Patent Application: US-11760999-A

Abstract:
the present invention is drawn to an isolated purified dna sequence from the promoter region of a microspore - specific gene of tobacco . the microspore - specific promoter can play a role in the expression of genes in microspores . the invention also is drawn to chimeric genes suitable for transforming plants comprising the microspore - specific promoter . further , the invention is also drawn to plants transformed with the chimeric genes .

Description:
plants of nicotiana tabacum l . cv . ‘ petit havana ’ sr1 [ 19 ], were grown in soil under greenhouse conditions . flower buds of selected length were collected to obtain pollen of different developmental stages . the determination of developmental stage and isolation of the anther content was carried out as previously described [ 28 ]. rna was isolated with guanidine thiocyanate as rnase inhibitor by the procedure of cathala [ 4 ] with minor modifications . all plant tissues including the microspores were frozen in liquid nitrogen and ground in a prechilled mortar . the ground material was placed in 2 ml lysis buffer and , after centrifugation at 3000 g for 10 min at 15 ° c ., the supernatant was collected . the duration of all centrifugation steps in the procedure was modified from the original procedure to 20 min . the mrna to be used for the synthesis of the cdna library was bound on the surface of oligo ( dt ) dynabeads ( dynal ) as described by the manufacturer [ 10 ]. the mrna used to synthesize a single - stranded cdna probe was isolated as reported [ 26 ]. plasmid dna was isolated according to standard procedures [ 26 ], genomic dna as reported [ 5 ] and phage dna was obtained with lambdasorb phage adsorbent according to the manufacturer &# 39 ; s protocol ( promega ). a cdna library in λ zap ii [ 31 ] was made from mrna isolated from microspores . the cloning kit and the gold packaging extract of stratagene were used according to the manufacturer &# 39 ; s protocols . the cdna library was amplified once prior to screening . differential screening was carried out with 32 p labelled single - stranded cdna probes derived from either mrna from microspores or young leaves . the screening procedure of stratagene was followed at a hybridisation and washing temperature of 55 ° c . the filters were washed in steps of 30 min in ssc with 0 . 1 % ( w / v ) sds ( 1 × ssc = 150 mm nacl , 15 mm na citrate , ph 7 ). the ssc concentration was lowered in steps ; 6 × ssc , twice in 2 × ssc followed by 0 . 5 × ssc and 0 . 2 × ssc . a primary library screening of 2 × 10 − cdna clones was done . the single - stranded cdna probes were made as described [ 251 ] with the following changes : 2 μg mrna , oligo ( dt ), 75 μci [ α - 32 p ] datp ( 3000 ci mmol − 1 ) and 400 units ‘ superscript ’ reverse transcriptase from brl were used to prime and radiolabel the probe . selected plaques were isolated and excised in vivo from the λ zap ii phagemid to form a pbluescript sk - plasmid according to the procedure of stratagene . the genomic library of nicotiana tabacum cv . samsun in bacteriophage λ charon 32 [ 12 ] was a generous gift of dr . r . b . goldberg [ 15 ] , e . coli k802 cells were used as hosts . a cdna of 240 bp comprising part of the 3 ′ end region was used to synthesize a random - primed probe labelled with [ α - 32 p ] datp as described previously [ 7 ]. the screening and hybridisation were carried out using standard techniques [ 26 ]. hybridisation and washings were done at 65 ° c ., with stringent washing up to 0 . 1 × ssc , 0 . 1 % sds . a positive clone of 11 kbp was digested with kpni and xhoi . a digested fragment of 3 kbp containing the sequence of interest was subcloned into the plasmid pgem 7zf + ( promega ) using standard methods [ 26 ]. samples of 25 μg of total rna were denatured , electrophoresed on a 1 % ( w / v ) agarose formaldehyde gel , and capillary - blotted on hybond - n membrane ( amersham ) as reported [ 26 ]. the rrna bands were used as size markers . the rna was bound to the membrane by baking for 2 hours at 80 ° c . the same probe was used as for the screening of the genomic library . hybridisation was carried out overnight at 65 ° c . in 6 × ssc , 5 × denhardt &# 39 ; s reagent , 0 . 5 % sds and 100 μg / ml denatured herring sperm dna . washing was done at the sane temperature during 30 min in 6 × ssc , 0 . 1 % sds followed by 2 × ssc , 0 . 1 % sds . an actin probe was hybridised to the stripped northern blot as a control . southern analysis of 10 μg genomic dna was done after restriction digestion and electrophoresis on a 0 . 