Study On Cloning And Soybean Transformation Of Methionine-rich Storage Protein Gene

Plant,especially plant seed,is an important source of protein consumed by humans and animals,but its nutritional quality is often not complete without some essential amino acids.Typically,gramineous crop seed proteins usually lack of tryptophan and lysine,but methionine was the first limiting amino acid in legumes.It is of great significance to obtain the methionine–rich protein(MRP)gene and then increase the Met content of the low sulfur plant especially the legume crop using it by genetic engineering.Based on homologous cloning techniques,some MRP genes from different plants in Gramineae and 5' promoter region of MRP gene in reed were cloned,and then it is proved that the MRP genes are storage protein genes by bioinformatics.Furthermore,the MRP gene in reed was transformed into soybean,and the molecular biological detection and analysis of amino acid content of the transformed regenerated soybean were done.The main results were as follows:1.Some length of 400-450 bp MRP genes,which are highly homologous,were cloned in rice,wild rice,reed,Echinochloa from different subfamilies and genera in Gramineae.The Met content of the polypeptides encoded by the MRP gene were more than 10% and far more than that in legume and other some sulfur deficient plants,and much higher than the average content of methionine required by WHO in food also.2.The results of multiple sequence alignment and online Blast alignment indicated that the cloned MRP genes in this study were high homology with other high methionine storage protein genes,and most likely seed.storage protein gene.3.The results about simulations of the protein secondary structure,motif,3-D and protein characteristic structural domains for amino acid sequence encoding by the MRP gene indicated that the MRPs also contains some characteristic seed storage protein domains such as 2S albumin,allergization domain,lipid-transfer protein and protease / amylase inhibitor,and their protein structure are very similar with some 10 kD prolamin in sorghum,rice and maize through the simulation of protein interaction.It showed that the cloned MRP genes should be the storage protein gene.4.In order to further confirm that the MRP gene is a storage protein gene,the primers were designed according to the sequence of reed MRP gene encoding region,and then it's 5' promoter region was successfully cloned from reed using the method of chromosome walking.The analysis of this sequence showed that the cloned 5' promoter region not only has some typical promoter element such as Kozak(ACACC ATG),initiator(CAT),TATA box(TATAAA),CAAT box(CCAAAT)and GC box(GGCGGC),but also highly conserved seed specific cis-acting elements such as repeats RY,GCN4 box,Prolamin box,E box,ABRE box,Skn-1 motif,P box,O2-site and GARE,etc.,and thus indicated that the cloned MRP gene from reed is a specific storage protein expressed in seed.The result of multiple sequences alignment of the promoter region also provide important evidence that the MRP sequence is storage protein gene sequence.The results of alignment for signal peptides showed that the MRP signal peptide with equal molecular weight was nearly identical,which may be related to the synthesis and secretion of the endosperm storage protein in the process of seed formation.5.The recombinant plasmid pBI121-LW constructed with the reed MRP gene and a binary vector pBI121 was transformed into Agrobacterium by liquid nitrogen cold stimulation method,and then infected the soybean cotyledonary node.After dark culture,induction of bud and hairy root,seedling,the explants grew up into new plants.The results of GUS histochemical staining,PCR assay,fluorescence quantitative PCR detection and amino acid content detection of some antibiotic-resistance seedlings showed that the MRP gene cloned from reed had been transferred into the soybean resistance seedlings,and expressed successfully.In different resistant seedlings,the methionine content increase by varying degrees in transgenic soybean plants,and the highest reached up to 51.11%,and the average value of methionine content was 27.81%.The cloned MRP gene in this study will further enrich the MRP gene,and also provide more target genes selection for the plant nutrition protein genetic engineering with the aim of increasing Met content.At the same time,it is a simple and easy way to find a simple and feasible method for the homologous cloning and batch amplification.The results of MRP gene transformation of soybean have laid a certain foundation for the next step of breeding work of soybean quality improvement,but also provide a theoretical support for the next step to the development of improved sulfur deficient plant protein quality gene engineering.