Publication Date

8-2006

Advisor(s) - Committee Chair

Shivendra Sahi, Sigrid Jacobshagen, Linda Brown

Comments

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Degree Program

Department of Biology

Degree Type

Master of Science

Abstract

This study describes the results of isolation and characterization of genes in response to heavy metals in Sesbania drummondii. Plants were grown in aqueous solution supplemented with lead and mercury to elucidate the molecular response. The suppression subtractive hybridization (SSH) method was used to capture and enrich rare transcripts expressed in Sebasnia drummondii as a consequence of exposure to lead as well as mercury.

Two-weeks old Sesbania drummondii seedlings were grown hydroponically in half strength Hoagland’s medium supplemented with 500 mg/L lead nitrate and 50 mg/L mercuric chloride separately. It was observed that seedlings of S. drummondii were able to tolerate up to 500 mg/L nitrate and 50 mg/L mercuric chloride. The only visible symptoms of lead (Pb) and mercury (Hg) toxicity were stunted growth of seedlings. Inhibition in roots and shoots elongation was shown in both Pb and Hg stressed plants in comparison to unstressed plants. For control, seedlings were grown in half strength Hoagland’s medium in the absence of lead nitrate and mercuric chloride. Following the 12 days of metal treatment, seedlings were harvested and RNA was isolated from both control and treated plants. Further mRNA was purified from RNA. All mRNA populations were then converted into cDNAs followed by Rsa I digestion and adaptor ligation. Both control and treated adaptor-ligated cDNAs were used for two rounds of hybridization. PCR amplification was performed to enrich the differentially regulated gene sequences. The primary PCR was performed with adaptor specific primers while the secondary PCR used nested PCR primers to further reduce any background PCR products. The SSH library enriched for differentially expressed cDNAs was constructed by cloning the subtracted secondary PCR products into the T/A cloning vector pCRscript.

After the SSH library was obtained, differential screening of the subtracted library using revers Northern analysis was performed before undertaking Northern blot analysis to eliminate false positives. Screening the library by reverse Northern analysis revealed that 20-25% of clones selected from the library were differentially regulated in lead treated plants and suggested higher levels of expression under oxidative stress rather than toxic metal-stress while 35-40% of clones were differentially regulated in mercury treated plants. Northern blot was performed to confirm that individual clones indeed represent differentially expressed genes in both lead and mercury treatments. It is anticipated that the information obtained will be helpful for further exploration of the potential for manipulation of the plants tolerant of toxic metal.

Disciplines

Biology | Botany | Life Sciences | Plant Biology | Plant Sciences

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