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