Patent Application: US-19382694-A

Abstract:
a method for altering self - compatibility / self - incompatibility in flowering plants is presented . the method comprises manipulation of the plant genome and production of the transonic plants . abolishment of self - incompatibility was achieved by inserting into plant genome as dna segment comprising anti - sense message of the allelic form of the s - gene responsible for self - incompatibility . this insert prove to be satisfactory to block expression of said allelic form of s - gene . introduction of self - incompatibility was achieved by inserting into plant genome dna construct comprising message coding for the s - protein responsible for self - incompatibility .

Description:
the present invention describes the evidence of correlation between self - compatibility and s - genes in petunia plants . during the course of detailed studies involving the investigation of plant self - incompatibility , the involvement of proteins encoded by s - genes was demonstrated . the support for this new finding and ways of using it commercially are described herein . a detailed embodiment of the present invention involving petunia plants is herein disclosed . however it is understood that the preferred embodiment is merely illustrative of the invention which may be embodied in various forms and applications . accordingly , specific structural and functional details disclosed herein are not to be interpreted as limiting , but merely as a support for the invention as claimed and as appropriate representation for the teaching one skilled in the an to variously employ the present invention in any appropriate embodiment . the applicant used both loss - of - function and gain - of - function approaches to ascertain whether the p . inflata s - proteins previously identified 9 do indeed control self - incompatibility interactions between pollen and pistil . in the former approach , he introduced antisense s 3 - cdna under the control of the promoter of the s 3 - gene into p . inflata plants of s 2 s 3 genotype by agrobacterium - mediated transformation ( fig1 a ). this allowed him to determine whether inhibition of s 3 - protein synthesis in the pistil abolished the ability of the transgenic plants to reject s 3 - pollen . forty - seven independent transgenic plants were self - pollinated to determine whether their self - incompatibility behavior had been affected . thirty plants were found to set variable numbers of seeds , and 6 of them consistently produced a number of seeds comparable to that obtained from compatible pollination of p . inflata plants ( approximately 200 per fruit ). these 6 transgenic plants ( as - 4 , as - 14 , as - 23 , as - 27 , as - 35 , and as - 37 ) were chosen for further analysis . genomic blot analysis of 4 of the plants revealed that , in addition to the 11 . 5 kb dna fragment corresponding to the endogenous s 3 - gene , they contained one to three insertions of the transgene ( fig1 b ). to determine whether the breakdown of self - incompatibility in the 6 self - compatible transgenic plants resulted from loss of their ability to reject s 3 - pollen , these plants were pollinated with pollen from s 2 s 3 and s 2 s 2 tester plants . as - 14 , as - 23 , and as - 37 rejected pollen from s 2 s 2 plants , but accepted pollen from s 2 s 3 plants , indicating that s 3 - allele had been rendered nonfunctional , but s 2 - allele was not affected . as - 4 , as - 27 , and as - 35 accepted pollen from both s 2 s 2 and s 2 s 3 plants , indicating that , in addition to s 3 - allele , s 2 - allele had also been rendered nonfunctional . as - 39 , a self - incompatible transgenic plant , rejected pollen from both s 2 s 2 and s 2 s 3 plants . next the applicant investigated whether the inability of the 6 self - compatible transgenic plants to reject s 2 - or s 3 - pollen was caused by inhibition of s 2 - or s 3 - protein synthesis . as shown in fig2 a , as - 14 , which rejected s 2 - allele but failed to reject s 3 - allele , contained a normal level of s 2 - rna , but a nondetectable level of s 3 - rna ; as - 27 , which failed to reject either s 2 - or s 3 - allele , contained nondetectable levels of s 2 - rna and s 3 - rna ; as - 39 , which rejected both s 2 - and s 3 - alleles , contained normal levels of s 2 - and s 3 - rna . the applicant then determined the amounts of s 2 - and s 3 - proteins in the pistils of the 6 self - compatible and 7 self - incompatible transgenic plants , and 7 parental s 2 s 3 plants ( fig2 b ). profile a in fig2 b is representative of these parental s 2 s 3 plants . as - 14 , as - 23 , and as - 37 all contained normal amounts of s 2 - protein , but drastically reduced amounts of s 3 - protein ( profile c in fig2 b shows as - 14 ). the amounts of both s 2 - and s 3 - proteins were drastically reduced in as - 4 , as - 27 , and as - 35 ( profile d shows as - 27 ). in fact , the amounts of s 3 - protein