Patent Application
20110107461
1. Field of the Invention
The present invention relates to cloning of novel gene sequences expressed and repressed during winter dormancy in the apical buds of Camellia sinensis L. (O.) Kuntze (hereinafter, referred to tea) bush. Particularly, this invention relates to identification, cloning and analysis of novel 3 prime (hereinafter called as 3′) ends of the genes (gene in the present invention refers to the deoxyribonucleic acid (hereinafter known as, DNA) sequences that are expressed and repressed in winter-dormant apical buds of tea. 3′ end refers to that end of DNA which has free hydroxyl group at 3rd position of the carbohydrate moiety of the DNA molecule.
2. Background and Prior Art References to the Invention
Tea (Camellia sinensis L. (O.) Kuntze) is a perennial plant grown in 31 countries located between 45° N-30° 5 and 150° E-60° W. Being perennial, the plant experiences several cycles of summer, winter and rainy seasons. Apical bud and the associated two leaves (hereinafter, referred to BATS) are used for tea manufacture. BATS are harvested at frequent interval, depending upon their availability. Prevailing environmental conditions (such as temperature, sun-shine hours and soil and atmospheric moisture etc.) and tea clone determine the availability of BATS for the purpose of harvesting.
A temperature of 30° C. and a day length of 13 hours are considered ideal for growth of the plant. As the temperature drops below 13° C. and day length becomes shorter than 11 hours and 15 minute, the growth and development of BATS is stopped. This phenomenon of suspension of the growth of BATS is termed as winter dormancy. Unlike deciduous plants, tea does not shed any leaf and remains green during dormancy period (Barua, D. N. 1989. Science and practice in tea culture. Tea Research Association, Calcutta, India. 509 p.). Winter dormancy is a universal to phenomenon occurring in tea crops grown under the environmental conditions described above (Barua, D. N. 1989. Science and practice in tea culture. Tea Research Association, Calcutta, India. 509 p.). The following Chart 1 illustrates the period of winter dormancy in various tea growing countries across the globe:
(Data adapted from Barua, D. N. 1989. Science and practice in tea culture. Tea Research Association, Calcutta, India. 509 p.; Dudeja, V. 1992. Tea Today 13: 1-5).
Temporary suspension of growth or the dormancy is not only limited to apical buds as in the case of tea but, is prevalent in other plats and systems as follows:
Modulation of dormancy has been and will continue to be a key issue in agriculture system. For example, if seeds or tubers or corms have reduced dormancy periods, before-time germination would lead to a loss to the growers if, the intention was to use them as sowing material. If intended for human or animal consumption, there will be a loss in the nutritional and or processing quality. In either of the cases, economic benefits will not be realized. On the contrary to this, an extended period of dormancy will lead to late germination or sprouting in the grower's field that would affect the crop performance and the crop yield.
Also, one of the major requirements of the tea industry is to have a tea plant with no or at least a reduced period of dormancy in the areas where this problem is prevalent. Nakayama, A. and Harada, S. (Bulletin of the Tea Research Station, Japan, 1962. 1: 28-39) and Barua, 1969 (Two and A Bud. 16: 41-45) pointed out that low temperature coupled with short day length were responsible for inducing winter dormancy in tea. Hence, application of gibberellic acid, a plant growth regulator, was recommended to break dormancy. But the action of gibberellic acid was not persistent. Moreover, the results varied with the clones (Das, S. 1977. Ph.D. Thesis, Gauhati University, Assam; Rustogi, P. N. 1980. Two and A Bud. 21: 33). Studies in seeds, tubers and corms also resulted the similar conclusions (Aalen, R. B., Opsahl-Ferstad, H-G., Linnestad, C. and Olsen, O-A. 1994. Plant J. 5: 385-396; Bagni, N., Malucelli, B. and Torrigiana, P. 1980. Physiol. plant. 49: 341-345; DeBottini, G. A., Bottini, R. and Tizio, R. 1982. Oyton. 42: 115-121; Bewley, J. D. and Black, M. 1985. Physiology of development and germination. Plenum Press, New York. pp 190-192; Delvallee, I., Paffen, A., and de Klerk, G. J. 1990. Physiol. Plant. 80: 431-436; Djilinov, D., Gerrits, M., Ivanova, A., Van Onikelen, H. A. and Dc Klerk, G. J. 1994. Physiol. Plant. 91: 639-644; Ginzberg, C. 1973. J. Exp. Bot. 24: 558-566; Kurashi, S., Daisuki, Y., Naoki, S, and Shigeki, H.1998. J. Plant Growth Regul. 8: 3-10).
