Patent Application: US-80783602-A

Abstract:
this invention provides a method of conferring osmoprotection to plants . plant plastid genomes , particularly the chloroplast genome , is transformed to express an osmoprotectant . the transgenic plants and their progeny display drought resistance . more importantly , such transgenic plants display no negative pleiotropic effects such as sterility or stunted growth .

Description:
this invention discloses a method of conferring drought tolerance to plants by transforming plants via the chloroplast with a vector that contains a dna sequence encoding a gene of interest that protects against water stress . in the preferred embodiment of this invention , the vector used is the universal vector as described by daniell in wo99 / 10513 , which is incorporated herein by reference . other vectors that are capable of chloroplast transformation such as puc , pbr322 , pbluescript , pgem and others described in u . s . pat . nos . 5 , 693 , 507 and 5 , 932 , 479 may be used . in the preferred embodiment of this invention , the osmoprotection is the yeast trehalose - 6 - phosphate synthase ( tsp1 ). other genes which are capable of conferring drought resistance or osmoprotection may also be used . it is known that the yeast trehalose - 6 - phosphate synthase gene can be expressed in nuclear transgenic plants . because chloroplasts are prokaryotic in nature , it is desirable to test expression levels of the eukaryotic yeast tps1 gene in e coli . because of the high similarity in the transcription and translation systems between e . coli and chloroplasts , expression vectors are , routinely tested in e . coli before proceeding with chloroplast transformation of higher plants . therefore , the tps1 gene from yeast was cloned into the e . coli expression vector pqe 30 ( see fig1 a for details of pqe - tps1 ) and expressed in a suitable e . coli strain m15 ( prep4 ). sds - page as shown in fig1 b shows the presence of tps1 protein in crude cell extracts , even with coomassie blue stain ( lane 1 ), indicating high levels of expression . western blot analysis using tps1 - antibody confirms the true identity of the expressed protein as shown in fig1 b , lane 41 . these results confirm that the codon preference of tps1 is compatible for expression in a prokaryotic compartment . hyper - expression also facilitated purification as shown in fig1 lanes 2 . 55 and preparation of polyclonal antibody for characterization of transgenic plants . having confirmed suitability for prokaryotic expression , the yeast tps1 gene was inserted into the universal chloroplast expression vector pct - tps1 as shown in fig2 b . this vector can be used to transform chloroplast genomes of several plant species because the flanking sequences are highly conserved among higher plants . this vector contains the 16 srrna promoter ( prm ) driving the aada ( aminoglycoside 3 ″- adenylyl transferase ) and tps1 genes with the psba 3 ′ region ( the terminator from a gene coding for photosystem ii reaction center component ) from the tobacco chloroplast genome . it is known that the 16 srrna promoter is one of the strong chloroplast promoters and the psba 3 ′ region stabilized transcripts to avoid hyper - expression of tps - 1 and associated pleiotropic effects . the yeast ribosme binding site ( rbs ) was used instead of the genome chloroplast rbs ( ggagg ). this construct integrates both genes into the spacer region between the chloroplast transfer rna genes coding for alanine and isoleucine within the inverted repeat ( ir ) region of the chloroplast genome by homologous recombination . for nuclear expression , the yeast tps1 gene was inserted into the binary vector phgtps1 ( fig2 c ), in which the tps1 gene is driven by the camv 35s promoter and the hph gene is driven by the nopaline synthase promoter . the expression cassette is flanked by both the left and right t - dna border sequences . the binary vector phgtps1 was mobilized into the agrobacterium tumafaciens strain lba 4404 by electroporation . transformed agrobacterium strain was introduced into nicotiana tabaccum var xanthi using the leaf disc transformation method . ninety two independent tps1 nuclear tranformants were obtained on hygromycin selection . seventeen confirmed nuclear tranformants were analyzed by northern blots . among tranformants showing various levels of transcripts , five tranformants with strong , moderate , weak , very weak and absence of