RNA TECHNOLOGY

DAYANANDA SAGAR COLLEGE OF ENGINEERING

DEPARTMENT OF BIOTECHNOLOGY

Seminar Presentation

Antisense RNA TechnologyS. Kiruthika

(14163PGDPGT0006)1st Semester, BTFS, DSI, Bengaluru-560 078

Under the Guidance of

Ms. Supreetha. KDepartment of Biotechnology

Dayananda Sagar College of Engineering

S. M. Hills, Kumaraswamy Layout, Bengaluru -560 078

INTRODUCTION

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When expression of a transgene is desired, the arrangement of

promoter and sequence to be expressed mimics the usual

arrangement of the gene, so that the promoter directs

expression of sense RNA that is in the correct orientation to

be translated, The simplest RNAi—related technology is to

produce so called antisense RNA.

In vectors, designed to produce antisense RNA, the sequence

to be expressed(which can be all or part of the coding

sequence of an endogenous gene) is placed in the reverse

orientation with respect to the promoter. The promoter

therefore directs the production of antisense RNA, which is

complementary to the endogenous mRNA.

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The complementarity between antisense RNA and mRNA results in the

production of dsRNA, RNAi can be deliberately induced to silence the

expression of specific genes in transgenic plants.

BASIS OF THE TECHNOLOGY:

A sense strand is a 5’ to 3’ mRNA molecule or DNA molecule. The

complementary strand to this strand is called an antisense. The antisense

technology works by binding the antisense strand with the targeted sense

strand through hydrogen bond. The double stranded molecule will be

recognized as the foreign molecule and is susceptible to degradation.

Though, as the DNA is double helix, it can be used in antisense

technology; resulting in the formation of triplex.

Antisense strand can be:

1. DNA: A DNA antisense strand is of approximately 17 bases long.

2. RNA (either catalytic or non-catalytic): 13 bases long RNA antisense

strand functions properly.

ANTISENSE RNA TECHNOLOGY FOR IMPROVING VASE LIFE OF

CUT FLOWERS

ETHYLENE BIOSYNTHESIS AND ITS REGULATION

Methionine

SAM Synthetase ATP

PPi + Pi

S-Adenosylmethionine (SAM)

ACC Synthase

1-Amino-cyclopropane-1-carboxylic acid (ACC)

ACC Oxidase O2

Ethylene

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The vase life of cut flowers can be extended by adding silver salts to the water, which blocks the response to ethylene.

Antisense ethylene technology is applicable to other systems triggered by ethylene like senescence of picked flowers.

The phytohormone ethylene is essential for the senescence of fruits and flowers. In the biosynthesis of ethylene in plants, the conversion of SAM into ACC and the conversion of ACC into ethylene is catalysed by ACC synthase and ACC oxidase respectively.

The introduction of antisense ACC synthase or ACC oxidase would have a similar effect of extending vase-life of cut flowers by suppressing ethylene synthesis.


CASE STUDY-1Agrobacterium-Mediated Transformation of Dendrobium secundum (Bl.) LindI with

Antisense ACC oxidase

Prontip et al., (2007), Asian Journal of Plant Sciences 6 (7): 1065-1071.

Objectives:

The objectives of this study were to improve the efficiency of regeneration and

transformation of Dendrobium secundum with antisense ACC oxidase gene using

Agrobacterium tumefaciens for prolonging the longevity and vase life of orchid flowers.

Design of study:

1. Agrobacterium mediated transformation: Agrobacterium tumefaciens strain LBA4404

(pCAMBIA 1305.1) containing Antisense ACC oxidase, β-glucuronidase (GUS), and

hygromycin resistance (hptII) gene with CaMV 35S promoter. Protocorms were used

as the explant for transformation.

2. Assay for β-glucuronidase (GUS) activity.

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Results:





RESULTS ON EFFECT OF ANTIBIOTICS

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Conclusion:

The protocorms with high proliferation efficiency capacity were cultured on

the modified VW medium with 0.5 mg/L NAA.

The efficiency of transformation was 40 min of co-cultivation of protocorms

with Agrobacterium tumefaciens that has been induced with 200 µM

acetosyringone.

Effect of antibiotics on Protocorms:

1. Hygromycin at 25 mg/L was effective for the transformant selection

2. Maximum concentration of cefotaxime that protocorms could tolerate

was 500 mg/L

Hygromycin resistant protocorms showed histochemical blue staining due to

GUS activity was 60%.

