Patent Application: US-8954302-A

Abstract:
the present invention relates to a cotton β - tubulin gene cftub2 , and active fragments thereof . these promoters show strong fiber - specific activity .

Description:
the cftub2 promoter is an active fiber - specific promoter in cotton . results of a northern blot analysis of cdnas from a variety of cotton tissues showed that a cdna clone comprising the cftub2 gene was strongly expressed in young fibers of 8 and 14 days postanthesis ( dpa ), and also expressed in young ovules of 4 , 8 and 14 dpa , but less or not at all in other tissues . sequencing of the cdna clone revealed that it was 1623 bp in length containing a open reading frame of 1338 bp ( fig1 ). comparing the nucleotide and predicted polypeptide sequences of the cotton cftub2 with the data banks , it was found that the cftub2 cdna shared 96 %– 98 % homology at the amino acid level and over 78 % homology at the nucleotide level with the known β - tubulin cdnas and genes from other plants ( such as arabidopsis , tobacco , rice , soybean , maize , potato , carrot , etc .) [ liaud et al ., 1992 ; snustad et al ., 1992 ; villemur et al , 1994 ; tonoike , et al ., 1994 ; taylor et al ., 1994 ; kang et al ., 1994 ; okamura et al ., 1997 ; chu et al ., 1998 ; okamura et al ., 1999 ]. the transcripts of the cftub2 gene exhibited the highest accumulation in cotton young fibers of 8 dpa , and then there was a visible decrease in the accumulation of the gene products ( mrna ) with further development of the fibers . comparison of gene expression in different developmental stages of cotton ovules also showed that the gene transcripts accumulated more in 8 dpa ovules than in 4 and 14 dpa , and there was a gradual and visible decrease to an undetectable level in the accumulation of gene products with fiber development from 8 dpa to 28 dpa . this suggests that the gene is specifically expressed with a strict regulation at the transcriptional level during cotton fiber and ovule development , as with other cotton fiber - specific genes [ whittaker and triplett , 1999 ; shin and brown , 1999 ; kawai et al ., 1998 ; john , 1996a ; song and allen , 1997 ; ma et al , 1997 ; rinehart et al ., 1996 ; john and crow , 1992 ]. two fragments in the promoter region were isolated and cloned into pgem - t vector , respectively . one fragment of the cftub2 promoter was 1433 bp in length ( fig2 ), and another was 984 bp long . both fragments functioned as active , fiber - specific promoters . the constructs of cftub2 promoter / gus fusion gene were used to transform tobacco and cotton by agrobacterium - mediated gene transfer , using the pbi121 vector containing camv35s promoter / gus fusion as a positive control . consistent with the results from northern blot analysis , the gus gene driven by cftub2 promoter specifically expressed in the young fibers , but not in other tissues , in all the 31 transgenic cotton plants studied , while the gus activity was detected in all the tissues of positive control cotton plants ( 35s : gus ). a total of 36 transformed cotton plants were obtained and transplanted in soil to grow to maturation . similarly , it was found that under the cftub2 promoter , gus gene activity was only detected in the seeds in all of the 15 transgenic tobacco plants studied , suggesting the cftub2 promoter activity was also tissue - specific in tobacco ( the cotton fiber , being an elongated hair of the seed coat , finds histological correspondence in the tobacco seed coat ). this result , together with the above northern blot analysis , indicates that the cftub2 promoter controls gene specific expression at the transcriptional level in cotton fibers . accordingly , one embodiment of the present invention is a fiber - specific promoter obtained from the cotton fiber β - tubulin gene cftub2 . another embodiment of the present invention is a fiber - specific promoter comprising a 1433 kb active fragment of the cotton fiber cftub2 gene promoter . another embodiment of the present invention is a fiber - specific promoter comprising a 984 kb active fragment of the cotton fiber cftub2 gene promoter . stll another embodiment of the present invention is a promoter that is cotton fiber - specific comprising an active fragment of the cftub2 promoter fragment of seq id no : 2 . an active fragment is a sequence of shorter length than seq id no : 2 which still retains activity as a fiber - specific promoter in cotton . a fragment can comprise excisions , deletions , truncations or substitutions of the sequence of seq id no : 2 , or