transcripts were chosen for further characterization . for chloroplast transformation , green leaves of n . tabacum var . burley were transformed with the chloroplast integration and expression vector by the biolistic process . bombarded leaf segments were selected on spectinomycin / streptomycin selection medium . integration of foreign gene into the chloroplast genome was determined by pcr screening of chloroplast tranformants , ( fig2 a ). primers were designed to eliminate mutants , nuclear integration and to determine whether the integration of foreign genes had occurred in the chloroplast genome at the directed site by homologous recombination . primers 5p / 5m land within the aada gene and should generate a 0 . 4 kbp fragment if the aada gene was present in transgenic plants and eliminates the possibility of mutation that could otherwise confer streptomycin / spectinomycin resistance . fig2 a shows the presence of 0 . 4 kbp pcr product in plants transformed with the universal vector alone ( pct ,) or the universal vector containing the tps1 gene ( pct - tps1 ), but not in control untransformed plants , confirming that these are transgenic plants and not mutants . the strategy to distinguish between nuclear and chloroplast transgenic plants was to land one primer ( 3p ) on the native chloroplast genome adjacent to the point of integration and the second primer ( 3m ) on the aada gene . this primer set generated 1 . 6 kbp pcr product in chloroplast tranformants obtained with the universal vector ( pct ) and the universal vector containing the tps1 gene ( pct - tps1 ). because this product can not be obtained in nuclear transgenic plants , the possibility of nuclear integration can be eliminated . another primer set was designed to test integration of the entire gene cassette . the presence of the expected size pcr products using 5p / 5m confirms that the entire gene cassette has been integrated and that there has been no internal deletions or loop outs during integration via homologous recombination . since there are no significant differences in the level of foreign gene expression among different chloroplast transgenic lines , one line was chosen to generate subsequent generations ( t 1 t 2 t 3 ). southern blot analysis was performed using total dna isolated from transgenic and wild type tobacco leaves . total dna was digested with a suitable restriction enzyme . presence of a bglii at the 3 ′ end of the flanking 16s rrna gene and the trna intron allowed excision of predicted size fragments in the chloroplast tranformants and untransformed plants . to confirm foreign gene integration and homoplasmy , individual blots were probed with the chloroplast dna flanking sequence ( probe p1 , fig2 a ). in the case of the tps1 integrated plastid tranformants ( t 1 t 2 ), the border sequence hybridized with 6 . 13 and 1 . 17 kbp fragments while it hybridized with a native 4 . 47 kbp fragment in the untransformed plants ( fig2 b ). the copy number ofthe integrated tps1 gene was also determined by establishing homoplasmy in transgenic plants . tobacco chloroplasts contain about 10 , 000 copies of chloroplast genomes per cell . if only a fraction of the genomes were transformed , the copy number should be less than 10 , 000 . by confirming that the tps1 integrated genome is the only one present in transgenic plants , one could establish that the tps1 gene copy number could be as many as 10 , 000 per cell . dna gel blots were also probed with the tps1 gene coding sequence ( probe p2 ) to confirm integration into the chloroplast genomes . in chloroplast transgenic plants ( t 1 t 3 ), the tps1 gene coding sequence hybridized with 6 . 13 and 1 . 17 kbp fragments which also hybridized with the border sequence in plastid transgenic lines ( fig2 b ). this confirms that the tobacco tranformants indeed integrated the intact gene expression cassette into the chloroplast genome and that there has been no internal deletions or loop out during integration via homologous recombination . for comparison of introduced gene expression between chloroplast and nuclear tranformants , northern blot analysis of transgenic tobacco at similar developmental stages was performed in t 1 , t , and t 2 plants . as shown in fig3 quantification of transcription level showed that the chloroplast transformant ( t2 ) expressed 16 , 960 - fold ( fig3 e , lane 5 ) more tps1 transcript than that of highly expressing nuclear ( t 1 ) transformant ( fig3 e , lanes 