CN1364905A - Suaeda liaotungensis kitag chloine monoxygenase gene and its cloning - Google Patents
Suaeda liaotungensis kitag chloine monoxygenase gene and its cloning Download PDFInfo
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- CN1364905A CN1364905A CN 01106075 CN01106075A CN1364905A CN 1364905 A CN1364905 A CN 1364905A CN 01106075 CN01106075 CN 01106075 CN 01106075 A CN01106075 A CN 01106075A CN 1364905 A CN1364905 A CN 1364905A
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Abstract
The present ivnention relates to the field of biological engineering and provides a kind of Suaeda liaotungensis kitag encoded choline monooxygenase gene(CMO), CMO cDNA complete sequence and its corresponding amino acid seqeunce, as well as CMO containing cloned carrier pUC19-CMO, plant expression carrier -CMO and E.coli JM109 cell JM109-pUC19-CMO and JM109-pB1121-CMO containing these two kinds of carrier separately. The present invention may be used in gene conversion to reach the aim of raising plant's salt tolerance, low temperature resistance, and drough tolerance.
Description
The present invention is Suaeda liaotungensis kitag chloine monoxygenase gene and clone thereof, belongs to bioengineering field.Specially refer to the gene and the clone thereof of Suaeda liaotungensis kitag (S.liaotungensis kitag.) coding chloine monoxygenase.
Generally adaptation to abiotic stress is the accumulation organic solute compatible with cellular metabolism, and extensively the most general permeate substance that exists is the tetravalence compound of glycerine, N.F,USP MANNITOL, proline(Pro), sucrose, trehalose and ammonium.Trimethyl-glycine is exactly a class quaternary ammonium compound, ubiquity in bacterium, algae, animal and various plants, and wherein the accumulation of plant such as Chenopodiaceae, Gramineae trimethyl-glycine under arid or salt stress is more obvious.Trimethyl-glycine also is present in microsome and the endochylema except that being positioned chloroplast(id).The existence that bacterium and Study on plants is all shown trimethyl-glycine is relevant with anti-osmotic stress.Trimethyl-glycine plays a part nontoxic osmotic protection agent, and its accumulation makes the important enzyme in many metabolism can keep active under osmotic stress.People such as Saneoka prove that the corn damage more suffered than absence type under salt stress that contains trimethyl-glycine obviously descends.Trimethyl-glycine is to be that substrate generates through two step enzymatic oxidations with the choline, that is: choline → betaine aldehyde chloride → trimethyl-glycine.In higher plant, that the catalysis the first step is reacted is chloine monoxygenase (choline monooxygenase CMO), and that second step of catalysis reacts is betaine-aldehyde dehydrogenase (betaine aldehyde dehydrogenase BADHE.C.1.2.1.8).The activity of these two enzymes is induced by salt and drought stress.Has only the chloine monoxygenase gene of the only a few several plant clone that checked order at present.Suaeda liaotungensis kitag (S.Liaotungensis kitag.) is the very strong halophytes of a kind of salt tolerance, and its chloine monoxygenase activity is higher.
The invention provides the gene C MO of Suaeda liaotungensis kitag (S.Liaotungensis kitag.) coding chloine monoxygenase, CMO cDNA complete sequence and amino acid sequence corresponding thereof are provided.Cloning vector pUC19-CMO that contains CMO and intestinal bacteria (E.coli) the JM109 cell JM109-pUC19-CMO that contains this carrier are provided.The plant expression vector pBI121-CMO and intestinal bacteria (E.coli) the JM109 cell JM109-pBI121-CMO that contains this carrier of CMO are provided.Utilize the present invention, can carry out plant gene and transform, improve plant salt tolerance, low temperature resistant and drought tolerance.
