Patent Application: US-47235895-A

Abstract:
the present invention relates to transgenic plants which produce poly - beta - d - hydroxybutyric acid and related polyhydroxyalkanoates . the production of phb is accomplished by genetically transforming the plants with modified genes from microorganisms . the genes encode the enzymes required to synthesize phb from acetyl - coa or related metabolites . phb is a very useful polymer which is biodegradable .

Description:
prior to setting forth the invention , it is helpful to set forth definitions of certain terms to be used hereinafter . transformation means the process for changing the genotype of a recipient organism by the stable introduction of dna by whatever means . a transgenic plant is a plant which contains dna sequences which are not normally present in the species , but were introduced by transformation . transcription means the formation of an rna chain in accordance with the genetic information contained in the dna . translation means the process whereby the genetic information in an mrna molecule directs the order of specific amino acids during protein synthesis . a promoter is a dna fragment which causes transcription of genetic material . for the purposes described herein , promoter is used to denote dna fragments that permit transcription in plant cells . the camv 35s - promoter is a dna fragment from the cauliflower mosaic virus that causes relatively high levels of transcription in many different tissues of many species of higher plants ( benfey , p . n . and chua , n . h . science 250 : 959 - 966 ( 1990 )). a poly - a addition site is a nucleotide sequence which causes certain enzymes to cleave mrna at a specific site and to add a sequence of adenylic acid residues to the 3 &# 39 ;- end of the mrna . phbc , phba , phbb are the gene symbols given to the a . eutrophus genes for phb synthase , 3 - ketothiolase and acetoacetyl - coa reductase , respectively ( peoples , o . p . and sinskey , a . j ., j . biol . chem . 264 : 15298 - 15303 ( 1989 )). in describing the progeny of transgenic plants , it is useful to adopt a convention which designates how many generations of self - pollination have elapsed since the introduction of dna . herein , we designate the original transformant the to generation . the progeny resulting from self - pollination of this generation is designated the t1 generation and so on . in the case of cross - pollination between two distinct parental plants , the resulting progeny from the initial cross - pollination event is designated the f1 generation . although the experiments discussed hereinafter concern the plant species arabidopsis thaliana ( l .) the process described is generally applicable to any higher plant for which a method of transformation is available . similarly , although the process described herein concerns the use of genes from a . eutrophus , the process described is generally applicable to the use of genes from any organism which is capable of synthesis of phb . it is also clear that , although the process described concerns the production of phb , the procedure is generally applicable to the production of any polyhydroxyalkanoate which is normally produced in microorganisms by the activity of polyhydroxyalkanoate ( pha ) synthase ( which includes phb synthase ), and for which the appropriate hydroxyacyl - coa substrate is produced in the particular plant . the production of phb in progeny of transformed plants requires the completion of a sequence of steps as follows : ( 1 ) the construction of a series of bacterial plasmids containing promoter fusions , ( 2 ) the transfer of these plasmids into agrobacterium tumefaciens , ( 3 ) the use of a . tumefaciens to introduce the genes into cells of the plant ( i . e ., a . thaliana in this example ), ( 4 ) the regeneration of transgenic plants ( 5 ) the selection of plants which are homozygous for the ectopic genes ( 6 ) analysis of the function of the ectopic genes in the transformed plants to ensure that they are expressed and that the gene products are functional ( 7 ) the production of hybrid plants containing two or more different ectopic genes by sexual crosses , ( 8 ) the analysis of the hybrid material for the presence of phb . these steps are described in detail in the following sections . in order to obtain transcription of the bacterial genes in higher plants , the bacterial genes must be modified by the addition of a plant promoter so that they are transcribed when introduced into higher plants . in addition , it is common practice to add a poly - a addition site to the 3 &# 39 ; region of bacterial genes in order to obtain proper expression of the genes in higher plants . both of these requirements were satisfied by cloning the phbc , phba and phbb genes from plasmid ptz18u - phb into the binary ti plasmid vector pbi121 ( clonetech , ca ). the nucleotide sequence of the phbc , phba and phbb genes contained within the plasmid ptz18u - phb is shown in seq id no : 1 . the relevant restriction enzyme sites used in the subclosing of the phb genes is shown in fig2 . a camv 35s - phba gene fusion was constructed by digesting the plasmid ptz18u - phb with restriction enzymes psti and ddei . the 1 . 3 kb restriction fragment containing the coding sequence of the 3 - ketothiolase gene was separated from other dna fragments by agarose gel electrophoresis . the dna fragment was recovered from the agarose using a deae cellulose membrane ( schleicher & amp ; schuell na - 45 deae membrane ). the staggered ends of the dna fragment were filled - in by incubating the purified restriction fragment with t4 dna polymerase and deoxynucleotide triphosphates . the blunt fragment was then cloned into the smai site in plasmid puc18 to produce the plasmid puc - thio . the 1 . 3 kb restriction fragment was excised from puc - thio plasmid by digestion with bamhi and saci , purified by electrophoresis using a deae cellulose membrane and ligated into plasmid pbi121 which had been previously digested with the same two enzymes . the resulting plasmid , designated pbi - thio , was found to have the a . eutrophus 3 - ketothiolase gene in the correct orientation , relative to the camv 35s promoter and poly - a addition site of pbi121 , so that it would be expected to be expressed in higher plants . a schematic summary of the steps involved in construction of pbi - thio is presented as fig3 . a camv 35s - phbc gene fusion was constructed by digesting the plasmid ptz18u - phb with restriction enzymes bstbi and tthllli . the 1 . 9 kb restriction fragment containing the coding sequence of the phb synthase gene was separated from other dna fragments by agarose gel electrophoresis . the dna fragment was recovered from the agarose using a deae cellulose membrane . the staggered ends of the dna fragment were filled in by incubating the purified restriction fragment with t4 dna polymerase and deoxynucleotide triphosphates . the blunt fragment was then cloned into the smai site in plasmid puc18 to produce plasmid puc - syn . the 1 . 