8 % agarose gel using standard methods [ 261 ] except the depurination , which was carried out by uv - irradiation . southern blots were hybridised overnight at 60 ° c ., and washed to a stringency of 0 . 4 × ssc at 60 ° c . the probe used in southern analysis was made of a fragment of 250 bp including partial 5 ′ noncoding and 5 ′ coding regions . filters were exposed to kodak x - omat ar film with intensifying screens at − 80 ° c . dna sequencing was performed by the dideoxy chain termination method [ 27 ] with the use of t7 polymerase ( pharmacia ). the genomic clone was subcloned in pgem 7zf + ( promega ). sequencing was performed with part single - stranded and double - stranded template dna [ 41 ]. sequence data were analysed with the pc / gene programme from intelligenetics inc . geneva ( switzerland ). anthers were collected for in situ hybridisation from tobacco flower buds of varying lengths ( 6 , 8 , 12 , 20 , 30 and 48 mm ) covering a broad range of pollen developmental stages , from pollen mother cells to mature pollen . by use of a light microscope , the selected pollen developmental stages are distinguished clearly by their morphological characteristicts visible after a dapi - staining [ 28 ]. the procedure as described by reijnen et al . [ 241 ] was used with the following modifications . prior to fixation the too of the anther was cut to optimise penetration of the fixative . fixation was done for 12 hours and initiad with degassing for 15 min . washing after hybridisation was started with 2 × ssc for 15 min at room temperature and coverslips removed . subsequently , an rnase a treatment 20 μg ml − 1 in 0 . 5 m nacl , 10 mm tris - hcl ( ph 7 . 5 ), 1 mm na 2 ed - ta ) was carried out for 45 min at 37 ° c . the slides were washed twice in 2 × ssc , 1 mm dtt ( dithiotreitol ) for 30 min at 37 ° c ., followed by 0 . 2 × ssc , 1 mm dtt for 1 hour at 45 ° c . the ntm19 rna probes were synthesised by in vitro transcription in sense and antisense orientation from a cdna subclone in pbluescript , that contained a 200 nt part of the coding region . the sense probe was used as a negative control . the protocol of promega was followed with the use of [ 5 , 6 - 3 h ] utp and for each slide 2 × 10 5 c . p . m . were used in hybridisation . after processing of the slides , anthers were stained in 1 μg ml − 1 ethidium bromide for 5 min and washed in distilled water for 20 sec . a confocal laser scanning microscope ( biorad mrc - 500 ) was used to analyse the sections . pictures were made combining those made from a fluorescent image resulting from ethidium bromide staining , with a reflection image that visualized silver grains which are formed if the probe used hybridises to the section . to determine the transcription start site , the primer extension technique was used as previously described [ 341 ]. a 30 - mer primer was chosen 30 nt downstream of the translation start point , with the help of a primer programme ( biosciences inc . plymouth , usa ) and the pc gene programme ( intelligenetics inc ., geneva , switzerland ). the latter programme was used to localise secondary structures in the rna . the longest possible secondary structure was prevented from forming by blocking this region using hybridisation of the 30 - mer primer . microspores of tobacco at an early stage were isolated as described in materials and methods . poly a + rna was prepared from these cells and used to synthesise a cdna library . the primary library contained 1 . 6 × 10 6 recombinants and was amplified once . differential screening was done with cdna from leaves as a negative probe . from the selected microspore - positive and leaf - negative clones , 10 clones were tested for tissue — and pollen developmental stage - specific expression by northern analysis . this analysis resulted in 2 microspore - specific cdna clones ; one of these clones called “ ntm19 ” ( nicotiana tabacum microspore - specific ) was characterized . fig1 shows the results of a northern blot containing total rna prepared from isolated microspores and pollen at different stages of development : hybridised with ntm19 . other plant tissues , namely : pistil , flower bud of which anthers were removed , seedling , leaf , root tips and stem were also included in this analysis . no hybridisation signal was observed in the lanes with rna from the bicellular pollen stages or any other plant tissue . the position of the signal corresponded to a transcript length of about 650 nt . the ntm19 cdna clone has a length of about 500 bp , and is therefore not a full length clone . to complete the sequencing of the transcript a corresponding genomic clone was isolated . the available and presumed 3 ′ non - coding