CN104328126A - Aquaporin gene specifically expressed by virtue of chorispora bungeana root as well as protein encoded by aquaporin gene and method for cloning aquaporin gene - Google Patents
Aquaporin gene specifically expressed by virtue of chorispora bungeana root as well as protein encoded by aquaporin gene and method for cloning aquaporin gene Download PDFInfo
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Abstract
The invention relates to an aquaporin gene specifically expressed by virtue of chorispora bungeana root as well as a protein encoded by the aquaporin gene and a method for cloning the aquaporin gene and belongs to the field of research on plant-resistant genes. The aquaporin gene specifically expressed by virtue of chorispora bungeana root disclosed by the invention is derived from chorispora bungeana; and under the condition of low-temperature stress, the expression quantity of the aquaporin gene in chorispora bungeana is significantly increased and the stress resistance of chorispora bungeana is enhanced due to the over-expression of the gene.
Description
Technical field
The present invention relates to a kind of high mountain ion mustard root specifically expressing water channel protein gene and coded protein thereof and gene clone method thereof, belong to stress resistance of plant gene studies field.
Background technology
Low temperature stress often makes some plant originating from tropical and subtropical region cannot, at northern area, temperate zone open country safe overwintering, bring serious disaster also to tropical, that subtropical plant is accidental low temperature stress because of chill sensitive.Therefore, the hereditary property transforming farm crop increases the long-sought target that winter resistance has become plant breeding scholar.Regrettably, the progress that traditional breeding method obtains in raising crop cold resistance is very micro-.From modern biotechnology and Development of Molecular Biology trend, transgenic technology solves one of most effective means of this difficult problem.Chilling injury is the seasonality of crop growth and zonal limiting factor, but the essence of low-temperature adaptation process is genes within cells expresses the result changed, cold condition is specifically induction of a series of with the freeze proof relevant genetic expression of cell (Weiser C J.Sciene.1970 in cell, 169:1269-1278), (Artus N N, Uemura M, Steponkus P L, et al.PNAS, 1996,93:13404-13409).
At present the research of high mountain ion mustard water channel protein gene is not also reported.In plant cold resistance transgenic research, (Wada H, the Gombos Z such as Davies, Murata N.Nature, 1990,347 (6289): 200-203), (Murata N I, Nashita O, Higashi S.Hayasha, et al.Nature, 1992,356:710-713) first the Antifreeze protein gene of winter flounder fish is imported tobacco, turmeric and rape at the end of the eighties in last century, find that transfer-gen plant obtains certain freezing tolerance.Afterwards, the cold acclimation protein such as the glycerol-3-phosphate acyl enzyme gene of desaturase gene, Arabidopis thaliana and Cor15a gene are utilized also to improve cold tolerance (the Steponkus P L of subject plant in varying degrees, Uemura M, Joseph R A, Gilmour S J and Thomashow M F.Proc.Natl.Acad.Sci.USA.1998,95:14570-14575).There are some researches show that the expression of aquaporin is coerced by high salt condition, drought, cold regulation and control of coercing, may be relevant to the resistance of plant, but regulatory mechanism also needs further research (Jang J Y, Kim Y, el al. I lanl Mol Biol.2004.54 (5): 713-725).Aquaporin is present in (Zhang Zhengbin in the multiple biology such as animal, plant in a large number.The physiological inheritting breeding basis [M] of crop drought resistance water saving. Beijing: Science Press, 2003.84-96).First the studies on plant aquaporins-TIP separates (Hofte G M, Hubbard L, Reizer J, et al.Plant Physiol, 1992,17 (256): 385-387) from Arabidopis thaliana, and it is positioned on vacuole skin.Subsequently, first plasmalemma of plant AQP-Arabidopis thaliana RD28 is also found (Daniels M J, Mirkov T E, Chrispeels M J.Plant Physiology, 1994,106 (4): 1325-1333).Up to now, find and clone to obtain aquaporin in the various plants such as Arabidopis thaliana, corn, tobacco, paddy rice, pea, wild cabbage, potato, spinach, tomato, strawberry.In addition, have also discovered a large amount of AQP homologues from albumen database, it is extensively present in courage unifacial leaf and dicotyledons, C3 and C4 metabolizing plants (Scheaffner A R.P lanta, 1998,204:131-139).The discovery of aquaporin is significant for the water relation of research plant, but up to the present, and the research for each AQP and whole plant AQP function under not too physiological condition is still less.