9 kb restriction fragment was excised from puc - syn by complete digestion with bamhi and partial digestion with saci , purified by electrophoresis using deae cellulose membranes and ligated into plasmid pbi121 which had been previously digested with the same two enzymes . the resulting plasmid , designated pbi - syn , was found to have the a . eutrophus phb synthase gene in the correct orientation , relative to the camv 35s promoter and poly - a addition site of pbi121 , so that it would be expected to be expressed in higher plants . a schematic summary of the steps involved in construction of pbi - syn is presented in fig4 . a camv 35s - phbb gene fusion was constructed by using a pair of synthetic oligonucleotides for primers in a polymerase chain reaction ( pcr ) to amplify the phbb gene from plasmid ptz18u - phb . the sequence of the oligonucleotide primers is presented in fig5 where they are designated pcr primer # 1 and pcr primer # 2 . the oligonucleotides were designed in such a way that the amplified dna sequence contained a synthetic bamhi site near the 5 &# 39 ;- end of the coding sequence and a synthetic kpni site at the 3 &# 39 ;- end of the sequence . the 790 base pair product of the polymerase chain reaction was separated and purified from agarose gel , restricted with bamhi and kpni and ligated into plasmid puc18 , which was previously restricted with the same two enzymes , to produce plasmid puc - red . the restriction fragment was excised from puc - red by digestion with bamhi and saci , purified by electrophoresis using a deae cellulose membrane and ligated into plasmid pbi121 which had been previously digested with the same two enzymes . the resulting plasmid , designated pbi - red , was found to have the a . eutrophus acetoacetyl - coa reductase gene in the correct orientation , relative to the camv 35s promoter and poly - a addition site of pbi121 , so that it would be expected to be expressed in higher plants . a schematic summary of the steps involved in construction of pbi - red is presented as fig5 . the plasmids pbi - syn , pbi - thio and pbi - red were transferred into agrobacterium tumefaciens strain c58 pgv3850 by electroporation . plasmid containing colonies were recovered by selection for expression of the kanamycin resistance gene present on the parental plasmid pbi121 . cells of a . thaliana were transformed by incubating sterile root tissue with cultures of a . tumefaciens carrying the recombinant binary ti plasmids described in the previous section . roots from sterile seedlings of a . thaliana race rschew were transformed as described by valvekens , d . et al ., proc . natl . acad . sci . usa 85 : 5536 - 5540 ( 1988 ). each of the three strains of a . tumefaciens carrying one of the modified phb genes was used to infect a . thaliana root pieces . this resulted in the recovery of approximately 50 kanamycin resistant callus tissues in each case . of these , 10 - 25 % gave rise to fertile shoots which produced seeds . each plant which produced seeds was assigned a different number to indicate that it represented a distinct transformation event . a total of 11 putative transgenic plants were recovered from tissues treated with a . tumefacienes carrying the plasmid pbi - red . these designated redb - 2a , - 2b , - 2c , - 2d , - 2e , - 2f , - 2g , - 2h , - 3a , - 5a and redd - 3a . all these transgenic plant lines , except redb - 2f , - 2h and - 5a , were analyzed in detail as described in the following sections . a total of 5 putative transgenic plants were recovered from tissues treated with a . tumefacienes carrying the plasmid pbi - thio . these were designated t3 - 2a , t4 - 2a , t4 - 3a , t4 - 3b and t4 - 3c . all these transgenic plant lines , except t4 - 3c , were analyzed in detail as described in the following sections . a total of 4 putative transgenic plants were recovered from tissue treated with a . tumefacienes carrying the plasmid pbi - syn . these were designated s8 - 1 - 2a , s8 - 1 - 2c , s12 - 3a and sspuc - 2a . all these trangenic plant lines were analyzed in detail as described in the following sections . the presence of t - dna in the putative transgenic plants was verified by sowing seed from the transgenic plants on agar - solidified mineral medium containing 50 μg / ml of kanamycin . this concentration of kanamycin prevents the growth of untransformed a . thaliana plants but permits plants containing the nptii gene carried on pbi121 or pbi121 - derived plasmids to grow normally . the seeds from transgenic plants t4 - 2a , redd - 3a and s8 - 1 - 2a are available from the american type culture collection , rockville , md 20852 . a minimum criterion used to produce homozygous transgenic lines was that all the progeny from an homozygous plant are expected to be kanamycin resistant . because the presence of multiple ectopic copies of the nptii gene from pbi121 at different locations in the genome may cause a similar phenotype , this criterion is most useful when the primary transformation event involves insertion of t - dna into only one chromosomal location . in order to identify putative homozygous lines , several kanamycin resistant t1 plants from each transgenic line were grown to maturity in reproductive isolation . the frequency of kanamycin resistance was then determined in samples of approximately 50 t2 seed from each line . if all of the t2 seed from a particular plant were kanamycin resistant , the line was provisionally considered to be homozygous . in order to verify the proper integration of the phb genes in the various trangenic plant lines produced , the genomic dna of the trangenic plants was analyzed . high molecular weight dna from control untransformed plants and from t3 transgenic plants transformed with the plasmids pbi - thio , pbi - red and pbi - syn was isolated . the dnas were digested with the restriction enzymes hindiii , the fragments separated by agarose gel electrophoresis and transferred onto a nylon filter . the restriction enzyme hindiii cuts only once at the 5 &# 39 ; end of the camv 35s promoter in plasmids pbi - thio , pbi - red and pbi - syn ( fig3 and 5 ). fragments detected using phb gene specific probes should therefore represent junction fragments of the ti vectors with the plant genomic dna , or internal fragments of concatamerized ti vectors . the inserts in plasmids puc - thio , pcu - red and puc - syn were excised by treatment with ecori and hindiii , purified by agarose gel electrophoresis using deae cellulose membranes and labeled with 32 p - deoxyribonucleotides by random priming . the labeled phb gene fragments were then used to probe the nylon filters . the filters were hybridized and subsequently washed under high stringency conditions . the result of these filter hybridizations is shown in fig6 . none of the three phb genes can be detected in untransformed control plants ( fig6 a , b and c , lane a ). the phba gene was detected in four of the transgenic lines produced by transformation with the plasmid pbi - thio ( fig6 a , lanes b to e ). the phbb gene was detected in seven of the transgenic plants produced by transformation