region of cdna clone ntm19 was used as a probe to screen a genomic tobacco library in λ charon 32 . in the non - coding parts of the transcript the restraint on evolutionary divergence is low and therefore this sequence accurately identifies the corresponding gene . the use of a non - coding region as a probe diminishes the chance of isolating a related gene from a gene family . the genomic clone corresponding to the ntm19 cdna clone was successfully isolated using this procedure . after the isolation of hybridising genomic clones , the entire cdna was used to probe restriction fragments of the selected clones . a subclone was made that covered the region of a genomic clone hybridising to the cdna clone . another subclone overlapping the former one and protruding in the 5 ′ direction was also constructed . to identify the ntm19 gene , a sequence analysis of the two genomic subclones and the cdna clone was performed ( fig2 ). the longest open reading frame covering the cdna region was selected . from the cdna region and from the corresponding genomic clone an open reading frame was deduced ranging from position + 40 to + 316 bp , relative to the transcription start , encoding a protein of 92 amino acids . the translation initiation region , 6 nucleotides immediately upstream and 3 nucleotides downstream of the atg start codon , conforms well to the consensus in other plant genes [ 16 ] and thus supported the choice of reading frame . the putative polyadenylation signal [ 12 ] is underlined in fig2 and is at a distance of 20 nucleotides followed by a poly ( a ) tail of 18 nucleotides in the cdna clone . the transcription start was determined by primer extension analysis ( fig3 ). the first transcribed nucleotide is an adenine ( a ) at a distance of 39 nt from the start codon , marked in fig2 by an arrowhead and denoted as position + 1 . this is in accordance with the fact that in plant genes , adenine is the most common nucleotide at the transcription start site [ 13 ]. the putative tata box is 35 nt upstream from the transcription start and underlined in fig2 . the distances between tata box , transcription start and first aug codon fit well within the range determined for a large number of plant genes [ 13 ]. the coding region of the genomic clone is almost identical to that present in the cdna . two nucleotide differences are noted at nucleotide positions + 74 and + 132 , and no intervening sequences were seen . the transcribed region of the genomic clone has a length of 658 bp , including the 18 nt poly ( a ) tail that starts at sequence position + 641 . this is in good agreement to the estimated transcript length of 650 nt determined from the northern blot . therefore it is inferred that no introns are present in the ntm19 gene . database searches did not reveal any significant homology to nucleotide sequences ( genbank , embl ) or polypeptide sequences ( swissprot , pir ). therefore the ntm19 gene represents a novel sequence . the properties of the ntm19 protein were deduced from computer analysis . in fig2 the deduced amino acid sequence of the ntm19 gene is given . the two differences in nucleotide sequence between the genomic and cdna clone result in a change of one amino acid . one c in the genomic clone is replaced by a t in the cdna clone , resulting in a change from alanine in the genomic sequence ( bracketed at amino acid 74 ) to valine in the cdna sequence . because both amino acids have a similar chemical character , this substitution would not greatly change the properties of the protein . the second difference is at nucleotide position 132 , a g in the genomic clone is replaced by a t in the cdna clone without affecting the encoded serine residue ( amino acid position 31 ). the sequence differences between genomic and cdna clone were repeatedly found in independent sequencing experiments . the cdna library was made from the cultivar petit havanna and the genomic clone from the cultivar samsun . this may be the explanation for the sequence differences . the function of the ntm19 protein is unknown . the precise temporal and spatial expression patterns of ntm19 were revealed by in situ hybridisation . this technique allows the accurate discrimination between sporophytic and gametophytic localisation of expression . fig4 shows the localisation of ntm19 mrna in longitudinal sections of tobacco anthers at different developmental stages . the antisense ntm19 rna probe hybridised specifically with the microspore rna and not with tapetum rna . a hybridisation signal was not found in anthers with pollen mother cells undergoing meiosis , nor in tetrads , nor in bicellular pollen . the ntm19 