2 , 3 ). similar results were obtained when t 1 chloroplast ( fig3 b , lane 7 ) and to nuclear transgenic plants ( fig3 , lanes 2 - 5 ) were compared . this large difference in tps1 expression between nuclear and chloroplast transgenic plants should be due to the presence of thousands of tps1 gene copies in each cell of transgenic tobacco . fig3 ( c , f ) show ethidium bromide stained rna gels before blotting ; this confirms that equal amount of rna ( 10 μg ) was loaded in all lanes . it is remarkable that the 16srrna promoter is driving both genes very efficiently , eliminating the need for inserting additional promoters for the gene of interest . polyclonal antibodies raised against the tps1 protein overexpressed and purified from e . coli ( see experimental protocol ) were used for immunoblotting ( fig3 a , d ). a 60 kda tps1 polypeptide was detected in the t 0 nuclear ( fig3 a , lanes 2 , 3 , 5 . ), t 1 nuclear ( 3d lanes 2 , 3 ) and t 1 plastid ( fig3 a , lane 7 ) and t 2 plastid ( fig3 d , lane 0 . 5 ) tranformants . however , no tps1 was detected in the untransformed control ( fig3 a , lanes 1 , 6 ; 3d 1 , 4 )) and transgenic plants which showed no tps1 transcript ( fig3 a , lane 4 ). as anticipated , western blots showed only a five or ten fold increase in tps1 protein in chloroplast over highly expressing nuclear transgenic plants . this is because of the fact that the chloroplast vector pct - tps1 was intentionally designed to lower translation by not inserting a chloroplast preferred ribosome binding site ( ggagg ), so that transgenic plants are not killed by hyper - expression of tps1 . this level expression was adequate to compare trehalose accumulation in cytosolic and chloroplast compartments and observe resultant phenotypic / physiological changes . t 1 nuclear and t 2 chloroplast transgenic plants had higher levels of tps1 protein ; this may be due to homozygous tps1 alleles or homoplasmy . trehalose formation is a two step process , involving trehalose - 6 - phosphate synthase and trehalose 6 - phosphate phosphatase . trehalose - 6 - phosphate was not detected in all tested chloroplast and nuclear transformers even though the tps2 , trehalose - 6 - phosphate phosphatase that converts t6p to trehalose , was not introduced ( table 1 ). conversion of t6p to trehalose should have been accomplished by endogenous tobacco trehalose phosphatase or by any non - specific endogenous phosphatase . simultaneous expression of both enzymes in transgenic plants resulted only in marginal increase of trehalose accumulation in previous studies , confirming that it is adequate to express only tps1 . leaf extracts from both nuclear and chloroplast transgenic plants catalyzed the synthesis of trehalose 6 - phosphate from glucose - 6 - phosphate and udp - glucose whereas untransformed tobacco had very low activity . to chloroplast and nuclear transgenic plants showed a 7 - 10 fold higher tps1 activity than untransformed control plants . the amount of trehalose present in untransformed control plants and to nuclear transgenic plants were similar whereas chloroplast transgenic plants accumulated a 17 - 25 fold mm trehalose than the best surviving nuclear transgenic plants ( table 1 ). t 1 nuclear transgenic plants accumulated less trehalose than control untransformed plants whereas t 1 chloroplast transgenic plants continued to accumulate high levels of trehalose ( table 1 ). observation of comparable tps1 activity in both nuclear and chloroplast transgenic plants but lack of trehalose accumulation in nuclear transgenic planes indicates that trehalose may be degraded in the cytosol by trehalase but not in the chloroplast compartment . this is consistent with previous studies on inhibition of trehalase activity that resulted in trehalose accumulation in the cytosol . chloroplast and nuclear tranformants were examined for drought tolerance and pleiotropic effects . after six weeks of growth in vitro , rooted shoots were transferred to pots and grown in the greenhouse . tps1 nuclear tranformants showed moderate to severe growth retardation , lancet - shaped leaves and infertility ( fig4 ). the chloroplast tranformants ( t 0 ) showed decreased growth rate and delayed flowering but all subsequent generations ( t 1 , t 2 ) showed similar growth rates and fertility as controls . the nuclear transgenic lines of stunted phenotype showed delayed flowering and produced fewer seeds compared to wild type or did not flower . this