The above results dictated the adoption of molecular approach. A few dormancy related cDNAs have been cloned from seeds of Bromus and Hordeum vulgare using differential screening (Goldmark, P. J., Curry, J., Morris, C. F. and Walker-Simmons, M. K. 1992. Plant Mol. Biol. 19: 433-441; Aalen, R. B., Opsahl-Ferstad, H-G., Linnestad, C. and Olsen, O-A. 1994. Plant J. 5: 385-396.). Clone pBS128 was isolated from dormant Bromus seeds and the application of ABA to non-dormant seeds resulted in enhanced expression of this transcript (Goldmark, P. J., Curry, J., Morris, C. F. and Walker-Simmons, M. K. 1992. Plant Mol. Biol. 19: 433-441). This pBS128 from Bromus secalinus, which is maintained at a high level in dormant, but not in non-dormant hydrated seeds, has been suggested to play a role in the maintenance of embryo dormancy. Sequence analysis revealed that the encoded to protein belongs to recently discovered group of antioxidants called peroxiredoxin (Li, B. and Forey, M. E. 1997. Trends in Plant Sci. 2: 384-389). The sequence analysis did not show any homology to the other reported genes. Interestingly, a transcript B15C present in the embryo of developing barley seeds showed 95% homology with the clone pBS128 (Aalen, R. B., Opsahl-Ferstad, H-G., Linnestad, C. and Olsen, O-A. is 1994. Plant J. 5: 385-396.).
Differentially expressed partial cDNA fragments have been cloned from dormant and non-dormant wild oat embryos (Johnson, R. R., Cranston, H. J., Chaverra, M. E. and Dyer, W. E. 1995. Plant Mol. Biol. 28: 113-122). Out of several such clones, two clones namely AFD4 and AFN5 were found to be very interesting. The protein coded by AFD4 (protein Z analogue) was presumed to be a repressor of germination that maintains the seeds in dormant state. AFN5, which was expressed very early during imbibition, might code for a protein to be needed to signal or initiate early steps in germination (Johnson, R. R., Cranston, H. J., Chaverra. M. E. and Dyer, W. E. 1996. Plant dormancy, Physiology, Biochemistry and Molecular Biology. CAB International, Wallingford, UK. pp. 293-300). Other embryo specific genes that are induced by the application of ABA have also been identified (Hatzopoulos, P., Fong, F. and Sung, Z. R. 1990. Plant Physiol. 94: 690-695; Vance, V. B. and Huang, A. H. C. 1988. 3. Biol. Chem. 263: 1476-1481), but their correlation with dormancy was not established.
The following Chart 2 illustrates the status of information available on the phenomenon of dormancy in plants:
polyrhiza)
menziesii(Mirb.)
fatua L. caryopses
thaliana C24 ecotype,
vulgare L.)
vinifera L.
sylvatica
secalinas (Cheat)
sylvatica
Hordeum cv. Triumph
Iedebouri cv. Golden
Triticum aestivum L.
Rosa hydrida L. cv.
Ruidrilo Vivaldi
esculentum Mill. Cv
tuberosum L. cv. King
Hordeum vulgare cv.
menziesii)
Menziesii)
persica L.)
Agrobacterium
rizogenes rolC and
There have been attempts to modulate dormancy process as follows:
Below is specifically given a state of art knowledge with reference to the dormancy related genes from apical buds of the plants:
Reference may be made to document (1) by Or, E., Vilozny, I., Eyal, Y. and Ogrodovitch, A. 2000. Plant Mol. Biol. 43: 483-494, wherein is described the identification of a grape dormancy-breaking-related protein kinase (hereinafter referred to GDBRPK) transcript, which was up-regulated upon chemical induction of dormancy release by hydrogen cyanamide. GDBRPK has a 976 base pair (hereinafter known as, bp) open reading frame that encodes for 325 amino acids. This is followed by a 241 bp 3′-untranslated region. GDBRPK, presumably, served as a sensor of stress signal.
Reference may be made to document (2) by Rohde, A., Montagu, M. V. and Boerjan, W. 1996. (In Somatic Cell Genetics and Molecular Genetics of Trees. eds Ahuja, M. R., Boerjan, W. and Neale, D. B. Kluwer Academic Publishers. Netherlands. pp 183-188), wherein it was concluded that the process of bud and seed dormancy may share similar events. It may be noted that the work did not include any differential gene expression pattern or protein profiling studies. The conclusion was based upon work on transgenic Populus harboring chimeric β-glucuronidase gene under the control of cell cycle specific promoters. The studies, therefore, did not reveal any mechanism of bud dormancy per se or yielded any gene(s) related to the bud dormancy.
The drawbacks in the prior art are:
The above drawbacks have been eliminated for the first time in a simple and reliable manner by the present invention, which is not so obvious to the person skilled in the art.
A benefit of the present invention is the cloning of novel DNA sequences expressed and repressed during winter dormancy in the apical buds of tea (Camellia sinensis L. (O.) Kuntze) bush growing under field conditions.
