Connecticut State University

2000 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:Construction of Partial Antisense-PHAN Transgenic Plants in Nicotiana sylvestris.

Amount Requested: $4000

Abstract (limit to 100 words):

The proposed research involves the molecular biology of plant leaf formation. Partial antisense fragments of Nicotiana tabacum cDNA homologous to the Antirrhinum PHAN gene required for leaf blade formation will be used to construct Nicotiana sylvestris transgenic plants through Agrobacterium-mediated transformation. The resulting plants will be compared to control plants transformed with full-length antisense PHAN. The results will help dissect whether the formation of ectopic blades in full-length antisense transformed plants is due to inhibition of a specific myb gene or to silencing of a range of myb genes.


Historical Background. As a result of work completed under my 1998 CSU-AAUP grant, I was able to win the financial support of the US Department of Agriculture for my sabbatic leave research last year. Under this USDA-CSREES-NRICGP-Career Enhancement Grant during my CSU sabbatic leave at the CT Agricultural Experiment Station and in months since, I have learned many new techniques in molecular biology and important new findings outlined below. I have also equipped my research space at ECSU to continue the project proposed herein.

What is Already Known. The PHAN gene from Nicotiana tabacum was fished out of a cDNA library using an Antirrhinum phan probe and sequenced. The sequence shows over 90% homology to PHAN and phan sequences from other species. The PHAN gene consists of a 5' untranslated region (UTR), a coding region, and a 3' UTR. The coding region begins with three repeated domains that are typical of myb genes and then a unique sequence from there to the stop codon. The 5' and 3' UTR are also gene-specific. The myb genes known to date code for proteins that regulate transcription of other genes. It is expected that PHAN protein is also a transcription regulator. The mutant Antirrhinum phan plants are bladeless, so it is expected that PHAN protein regulates transcription of genes involved in leaf formation.

In my previous AAUP project and the USDA project, I cloned full-length sense and antisense PHAN into vectors for constitutive and inducible expression. These constructs were transformed into Nicotiana sylvestris which was heterozygous for the leaf development gene, lam-1. It was determined that PHAN and lam-1 are not allelic, so further consideration of this relationship was terminated. Rather, the expression of PHAN was studied through Northern analysis and in situ hybridization of wild-type and transgenic plants.

Critical to the proposed research are results obtained from antisense transgenic plants. By inserting a copy of PHAN DNA backwards (in antisense orientation), the antisense strand is used as the template for making mRNA. By putting this gene under a constitutive promoter, the antisense mRNA is made continuously and without regulation. When this construct is present in a plant, the antisense mRNA combines with any sense mRNA being produced by the normal genomic DNA. The resulting double-stranded RNA cannot be translated by ribosomes, so the normal genomic PHAN of the transgenic plants is effectively "silenced". This approach was taken during my sabbatic leave.

A most interesting phenotype was uniformly produced in full-length antisense PHAN transgenics (confirmed by Southern analysis). Such plants produced ectopic blades along the midribs of each leaf, and the midrib was expanded laterally. This phenotype became more severe with the transition to flowering. Basal cauline leaves were progressively peltate and subfloral leaves were ultimately bladeless. The auricles in these transgenics were exaggerated, ultimately reaching sizes approaching those of full blades. It would appear that the PHAN gene, silenced by the constitutive antisense expression, is indeed involved with ending leaf blade expansion. The phan mutant of Antirrhinum is bladeless and silencing the PHAN gene of tobacco gives ectopic vegetative blade formation and cauline leaf bladelessness.

The Research Problem. The problem with this progress is that it cannot be published until I produce some additional transgenic plants. As the family of myb genes share the three repeated domains, it cannot be assured that our full-length antisense construction is silencing only the PHAN gene. It could be silencing several or even all of the other myb genes to give the observed phenotype. Southern autoradiograms of HinDIII fragments of genomic wild-type DNA consistently showed an exceedingly strong signal for one specific band which I interpret as a fragment containing the native PHAN gene. When a blot was washed less stringently, the autoradiogram also showed some faint signals in a specific pattern of what may be fragments containing other myb genes hybridized to the full-length PHAN probe. Any hybridization on the blot suggests that antisense PHAN mRNA could interfere with expression of these other genes.