with the plasmid pbi - red ( fig6 b , lanes f to l ). finally , the phbc gene was detected in three of the transgenic plants produced by transformation with the plasmid pbi - syn ( fig6 c , lanes m to p ). although the plant line s12 - 3a was resistant to 50 μg / ml of kanamycin , suggesting the integration of the nptii gene , no phbc gene could be detected . it is likely that only the fragment of the ti vector harboring the nptii gene , and not the phbc gene , was integrated in the genomic dna of plant line s12 - 3a . in order to determine if the a . eutrophus phb genes were expressed in the various transgenic lines , the cloned genes present in plasmids puc - thio , puc - red and puc - syn were used as probes in filter hybridization experiments . total rna was extracted from untransformed control and t3 transgenic plants . the rna was resolved by electrophoresis in formaldehyde - containing agarose gels and transferred to nylon filters by established procedures . the inserts of plasmids puc - thio , puc - red and puc - syn were excised by treatment with ecori and hind iii , purified by electrophoresis using deae cellulose membranes and labeled with 32 p - deoxyribonucleotides by random priming . the labeled phb genes were used to probe the nylon filters . these experiments showed that none of the three phb probes hybridized to any rna in the untransformed control plant ( fig7 lane e , j and r ). by contrast , transgenic plants produced by transformation with pbi - thio had rna of 1 . 6 kbp which was complementary to the 3 - ketothiolase gene ( fig7 a , lanes a to d ). the camv 35s promoter and poly - a addition sequences present on the pbi121 - derived plasmids contribute approximately 300 bp to the final length of the mrnas produced from the phb fusion genes . the level of 3 - ketothiolase mrna was low in plant line t4 - 3a relative to the other plant lines . similarly , three of the transgenic lines produced by transformation with pbi - syn had mrna of 2 . 1 kbp corresponding to the phb synthase gene ( fig7 b , lanes f , g , and i ). transgenic line s12 - 3a had no detectable mrna hybridizing to the phbc probe ( fig7 b , lane h ). this result is in accordance with the southern blot analysis showing no integration of the phbc gene in the genomic dna of line s12 - 3a ( fig6 c , lane m ). finally , seven transgenic lines produced by transformation with the plamid pbi - red had mrna of 1 . 1 kbp which was complementary to the acetoacetyl - coa reductase gene ( fig7 c , lanes k to q ). thus , for each of the three phb genes , at least three independent transgenic plants were obtained which expressed complementary rna of the expected size . although the presence of rna indicates that the genes are transcribed , it does not provide any information that they are translated or that the translation product is functional . this was examined by assaying the transgenic plants for enzyme activity . transgenic plants produced by transformation with pbi - thio were assayed for 3 - ketothiolase activity by minor modifications of the assay described by senior , p . j . and dawes , e . a ., biochem . j . 134 : 225 - 238 ( 1973 ). frozen leaf tissues from t3 plants were homogenized in tris buffer and the clarified crude extracts were assayed for 3 - ketothiolase activity . the results of these experiments are presented in table 1 . extracts from untransformed a . thaliana plants had very low levels of 3 - ketothiolase activity under the assay conditions . by contrast , each of the transgenic plants found to transcribe the phba gene had substantially increased levels of thiolase activity . this indicated that the bacterial thiolase gene is functional when expressed in transgenic plants . however , the specific activity of 3 - ketothiolase detected in the various transgenic plants was significantly lower compared to extracts prepared from e . coli harboring the phba gene on the plasmid ptz18u - phb . table 1______________________________________levels of 3 - ketothiolase activity in a . thaliana transgenic plantssample 3 - ketothiolase activity . sup . a______________________________________dh5alpha / phb . sup . b 9 . 5wild type a . thaliana 0 . 019t4 - 3a transgenic 0 . 057t3 - 2a transgenic 0 . 42t4 - 2a transgenic 0 . 43t4 - 3b transgenic 0 . 54______________________________________ . sup . a micromoles of acetoacetylcoa degraded per minute per milligram of protein . values are an average of two to four measurements . . sup . b e . coli dh5alpha containing the plasmid ptz18uphb harboring the ph operon . transgenic plants produced by transformation with plasmid pbi - red were assayed for acetoacetyl - coa reductase activity by minor modifications of the assay described by senior , p . j . and dawes , e . a ., biochem . j . 134 : 225 - 238 ( 1973 ). leaves from t3 plants were homogenized in potassium phosphate buffer and the clarified extracts were assayed for acetoacetyl - coa reductase activity . the results of these experiments are presented in table 2 . extracts from untransformed a . thaliana plants had undetectable levels of acetoacetyl - coa reductase activity under the assay conditions . by contrast , each of the transgenic plants found to transcribe the phbb gene had high levels of acetoacetyl - coa reductase activity . this indicates that the bacterial acetoacetyl - coa reductase gene is functional when expressed in transgenic plants . furthermore , the specific activity of acetoacetyl - coa reductase detected in six of the seven transgenic plants analyzed was significantly higher than in extracts from e . coli harboring the phbb genes on the plasmid ptz18u - phb . table 2______________________________________levels of acetoacetyl - coa reductase in a . thaliana transgenic plantssample acetoacetyl - coa reductase activity . sup . a______________________________________dh5alpha / phb . sup . b 1 . 4wild type a . thaliana & lt ; 0 . 03redb - 2a transgenic 12 . 5redb - 2b transgenic 16 . 2redb - 2c transgenic 9 . 1redb - 2d transgenic 8 . 8redb - 2e transgenic 1 . 6redb - 2g transgenic 5 . 2redd - 3a transgenic 2 . 