Yet another benefit of the present invention is to ensure the same genetic make up of the plant considered for the purpose of identification and cloning of novel DNA sequences expressed and repressed during winter dormancy in the apical buds of tea (Camellia sinensis L. (O.) Kuntze) bush growing under field conditions.
Yet another benefit of the present invention is to generate a spectrum of the gene(s) expressed and repressed during the process of winter dormancy in the apical buds of tea (Camellia sinensis L. (O.) Kuntze) bush growing under field conditions.
Yet another benefit of the present invention is the identification of 3′ ends of the gene(s) expressed and repressed during the process of winter dormancy in the apical buds of tea bush growing under field conditions.
Yet another benefit of the present invention is the confirmation of the identified 3′ ends of the differentially expressed gene(s) for establishing differential expression during winter dormancy in the apical buds of tea bush growing under field conditions.
Still another benefit of the present invention is the cloning of the identified 3′ ends of the differentially expressed gene(s).
Yet another benefit of the present invention is the sequencing of the identified 3′ ends of the cloned gene.
Accordingly, the present invention provides:
(a) novel DNA sequences expressed and repressed during winter dormancy in the apical buds of Camellia sinensis L. (O.) Kuntze (tea) bush under field conditions,
(b) cloning of such sequences have been from the same generic make up of the tea bush,
(c) a spectrum of 3′ ends of the expressed and repressed genes in non-dormant and dormant apical buds of tea bush growing under field conditions,
(d) confirmation of the identified 3′ ends of the differentially expressed gene(s) for establishing differential expression during winter dormancy in the apical buds of tea bush growing under field conditions,
(e) method to correlate the identified gene with the dormancy of tea buds under field conditions, and
(f) sequencing of the cloned 3′ ends of the differentially expressed gene(s) showed uniqueness in terms of novel sequences not deposited in the data bank so far.
Accordingly, the present invention provides novel DNA sequences expressed and repressed during winter dormancy in the apical buds of Camellia sinensis L. (O.) Kuntze (tea) bush or a tree species, said sequences comprising SEQ ID NOs: 1 to 4.
In an embodiment of the invention, the novel DNA sequences, which are cloned from the tea bush of the same, genetic make up.
In another embodiment of the invention, the novel DNA sequences, which are cloned from the tea bush of the same genetic make up growing under field conditions.
In another Novel DNA sequences as claimed in claim 1, are associated with winter dormancy in tea.
In still another embodiment of the invention, the novel DNA sequences, which are cloned by any methods but not limited to, subtractive hybridization and differential screening.
In yet another embodiment of the invention, the nucleotide sequence of the DNA is given in SEQ ID NO 1.
In yet another embodiment of the invention, the nucleotide sequence of the DNA as is given in SEQ ID NO 1 is overexpressed only in non-dormant apical buds of tea.
In yet another embodiment of the invention, the nucleotide sequence of the DNA is given in SEQ ID NO 2.
In yet another embodiment of the invention, the nucleotide sequence of the DNA as is given in SEQ ID NO 2 is expressed only in non-dormant apical buds of tea.
In yet another embodiment of the invention, the nucleotide sequence of the DNA is given in SEQ ID NO: 3.
In yet another embodiment of the invention, the nucleotide sequence of the DNA as is given in SEQ ID NO 3 is expressed only in non-dormant apical buds of tea.
In yet another embodiment of the invention, the nucleotide sequence of the DNA is given in SEQ ID NO: 4.
In yet another embodiment of the invention, the nucleotide sequence of the DNA as is given in SEQ ID NO 4 is expressed only in dormant apical buds of tea.
In yet another embodiment of the invention, the novel sequences are capable of being cloned to full-length cDNA.
In yet another embodiment of the invention, the novel sequences are capable of being cloned to full length genomic DNA.
In yet another embodiment of the invention, the novel sequences are capable of being cloned to important sequences, such as but not limited to, promoter sequences and regulatory sequences etc.
Tea bushes, belonging to chinary type, growing and maintained in the tea farm of the Institute of Himalayan Bioresource Technology, Palampur (32° 04′ N, 76° 29′ E; altitude, 1300 m) were selected. Tea plants belonging to other types namely, assamica and combod could have also been selected. However, due to predominance of the chinary type in Kangra region of the Himachal Pradesh, this particular clone was is selected.
