The Study On The Dwarfing Mechanism Of An Upland Cotton(Gossypium Hirsutum L.) Dwarf Mutant LA-1 Based On ITRAQ

As an important economic crop and national strategic material in our country,cotton is confronted with the restrictive problems in industry development at present,such as the shortage of labor and the high cost of planting.Therefore whole-course mechanization is the inevitable choice to solve these restrictive factors.Breeding cotton dwarf varieties which are lodging-resistant,suitable for high density planting and have high harvest index,with contribute to meet the whole-course mechanization production needs.Extensively digging and accurately using of dwarf genes are the keys to breed dwarf varieties.iTRAQ?isobaric tags for relative and absolute quantification?has been widely used in screening of specific proteins on a large scale,because it is high throughput,high sensitivity and high accuracy.The upland cotton?Gossypium hirsutum L.?dwarf line LA-1 and near-isogenic line?NIL?LH-1 were used as research materials,we screened dwarf-related differential expression protein through applying the method of iTRAQ combing LC-MS/MS quantitative proteomic technology and bioinformatic analysis,verified gene transcriptional expression by real time PCR,and furthermore,analyzed and combined the results of tissue-cytology examination and photosynthetic characteristics indexes.Those results unfolded the molecular regulated network of dwarf,provided referrals for make sure the key genes that regulated plant height in LA-1 and dwarfism breeding in cotton.The main results are as follows:1.By iTRAQ quantitative proteomic analysis of the stem terminal buds from the upland cotton?Gossypium hirsutum L.?dwarf line LA-1 and NIL LH-1,the results showed that 14417 unique peptide fragments,4849 proteins,697 differential expression proteins were identified,including 458 up-regulated proteins and 239 down-regulated proteins in LH-1 VS LA-1.GO analysis of different expression proteins revealed that 75.21% protein were located in cell,cell part,membrane and organelle;Most of the proteins functioned as catalytic,binding and transporter activity,they were about 90.06%;More than a half proteins were involved in cellular process,metabolic process,single-organism process and response to stimulus pathway.Pathway analysis revealed that 525 proteins were involved in 98 pathways,including metabolism,secondary metabolism,amino acid and nucleotide sugar metabolism,photosynthesis,synthesis and metabolism of hormones,lipid synthesis and metabolism pathways and so on.2.The results of histological detection showed that the number of longitudinally cells in ordinal 3rd internode was less than LH-1,the width ratio of vascular layer to cortical in LA-1 were significantly more than LH-1 from top of LA-1in ordinal 1st to 6th internode,the number of vessel in LA-1 was obviously more than LH-1,the development of vascular layer was earlier than LH-1.Ligin is the basis of forming vascular.Through the analysis of different expression proteins,there were several different expression proteins in phenylalanine synthesis metabolic pathway which are the synthetic raw material of ligin,trp D is the key regulatory element in phenylalanine synthesis,which was down-regulated in LA-1;4CL,CCo AOMT and CYP73 A are the key regulatory elements in phenylalanine metabolic pathway,which were up-regulated in LA-1.All of the genes had the same trend in transcriptional level through real time PCR.All of the results reveal that the dwarfism of LA-1 is related to phenylalanine synthesis and metabolic pathway.3.It revealed that the key signal elements Psb O,Psa E,Psa H,Pet F-1 and Pet F-2 were down-regulated,Pet C and delta were up-regulated in LA-1 through the further analysis of the different expression protein of photosynthesis system element.The results of real time PCR indicated that the transcriptive level of delta was the same in the two materials,the others had the same tendency as the protein level.Through the method of detecting the photosynthesis and chlorophyll fluorescence of the two samples,it revealed that the net photosynthetic efficiency?Pn?,conductance?Cond?,transpiration rate?Tr?in LA-1 were all lower than LH-1,but the inter-cellular CO2 concentration?Ci?had no significant differences after the budding,it indicated that the non-stomatal factor leaded to the decreasing of the net photosynthetic rate in LA-1.The Chlorophyll fluorescence detection revealed that the Y?II?was about 23.2% lower;NPQ was about 23.3% higher;Fv/F0 was about 25.5%?P< 0.05?lower in LA-1 than LH-1.However,the F0,Fm,Fv/Fm,q P,q N had no significant differences.Those results suggest that the down regulated proteins in photosynthesis result in the reduction of photosynthetic capacity of LA-1,thus generate the dwarfism.4.The results of analyzing the hormone related different expression proteins showed that,there were six different expression proteins which were down-regulated in LA-1 compared to LH-1 in hormone anabolic and signaling pathways,including two gibberellin?GA?receptors?GID1-1,GID1-2?,two cytokinin?CTK?receptors?CRE1-1,CRE1-2?,CTK synthetase CKX and UGT76C1₂.GA3ox1 and GA20 ox which are the negative regulator in signal pathway of GA,were up-regulated in LA-1compared to LH-1.The results of real time PCR revealed that all of the genes had the same tendency at the protein level except CRE1-4.In ubiquitin-proteasome system,the ubiquitin-activating enzyme UBE1 was down-regulated in LA-1 compared to LH-1,ubiquitin conjugating enzyme UBE2 DE and UBE2G1 were up-regulated in LA-1.The protein expression of E3 ubiquitin ligases were the same in the two materials.The results of real time PCR showed that all of those genes had the same expression tendency in the m RNA level and protein level.Our results suggest that the DELLA-independent GA signaling pathway is the primary cause of phytohormone-mediated dwarfism in LA-1 and CRE1-2,GID1-1,GID1-2,CUIKX,GA20 ox are potential indicators of dwarf cotton.