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Endogenous Gene Expression in Plants
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In plants, certain genes are responsible for certain characters. Plants processes are often modified so that they can be made of use commercially. In these cases, it is often seen that upon eliminating the gene expression of some of the endogenous genes, the required traits was improved. Several mechanisms responsible for these are:
(i) Anti sense gene approach: A DNA consists of two strands complementary to each other. The strand which run from 5’ to 3’ is known as sense strand and the genes in the same is known as sense gene. Similarly the complementary strand to the sense strand is known as anti sense strand and runs from 3’to 5’ end. The genes in the latter are known as antisense genes. The mRNA produced by transcribing the anti sense strand are therefore similar to the sense strand.

In order to produce an antisense gene, the protein encoding region of the gene is inverted so that it becomes oriented in 3’ -5’ direction. The RNA produced upon transcription of the same is therefore similar to the original anti sense strand and is known as anti sense RNA. In a nucleus which consists of normal endogenous gene as well as altered anti sense gene, the transcription of both yields mRNA molecule which are in turn complementary to each other. Thus, the two RNA molecule pairs with each other resulting in the formation of a double stranded RNA molecule. The double stranded RNA molecule so produced is not available for translation and the protein formation is altered. It may also be attacked by RNAases which are double stranded specific and hence attacks only the altered mRNA molecules. Another effect can be methylation of the promoter as well as coding regions of such genes resulting in the silencing of endogenous genes.

Production of Flavr Savr tomato:
The technique of anti sense gene approach has found an important application in improving the quality of tomatoes. In tomatoes, an enzyme called polygalacturonase is produced which is responsible for degrading pectin. The pectin is major component of cell wall and the degradation of the same results in ripening and deterioration of the fruit. In order to improve the shelf life of tomato, anti sense gene therapy is utilized wherein anti sense gene construct of the enzyme polygalacturonase is produced in such modified tomato so that the over ripening of the fruit is seen to reduce rapidly and thus the general quality of the tomato is increased.

(ii) Co suppression of endogenous genes
It is found that, over expression of sense RNA in some plants was responsible for drastically reducing the expression of the corresponding genes. This process of suppression of gene expression is known as co suppression.

This co suppression can be achieved by introduction of homologous sense construct into the nucleus so that it produces target mRNA along with the endogenous mRNA leading to an over expression and consecutive suppression of the gene expression.

It is proposed that, accumulation of RNA transcripts activates production of aberrant RNA from endogenous gene. These aberrant RNA produced activates RNA dependent RNA polymerase which inturn transcribes RNA leading to production of antisense RNA. This will form duplex of RNA molecule resulting in the degradation and reduction of expression of the corresponding RNA. An important example is that of ethylene in fruit ripening. Ethylene is a phytohormone actively involved in ripening of fruits. It is produced from methionine by an enzyme called as ACC synthase into aminocyclocarboxylic acid (ACC). This, when acted upon by ACC oxidase forms ethylene. The production of ethylene can be controlled by integration of antigenic constructs of any of the two enzymes ACC synthase or oxidase or co suppression of any of the enzymes. Thus a reduced ethylene production results in delayed petal senescence as well as delayed fruit over ripening.

(iii) RNA –mediated interference (RNA i):
This technique involves silencing of homologous gene expression activated by presence of double stranded RNA molecule. The double stranded molecule gets degraded by specific degradation mechanism. The long double stranded RNA molecule is cleaved by an enzyme called as dicer. This breaks up long double stranded DNA molecules into small molecules called as small interfering RNA (si RNA). These bind together to form a complex known as RNA-induced silencing complex. The antisense RNA molecule so generated pair with the target RNA molecules and hydrolyse it by initiating an endonuclease activity thus resulting in controlling the respective gene expression.
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