Identifying and studying of aquaporin provides new approaches for inquiring into Plant water relation further.Moisture transports the modification by the expression of specific water channel protein gene, regulation and control and Subcellular Localization in plant materials.Aquaporin regulates moisture regulating moisture at transcellular transport and the molecular basis that conducts in plant, and and the resistance of plant is as also existed the relation mutually regulated and controled between salt resistance, winter resistance, frost resistance, therefore can express the transfer-gen plant of different membranin by research, molecular biology is combined with plant physiology determine the effect of AQP in plant Water Transportation and and stress resistance of plant between relation.At present also relevant report is not had for high mountain ion mustard, therefore, to the clone of high mountain ion mustard water channel protein gene and the research to its expression, to the molecule mechanism of clear and definite water channel protein gene effect be contributed to, obtain the genetic resources of the frost resistance improvement that can be applicable to the plant for cold sensitive of other tropical and subtropical region simultaneously.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of high mountain ion mustard root specifically expressing water channel protein gene and coded protein thereof and gene clone method thereof, wherein, described high mountain ion mustard root specifically expressing water channel protein gene called after ChlRB7 gene, from high mountain ion mustard.
High mountain ion mustard root specifically expressing water channel protein gene provided by the invention is from high mountain ion mustard.
The technical scheme that the present invention solves the problems of the technologies described above is as follows: a kind of high mountain ion mustard root specifically expressing water channel protein gene, its nucleotide sequence is as shown in SEQ ID NO:1.
The technical scheme that the present invention solves the problems of the technologies described above is as follows: a kind of protein of high mountain ion mustard root specifically expressing water channel protein gene coding, its aminoacid sequence is as shown in SEQ ID NO:2.
The technical scheme that the present invention solves the problems of the technologies described above is as follows: a kind of cloning process of high mountain ion mustard root specifically expressing water channel protein gene, comprising:
1) select high mountain ion canola seed as experiment material, after low temperature induction, get plant tissue be placed in mortar, Trizol reagent extracted total RNA is adopted under room temperature, and adopt Dnase I process, to remove the contaminating genomic DNA in sample, rna content and quality adopt agarose gel electrophoresis to detect;
2) the RT-PCR system reverse transcription of Promega is utilized to obtain cDNA;
3) according to EST information design high mountain ion mustard water root specifically expressing channel protein gene coding region primer;
Forward primer: 5 '-TTG CTG GDG TTG GGT CHG C-3 ' SEQ ID NO:3
Reverse primer: 5 '-TCC GCG GCY GTK GCR TAA ACI-3 ' SEQ ID NO:4
Reaction system is: 10 × buffer 8.0 μ l, rTaq 1.0 μ l, dNTPs 1.0 μ l, primers 2.0 μ l, dH
2o 8 μ l, totally 20 μ l reaction systems;
PCR response procedures: 94 DEG C of denaturation 4min; 94 DEG C of sex change 30s, 58 DEG C of renaturation 40s, 72 DEG C extend 60s, 30 amplification cycles; 72 DEG C extend 10min, 4 DEG C of preservations;
4) PCR reaction product detects on the Agarose glue of 1.0%, reclaims object fragment, be connected into pGEM mono-carrier T (Promega) with GelExtractionKit.Plasmid transformation escherichia coli DH5a competent cell after connection, after blue hickie screening, picking hickie extracts plasmid, is verified as after positive colony and checks order, obtain high mountain ion mustard root specifically expressing water channel protein gene through PCR.
The invention has the beneficial effects as follows:
High mountain ion mustard root specifically expressing water channel protein gene provided by the invention is from high mountain ion mustard, and under low temperature stress condition, the expression amount in high mountain ion mustard all obviously rises, the overexpression of this gene, thus strengthens the resistance of high mountain ion mustard.Its genetically engineered recipient plant had both been suitable for the monocotyledonss such as paddy rice, corn, wheat, sugarcane, was also suitable for the dicotyledonss such as soybean, cotton, tobacco simultaneously.
Embodiment
Be described principle of the present invention and feature below, example, only for explaining the present invention, is not intended to limit scope of the present invention.
