ITRAQ-based Proteomic Analysis Of Green-leaf,Mosaic-leaf And Yellow-leaf Cassava Leaves

Cassava(Manihot esculenta Crantz)is a staple food for nearly one billion people in the tropical regions of the world.It is the fourth largest food crop in the world and is widely grown in 105 countries.Cassava is an important raw material for producing starch,modified starch,fuel ethanol and other chemical products as well as feed in China.According to the development demand of cassava industry,it is urgent to select and breed cassava varieties with high efficiency and high starch accumulation.However,the whole genome of cassava is highly heterozygous,the descendants are separated seriously,and the breeding cycle to select new varieties is long.Those factors seriously block the development of cassava breeding.Therefore,this study uses iTRAQ to reveal the molecular mechanism of cassava high photosynthetic efficiency from the molecular level,find the key proteins related to cassava leaf high photosynthetic efficiency and their interaction regulated networks,and provide a new clue for breeding backbone parent materials of new cassava varieties.The main research results were as follows:1.In the present study,cotyledons of green-leaf cassava MBRA206 were induced by Co60-radiation,and stable mosaic-leaf cassava ZM-IOJ and yellow-leaf cassava ZM-11K strains were obtained through tissue culture of many generations.Tissue culture seedlings of cassava MBRA206,ZM-10J and ZM-11K were used as test materials.The relative quantitative analysis of proteome of MBRA206,ZM-10J and ZM-11K leaves was carried out by iTRAQ technology.A total of 394,498 spectrograms were obtained and 71189 matched spectrograms were obtained,including 54441 unique spectrograms.A total of 6.094 proteins and 27,762 peptide segments were identified,including 23,090 unique peptide segments.From the molecular functional classification,it was found that differential proteins were mainly catalytic proteins and binding proteins.Differential proteins participate in 106 metabolic pathways,which are divided into 12 categories according to their functions.The String on-line software was used to construct a differential protein-protein interaction regulation network for carbon and energy metabolism.It is speculated that GLT1,ACLB-2,PETC,SPBASE,SHM1,AT5G58330,CA1,PGK.FBA2,FBRFNR1 and ACP-4 have more and stronger interaction relationships,which can be used as the core protein in the center of the whole regulation network to regulate leaf photosynthesis.2.Analysis of transcription level and protein expression level of key proteins in carbon and energy metabolism after girding.The results showed that at transcription level,9 proteins were up-regulated in green-leaf cassava and 7 proteins were up-regulated in mosaic-leaf cassava after girding of cassava seed stem.By analyzing the chlorophyll content of cassava leaves after girding,it was found that the chlorophyll a,chlorophyll b and total chlorophyll content of green-leaf and mosaic-leaf cassava leaves were significantly decreased.Western blot analysis showed that the expression levels of OEC.Rbcl and PRXQ in the leaves of green-leaf cassava and mosaic-leaf cassava were significantly reduced,and the expression level of D1 in the leaves of mosaic-leaf was also significantly reduced.3.Response analysis of cassava leaves after girding.The results showed that after girding,there were 15 differential proteins in the leaves of green-leaf cassava and 9 differential proteins in mosaic-leaf cassava compared to control.Most of the expression levels of the differential proteins after girding were significantly lower than those of the control.The differential proteins included carbon and energy metabolism,molecular chaperone.structure,protein synthesis,DNA and RNA metabolism related proteins and defense proteins according to their functions.A protein-protein interaction regulation network was constructed by String online software.The results showed that phosphoglycerate kinase(point 7)and glyceraldehyde-3-phosphate dehydrogenase(point 8),cytochrome b6-f complex iron sulfur subunit(point 19)and fructose diphosphate aldolase polysaccharide enzyme 2(point 1)had the maximum interaction relationships,but down-regulated.