The Construction Of DNA Library Of The Peanut And The Clone Of The Upstream Sequence Of Seed-specific Expression Gene Arachin

The key problem of low expression of gene in transgenic Plant is the promoter and choose the rightpromoter is the crucial way to enhance the expression of differential gene. The most frequently used promoter for regulating the interested genes expression is CaMV35Spromoter Ubiquitin promote and Actin1 promoter from rice which all can over-express the interestedgenes, but the expression is neither environment, tissue nor time specific, it is constitutive. Theexpression of protein wastes the energy in normal environment and the accumulation of the protein caninhibit the plants normal metabolism, causing the morphology change of the plant, resulting in growthand development of transgenic plants. In order to solve the problem as above, people can use the stressspecific induced promoters, but the activity of these promoters identified as far still can't content theneed of related research. So the experiment attempts to construct artificial promoters which can bestrongly induced by conditions. The specific promoters have been found and searched are mainly thetissue-specific promoter and induced-specific promoter, such as seed-specific promoter, fruit-specificpromoter, root-specific promoter, wound induced-specific promoter and others. The clone and use ofthese tissue-specific promoter and condition induced-specific promoter are the ground of gene's specificexpression. The nature promoter are not enough in the study of transgenic plant, especially when the gene needto express specifically. Constructing a multiple promoter is a way to solve the problem above. Spatially and temporally regulated gene expression programs are the basis for development andmorphology. The strictly seed-specific and development expression of seed storage promoter genesprovides a suitable experimental system to study differential gene activation in plants. It is generallyaccepted that the seed specificity of storage protein gene expression is primarily regulated at thetranscriptional level, although post-transcriptional processes can modulate the final amount oftranslational products widely. Current ideas imply complex interactions between specific trans-actingtranscription factors with their cis-acting target DNA sequences as the principal mechanism fortranscription regulation. Several DNA fragments derived from the 5'-flanking regions of different seedprotein genes have been shown to bind defined nuclear protein factors. However, in most cases a causalrelationship connecting trans factor binding with regulated promoter activity has not been demonstrated.The availability of extensive sequence data from 5' flanking regions of storage protein genes isolatedfrom several different species has prompted the search for conserved sequence motifs, assuming thatthese elements might be involved in trans-factor binding and therefore in the regulation of seed proteingene expression.Peanut seed storage protein gene Arachin is seed-specific expressed in seed, it can be supposed thatthe promoter of the gene can promote the seed-specific expression of differential gene.In this experiment peanut genome DNA is centrifuged by CTAB protocol, and is partial digested.Construct the DNA library using the vector, lambdaGEM-11.TAIL-PCR is one protocol to amplify the flanking sequence of known DNA sequence. We get thegene Arachin part sequence of DNA . We Design the primer in DNA sequence of the gene and get theupstream sequence of the gene.