Patent Application: US-201414546352-A

Abstract:
the invention relates to a pathogen - inducible synthetic promoter which is suitable for regulating the transcription of a nucleic acid , and includes a minimal promoter , characterized in that the minimal promoter includes a sequence motif a ) dbrmwa or b ) twcccmt which is disposed downstream from a tata region and in front of a transcription starting point which is located on the minimal promoter and at which transcription of the nucleic acid to be regulated starts .

Description:
the symbols of the sequence motive as used herein have the following meaning : in the sense of the invention a “ minimal promoter ” is a dna - sequence of the promoter , which is necessary for promoter function . general transcription factors such as for example tfii - d , tfii - a , tfii - b , tfii - e and tfii - f could bond at this dna - sequence , and form the platform for the bonding of the rna - polymerase 11 / tfii - f complex . since the transcription of the dna into the mrna starts in this region , the transcription start point ( ts ) lies within the minimal promoter and is identified as position + 1 . the minimal promoter encompasses the ts and can extend for example from position − 50 through position + 15 . frequently a so - called tata - box is found at the position − 30 , which however does not occur in all promoters . the tata - box is a region of a sequence of thymine and adenine bases . the tata - box is the binding location for the tata - box binding protein ( tbp ). characterized as “ synthetic promoters ” are those promoters which do not occur in nature , are assembled from multiple elements and contain a minimal promoter as well as , upstream of the minimal promoter , at least one cis - regulatory element , which serves as the bonding location for special transcription factors . synthetic promoters are designed according to the desired requirements and are induced or repressed by various factors . “ derivatives ” of a promoter are shortened or lengthened or partially identical versions of this promoter or homologs with the same , modified or singular characteristics . the expression “ homology ” herein means a homology of at least 70 % based on dna , which can be determined by known processes , for example , a computer supported sequence comparison ( altschul , s . f . et al ., 1990 ). the inventive pathogen inducible synthetic promoter results after transient biologic transformation in a reduced base activity in the leaf tissue of the respective plants in comparison to conventionally employed promoters with a minimal promoter such as the 35s - minimal promoter in dicotyledonous , and the corn - ubi1 - minimal promoter in monocotyledonous , plants . beyond this it was discovered that in the inventive pathogen inducible synthetic promoters the induction rate is also higher . the inventive pathogen inducible synthetic promoters can thus be employed for production of transgenic plants which have a broad resistance against numerous pathogens , such as fungi , oomycetes , bacteria , virus , insects and nematodes . the sequence motives dbrmwa and twcccmt lie in sense orientation on the codogenic strand between the tata - box and the transcription start point and can also occur two or more times . preferred sequences for minimal promoters are indicated in seq id nos : 1 through 9 . cis - regulatory elements for production of pathogen inducible synthetic promoters are primarily those elements which occur in natural pathogen inducible promoters and they are responsible for pathogen induction . their identification is described in rushton et al . ( 2002 ). preferred cis - regulatory elements for production of synthetic promoters with use of the inventive minimal promoters are also described in wo 00 / 29592 . from the cis - regulatory elements mentioned there , the d - box ( seq id no : 10 ) is particularly suitable , in particular in the combination 2xs / 2xd ( seq id no : 11 ), as well as the gst1 - element , preferably in the combination 4xgst1 ( seq id no : 12 ). preferred cis - element combinations include in general combinations of the d - box ( seq id no : 10 ) with the s - box or , as the case may be , the gst1 - element . particularly preferred are , besides the above - mentioned combination 2xs / 2xd ( seq id no : 11 ), the combination 2xs / 4xd ( seq id no : 13 ); 4xs / 2xd ( seq id no : 14 ) and 2xgst1 / 2xd ( seq id no : 15 ). the combination of the 2xs / 4xd element ( seq id no : 13 ) with the minimal promoter according to seq id no : 2 shows in transgenic potatoes following infection with phytophthora infestans an average elevation of the reporter gene activity by a factor of 253 , 000 in comparison to a non - infected control . if the element 4xs / 2xd ( seq id no : 14 ) was cloned ahead of the minimal promoter ( seq id no : 2 ), an average increase in the reporter gene activity by a factor of 2 , 892 could be detected . with element 2xgst1 / 2xd ( seq id no : 15 ) an average increase by a factor of 2 , 967 in comparison to control was achieved . with the inventive promoters transgenic plant cells can be produced , which can be regenerated to complete plants with improved defensive characteristics against pathogens . the inventive promoters are likewise contained in the seeds of such transgenic plants . the invention is not limited to particular types of plants . the present invention is thus concerned with the process for production of a plant resistant against pathogens , in which a gene suitable for production of a pathogen resistance is introduced into a plant cell , which is under the control of a pathogen inducible synthetic promoter , and subsequently this plant cell is regenerated into a plant , characterized in that the pathogen inducible synthetic promoter is a pathogen inducible synthetic promoter as described above . fig1 shows a sequence comparison between the preferred minimal promoters ( seq id nos : 1 through 7 ) for dicotyledonous plants with the conserved tata - regions and the dbrmwa - motives as well as the cleavage site pstl and xhol employed for cloning in the plasmid pms23luc +. fig2 shows a sequence comparison between the minimal promoters ( seq id nos : 8 and 9 ), preferred for monocotyledonous plants , which are employed for the transient transformation of wheat leaves . in addition to the tata - region the sequence motive twcccmt is shown as conserved region . it could be shown that the minimal promoters stgst ( seq id no : 6 ), nttgaa ( seq id no : 5 ), stpsbr ( seq id no : 7 ), npcabe ( seq id no : 2 ), ntrbs ( seq id no : 3 ), npatp2 ( seq id no : 1 ) and nt5eas ( seq id no : 4 ) exhibited a clearly reduced activity (& lt ; 70 %) in comparison to the 35s - minimal promoter . fig3 shows an overview of the average reporter gene activity of non - infected , transgenic potato plants with a synthetic promoter comprised of the 4xgst1 element ( seq id no : 12 ) and the indicated minimal promoters , cloned ahead of the luciferase gene from photinus pyralis as reporter gene ( rlu = relative light unit ). stable , transgenic lines with the minimal promoters , which carry the sequence motive dbrmwa showed under controlled conditions a clearly reduced expression of the reporter gene in comparison to the 35s - minimal promoter . the smallest average activity was achieved with use of the minimal promoter of the npatp2 gene ( seq id no : 1 ). in these plants only 9 . 7 % of the average activity of the 35s - minimal promoter could be measured . with use of the minimal promoters stpsbr ( seq id no : 7 ), nttgaa ( seq id no : 5 ) or stgst ( seq id no : 6 ) 18 % of the activity of the 35s - minimal promoter was measured , with ntrbs - minimal promoter ( seq id no : 3 ) 26 %, with npcabe - minimal promoter ( seq id no : 2 ) 39 % and with nt5eas - minimal promoter ( seq id no : 4 ) 41 %. for the person of ordinary skill the manufacture of suitable constructs for transformation of plants with the inventive promoters is no problem . thus for example the binary vectors p4xgst1 - luc - kan ( fig8 ) could be produced , which was used for the stable transformation of potato plants of the variety “ baltica ”. this vector is a derivative of the binary vector pgptv ( becker et al ., 1992 ). the binary vector p4xgst1 luc - kan carries the luciferase gene from photinus pyralis under the control of the synthetic promoter 4xgst1 : 35s minimal promoter ( rushton et al ., 2002 ). as the termination sequence the plasmid is given the terminator of the nopalinsynthase gene from agrobacterium tumefaciens . the described expression cassette is localized on the t - dna together with a functional expression cassette for the neomycinphosphotransferase gene ( nptll ) as selection marker . the neomycinphosphotransferase imparts to the transgenic plants resistance against kanamycin or paromycin . in order to exchange the 35s - minimal promoter with the above - described minimal promoters , the binary vector p4xgst1 luc - kan was digested with xhol / sall , whereby the 35s - minimal promoter was removed , the tetramer of the gst1 - element however remained intact . the sall cleavage location was filled with the aid of the enzyme klenow polymerase and dntp &# 39 ; s in order to achieve a blunt end . the minimal promoters , cloned in the plasmid pms23luc +, were excised using pdil / xhol - digestion and ligated in the binary vector and subsequently transformed in e . coll . binary vectors with the new sequence were transformed in the agrobacterium type gv3101 :: pmp90 ( koncz and schell , 1986 ) ( an , 1987 ) and selected using the antibiotic kanamycin ( 50 mg / l ). the transgenic agrobacterium were employed for the transformation of potatoes of the type “ baltica ” ( dietze et al ., 1995 ). fig4 shows a overview of the individual inductions following in vitro infection of stable , transgenic potato plants with the synthetic promoter comprised of the 4xgst1 element ( seq id no : 12 ) and the indicated minimal promoters . the infection occurred in in vitro plants with a zoospore suspension of phytophthora infestans . at various times following inoculation leaf samples of in vitro plants were removed , the sample weight was determined and 10 volumes ixcclr buffer ( promega , mannheim ) was added . the material was homogenized with the aid of a ria / 90 hnrnflga . ni & gt ; . c ′ r oka labortechnik , staufen ) in buffer on ice . by centrifugation at & gt ; 10 , 000 × g for 10 minutes the homogenate was clarified and 10 pl of the supernatant was suspended with 50 pl of the substrate lar ( promega , mannheim ) in a luminometer tube and the light emission was determined as value for