in the former 3 plants , and the amounts of s 2 - and s 3 - proteins in the latter 3 plants were lower than those present in immature buds which are fully receptive to serf pollen 9 . the amounts of s 2 - and s 3 - proteins of the 7 self - incompatible transgenic plants were comparable to those of the parental s 2 s 3 plants ( profile b shows as - 39 ). the effect of the antisense s 3 - gene was inheritable , because self - compatible plants were found in selfed progeny of as - 14 and as - 27 . as in the parental transgenic plants , the amounts of s 2 - and s 3 - proteins also correlated perfectly with breeding behavior ( results not shown ). thus , it was concluded from the antisense experiments that s - proteins are necessary for the pistil to reject self pollen . the applicant used the gain - of - function approach to ascertain whether s - proteins alone are sufficient for the pistil to reject serf pollen . a 3 . 6 kb dna fragment ( fig3 a ) containing the gene for s 3 - protein 9 , 18 was introduced into p . inflata plants of s 1 s 2 genotype , and it was determined whether expression of the s 3 - gene could confer on the transgenic plants a new s 3 - allele specificity . eighty - one transgenic plants were pollinated with pollen from s 3 s 3 plants , and 4 plants , gs3 - 13 , gs3 - 16 , gs3 - 41 , and gs3 - 55 , were found to completely reject s 3 - pollen . the rest set fruits of variable sizes with seed numbers ranging from 20 to 200 , indicating that they were either partially or fully compatible with s 3 - pollen . the 4 transgenic plants that completely rejected s 3 - pollen also rejected pollen from s 1 s 2 plants . however , they set large fruits when selfed at the immature - bud stage when self - incompatibility is not yet expressed . thus , the failure of the 4 plants to set fruits when pollinated with s 1 -, s 2 - and s 3 - pollen was a true self - incompatible response , and not due to female sterility resulting from tissue culture manipulations . genomic blot analysis revealed that all 4 plants contained a single insert of the transgene ( fig3 b ). two other transgenic plants were also included in the analysis : gs3 - 75 , a plant partially compatible with s 3 - pollen ( producing approximately 50 seeds per fruit , and gs3 - 113 , a plant fully compatible with s 3 - pollen ( producing approximately 200 seeds per fruit ). gs3 - 75 contained two inserts and gs3 - 113 contained one insert ( fig3 b ). to investigate whether the new s 3 - allele specificity acquired by the 4 transgenic plants resulted from expression of the s 3 - transgene in the pistil , the applicant first examined the level of s 3 - rna in the pistils of representative transgenic plants . as shown in fig4 a , gs3 - 16 and gs3 - 55 , which completely rejected s 3 - pollen , contained a normal level of s 3 - rna ; gs3 - 75 and gs3 - 109 , which were partially compatible with s 3 - pollen , contained approximately 30 % the normal level of s 3 - rna ; gs3 - 113 , which was fully compatible with s 3 - pollen , did not contain any detectable s 3 - rna . the applicant examined the mounts of s 3 - protein in the pistils of 31 transgenic plants which included all the ones described above ( fig4 b ). all 4 plants that completely rejected s 3 - pollen were found to produce a normal level of s 3 - protein , in addition to producing normal levels of s 1 - and s 2 - proteins ( compare the profile of gs3 - 55 with those of s 1 s 2 and s 2 s 3 plants ). all 17 plants that were partially compatible with s 3 - pollen produced the amounts of s 3 - protein that were invariably much lower than normal ( compare the profile of gs3 - 75 with that of gs3 - 55 ), and were comparable to or less than those present in immature buds of nontransgenic plants . all 10 plants that were fully compatible with s 3 - pollen did not produce any detectable amount of s 3 - protein ( see the profile of gs3 - 113 ). thus , it was concluded that the production of a normal level of s 3 - protein in the pistil of the 4 transgenic plants conferred on them the ability to completely reject s 3 - pollen . the finding that low level expression of the s 3 - transgene in the pistil of mature flowers was not sufficient for rejection of s 3 - pollen is similar to the previous findings that a low level of s - gene expression in immature buds of petunia plants correlated with their inability to reject self pollen 9 , 19 . both the s 3 - transgene and the antisense s 3 - gene did not affect the self - incompatibility behavior of the pollen of the transgenic plants ( data not shown ). these results are consistent with the model of the s - locus proposed by lewis which states that separate but closely linked genes control the self - incompatibility phenotype of the pollen and pistil 20 . in conclusion , the results presented in this invention provide direct in vivo evidence that the s - proteins of p . inflata are necessary and sufficient for