sense rna probe used as a negative control in all experiments gave no hybridisation signal . the genomic clone of ntm19 was digested with bamhi and hincii , and subcloned into bamhi and hincii cut pgem - 3zf (+) ( promega corporation ). the resulting plasmid was digested with kpni and ncoi to release the ntm19 promoter . this fragment was used to replace the kpni / ncoi lat52 promoter fragment from plasmid plat52 - 7 [ 36 ] to give plasmid pmo4 . pmo4 was digested with kpni and saci to release the , ntm19 - gus fusion , which was used for ligation into xpni / saci cut pembl19 (−) ( boehringer mannheim ). the resulting plasmid pembl - ntm19gus was digested with hindiii and saci to release the ntm19 - gus fusion . this fragment was used to replace the hindiii / saci 35s - camv promoter - gus fragment of the binary plasmid pbi121 [ 11 ] to give the binary plasmid pntm19 - gus . pntm19 - gus was transferred from escherichia coli to the agropine agrobacterium tumefaciens strain ag10 [ 17 ] for use in plant transformation experiments . two oligo nucleotide primers were designed to fit the 5 ′- untranslated region of the bp4a gene , spanning nucleotides 4 - 265 [ 1 ], and containing overhanging sali and hindiii restriction sites at the 5 ′- end , and xbai and bamhi restriction sites at the 3 ′- end . the sequences of these primers are as follows . primer 1 : 5 ′- gtc gac aag ctt cta aaa ata gca ata act - 3 ′, seq id no : 4 and primer 2 : 5 ′- gga tcc tct aga aag aga tga agt att cta - 3 ′, seq id no : 5 . after pcr amplification of the bp4a promoter fragment from genomic dna isolated from brassica napus cv ‘ topas ’, the resulting 282 bp dna fragment was digested with hindiii and xbai , and cloned into hindiii / xbai cut pembl19 (−) ( boehringer mannheim ). the resulting plasmid , pembl - bp4 , was used as bp4 promoter source for all further experiments . the sequence of the isolated promoter was determined and compared to the published sequence derived from cv ‘ westar ’ [ 1 ]. two differences were detected : 1 ) a cytosine residue at position 203 was replaced by a guanine , and 2 ) a guanine at position 122 was replaced by cytosine [ 1 ]. the bp4 promoter was exsised from pembl - bp4 with hindiii and xbai . this fragment was used to replace the hindiii / xbai 35s - camv promoter fragment of the binary plasmid pbi121 [ 11 ] to give the binary plasmid pbp4 - gus . pbp4 - gus was transferred from escherichia coli to the agropine agrobacterium tumefaciens strain ag10 for use in plait transformation experiments . a xbai / ecorv fragment from plasmid pwp126 , containing an engineered barnase - barstar operon from bacillus amyloliquefaciens and the camv polyadenylation sequence identical to pwp127 [ 23 ] was inserted into xbai / smai cut pucap to give plasmid pr - barnase [ 38 ]. the sali / ncoi ntm19 promoter fragment from pembl - ntm19gus was ligated into sali / ncoi cut pr - barnase to yield puc - ntm19barnase . the entire ntm19 promoter - barnase - camv polyadenylation sequence cassette was excised from puc - ntm19barnase with asci and paci , and transferred to asci / paci cut pbinplus [ 38 ]. the resulting binary plasmid , pntm19 - barnase was used for plant transformation after transfer to the agropine agrobacterium tumefaciens strain ag10 for use in plant transformation experiments . the bp4 promoter from pembl - bp4 was excised with hindiii / xbai , and transferred to hindiii / xbai cut pucap . the asci / xbai bp4 - promoter fragment from this plasmid was transfered to asci / xbai cut pr - barnase , to give puc - bp4barnase . the entire bp4 promoter - barnase - camv polyadenylation sequence cassette was excised from puc - bp4barnase with asci and paci , and transferred to asci / paci cut pbinplus [ 38 ]. the resulting binary plasmid , pbp4 - barnase was used for plant transformation after transfer to the agropine agrobacterium tumefaciens strain ag10 for use in plant transformation experiments . for allowing functional analysis , the promoter constructs were introduced into tobacco ( nicotiana tabacum cv petit havana sr1 ), using the leaf - disc transformation method reported by [ 9 ]. inocula of agrobacterium tumefaciens ag10 with the various promoter constructs were prepared by overnight culture , until od 1 ( 550 mn ), in liquid lb medium supplemented with 50 mg / l rifampicin + 50 mg / l kanamycin . leaf discs were punched from in vitro grown stock plants and immersed in inoculum diluted to od 0 . 