The present invention is for referencial use with the chloine monoxygenase gene CMO nucleotide sequence of spinach (Spinacia oleracea L.), beet (Beta vulgaris), zone design and synthetic two Oligonucleolide primers according to better homology, extracting the total RNA of Suaeda liaotungensis kitag (S.Liaotungensis kitag.) is template, the part fragment of the synthetic CMO of RT-PCR method, and to this PCR product mensuration nucleotide sequence.According to this sequence, designing a reverse transcription primer near 5 ' end, one side, two pairs of nested PCR primers, 5 ' end fragment of the synthetic CMO of 5 ' RACE method carries out the order-checking of PCR product to this fragment.According to first fragments sequence, designing a primer near 3 ' end, one side, 3 ' end fragment of the synthetic CMO of 3 ' RACE method carries out the order-checking of PCR product to this fragment.Obtained Suaeda liaotungensis kitag (S.Liaotungensis kitag.) CMO cDNA complete sequence like this, according to CMO cDNA complete sequence, design two primers, the RT-PCR method is synthesized the CMO gene coding region, and the CMO gene coding region is cloned into cloning vector pUC19, form CMO cloning vector pUC19-CMO.The thermal shock method imports intestinal bacteria (E.coli) JM109 competent cell with this cloning vector, forms the Bacillus coli cells JM109-pUC19-CMO that contains pUC19-CMO.PUC19-CMO and plant expression vector pBI121 are cut with restriction enzyme respectively, form the complementary sticky end, T
4Dna ligase connects, and forms the plant expression vector pBI121-CMO of CMO, and the thermal shock method imports intestinal bacteria (E.coli) JM109 competent cell with this expression vector, forms the Bacillus coli cells JM109-pBI121-CMO that contains pBI121-CMO.
Suaeda liaotungensis kitag (S.liaotungensis kitag.) CMO cDNA nucleotide sequence is as follows:
Wherein the lowercase of front is represented 5 ' end non-coding region, and middle capitalization is coding region (both open reading frame), and the lowercase of back is represented 3 ' end non-coding region.
Be translation initiation password,
Be translation stop codon,
For adding poly (A) signal.
According to above nucleotide sequence, derivation Suaeda liaotungensis kitag (S.Liaotungensis kitag.) the proteic aminoacid sequence of chloine monoxygenase is as follows:
DAQTFDPKEYGLKPLKVAVWGPFVLISLDKTLPESDVGTEWLGSSAEDVKAHAFDP SLKFIHRSEFPMECNWKVFSDNYLDSSYHVPYAHKYYATELDFDTYDTQTIGKVVI QRVGSNTNRPDGFDRLGEKAFYAFTYPNFAVERYGPWMTTMHVQPIAQRKCKLVVD YYIEDSLLDNKDYIEKGIAINDNVQKEDKVLCESVQKGLETPAYRSGRYVMPIEKG IHHFHCWLHQILK
CMO cDNA total length 1820bp, 5 ' end non-coding region 123bp, 3 ' end non-coding region 368bp, open reading frame 1329bp, coding 443aa wherein has the proteic conservative Cys-His of Rieske-type (2Fe-S) right
Contain CMO coding region complete sequence among the cloning vector pUC19-CMO.Contain cloning vector pUC19-CMO among the acceptor Bacillus coli cells JM109-pUC19-CMO.Contain CMO coding region complete sequence among the expression vector pBI121-CMO.Contain expression vector pBI121-CMO among the acceptor Bacillus coli cells JM109-pBI121-CMO.
CMO is the chloine monoxygenase gene of the Suaeda liaotungensis kitag (S.Liaotungensis kitag.) that is extracted first, checks order and clone.Because of its coded chloine monoxygenase can the catalysis trimethyl-glycine synthetic, so this expression carrier can be used for the gene transformation of plant, improve the salt tolerant of plant, cold-resistant, drought-resistance ability.
Below be most preferred embodiment of the present invention:
The acquisition of embodiment 1:S.Liaotungensis kitag. chloine monoxygenase cDNA complete sequence
Gather Suaeda liaotungensis kitag (S.Liaotungensis kitag.), get leaf and carry out liquid nitrogen grinding, TRIZOL test kit (GIBICOL company product) extracts total RNA.
1., the segmental acquisition of CMO cDNA part
It is for referencial use to add single enzyme gene C MO nucleotide sequence with the choline of spinach (Spinacia oleracea L.), beet (Beta vulgaris), and according to zone design and synthetic two Oligonucleolide primers of better homology, the RT-PCR method obtains the part fragment.
Primer is as follows:
C1：5′-AAAGGATGGCAAGTTGCAGG-3′
C2：5′-GGGCCATACCTTTCCACAGC-3′
The RT reaction conditions is as follows:
MgCl
2 4ul
10×RNA?PCR?Buffer 2ul
RNase?Free?H
2O 8.5ul
dNTP?Mixture 2ul
RNase?Inhibitor 0.5ul
AMV?Reverse?Transcriptase 1ul
Oligo?dT-adapter?Primer 1ul
Template?RNA 1ul
20ul
42℃ 30min
99℃ 5min
5 ℃ of 5minPCR reaction conditionss are as follows:
MgCl
2 6ul
10×RNAPCRBuffer 8ul
Sterile purified water 61.5ul
TaKaRa?Taq 0.5ul
C1(10pM) 2ul
C2(10pM) 2ul
cDNA 20ul
100ul
94℃ 2min
72℃ 7min
4℃
Pcr amplification product is behind agarose gel electrophoresis, reclaim the dna fragmentation that test kit (TAKARA company product) reclaims about 700bp with NaI, this fragment is measured its nucleotide sequence with the Sanger chain termination method, be the chloine monoxygenase gene partial sequence of Suaeda liaotungensis kitag (S.Liaotungensis kitag.).