In a preferred embodiment of the present invention the genetic make of the plant, considered for the purpose of identification and cloning of novel DNA sequences expressed and repressed during winter dormancy in the apical buds of tea (Camellia sinensis L. (O.) Kuntze) bush growing under field conditions was ensured to be the same. This is important for a plant like tea. Tea is a highly cross-pollinated plant and grown by the seed. This leads to enormous plant to plant heterogeneity due to differential genetic make up. Each seed raised plant of tea is regarded a clone in itself. Particularly, for the inventions related to compare gene expression pattern for the purpose of cloning the differentially expressed gene in response to an altered environmental condition, such as is the purpose of the present invention of comparing and cloning the expressed and repressed gene(s) from the control and a winter dormant plant, it is absolutely essential that the plants experiencing altered environmental conditions have the same genetic make up. Having realized this important aspect, only one bush of tea belonging to chinary type was selected.
In another embodiment of the present invention the apical buds of tea measuring 1.2-1.5 cm (in length); 0.5-0.6 cm (perimeter) and were collected during non-dormant season (in the month of April) at 10.00 O'clock in the morning from only one bush. Apical buds during non-dormant season grow to open into the leaves. These are referred to as non-dormant (hereinafter known as ND) buds and are considered “control” within the scope of the present invention. The bush yielded around 100 numbers of apical buds. Apical buds were washed with diethyl pyrocarbonate (hereinafter known as DEPC) treated water [to prepare DEPC treated water, DEPC was added in distilled water to a final concentration of 0.1% followed by autoclaving (i.e. heating at 121° C. under a pressure of 1.1 kg per square centimeters) after an overnight incubation.], harvested and immediately dipped in liquid nitrogen to freeze the cellular constituents for ceasing the cellular activities.
Tea bush became dormant from the month of November onwards. The apical buds during this period do not grow in the size to open into the leaves and are referred to as dormant (hereinafter known as D) buds. The same bush, from which the ND buds were collected, was used to collect the D buds. D buds, measuring the similar dimensions as for the ND buds, were plucked and stored essentially as described for ND buds.
Collection of ND and D buds from the same bush ensured the same genetic make up of the tissue under consideration.
In another embodiment of the present invention total RNA from ND and D buds was isolated and the “differential display technique” (Liang, P., Zhu, W., Zhang, X., Guo, Z., O'Connell, R., Averboukh, L., Wang, F. and Pardee, A. B. 1994. Nucleic acids Res. 22: 1385-1386) was employed to generate a spectrum of 3′ ends of the expressed and repressed genes in ND and D buds of tea.
In an advantageous embodiment of the present invention 3′ ends of the expressed and repressed genes in ND and D buds of tea were ligated into a vector to yield a recombinant plasmid, which upon transformation into a suitable E. coli host resulted into a clone. Vector, in the present invention refers to the sequence of DNA capable of accepting foreign DNA and take the form of a circular plasmid DNA that shows resistance to a given antibiotic.
In yet another embodiment of the present invention the gene cloned was tested for its expression or repression in ND and D buds of tea to define association of the cloned gene with the dormancy process.
In another embodiment of the present invention the gene was sequenced using the dideoxy chain termination method (Sanger, F. S., Nicklen, S, and Coulson, A. R. 1977. Proc. Natl. Acad. Sci. USA. 74: 5463-5467) to figure out the uniqueness of the gene.
s One more embodiment of the invention, wherein the sequence data is used for obtaining, important information on the gene regulation.
In another embodiment of the invention, the novel genes are used to modulate winter dormancy in plants after transferring these genes using the techniques such as, but not limited to, Agrobacterium mediated transformation and Biallistic medited transformation.
In another embodiment of the invention, it is possible to modulate winter dormancy using the novel genes in the plants such as, but not limited to, tea, plums, cherries, peaches, Taxus, apples, peers, vines, grapes, olives, Kiwi fruit, figs, morus, strawberries, raspberries, cranberries, blackberries, loganberries, almonds, walnuts and chestnuts after transferring these genes using the techniques such as, but not limited to, Agrobacterium mediated transformation and bialistic medited transformation.
In still another embodiment of the invention, use of sequence data of the novel genes for obtaining important information on the gene regulation to be exploited to regulate gene expression in transgene.
In still another embodiment of the invention relates to use of cDNAs and the genomic DNAs for synthesizing unique proteins and in addition use of unique proteins for raising antibodies.
In yet another embodiment of the invention relates to use of the present antibodies as probe to look for the similar proteins in other plants, animal and/or microbial systems or the like.
In yet another embodiment of the invention relates to use of novel sequences, cDNAs and the genomic DNAs of the present invention as probe to look for the sequences of nucleotides in other plants, animal and/or microbial systems and the like.
In yet another embodiment of the invention relates to use of novel sequences, cDNAs and the genomic DNAs of the present invention, as probe to look for the expression of these sequences of nucleotides in other plants, animal and/or microbial systems and the like.
In yet another embodiment of the invention relates a method to correlate the identified gene with the process of dormancy of tea buds as described for sequence ID 1 is unique.
In yet another embodiment of the invention relates a method which, can be applied to other sequence ID as well.