the pistil to reject self pollen . this study also demonstrates the feasibility of using the antisense rna approach to break down self - incompatibility . furthermore , demonstration that the s - phenotype of a self - incompatible plant can be altered by the introduction of a single gene , the s - gene , should allow future dissection of the functional domains of the s - protein through mutagenesis studies . transformation of p . inflata plants with antisense s 3 - gene , and analysis of transgenic plants for presence of the transgene the construct is presented in fig1 a . the promoter used to express the antisense s 3 - cdna was contained in a dna fragment spanning from position - 2 , 032 bp to position + 15 bp of s 3 - gene 18 . this dna fragment had already been shown to confer pistil expression of the gene encoding b - glucuronidase ( gus ) in transgenic p . inflata plants ( unpublished results ). the ecori - ndei fragment of the s 3 - cdna used in the construct contained approximately 70 % of the full - length s 3 - cdna previously reported 9 . sense s 3 - cdna is indicated by an arrow pointing to the right ; antisense s 3 - cdna is indicated by an arrow pointing to the left . the transcriptional termination signal was provided by the nopaline synthase terminator ( nos - ter ) present on a binary ti - plasmid pbi101 ( clontech ). the nptii gene , which encodes neomycin phosphotransferase and confers kanamycin resistance in transgenic plants , is expressed by the promoter of the nos gene ( nos - pro ). the 2 , 047 bp s 3 - promoter fragment was cloned into the hindiii and smai sites of pbluescript ks + ( stratagene ) to yield ps3 . ps3 was digested with bamhi , and klenow enzyme was used to create blunt ends . in the same fashion , blunt ends were created on an ecori - ndei fragment of the s 3 - cdna , and this fragment was ligated in antisense orientation to the s 3 - promoter in ps3 . the s 3 - promoter - s 3 - cdna fusion product was released by double digestion with xhoi and saci , and the fragment was ligated into the sali and saci sites of pbi101 . ( the gus gene present on pbi101 was removed during this step .) the recombinant ti - plasmid , designated pas3 , was electroporated into agrobacterium tumefaciens strain lba4404 . leaf discs of p . inflata with s 2 s 3 genotype were infected with the agrobacterium by the co - cultivation method 21 on ms medium supplemented with benzylaminopurine ( 1 . 0 mg / l ) and naphthalene acetic acid ( 75 mg / l ). shoots were regenerated on fresh ms medium 22 supplemented with kanamycin ( 100 mg / ml ) and carbenicillin ( 500 mg / ml ). regenerated shoots were transferred to hormone - free ms medium containing the same concentrations of antibiotics to induce root formation . the level of protein present in the transgenic plants is represented in fig1 b , which is a radiograph of a filter from a genomic southern blot . the filter containing ecori digests of genomic dna was hybridized to a probe containing the full - length s 3 - cdna 9 . genomic dna was isolated from 2 g of frozen leaves with an elu - quick kit ( schleicher & amp ; schell ) following the manufacturer &# 39 ; s protocol . genomic dna ( 5 mg ) was digested with ecori , separated on a 0 . 8 % agarose gel , and transferred to a biotrans (+) nylon membrane ( icn ). the membrane was prehybridized in a solution containing 5 × ssc , 5 × denhardt &# 39 ; s , 0 . 1 % sds , and 100 mg / ml of sheared and denatured fish sperm dna for 2 hours at 65 ° c . hybridization was carried out in the same solution with the addition of 32 p - labelled s 3 - cdna for 16 hours at 65 ° c . the membrane was washed in 0 . 1 × ssc and 0 . 1 % sds at 65 ° c . for 1 hour and exposed on x - ray film with an intensifying screen for 2 days at - 70 ° c . analysis of the amounts of s 2 - and s 3 - rna , and s 2 - and s 3 - proteins in a parental s 2 s 3 plant and transgenic plants the analysis of the amount of the rna was performed using northern blots . the results are presented in fig2 a . two identical rna blots each containing total pistil rna ( 10 μg per lane ) of a parental s 2 s 3 plant , as - 39 , as - 14 , and as - 27 , were hybridized with two radiolabelled probes separately : s 2 - oligo , an oligonucleotide specific to sense s 2 - rna , and s 3 - oligo , an oligonucleotide specific to sense s 3 - rna . after autoradiography , the bound radiolabelled probes were removed from the blots , and the blots were hybridized with the third probe , rdna , which encodes 25s rrna of p . inflata . that step was performed in order to eliminate a possibility that a lack of signal is due to rna degradation . total rna was isolated from pistils as previously described 10 . rna samples were electrophoresed on 1 . 