Patent Application
20110099648
This application claims the benefit of Chinese application No. 200910272539.1, filed Oct. 27, 2009.
1. Field of the Invention
The invention relates to the field of genetic breeding for aquatic animals, in particularly to a recombinant gene which enhances the hypoxia tolerance. The invention also relates to the use of such recombinant gene for genetically improving farmed species.
2. Description of the Related Art
Dissolved oxygen (DO) is the most important environmental factor in aquiculture, which may be affected by temperature, diurnal rhythm, seasonal variation and eutrophication, etc. Fish are very sensitive to the variation of DO condition in the water. Low DO reduces the growth rate of fish, induces endocrine disorders of fish, impacts metabolism and reproduction features of fish, changes the behavior and distribution of fish, and even results in the death of fish. Therefore, the abundance, diversity and capture amount of fish are all severely impaired in the water areas affected by low DO. As a result, it is practically worth to develop novel farmed species with the hypoxia tolerance using transgenic technology.
Currently, the study of improving hypoxia tolerance in fish using transgenic technology is still a presumption. The hypoxia tolerance in fish mainly relies on the contents and types of globulin families and oxygen affinity (Giles M. A (1991). Strain differences in hemoglobin polymorphism, oxygen consumption, and blood oxygen equilibria in three hatchery broodstocks of Arctic charr, Salvelinus alpinus. Fish Physiology and Biochemistry 9: 291-301. 2, Terwilliger N. B (1998). Functional adaptations of oxygen-transport proteins. J. Exp. Biol. 201(8): 1085-1098. 3, Skjæaasen J. E., Nilsen T., Meager J. J., Herbert N. A., Moberg O., Tronci V., Johansen T., Salvanes A. G. V (2008). Hypoxic avoidance behaviour in cod (Gadus morhua L.): The effect of temperature and haemoglobin genotype. Journal of Experimental Marine Biology and Ecology; 358; 70-77.). Four types of globulins were found in human and other vertebrates: haemoglobin (Hb), myoglobin (Mb), neuroglobin and cytoglobin. These four types of globulins vary to some extent in structure, tissue distribution and function (4, Pesce A., Bolognesi M., Bocedi A., Ascenzi P., Dewilde S., Moens L., Hankeln T., Burmester T. (2002). Neuroglobin and cytoglobin. Fresh blood for the vertebrate globin family. EMBO Rep. 3(12):1146-1151.). In mammals, haemoglobin (Hb) and myoglobin (Mb) may be induced by low oxygen and hypoxia (5, Hoppeler H., Vogt M. (2001). Muscle tissue adaptations to hypoxia. J. Exp. Biol. 204: 3133-3139. 6, Vogt M., Puntschart A., Geiser J., Zuleger C., Billeter R., and Hoppeler H. (2001). Molecular adaptations in human skeletal muscle to endurance training under simulated hypoxic conditions. J. Appl. Physiol. 91: 173-182. 7, Nitta T., Xundi X., Hatano E., Yamamoto N., Uehara T., Yoshida M., Harada N., Honda K., Tanaka A. & Sosnowski D. (2003). Myoglobin gene expression attenuates hepatic ischemia reperfusion injury. J. Surg. Res. 110: 322-331.).
Strictly speaking, Vitreoscilla stercoraria is an aerobe, but they are able to adapt to hypoxia by expressing soluble haemoglobin (Vitreoscilla hemoglobin, VHb) which consists of 2 identical subunits of 15,775 Da and 2 heme molecules (Wakabayashi S., Matsubara H., Webster D. A. (1986). Primary sequence of a dimeric bacterial hemoglobin from Vitreoscilla. Nature 322:481-483.). Although VHb does not increase intracellular oxygen concentration, it can accelerate oxygen delivery in a low oxygen condition due to the high dissociation rate constant between VHb and oxygen molecules, and thus improves respiration and energy metabolism. Therefore, hypoxia tolerance in fish may be prospectively enhanced by introducing VHb gene into fish, so as to develop novel farmed species with hypoxia tolerance.