In yet another embodiment of the invention relates to a method which, can be applied to other crops such as, but not limited to plums, cherries, peaches, Taxus, apples, peers, vines, grapes, olives, Kiwi fruit, figs, morus, strawberries, raspberries, cranberries, blackberries, loganberries, almonds, walnuts and chestnuts as well for correlating similar genes.
The present invention will be illustrated in greater details by the following examples. These examples are presented for illustrative purposes only and should not be construed as limiting the invention, which is properly delineated in the claims.
To ensure a high quality of ribonucleic acid (hereinafter known as, RNA) from ND and D buds of tea bush, RNeasy plant mini kits (purchased from M/s. Qiagen, Germany) were used. Manufacturer's instructions were followed to isolate RNA. RNA was quantified by measuring absorbance at 260 nm and the purity was monitored by calculating the ratio of absorbance measured at 260 and 280 nm. A value >1.8 at 260/280 nm was considered ideal for the purpose of present investigation. The formula used to calculate RNA concentration and yield was as follows:
Concentration of RNA (μg/ml)=A260(absorbance at 260 nm)×40×dilution factor
Total yield (μg g)=concentration×volume of stock RNA sample
To check the intigrity of RNA, 5-6 lg of RNA in 4.5 μl was diluted with 15.5 μl of M1 solution (2 μl of 5×MOPS buffer, 3.5 μl of formaldehyde, and 10 μl of formamide [5×MOPS buffer: 300 mM sodium acetate, 10 mM MOPS (3-{N-morpholino]propanesulfoni-c acid}, 0.5 mM ethylene diamine tetra-acetic acid (EDTA)] and incubated for 15 minutes at 65° C. RNA was loaded onto 1.5% formaldehyde agarose-gel after adding 2 μl of formaldehyde-gel loading buffer [50% glycerol, 1 mM EDTA (pH, 8.0), 0.25% bromophenol blue, 0.25% xylene cyanol FF], and electrophoresed at 72 volts in 1×MOPS buffer (60 mM sodium acetate, 2 mM MOPS, 0.1 mM EDTA), (Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
To remove the residual DNA, RNA (10-50 lg) was digested using 10 units of DNase I, in 1×reaction buffer [10×reaction buffer: 100 mM Tris-Cl (pH, 8.4), 500 mM KCl, 15 mM MgCl.sub.2, 0.01% gelatin] at 37° C. for 30 minutes (Message clean kit from M′ s. GenHunter Corporation, USA). DNase I was precipitated by adding PCI (phenol, chloroform, isoamylalcohol in ratio of 25:24:1) and RNA present in the aqueous phase was precipitated by adding 3 volumes of ethanol in the presence of 0.3 M sodium acetate. After incubating for 3 hours at −70° C., RNA was pelleted, rinsed with chilled 70% ethanol and finally dissolved in 10 μl of RNase free water. DNA-free-RNA thus obtained was quantified and the integrity was checked as above. The quality of RNA is depicted in
DNA-free-RNA (0.2 μg) from dormant and non-dormant samples was reverse transcribed in separate reactions to yield cDNAs using an enzyme known as reverse transcriptase. The reaction was carried out using 0.2 μM of T11M primers (M in T11M could be either T11A, T11C or T11G), 20 μM of dNTPs, RNA and RT buffer [25 mM Tris-Cl (pH, 8.3), 37.6 mM KCl, 1.5 mM MgCl2 and 5 mM DTT]. In the present invention dNTP refers to deoxyadenosine triphosphate (hereinafter referred to dATP), deoxyguanosine triphosphate (hereinafter referred to dGTP), deoxycytidine triphosphate (herein after referred to dCTP) and deoxythymidine triphosphate (hereinafter referred to dTTP). Three RT reactions were set per RNA sample for the corresponding T.sub.11M primer. The reactions were carried out in a thermocycler (model 480 from M/s Perkin-Elmer). Thermocycler parameters chosen for transcription were 65° C. for 5 minutes, →37° C. for 60 minutes, →75° C. for 5 minutes, →4° C. 100 units of reverse transcriptase was added to each reaction after 10 minute incubation at 37° C. and reaction then continued for rest of the 50 minutes. This yielded cDNAs as is shown in
Different sub-classes of cDNA from dormant and non-dormant RT product as obtained in Example 2 were amplified in the presence of a radiolabelled dNTP to label the amplified product through polymerase chain reaction (hereinafter known as PCR; PCR process is covered by patents owned by Hoffman-La Roche Inc.). Radioactive PCR was carried out in 20 μl reaction mix containing a (1) reaction buffer [10 mM Tris-Cl (pH, 8.4), 50 mM KCl, 1.5 mM MgCl.sub.2, 0.001% gelatin], (2) 2 μM dNTPs, (3) 0.2 μM T1111M and (4) 0.2 μM arbitrary primers (chemicals 1 to 4 were purchased from M/s. GenHunter Corporation, Nashville, USA as a part of RNAimage kit), 0.2 μl α[33P] dATP (˜2000 Ci/mmole, purchased from JONAKI Center, CCMB campus Hyderabad, India), and 1.0 units of Thermus aqueticus (hereinafter referred to Taq) DNA Polymerase (purchased from M/S. Qiagen, Germany). 30 μl of autoclaved mineral oil was overlaid at the top of each reaction to avoid alteration in volume due to evaporation. T11M primer in each reaction was the same that was used to synthesize cDNA. Parameters chosen were: 40 cycles of 94° C. for 30 seconds, →40° C. for 2 minutes, →72° C. for 30 seconds; and 1 cycle of 72° C. for 5 minutes and final incubation at 4° C.