2 % agarose / formaldehyde gels and transferred to biotrans (+) membranes . the probe specific to sense s 2 - rna was a 51 - mer with sequence : 5 &# 39 ;- cagaacattgattatattatcttctttffaaaacgcgaatacttgtcgccagt - 3 &# 39 ; ( seq . id no : 1 ). this sequence is complementary to a segment of s 2 - cdna encoding amino acid residues 48 to 64 of s 2 - protein 9 , which is located in the hypervariable region hva of s - proteins 25 . the probe specific to sense s 3 - rna was a 54 - mer with sequence : 5 &# 39 ;- caaatcattgacaattctatcttttaagctgaacgacacaaacttatctccatc - 3 &# 39 ; ( seq . id no : 2 ). this sequence is complementary to a segment of s 3 - cdna encoding amino acid residues 48 to 65 of s 3 - protein 9 , which is also located in the hva region . the two oligonucleotides were 32 p - labelled at their 5 &# 39 ; ends with t4 polynucleotide kinase . for hybridization with s 2 - or s 3 - oligo , the membranes were prehybridized as described in fig1 b , except at 45 ° c . for 2 hr , and hybridized in the prehybridization solution containing s 2 - or s 3 - oligo probe at 45 ° c . overnight . the membranes were twice washed in 0 . 1 × ssc , 0 . 1 % sds at room temperature for 10 min each , and then washed with the same solution at 40 ° c . for 5 min . autoradiography was carded out at - 70 ° c . for 16 hr with an intensifying screen . the bound radiolabelled probes were removed from the membranes by boiling in 0 . 1 × ssc and 0 . 1 % sds . for hybridization with the rdna probe , the membranes were prehybridized as described for fig1 b in example 1 , and hybridized with the prehybridization solution containing 32 p - labelled rdna probe at 65 ° c . overnight . the membranes were washed in 0 . 1 × ssc , 0 . 1 % sds at 65 ° c . for 1 hr , and autoradiographed at - 70 ° c . for 1 hr with an intensifying screen . analysis of the amounts of s 2 - and s 3 - proteins the protein level was measured using fplc profiles . the results are presented on fig2 b . s 2 - and s 3 - proteins cannot be separated by sds - page due to their similar molecular weight 9 , but can be separated from each other and from other pistil proteins by cation - exchange chromatography on a mono - s column using the fplc system ( pharmacia ) 14 . the majority of pistil proteins flowed through during loading and washing of the column with 50 mm sodium phosphate ( ph 6 . 0 ). the bound proteins that were eluted with the salt gradient consisted mainly of s - proteins and a pistil - specific ribonuclease , x2 14 , 23 . the observed concomitant reduction of the level of s 2 - protein in as - 27 ( as well as in as - 4 and as - 35 , not shown ) and of the level of x2 in as - 27 by the antisense s 3 - gene may be due to the sequence similarity between the 585 bp fragment of s 3 - cdna used in the antisense construct and the corresponding regions in the genes for s 2 - protein 9 and rnase x2 23 . it has been previously observed that antisense rna can inhibit the mrna production from a target gene , as well as from genes with sequence similarity to the target gene 24 . pistils were collected from freshly opened flowers of each plant and stored at - 70 ° c . until use . forty milligrams of pistils from each plant were ground to a fine powder in liquid nitrogen , with further grinding after the addition of 1 ml of the extraction buffer containing 50 mm tris - hcl , ph 8 . 5 , 10 mm edta , 1 mm pmsf , 1 mm cacl 2 , and 1 mm dtt . the crude extract was centrifuged at 12 , 000 g for 10 min , and the supernatant filtered through a 0 . 45 mm millex - gv filter ( millipore ) to remove unsedimented fine particles . the filtrate was applied to a mono - s column ( hr 5 / 5 ) which had been equilibrated with 50 mm sodium phosphate ( ph 6 . 0 ). the bound proteins were eluted with a linear gradient of 0 to 500 mm nacl in the same buffer at a flow rate of 0 . 5 ml / min . proteins were monitored at a 280 nm with the sensitivity of the detector set to 0 . 1 aufs . no discernible differences in the fplc profiles for each plant were detected when two separate pistil extractions were used . the profile shown for each plant is one of the two runs . transformation of p . inflata plants with s 3 - gene , and analysis of transgenic plants for presence of the transgene schematic representation of the genomic dna used in transformation experiments is presented in fig3 a . the dna fragment , gs3 , spans from position - 2 , 032 bp to position + 1 , 553 bp of the s 3 - gene 18 . open boxes denote the exons ; the hatched box denotes the intron . restriction enzyme sites are indicated with one - letter abbreviations : h , hindiii ; n , nde i ; s , stu i ; b , bsu 96i . the 3 . 6 kb genomic dna ( gs3 ) was released from the cloning vector pbluescript ks + ( stratagene ) by digestion with xho i and sac i , and the fragment was ligated into sal i and sac i sites of a binary ti - plasmid vector pbi101 . the gus gene present on pbi101 was removed during this step . the recombinant ti - plasmid was introduced into p . inflata plants by agrobacterium - mediated transformation as described in fig1 b . the analysis was performed using the genomic southern blot technique . the falter containing hindiii digests of genomic dna was hybridized with a radiolabelled probe of the full - length s 3 - cdna 9 . the genomic dna was isolated from 1 nontransgenic plant s 1 s 2 and 6 transgenic plants , as indicated above the blot . the results are presented on fig3 b . the arrow marks a weakly hybridizing dna fragment of 9 . 