3______________________________________ . sup . a micromoles of nadph reduced per minute per milligram of protein . values are an average of two to four measurements . . sup . b e . coli dh5alpha containing the plasmid ptz18uphb harboring the ph operon . transgenic plants obtained by transformation with the plasmid pbi - syn were not assayed for the presence of phb synthase activity because of technical difficulties in measuring the activity of this enzyme in the absence of thiolase and reductase activities . because higher plants contain an endogenous cytoplasmic 3 - ketothiolase activity , the only additional enzymes required to produce phb are acetoacetyl - coa reductase and phb synthase . these two genes were introduced into the same plant by cross - pollinating a transgenic line which was judged to be homozygous for the acetoacetyl - coa reductase gene with a transgenic line which was judged to be homozygous for the phb synthase gene . the hybrid seeds resulting from these crosses were grown in soil for two to three weeks before assaying for the presence of phb . in order to determine if the presence in plants of the acetoacetyl - coa reductase and phb synthase genes was sufficient for production and accumulation of phb , extracts of chloroform - soluble material were made from control plants and hybrid plants containing both of these genes . the presence of phb within these extracts was analyzed by gas chromatography ( gc ). two methods were used to prepare plant extracts for gc analysis . these methods exploit both the highly polymerized nature of phb ( 10 6 daltons on average for phb produced from a . eutrophus ) and its selective solubility in chlorinated hydrocarbons such as chloroform . briefly , in method # 1 , whole leaves are placed in a 1 : 1 solution of chloroform and water and shaken by inversion for 16 hours at 65 ° c . because molecules larger than approximately 50 , 000 daltons cannot pass through the plant cell wall , only low molecular weight water or chloroform - soluble products are extracted from the leaves under these conditions . the putative high molecular weight phb is then extracted from the leaves by homogenizing the remaining tissue to disrupt the cell wall , and re - extracting it in a solution of 1 : 1 chloroform and water for 12 hours at 65 ° c . in method # 2 , whole leaf samples are successively extracted for 2 hours at 55 ° c . in 50 % ethanol , 2 hours at 55 ° c . in 100 % methanol and 15 minutes at 20 ° c . in 100 % diethyl ether . the remaining tissue is then homogenized and extracted in chloroform at 100 ° c . for 4 hours . the products present in the final chloroform extract obtained from both of these methods were transesterified with ethanol and hydrochloric acid and analyzed by gas chromatography . the retention time of the transesterified plant products were compared to transesterified commercial phb purified from a . eutrophus ( sigma chemical co ., mo ). transgenic plants s8 - 1 - 2a and / or s8 - 1 - 2c were cross - pollinated with transgenic plants redb - 2a , - 2c , - 2d , - 2g and redd - 3a . the resulting f1 seeds were sowed in soil and leaf samples or whole shoots of 2 - 3 week - old plants were collected and analyzed for the presence of phb . an example of the results obtained using purification method # 1 are shown in fig8 . a product present in the extracts of f1 plants having both the acetoacetyl - coa reductase and the phb synthase transgenes has a retention time identical to ethyl - hydroxybutyrate , as determined by comparison with the retention time of the transesterified product of commercial phb . this new product , tentatively identified as ethyl - hydroxybutyrate , was only detected in f1 hybrid plants having both an active acetoacetyl - coa reductase transgene and a phb synthase transgene . a similar product could not be detected in transgenic plants having only one of the above mentioned genes or in untransformed a . thaliana plants . furthermore , this product could not be detected in chloroform extracts of plant tissues which had not been previously homogenized . this indicates that the ethyl - hydroxybutyrate is derived from a high molecular weight precursor . the identity of the new plant product having a retention time identical to ethyl - hydroxybutyrate was analyzed by gas chromatography - mass spectrometry ( gc - ms ). gc - ms analysis was performed by the msu - nih mass spectrometry facility . fig9 a shows the mass spectrometric spectra of ethyl - hdydroxybutyrate prepared from authentic phb . fig9 b shows the mass spectrum of the putative ethyl - hydroxybutyrate extracted from an f1 hybrid plant which resulted from a cross between transgenic plants s8 - 1 - 2a and redb - 2c . the results indicated that the new plant product eluting with the same retention time as ethyl - hdyroxybutyrate also has the same fragmentation pattern as an authentic sample of ethyl - hyroxybutyrate . the fact that this new product can only be detected in extracts from leaf tissue which has previously been homogenized indicates that the ethyl - hydroxybutyrate is derived from material having a molecular weight greater than approximately 50 , 000 daltons ( the approximate porosity of plant cell walls ). together , these data indicate that transgenic plants containing both the acetoacetyl - coa reductase and the phb synthase genes accumulate polyhydroxybutyrate . table 3 shows a summary of the f1 plants that were analyzed by gc and gc - ms . based on the gc analysis , the amount of phb accumulated in leaves of f1 hybrids ranged from approximately 5 μg of phb per gram of fresh weight of leaves for f1 hybrids between redd - 3a and s8 - 1 - 2c , to approximately 100 μg of phb per gram fresh weight of leaves from f1 hybrid between redb - 2c and s8 - 1 - 2a . table 3______________________________________summary of evidence for production of phb in f1 hybrid plants parental transgenic line . sup . aparental transgenic line . sup . a s8 - 1 - 2c s8 - 1 - 2a______________________________________redd - 3a gc . sup . b ms . sup . c tem . sup . d ( leaf ) redb - 2a gc tem ( seed ) tem ( leaf , seed ) redb - 2g gc ms tem ( leaf ) redb - 2c gc ms tem ( leaf ) redb - 2d tem ( leaf ) ______________________________________ . sup . a transgenic lines harboring the acetoacetylcoa reductase transgene were crosspollinated with transgenic lines harboring the phb synthase . th resulting f1 hybrids were analyzed for production of phb . . sup . b evidence for production of phb by gas chromatography analysis . sup . c evidence for production of phb by gas chromatographymass spectrometry . . sup . d detection of electronlucent granules by transmission electron microscopy . in parenthesis is indicated the plant tissue analyzed . . sup . e all blank spaces indicate that the analysis has not been performed transmission electron microscopy ( tem ) of bacteria accumulating phb revealed electron - lucent granules of 0 . 2 to 0 . 5 μm in diameter surrounded by a membrane coat of about 2 nm thick ( lundgren , d . g ., pfister , r . m . and merrick , j . m ., j . gert . microbiol . 34 : 441 - 446 ( 1964 )). to determine if similar granules could be detected in hybrid plants shown to be positive for phb production by gc - ms analysis , plant tissues were examined by transmission electron microscopy . transgenic plants s8 - 1 - 2a and / or s8 - 1 - 2c were cross - pollinated to transgenic redb - 2a , - 2c , - 2d , - 2g and redd - 3a . the resulting f1 hybrid seeds and mature leaf material were fixed for analysis by transmission electron microscopy . briefly , tissues were fixed in 3 % glutaraldehyde and 1 % osmium tetroxide and embedded in epoxy resin . sections of 80 - 90 nm were stained with 5 % uranyl acetate and lead citrate . in one series of experiments , the f1 seeds were sowed in soil and leaves from 2 - 3 week - old plants were collected for tem analysis . inspection of the cells present in the leaves revealed the presence of agglomerations of electron - lucent granules . these granules were detected in all analyzed f1 hybrid plants resulting from crosses between transgenics having the phb synthase gene and transgenics having the acetoacetyl - coa reductase gene . examples are shown in fig1 a through 10f . similar granules were never detected in the parental transgenic lines having only the phb synthase or the acetoacetyl - coa reductase genes , nor was it detected in untransformed a . thaliana . in f1 hybrid leaf tissues , the granules were detected in mesophyll cells ( fig1 a to 10e ). the agglomerate of electron - lucent granules were detected most frequently in the nucleus ( fig1 a to 10c ), but similar structures were also detected in the cytoplasm ( fig1 e ) and the vacuole ( fig1 d ) of the f1 hybrid leaf tissues . in the nucleus and cytoplasm , individual granules could reach a maximum size of approximately 0 . 