the activity of the luciferase in the luminometer ( sirius , berthold detection system gmbh , pforzheim ). for control or comparison in vitro plants were employed , which were raised under the same conditions and , in place of zoospores , were subject to a sham treatment with water . the average value of the quotients , in 5 independent lines , of the luciferase activity of the infected to the sham treated variants , indicates the induction of the synthetic promoter by the infection . as can be seen in fig4 , with use of the 35s - minimal promoter , a maximal induction of the luciferase activity by a factor of only 10 could be achieved 72 hours after infection . all new minimal promoters in contrast showed a clearly improved induction . the strongest induction after infection with a factor 395 was achieved 72 hours post - infection with the stpsbr minimal promoter ( seq id no : 7 ). in general , the induction by use of the new minimal promoters could be improved at time 72 hours post - induction by a factor of 3 . 5 with stgst minimal promoter ( seq id no : 6 ) to a factor of 39 . 5 with stpsbr minimal promoter ( seq id no : 7 ) in comparison to 35s - minimal promoter . interestingly , clear differences in the kinetics of the induction following pathogen induction exist between the minimal promoters . while the most discernible induction is measurable with use of the 35s - minimal promoter 72 hours post induction , this also applies with use of the stpsbr , nttgaa , stgst , ntrbs as well as npatp2 minimal promoter . for npcabe and nt5eas promoters in comparison a strong activation is already detectable at time interval 9 and the induction remains at approximately the achieved level over the remaining test period . the preferability of the new minimal promoters was shown following fusion with the cis - element combination 2xs / 2xd . for this , potato plants were stably transformed with the binary vectors p2xs / 2xdluc - kan , p2xs / 2xdnpcabeluc - kan and p2xs / 2xdnttgaaluc - kan . the binary vectors were produced in that the 4xgst1 - element from the above - described binary vector with the new minimal promoter and the 4xgst1 - element were eliminated via bcul / eco1471 - digestion and the element 2xs / 2xd ( seq id no : 11 ) was introduced as bcul / eco32l - fragment . binary vectors with the new sequence were transformed in the agrobacterium type gv3101 :: pmp90 ( koncz and schell , 1986 ) ( an , 1987 ) and selected using the antibiotic kanamycin ( 50 mg / l ). the transgenic agrobacterium were employed for the transformation of potato of the type “ baltica ” ( dietze et al ., 1995 ). transgenic sprouts were multiplied and inoculated under in vitro conditions with the zoospore suspension ( 50 , 000 spores / ml ) of phytophthora infestans . it could be shown , that also with use of the cis - regulatory element 2xs / 2xd ( seq id no : 11 ) a reduced background activity could be achieved with the inventive minimal promoters in comparison to 35s - minimal promoters ( fig5 ). at the same time a stronger induction of the synthetic promoters following inoculation of the transgenic potatoes with p . infestans could be observed ( fig6 ). the amplification of the induction was not so pronounced at the later time ( 3 days post infection = 3 dpi ) as could be observed following use of the 4xgst1 - element . two days following infection however by the use of the new minimal promoter a clearly stronger induction following pathogen attack could be observed . herewith the use of these minimal promoters has as a consequence an improvement of the kinetics of the synthetic promoter , so that the reaction to pathogen attack occurs earlier , in comparison to the synthetic promoter using the 35s - minimal promoter . fig7 shows a comparison of the normalized activity of pathogen inducible synthetic promoters comprised of an element 2xs / 2xd ( seq id no : 11 ) and the minimal promoters ubi1 ( comparison promoter ), tapal ( seq id no : 9 ) and taacs ( seq id no : 8 ) following biologic transformation in primary leaves of the wheat type “ taifun ”. as can be seen , the new minimal promoters tapal and taacs in wheat have a reduced base activity in comparison to ubi1 - minimal promoter . while a normalized activity of 0 . 17 was measured with the ubi1 - minimal promoter , with use of the tapal - minimal promoter this could be reduced to 0 . 072 , and with use of the taacs - minimal promoter it could be reduced to 0 . 13 . fig9 shows the plasmid pubitatarucll , which contains the cdna with the luciferase gene from renilla reniformis , as it exists in the commercially available plasmid prl - null . the cdna is under the control of the ubi1 - minimal promoter . the ubi1 - minimal promoter includes the sequence range from − 45 through + 76 relative to the transcription start point . for elevating the expression strength the first intron of the ubi1 - gene is maintained in its natural context in the plasmid ahead of the reporter gene . the plasmid serves for cloning the cis - regulatory element 2xs / 2xd ( seq id no : 11 ), in order thereby to produce a pathogen inducible synthetic promoter . the ubi1 - minimal promoter was exchanged for the new minimal promoter for improving the characteristics of the synthetic promoter . an , g . 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