result is consistent with prior observations which demonstrated that e . coli otsa ( tps1 ) and s . cerevisiae tps1 transgenic plants exhibited - stunted plant growth and other pleiotropic effects . the nuclear transgenic line showing severe growth retardation did not flower . t 1 nuclear transgenic plants that survived showed no growth retardation and trehalose accumulation . therefore , these plants could not be used for appropriate comparison with chloroplast transgenic plants . when the seeds of chloroplast transgenic plant ( crossed between transgenic female and untransformed male ) and wild type seeds were germinated on ms medium containing spectinomycin , all chloroplast transgenic progeny were spectinomycin resistant while all wild type seedlings were sensitive to spectinomycin ( fig5 ). because tps1 transgenic lines showed accumulation of trehalose , they were tested for drought tolerance . seeds of chloroplast and nuclear transgenic plants were germinated on the ms medium containing polyethylene glycol . as shown in fig6 chloroplast transformant seedlings showed resistance to medium containing 3 % and 6 % peg whereas control and nuclear transgenic seedlings exhibited severe dehydration , necrosis and severe growth retardation , ultimately resulting in death . three - week - old seedlings were chosen to study drought tolerance by dehydration and subsequent rehydration . when seedlings were dried for 7 hours at room temperature in 50 % relative humidity , they were all affected by dehydration . however , when dehydrated seedlings were rehydrated for 48 hours in ms medium , all chloroplast transgenic lines recovered while all control seedlings were bleached ( fig7 ). even the couple of control seedlings that partly survived ( because of uneven drying of seedlings on filter papers ) eventually died . these results suggest that the loss of water from tps1 transgenic plants may not be decreased but the ability to recover from drought was dramatically enhanced . this is consistent with existing understanding that trehalose functions by protecting biological membranes rather than regulating water potential ( iwahashi et al ., 1995 ). mature leaves from fully - grown plants were tested for their ability to regulate water loss under drought conditions . when detached leaves were air dried , control and chloroplast transgenic plants lost water to the same extent ( fig8 ). control and chloroplast transgenic potted plants were not watered for 24 days . again , both showed dehydration to the same extent ( fig9 a , b ). however , upon rehydration , fully dehydrated leaves ( indicated by arrows , fig9 c , d ) recovered in chloroplast transgenic plants but not in controls . plant , a . tumefaciens and e . coli culture : for transformation experiments , nicotiana tabacum var . xanthi and burley were grown in ms medium in the magenta culture box ( sigma , usa ). for drought tolerance assays of transgenic tobacco plants , the rooted young plants were transferred to pre - swollen jiffy - 7 peat pellets ( jiffy products , norway ) inside the greenhouse . plants used for enzyme assays were grown and kept in magenta culture boxes . seven or 8 leaf stage plants were used for enzyme assays . two to three - week old young transgenic tobacco plants were used for stress analyses . ( agrobacterium tumefaciens strain lba4404 was grown in the yep medium at 29 ° c . in a shaking incubator . other e . coli strains were cultured and maintained as described in sambrook et al . plasmid construction and antibody production : for hyper - expression of the tps1 in e . coli for antibody production , the yeast tps1 gene was cloned into plasmid pqe30 ( qiagen ) and subsequently transformed into e . coli strain m15 [ prep4 ]. the resulting e . coli transformant was grown at 37 ° c . to an a 600 of 0 . 5 - 0 . 8 and induced by 2 mm isopropyl - p - β - thiogalactopyranoside ( iptg ) for 1 - 5 hours . the induced cells were harvested and lysed by sonication . sds - page analysis showed the presence of tps1 protein in crude cell extracts , even with coomassie blue stain , indicating high levels of expression . western blot analysis using tps1 antibody confirmed the true identity of the expressed protein ( data not shown ). the recombinant protein was purified using ni 2 + resin , using the procedures provided by the manufacturer . affinity column purified recombinant protein was analyzed for purity by sds - page . protein concentrations were determined using ‘ the bio - rad ( usa ) protein assay kit with bsa as a standard . polyclonal antibody was generated using the purified tps1 protein by the takara shuzo co . ( japan ). vector construction for plant transformation : the yeast 1 . 