The Proposed Research. To address this problem, I have amplified the unique 5' UTR and the unique non-myb coding region plus 3' UTR DNA by using custom oligonucleotides in the polymerase chain reaction (PCR). These two fragments have been verified and must now be cloned into the pFF-19 vector in antisense orientation to place them under a constitutive promoter (35S) and then recloned into the ti-plasmid (pPZP-221) for transformation via Agrobacterium. Gentamicin-resistant putative-transgenic Nicotiana sylvestris clones will be verified by Southern analysis and phenotypes compared.

The 5' UTR, the non-myb coding fragment, and the 3' UTR should have no meaningful homology with non-PHAN myb genes and should not hybridize with non-myb fragments in Southern analysis. Using probes produced from these fragments, I expect to find only the one genomic signal on membranes from HinDIII-fragmented wild-type Nicotiana sylvestris genomic DNA, and only two signals on similarly-fragmented genomic DNA from our transgenic plants. If this prediction holds with low-stringency washing of the probed membrane, then any phenotype elicited by antisense orientation of these fragments under constitutive promoters is likely due to silencing of the PHAN gene alone.

Constructing the Transgenic Plants. The 5'-UTR and non-myb coding region plus 3'-UTR fragments have a BamH1 site at one end and a SacI site at the other (designed through the custom oligonucleotide primers). These will be trimmed with those enzymes and ligated into matching sites of the pFF-19 vector. The vector will be transformed into DH5a Escherichia coli. The multiple-cloning site on this vector is contained in a LacZ gene. This allows me to identify putative fragment insertions by blue-white screening. White colonies likely contain the vector with the desired insert. This will be verified by restriction fragment length analysis. Digestion of the cloned vector with BamH1/SacI enzymes should release the insert, to be revealed by electrophoresis.

E. coli clones having vectors with successfully inserted PCR products will be ramped up and digested with HinDIII and EcoRI enzymes. This releases a fragment containing the PCR insert in antisense orientation between the 35S constitutive promoter and its terminator. The fragment will be cloned into similarly digested pPZP-221 vector. The insertions will be verified with diagnostic restriction fragment length analysis. Successful insertion will place the constitutive antisense PCR fragments between the left and right borders of the ti-plasmid required for Agrobacterium transformation.

The verified vector will be ramped up and transformed into Agrobacterium tumiefaciens (pGV-2260) cells. Successful transformation is accomplished in all transformants demonstrating kanamycin resistance.

Transformed Agrobacterium will be used to infect wild-type Nicotiana sylvestris leaf explants. The bacterium inserts its plasmids into the leaf cells. In the leaf cells, the left and right borders of the plasmids recombine into the Nicotiana genome, carrying the constitutive antisense PCR fragments with them. As the pPZP-221 vector also includes a gentamicin resistance gene, leaf tissues that survive gentamicin tissue culture regenerate into putative transgenic Nicotiana sylvestris plants.

The regenerated plants will be grown up to observe any phenotypes comparable to those obtained after transformation with full-length constitutive antisense PHAN constructs. Whether I observe ectopic blades, reduction of cauline blades, and exaggerated auricle development will demonstrate whether further analysis is desirable. This much work will consume both the budget and the funded period.

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 would be an author. I anticipate a pre-print of this work would be placed on my website (http:/ well before that time. I will be presenting the findings of all research completed at the national meeting of either the American Society of Plant Physiologists or Botanical Society of America this summer.

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, Forceps, Media$ 350
Recombinant DNA Techniques
Chemical Reagents, Enzymes, Buffers1500
Supplies, disposable plasticware2050

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

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