2., the acquisition of CMO cDNA 5 ' terminal sequence
According to sequence 1., designing a reverse transcription primer near 5 ' end, one side, two pairs of nested PCR primers, 5 ' end fragment of the synthetic CMO of 5 ' RACE method.
Primer is as follows:
RT-primer：5′-(p)GAGAACGAATGGTC-3′
F1：5′-CCTGCTTTGTGTGCCCTTAC-3′
R1：5′-GACTTTITGCCACTTCCACA-3′
F2：5′-ACAAAAGCCACCCAAACAAC-3′
R2：5′-TTCACCATCTCGGCTCACCA-3′
The RT reaction conditions is as follows:
Total RNA 1ul
10×RT?buffer 1.5ul
Rnase?Inhibiter(40U/ul) 0.5ul
AMV?Reverse?Transcriptase?XL(5U/ul) 1ul
RT-primer(1ug/ul) 1ul
15ul
30℃ 10min
50℃ 30min
80℃ 2min
The decomposition of 4 ℃ of Hybrid RNA:
RT reaction solution 15ul
5×Hybrid?RNA?degenaration?buffer 15ul
DEPC water 45ul
Rnase?H(60U/ul) 1ul
30 ℃ were reacted 1 hour, and carried out ethanol sedimentation then.By ligation with strand cDNA cyclisation:
The strand cDNA of ethanol sedimentation
5×RNA?Ligation?Buffer 8ul
40％PEG#6000 20ul
DEPC water 12ul
T
4RNA?ligase 1ul
41ul
16 ℃ were reacted 15～18 hours.1st PCR reaction:
Above-mentioned connection liquid 1ul
10×LA?PCRBuffer 5ul
dNTP?Mixtme 8ul
TaKaRa?LA?Taq(5U/ul) 0.5ul
F1(20pmol/ul) 0.5ul
R1(20pmol/ul) 0.5ul
ddH
2O 34.5ul
50ul
94℃ 1min
4 ℃ of 2nd PCR reactions:
1st PCR reaction product 1ul
10×LA?PCR?Buffer 5ul
dNTP?Mixtrue 8ul
TaKaRa?LA?Taq(5U/ul) 0.5ul
F2(20pmol/ul) 0.5ul
R2(20pmol/ul) 0.5ul
ddH
2O 34.5ul
50ul
4℃
Reaction obtains a treaty 700bpDNA fragment, reclaims, and order-checking is chloine monoxygenase gene 5 ' terminal sequence of Suaeda liaotungensis kitag (S.Liaotungensis kitag.).
3., the acquisition of CMO cDNA 3 ' terminal sequence
According to sequence 1., designing a primer near 3 ' end, one side, 3 ' end fragment of the synthetic CMO of 3 ' RACE method.
Primer is as follows:
C3：5′-CTAGTGCCGAAGATGTTAAG-3′
The RT reaction conditions is as follows:
MgCl
2 4ul
10×RNA?PCR?Buffer 2ul
RNase?Free?H
2O 8.5ul
dNTP?Mixture 2ul
RNase?Inhibitor 0.5ul
AMV?Reverse?Transcriptase 1ul
Oligo?dT-adapter?Primer 1ul
Template?RNA 1ul
20ul
42℃ 30min
99℃ 5min
5 ℃ of 5minPCR reaction conditionss are as follows:
cDNA 2ul
TaKaRa?LA?Taq 1ul
10×LA?PCR?Buffer 10ul
dNTP?Mixtrue 16ul
C3(20pM) 1ul
M13primer?M4(20pM) 1ul
ddH
2O 69ul
100ul
94℃ 1min
72℃ 7min
4℃
Reaction obtains a treaty 1kbDNA fragment, reclaims, and order-checking is chloine monoxygenase gene 3 ' terminal sequence of Suaeda liaotungensis kitag (S.Liaotungensis kitag.).
Above-mentioned three sequences are coupled together, and both have been the chloine monoxygenase gene complete sequence of Suaeda liaotungensis kitag (S.Liaotungensis kitag.).
