GENE SUBTRACTION

PRESENTED BYHARIS.P & ARUN C.S

MSC.BIOTECHNOLOGYMAC & AL-AZHAR

ERNAKULAM

INTRODUCTION

• GENE CLONING ENABLES DIRECTED CHANGES IN THE GENOTYPE OF PLANTS.

• PROJECTS ARE CARRIED OUT BY EXPLOITING THE POTENTIAL OF GENE ADDITION AND GENE SUBTRACTION.

• GENE ADDITION,CLONING IS USED TO ALTER PLANT CHARACTERISTICS BY PROVIDING MORE NEW GENES.

• GENE SUBTRACTION,INACTIVATION OF EXISTING GENES.

GENE SUBTRACTION

• NOT THE REMOVAL OF GENE BUT MERELY ITS INACTIVATION• GENE SUBTRACTION IS DONE BY ANTISENSE TECHNIQUE • RNA PRODUCED BY THIS TECH IS ANTISENSE RNA(asRNA)

ANTISENSE TECHNOLOGY

• GENE TO BE CLONED IS LIGATED INTO VECTOR IN REVERSE ORIENTATION.

• WHEN THIS CLONED GENE IS TRANSCRIBED,THE RNA SYNTHESISED IS THE REVERSE COMPLEMENT OF THE mRNA FROM THE NORMAL SEQUENCE

• THIS ANTISENSE RNA CAN PREVENT SYNTHESIS OF THE PRODUCT OF THE GENE WHICH IS DIRECTED AGAINST,THIS IS DUE TO THE HYBRIDIZATION BETWEEN ANTISENSE AND SENSE COPIES

• THIS BLOCK THE EXPRESSION BY DEGRADING DOUBLE STRANDED RNA BY CELLULAR RIBONUCLEASES

• PREVENTS RIBOSOMAL ATTACHMENT TO SENSE STRAND.

ANTISENSE RNA Gene in the correct orientation

promoter transcription

mRNA

Gene in the reverse orientation promoter

transcription Antisense RNA

INHIBITION OF GENE EXPRESSION BY ANTISENSE RNA

mRNA

antisense RNA

• I I I I I I I I I I I I I I I I mRNA• I I I I I I I I I I I I I I I I as RNA

• degraded by ribosomes• ribonucleases cannot attach

APPLICATION SLOW RIPENING TOMATOS

1. INACTIVATION OF POLYGALACTURONASE GENE • POLYGALACTURONASE ENZYME BREAKS DOWN THE

POLYGALUCTURONICACID COMPONENT OF CELLWALL.• PARTIAL INACTIVATION INCREASES THE TIME BETWEEN FLAVOUR

DEVELOPMENT AND SPOILAGE.

METHOD • OBTAIN 730bp RESTRICTION FRAGMENT FROM NORMAL

POLYGALACTURONASE GENE.• ORIENTATION OF FRAGMENT WAS REVERSED.• PROMOTER LIGATED TO THE START OF SEQUENCE

•

• POLYADENYLATION SIGNAL IS ATTACHED TO THE END.

• INSERTED THE CONSTRUCTION INTO A PLASMID VECTOR.

• INSIDE THE PLANT IT SYNTHESIS ANTISENCE RNA COMPLEMENTARY TO POLYGALACTURONACE mRNA .

• THIS PREVENTS TRANSLATION OF TARGET mRNA.

CONSTRUCTION OF ANTISENSE POLYGALACTURONASE.

polygalacturonase gene

R R

restriction ,ligation to control sequences in the reverse orientation

promoter antisense polygalacturo- polyadenylation signal nase gene

2. INACTIVATION OF ETHYLENE SYNTHESIS• ETHYLENE ACTS AS A HORMONE INVOLVING IN

THE LATER STAGE OF RIPENING.• METHOD S-ADENOSYLMETHIONINE ACC SYNTHASE

1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID (ACC)

ETHYLENE

• INACTIVATION OF ACC SYNTHASE ENZYME LEADS TO THE DECREASE IN PRODUCTION OF ETHYLENE.

OTHER EXAMPLES TARGET GENE

1. POLYGALACTURONASE

2. OXIDASE

3. STARCH SYNTHASE

MODIFIED CHARACTERISTICS

DELAY OF FRUIT SPOILAGE IN TOMATO

PREVENTION OF DISCOLORATION IN FRUITS AND VEGETABLES.

REDUCTION OF STARCH CONTENT IN VEGETABLES.