Connecticut State University

2001 University Research Grant Proposal Cover Sheet

Name: Ross E. Koning

University: Eastern CSU       Department: Biology

E-Mail:       Phone: 860-465-5327

Campus Address: Goddard Hall, Willimantic, CT 06226

Is this a joint proposal submitted with (an)other faculty member(s)? No

If YES, who are the other proposers? NA

Project Title:PCR Verification of Full and Partial Antisense-PHAN Transgenic Plants in Nicotiana sylvestris.

Amount Requested: $4000

Abstract (limit to 100 words):

The proposed research will develop a PCR technique to verify if putative transformed plants are indeed transgenic without the lengthy, expensive, and radioactive aspects of traditional Southern blots. Verified and putative transgenic Nicotiana sylvestris plants from previous CSU-AAUP projects are available to test and to apply the new method. The lack of introns and presence of specific restriction endonuclease recognition sites in the Nicotiana tabacum PHAN cDNA insert should permit analysis of PCR products to distinguish the insert from the genomic homolog. The new method may also give further information about the genomic homolog of Nicotiana sylvestris.


Pertinent Historical Background. As a result of work completed under my 1998 CSU-AAUP grant, my 1999 USDA-CSREES-NRICGP-Career Enhancement Grant used for my sabbatic leave research, and my 2000 CSU-AAUP grant, I have produced transformed plants of Nicotiana sylvestris. Some of these are full-length antisense PHAN true transgenic plants, verified by traditional Southern blot analysis at the CT Agricultural Experiment Station during my sabbatical. Plants produced more recently at ECSU are untested putative transgenic plants that should contain one of two specific fragments of the PHAN gene. The next natural step for my research program is to verify that these putative transgenic plants indeed contain the inserted DNA. Under current NRC licensing, this cannot be done at ECSU using 32P-labelling for probes found in traditional Southern analysis.

The Verification is Critical. The verified full-length antisense PHAN transgenics show a common phenotype: ectopic blades, reduction of cauline blades, and exaggerated auricle development. It is hoped that this phenotype is due to silencing of the genomic homolog of PHAN by the antisense insert. However, the base-sequence of PHAN reveals shared conservative domains (myb regions) that are found in a family of transcription-regulating genes. The phenotype observed could be the result of silencing one or more of the other members of the myb-gene family. The unverified putative transgenics produced through my 2000 CSU-AAUP grant should have an antisense insert of either the 5' untranscribed region (5' UTR) or the non-myb coding region plus 3' UTR. To date, the putative transgenics show no phenotype like that of the full-length antisense PHAN transgenics. There are two possible reasons for this failure.

It is possible that the partial inserts which should be specific to PHAN are not silencing the non-PHAN myb genes, and that silencing these other myb genes was responsible for the observed phenotype in the full-length antisense transgenics. This possible outcome would totally revise current hypotheses on the function of the PHAN gene. This reason for the failure would be indicated if our no-PHAN-phenotype putative transgenics are indeed found to contain the antisense insert.

A second possible reason for the lack of phenotype could be that all of the putative transgenics I have recovered are false-positives. A false positive plant is somehow able to escape the antibiotic screening procedure. The inserts include a gentamicin resistance gene, but a false positive is able to grow in the presence of gentamicin. Generally false-positives are low-frequency outcomes, but the frequency of unexpected results can be very high in tissue cultures. This reason for the failure would be indicated if our no-PHAN phenotype putative transgenics are indeed found NOT to contain the antisense insert.

Before this program goes any further it is essential to distinguish whether the putative transgenics are false positives or true transgenics. If the plants are false positives, then further and more-stringent screening must be initiated. If the plants are true transgenics, then the interaction with other genes in the myb family should be the new avenue for research. I am at a critical dichotomy, then, where verification of the putative transgenics is critical. However, ECSU is not licensed to carry out the necessary Southern analyses.