537 kbp tps1 gene was inserted into the xbal site of pct vector generating pct - tps1 ( fig2 b ). for the nuclear transformation , the yeast tps1 gene was inserted into the phgtps1 vector in which the tps1 gene is driven by the camv 35s promoter . the resulting vector confers hygromycin resistance because of the hygromycin phosphotransferase gene driven by the nos promoter . chloroplast and nuclear transformation : for chloroplast transformation , particle bombardment was carried out using a helium driven particle gun , biolistic pdh1000 . briefly , chloroplast vectors , pct and pct - tps1 were delivered to tobacco leaves ( burley ) using 0 . 6 μm gold microcarriers ( bio - rad ) at 1 , 100 psi with a target distance of 9 cm . for nuclear transformation , phgtps1 was mobilized into the acrobacterium tumefaciens strain lba4404 by electroporation using gene pulsar ( bio - rad . usa ). the resulting agrobacterium strain was used in leaf disc transformation of wild type n . tabacum var . xanthi . chloroplast dna isolation and pcr : total dna was extracted from leaves of wild type and transformed plants using ctab extraction buffer described . pcr was carried out to confirm spectinomycin resistant chloroplast tranformants using peltier thermal cycler ptc - 200 ( mj research , usa ). three primer sets , 2p ( 5 ′- gcgcctgaccctg agatgtggatcat - 3 ′)- 2m ( 5 ′- tgactgcccaacctgagagcggaca - 3 ′), 3p ( aaaacccgtcctcagttcggattgc )- 3m ( ccgcgttgtttcatca agccttacg ) and - 5p ( ctgtagaagtcaccattgttgtgc ), 5m ( gtccaagat aagcctgtctagcttc ) were used for the pcr . pcr reactions were carried out as described elsewhere ( daniell et al ., 1998 ; guda et al ., 2000 ). rna isolation and northern slot analysis : total rna was extracted from transgenic tobacco plants using tri reagent ( mrc , usa ) following manufacturer &# 39 ; s instruction . for northern blots , rna samples ( 10 μg of total rna per lane ) were electrophoresed on a 1 . 5 % agarose - mops gel containing formaldehyde . uniform loading and integrity of rnas were confirmed by examining the intensity of ethidium bromide bound ribosomal rna bands under uv light . rnas on the gel were transferred onto hybond - n membrane ( amersham , usa ). the membrane was hybridized to radiolabeled tps1 probe and washed at 65 ° c . in a solution of 0 . 2 × ssc and 0 . 1 % sds for 20 min twice . the blot was exposed to an x - ray film at − 70 ° c . overnight . transcripts were quantified using the biold ++ program with vilber lourmat image analyzer ( bioprofil , france ). western blot analysis : tobacco total protein extracts were prepared by modified methods described by ausubel et al . the total extracts were fractionated on a 10 % one - dimensional sds - page , transferred to biotrace pdvf nitrocellulose membrane ( gelman sciences , usa ), and immunostained using renaissance western blot chemiluminescence reagent ( nen life science products , usa ) according to manufacturer &# 39 ; s instructions . each lane was loaded with 100 μg of total protein . the primary antibody used was anti - tps1 at a 5000 - fold dilution . the secondary antibody was anti - rabbit igg hrp conjugate at a 2000 - fold dilution ( promega , usa ). drought tolerance and biochemical characterization : for analyses of drought tolerance , 2 - 3 week old transgenic tobacco plants were used . seeds of chloroplast and nuclear tranformants were germinated on ms plates containing 3 % or 6 % peg ( mw 8 , 000 ). tps1 enzyme assay was performed spectrophometrically by the method described by londesbrough and vuorio . for quantitative determination of t6p and trehalose , carbohydrates were extracted from aerial parts of transgenic or wild type tobacco plants by treatment in 85 % ethanol at 60 ° c . for 1 hour . the amount of t6p and trehalose were measured by high - performance liquid chromatography ( hplc ) on a waters system equipped with a waters high performance carbohydrate column ( 4 . 6 × 250 mm ) and a refractive index detector . the insoluble phase system was 75 % acetanitrile - 25 % h 2 o with a flow rate of 1 . 0 ml / min . thevelein , j . m . & amp ; hohmann , s . trehalose synthase : guard to the gate of glycolysis in yeast ? 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