Patent Application: US-201013520350-A

Abstract:
control of transgene expression in planta is dependent upon genetic elements that affect both transcription and translation of mrna transcripts . the disclosed invention describes the combination of dna elements from four different plant viruses that function as an activator of transcription and enhancer of translation of mrna transcripts in transgenic plants .

Description:
the synthetic plant promoter nucleotide sequences and methods disclosed herein are useful in regulating expression of any heterologous nucleic acid sequences in a host plant in order to alter the phenotype of a plant . various changes in phenotype are of interest including , but not limited to , modifying the fatty acid composition in a plant , altering the amino acid content of a plant , altering a plant &# 39 ; s pathogen defense system , and the like . these results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants . alternatively , the results can be achieved by providing for a reduction of expression of one or more endogenous products , particularly enzymes or cofactors in the plant . these changes result in a change in phenotype of the transformed plant . genes of interest are reflective of the commercial markets and interests of those involved in the development of the crop . crops and markets of interest change , and as developing nations open up world markets , new crops and technologies will emerge also . in addition , as our understanding of agronomic characteristics and traits such as yield and heterosis increase , the choice of genes for transformation will change accordingly . categories of transgenes , also known as heterologous genes , for example , include , but are not limited to , genes encoding important agronomic traits , insect resistance , disease resistance , herbicide resistance , sterility , grain or seed characteristics , and commercial products . genes of interest include , generally , those involved in oil , starch , carbohydrate , or nutrient metabolism as well as those affecting seed size , plant development , plant growth regulation , and yield improvement . plant development and growth regulation also refer to the development and growth regulation of various parts of a plant , such as the flower , seed , root , leaf , and shoot . other commercially desirable traits are genes and proteins conferring cold , heat , salt , and drought resistance . disease and / or insect resistance genes may encode resistance to pests that have great yield drag such as for example , anthracnose , soybean mosaic virus , soybean cyst nematode , root - knot nematode , brown leaf spot , downy mildew , purple seed stain , seed decay , and seedling diseases commonly caused by the fungi pythium sp ., phytophthora sp ., rhizoctonia sp ., diaporthe sp . bacterial blight caused by the bacterium pseudomonas syringae pv . glycinea . genes conferring insect resistance include , for example , bacillus thuringiensis toxic protein genes ( u . s . pat . nos . 5 , 366 , 892 ; 5 , 747 , 450 ; 5 , 737 , 514 ; 5 , 723 , 756 ; 5 , 593 , 881 ; and geiser et al ( 1986 ) gene 48 : 109 ); lectins ( van damme et al . ( 1994 ) plant mol . biol . 24 : 825 ); vegetative insecticidal proteins ( vip3c , u . s . pat . no . 7 , 378 , 493 ); and the like . herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase ( als ), in particular the sulfonylurea - type herbicides ( e . g ., the acetolactate synthase als gene containing mutations leading to such resistance , in particular the s4 and / or hra mutations ). the als - gene mutants encode resistance to the herbicide chlorosulfuron . glyphosate acetyl transferase ( gat ) is an n - acetyltransferase from bacillus licheniformis that was optimized by gene shuffling for acetylation of the broad spectrum herbicide , glyphosate , forming the basis of a novel mechanism of glyphosate tolerance in transgenic plants ( castle et al . ( 2004 ) science 304 , 1151 - 1154 ). other herbicide resistance traits , including , but not limited to , epsps ( u . s . pat . no . 6 , 248 , 076 ), bar ( u . s . pat . no . 6 , 025 , 545 ), and hppd ( u . s . pat . no . 