1 for 5 minutes , and then were blotted dry on filter paper . they were co - cultivated upside - down on agar - solidified ms - 20 plates supplemented with 1 mg / l ba and without antibiotics for two days . thereafter , the discs were transferred to the same medium supplemented with 250 mg / l cefotaxime , 200 mg / l vancomycin and 100 mg / l kanamycin . after 4 to 8 weeks , shootlets that developed from the leaf - disc edges were transplanted to ms - 20 plates with the same antibioticum cocktail but without phytohormones , to allow shoot elongation and root formation . plants that rooted on the kanamycin medium were considered to be transgenic , and were transferred into a glasshouse and grown to flowering . kanamycin - resistant plants were analysed for the distribution of β - glucuronidase activity ( gus ) using the method described by [ 11 ] with modifications as reported by [ 37 ]. different plant tissues , including roots , stems and leaves , and all different parts of the flower , taken from a number of independent transgenic plants were analyses . for qualitative assessment of gus activity in the microspores and pollen of successives stages of development ( from microspore release from the tetrad until mature pollen ), these were collected by sectioning anthers in gus - extraction buffer and filtering through a 50 μm nylon mesh sieve . to enable exact determination of the microspore or pollen developmental stages , counter - staining was carried out by adding dapi to a final concentration of 1 . 25 μg / ml followed by incubation at 45 ° c . for 8 - 16 h . microspores or pollen were collected as described above in m 1 s medium [ 42 ], washed once , counted , and pelleted . the pellet was stored frozed (− 80 ° c .) untill used for quantitative enzymatic analysis . gus activity was determined as described previously , using methylumbelliferyl glucuronide ( mug ) as a substrate [ 22 ]. the x - gluc assay is not quantitative , but is used to be able to correlate the β - glucuronidase activity with the precise developmental stage by comparing the x - gluc staining with a dna staining used here for assessing the developmental stage . the ntm19 promoter gave a very strong blue staining from mid - unicellular microspore stage up to mature pollen . when the pollen were germinated on bk - medium staining decreased rapidly and disappeared after 5 hours . other tissues were not stained , except for the top cells or trichomes in some transgenic plants . the bp4 promoter became only active in the mid - bicellular stage and continued to be active up to the mature pollen stage . when the pollen were germinated staining disappeared within 1 hour . the promoter provided a staining which was substantially constant in all stages in which the promoters were active . however , the intensity of the staining produced by ntm19 was much stronger than that produced by bp4 . mug assay was carried out to determine quantitative differences in the amount of gus protein . the activity of the ntm19 and bp4 promoters started at the same stage as observed in the x - gluc assays . the activity of ntm19 increased to a maximum at a bud length of about 16 mm ( late unicellular up to first pollen mitosis ) and subsequently decreased . the microspore mitosis occurs between 16 and 18 mm buds . the bp4 promoter showed some activity from the 18 mm bud length stage , which was only significant from the 22 mm bud length stage . a maximum was reached which continued up to the mature pollen stage . the activity of the bp4 promoter , however , was much lower than that of the ntm19 promoter . fig5 shows the pattern of gus activity . when using the promoter - barnase constructs containing the ntm19 promoter or the bp4 promoter , in both cases transgenic plants were formed . consequently , the ntm19 and bp4 promoters are not active during regeneration . when first callus was made on medium containing 2 , 4 - d or naa 1 mg / l + ba 0 . 25 mg / l and thereafter ba 1 mg / l , plants were formed too . said assay was carried out starting from transgenic plant leaves . as regards the phenotype , when using the ntm19 promoter cells were killed in the mid - unicellular stage . dead microspores are visible between mature pollen and during in vitro pollen germination . when using the bp4 promoter cells were killed only after having reached the mid - bicellular stage . on the basis of the percentages of killed microspores and pollen , respectively , plants having one locus and plants having more loci were found . in all transgenic plants female fertility was maintained . all plants formed seed after self - pollination , but seed production decreased in plants having a high number of introduced copies . said last mentioned plants did form the normal amount of seed after pollination with wild - 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