Zebrafish (Danio rerio) is a fish model currently widely used in many investigations such as Developmental Biology and Hydrobiont Technology (Westerfield M. (1993). The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio). University of Oregon Press, Eugene, Oreg.). A new method, where zebrafish is used as a model to study the improvement of the ability of recipient fish to tolerate low oxygen stress, results in an instructive significance for breeding novel economically farmed species with tolerance to low DO stress. Based on breeding of the transgenic zebrafish with the hypoxia tolerance, the recombination technique is further applied to other important economically farmed species such as blunt snout bream (Megalobrama amblycephala) and common carp (Cyprinus carpio L.). The resultant vhb transgenic blunt snout bream and common carp also have the hypoxia tolerance.
The purpose of the invention is to provide a recombinant gene which enhances the hypoxia tolerance in fish (the recombinant gene of Vitreoscilla hemoglobin (VHb) driven by carp β-actin gene promoter). Vitreoscilla hemoglobin consists of 2 identical subunits of 15,775 Da and 2 heme molecules (Wakabayashi S., Matsubara H., Webster D. A. (1986). Primary sequence of a dimeric bacterial hemoglobin from Vitreoscilla. Nature 322:481-483.). Due to the high dissociation rate constant between VHb and oxygen molecules and the strong regulatory ability of the β-actin gene promoter, the recombinant gene can ensure the acceleration of oxygen delivery and the enhancement of respiration and energy metabolism in the recipient animals under hypoxia.
Another purpose of the invention is to provide a use of the recombinant gene which enhances hypoxia tolerance in fish. The recombinant vhb gene driven by carp β-actin gene promoter is microinjected into zygotes of zebrafish. Then the 7-day transgenic fish obtained by screening are subjected to low oxygen stress treatment in a low DO (0.91 mg/l) stress condition for 156 h. The survival rate of these transgenic fish is 92%, which is significantly different from the survival rate of 65% of the control sister fish group. The introduction of the recombinant gene enhances the hypoxia tolerance and provides a commonly applicable method for breeding novel economically farmed species with hypoxia tolerance. The transgenic blunt snout bream and transgenic common carp bred by same method have the feature of hypoxia tolerance.
A further purpose of the invention is to provide a fish species with enhanced hypoxia tolerance, wherein said fish comprises the above recombinant Vitreoscilla hemoglobin (VHb) driven by carp β-actin gene promoter. In one embodiment, said fish comprises a vector with a sequence shown as SEQ ID NO: 5. In another embodiment, said fish is an economically farmed species. In a further embodiment, said fish is zebrafish, blunt snout bream or common carp.
To achieve the above purposes, the following technical approaches are used in the invention.
Carp actin gene promoter (cloned from the genomic bank of carps, a conventional protocol, Molecular Cloning A Laboratory Mannual, 2nd ed., Sambrook, J. et al. Science Press, 1993) is used as a promoter; and Vitreoscilla hemoglobin (VHb) is used as a target gene (VHb is cloned from Vitreoscilla stercoraria commercially purchased from ATCC center, USA, with the ATCC number 15128). The 5′ upstream primer is CCATGGTAGA CCAGCAACC (SEQ ID NO: 6), and the 3′ downstream primer is GGGTAACCTT TATTCAACCG (SEQ ID NO: 7). PCR condition is shown as follows: pre-denaturalizing at 94° C. for 10 min; 25 cycles of denaturalizing at 94° C. for 30 sec, annealing at 52° C. for 45 sec, extending at 72° C. for 45 sec; then 72° C. for 7 min. A 441 bp fragment is amplified and recovered, and then cloned into a pGEM-T-easy vector (Tiangen Co.), so as to construct an expression vector of Vitreoscilla hemoglobin (VHb) gene driven by carp β-actin gene promoter (a conventional protocol, Molecular Cloning A Laboratory Mannual, 2nd ed., Sambrook, J. et al. Science Press, 1993) and thus develop transgenic fish with the hypoxia tolerance.
The invention includes the following steps:
The recombinant Vitreoscilla hemoglobin (VHb) gene driven by the carp β-actin gene promoter is constructed. The detailed steps are shown as follows.