7 , 312 , 379 ), would be obvious to use to one skilled in the art . the present invention includes the transformation of a recipient cell with at least one advantageous transgene . two or more transgenes can be supplied in a single transformation event using either distinct transgene - encoding vectors , or a single vector incorporating two or more gene coding sequences . any two or more transgenes of any description , such as those conferring herbicide , insect , disease ( viral , bacterial , fungal , and nematode ) or drought resistance , oil quantity and quality , or those increasing yield or nutritional quality may be employed as desired . the synthetic plant promoter sequence of the present invention can be modified to provide a range of constitutive expression levels of the heterologous nucleotide sequence . thus , less than the entire synthetic plant promoter regions may be utilized and the ability to drive expression of the coding sequence retained . however , it is recognized that expression levels of the mrna may be decreased with deletions of portions of the synthetic plant promoter sequences . therefore , fragments of seq id no : 3 which are 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identical to seq id no : 3 may still function as exemplified by this description . embraced by the present invention are also functional equivalents of the synthetic plant promoters of the present invention , i . e . nucleotide sequences that hybridize under stringent conditions to any one of seq id no : 3 , seq id no : 4 , seq id no : 5 , seq id no : 6 , seq id no : 7 , seq id no : 8 , or seq id no : 9 . a stringent hybridization is performed at a temperature of 65 ° c ., preferably 60 ° c . and most preferably 55 ° c . in double strength ( 2 ×) citrate buffered saline ( ssc ) containing 0 . 1 % sds followed by rinsing of the support at the same temperature but with a buffer having a reduced ssc concentration . such reduced concentration buffers are typically one tenth strength ssc ( 0 . 1 × ssc ) containing 0 . 1 % sds , preferably 0 . 2 × ssc containing 0 . 1 % ssc and most preferably half strength ssc ( 0 . 5 × ssc ) containing 0 . 1 % sds . combining viral enhancer elements and plant components to create synthetic plant promoters to express a synthetic oat ( avena sativa ) 4 - hydroxyphenylpyruvate dioxygenase ( cavhppd ) in stably transformed soybean , viral transcriptional and translational enhancers , and a minimal promoter were created by pcr or direct dna synthesis and combined by standard dna restriction digestion and ligation reactions . a synthetic plant promoter comprising defined components efmv ( seq id no : 1 ), etmv ( seq id no : 2 ), the cauliflower mosaic virus 35s enhancer region ( e35s ) and a minimal promoter ( pr35scmp : cestrum yellow leaf curl virus tata - box motif ; no caat 35s - proximal promoter sequence ) were combined to create seq id no : 3 . subsequently , seq id no : 3 was modified by digestion with a dna restriction enzyme xhoi to remove defined components e35s , pr35scmp ( including the tata - box motif ) followed by a standard ligation reaction to create seq id no : 4 . the seq id no : 3 was again modified by the ligation of the first intron ( iubq3 ) derived from the arabidopsis ubiquitin promoter as a bgl ii ( 5 - prime end ) and bamhi ( 3 - prime end ) dna fragment to the bamhi site to create seq id no : 5 . finally , seq id no : 3 was modified by the ligation of a 1092 base pair dna fragment of an arabidopsis constitutive promoter ( prac26 ) as a bgl ii ( 5 - prime end ) and bamhi ( 3 - prime end ) to the bamhi site to create seq id no : 6 . the completed gene cassettes harboring individual synthetic plant promoters comprising seq id : 3 , seq id : 4 , seq id : 5 or seq id : 6 , the cavhppd coding region and nos terminator ( tnos ) were subsequently ligated to binary vectors containing the appropriate selectable markers for soybean transformation experiments . table 1 indicates the arrangement of subelements in the above described synthetic plant promoters . one skilled in the art would readily recognize other subelements which would be suitable to use . the plasmids containing the synthetic plant promoter expression cassettes were transformed into soybean using agrobacterium tumefaciens . t0 events were cultivated and selected for cavhppd expression by application of mesotrione spray . leaf samples of surviving t0 plants were tested for zygosity by taqman ® assay . expression of cavhppd of surviving t0 plants was quantified by elisa ( engvall e , perlman p ( 1971 ). “ enzyme - linked immunosorbent assay ( elisa ). quantitative