a combination of these . a preferred active fragment is the fragment consisting of nucleotides 449 – 1433 of seq id no : 2 . the promoters of the present invention are useful in creating transgenic cotton having altered fiber characteristics . the use of the fiber - specific promoters of the present invention permits selective expression of a transgene in the cotton fiber , permitting greater latitude in the types of transgenes employed . selective expression avoids problems such as the metabolic burden imposed on a transgenic plant by systemic expression of a transgene , or the adverse effects of the expression of a transgene in non - fiber tissues . examples for expressing desirable genes in cotton fiber , but not in other parts of the cotton plants include : ( 1 ) anthocynin genes for colored cotton , ( 2 ) silk protein genes from silk worm or spiders for increased strength of cotton fiber , ( 3 ) and biosynthesis of polyhydroxybutrate in cotton fiber for improved thermal properties and insulating characteristics [ john , et al ., 1996 ]. there are numerous examples in the art of fiber - enhancing genes that could be advantageously linked to the promoters of the present invention , and used to transform cotton using well - known techniques ( see , e . g ., umbeck , 1992 ), to achieve expression of the transgene in transgenic cotton fibers . see e . g ., john , 1996b , 1997a , 1997b ; john et al ., 1996 . isolation of fiber - specific cdna encoding cftub2 sequences expressed early during fiber development of cotton cotton seeds were surface - sterilized with 70 % ethanol for 30 – 60 seconds and 10 % h 2 o 2 for 30 – 60 minutes , followed by washing with sterile water . the seeds germinated on ½ ms medium on light at 28 ° c . in a culture room , and cotyledons and hypocotyls cut from sterile seedlings were used as transformation explant materials . cotton plants were grown in pots for dna and rna extraction . total rna was extracted from young fibers , ovaries , anthers , petals , sepals , leaves and roots of cotton by using the guanidinium thiocyanate method or sv total rna isolation system ( promega ). poly ( a )+ rna was purified by using oligo ( dt )- cellulose spin columns from an mrna purification kit ( pharmacia biotech ). cotton cdna was synthesized by using a cdna synthesis kit ( pharmacia biotech ). cotton cdna libraries were constructed by inserting the cdna fragments into the zap express vector ( stratagene ). poly ( a )+ rnas from cotton young fibers of about 8 and 14 days postanthesis ( dpa ), respectively , were converted to cdnas which were used to construct cotton cdna libraries . from the fiber cdna libraries , about 200 cdna clones were randomly picked out and subsequently sequenced . some clones with potential involvement in cell expansion were selected according to the sequence data . to find cdna clones whose transcripts are specifically expressed in cotton fibers , the expression pattern of the selected cdna clones was analyzed by northern blot hybridization with total rnas isolated from cotton fibers , ovules , anthers , petals , sepals , squares , leaves and roots , using probes from the clones . rna samples from the different cotton tissues were separated on agarose - formaldehyde gels , and transferred onto hybond - n nylon membranes by capillary blotting . rna northern blots were hybridized in expresshyb solution ( clontech ) at 68 ° c . with 32 p cdna probes prepared by random labeling ( promega prime - a - gene labeling system ). after hybridization , the blots were washed at 68 ° c . in 0 . 1 × ssc , 0 . 5 % sds for 30 – 60 minutes . the experimental results showed that one cdna clone strongly expressed in young fibers of 8 and 14 dpa , and also expressed in young ovules of 4 , 8 and 14 dpa , but less or not at all in other tissues . pcr fragments and cdna fragments were subcloned into vectors , and plasmid dna prepared with a qiagen plasmid kit was used as templates in pcr reactions . the pcr products were sequenced by autosequencer . sequencing of the cdna clone revealed that it was 1623 bp in length containing a open reading frame of 1338 bp , and identical to the β - tubulin gene ( fig1 ). this is the first cftub2 cdna clone isolated from cotton . comparing the nucleotide and predicted polypeptide sequences of the cotton cftub2 with the data banks , it was found that the cftub2 cdna shared 96 %– 98 % homology at the amino acid level and over 78 % homology at the nucleotide level with the known β - tubulin cdnas and genes from other plants ( such as arabidopsis , tobacco , rice , soybean , maize , potato , carrot , etc .) [ liaud et al ., 1992 ; snustad et al ., 1992 ; villemur et al , 1994 ; tonoike , et al ., 1994 ; taylor et al ., 1994 ; kang et al ., 1994 ; okamura et al ., 1997 ; chu et al ., 1998 ; okamura et al ., 1999 ]. total rnas from different tissues of cotton were used to reverse - transcribe first - strand cdnas which were used as templates in differential display pcr reactions . differential display analysis was carried out by using a differential display kit ( clontech ). first - strand cdna was synthesized with 2 pg total rna as starting materials of reverse transcription and oligo ( dt ) as primers at 42 ° c . for 1 hour . differential display pcr reactions were carried out with a initial cycle consisting of 94 ° c . for 5 minutes , 40 ° c . for 5 minutes and 68 ° c . for 5 minutes , followed by two cycles consisting of 94 ° c . for 2 minutes and 40 ° c . for 5 minutes and 68 ° c . for 5 minutes , and then 25 cycles consisting of 94 ° c . for 1 minute and 60 ° c . for 1 minute and 68 ° c . for 2 minutes , and a final extension at 68 ° c . for 7 minutes . target differential display bands were excised and reamplified for further analysis . reproducible fiber - specific differential display products were targeted for further analysis . the cdna in each target band was harvested and regenerated by pcr amplification . the isolated cdna was subsequently subcloned into vectors and sequenced . the northern blot analysis showed that the transcripts of the cftub2 gene exhibited a highest accumulation in cotton young fibers of 8 dpa , and then there was a visible decrease in the accumulation of the gene products ( mrna ) with further development of the fibers . comparison of gene expression in different developmental stages of cotton ovules also showed that the gene transcripts accumulated more in 8 dpa ovules than in 4 and 14 dpa , and there was a gradual and visible decrease to an undetectable level in the accumulation of gene products with fiber development from 8 dpa to 28 dpa . this suggests that the gene is specifically expressed with a strict regulation at the transcriptional level during cotton fiber and ovule development , as seen with other cotton fiber - specific genes [ whittaker and triplett , 1999 ; shin and brown , 1999 ; kawai et al ., 1998 ; john , 1996a ; song and allen , 1997 ; ma et al , 1997 ; rinehart et al ., 1996 ; john and crow , 1992 ]. based on the screened cftub2 cdna sequence , the cftub2 promoter was isolated from cotton genome walker libraries by genome walker pcr . total dna was extracted and purified from leaves of cotton plants by using the following method . liquid n 2 was added to 4 g of leaf tissues , and the leaves were homogenized thoroughly . 20 ml ice - cold extraction buffer ( 63 g / l glucose , 0 . 1 m tris . hcl ( ph 8 . 0 ), 5 mm edta , 20 g / l pvp - 40 , 1 g / l dieca , 1 g / l ascorbic acid , 2 ml / l , betamercaptoethanol ) was added to the homogenized tissues in a 50 ml tube and centrifuged at 2500 rpm for 15 minutes . after removing the supernatant , 10 ml lysis buffer was added to each tube . the resuspended pellets were incubated at 65 ° c . for 30 minutes . 10 ml chloroform was added to each tube , mixed with the samples and centrifuged at 3500 rpm for 10 minutes . the supernatant was transferred to a clean tube , and chloroform extraction was repeated one more time . the supernatant was transferred to a clean tube , and 0 . 6 volume isopropanol was added to each tube for dna precipitation . after centrifuging at 3500 rpm for 30 minutes , the dna was washed with 70 % ethanol . the isolated genomic dna was then dissolved in sterile water or te ( 10 mm tris . hcl , 1 mm edta ) for use . cotton genomic dna libraries were constructed from leaves of cotton plants . dna was partially digested with bamh i , and the dna fragments were cloned in the bamh i site of the zap expression vector ( stratagene ). genome walker libraries were constructed by using universal genome walker kit ( clontech ). genomic dna from leaves of cotton plants was digested with five restriction enzymes respectively , and then purified by phenol / chloroform and precipitated by ethanol . digested dna was ligated to genome walker adaptors . two rounds of genome walker pcr reactions were carried out successively . 