diameter continue to increase.90-150 days after planting is their rapid growth period,completing about 80%of the growth in mass,length,and diameter during the entire expansion period.The growth rate slows down in the later stage of expansion,and the difference is not significant.（2）The accumulation of total starch,amylose,and amylopectin in tubers showed a trend of first increasing and then decreasing,and there were significant differences in the content of various types of starch after planting at 120（D-120）,150（D-150）,and 180days（D-180）;D-150 amylopectin accounts for 88.35%of the total starch content,and the increase in total starch content is mainly due to the accumulation of amylopectin.There is a negative correlation between various starch contents and sucrose content.The sucrose content during the initial stage of swelling is very high(about 0.9 mg·g^-1),and then significantly decreases for the development and transformation of tuber cells into starch.The total starch and amylopectin content of tubers during the swelling period are highly significantly positively correlated with morphological indicators,indicating that starch accumulation plays a key role in tuber swelling,and starch is an important contributing factor to yam tuber swelling.（3）The SUS enzyme activity in tubers shows a continuous decreasing trend,and the sucrose content is highly significantly positively correlated with it（r=0.937）.AGPase and SBE activities are highly significantly positively correlated with total starch and amylopectin content,and are important functional enzymes directly involved in the accumulation of starch in tubers.（4）LFQ proteomics analysis was carried out on the key stages of yam tuber expansion:D-120 in the initial stage,D-150 in the middle stage and D-180 in the later stage.A total of 37947 peptide segments and 4599 proteins were identified through mass spectrometry analysis.807,1017 and 352 differentially expressed proteins（DEPs）were identified in the three comparative groups of tuber expansion D-150vs D-120,D-180vs D-120,D-180vs D-150,of which 55/752,88/929 and 120/232 were up-regulated/down-regulated proteins,and the number of down-regulated proteins was higher than that of up-regulated proteins;Gene ontology and pathway enrichment analysis showed that these DEPs participated in processes including photosynthesis,starch and sucrose metabolism,fatty acid biosynthesis,plant hormone signal transduction and MAPK signal pathway.（5）During the expansion of yam tubers,a large number of differential expressions of proteins related to plant hormone signal,light signal,calcium signal,MAPK signal transduction,starch and sucrose metabolism,lipid metabolism,transcription factors and so on were identified.It is speculated that these signal transduction pathways and proteins related to starch and lipid metabolism play an important role in the expansion of yam tubers.（6）The results of qRT-PCR validation showed that the LFQ proteomics data was highly consistent with the m RNA expression level.The coincidence degree of up or down regulation in the three comparison groups D-150vs D-120,D-180vs D-120 and D-180vs D-150 was 91.67%（11/12）,91.67%（11/12）and 75%（9/12）,indicating the reliability of the LFQ proteomics data.