assay of immunoglobulin g ”. immunochemistry 8 ( 9 ): 871 - 874 ). the first generation transgenic soybean events ( t1 ) harboring seq id no : 3 , seq id no : 4 , seq id no : 5 , seq id no : 6 , or seq id no : 7 were characterized for segregation analysis , oat hppd protein expression and tolerance to mesotrione herbicide spray . the green leaf tissues from the first trifoliate of five independent events were sampled to determine the segregation ratios ( homozygous , heterozygous or null ) of the individual seedlings as determined by zygosity taqman ® assays and oat hppd protein expression by elisa . at the v2 stage , the seedlings were sprayed with the hppd inhibiting herbicide mesotrione and tolerance rating determined approximately 10 days post - application . the results from analysis of transgenic soybean events harboring seq id no : 3 showed significant initial damage to the homozygous ( hom ) compared to the heterozygous ( het ) siblings . these data are consistent with the elisa data showing relatively low level of oat hppd protein expression in the hom seedlings (& lt ; 20 ng / mg total protein ) compared to het siblings for each independent event ( table 2 ). collectively , these results are indicative of transgene silencing whereby overexpression of a transgene in hom siblings activates a micro - rna mediated methylation which results in very low expression of the transgene ( martienssen r a , colot v ( 2001 ). dna methylation and epigenetic inheritance in plants and filamentous fungi . science 293 ( 5532 ): 1070 - 1074 ). the analyses of t1 generation soybean events harboring seq id no : 4 or seq id no : 5 showed more consistent expression of oat hppd protein as would be expected in hom siblings ( table 3 , table 4 , respectively ). these data reveal that seq id no : 4 and seq id no : 5 modulate oat hppd expression to such an extent as to relieve transgene silencing resulting and improved tolerance to the hppd inhibitor herbicide mesotrione . however , the modification as exemplified in seq id nos : 6 and 7 did not relieve transgene silencing in hom plants ( tables 5 and 6 ). a similar strategy for building synthetic plant promoters for use in maize was implemented . in addition to the promoters illustrated in table 7 , below , seq id no : 3 was also successfully used to promote the expression of a heterologous sequence in maize . seq id no : 8 was synthesized by gene art as a sandi / bamhi fragment then ligated directly into a cloning vector harboring the epsps gene ( czmepspsct - 01 ) to confer glyphosate tolerance ( terada , et al ., ( 1995 ) a type i element composed of the hexamer ( acgtca ) and octamer ( cgcggatc ) motifs plays a role ( s ) in meristematic expression of a wheat histone h3 gene in transgenic rice plants . plant molecular biology 27 : 17 - 26 ). seq id no : 9 was created by ligation of the xzmh3cis dna elements to the prcmp promoter as an nhei fragment such that these elements are 5 ′ to the tata - box ( brignon , et al ., ( 1993 ) nuclease sensitivity and functional analysis of a maize histone h3 gene promoter . plant molecular biology 22 : 1007 - 1015 ). data indicate that seq id nos : 8 and 9 were as efficient in promoting the expression of an operably linked heterologous sequence as an unmodified cestrum virus promoter ( seq id no : 10 ). see table 9 for glyphosate phytotoxicity , in terms of percent injury . plants were sprayed with an appropriate amount of glyphosate ( i . e . 4 × touchdown ®) at the v4 stage and the v8 stage . percent injury was measured at 7 and 14 days after the v4 stage glyphosate spray , as well as 7 and 14 days after v8 stage glyphosate spray . it is clear from these results that the synthetic plant promoters embodied in seq id no : 8 and seq id no : 9 function at least as well on average as the unmodified cestrum virus promoter . additionally , seq id nos : 8 and 9 show no evidence of hdgs and hdms . if there were silencing , these plants would not be as tolerant to glyphosate as the unmodified prcmp . secondly , maize histone h3 and h4 genes are organized into multigene families of 40 - 50 and 50 - 60 copies , respectively . hdgs may be induced by the use of repetitive promoter or cis - elements . however , as maize already has 40 - 50 copies of endogenous histone promoter cis - elements , the potential to induce hdgs with either the wheat or maize h3 elements