18 μm . in the vacuoles , the granules were generally larger , reaching a maximum diameter of approximately 0 . 55 μm . at higher magnification , the nuclear granules appear to be surrounded by electron - dense material . both the size and apparent structure of these granules are very similar to granules observed in bacteria which accumulate phb . in a second series of experiments , f1 seeds were soaked in water for 24 hours , the embryos were dissected out of the seed coat and tissues were fixed . analysis of the embryonic cotyledons revealed the presence of agglomeration of electron - lucent granules in the nucleus ( fig1 f ). the granules could reach a maximum diameter of 0 . 18 μm . these granules could only be detected in the nucleus of f1 hybrid embryos resulting from crosses between transgenics having the phb synthase gene and transgenics having the acetoacetyl - coa reductase gene . no granules could be detected in either of the parental transgenic plants having only one of the ectopic genes , or in untransformed wild type a . thaliana . table 3 shows a summary of the f1 plants that were analyzed by tem . the data described above show a positive correlation between detection of phb by gc - ms and the presence of granules at the electron microscope level . the size , shape and presence of electron - dense material surrounding the individual granules very closely resembles the granules present in bacteria producing phb . finally , both the detection of phb by gc - ms and the presence of electron - lucent granules are only observed in hybrid plants possessing both the acetoacetyl - coa reductase and the phb synthase transgenes . together , these data indicate that the granules observed in f1 hybrid plants are composed of polyhydroxybutyrate . in these studies , it has been demonstrated that bacterial genes encoding enzymes required for phb synthesis can be stably introduced into a higher plant in such a way that the genes are transcribed and produce transcripts of the expected size . it was further shown that , in the case of the phba and phbb genes , the presence of these genes in transgenic plants confers an increase in the level of 3 - ketothiolase or acetoacetyl - coa reductase enzyme activity , respectively . thus , it is clear that these two gene products are functional when translated in the plant . because of technical difficulties associated with assaying phb synthase activity directly , the amount of phb synthase activity in the transgenic plants was not determined . it was shown that only plant extracts from f1 transgenic plants expressing both the acetoacetyl - coa reductase and phb synthase produce a new high molecular weight chloroform - soluble compound , which upon transesterification with ethanol and hydrochloric acid , produces ethyl - hydroxybutyrate . these data indicate that the new compound is polyhydroxybutyrate . in addition , these data are an indirect evidence for the production of a functional phb synthase in transgenic plants . this is important since an in vitro assay for the phb synthase activity could not be performed . furthermore , production of phb also indirectly indicate that d (-)- hyroxybutyryl - coa , the substrate for the phb synthase , is produced in plants . this hydroxyacyl - coa is not naturally found in plants . transmission electron microscopy further substantiates the claim that phb is produced in transgenic plants . analysis of embryonic cotyledons and mature leaves of f1 transgenic plants expressing both the acetoacetyl - coa reductase and the phb synthase revealed agglomerates of electron - lucent granules having a size and structure very similar to granules found in bacteria producing phb , such as a . eutrophus . these granules were found most frequently in the nucleus , but were also detected in the vacuole and the cytoplasm of f1 hybrid plants . in the experiments described in this work , the products of the phba , phbb , and phbc genes from a . eutrophus are most likely expressed in the cytoplasm , since no specific amino acid sequences were added to the proteins to target them specifically into any organelles . since the cytoplasm of plant cells already contains a 3 - ketothiolase , only the additional expression of the acetoactyl - coa reductase and phb synthase was required to produce phb . the fact that granules are found in the nucleus and vacuoles is not necessarily contradictory with the expression of the enzymes in the cytoplasm . since nuclear membranes dissemble and reassemble during cell division , phb granules initially produced in the cytoplasm could be entrapped within the newly reforming membranes of the nucleus . alternatively , because of their hydrophobicity , phb granules could pass through the membranes of the nucleus or vacuole . in an alternative approach , phb production could be localized to a specific plant cell organelle through targeted expression of the enzymes involved in phb synthesis to the organelle . in this case , if the targeted organelle does not express an active 3 - ketothiolase , expression of an exogenous 3 - ketothiolase activity would be required , in addition to the acetoacetyl - coa reductase and the phb synthase , for the production of phb . the long term goal of phb or pha production in higher plants is to divert carbon away from major storage compounds such as lipid , starch or terpenoids , to channel it towards pha synthesis . this goal will require tissue - specific expression as well as potentially organelle - specific expression of the enzymes involved in pha synthesis . oil producing crops are likely targets for genetic engineering . lipids are synthesized in the plastid using acetyl - coa , the same precursor used in synthesis of phb and other pha . therefore , genetic engineering of oil crops will require targeting the pha biosynthetic enzymes into the plastid . examples of oil crops that could be engineered for pha production are rapeseed , sunflower and oil palm . rapeseed and sunflower are major crops in north america and can be transformed with foreign dna . alternatively , pha production could be targeted into the mesocarp of the oil palm fruit . because lipids produced in the mesocarp are not essential for the survival of the tree or the embryo , the production of pha should have no deleterious effects