Amplified products were fractionated onto a 6% denaturating polyacrlamide gel. For the purpose 3.5 μl of each of amplified product was mixed with 2 μl of loading dye [95% formamide, 10 mM EDTA (pH, 8.0), 0.09% xylene cyanol FF and 0.09% bromophenol blue], incubated at 80° C. for 2 minutes and loaded onto a 6% denaturating polyacrlamide gel [denaturating polyacrlamide gel: 15 ml of acrylamide (40% stock of acrylamide and bisacrylamide in the ratio of 20:1), 10 ml of 10×TBE, 40 ml of distilled water and 50 g urea]. Electrophoresis was performed using 1×TBE buffer [10×TBE: 108 g Tris base, 55 g boric acid and 40 ml of 0.5 M EDTA (pH, 8.0)] as a running buffer at 60 watts until the xylene cyanol (the slower moving dye) reached the lower end of the glass plates. Size of the larger plate of the sequencing (denaturing polyacylamide) gel apparatus was 13×16 inch. After the electrophoresis, one of the glass plates was removed and the gel was transferred onto a 3 mM Whattman filter paper. Gel was dried at 80° C. overnight and exposed to Kodak X-ray film for 2-3 days. Before exposing to X-ray film, corners of the dried gel were marked with radioactive ink for further alignment.
Sequences of the Primers Used for Differential Display were purchased from M/s. GenHunter Corporation, USA as part of an RNA Image Kit. Anchored primers were T11A (SEQ ID NO: 5), T11C (SEQ ID NO: 6), and T11G (SEQ ID NO: 7), and arbitrary primers were AP1-8 (SEQ ID NOs: 8-15, respectively), A33-40 (SEQ ID NOs: 16-23, respectively), and AP65-72 (SEQ ID NOs: 24-31, respectively).
Cloning the differentially expressed bands required elution of the same from the denaturating polyacrylamide gel and further amplification to yield substantial quantity of DNA for the purpose of cloning. Autoradiogram (developed X-ray film) was oriented with the dried gel aided with radioactive ink and the identified differentially expressed band (along with the gel and the filter paper) was cut with the help of a sterile sharp razor. DNA was eluted by incubating in 100 μl of sterile dH20 for 10 min in an eppendorf tube, followed by boiling for 10 minutes. Paper and gel debris were pelleted by spinning at 10,000 rpm for 2 min and the supernatant containing DNA was transferred into a new tube. DNA was precipitated with 10 μl of 3M sodium acetate, pH, 5.5, 5 μl of glycogen (stock; 10 mg/ml) and 450 μl of ethanol. After incubation at −70° C. for overnight, centrifugation was performed at 10,000 rpm for 10 min at 4° C. and pelleted DNA was rinsed with 85% ethanol. DNA pellet was dissolved in 10 μl of sterile distilled water.
Eluted DNA was amplified using the same set of T11M and arbitrary primer that was used for the purpose of performing differential display as in the Example 3. Also, the PCR conditions were the same except that dNTP concentration was 20 μM instead of 2 μM and no isotopes was added. Reaction was up-scaled to 40 μl and after completion of PCR, 30 μl of PCR sample was run on 1.5% agarose gel in TAE buffer (TAE buffer: 0.04 M Tris-acetate, 0.002 M EDTA, pH 8.5) containing ethidium bromide (final concentration of 0.5 μg/ml). Rest of the amplified product was stored at −20° C. for cloning purposes (see
Re-amplified PCR products as obtained in example 4 were ligated in 300 ng of insert-ready vector called as PCR-TRAP® vector using 200 units of T4 DNA-ligase in 1×ligation buffer (10× ligase buffer: 500 mM Tris-Cl, pH 7.8, 100 mM MgCl2, 100 mM DTT, 10 mM ATP, 500 μg/ml BSA). Vector and the other chemicals required were purchased from M/s. GenHunter Corporation, Nashville, USA as PCR-TRAP® cloning system. Ligation was performed at 16° C. for 16 hours in a thermocycler model 480 from M's. Perkin Elmer, USA. Ligation of the PCR product into a vector such as above yields to a circularized plasmid. The process of ligation of the foreign DNA, such as the PCR product in the present invention, into a suitable vector, such as PCR-TRAP®. vector in the present invention, is known as cloning. There is a range of other vectors that are commercially available or otherwise that suits the cloning work of PCR products and hence may be used. The plasmid, as per the definition, is a closed circular DNA molecules that exists in a suitable host cell such as in Escsherichia coli (hereinafter referred to E. coli) independent of chromosomal DNA and may confer resistance against an antibiotic. PCR-TRAP® vector resulting plasmid confers resistance against tetracycline.
