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RNAi-DIRECTED SILENCING OF POTENT STRESS TOLERANT GENE(S)
AND ITS EFFECT ON STRESS TOLERANCE IN PLANTS
Indrani Baruah
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PREAMBLE
RNA interference (RNAi)
RNA interference mechanism
Effects of Stress in plants
Glycine Betaine (GB)
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RNA interference(RNAi)
A general endogenous mechanism in many organisms including plant that is used to
silence the expression of the genes that control various events in the cell
In 1990, In an attempt to enhance the flower color in petunias, researchers introduced
additional copies of a gene encoding chalcone synthase
Over expressed gene was expected to result in darker flowers, but instead produced less
pigmented, fully or partially white flowers
Andrew Fire and Craig C. Mello shared 2006 Nobel Award in Medicine or Physiology for
their work in RNA interference in Caenorhabditis elegans
Micro ribonucleic acid (miRNA) and small interfering RNA (siRNA) are central to RNA
interference
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Advantages:
• This natural mechanism for sequence-specific gene silencing may have important
practical application in functional genomics, therapeutic intervention, agriculture
and other areas
• RNA interference provides defence to the cell against parasitic nucleotide
sequence e.g. virus
•RNA interference can also be mediated artificially by inserting a dsRNA into the
cell
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Effect of stress on plants
Biotic stress: is imposed by other organism
Abiotic stress: arises from an excess or deficit of the physical or
chemical environment like drought, water logging, excess low
temperature or high temperature and excess soil salinity
Abiotic factors provide the major limitation to crop production worldwide
Affects plant growth and development
Therefore there is need of stress resistance in plants
Stress resistance or sensitivity depends on the genotype, species and
developmental age of the plant
Glycine Betaine (GB): as an osmoprotectant
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N,N,N- trimethylglycine (GB) is an amphoteric quarternary amine
Provides osmotic adjustment to the plant under stressed condition
GB is synthesized in plants through a two stepped process
Choline monooxygenase (CMO) and Betaine aldehyde
dehydrogenase (BADH) enzyme catalyze the first and second step
respectively
Rice (Oryza sativa) has two homologs of BADH gene.viz-BADH1
and BADH2
Figure: Schematic representation of glycine betaine (GB) biosynthesis
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CMO- Choline Monooxygenase enzymeBADH- Betaine Aldehyde Dehydrogenase enzyme
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OBJECTIVES
To demonstrate a pivotal role of OsBADH1 in stress tolerance using
RNA interference technology (RNAi) without affecting GB biosynthesis
capacity
Stress tolerance analysis of japonica transgenic lines downregulating
OsBADH1 by giving NaCl, drought and cold stress treatments
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METHODOLOGY
Full length cloning of OsBADH1 cDNA
Construction of pHB-OsBADH1-RNAi expression plasmid
Analysis of gene expression of OsBADH1 by reverse transcription-
quantitative real time PCR (RT-q PCR)
Identification of japonica transgenic plants
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METHODOLOGY
Analysis of BADH activity
Assay of tolerance to abiotic stress
Assay of MDA and H2O2
• using leaf tissues
Determination of glycine betaine (GB)
Fig. 1 Expression pattern of OsBADH1 in various tissues.•Expression abundance in root, stem, leaf, internodes, immature flower, seedling leaf and seedling root of a japonica rice variety Nippobare is shown
RESULT AND DISCUSSION
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Figure 2: Semi-quantitative and real-time qPCR analyses of (A)OSBADH1, (B) OSBADH2 and (C) Expression levels of OsBADH1 and OsBADH2 of transene positive and negative were indicated comparing with the internal control Actin 13
