Progress 08/01/02 to 07/31/05
Outputs
Objective 1: Monitor transposition rates and frequency of unlinked transposition of the maize Ds element in soybean. The initial binary plasmid that carried the cDNA of the Ac (pPTN258) was found to contain an out of frame ATG. A new vector was assembled that corrected this mistake and is designated pPTN398. A total of 25 transgenic soybean events have been generated with pPTN398. Among these events 18 were confirmed to be co-expressing Ac based on northern blot analysis. A set of antibodies derived from either N-terminal of C-terminal peptides of Ac were ordered. Although there was a significant amount of background observed, the C-terminal derived antibodies were able to detect a 91 kDa protein which correlated with the northern data. A series of crosses were set-up to pyramid Ac with Ds delineated elements in the soybean genome. To date a total of twenty populations have been established derived from these crosses. In three of the populations somatic Ds
transposition has been observed in F2 individuals, which carry both transgenes. The F3 generations derived from the F2 are currently being characterized to monitor for germinal transposition. Accomplishment: This is the first whole plant demonstration that Ds transposes in the soybean genome. Objective 2: Generate soybean events that carry gene trap and enhancer trap constructs delineated by Ds termini. A total of 150 events have been generated that carry either gene trap (vector designated pPTN336) or enhancer trap (vector designated pPTN335) elements. To date 75 were monitored for GUS activation which should imply the T-DNA element landed proximal to a endogenous promoter (enhancer trap) or within a gene (gene trap). Tissue samples were assayed from roots, leaves, flowers, pods and seeds. Among the 75 events screened 15 displayed GUS expression in at least one of the tissues sampled. Junction fragments have been cloned from either the LB and/or RB region in four of the 15 events,
and significant matches have been identified in genbank. Objective 3: Generate soybean events carrying seed specific activation tagging elements delineated by Ds termini In collaboration with the University of Missouri and Iowa State University, a set of 12 activation-tagging elements was assembled. This collaboration is being supported through monies provide by the United Soybean Board. To date approximately 350 transgenic soybean events have been generated. The goal within two years is to have in-hand 1600 events that carry activation-tagging elements.
Impacts
This research will help determine the utility of the Ac/Ds transposon system as tool for functional genomics in soybean
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
To gather information on the frequency of Ds transposition in soybean we pyramided the Ac transposase and various Ds delineated elements via sexual crossing. The original binary plasmid carrying the Ac cassette designated pPTN258 contained an out of frame ATG. We therefore remade the plasmid and the resultant binary plasmid carrying the 35s CaMV Ac cassette is referred to as pPTN398. To date 18 transgenic soybean lines have been characterized from transformations conducted with the binary plasmid pPTN398. Among the 18 lines, 13 were confirmed to be expressing Ac based on northern blot analysis. Antibodies derived from the C-terminal region of the Ac gave reliable data in western blot analysis. Although a number of non-specific bands were detected using the C-terminal derived antibodies, an approximate 97 kDa band, which correlated with northern blot data, was observed in the transgenic lines and absent in controls. To date 37 crosses have been attempted with various
Ac lines by either gene trap or enhancer trap lines (see below). The F2 progeny derived from the crosses will be characterized to ascertain germinal transposition of the Ds delineated elements. Binary plasmids referred to as pPTN335 and pPTN336 correspond to the enhancer trap and gene trap constructs, respectively. Within these constructs Ds termini delineate the respective traps. To data over 130 transgenic soybean lines have been generated from these constructs. Monitoring for activation of the visual marker gene GUS was conducted on T1 progeny derived from 39 of the transgenic lines carrying either pPTN336 or pPTN335. Data was ascertained by monitoring tissue from leaf, petiole, root, flowers, and pods for GUS using the histochemical assay for the protein. Among the 39 lines screened, nine displayed GUS activity in at least one tissue type monitored. The junction fragments about the left (LB) and right (RB) elements have been ascertained from a subset of the nine lines in which
activation of GUS was observed. Analyses of the sequences about the LB region of lines 457-13 and 453-56 and RB region of line 456-1 reveled that the border elements were not completely respected. For example, only two bp of the RB resides in line 456-1, and nine bp of the LB region is present in line 457-13, while a 30 bp deletion proximal to the LB of the T-DNA element occurred in line 453-56. A total of 968 bases and 309 bases of sequence information were ascertained about the LB and RB region of line 457-13. A blast search suggested that the T-DNA element in this line tagged a gene corresponding to soybean EST designated GM-c1065 (Genbank No. BM093179). Confirmation of this tag, however, will require additional molecular characterization, which is underway. Sequence information was acquired on 370 bases proximal to the truncated LB region of line 453-56, a blast search revealed significant homology to a soybean EST designated GM-1030-1440 (Genbank No. AW508443). The junction
sequence proximal to the RB in this line has not been determined. No significant match was found with the sequence data gathered on the junction fragment proximal to the RB from line 456-1.