Ligated product or the plasmid needs to be placed in a suitable E. coli host for its multiplication and propagation through a process called transformation. Ligated product (10 μl) as obtained above was used to transform 100 μl of competent E. coli cells (purchased from M/s. GenHunter Corporation USA as a part of PCR-TRAP cloning system). Competent means the E. coli cells capable of accepting a plasmid DNA. For the purpose, ligated product and competent cell were mixed, kept on ice for 45 minutes, heat shocked for 2 minutes and cultured in 0.4 ml of LB medium (LB: for 1 litre: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride) for 4 hours. 200 μl of transformed cells were plated onto LB-tetracyclin (for 1 litre: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and tetracyclin added to a final concentration of 20 μg/ml) plates and grown overnight at 37° C. Colonies were marked and single isolated colonies were restreaked on to LB-tetracyclin plates to get colonies of the same kind. Conferral of tetracycline resistance to E. coli cells apparently suggests that the PCR product i.e. the identified gene has been cloned.
In whole of the above process, the selection of T11M primer will amplify the poly A tail region of mRNA. Poly A tail is always attached to 3′ end of the gene and hence T11M primer in combination with an arbitrary primer would always yield 3′ region of the gene.
Once the gene has been cloned and the E. coli has been transformed, it becomes imperative to check if the plasmid has received right size of the PCR product. This can be accomplished by performing colony PCR wherein the colony is lysed and the lysate is subsequently used to perform PCR using the appropriate primers. Amplified product is then analysed on an agarose gel.
Single isolated colonies were picked up from re-streaked plates (Example 5) and lysed in 50 μl colony lysis buffer (colony lysis buffer: TE (Tris-Cl 10 mM, 1 mM EDTA, pH 8.0) with 0.1% tween 20) by boiling for 10 minutes. Cell debris were pelleted and the supernatant or the colony lysate containing the template DNA was used for PCR. PCR components were essentially the same as in example 4 except that in place of T11M and arbitrary primers, Lgh (5′-CGACAACACCGATAATC-3′) (SEQ ID NO:32), and Rgh (5′-GACGCGAACGAAGCAAC-3′) (SEQ ID NO:33) primers (specific to the vector sequences flanking the cloning site) were used and 2 μl of the colony lysate was used in place of eluted DNA. Also the reaction volume was reduced to 20 μl. PCR conditions used for colony PCR were, 94° C. for 30 seconds, →52° C. for 40 seconds, →72° C. for 1 minute for 30 cycles followed by 1 cycle of 5 min extension at 72° C. and final soaking into 4° C. Amplified product are run on 1.5% agarose gel along with molecular weight marker and analyzed for correct size of insert. While using Lgh and Rgh flanking primers, the size of the cloned PCR product was larger by 120 bp due to the flanking vector sequence being amplified (See
PCR products cloned above represent 3′ end of the differentially expressed genes. Within the scope of the present invention, these cloned fragments of DNA will be called as genes. Since differential display invariably leads to false positives i.e. apparently differentially expressed genes (Wan, J. S, and Erlander, M. G. 1997. Cloning differentially expressed genes by using differential display and subtractive hybridization. In Methods in Molecular Biology. Vol. 85: Differential display methods and protocols. Eds. Liang, P. and Pardee, A. B. Humana press Inc., Totowa, N.J., pp 45-68), a confirmatory test through northern analysis is mandatory to ascertain is differential expression between ND and D apical buds. Northern analysis requires preparation of a radio-labelled probe followed by its hybridization with denatured RNA blotted onto a membrane.
Amplified products as in Example 6 were used as a probe in northern analysis After visualizing the amplified products on 1.5% agarose gel these were cut from the gel and the DNA was eluted from the gel using QIAEX II gel extraction kit from M/s. Qiagen, Germany following the manufacturer's instructions.
Purified fragments were radiolabelleled with α[32P]dATP (4000 Ci/mmole) using HotPrime Kit from M/s. GenHunter Corporation, Nashville, USA following their instructions. Radio-labelled probe was purified using QIAquick nucleotide Removal Kit (QIAGEN, Germany) to remove unincorporated radionucleotide.