4., the acquisition of CMO gene coding region
According to above-mentioned complete sequence design primer, the RT-PCR method is obtained the CMO gene coding region.
Primer is as follows:
CMO?ORF?F?primer：5′-ggGGATCGAATTTAAGGTCTTGTTTGATGGCTG-3′
CMO?ORF?R?primer：5′-ggGAGCTC5′TCACTTCAAAATTTGGTGCAACC-3′
The RT reaction conditions is as follows:
MgCl
2 4ul
10×RNA?PCR?Buffer 2ul
RNase?Free?H
2O 8.5ul
dNTP?Mixture 2ul
RNase?Inhibitor 0.5ul
AMV?Reverse?Transcriptase 1ul
Oligo?dT-adapter?Primer 1ul
Template?RNA 1ul
20ul
42℃ 30min
99℃ 5min
5 ℃ of 5minPCR reaction conditionss are as follows:
cDNA 2ul
TaKaRa?LA?Taq 1ul
10×LAPCRBuffer 10ul
dNTP?Mixtrue 16ul
CMO?ORF?F?primer(20pM) 1ul
CMO?ORF?R?primer(20pM) 1ul
ddH
2O 69ul
100ul
94℃ 1min
72℃ 7min
4℃
Reaction obtains a treaty 1.3kbDNA fragment, reclaims, and order-checking is the chloine monoxygenase gene coding region complete sequence of Suaeda liaotungensis kitag (s.Liaotungensis kitag.).The reorganization of embodiment 2:CMO and cloning vector pUC19 and transformed into escherichia coli JM109CMO and cloning vector pUC19 use BamH I/Sac I double digestion respectively.The enzyme tangent condition is as follows: Eppendorf A Eppendorf BCMO gene, (0.2ug/ul) 35ul pUC19, (2ug/ul) 5ul0.5 * K buffer 7.5ul 0.5 * K buffer 10ulBamH I 6ul BamH I 9ulSac I 7ul Sac I 7ulH
2O 94.5ul H
2O 166ul
150ul 200ul
37 ℃ of enzymes were cut 3 hours, reclaimed two purpose fragments behind the electrophoresis respectively, used T
4Dna ligase connects.Condition of contact is as follows:
pUC19(0.05ug/ul) 1ul
CMO?gene(0.05ug/ul) 3ul
10 * T
4Dna ligase Buffer 1ul
T
4Dna ligase (1U/ul) 1ul
ddH
2O 4ul
16 ℃ connect 2 hours, heat shock method transformed into escherichia coli JM109, and the PCR method detects positive colony JM109-pUC19-CMO.From JM109-pUC19-CMO, extract plasmid, measure the nucleotide sequence of external source insertion sequence CMO with the universal primer of pUC19, consistent with embodiment 1 described sequencing result.
The reorganization of embodiment 3:CMO and expression vector pBI121 and transformed into escherichia coli JM109
From JM109-pUC19-CMO, extract plasmid pUC19-CMO, expression vector pBI121 and use BamH I/Sac I double digestion respectively, connect, form JM109-pBI12-CMO, heat shock method transformed into escherichia coli JM109, form JM109-pBI12-CMO, condition is with embodiment 2.
Claims (6)
1, a kind of chloine monoxygenase gene CMO is characterized in that it is Suaeda liaotungensis kitag (S.Liaotungensis
Kitag.) Bian Ma chloine monoxygenase gene.
2, the nucleotide sequence of the described chloine monoxygenase gene CMO of claim 1:
3, according to the cloning vector pUC19-CMO of the described chloine monoxygenase CMO of claim 1.
4, according to intestinal bacteria (E.coli) the recipient cell JM109-pUC19-CMO of the described cloning vector pUC19-CMO of claim 3.
5, according to the expression vector pBI121-CMO of the described chloine monoxygenase CMO of claim 1.
6, according to intestinal bacteria (E.coli) the recipient cell JM109-pBI121-CMO of the described expression vector pBI121-CMO of claim 5.
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| CN 01106075 CN1364905A (en) | 2001-01-12 | 2001-01-12 | Suaeda liaotungensis kitag chloine monoxygenase gene and its cloning |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971772A (en) * | 2019-03-12 | 2019-07-05 | 中国农业科学院生物技术研究所 | A kind of breeding method of low temperature resistant cotton variety |
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2001
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971772A (en) * | 2019-03-12 | 2019-07-05 | 中国农业科学院生物技术研究所 | A kind of breeding method of low temperature resistant cotton variety |
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