on palm trees . unfortunately , no transformation techniques are yet available for oil palm . pha production could also be targeted to the roots and tubers of sugar beets and potatoes , crops which accumulate large amounts of starch . the major problem with this approach is that since starch and pha do not use the same precursors , potentially multiple modifications in carbon metabolism will be required before carbon could be diverted away from starch into pha . possibly the most direct approach to pha production would be to use crops accumulating large amounts of terpenoids , such as carrot which accumulates carotenoids , or the mexican yam which accumulates sterols . since terpenoids use the same precursors as pha ( acetyl - coa and acetoacetyl - coa ), diverting carbon into pha production could be more easily achieved . e . coli strain dh5alpha harboring plasmids were grown in lb broth supplemented with kanamycin ( 50 μg / ml ) or ampicillin ( 50 μg / ml ). large - scale preparations of plasmid dna was done by the alkaline lysis and polyethylene glycol precipitation procedure as described by sambrook , j ., fritsch e . f . and maniatis , t ., molecular cloning , a laboratory manual , cold spring harbor laboratory press ( 1989 ). plasmid dna was cleaved with restriction endonucleases according to the manufacturer &# 39 ; s recommendations ( new england biolabs , mass ; promega corp ., wi ; boehringer mannheim biochemicals , in ; stratagene , ca ), separated by agarose gel electrophoresis and visualized by ethidium bromide staining as described by sambrook , j ., fritsch , e . f . and maniatis , t ., molecular cloning , a laboratory manual , cold spring harbor laboratory press ( 1989 ). the dna fragments were recovered from the agarose gel with deae membranes ( na - 45 deae membrane , schleicher and schuell , inc ., nh ). briefly , dna is electrophoresed onto a strip of na - 45 and the membrane is washed in 0 . 15m nacl , 0 . 1 mm edta and 20 mm tris - hcl ( ph 8 ). the dna is then eluted in 1 . 0m nacl , 0 . 1 mm edta and 20 mm tris - hcl ( ph 8 ) at 65 ° c . for 1 to 2 hours . the dna is further purified by phenol - chloroform extraction and ethanol precipitation . in some experiments , the recessed 3 &# 39 ; termini of dna fragments were converted into blunt ends with t4 dna polymerase using the protocol described in sambrook , j ., fritsch , e . f . and maniatis , t ., molecular cloning , a laboratory manual , cold spring harbor laboratory press ( 1989 ). ligation of dna fragments with cohesive or blunt ends was done at 14 ° c . for 16 hours in buffer containing 50 mm tris - hcl ( ph 7 . 6 ), 5 mm mgcl 2 , 5 % ( w / v ) polyethylene glycol 8000 , 0 . 5 mm atp and 5 mm dithiothreitol as described by sambrook , j ., fritsch , e . f . and maniatis , t ., molecular cloning , a laboratory manual , cold spring harbor laboratory press ( 1989 ). a fraction of the ligation reaction was transferred into e . coli by the rubidum chloride method as described by hanahan , d ., j . mol . biol . 166 : 557 - 580 ( 1983 ). the transformed bacteria were plated on agar plates containing lb broth and either 50 μg / ml kanamycin or 50 μg / ml ampicillin . bacterial colonies containing recombinant plasmids were identified by hybridization with 32 p - labeled dna probes as described by sambrook , j ., fritsch , e . f . and maniatis , t ., molecular cloning , a laboratory manual , cold spring harbor laboratory press ( 1989 ), except that nylon membranes ( hybond - n , amersham , il ) were used instead of nitrocellulose membranes . preparation of radiolabeled dna probes and hybridization are described in a following section . small - scale preparation of plasmid dna was done by the alkaline lysis method as described by sambrook , j ., fritsch , e . f . and maniatis , t ., molecular cloning , a laboratory manual , cold spring harbor laboratory press ( 1989 ). oligonucleotides were synthesized on an applied biosystems 380a dna synthesizer according to the manufacturer &# 39 ; s instructions ( applied biosystems , ca ). the oligonucleotides with a dimethoxytrityl group attached to the 5 &# 39 ; ends were purified on a varian 5000 hplc equipped with a c18 column ( varian instrument group , tx ). the oligonucleotides were resuspended in 0 . 1m triethylamine and injected onto a c18 column preequilibrated with 12 % acetonitrile / 88 % 0 . 1m triethylamine - acetate ( ph7 ) ( solvent a ). the hplc program was set as follows : flow rate , 0 . 9 ml / min ; maximum pressure , 200 psi ; time 0 min , 88 % solvent a / 12 % solvent b ( acetonitrile ); time 3 min , 88 % solvent a / 12 % solvent b ; time 21 min , 65 % solvent a / 35 % solvent b ; time 25 min , 65 % solvent a / 35 % solvent b . the purified oligonucleotides were detritylated in 80 % acetic acid for 10 min and dried under nitrogen . the oligonucleotides were dissolved in 10 mm tris - hcl ( ph 7 . 5 ) and 0 . 1 mm edta , extracted three times with equal volumes of ethyl acetate and precipitated with ethanol . polymerase chain reaction ( pcr ) was performed using a perkin - elmer cetus dna thermal cycler ( perkin - elmer , ct ). the reaction mixture contained 100 pmoles of oligonucleotides , pct primer # 1 and # 2 ( see fig5 ), 200 ng of plasmid ptz18u - phb linearized with the restriction enzyme ecori , 125 μm dntp , 50 mm kcl , 10 mm tris - hcl ( ph 8 . 3 ), 2 . 5 mm mgcl 2 , 0 . 01 % gelatin and 2 . 5 units of taq polymerase ( perkin - elmer , ct ). the dna thermal cycler program was as follows : 3 min at 94 ° c ., 40 cycles of the sequence 1 min at 94 ° c .-- 3 min at 55 ° c .-- 3 min at 72 ° c ., and finally 7 min at 72 ° c . the pcr product was isolated by agarose gel electrophoresis and elution with deae cellulose membrane . the ti plasmid vectors used to produce transgenic plants were first transferred into agrobacterium tumefaciens strain c58 - pgv3850 by electroporation ( zabrisky , p . et al ., embo 2 : 2143 - 2150 ( 1983 ); and sambrook , j ., fritsch , e . f . and maniatis , t ., molecular cloning , a laboratory manual , cold spring harbor laboratory press ( 1989 )). arabidopsis thaliana race rschew were grown aseptically on vertical petri plates containing mineral elements , 0 . 8 % agar ( difco ) and 1 % sucrose as described by estelle , m . a . and somerville , c ., mol . gen . genet . 206 : 200 - 206 ( 1987 ) and schiefelbein , j . w . and somerville , c . r ., plant cell 2 : 235 - 243 ( 1990 ). roots from 10 to 12 day - old plants were excised and used for transformation as described by valvekens , d ., van montagu , m . and van lijsebettens , m ., proc . natl . acad . sci usa 85 : 5536 - 5540 ( 1988 ). seeds from t0 and t1 transgenics plants were grown on media containing mineral elements , 1 % sucrose , 0 . 