Preparation Of Spi And Sbei Polyclonal Antibodies And Interaction Of Protein Complexes

Starch is an important storage polysaccharide in plants and is the main component of plant seeds.Starch can provide 80% energy for food and is an important part of food.Starch is a major ingredient in the production of various crop plants and a sustainable source for many industrial applications.Starch synthesis and decomposition are regulated by a series of enzymes,including starch phosphorylase(SP),starch synthase(SS),starch branching enzymes(SBEI),starch debranching enzymes.(starch-Debranching Enzymes,DBE),ADP-glucose pyrophosphorylase(AGPase),etc.Starch phosphorylase and starch branching enzyme are both enzymes that play an important role in the biosynthesis of plant starch.SP can participate in the side chain modification reaction of amylopectin in plant starch storage organs.SBE can catalyze the formation of branching structures of starch by catalyzing the cleavage of polysaccharide chains into-1,4 glycosidic bonds,and the formation of-1,6 glycosidic bonds on specific glycosyl groups of glucose chains to join the chopped sugar chains,and the addition of non-reducing ends for the further synthesis of starch.Previous studies have focused on the functions of SBEI and SPI in starch synthesis and metabolism,but the direct interaction of SBEI and SPI proteins has not been determined,and the distribution of SBEI and SPI in protein tissues has not been reported.In this study,SBE1 and SPI polyclonal antibodies were prepared and their tissue expression distribution was clarified.The interaction proteins of SBE1 and SPI were studied by double hybridization with IP and yeast.New multienzyme complex combinations were proposed in maize.Mainly obtained the following results:(1)Prokaryotic expression vectors of pGEX-6T-1-SPI-C(768-984aa)and pGEX-6T-1-SBEI-C(649-823aa)were constructed.The maize backbone 08-641(R08)from Southwest China was selected from the maize research of Sichuan Agricultural University as the material to prepare cDNA,design primers,and clone the complete SPI(full length 2955bp)and SBEI(full length 2472 bp)by NCBI sequence PCR coding region sequence.Due to the long SPI and SBEI genes,it is difficult to express proteins in microorganisms;in order to prepare SPI and SBEI antigens,the GST-tagged C-terminal V domain prokaryotic expression vector pGEX-6T-1-SPI-C was constructed(768-984aa),651 gene C-terminal domain gene fragment 651bp;pGEX-6T-1-SBEI-C(649-823aa),SBEI gene C-terminal gene fragment 525 bp,further sequencing verification,indicating that the expression vector was successfully constructed.(2)Preparation and evaluation of SBEI and SPI rabbit-derived polyclonal antibodies.BL21(DE3)was transformed with a successful prokaryotic expression vector,and IPTG induced protein expression and GST purification.New Zealand white rabbits were immunized with purified GST-SBEI-C and GST-SPI-C fusion proteins as antigens to prepare SBEI and SPI polyclonal antibodies.Western blot analysis with this antibody showed that the prepared SBEI and SPI antibodies have high specificity and sensitivity and can be used to detect nanogram antigen proteins.Western blot analysis was performed on pre-immune serum and post-immune serum and SPI and SBEI as antibodies for 20 days post-pollination of maize endosperm protein.The results showed that the pre-immune serum could not detect SBEI and SPI protein,and the serum could detect SBEI and SPI protein after immunization,which proved that the antibody was successfully prepared.The SBEI and SPI antibodies immunoprecipitated the 20-day endosperm protein after pollination and were subjected to Western blot analysis.The results showed that the antibody was able to immunoprecipitate its protein.In summary,SBEI and SPI antibodies were successfully prepared,and antibodies can be used for Western blot and immunoprecipitation(IP)experiments.(3)Expression of SPI and SBEI in different tissues after corn pollination.In vitro,SPI and SBEI antibodies recognize their antigens,but it is unclear whether they recognize proteins in other tissues in corn.In order to select appropriate corn tissue for in vivo validation,qRT-PCR was first performed on SPI and SBEI,and the expression of endosperm,grain and roots,stems and leaves in 15 days after pollination was analyzed.The results were shown in mRNA.At the level,the expression level was higher in the grain and endosperm in 15 days,the expression in the roots and leaves was lower,and the endosperm was higher than the grain,indicating that SPI and SBEI were mainly expressed in the endosperm.To further verify whether SPI and SBEI antibodies can recognize endogenous SPI and SBEI proteins in maize,the endosperm and total protein of maize were extracted from corn 08-641 15 days after pollination and Western blot experiments were performed.The results showed that SPI and SBEI were pollinated for 15 days.Protein bands were detected in the endosperm,stem and leaves of the 15 days,but the expression in the roots was very small.The molecular weight of the SPI protein was about 112 kD,and the molecular weight of the SBEI protein was about 84 kD,which was consistent with the mRNA level detection;because of ?-actin The amount of sample loading was lower than that of the grain,so the endosperm expression was slightly higher than that of the grain,indicating that SPI and SBEI were mainly expressed in the endosperm and mainly accumulated in the endosperm.These results indicate that SPI and SBEI polyclonal antibodies specifically recognize SPI and SBEI proteins in maize,and that SPI and SBEI proteins are specific for tissue expression.(4)Expression of SPI and SBEI at different stages after corn pollination.SPI and SBEI antibodies have good specificity,but at the mRNA and protein levels,their protein expression and tissue expression distribution are still unclear.In order to clarify the protein expression and tissue expression distribution in maize,the expression of mRNA in the endosperm of SBEI and SPI after 5-35 days of pollination was first analyzed by qRT-PCR.The results showed that SPI in endosperm was 5-20 days after pollination.And SBEI mRNA expression increased,and gradually decreased in 20-35 days.Secondly,the endosperm at different stages after pollination was separated,and the total protein was extracted and detected by Western blot.The results showed that the SPI protein content was very small within 10 days of pollination,and the expression of SPI and SBEI protein increased sharply within 10-15 days.The signal was strongest at 15 days and 20 days,and then gradually weakened.This result was consistent with the expression pattern of mRNA levels and the accumulation pattern of starch.(5)Study on multi-enzyme complexes during starch synthesis.The yeast-double-hybrid experiment was used to study protein-protein interactions,and recombinant yeast primers were designed to transform the constructed vector plasmid into yeast,so that each proteome was fused with GAL4-AD or GAL4-BD.In addition,each gene was The fusion plasmid was combined with an empty plasmid as a negative control,and 1034-AD and q146-BD were used as positive controls.Previous studies have reported that SSI,SSIIa and SBEIIb constitute heterotrimers,and large protein complexes in which SSI interacts with SSIIa,SBEIIa and SBEIIb also exist in a phosphorylation-dependent manner,but the binding relationship between protein complexes It is not clear that experimental studies have shown that SBEIIa interacts with SBEIIb,SBEIIa and SSI,SBEI and SSI,SBEIIb,SSIIa and SBEIIb.It is indicated that in the process of starch synthesis,SSI,SSIIa,SBEI,SBEIIa and SBEIIb exist in the form of SBEI-SSI-SBEIIa-SBEIIb-SSIIa complex,and there is no SPI in this complex.