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Home >> Expression of Cloned Genes - Introduction >>Antisense RNA
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Antisense RNA - This technique is based on blocking the informational flow from DNA via RNA to protein by the introduction of an RNA sequence complementary to the sequence of the target, mRNA.

Thus an RNA duplex is formed between mRNA and antisense RNA. This duplex RNA is either rapidly degraded or the mRNA is impaired in nuclear processing or it is blocked for translation.

Studies done to date indicate that the expression of both sense and antisense a amylase transcripts is developmentally regulated and both RNAs are present in about an equal amount in barley aleurone tissue.

The importance of antisense RNA, which is complementary to mRNA, lies in its ability to neutralize the function of a specific gene sequence. Antisense RNA exerts its effect in two ways:

(1) it forms a stable base paired structure with the mRNA and thus prevents translation and

(2) because many cells contain ribonucleases which attack double stranded RNA, the antisense RNA-mRNA base paired structure is degraded by endogenous ribonucleases.

Antisense RNA for a specific gene sequence can be generated by reversing the coding sequence under the control of a promoter in the normal orientation.

The antisense RNA approach has been successfully used for reducing polygalacturonase, which degrades pectic polymers.

Higher concentrations of this enzyme cause rapid fruit softening and adversely affect transportation and shelf life of tomatoes.

A cDNA clone containing the coding sequences for polygalacturonase was spliced in reverse orientation between a cauliflower mosaic virus 35-S promoter and Nos termination signals.

This gene construct was introduced into tomato through Agrobacterium mediated transformation.

The resultant transgenic plants had variable levels of expression, with some exhibiting as much as 90% reduction in the level of polygalacturonase enzyme.

What has been achieved so far is path breaking but unfortunately does not prevent fruit softening, either because the remaining 10% enzyme level suffices to degrade pectin to soften cell wall or because other enzymes are also involved in fruit softening.
 
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