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Cloning and Functional Analysis of Maize Starch Branching Enzyme Gene SBEⅡb Promoter

Author: XuYaWei
Tutor: ChaiXiaoJie
School: Jilin Agricultural University
Course: Biophysics
Keywords: Branching Enzyme SBE Ⅱ b Promoter Clone GUS
CLC: S513
Type: Master's thesis
Year: 2005
Downloads: 338
Quote: 3
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Abstract

Currently, the main problems in genetic engineering is the expression level of exogenous gene expression site, and other issues, therefore, one of the key components of the promoter as the regulation of gene expression has become the key to the study of genetic engineering. Promoter is a constitutive promoter (constitutive promoter), sub (tissue-specific promoter) and inducible promoter (inducible promoter) three kinds of tissue-specific promoter. To date, the plant expression vector the most widely used is the composition of the CaMV 35S promoter, which causes the exogenous gene expression in transgenic plants of all parts and all developmental stages, not only result in a waste of energy but also cause the plant morphological changes affect plant growth and development. This makes people more and more emphasis on research and application of specific expression promoter, the promoter gene expression in specific tissues and developmental stages start, both to ensure that the foreign genes in plants effectively reduce the plant adverse impact. Although there are many specific promoter have been cloned successfully, but better, really can be applied to production practice tissue-specific promoter is not much, seed-specific promoters less. This article cloned corn starch branching enzyme sbe Ⅱ b gene promoter is a seed-specific promoter. Corn starch branching enzyme (SBE) is one of the key regulatory enzyme in corn starch synthesis process, sbe Ⅰ sbe Ⅱ a sbe Ⅱ b three isozymes sbe Ⅱ b amylose content of endosperm. Therefore, to improve the level of expression sbe Ⅱ b become a hot spot of research in plant genetic engineering today. Currently, only retrieved research about barley sbe Ⅱ b gene promoter, start the substudy reported no published corn sbe Ⅱ b. The purpose of this paper is to tissue-specific promoter sbe Ⅱ b to replace the CaMV 35S constitutive promoter, to build a new plant expression vector, to start corn starch branching enzyme gene-specific expression in plants, improve amylose corn starch content and the basis for changing the quality of corn starch. This study were ordinary corn and yellow, white sticky corn of genomic DNA as template, known corn sbe II b gene promoter sequence (GenBank accession: AF072725), designed one pair of primers material, the LA-PCR amplification of the sbe Ⅱ b gene start sub-part of the fragment. Yellow sticky corn Kat waxy No. 1 \Blue-white screening recombinant plasmid E. coli transformed 17 leukoplakia 32 locus coeruleus. The plasmid extract positive colonies PCR and restriction enzyme digestion, consistent with the results obtained fragment size and purpose fragments. The identified strains be sent to the Takara sequencing and to utilize DNAMAN biological software for analysis. The results showed that the cloned insert fragment length of 934bp, the same size and is expected to design, only 14 base mutations, 98.52% homology. Found the core sequence unique to higher plants promoter TATA-box (TATAATA) and CAAT-box sequence. GC box basic regulatory elements as the eukaryotic promoter is as much as six copies. In addition, the sequence of many seed-specific expression of specific regulatory elements required, such as at-865bp endosperm expression of cis-acting elements of GCN4 (TGAGTTTC),;-113bp at the role of seed-specific factor RY-element (CATGCA) ;

Cloning and Functional Analysis of Maize Starch Branching Enzyme Gene SBEⅡb Promoter

Abstract

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