8 % agar ( difco ) and 50 μg / ml kanamycin . after 10 to 14 days of growth , kanamycin resistant ( km r ) transgenic plants had green leaves while untransformed kanamycin sensitive ( km s ) plants had yellow leaves . at this stage , km r plants could be removed from the agar plates and transplanted into fertilized soil . wild type and transgenic plants were grown in soil for 2 to 3 weeks and approximately 5 g of leaf material was collected and frozen in liquid nitrogen . high molecular weight dna was extracted from the frozen plant tissues as described by rogers , s . c . and bendich , a . j ., plant molecular biology manual a6 : 1 - 10 ( 1988 ). restriction endonuclease cleavage with the enzyme hindiii was performed under the conditions recommended by the manufacturer ( new england biolabs inc ., mass ). dna analysis by agarose gel electrophoresis and transfer to nylon membranes ( hybond - n , amersham , il ) were done using established procedures described by southern , e . m ., j . mol . biol . 38 : 503 - 517 ( 1975 ) and sambrook , j ., fritsch , e . f . and maniatis , t ., molecular cloning , a laboratory manual , cold spring harbor laboratory press ( 1989 ). specific cloned dna fragments to be used as probes were excised from the vector with appropriate restriction endonucleases , the inserts were purified from the vector by agarose gel electrophoresis and electroelution using deae cellulose membranes . probes were labeled with 32 p - deoxyribonucleotides by the random primer extension method using hexamers as described by feinberg , a . p . and volgelstein , b ., anal . biochem . 136 : 6 - 13 ( 1983 ). nylon filters were hybridized with labeled probes and exposed on film as described by poirier , y . and jolicoeur , p ., j . virol . 63 : 2088 - 2098 ( 1989 ). total rna was isolated from frozen leaf samples as described by puissant , c . and houdebine , l . m ., biotechniques 8 : 148 - 149 ( 1990 ). the isolated rna was separated by electrophoresis in agarose gel containing formaldehyde and transferred onto nylon membranes ( hybond - n , amersham , il ) as described by sambrook , j ., fritsch , e . f . and maniatis , t ., molecular cloning , a laboratory manual , cold spring harbor laboratory press ( 1989 ). the nylon filters were hybridized with labeled probes as described in the previous section . one gram of frozen leaf samples were homogenized in 2 ml of ice - cold buffer containing 100 mm tris - hcl ( ph 8 . 0 ), 40 mm mgcl 2 and 5 mm beta - mercaptoethanol . the homogenate was clarified by centrifugation at 10000 × g for 2 min and the supernatant transferred to a fresh tube . the protein content of the extract was measured by the bradford assay using the biorad protein assay kit ( biorad laboratories , ca ). between 3 to 30 μg of plant protein extracts were used per assay . protein extracts were also prepared from bacteria . in this case , stationary cultures of bacteria were pelleted by centrifugation , washed once with ice - cold assay buffer and resuspended in 200 μl of the same buffer . the bacterial suspension was lysed by sonication , the homogenate clarified by centrifugation and the protein content of the extract determined by the bradford assay . between 0 . 2 to 1 μg of bacterial protein extract was used per assay . activity of the 3 - ketothiolase enzyme in the different extracts was assayed according to the procedure of senior , p . j . and dawes , e . a ., biochem . j . 134 : 225 - 238 ( 1973 ). one gram of frozen leaf samples were homogenized in 2 ml of ice - cold buffer containing 100 mm kh 2 po 4 ( oh 5 . 5 ), 0 . 02 mm mgcl 2 and 4 . 0 mm beta - mercaptoethanol . the homogenate was clarified by centrifugation at 10000 × g for 2 min and the supernatant transferred to a fresh tube . the protein content of the extract was measured by the bradford assay using the biorad protein assay kit . between 0 . 8 to 10 μg of plant protein extract was used per assay . bacterial extracts were also prepared in the assay buffer essentially as described in the previous section . activity of the acetoacetyl - coa reductase enzyme was assayed according to the procedure of senior , p . j . and dawes , e . a ., biochem . j . 134 : 225 - 238 ( 1973 ). two methods were used to prepare plant extracts for gc analysis . in method # 1 , between 0 . 005 and 0 . 05 g of fresh or frozen plant material ( leaves or whole shoots ) was extracted in 1 to 2 ml of a 1 : 1 solution of chloroform and water at 65 ° c . for 16 hours with constant agitation . the plant material was then homogenized in water and re - extracted in a 1 : 1 solution of chloroform and water for 16 hours at 65 ° c . with constant agitation . the chloroform phase was transferred to a new tube and extracted once with an equal volume of water . the final volume of the chloroform phase was adjusted to 0 . 5 ml and used for transesterification with ethanol and hcl as described below . in method # 2 , between 0 . 005 to 0 . 15 g of frozen or fresh plant material was successively extracted in 50 % ethanol at 55 ° c . for 2 hours , 100 % methanol at 55 ° c . for 2 hours and 100 % diethylether for 15 minutes at room temperature . the remaining tissue was then homogenized in water , dried and extracted in 0 . 5 ml of chloroform for 4 hours at 100 ° c . the final chloroform extracts ( 0 . 5 ml ) obtained by method # 1 and # 2 was transesterified by adding 0 . 2 ml of concentrated hcl and 1 . 7 ml of 100 % ethanol and heating at 100 ° c . for 2 hours . the reaction mixture was then cooled down to room temperature , the chloroform phase extracted twice with 2 ml of 0 . 9 % nacl ( w / v ) and the final organic phase reduced to 100 μl . as a standard , commercial phb ( sigma chemical co ., mo ) was dissolved in warm chloroform and 1 mg was transesterified as described above . the chloroform phase containing the ethyl esters were transferred to a gc vial for injection of 1 μl into a hewlett packard 5890 series ii gc equipped with a programmable autosampler and a sp - 2330 glass capillary column ( supelco , pa ). the approximate linear velocity was 20 cm / s with helium as the carrier gas . the temperature of the injection port was set at 220 ° c ., and that of the flame ionization detector port was set at 220 ° c . the following temperature profile was used : 4 minutes at 65 ° c ., followed by a temperature increase rate of 20 ° c ./ minute up to 195 ° c ., 3 . 5 minutes at 195 ° c ., a post - run temperature decrease rate of 20 ° c ./ minute down to 65 ° c . the identity of peaks of interest was established by gc - mass spectrometry . electron impact mass spectral data was obtained on a jeol jms - ax505h mass spectrometer coupled with a hewlett packard 5890 gc . the following parameters were used : source temperature , 200 ° c . ; ionization current , 100 μa ; accelerating voltage , 3 kev . a j & amp ; w scientific co . column db - 225 was directly inserted into the mass spectrometer source and helium was used as carrier . the splitless injector was held at 260 ° c . and the transfer line at 260 ° c . the same gc oven temperature profile was used ( see previous paragraph ). plant tissues were fixed with 3 % glutaraldehyde in 0 . 1m phosphate buffer ( ph 7 . 2 ) for 1 . 5 - 2 hours at room temperature . the samples were washed 4 times in 0 . 1m phosphate buffer ( ph 7 . 