9 kb which corresponds to the endogenous s 2 - gene . the endogenous s 1 - gene did not cross - hybridize with the s 3 - cdna probe under the conditions used . the dna size markers are indicated . two fragments seen in gs3 - 75 each resulted from one cut by hindiii within the integrated s 3 - gene and a second cut in the genome . genomic dna was isolated from 2 g of frozen leaves as described in example 1 for fig1 b . genomic dna ( 5 mg ) was digested with hindiii , separated on a 0 . 8 % agarose gel , and transferred to a biotrans (+) nylon membrane ( icn ). prehybridization , hybridization , and washing of the falter were carded out as described in example 1 for fig1 b . the filter was exposed on x - ray film at - 70 ° c . for 48 h with an intensifying screen . the amount of the rna was measured by northern blot . the results are presented in fig4 a . each lane of the rna blot contains 10 μg of total pistil rna . of the 7 plants analyzed , s 2 s 3 and s 1 s 2 are nontransgenic plants and the other 5 are transgenic plants . the blot was first hybridized with a radiolabelled oligonucleotide probe ( s3 ) specific to s 3 - rna . after autoradiography , the bound radiolabelled probe was removed , and the blot was hybridized with a second probe ( rdna ) which encodes 25s rrna of p . inflata . the absence of a hybridizing fragment in the s 1 s 2 sample confirms the specificity of the s3 probe used . the procedures for isolation of total rna and for northern analysis were identical to those described in example 2 . the s 3 - rna specific probe and the rdna probe used were described in example 2 . after washing , the amount of radioactivity associated with each hybridizing band was determined using a betagen betascope . the relative amount of s 3 - rna in each transgenic plant to that of s 2 s 3 plant was calculated after correction for the differences in the total amount of rrna . the filters were then exposed on x - ray films at - 70 ° c . for 30 min ( rdna probe ) and 5 h ( s3 probe ) with intensifying screens . the fplc was used to measure the mounts of proteins . the profiles are presented in fig4 b . total pistil protein of each plant was separated by a mono - s column . only the s - protein fractions are shown . extractions of total pistil protein and conditions for column chromatography were as described in example 2 , except for three modifications : 5 mg of the pistils from each plant were used for extractions ; the gradient was 2 times shallower ; proteins were monitored with the sensitivity of the detector set to 0 . 02 aufs . in addition , a different mono - s column with the same dimension was used . some of these modifications may account for differences in the salt concentration at which the same s - protein was eluted in the profiles shown here and in those shown in example 2 . thus , while i have illustrated and described the preferred embodiment of my invention , it is to be understood that this invention is capable of variation and modification , and i , therefore , do not wish or intend to be limited to the precise terms set forth , but desire and intend to avail myself of such changes and alterations which may be made for adopting the invention of the present invention to various usages and conditions . accordingly , such changes and alterations are properly intended to be within the full range of equivalents and , therefore , within the purview of the following claims . the terms and expressions which have been employed in the foregoing specification are used therein as terms of description and not of limitation , and thus there is no intention in the use of such terms and expressions of excluding equivalents of features shown and described or portions thereof , it being recognized that the scope of the invention is defined and limited only by the claims which follow . thus is described my invention and the manner and processing of making and using it in such full , clear , concise , and exact terms so as to enable any person skilled in the art to which it pertains , or with which it is most nearly connected , to make and use the same . 1 . frankel , r . & amp ; galun , e . in monographs on theoretical and applied genetics ( eds frankel , r ., gall , g . a . e . & amp ; linskens , h . f .) ( springer , berlin , 1977 ) 2 . de nettancourt , d . in monographs on theoretical and applied genetics ( eds frankel , r ., gall , g . a . e . & amp ; linskens , h . f .) ( springer , berlin , 1977 ) 3 . bredemeijer , g . m . m . & amp ; blass , j . 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