(1) Carp actin gene promoter is used as a promoter (cloned from the genomic bank of carps, a conventional protocol, Molecular Cloning A Laboratory Mannual, 2nd ed., Sambrook, J. et al. Science Press, 1993; its nucleotide sequence is shown as SEQ ID No. 1, see Sequence Listing); and Vitreoscilla hemoglobin gene is used as a target gene, which is cloned from Vitreoscilla stercoraria commercially purchased from ATCC center, USA, with the ATCC number 15128. The 5′ upstream primer is CCATGGTAGA CCAGCAACC (SEQ ID NO: 6), and the 3′ downstream primer is GGGTAACCTT TATTCAACCG (SEQ ID NO: 7). PCR condition is shown as follows: pre-denaturalizing at 94° C. for 10 min; 25 cycles of denaturalizing at 94° C. for 30 sec, annealing at 52° C. for 45 sec° C. extending at 72° C. for 45 sec; then 72° C. for 7 min. A 441 bp fragment with a nucleotide sequence shown as SEQ ID No. 2 (see Sequence Listing) is amplified and recovered, and then cloned into a pGEM-T-easy vector (Tiangen Co.). Simian virus 40 poly A (Clontech Co.) is used as a terminating sequence, the nucleotide sequence of which is shown as SEQ ID No. 3 (see Sequence Listing).
(2) The enhanced green fluorescent protein eGFP (Clontech Co.) driven by cytomegalovirus CMV (Clontech Co.) promoter is used as a reporter gene, the nucleotide sequence of which is shown as SEQ ID No. 4 (see Sequence Listing).
(3) After digested by restriction enzymes (XhoI, SmaI, Not I and Hind III), the above four gene fragments (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) are ligated with plasmid vector PUC118 (SABC Co.) (a conventional protocol, Molecular Cloning A Laboratory Mannual, 2nd ed., Sambrook, J. et al. Science Press, 1993). An expression vector containing the recombinant gene is obtained and named as pCVCG, the nucleotide sequence of which is shown as SEQ ID NO: 5. In such recombinant gene, Vitreoscilla hemoglobin consists of 2 identical subunits of 15,775 Da and 2 heme molecules (Wakabayashi S., Matsubara H., Webster D. A. (1986). Primary sequence of a dimeric bacterial hemoglobin from Vitreoscilla. Nature 322:481-483.). The association rate constant (kon 78 μM−1s−1) between Vitreoscilla hemoglobin and oxygen molecules is similar to that for other hemoglobins; whereas the dissociation rate constant (koff 5000 s−1) between Vitreoscilla hemoglobin and oxygen molecules is higher than that for other hemoglobins by hundreds of folds (Orii Y., Webster D. A. Photodissociation of oxygenated cytochrome o(s) (Vitreoscilla) and kinetic studies of reassociation. J Biol Chem 1986; 261:3544-3547.). Therefore, oxygen delivery may be accelerated in a low oxygen condition, and thus respiration and energy metabolism may be improved.
The expression vector pCVCG containing the recombinant gene is microinjected into zygotes of fish (Zhu Z, Li G, He L, et al. Novel gene transfer into the fertilized eggs of goldfish (Carassius auratus L. 1758). Z angew Ichthyol, 1985, 1:31-34).
(2) Screening of the Transgenic Fish with the Hypoxia Tolerance
The green fluorescent protein expression is observed under a fluorescent dissecting microscope (Olympus Model SZX12). The recombinant gene is tested by PCR and the transgenic fish with the hypoxia tolerance is obtained by a low DO screening experiment.
The invention has the following advantages and effects. The invention provides a genetically improved new breeding technique for enhancing the ability of fish to tolerate low DO stress. The 7-day transgenic zebrafish bred with such method is subject to low oxygen stress treatment in a low DO (0.91 mg/l) stress condition for 156 h. The survival rate of those transgenic fish is 92%, which is significantly different from survival rate of 65% of the control sister fish group. The transgenic blunt snout bream and transgenic carps bred by such method both have the hypoxia tolerance. This genetically improved breeding technique may be widely used for breeding new excellent farmed species with the hypoxia tolerance.