1 μl of each genome walker dna library was used as templates in the primary pcr , and the primary pcr products were used as templates in secondary pcr . the pcr was started at 95 ° c . for 1 minute , followed by 35 cycles consisting of 95 ° c . for 15 seconds and 68 ° c . for 4 minutes , and a final extension at 68 ° c . for 6 minutes . target pcr bands were cut out and purified by geneclean kit ( bio 101 ). two fragments in the promoter region were isolated and cloned into pgem - t vector , respectively . one fragment of the cftub2 promoter was 1433 bp in length fig2 , and another was 984 bp long . fig2 . a hind iii site and a bamh i site were created at the 5 ′- end and 3 ′- end of the 0 . 9 kb cftub2 promoter fragment of cotton respectively by pcr method . the hind iii / bamh i fragment was initially subcloned into pgem - t vector ( promega ). plasmid dna containing the cftub2 promoter fragment was digested with hind iii and bamh i , and the digested fragment was isolated by agarose gel electrophoresis . a chimeric cftub2 promoter / gus construct was generated by insertion of the fragment , replacing camv 35s promoter , into the hind iii / bamh i sites of pbi121 vector . the 1 . 3 kb of bamh i / bamh i cftub2 promoter fragment was initially subcloned into the pgem - t vector ( promega ). plasmid dna containing the cftub2 promoter fragment was digested with bamh i , and the digested fragment was isolated by agarose gel electrophoresis . a chimeric cftub2 promoter / gus construct was generated by insertion of the fragment into the bamh i site of pbi101 vector . in order to characterize the function of cftub2 promoter in fiber - specific expression of the cftub2 gene , a 1 . 3 kb of fragment and a 0 . 9 kb of fragment of the cftub2 promoter were fused with gus coding sequence in the gene expression vector pbi101 or pbi121 ( deleting camv35s promoter ), respectively ( fig3 ). the constructs of cftub2 promoter / gus fusion gene were used to transform tobacco and cotton by agrobacterium - mediated gene transfer , using the pbi121 vector containing camv35s promoter / gus fusion as a positive control . the camv35s promoter is active in all the tissues of cotton and other plants and is a constitutive promoter [ odell et al ., 1985 ; ow et al ., 1987 ; mccabe and martinell , 1993 ]. a binary vector containing either a cftub2 promoter / gus fusion gene or the camv35s promoter / gus control control was transferred into agrobacterium tumefaciens strain lba 4404 . cotton explants for transformation were obtained from cotton seedlings grown as in example 1 . tobacco explant material was obtained from tobacco seedlings . tobacco seeds were surface - sterilized with 70 % ethanol for 30 – 60 seconds and 0 . 1 % hgcl 2 for 15 minutes , followed by washing with sterile water . the seeds germinated on ½ ms medium on light at 28 ° c . in culture room , and leaves cut from sterile seedlings for use as explants for transformation . cotton cotyledon and hypocotyl explants and tobacco leaf explants were transformed by the agrobacterium with the vectors , and transformed plants were transplanted to soil in greenhouse for growing to maturity . tobacco leaves were cut into about 2 × 2 cm pieces , and immersed in agrobacterium suspension for 5 minutes . the infected tobacco explants were cultivated on ms medium with 1 mg / l 6 - ba for 48 hours at 28 ° c ., and then transferred onto selection ms medium containing 100 mg / l kanamycin and 1 mg / l 6 - ba for 20 – 30 days for selecting transformed shoots ( kanamycin - resistant shoots ). the transformed shoots were cut from the calli and rooted on ms medium with 50 – 100 mg / l kanamycin . the transformed tobacco plants were transplanted to soil in greenhouse for growing to maturity . the cotyledon and hypocotyl were used as explants for cotton transformation . cotton seeds were surface - sterilized with 70 % ethanol for 30 seconds and 10 % h 2 o 2 for 60 minutes , followed by washing with steril water . these seeds were incubated in the sterile water at 28 ° c . after over night , the seeds sprouted . the embryos were taken out and put on the im medium ( ½ { ms ( macronutriants , micronutrients , edta - fe )+ vb1 10 mg / l + vb6 1 mg / l + vpp 1 mg / l + myo - insitol 100 mg / l }+ phytogel 2 g / l ph = 6 . 4 ) at 28 ° c . for 7 days . the cotyledon and hypocotyl of cotton were used as explants for transformation . after cutting into 5 mm 2 ( mm ) piece , the explants were soaked in the agrobacterium tumefaciens strain lba 4404 suspension ( od 600 = 0 . 2 – 0 . 4 ) for 15 minutes . then the explants were put on cm medium ( ms ( macronutriants , micronutrients , edta - fe )+ vb1 10 mg / l + vb6 1 mg / l + vpp 1 mg / l + myo - instiol 100 mg / l + 2 . 4 - d 0 . 1 mg / l + kt 0 . 1 mg / l + glucose 30 g / l + mgcl 2 0 . 