For blotting, 20 μg of RNA was run on 1.0% formaldehyde agarose gel essentially as described in Example 1. Once the run was completed, gel was washed twice in DEPC treated autoclaved water for 20 minutes each with shaking. Gel was then washed twice in 10×SSPE (1.5 M sodium chloride, 115 mM NaH2PO4, 10 mM EDTA) for 20 minutes each with shaking. In the mean time nylone membrane (boehringer mannheim cat. no. #1209272) was wetted in DEPC water and then soaked in 10×SSPE (1.5 M sodium chloride, 115 mM NaH2PO4, 10 mM EDTA) for 5 minutes with gentle shaking. RNA from the gel was then vacuum-blotted (using pressure of 40 mbar) onto nylon membrane using DEPC-treated 10×SSPE (1.5 M sodium chloride, 115 mM NaH2PO4, 10 mM EDTA) as a transfer medium. Transfer was carried out for 4 hours. Pressure was Increased to 70 mbar for 15 minutes before letting out the gel from the vacuum blotter.
After the transfer, gel was removed, and the location of RNA marker was marked on the nylon surface under a UV light source. Membrane was dried and baked at 80° C. for 45 minutes. After a brief rinse in 5×SSPE (20×SSPE: 3M sodium chloride, 230 mM sodium phosphate, 20 mM EDTA) membrane was dipped into prehybridization solution (50% formamide, 0.75 M NaCl, 50 mM sodium phosphate, pH 7.4, 5 mM EDTA, 0.1% Ficoll-400, 0.1% BSA, 0.1% polyvinypyrollidone, 0.1% SDS solution and 150 ug/ml freshly boiled salmon sperm DNA) for 5 hours.
Radiolabelled probe synthesized earlier was denatured by boiling for 10 minutes followed by addition to the prehybridization solution dipping the blotted is membrane. Hybridization was carried out for 16 hours. Solution was removed and the membrane was washed twice with 1×SSC (20×SSC; 3M sodium chloride and 0.3M sodium citrate dihydrate, pH, 7.0) containing 0.1% SDS at room temperature for 15 minutes each. Final washing was done at 50° C. using pre-warmed 0.25×SSC containing 0.1% SDS for 15 minutes. Membrane was removed, wrapped in saran wrap and exposed to X-ray film for 12-240 hours depending upon the intensity of the signal.
While performing northern hybridization, RNA from ND and D apical buds are blotted on the membrane and tested for the probe of choice.
21.2 (T11C, AP7) which is basically a 3′ end region of the gene, hybridized to the transcripts 0.96 kb size on northern blot as in
53.1 (T11A, AP34) which is basically a 3′ end region o the gene, hybridized to the transcripts 0.95 kb size on northern blot as in
44.3 (T11G, AP33) which is basically a 3′ end region of the gene, hybridized to the transcripts 1.75 kb size on northern blot as in
Above Example 6 identified 4 differentially expressed genes cloned by us. To further correlate these differentially expressed genes with the phenomenon of dormancy, the dormant bush during winter month was forced to break bud dormancy using a plant growth regulator gibberrellic acid (hereinafter referred to GA3). GA3 was dissolved in 5% ethanol to yield a final concentration of 5 μM. The solution was applied onto each dormant bud with the help of a paint brush at least 4 times on the same day during the month of December. Bud length and its perimeter was recorded regularly as an indicator of its growth. Out of 20 buds only 10 buds showed increase in growth (dormancy break) in response to GA3 application. These were termed as forced ND apical buds. These were collected and stored as mentioned in Example 1 and used for the purpose of RNA isolation to be used in northern analysis.
For such an experiment, RNA from ND, D and forced ND was blotted onto a is nylon membrane and probed with ND (31.2) probe. Various procedures involved are already mentioned in Example 6.
As can be seen from
Each clone was sequenced manually using a T7 sequence version 2 sequencing kit from Amersham Pharmacia Biotech, USA. Sequencing primers used were Lgh (5′-CGACAACACCGATAATC-3′) (SEQ ID NO:32), and Rgh (5′-GACGCGAACGAAGCAAC-3′) (SEQ ID NO:33). Sequences for the differentially expressed clones are listed as SEQ ID NOs: 1-4.
Each clone was subjected to BLAST analysis and the four clones were found to be unique.
This application is a divisional application of U.S. patent application Ser. No. 11/516,532, filed Sep. 6, 2006 which is a divisional application of U.S. patent application Ser. No. 09/823,887, filed Mar. 3, 2001, the contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11516532 | Sep 2006 | US |
| Child | 12975918 | US | |
| Parent | 09823887 | Mar 2001 | US |
| Child | 11516532 | US |