is unlikely ( chaubet et al ., ( 1987 ) histone genes in higher plants : organization and expression . developmental genetics 8 : 461 - 473 ). in view of the results presented here , an embodiment of the present invention is a synthetic plant promoter functional in a plant cell , wherein a 5 ′ terminus of the synthetic plant promoter is an enhancer from figwort mosaic virus or an enhancer from tobacco mosaic virus and wherein a 3 ′ terminus of the synthetic plant promoter is an enhancer from the tobacco mosaic virus when the 5 ′ terminus is the enhancer from figwort mosaic virus or the 3 ′ terminus is the enhancer from the figwort mosaic virus when the 5 ′ terminus is the enhancer from the tobacco mosaic virus . in another embodiment , the synthetic plant promoter has an optional kozak sequence which extends beyond the 3 ′ terminus of the synthetic promoter . in another embodiment of the present invention , the enhancer from a figwort mosaic virus comprises seq id no : 1 and the enhancer from a tobacco mosaic virus comprises seq id no : 2 . in yet another embodiment of the present invention , the synthetic plant promoter comprises any of seq id no : 3 , 4 , 5 , 6 , 7 , 8 , or 9 . an embodiment of the present invention is a method of constructing a synthetic plant promoter functional in a plant comprising the steps of : ( a ) obtaining an enhancer from a figwort mosaic virus and an enhancer from a tobacco mosaic virus and optionally one or more nucleotide sequences selected from the group consisting of enhancers , promoters , exons , introns , and other regulatory sequences ; and ( b ) operably linking the enhancer from the figwort mosaic virus , the one or more optional nucleotide sequences , and the enhancer from the tobacco mosaic virus thus creating the synthetic plant promoter functional in a plant , wherein a 5 ′ terminus of the synthetic plant promoter is the enhancer from figwort mosaic virus or the enhancer from tobacco mosaic virus and wherein a 3 ′ terminus of the synthetic plant promoter is the enhancer from a tobacco mosaic virus when the said 5 ′ terminus is the enhancer from the figwort mosaic virus or the 3 ′ terminus of the promoter is the enhancer from the figwort mosaic virus when the said 5 ′ terminus is the enhancer from the tobacco mosaic virus , and wherein the one or more optional nucleotide sequences are positioned between the enhancers . another embodiment of the present invention provides the method above , wherein the enhancer from the figwort mosaic virus comprises seq id no : 1 and the enhancer from the tobacco mosaic virus comprises seq id no : 2 . in yet another embodiment , the product of step ( b ) comprises seq id no : 3 . in still yet another embodiment , the product of step ( b ) comprises seq id no : 4 . in another embodiment , the product of step ( b ) comprises seq id no : 5 . in yet another embodiment , the product of step ( b ) comprises seq id no : 6 . in still yet another embodiment , the product of step ( b ) comprises seq id no : 7 . in further yet another embodiment , the product of step ( b ) comprises seq id no : 8 . in another embodiment , the product of step ( b ) comprises seq id no : 9 . an embodiment of the present invention is a method of expressing a heterologous gene in a plant , plant cell , or plant tissue , comprising : ( a ) constructing a synthetic plant promoter according to the method of constructing a synthetic plant promoter functional in a plant comprising the steps of : ( i ) obtaining an enhancer from a figwort mosaic virus and an enhancer from a tobacco mosaic virus and optionally one or more nucleotide sequences selected from the group consisting of enhancers , promoters , exons , introns , and other regulatory sequences ; and ( ii ) operably linking the enhancer from the figwort mosaic virus , the one or more optional nucleotide sequences , and the enhancer from the tobacco mosaic virus thus creating the synthetic plant promoter functional in a plant , wherein a 5 ′ terminus of the synthetic plant promoter is the enhancer from figwort mosaic virus or the enhancer from tobacco mosaic virus and wherein a 3 ′ terminus of the synthetic plant promoter is the enhancer from a tobacco mosaic virus when the said 5 ′ terminus is the enhancer from the figwort mosaic virus or the 3 ′ terminus of the promoter is the enhancer from the figwort mosaic virus when the said 5 ′ terminus is the enhancer from the tobacco mosaic virus , and wherein the one or more optional nucleotide sequences are positioned between the enhancers ; ( b ) operably linking the synthetic plant promoter to the heterologous gene , thereby creating an expression cassette , wherein the expression cassette is functional in a plant , plant cell , or plant tissue ; and ( c ) creating a plant , plant cell , or plant tissue or a portion thereof comprising the expression cassette , wherein the heterologous gene is expressed . in another embodiment , the heterologous gene comprises a nucleotide sequence encoding an herbicide resistance trait . in yet another embodiment , the nucleotide sequence encoding an herbicide resistance trait comprises a nucleotide sequence encoding hppd resistance . in still yet another embodiment , the synthetic plant promoter is manipulated to optimize expression . in another embodiment , the synthetic plant promoter is manipulated to reduce expression . in further yet another embodiment , the synthetic plant promoter is manipulated to increase expression . in still yet another embodiment , the plant , plant cell , or plant tissue or a portion thereof is a monocot . in another embodiment , the plant , plant cell , or plant tissue or a portion thereof is maize . in yet another embodiment , the plant , plant cell , or plant tissue or a portion thereof is a dicot . in still yet another embodiment , the plant , plant cell , or plant tissue or a portion thereof is soybean . an embodiment of the present invention is a method of selecting for male sterile plants comprising : ( a ) constructing an expression cassette comprising a synthetic plant promoter operably linked to a heterologous gene , wherein a 5 ′ terminus of the synthetic plant promoter comprises seq id no : 1 or seq id no : 2 and wherein a 3 ′ terminus of the synthetic plant promoter comprises seq id no : 2 when the 5 ′ terminus is seq id no : 1 or the 3 ′ terminus of the synthetic plant promoter is seq id no : 1 when the 5 ′ terminus is seq id no : 2 , and wherein the synthetic plant promoter is functional in a plant cell ; ( b ) creating a plant , plant cell , or plant tissue or a portion thereof comprising the expression cassette , wherein the heterologous gene is overexpressed and wherein such overexpression induces male sterility ; and ( c ) selecting for the male sterile plants . in another embodiment , the synthetic plant promoter is selected from the group consisting of : seq id nos : 4 and 6 . in yet another embodiment , the heterologous gene comprises a nucleotide sequence encoding an herbicide resistance trait . in still yet another embodiment , the nucleotide sequence encoding an herbicide resistance trait comprises a nucleotide sequence encoding hppd resistance . an embodiment of the present invention is a method of selecting for heterozygous plants comprising : ( a ) constructing an expression cassette comprising a synthetic plant promoter operably linked to a heterologous gene , wherein a 5 ′ terminus of the synthetic plant promoter comprises seq id no : 1 or seq id no : 2 and wherein a 3 ′ terminus of the synthetic plant promoter comprises seq id no : 2 when the 5 ′ terminus is seq id no : 1 or the 3 ′ terminus of the synthetic plant promoter is seq id no : 1 when the 5 ′ terminus is seq id no : 2 , and wherein the synthetic plant promoter is functional in a plant cell ; ( b ) creating a plant , plant cell , or plant tissue or a portion thereof comprising the expression cassette , wherein the heterologous gene is overexpressed in homozygous plants and wherein such overexpression induces gene silencing ; and ( c ) selecting for the heterozygous plants . in another embodiment , the synthetic plant promoter is selected from the group consisting of : seq id nos : 4 and 6 . in yet another embodiment , the heterologous gene comprises a nucleotide sequence encoding an herbicide resistance trait . in still yet another embodiment , the nucleotide sequence encoding an herbicide resistance trait comprises a nucleotide sequence encoding hppd resistance . iyer m ., wu l ., et al . v ( 2001 ) two step transcriptional amplification as a method for imaging reporter gene expression using weak promoters pnas 98 ( 25 ): 14595 - 14600 . larkin , j . c ., oppenheimer , d . g ., pollock , s ., and marks , m . d . 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