7 mg / l + phytogel 2 g / l ph = 6 . 4 ) at 24 ° c . for 2 days . after washing with liquid ms medium , the explants were put on the sm medium ( ms ( macronutriants , micronutrients , edta - fe )+ vb1 10 mg / l + vb6 1 mg / l = vpp 1 mg / l + myo - insitol 100 mg / l + 2 . 4 - d 0 . 1 mg / l + kt 0 . 1 mg / l + glucose 30 g / l + mgcl 2 0 . 7 mg / l + phytogel 2 g / l + kanamycin 50 mg / l + cefutoxime 200 mg / l ph = 6 . 4 ) on light at 28 ° c . in culture room for selecting and the subculture was per month . after 2 – 3 months subculturing on sm , the calli were induced from explants . the calli were transferred on dm medium ( ms ( macronutriants , micronutrients , edta - fe )+ vb1 10 mg / l + vb6 1 mg / l + vpp 1 mg / l + myo - insitol 100 mg / l + kno 3 19 g / l + mgcl 2 0 . 7 mg / l + glucose 30 g / l + phytogel 3 g / l ph = 6 . 4 ) and subcultured per month . after about 5 months , the somatic embryos begin to form . continuing to culture the young embryos on dm medium until they develop into maturity . the mature embryos were transferred on gm medium ( ½ { ms ( macronutriants , micronutrients , edta - fe )+ vb1 10 mg / l + vb6 1 mg / l + vpp 1 mg / l + myo - insitol 100 mg / l )+ naa 0 . 01 mg / l + glucose 30 g / l + phytogel 3 . 5 g / l ph = 6 . 4 ) in the box for developing into plantlets . and then the plantlets were transplanted in the soil for the plant growing and collecting the transgenic seeds . transgenic tobacco and cotton plants possessing the chimeric cftub2 promoter / gus gene ( or 35 s : gus gene ), and non - transformed plants as negative controls , were analyzed by dna southern blot hybridization and by gus histochemical assay . total genomic dna from cotton and tobacco leaves were digested with restriction enzymes , separated on agarose gels , and transferred onto hybond - n nylon membranes by capillary blotting . dna southern blots were hybridized in expresshyb solution ( clontech ) at 68 ° c . with 32 p - dna probes prepared by random labeling ( promega prime - a - gene labeling system ). after hybridization , the blots were washed at 68 ° c . in 0 . 1 × ssc , 0 . 5 % sds for 30 – 60 minutes . the 32 p - labeled nylon membranes were exposed to x - ray film at − 70 ° c . for autoradiography . the results of southern blot analysis demonstrated that cftub2 promoter / gus gene was integrated into tobacco and cotton genomes . total of 325 transformed cotton plants , which belong to 31 transformed lines , were obtained and transplanted in soil to grow to maturation . histochemical assays for gus activity in transgenic tobacco and cotton plants were conducted according to the protocol described previously by jefferson et al . ( 1987 ) with some modifications . fresh tissues from the plants were incubated in x - gluc ( 5 - bromo - 4 - chloro - 3 - indolylglucuronide ) solution consisting of 0 . 1 m sodium phosphate ( ph 7 . 0 ), 10 mm ethylene diaminetetraacetic acid ( edta ), 0 . 5 mm potassium ferrocyanide and 0 . 5 mm potassium ferricyanide , and 0 . 1 % x - gluc ( clontech chemical ) overnight . the stained plant materials were then cleared and fixed by rinsing with 100 % and 70 % ethanol successively , and the samples were examined and photographed directly or under a microscope . consistent with the results from northern blot analysis , the gus gene driven by cftub2 promoter specifically expressed in the young fibers , but not in other tissues , in all the 31 transgenic cotton plants studied , while the gus activity was detected in all the tissues of positive control cotton plants ( 35 s : gus ). a total of 36 transformed cotton plants were obtained and transplanted in soil to grow to maturity , all of which had detectable gus activity only in the young fibers , not in the flower organs such as anthers , petals and sepals , or in leaves and roots . similarly , it was found that under the cftub2 promoter , gus gene activity was only detected in the seeds in all of the 15 transgenic tobacco plants studied , suggesting the cftub2 promoter activity was also tissue - specific in tobacco . this result , together with the above northern blot analysis , indicates that the cftub2 promoter controls gene specific expression at the transcriptional level in cotton fibers . an y q , huang s , mcdowell j m , 1996 . conserved expression of the arabidopsis act1 and act3 actin subclass in organ primordia and mature pollen . plant cell , 8 ( 1 ): 15 – 30 . arthur j c , 1990 . in polymers : fibers and textile , a compendium , ed . kroschwitz , j i . 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