Embodiment 1
1) high mountain ion mustard root Total RNAs extraction and detection
With reference to the using method of Invitrogen company's T rizol reagent, extract total serum IgE from high mountain ion mustard root and according to the using method of M-MLV Reverse Transcriptase (TakaRa) ThermoScript II by its reverse transcription be cDNA in-20 DEG C of preservations, for subsequent use.
2) high mountain ion mustard root specifically expressing water channel protein gene ChlRB7 first paragraph sequence obtains
Download to have cloned from Genbank and obtain genes involved as tobacco, Radix Dauci Sativae and Sunflower Receptacle gene order, carry out sequence alignment.According to comparison result at CDS conserved regions design degenerated primer, between utilization and primer obtains the band of two about 750bp and 391bp sizes respectively after pcr amplification.Wherein the fragment of 391bp conforms to expected results, and T-cloning and sequencing result carries out sequence alignment discovery on NCBI, reaches 100% with the homology of Arabidopis thaliana δ-TIPs.First paragraph sequence is as follows through T-cloning and sequencing result: TTGCTGGTGTTGGGTCTGCCATTGCCTACGCGAAGCTGACGTCAGACGCTGCTCTT GACACGCCCGGACTAGTGGCCATCGCGGTGTGCCATGGATTCGCCCCCTTCGTCGC CGTCGCAATCGGAGCCAACATCTCCGGTGGCCACGTGAACCCAGCCGTCACTTTTG GCCTAGCCGTCGGTGGTCATATCACAGTCATCACCGGAGTTTTCTACTGGATCGCT CAGCTTCTCGGCTCCACCGCCGCTTGCTTCCTCCTCAAATACGTCACCGGTGGTTT GGCTGTTCCAATCCACAGCGTTGGGGCTGGACTAGGTTCACTAGAAGGAGTAGTGA TGGAGATCATCATCACTTTCGCTTTGGTCTACACCGTTTATGCAACGGCCGCGGA, SEQ ID NO:5.
3) high mountain ion mustard root specifically expressing water channel protein gene ChlRB7 second segment sequence obtains
According to obtaining the interpretation of result of first fragment sequence in NCBI comparison, this sequence and Arabidopis thaliana TIPs genetic homology higher, design degenerated primer further in conserved sequence district thus, after pcr amplification, obtain the single band that size is about 270bp.Second segment sequence is as follows through T-cloning and sequencing result: TCGCTTTGGTCTACACCGTTTACGCCACAGCAGCTGATCCCAAGAAGGGATCTCTC GGAACCATCGCTCCTCTAGCCATTGGTCTCATTGTTGGTGCCAACATCCTAGCCGC CGGTCCATTCTCAGGTGGATCCATGAACCCGGCTCGTTCCTTTGGACCAGCTGTTG CAGCCGGTGACTTCTCTGGTCACTGGGTTTACTGGGTGGGACCACTCATCGGTGGT GGACTTGCCGGAGCTGTTTACGGAAATGTCTTCATGGGTTCTTCCGAA, SEQ ID NO:6.
4) the RACE reaction of high mountain ion mustard root specifically expressing water channel protein gene ChlRB7
According to Invitrogen GeneRacerTM RACE Ready cDNA kit test kit operational manual, in known array, devise the special primer of 5 ', 3 ' RACE respectively.The method being template reference reagent box with high mountain ion mustard root total serum IgE and tube-nursery cDNA, use the special primer of 5 ', 3 ' to carry out pcr amplification according to this test kit specification sheets respectively, obtain the single fragment of two about 270bp respectively.Through T-cloning and sequencing, analytical results shows the sequence containing initiator codon ATG and known iteron in 5 ' RACE sequence.Containing iteron sequence, terminator codon TAA and poly (A) structure in 3 ' RACE sequence, resulting in the full length sequence of ChlRB7 gene.5 ' RACE sequence is as follows: GGACACTGACATGGACTGAAGGAGTAGAAAATCTTCTTCATTAAACAAAAACTTTC AAAACACATTTTCTCCTTCTTCCTCATTCTTCTTCTTCATTTACAACAATGGCAGG AGTAGCCTTCGGTTCCTTTGACGATTCCTTCAGCTTGTCTTCTCTAAGAGCTTACC TCGCTGAGTTCATCTCCACTTTGCTCTTTGTTTTCGCTGGTGTTGGCTCTGCCATT GCCTACGCAAAGCTGACGTCAGACGCTGCTCTTGACACGCCCGGACTAG, SEQ ID NO:7.
