Construction Of RNAi Seed-specific Expression Vector On Soybean Lipoxygenase Gene

Soybean is one of the main sources of plant protein for human beings and animals,and it is not only the main grain crops,but also the main oil-bearing crops for our country,it has the extremely high nutritional value.However,there are many nutritional inhibitory factors influence the quality of soybean,such as soybean Lectin,Lipoxygenase,Trypsininhibitor and so on.The soybean lipoxygenase is one of the main nutrition inhibiting factors in soybean.The lipoxygenase is called Lox,is one kind of the protein containing non-heme iron,it catalysises the respond with oxygen singlely which has syn-1,4 pentadiene structure's multivariant unsaturated fatty acid,generate the fatty acid hydrogen hyperoxide having the conjugation double bond,then produce the short carbon chain fugacity matter,such as alcohol,ketone,aldehyde and so on by the enzyme decomposition.These fugacity matter lead soybean and their products to produce beany flavor, the main fishy smell matter is caproaldehyde and hexanol.The lipoxygenase is one of the main inhibitory factors influencing soybean quality.RNA Interference(RNAi) technique is a direct and effective method which can control the level of gene expression,block specific gene expression and cause post-transcriptional gene silencing(PTGS).Thus,RNAi technique has been applied to improve plants quality frequently.Seed-specific promoter can control the expression of the heterogeneous gene in plant seeds,avoid the expression of heterogeneous gene in other parts of plants and reduce the adverse effects of plants.It is very important both in theory and practice to control the gene expression promoted by seed-specific promoter genes.In this study,soybean lipoxygenase gene was cloned.And a RNAi expression vector was constructed using seed-specific promoter.Then the vector was transformed into soybean variety by Pollen Tube Pathway Method,and obtained transgenic plants.Moreover, the main purpose of this study is to reduce lipoxygenase content and increase protein or oil content,which could explore new ways to improve soybean quality.The main results are listed as following:1.According to the data of soybean lipoxygenase gene sequence published in GENE BANK,corresponding primers was designed,and cDNA of soybean variety "Jinong 18" was selected as the template which was obtained by reverse transcription with total RNA.The exon fragment of 357bp was cloned which the homology is the highest partial in three kinds of lipoxygenase genes(Lox1,Lox2,Lox3).The entire mature peptide gene of soybean lipoxygenase sequence was expanded by polymerase chain reaction.The sequencing result showed that the fragment length is 357bp.The recombinant plasmid was obtained by inserting the objective fragment which was amplified by PCR into pMD18-T Vector.After being transformed into Escherichia coli,it was screened by blue floccosoids,then took the positive colon to carry on the PCR examination and the enzyme cutting identification,finally the obtained fragment and the objective fragment is consistent.2.The recombinant plasmid and plant expressing vector pCAMBIA 1301 were cut after the restrictive enzyme double enzyming.With T4 DNA ligase,we inserted seed-specific promoter(which was constructed by Yu Zhang from Biotechnological Laboratory of Jilin Agricultural University) and the anti-sense and sense gene fragments of soybean lipoxygenase gene into pCAMBIA1301,the RNAi expressing vector pCAMBIA1301-LoxRt was constructed.Then we extracted the recombinant plasmid of pCAMBIA1301-LoxRt.3.We transformed RNAi expression vector with Lox gene into Soybean by Pollen Tube Pathway Method,got the seeds of T0 generation.After sprouting we took the leaf of single adult plant's total DNA as the template, carried on PCR to expand.We got the specific strip belts of 960bp from three plants,but the non-transformed plant had not this fragment of 960bp.