In the expression vector of Vitreoscilla hemoglobin gene, carp actin gene promoter with a length of 1213 bp was used as a promoter (SEQ ID NO: 1, which was screened from the genomic bank of carps, a conventional protocol, Molecular Cloning A Laboratory Mannual, 2nd ed., Sambrook, J. et al. Science Press, 1993). Vitreoscilla hemoglobin gene with a length of 441 bp, as a target gene (SEQ ID NO: 2, which was cloned from Vitreoscilla stercoraria commercially purchased from ATCC Center, USA, with the ATCC number 15128. The 5′ upstream primer was CCATGGTAGA CCAGCAACC (SEQ ID NO: 6), and the 3′ downstream primer was GGGTAACCTT TATTCAACCG (SEQ ID NO: 7). PCR condition was shown as follows: pre-denaturalizing at 94° C. for 10 min; 25 cycles of denaturalizing at 94° C. for 30 sec, annealing at 52° C. for 45 sec, extending at 72° C. for 45 sec; then 72° C. for 7 min. The amplified fragments with a length of 441 bp were recovered, and then cloned into a pGEM-T-easy vector) was inserted downstream of the promoter, followed by simian virus 40 poly A (Clontech Co.) as a terminating sequence and CMV promoter (Clontech Co.) driven enhanced green fluorescent protein eGFP (Clontech Co.) as a reporter gene. The vector frame was PUC118 (SABC Co.).
1) The detailed construction process is shown as follows.
The carp actin gene promoter was completely digested by XhoI and SmaI; Vitreoscilla hemoglobin gene was completely digested by SmaI and Not I; simian virus 40 poly A was completely digested by Not I and Hind III; CMV promoter-driven enhanced green fluorescent protein eGFP gene was completely digested by Hind III and XbaI; and PUC118 plasmid was completely digested by XhoI and XbaI. The resultant fragments were ligated with T4 ligase at 16° C. for 4 hours, respectively. The restriction enzymes and ligase used above were all purchased from Takara Co.
E. coli (E. coli TOP10, purchased from Invitrogen, with a genotype of F-mcrAΔ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15ΔlacX74 deoR recA1 araD139α(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG) was transformed by the above ligation solution; cultured at a constant temperature of 37° C.; and inoculated and cultured in LB broth for further identification.
The above PCR, digestion, ligation, DNA transformation, bacterial culturing and culture broth are all conventional protocols in Molecular Biology, which were performed according to “Molecular Cloning A Laboratory Manual, 2nd ed., Sambrook, J. et al. Science Press, 1993”, so as to produce the expression vector pCVCG containing Vitreoscilla hemoglobin gene.
2) Identification of the Expression Vector pCVCG Containing Vitreoscilla Hemoglobin Gene
The expression vector pCVCG (constructed in the invention) containing Vitreoscilla hemoglobin gene was identified by PCR amplification. The bacterial solution (2 μL) cultured in LB broth at 37° C. for 5 h was used as a template, and PCR amplification was carried out in a 25 μL, system. The primers were VCG-U1 (GCCGCAACCGATGACA, SEQ ID NO: 8) and VCG-D1 (GCCAAGTGGGCAGTTTACC, SEQ ID NO: 9). The target fragment was ˜563 bp in length (
(2) Digestion and identification
Plasmid pCVCG was extracted from the positive clones identified as above with the Plasmid Miniprep (˜40 μg) kit (Axygen), and further identified to be pCVCG by digestion with a single enzyme HindIII (target fragment of 6627 bp) and by double enzymes XhoI+HindIII (target fragments of 1952 bp and 4675 bp) (
Zebrafish AB strain was used. The plasmid DNA was extracted from the expression vector pCVCG containing Vitreoscilla hemoglobin gene using the Plasmid Miniprep kit (Axygen) and dissolved in ST solution (88 mmol/l NaCl, 10 mmol/l Tris-HCl, pH 7.5) to a final concentration of 85 ng/μl. Then the DNA solution was microinjected (Zhu Z, Li G, He L, et al. Novel gene transfer into the fertilized eggs of goldfish (Carassius auratus L. 1758). Z angew Ichthyol, 1985, 1:31-34) into the animal poles of zebrafish zygotes before the first cleavage. The DNA injection dose was 1-2 nl/zygote. The resultant zygotes were incubated and bred in 28.5° C. water.
