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Improvement of Forage Quality by Downregulation of Maize O-Methyltransferase

^a Dep. of Biological Sciences, Simon Fraser Univ., Burnaby, B.C. V5A 1S6, Canada
^b Animal Sciences, Univ. of Florida, Gainesville, FL
^c Agronomy Dep., Univ. of Florida, Gainesville, FL 32611

View larger version (9K): [in a new window]   Fig. 1. Schematic representation of an antisense sorghum omt vector used for maize transformation. The vector contained the CaMV 35S promoter to drive bar with the nos terminator; and the maize Ubi-1 promoter (plus first intron and first exon) to drive the sorghum omt full-length cDNA (1600 bp), or the 995-bp 5'-fragment, in the antisense orientation with the nos terminator. Restriction sites labeled are; H = HindIII, B = BamHI, and E = EcoRI. Digestion with EcoRI releases a 2.5-kb fragment with the full-length omt or a 1.9-kb fragment with the 995-bp omt vectors.

View larger version (37K): [in a new window]   Fig. 2. Endogenous OMT activities and OMT mRNA levels distributed within maize control plants. Maize plants were sampled 7 wk after planting in the greenhouse. Lanes 1 through 8 represent leaf samples from the second youngest to the ninth youngest leaf; Lane 9, young internodes; Lane 10, extending internodes; Lane 11, leaf sheaths; Lane 12, sorghum stem. (A) OMT activity was measured on each maize plant part and the highest OMT activity (0.76 pkat/mg protein) was considered as 100%. (B) Relative OMT mRNA steady state levels were determined by RNA quantification of the northern blot data. (C) Northern blot analysis of the OMT steady state levels using the maize omt cDNA probe. The blot was also hybridized with the 28S rDNA probe to standardize RNA loading and transfer.

View larger version (44K): [in a new window]   Fig. 3. OMT activity of 2-wk-old (5-leaf stage) plants: (A) Control plants, (B) A-group T1 transgenic plants. Plants initiated from five seeds resulting from self-pollination of 33 selected primary transgenic (T0) plants were established and grown in the greenhouse. The second youngest leaf from transgenic and control plants were assayed for OMT activity two weeks after planting. The mean of 10 control plants was considered as 100% (0.10 pkat/mg protein). The 33 plant families are labeled under the graph and individuals within a family were designated A1 to A5 sequentially from left to right (not labeled on the Fig.). A bm in the family designation indicates that the T0 parent had a brown midrib phenotype. Independent events are signified by nonconnecting lines below the family designations.

View larger version (73K): [in a new window]   Fig. 4. Northern blot analysis with the strand-specific omt antisense riboprobe to detect endogenous OMT mRNA steady state levels. (A) The five A-group T1 plants that maintained low OMT activity through the 7-wk stage. Third youngest leaf was sampled. The 1.7-kb band is the endogenous OMT mRNA and is not visible in the transgenic plants but shows a strong band in the control. (B) B-group T1 plants sampled at the flag-leaf stage. The 1.7-kb band is strongly present in the controls and bm17-B3, which behaved as a control as it had lost the antisense omt. The endogenous 1.7-kb band is present at a reduced level in some transgenics and is not visible in others. The blot was sequentially hybridized with the strand-specific maize antisense omt riboprobe and the 28S rDNA probe. The reduced expression of the endogenous OMT suggests that antisense omt expression may promote instability of the endogenous OMT mRNA. The 1.3 kb unknown RNA has strand-specific binding with the omt antisense probe. That RNA did not hybridize when an omt cDNA probe or the sense riboprobe was used. A bm in the plant designation indicates that T0 parent had the brown midrib phenotype. Each plant family is generated from an independent event.

View larger version (35K): [in a new window]   Fig. 5. OMT activity of the B-group T1 plants sampled at the flag-leaf stage. Plants were initiated using seed from 10 self-pollinated primary transgenic plants selected on the basis of reduced family OMT activity (Fig. 3). Family members are designated B1 through B6. The young flag leaves from transgenic and control plants of these 7-wk-old plants were assayed for OMT activity. The mean of 10 control plants was considered as 100% (0.32 pkat/mg protein). A bm in the plant designation number under the graph indicates the T0 parent had a brown midrib phenotype. Bm17-B3 could be viewed as a control as it lost the antisense omt. Families are generated from independent events, except that families bm8 and bm9 are not independent.

View larger version (110K): [in a new window]   Fig. 6. Southern blot analysis of the B-group T1 plants. Genomic DNA (10 µg) was digested with EcoRI to release the 2.5- or 1.9-kb fragment of the sorghum omt antisense expression cassette (full-length or 5'-fragment of the sorghum cDNA, respectively) and hybridized with a sorghum omt cDNA probe. The sorghum omt antisense expression cassette includes part of the Ubi-1 promoter first intron region (about 600 bp), sorghum antisense omt (full-length or 5'-fragment of the sorghum omt cDNA), and nos terminator (see Fig. 1). -DNA (HindIII) and X174 (HaeIII) were used as DNA size markers. Bm designations indicate the T0 parent had brown midrib phenotype. Families were generated from independent events.

View larger version (44K): [in a new window]   Fig. 7. Northern blot analysis of the T1 transgenic plants. Total RNA (10 µg per lane) was loaded. The blotted membrane was sequentially hybridized with the sorghum omt, bar, and 28S rDNA probes. (A) A-group T1 plants were sampled at the 2-wk-old stage (5-leaf stage) and (B) B-group T1 plants were sampled at the flag-leaf stage. The approximate sizes of the transcripts are marked. The 1.7-kb transcript is the maize endogenous OMT mRNA. The 1.6- and 1.0-kb transcripts represent the full-length, and 5'-fragment of the sorghum antisense omt vector, respectively. Families were generated from independent events, except that families bm8 and bm9 are not independent.

View larger version (94K): [in a new window]   Fig. 8. Lignin histochemical staining. Shown are the seventh and tenth internodes (from the top), of bm128-B3 transgenic and control plants. Plants were sliced into 1-mm free-hand sections from the top region of the internodes and subjected to (A) phloroglucinol-HCl, and (B) potassium permanganate-HCl staining. Note the more intense staining of the control sections.