Gene Subtraction

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THESE FILE CLARIFY YOUR DOUBT ABOUT THE GENE SUBSTRACTION METHODS....

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Gene Subtraction

  1. 1. GENE SUBTRACTION PRESENTED BY HARIS.P & ARUN C.S MSC.BIOTECHNOLOGY MAC & AL-AZHAR ERNAKULAM
  2. 2. INTRODUCTION <ul><li>GENE CLONING ENABLES DIRECTED CHANGES IN THE GENOTYPE OF PLANTS. </li></ul><ul><li>PROJECTS ARE CARRIED OUT BY EXPLOITING THE POTENTIAL OF GENE ADDITION AND GENE SUBTRACTION. </li></ul><ul><li>GENE ADDITION,CLONING IS USED TO ALTER PLANT CHARACTERISTICS BY PROVIDING MORE NEW GENES. </li></ul><ul><li>GENE SUBTRACTION,INACTIVATION OF EXISTING GENES . </li></ul>
  3. 3. GENE SUBTRACTION <ul><li>NOT THE REMOVAL OF GENE BUT MERELY ITS INACTIVATION </li></ul><ul><li>GENE SUBTRACTION IS DONE BY ANTISENSE TECHNIQUE </li></ul><ul><li>RNA PRODUCED BY THIS TECH IS ANTISENSE RNA(asRNA) </li></ul>
  4. 4. ANTISENSE TECHNOLOGY <ul><li>GENE TO BE CLONED IS LIGATED INTO VECTOR IN REVERSE ORIENTATION. </li></ul><ul><li>WHEN THIS CLONED GENE IS TRANSCRIBED,THE RNA SYNTHESISED IS THE REVERSE COMPLEMENT OF THE mRNA FROM THE NORMAL SEQUENCE </li></ul><ul><li>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 </li></ul><ul><li>THIS BLOCK THE EXPRESSION BY DEGRADING DOUBLE STRANDED RNA BY CELLULAR RIBONUCLEASES </li></ul><ul><li>PREVENTS RIBOSOMAL ATTACHMENT TO SENSE STRAND. </li></ul>
  5. 5. ANTISENSE RNA <ul><li>Gene in the correct orientation </li></ul><ul><li>promoter transcription </li></ul><ul><li>mRNA </li></ul><ul><li>Gene in the reverse orientation </li></ul><ul><li>promoter </li></ul><ul><li>transcription </li></ul><ul><li>Antisense RNA </li></ul>
  6. 6. INHIBITION OF GENE EXPRESSION BY ANTISENSE RNA <ul><li>mRNA </li></ul><ul><li>antisense RNA </li></ul><ul><li>I I I I I I I I I I I I I I I I mRNA </li></ul><ul><li>I I I I I I I I I I I I I I I I as RNA </li></ul><ul><li>degraded by ribosomes </li></ul><ul><li>ribonucleases cannot attach </li></ul>
  7. 7. APPLICATION <ul><li>SLOW RIPENING TOMATOS </li></ul><ul><li>INACTIVATION OF POLYGALACTURONASE GENE </li></ul><ul><li>POLYGALACTURONASE ENZYME BREAKS DOWN THE POLYGALUCTURONICACID COMPONENT OF CELLWALL. </li></ul><ul><li>PARTIAL INACTIVATION INCREASES THE TIME BETWEEN FLAVOUR DEVELOPMENT AND SPOILAGE. </li></ul><ul><li>METHOD </li></ul><ul><li>OBTAIN 730bp RESTRICTION FRAGMENT FROM NORMAL POLYGALACTURONASE GENE. </li></ul><ul><li>ORIENTATION OF FRAGMENT WAS REVERSED. </li></ul><ul><li>PROMOTER LIGATED TO THE START OF SEQUENCE </li></ul>
  8. 8. <ul><li>POLYADENYLATION SIGNAL IS ATTACHED TO THE END. </li></ul><ul><li>INSERTED THE CONSTRUCTION INTO A PLASMID VECTOR. </li></ul><ul><li>INSIDE THE PLANT IT SYNTHESIS ANTISENCE RNA COMPLEMENTARY TO POLYGALACTURONACE mRNA . </li></ul><ul><li>THIS PREVENTS TRANSLATION OF TARGET mRNA. </li></ul>
  9. 9. CONSTRUCTION OF ANTISENSE POLYGALACTURONASE. <ul><li>polygalacturonase gene </li></ul><ul><li>R R </li></ul><ul><li>restriction ,ligation to control sequences in the reverse orientation </li></ul><ul><li>promoter antisense polygalacturo- polyadenylation signal </li></ul><ul><li>nase gene </li></ul>
  10. 10. <ul><li>2. INACTIVATION OF ETHYLENE SYNTHESIS </li></ul><ul><li>ETHYLENE ACTS AS A HORMONE INVOLVING IN THE LATER STAGE OF RIPENING. </li></ul><ul><li>METHOD </li></ul><ul><li>S-ADENOSYLMETHIONINE </li></ul><ul><li>ACC SYNTHASE </li></ul><ul><li>1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID (ACC) </li></ul><ul><li>ETHYLENE </li></ul>
  11. 11. <ul><li>INACTIVATION OF ACC SYNTHASE ENZYME LEADS TO THE DECREASE IN PRODUCTION OF ETHYLENE. </li></ul>
  12. 12. OTHER EXAMPLES <ul><li>TARGET GENE </li></ul><ul><li>1. POLYGALACTURONASE </li></ul><ul><li>2. OXIDASE </li></ul><ul><li>3. STARCH SYNTHASE </li></ul><ul><li>MODIFIED CHARACTERISTICS </li></ul><ul><li>DELAY OF FRUIT SPOILAGE IN TOMATO </li></ul><ul><li>PREVENTION OF DISCOLORATION IN FRUITS AND VEGETABLES. </li></ul><ul><li>REDUCTION OF STARCH CONTENT IN VEGETABLES. </li></ul>

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