(2) Breeding and Screening of the Transgenic Zebrafish Family which Stably Inheriting and Expressing the VHb Gene
After vector pCVCG was introduced into zebrafish zygotes by micromanipulation, the zebrafish zygotes were screened for the embryos (P0) expressing green fluorescence under a fluorescent microscope, as shown in
DNA was extracted by the conventional phenol/chloroform method from about 0.1-0.2 cm2 tail fin tissue of the transgenic zebrafish F1 in the above different families. The conventional phenol/chloroform method was performed as follows: 0.4 ml DNA extraction solution (10 mmol/L EDTA, 10 mmol/L Tris.HCl, 300 mmol/L NaCl, 2% (weight/volume, g/L) SDS) was added into a 1.5 ml tube; after incubated in a 55° C. water bath for 1-2 h, the tissue was digested in a 37° C. water bath overnight; the digested tissue was then extracted with phenol, phenol/chloroform and chloroform, respectively; 2.5 volumes of ethanol was added, and the formed precipitate was transferred to another centrifuge tube immediately; the precipitate was washed once with 70% (volume ratio) ethanol and centrifuged at high speed for 1 min; the pellet was kept in a 37° C. incubator for 15 min after ethanol was completely poured out; the DNA pellet was dissolved in a proper volume of TE containing RNase (20 mg RNase/ml). The integration of pCVCG in the transgenic fish was confirmed by PCR technique, wherein the forward detecting primer (5′-ATCTGCCTGTAACCCATTCT-3′, SEQ ID NO: 10) was located on carp β-actin promoter, reverse detecting primer (5′-AATACTTCTTTAATCGCACCC-3′, SEQ ID NO: 11) was located on the target gene, i.e., Vitreoscilla hemoglobin gene, and PCR condition was shown as follows: pre-denaturalizing at 94° C. for 5 min; amplifying for 35 cycles (94° C., 30 s; 58° C., 30 s; 72° C., 30 s); finally extending at 72° C. for 5 min. The target fragment was 383 bp in length, as shown in
Total RNA was extracted from GFP-positive F1 fry of different zebrafish families and from the control sister fish group (TRIzol method). The extraction process was detailed as follows: (1) One zebrafish fry was homogenized thoroughly in 1 ml Trizol (Invitrogen) with an electro-homogenizer. (2) ⅕ volume of chloroform was added and the mixture was mixed thoroughly by inversing the container upside down for ˜1 min. The mixture was kept at room temperature (20-25° C., same as below) for 5 min followed by centrifuging at 4° C., 12,000 rpm for 15 min. (4) The supernatant was carefully sucked out without touching the intermediate layer and transferred into a fresh 1.5 ml centrifuge tube. Same volume of isopropanol was added to and mixed with the supernatant by inversing the tube upside down gently, and then kept at room temperature for 5 min. (5) The mixture was centrifuged at 4° C., 12000 rpm for 10 min. (6) The supernatant was removed, and ⅖ volume of 70% (% volume) ethanol was added to the remained pellet. Then centrifugalization was carried out at 4° C., 12000 rpm for another 15 min to wash the pellet. (7) After removing the supernatant, the pellet was naturally dried out at room temperature, and then dissolved in a suitable amount of RNase-free water by pipetting up and down. (8) 1-2 μL of the resultant RNA solution was diluted to 100 uL. The diluent was detected by a spectrophotometer (Beckman DU-70) for OD260, OD280 and RNA concentration. The RNA sample was stored at −80° C. until later use. After that, cDNA was synthesized from lug total RNA by Random 9mers reverse transcription using the ReverTra Ace kit (TOYOBO) (the reaction system comprises lug total RNA, Random primer, 2 ul 5×Buffer, 1 ul 10 mM dNTPs, 0.5 ul ReverTra Ace (100 U/ul), 10 U RNase Inhibitor, and DEPC water to make a total volume of 10 ul). The cDNA sample was diluted by 1:4 before PCR, and then 1-2 ul of the diluent was subjected to RT-PCR. The primers for detecting VHb gene expression were (5′-CGTTACCATTACCACGACTTT-3′, SEQ ID NO: 12) and (5′-GCATCGCCCAATACTTCTT-3′, SEQ ID NO: 13), and the target fragment was 276 bp in length. It was confirmed by RT-PCR that VHb mRNA was highly efficiently expressed in zebrafish. It was also found that VHb mRNA was expressed in GFP-positive individuals but not in GFP-negative individuals (
After confirmed by PCR, the transgenic zebrafish F1 of different families were crossed with wildtype zebrafish. The offspring were screened by the green fluorescence tag for the positive fish, i.e., the transgenic zebrafish F2. The embryos of F2 were bred till sexual maturity and then crossed with wildtype fish, so as to reproduce offspring F3. Then transgenic zebrafish family that stably inheriting and expressing VHb gene was obtained by PCR and RT-PCR confirmation.