2 ) and fixed in 1 % oso 4 in phosphate buffer for 2 hours at room temperature . the tissues were then dehydrated in a graded ethanol series and embedded in spurrs epoxy resin . sections of 80 - 90 nm were cut , placed on a copper grid and stained in 5 % uranyl acetate for 30 to 45 minutes , followed by staining in reynolds lead citrate for 3 to 4 minutes . sections were viewed in a jeol 100cxii transmission electron microscope operated at 80 kv . although the specific example of the invention described here involved the plant arabidopsis thaliana and genes from alcaligenes eutrophus , the invention is of general utility . the claims pertaining to production of polyhydroxybutyrate and / or polyhydroxyalkanoate in plants is not limited to arabidopsis thaliana , or linked specifically to the use of genes from alcaligenes eutrophus . the claims described below describe a general method for the production of polyhydroxyalkanoate in plants through the introduction of foreign dna material into plant cells . such plants include the plants discussed previously and carrot , sunflower , tobacco , tomato and potato , for instance . the seeds from the various lines of plants have been deposited under the budapest treaty with the american type culture collection , 12301 parklawn drive , rockville , md 20852 . these lines include redd - 3a ( atcc 75044 ) containing the acetoacetyl - coa reductase gene ; s8 - 1 - 2a ( atcc 75043 ) containing the phb synthase gene and t4 - 2a ( atcc 75042 ) containing the 3 - ketothiolase gene . the genes are each shown in sequence id no : 1 . the foregoing specific description is only illustrative of the present invention and it is intended that the present invention be limited only by the hereinafter appended claims . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 1 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 4983 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type :( a ) description : genomic dna ( iii ) hypothetical : no ( iv ) anti - sense : no ( vi ) original source :( a ) organism : alcaligenes eutrophus ( vii ) immediate source :( a ) library : genomic ( xi ) sequence description : seq id no : 1 : cccgggcaagtaccttgccgacatctatgcgctggcgcgcacgcgcctgg50cgcgcgccggctgtaccgaggtctacggcggcgacgcctgcaccgtggcc100gacgccggtcgcttctactcctatcggcgcgatggcgtgaccggccgcat150ggccagcctggtctggctggcggactgagcccgccgctgcctcactcgtc200cttgcccctggccgcctgcgcgcgctcggcttcagccttgcgtcggcggc250ggccgggcgtgcccatgatgtagagcaccacgccaccggcgccatgccat300acatcaggaaggtggcaacgcctgccaccacgttgtgctcggtgatcgcc350atcatcagcgccacgtagagccagccaatggccacgatgtacatcaaaaa400ttcatccttctcgcctatgctctggggcctcggcagatgcgagcgctgca450taccgtccggtaggtcgggaagcgtgcagtgccgaggcggattcccgcat500tgacagcgcgtgcgttgcaaggcaacaatggactcaaatgtctcggaatc550gctgacgattcccaggtttctccggcaagcatagcgcatggcgtctccat600gcgagaatgtcgcgcttgccggataaaaggggagccgctatcggaatgga650cgcaagccacggccgcagcaggtgcggtcgagggcttccagccagttcca700gggcagatgtgccggcagaccctcccgctttgggggaggcgcaagccggg750tccattcggatagcatctccccatgcaaagtgccggccagggcaatgccc800ggagccggttcgaatagtgacggcagagagacaatcaaatcatgatggcg850metalaaccggcaaaggcgcggcagcttccacgcaggaaggcaagtcccaaccatt900thrglylysglyalaalaalaserthrglngluglylysserglnproph51015caaggtcacgccggggccattcgatccagccacatggctggaatggtccc950elysvalthrproglypropheaspproalathrtrpleuglutrpsera20253035gccagtggcagggcactgaaggcaacggccacgcggccgcgtccggcatt1000rgglntrpglnglythrgluglyasnglyhisalaalaalaserglyile404550ccgggcctggatgcgctggcaggcgtcaagatcgcgccggcgcagctggg1050proglyleuaspalaleualaglyvallysilealaproalaglnleugl556065tgatatccagcagcgctacatgaaggacttctcagcgctgtggcaggcca1100yaspileglnglnargtyrmetlysasppheseralaleutrpglnalam70758085tggccgagggcaaggccgaggccaccggtccgctgcacgaccggcgcttc1150etalagluglylysalaglualathrglyproleuhisaspargargphe9095100gccggcgacgcatggcgcaccaacctcccatatcgcttcgctgccgcgtt1200alaglyaspalatrpargthrasnleuprotyrargphealaalaalaph105110115ctacctgctcaatgcgcgcgccttgaccgagctggccgatgccgtcgagg1250etyrleuleuasnalaargalaleuthrgluleualaaspalavalglua120125130135ccgatgccaagacccgccagcgcatccgcttcgcgatctcgcaatgggtc1300laaspalalysthrargglnargileargphealaileserglntrpval140145150gatgcgatgtcgcccgccaacttccttgccaccaatcccgaggcgcagcg1350aspalametserproalaasnpheleualathrasnproglualaglnar155160165cctgctgatcgagtcgggcggcgaatcgctgcgtgccggcgtgcgcaaca1400gleuleuilegluserglyglygluserleuargalaglyvalargasnm170175180185tgatggaagacctgacacgcggcaagatctcgcagaccgacgagagcgcg1450etmetgluaspleuthrargglylysileserglnthraspgluserala190195200tttgaggtcggccgcaatgtcgcggtgaccgaaggcgccgtggtcttcga1500phegluvalglyargasnvalalavalthrgluglyalavalvalphegl205210215gaacgagtacttccagctgttgcagtacaagccgctgaccgacaaggtgc1550uasnglutyrpheglnleuleuglntyrlysproleuthrasplysvalh220225230235acgcgcgcccgctgctgatggtgccgccgtgcatcaacaagtactacatc1600isalaargproleuleumetvalproprocysileasnlystyrtyrile240245250ctggacctgcagccggagagctcgctggtgcgccatgtggtggagcaggg1650leuaspleuglnprogluserserleuvalarghisvalvalgluglngl255260265acatacggtgtttctggtgtcgtggcgcaatccggacgccagcatggccg1700yhisthrvalpheleuvalsertrpargasnproaspalasermetalag270275280285gcagcacctgggacgactacatcgagcacgcggccatccgcgccatcgaa1750lyserthrtrpaspasptyrilegluhisalaalaileargalaileglu290295300gtcgcgcgcgacatcagcggccaggacaagatcaacgtgctcggcttctg1800valalaargaspileserglyglnasplysileasnvalleuglyphecy305310315cgtgggcggcaccattgtctcgaccgcgctggcggtgctggccgcgcgcg1850svalglyglythrilevalserthralaleualavalleualaalaargg320325330335gcgagcacccggccgccagcgtcacgctgctgaccacgctgctggacttt1900lygluhisproalaalaservalthrleuleuthrthrleuleuaspphe340345350gccgacacgggcatcctcgacgtctttgtcgacgagggccatgtgcagtt1950alaaspthrglyileleuaspvalphevalaspgluglyhisvalglnle355360365gcgcgaggccacgctgggcggcggcgccggcgcgccgtgcgcgctgctgc2000uargglualathrleuglyglyglyalaglyalaprocysalaleuleua370375380385gcggccttgagctggccaataccttctcgttcttgcgcccgaacgacctg2050rgglyleugluleualaasnthrpheserpheleuargproasnaspleu390395400gtgtggaactacgtggtcgacaactacctgaagggcaacacgccggtgcc2100valtrpasntyrvalvalaspasntyrleulysglyasnthrprovalpr405410415gttcgacctgctgttctggaacggcgacgccaccaacctgccggggccgt2150opheaspleuleuphetrpasnglyaspalathrasnleuproglyprot420425500505ggtactgctggtacctgcgccacacctacctgcagaacgagctcaaggta2200rptyrcystrptyrleuarghisthrtyrleuglnasngluleulysval510515520ccgggcaagctgaccgtgtgcggcgtgccggtggacctggccagcatcga2250proglylysleuthrvalcysglyvalprovalaspleualaserileas525530535cgtgccgacctatatctacggctcgcgcgaagaccatatcgtgccgtgga2300pvalprothrtyriletyrglyserarggluasphisilevalprotrpt540545550555ccgcggcctatgcctcgaccgcgctgctggcgaacaagctgcgcttcgtg2350hralaalatyralaserthralaleuleualaasnlysleuargpheval560565570ctgggtgcgtcgggccatatcgccggtgtgatcaacccgccggccaagaa2400leuglyalaserglyhisilealaglyvalileasnproproalalysas575580585caagcgcagccactggactaacgatgcgctgccggagtcgccgcagcaat2450nlysargserhistrpthrasnaspalaleuprogluserproglnglnt590595600605ggctggccggcgccatcgagcatcacggcagctggtggccggactggacc2500rpleualaglyalailegluhishisglysertrptrpproasptrpthr610615620gcatggctggccgggcaggccggcgcgaaacgcgccgcgcccgccaacta2550alatrpleualaglyglnalaglyalalysargalaalaproalaasnty625630635tggcaatgcgcgctatcgcgcaatcgaacccgcgcctgggcgatacgtca2600rglyasnalaargtyrargalailegluproalaproglyargtyrvall640645650655aagccaaggcatgacgcttgcatgagtgccggcgtgcgtcatgcacggcg2650ysalalysalaccggcaggcctgcaggttccctcccgtttccattgaaaggactacacaat2700me1gac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