Binary vector construction method for improving corn amylose content by utilizing genetic engineering

Abstract

The invention discloses a binary vector construction method for improving corn amylose content by utilizing genetic engineering. The binary vector construction method comprises: designing a primer to amplify the ninth extron and intron part sequence (315bp) and the ninth extron antisense sequence (155bp), and also to amplify sbe2b promoter, simultaneously linking an integrated DNA fragment containing sense and antisense extrons and introns and the promoter into multiple cloning sites (MCS) of a pCAMBIA3301 carrier; and then replacing 35S and GUS fragments of the pCAMBIA3301 carrier containing sbe2b RNAi three fragments in MCS by a ss1 promoter fragment and a ss1 gene cDNA fragment, so that the expression carrier contains both antisense sbe2b cDNA fragment controlled by sbe2b promoter and ss1 full-length cDNA fragment controlled by ss1 promoter. The sbe2b RNAi fragment contained in the expression carrier provided by the invention is capable of reducing activity of SBEIIb enzyme and reducing branch number when starch is synthesized, and thus the relative content of amylose is improved; and also the ss1 gene expression fragment contained in the carrier is capable of improving the activity of corn endosperm SSI enzyme, and thus the total starch content is improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a method for constructing a binary carrier for improving corn amylose content through genetic engineering. Background technique [0002] To carry out the genetic improvement mediated by the target gene, the corresponding expression vector should be constructed first and then the genetic transformation should be carried out. Fromm et al. (1986) first carried out the genetic transformation work of maize, using electric shock method to induce maize protoplasts to absorb the plasmid DNA carrying npt II (neomycin phosphotransferase II, chloramphenicol acetyltransferase gene). expression in maize cells, and further transfer the npt II gene into maize protoplasts to obtain stably transformed resistant callus. Rhodes et al. (1988) transferred the npt II enzyme gene into maize protoplasts by electric shock method, successfully established a maize protoplast transformation sys...

AI Technical Summary

Problems solved by technology

The expression of starch branching enzyme gene was reduced by RNA interference or antisense RNA technology, resulting in an increase in the relative content of amylose (amylose ratio) in the grain, but due to the change in the conformation of the functional complex, the activity of other related proteins At the same time, due to RNAi technology, the sbe2b gene cannot be expressed normally, which reduces the non-reducing end of dextran in the starch synthesis process, and also causes the decline in the activity of key enzymes such as SSI, resulting in insufficient sugar chain elongation ends, causing A reduction in the rate of overall starch synthesis, reducing the total starch content

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