Male fish of the transgenic zebrafish family that stably expressing VHb gene were crossed with wildtype female zebrafish. After 24 h fertilization, the offspring embryos were divided into two groups, i.e., GFP-positive and GFP-negative embryos under a fluorescent microscope. The two groups of zebrafish were bred separately under the same condition according to standard protocols, among which the embryos without GFP expression were used as control fish. Seven-day transgenic fish and control fish, 100 each, were placed into an incubator (3131/Thermo; Form a Scientific, Inc., Marietta, Ohio) simultaneously and co-exposed to a condition of 2.5% O2 (volume ratio), 97.5% (volume ratio) N2 and 28.5° C. (0.91 mg/L, dissolved oxygen concentration in water). Additionally, another 40 transgenic fish and 40 control fish were routinely bred in a condition of normal oxygen concentration (7.6 mg/L). During the experiment, the fish were observed every 12 hours, and at the same time the death was recorded and the dead fish were removed.
It was found from the low DO stress test that the vhb transgenic zebrafish and the control fish both had a survival rate of 100% within 156 h in the normal DO condition (7.6 mg/L, dissolved oxygen concentration in water). However after 156 h low oxygen stress under a condition of 2.5% (volume ratio) O2 (0.91 mg/l, dissolved oxygen concentration in water), the vhb transgenic zebrafish had a survival rate of 92% which was significantly higher than the survival rate of 65% of the control fish (
The important economically farmed species in China, i.e., blunt snout bream and common carp were used for preparing vhb transgenic fish. The plasmid DNA was extracted from the expression vector pCVCG containing Vitreoscilla hemoglobin gene using the Plasmid Miniprep kit (Axygen) and dissolved in ST solution (88 mmol/l NaCl, 10 mmol/l Tris-HCl, pH 7.5) to a final concentration of 85 ng/μl. Then the DNA extraction solution was microinjected (Zhu Z, Li G, He L, et al. Novel gene transfer into the fertilized eggs of goldfish (Carassius auratus L. 1758). Z angew Ichthyol, 1985, 1:31-34) into the animal poles of zygotes of blunt snout bream (common carp) before the first cleavage. The DNA injection dose was 1-2 nl/zygote. The resultant zygotes were incubated and bred according to the conventional methods.
(2) Screening of the Transgenic Fish with the Hypoxia Tolerance
After vector pCVCG was introduced into zygotes of blunt snout bream (common carp) by micromanipulation, the zygotes were screened for the embryos expressing green fluorescence under a fluorescent microscope (as shown in
Following observation under fluorescence and PCR detection, the obtained vhb transgenic blunt snout bream (common carp) P0 and the control blunt snout bream (common carp) were bred separately in the pounds without any oxygenating equipment according to the conventional breeding process. After bred in such pounds for 1 year, the control blunt snout bream and control carp were dead because of hypoxia; whereas about 10% of the vhb transgenic blunt snout bream and vhb transgenic carps survived due to the hypoxia tolerance.
After further breeding till sexual maturity in the pounds, the survived vhb transgenic blunt snout bream and common carp were crossed with control fish. The transgenic fish (F1) globally expressing GFP were screened out and continuously bred, so as to obtain the transgenic fish family with the hypoxia tolerance. Blunt snout bream and common carp were both bred and propagated according to conventional methods.
| Number | Date | Country | Kind |
|---|---|---|---|
| 200910272539.1 | Oct 2009 | CN | national |