3 ' RACE sequence is as follows: TGCCGGAGCTGTTTACGGAAATGTCTTCATGAGTTCTTCCGAACATGCTCCTCTTG CTTCTTCTGATTTCTAAGCTAAAAAAACATGTCATGATGATGATGATTATTGATTC ATGTTTTTTTATGTGTTCGTTTGGTTTCTTCTTGCTGCGTTTTGGACTTTTGGTCC GGTTTCGTTGTAATTCTTGTATGAATATCATTTAATGGAATTGGCTGTTTGGCTAA AAAAAAAAAAAAAAAAAAAAAACACTGTCATGCCGTTACGTAGCG, SEQ ID NO:8.
5) high mountain ion mustard root specifically expressing water channel protein gene ChlRB7 is long obtains
The full length gene sequence (GeneBank Accession No.EU636988) of 1004bp is obtained according to RACE sequencing result compare of analysis and first and second section of sequence assembly, wherein open reading frame (ORF) has 753bp, 251 amino acid whose protein of encoding.Before initiator codon ATG, there is the 5 ' non-translational region (5 ' UTR) of one section of 104bp, after terminator codon TAA, have 3 ' the Fei Fan district (3 ' UTR) of one section of 173bp, comprising the poly (A) of 24bp.Thus, we obtain the full length gene sequence not comprising intron.This gene C DS district complete sequence comparison result analysis on NCBI is shown, clone the homology of gene and the Arabidopis thaliana TIPs gene obtained up to 92%, be 65% with Radix Dauci Sativae (Daucus carota) DcRB7 genetic homology, be 64.1% with the TobRB7 genetic homology of tobacco (N.tabacum), be 62.2% with Sunflower Receptacle (common sunflower) SunRB7 homology, be 63.6% with tomato (Lycopersicon esculentum) RB7 homology, be 64% with potato (Solanum tuberosum) potRB7 homology.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. a high mountain ion mustard root specifically expressing water channel protein gene, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:1.
2. a protein for high mountain ion mustard root specifically expressing water channel protein gene coding, it is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:2.
3. a cloning process for high mountain ion mustard root specifically expressing water channel protein gene as claimed in claim 1, is characterized in that, comprising:
1) select high mountain ion canola seed as experiment material, after low temperature induction, get plant tissue be placed in mortar, Trizol reagent extracted total RNA is adopted under room temperature, and adopt Dnase I process, to remove the contaminating genomic DNA in sample, rna content and quality adopt agarose gel electrophoresis to detect;
2) the RT-PCR system reverse transcription of Promega is utilized to obtain cDNA;
3) according to EST information design high mountain ion mustard root specifically expressing water channel protein gene coding region primer;
Forward primer: 5 '-TTG CTG GDG TTG GGT CHG C-3 ' SEQ ID NO:3
Reverse primer: 5 '-TCC GCG GCY GTK GCR TAA ACI-3 ' SEQ ID NO:4
Reaction system is: 10 × buffer 8.0 μ l, rTaq 1.0 μ l, dNTPs 1.0 μ l, primers2.0 μ l, dH
2o 8 μ l, totally 20 μ l reaction systems;
PCR response procedures: 94 DEG C of denaturation 4min; 94 DEG C of sex change 30s, 58 DEG C of renaturation 40s, 72 DEG C extend 60s, 30 amplification cycles; 72 DEG C extend 10min, 4 DEG C of preservations;
4) PCR reaction product detects on the Agarose glue of 1.0%, object fragment is reclaimed with GelExtractionKit, be connected into pGEM-T carrier Promega, plasmid transformation escherichia coli DH5a competent cell after connection, after blue hickie screening, picking hickie extracts plasmid, be verified as after positive colony through PCR and check order, obtain high mountain ion mustard root specifically expressing water channel protein gene.
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| CN101671677A (en) * | 2008-09-09 | 2010-03-17 | 山东省花生研究所 | Peanut aquaporin gene AhAQ1 and coded protein and gene cloning method thereof |
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| CN103361366A (en) * | 2012-03-26 | 2013-10-23 | 兰州大学 | Chorispora bungeana alcohol dehydrogenase gene CbADH and preparation method thereof and amino acid sequence of encoding product of Chorispora bungeana alcohol dehydrogenase gene CbADH |
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