PCR analysis of fragment sizes 195, 180 and 843 bp for the 35S, NOS and

Antisense ACC oxidase respectively confirmed the successful transformation.

This methodology can be proposed as one of the efficient method used to

transform novel genes into the other orchid species.

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CASE STUDY-2Antisense ACC oxidase RNA delays Carnation petals Senescence.

Keith et al., (1995), Hortscience, Vol 30(5): 970-972.

Objective:

The transgenic carnation plants producing antisense ACC oxidase RNA were generated

to create a carnation line with low ethylene synthesis and delayed petal senescence, and to

more clearly define the role of ethylene in the senescence of carnation petals.

Design of study:

1. Agrobacterium mediated transformation for the production of transgenic carnation

plant: T-DNA based expression vector (pCGP407) has carnation ACO cDNA fragment

contained within pCGP363 was inserted in reverse orientation into a binary vector

pCGP293 between MAC promoter and mas 3’ end (Agrobacterium mannopine

synthase gene). MAC is a strong constitutive promoter and is able to direct the

expression of reporter gene (uidA).

2. Southern Analysis.

3. Northern Analysis

4. Gas chromatography

Results:

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Conclusion:

• The antisense ACO gene has succeeded in suppressing the level of ACO

mRNA and hence all but eliminated ethylene production and extended

carnation flower vase life.

• Expression of ACS and ACO genes in petals must be dependent on the

presence of ethylene.

• The generation of transgenic carnation plants in which the ability to

synthesis ethylene has been reduced or eliminated by use of antisense

technique reflects similar findings in tomato and reinforces the concept

of improving properties, such as postharvest qualities, by genetic

engineering rather than by classical long term breeding or by

application of chemicals.

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Page No: 18

Conclusion

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Antisense technology was successfully used to reduce or eliminate the

softening and ripening process in fruits and vegetables which results in the

improvement of the shelf-life.

Examples:

o Calgene’s Flavrsavr fresh tomato with antisense polygalacturonase,

antisense phytoene synthase, and antisense ACC oxidase gene for delayed

softening, red colouration and ripening respectively.

o Down regulation of Pectin Methyl Esterase by Antisense PME delayed the

softening process in apple (MacIntosh).

Similarly, the antisense RNA technology can be used for the down

regulation of the ethylene synthesis which would enhance the vase-life of

the cut flowers after post harvesting. This technology could be an effective

means rather than using chemicals for improving the vase-life of the cut

flowers.

Future Perspective


1. Application in Crop Improvement.

2. Artificial Antisense RNA Regulation of Gene expression.

3. Virus resistance by Antisense RNA.

4. Antisense Technology in the Treatment of Genetic

disorder.

5. Development of New methods of Drug Design.

References


1. Prontip Atichart, Sumontip Bunnag, and Piyada Theerakulpisut (2007).

Agrobacterium-Mediated Transformation of Dendrobium secundum (Bl.) LindI

with Antisense ACC oxidase. Asian Journal of Plant Sciences 6 (7): 1065-1071.

2. Keith W. Savin, Stanley C. Baudinette, Michael W. Graham, Michael Z. Michael,

Greg G. Nugent, Chin – Yi Lu, Stephen F. Chandler, and Edwina C. Cornish (1995).

Antisense ACC oxidase RNA delays Carnation petals Senescence. Hortscience, Vol

30(5): 970-972.

3. Adrian slater, Nigel W. Scott, and Mark R. Fowler : Plant Biotechnology, the

genetic manipulation of plants (Second Edition), Oxford University Press, ISBN

9780199560875.

Acknowledgement


1. Dayananda Sagar Institutions, S. M. Hills, Kumarasamy Layout,

Bangalore – 560078.

2. Dr. G. A. Ravi Shankar, Vice President, Research and Development

Life Sciences, Dayananda Sagar Institutions, S. M. Hills,

Kumarasamy Layout, Bangalore – 560078.

3. Dr. Kiran, BTFS Co-ordinator and Head of Department, Department

of Biotechnology, Dayananda Sagar College of Engineering, S. M.

Hills, Kumarasamy Layout, Bangalore – 560078.

4. Ms. Supreetha. K, Assistant Professor, Department of Biotechnology,

Dayananda Sagar College of Engineering, S. M. Hills, Kumarasamy

Layout, Bangalore – 560078.

Thank You

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