A Novel Approach. The PHAN genes and gene fragments used for the transformations were produced by the polymerase chain reaction (PCR). To prepare these inserts, the PHAN base sequence of the original cDNA from Nicotiana tabacum was determined. From this sequence, forward and reverse primers were designed, synthesized commercially at Yale, and used to initiate the PCR. These primers, already designed and synthesized, now provide a way around the problem of verifying the transgenic plants at ECSU.

Total genomic DNA will be extracted from a leaf disc of both verified and putative transgenic plants using a one-day, one-tube process developed at Sigma. This method requires no grinding of tissue or toxic phenol-chloroform steps. The DNA extract will be added to a PCR reaction mix with the previously-synthesized PHAN primers. These primers will settle down on both the insert (if present) and also any sufficiently-conserved regions of the genomic homolog. The PCR will certainly amplify any inserted fragments, and may also amplify the genomic homolog in the tube by many orders of magnitude.

When these PCR products are separated by agarose gel electrophoresis, the amplified inserts and any genomic homologs will probably have different size because of introns likely found in the genomic PCR product but which are not found in the cDNA-derived insert PCR products. These PCR products can be further distinguished by cleaving them at restriction endonuclease recognition sites that are known to be in the PCR products from the Nicotiana tabacum cDNA inserts but are less than likely to also be in the PCR products from the Nicotiana sylvestris genomic homolog. In the full-length cDNA PHAN inserts there are NcoI and XbaI restriction sites; the two partial-gene cDNA inserts that I have produced and used in the transformation attempts each contain one of these sites.

Advantages of the new method. The PCR method is a two-day process as opposed to a two-week Southern analysis. The PCR method is considerably less-expensive, uses minimal reagents, and produces almost no toxic waste compared to Southern analysis. The PCR method uses no radioisotope nor its inherent handling, processing, monitoring, and waste-storage requirements. Many PCR analyses can be accomplished side-by-side quite rapidly. The PCR method is amenable to safe student-centered research at ECSU.

Possible pitfalls of the new method. There is a remote chance that the PCR products from both the inserts and the genomic homolog are identical. This remote chance requires the absence of an intron from the genomic homolog, an unlikely occurrence. It also requires that the base sequences through the two Nicotiana tabacum restriction sites are identical in the Nicotiana sylvestris genomic homolog, an unlikely occurrence. It further requires that the PCR primers settle down properly on the Nicotiana sylvestris genomic homolog, a further unlikely occurrence. Thus it seems highly unlikely that this method will fail to distinguish a known transgenic from a known untransformed control. However unlikely a failure may be, the proposed research will be able to demonstrate either the success or failure of the new method because of the known positive and negative controls.

Possible additional benefit of the new method. Should a PCR product be produced from the genomic homolog using the full-length primers, this method will have captured the genomic homolog of PHAN in Nicotiana sylvestris. That remarkable outcome would provide a tremendous boost to my research program. It would achieve that capture without creating a library and screening it with radioisotope probes and hoping for a full-length match. Even better, it would capture the gene with any introns it may have intact. This would provide insight into pre-translational processing in the expression of PHAN.

Publication and Communication. It is hoped that this additional work along with my USDA-funded work at the CT Ag Expt Station will result in a publication on which I will be an author. My host and I are preparing a presentation for the 2001 national meeting of the American Society of Plant Physiologists. This will be submitted in the next month and the abstract for this presentation will appear on my website (http:/ shortly. It will also be published in the ASPP journal, Plant Physiology.

The findings of the research proposed here will be presented at the 2002 meeting of the same society, and will be a critical part of the paper mentioned above.

Concluding Remarks. In summary, the proposed research meets two of the priorities of the University Grants Committee. The project continues and brings to publication a newly established pathway of research, and the project should improve my chances of additional external funding from the USDA.


Plant Tissue Culture Supplies
Culture Dishes, Media$ 350
Soil, Pots, Markers50

Recombinant DNA Techniques
DNA extraction reagents, PCR reagents,
Restriction enzymes and buffers,
Agarose Gel electrophoresis reagents
Supplies, disposable plasticware1000

Publication Costs
Graphic Arts Films, Processing, Mounts100
Total Amount REQUESTED$ 4,000

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