Screening Of Alkaline Neutral Invertase Interaction Protein In Cassava (Manihot Esculenta Crantz)

Cassava roots are rich in starch that the accumulation is related to the sucrose content in roots.Invertases are the key enzymes in catabolism of sucrose,which irreversibly catalyze sucrose into glucose and fructose.Our preliminary study on the expression analysis of the alkaline/neutral invertase genes revealed that the expression level of the alkaline/neutral invertase MeNINV1(in the a group,located in the plastid)was much higher than that of the other members,which indicates that it is a key gene for sucrose catabolism in the roots of cassava.The alkaline/neutral invertase MeNINV1 function for an in-depth study will help us to analysis alkaline/neutral invertase regulation of cassava sucrose metabolish mechanism,and provide a theoretical basis for the cultivation of high yield and high starch content of cassava varieties,but also for screening high starch yield of transgenic cassava.At present,the mechanism of the regulation of the enzyme activity of the ? group alkaline/neutral invertase has been preliminaryly analyzed:AtCINV1 protein has a 14-3-3 protein binding region at the C-terminus,the calcium-dependent protein kinase gene family members CPK3 and CPK21 can cooperate with 14-3-3 protein,phosphorylation of Serine Residues in the Region Regulates AtCINV1 Protein Activity;a member of the PIP5K family,PIP5K9,interferes with the interaction of 14-3-3 protein and AtCINV1 protein and negatively regulates AtCINV1 activity.The C-terminus of the a group alkaline/neutral invertase does not have a binding region for the 14-3-3 protein,and their mechanism of enzyme activity regulation is not clear,therefore,we selected MeNINV1(a group)for in-depth studies to identify proteins that interact with MeNINV1 using yeast two-hybrid technology and two-molecule fluorescence complementary techniques;subcellular localization of validated successful interaction proteins was performed;and the interaction protein was encoded by qRT-PCR.Relative quantitative analysis of gene expression levels was performed to analyze their expression patterns in cassava in different tissues and stresses.The main findings are as follows:1.The point-to-point verification of MeNINV1 and Me 14-3-3 proteins was performed:The AH 109 bait vector pGBKT7-MeNINV1 was constructed,and it was confirmed to have no toxic effect and self-activation in AH 109.Bait plasmids were co-transformed with pGADT7 into yeast and the recombination yeast strain was confirmed to have no self-activation on SD/-Trp-Leu-His-Ade medium plate.We transformed pGADT7-Me 14-3-3 plasmids with yeast containing bait vetor pGBKT7-MeNINV1 and screened recombinant yeast strains using SD/-Trp-Leu-His-Ade+5 mM 3-AT+X-Gal medium plate.The results showed that the enzyme activity of MeNINV1 was not regulated by Me 14-3-3 protein..2.Screening MeNINV 1 interactive proteins by AH 109:The yeast strain AH109 was co-transformed with pGBKT7-MeNINV1 bait vector and yeast two-hybrid library plasmids for library screening.44 initial positive clones were screened on the SD/-Trp-Leu deficient plate and tested using the four missing plates SD/-Trp-Leu-His-Ade and ?-galactosidase activity.Finally,14 colonies were selected as candidate positive clones3.Validation of MeNINV1 interacting protein in yeast cells:In order to verify the screening result of MeNINV1 interacting preliminarily proteins,the full-length gene coding sequences of the interacting proteins were cloned and constructed with pGADT vector.The result vectors were named as pGADT7-CSN5B?pGADT7-CSN5A?pGADT7-Pti5?pGADT7-Cox11?pGADT7-UP1?pGADT7-PHOS34?pGADT7-FKBP20-1/pGADT7-BFNl/pGADT7-MBD4;then were co-transformed to AH109 with pGBKT7-MeNINV1 to screen the recombinant yeast strains in SD plates.The result showed that MeNINV1 interacted with CSN5B.4.Validation of MeNINV1 interacting protein by using BiFC:The bait protein(MeNINV1 and the interaction protein(CSN5B)were fused with the yellow fluorescent protein pSAT4-nEYFP-N1 and the pSAT4-cEYFP-N1 BiFC expression vector fragment,respectively.The two-molecule fluorescent carriers MeNINV1-nEYFP and CSN5B-cEYFP were constructed,then transformed into tobacco protoplasts using PEG-CaC12 method.After being placed under low light at 25? overnight,observation of fluorescence under a laser confocal microscope,the results of tobacco protoplast expression experiments showed that MeNINV1 interacts with CSN5B in plant living cells.5.Sub-cellular localization of MeNINV1 interacting protein:In order to define the sub-cellular localization of MeNINV1 interacting protein,the pCAMBIA 1300-GFP fusion expression vector was constructed and named as pCAMBIA1300-CSN5B-GFP.The expression vector plasmid DNA was transformed into tobacco protoplasts using PEG-CaCl2.The fluorescence was observed under a confocal laser scanning microscope.Experimental result showed that CSN5B was mainly located in the cytoplasm of tobacco cells.6.Analysis of the expression characteristics of CSN5B gene:qRT-PCR was used to analyze the expression characteristics of CSN5B gene in different tissues and stressed conditions in cassava.The experimental results showed that the CSN5B gene was expressed in all parts of the cassava,but the expression was highest in the tuberous roots and the lowest in the stems.The expression patterns of CSN5B gene induced by MeJA,GA,ABA,IAA and SA were studied.The results showed that the gene the expression of CSN5B was regulated by hormones.After treatment,the expression of CSN5B in tubers and stems decreased significantly.The expression of CSN5B in leaves showed a tendency of decreasing first and then increasing.