ITRAQ-based Proteomic Analysis Of Tuberous Root In High Amylose And High Amylopectin Of Cassava

Cassava(Manihot esculenta Crantz),a plant of the Manihot genus in Euphorbiaceae,is one of the most important food crops in the world.It is called the three largest root and tuber crops in the world together with potato and sweet potato.Among many starch crops,cassava has high starch content(20%-40%)in tuberous root with developing potential.Starch is widely used in people’s daily life.There are two forms including amylose and amylopectin in the starch,in which amylose becomes blue,and amylopectin becomes purple when adding iodine.Because of its especial functions and characteristics,starch was used in different processing fields.In this study,in cooperation with the International Center for Tropical Agriculture(CIAT),the cassava cultivars of high amylose(5G160-13)and high amylopectin(A1288-17 and AM206-5)were introduced as the experimental materials,and South China No.5(SC5),which was extensively planted in Hainan province,was used as the control.The metabolic pathway of starch biosynthesis in cassava tuberous root was studied from.the global proteins using iTRAQ technology.The biological interaction network of differential proteins was constructed by online String software.The yeast two-hybrid screening,bimolecular fluorescence complementation(BiFC)and qRT-PCR techniques were used to determine the key proteins to control the metabolic pathways of amylose and amylopectin biosynthesis in cassava tuberous root..It would provide a theoretical basis for the breeding of cassava varieties with high amylose and high amylopectin.The main results were as follows:1.Cut out the transverse section of fresh tuberous roots of cassava 4 varieties including 5G160-13,AM-1288-17,AM206-5 and SC5,sprayed the whole transverse section with potassium iodide,and observed the color change on the transverse section.The result showed that t the color on transverse section of 5G160-13 and SC5 became blue,and AM206-5 and AM1288-17 were purple.2.In the present study,the total number of the secondary spectrum maps was 329085,the number of matching spectra was 35065,the number of matching specific peptide spectrum was 24725;the number of peptide fragments was 13742,of which the number of specific peptide fragments was 10645,the number of identified proteins was 3971.SC5 was used as control,there were 808 differentially expressed proteins in 5G160-13 including 397 up-regulated proteins and 411 down-regulated proteins;495 differentially expressed proteins were found in AM1288-17,in which 299 proteins were up-regulated and 266 proteins were down-regulated;there were 729 differentially expressed proteins in AM206-5,and 345 up-regulated proteins and 384 down-regulated proteins were found.3.Using KEGG database as a reference,884 differentially expressed proteins in 103 metabolic pathways were classified into 10 groups,includingcarbon metabolism and energy metabolism(15 pathways;284 proteins);lipid metabolism(10 pathways;42 proteins);amino acid metabolism(17 pathways;142 proteins);auxiliaries and vitamins metabolism(7 pathways;40 proteins);nucleotide metabolism(2 pathways;38 proteins);other secondary metabolites(13 pathways;50 proteins);genetic information processing,folding,classification,degradation,replication and repair(11 pathways;88 proteins);intracellular transport and metabolism(3 pathways;35 proteins);genetic information processing,translation,transcription(6 pathways;130 proteins);Environmental information processing,membrane transport,signal transduction,biological system and environmental adaptation(7 pathways;35 proteins).4.The key proteins related with amylose and amylopectin biosynthesis in carbon and energy metabolism were studied.These proteins mainly included AT5G42740,PGI1,PGK,PGK1,ENO1,ENOC,MDH,mMDH1,PMDH1,ACLB-2,TIM,TPI,GAPC1,PKP-ALPHA,AT5G08570,AT4G17260,mtLPD1,MAB1,ATCS,NADP,and PAT5G58330.They play key roles in starch accumulation,amylose and amylopectin synthesis.5.Using qRT-PCR to verify the results of iTRAQ data Analysis.Proteins such as mMDH1,PMDH1,PGI,ENO1,GAPC1 and TIM had played key roles in accumulation of amylose and amylopectin in the pathway of carbon and energy metabolism.6.The online String software was used to construct the biological regulatory interaction network of 10 kinds of differential proteins and find the proteins with a large number of wires,mainly catalytic proteins and binding proteins.7.The yeast two-hybrid screening and bimolecular fluorescence complementation were used to determine the protein-protein.interaction.The results showed that there is not interaction between GBSSI and APL3 testing with yeast two-hybrid screening.However,the weak interactions between GBSSI,APL3,and SBE2.2 were found,suggesting the interactions between these proteinswere weak.