Impacts
This project will build upon the investments being made by the public sector in the area of soybean genomics. The development of transposon based methodologies are very powerful tools for functional genomics programs
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
Unexpected Problem: The Post Doc originally assigned to this project had to return to China in November of 2002. A new Post Doc did not begin working on this project until July 2003. Obj. 1: Monitor transposition rates and frequency of unlinked transpositions of the maize Ds element in soybean. We initially had on hand 35 lines that carry the T-DNA element of the binary vector pPTN235, that harbors a E35S CaMV-GUS cassette interrupted by a mini-Ds element. Ten soybean lines were established that contained the maize Ac under the control of the E35S promoter coupled with the tobacco etch translational enhancer element. The binary vector referred to as pPTN258. A series of crosses were made to bring together the maize Ac and Ds element. To date from these crosses we have F2 seed from 10 successful crosses. A total of 53 seed derived from 20 additional crosses using line 368-1 (Ac transposase line) as one of the parents with either gene trap or enhancer trap transgenic
soybeans are currently growing in the greenhouse. Molecular analysis will be conducted shortly on these plants to confirm the cross. Molecular characterizations including PCR and Southern blot analysis will be conducted to monitor for Ds mobilization. The 35S Ac cassette within the plasmid pPTN258 contains an ATG approximately 150 bp upstream of the actual translational start site. This upstream ATG could theoretically be used for initiation of translation that would result in an out of frame start. We have made an additional binary vector referred to as pPTN398 in which the upstream ATG has been deleted. Soybean transformations have been initiated with this new Ac cassette binary plasmid. Obj. 2: Generate soybean lines that carry gene trap and enhancer trap constructs delineated by Ds termini. Two binary plasmids were generated that carry gene trap and enhancer trap elements delineated by Ds wings. These plasmids are referred to as pPTN335 and pPTN336 for the enhancer trap and gene
trap plasmids, respectively. To date we have collected T1 seed derived from primary transformants from 130 lines of pPTN336 and 145 lines of pPTN335. Activation of GUS in T1 seed has been monitored for in progeny from 20 lines of pPTN335 and 11 lines of pPTN336. Among the 31 lines in which GUS activation has been monitored, 7 lines displayed GUS expression in either leaf, stem, flower or embryo tissues (5 pPTN335 and 2 pPTN336). We have preliminary molecular data on one of these lines 453-56. Southern blot data showed an approximate 9 kb Bam HI GUS hybridization fragment. We were successful in obtaining 371 bp of DNA sequence adjacent to the left border element within line 453-56 employing a PCR walking technique. A data base search revealed a significant match to a soybean EST si40h12.y1 (LIS designation CON 001 334840). Plans for Coming Year: Continue characterizations on the Ac/Ds crosses. Set-up crosses with new Ac transformants. Screen through remaining gene trap and enhancer
trap transgenic lines for GUS activation. Obtain sequence data about the right border and left border elements on all GUS positive lines derived from the gene trap and enhancer vectors.
Impacts
This project will build upon the investments being made by the public sector in the area of soybean genomics. The development of transposon based methodologies are very powerful tools for functional genomics programs
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
This project started on August 1, 2002, therefore we have not anything to report on the two month period.
Impacts
(N/A)
Publications
- No publications reported this period
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