Figure3: Shows the BADH activity in transgene positive and transgene negative plant leaves by using (D) betaine aldehyde and (E) acetaldehyde respectively
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Figure4: Abiotic stress tolerance of OsBADH1-RNAi transgenic rice.(A)0 mM NaCl, (B) 50 mM NaCl, (C) 100 mMNaCl, (D) 200 mM mannitol, (E) 300 mM mannitol and (F) cold (4 °C)
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Figure 5: Measurements of (A) root length (B) shoot length (C) seedlingweight (c) in transgene-negative plants (WT) and transgene-positive plants (B1-a, B1-c,B1-e)• The asterisk (*) above each column indicates there was a significant difference (p< 0.05) between transgene- positive and transgene – negative plants
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Figure 6: The phenotype of OsBADH1-RNAi transgenicRice (B1-a, B1-c, B1-e) and wild type (WT)
(A)before 100 mM NaCl treatment
(B) under 100 mM NaCl treatment
(C) Primary plants in field 17
Figure 7 : Detection of salt/ drought/ cold stress-induced H2O2 production by Diaminobenzidiene (DAB) staining in leaves of transgene negative (WT) and transgene positive plants (B1-a,B1-c,B1-e)
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Figure8: Malondialdehyde (MDA) contents in the leaves of transgene negative (WT) and transgene positive plants (B1-a,B1-c,B1-e)• The asterisk(*) above each column indicates a significant difference (P < 0.05) between WT and BI-a, B1-c,B1-e
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Figure 9: The (A) unhusked and (B) husked grains of transgene-negative (WT) and transgene positive plants (B1-a,B1-C,B1-e)
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Highest expression of OsBADH1 gene in roots and leaves, least expression
level in stem (figure1)
reduced abiotic stress tolerance and crop productivity in OsBADH1
Downregulated plants (figure 4,5,6 and 9)
Glycine betaine (GB) content was not affected (figure 3)
The downregulation of OsBADH1 altered the ROS scavenging capacity of the
transgenic plant without changing GB content (figure 7 and 8)
Therefore OsBADH1 has a pivotal role in stress tolerance without altering
GB (Glycine betaine) biosynthesis capacity
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CONCLUSION
OsBADH1 gene confers stress tolerance and also increase crop
productivity
Downregulation of OsBADH1 gene significantly alters scavanging of
ROS(reactive oxygen species) like H2O2 and lipid perozidation product
like MDA
Therefore, it can be concluded that OsBADH1 gene is very
essential for stress tolerance and crop productivity of rice plant
REFERENCES
1. Hasthanasombut S, Supaibulwatana K (2011) Genetic manipulation
of Japonica rice using the OsBADH1 gene from Indica rice
to improve salinity tolerance. Plant Cell Tissue and organ cult
104:79–89.
2. Hasthanasombut S, Ntui V, Supaibulwatana K (2010) Expression of
Indica rice OsBADH1 gene under salinity stress in transgenic
tobacco. Plant Biotechnol Rep 4:75–83.
3. Huang W, Ma X, Wang Q, Gao YF, Xue Y, Niu XL, Yu GY, Liu YS
(2008) Significant improvement of stress tolerance in tobacco
plants by overexpressing a stress-responsive aldehyde dehydrogenase
gene from maize (Zea mays). Plant Mol Biol 68:451–463.
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4. Luo D, Niu X, Yu J, Yan J, Gou X, Lu BR, Liu YS (2012) Rice choline
monooxygenase (OsCMO) protein functions in enhancing
glycine betaine biosynthesis in transgenic tobacco but does not
accumulate in rice (Oryza sativa L. ssp. japonica). Plant Cell Rep
31:1625–1635.
5. Hasthanasombut S, Supaibulwatana K (2011) Genetic manipulation
of Japonica rice using the OsBADH1 gene from Indica rice
to improve salinity tolerance. Plant Cell Tissue and organ cult
104:79–89.
6. Hasthanasombut S, Ntui V, Supaibulwatana K (2010) Expression of
Indica rice OsBADH1 gene under salinity stress in transgenic
tobacco. Plant Biotechnol Rep 4:75–83.
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Thank you