CN101705242B - Application of gene promoter region sequence of glycerophospholipid peptidy transeferace in crop genetic improvement - Google Patents
Application of gene promoter region sequence of glycerophospholipid peptidy transeferace in crop genetic improvement Download PDFInfo
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- CN101705242B CN101705242B CN200910193929XA CN200910193929A CN101705242B CN 101705242 B CN101705242 B CN 101705242B CN 200910193929X A CN200910193929X A CN 200910193929XA CN 200910193929 A CN200910193929 A CN 200910193929A CN 101705242 B CN101705242 B CN 101705242B
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Abstract
The invention discloses a gene promoter region sequence of glycerophospholipid peptidy transeferace (LPAAT) and application in crop genetic improvement. The gene promoter region sequence of glycerophospholipid peptidy transeferace is shown as SEQ ID NO:1. The application method of the gene promoter of glycerophospholipid peptidy transeferace in crop genetic improvement comprises the following steps of: (1) inserting the gene promoter of glycerophospholipid peptidy transeferace into a promoter verifying expression vector pBI101.3 and starting the expression of a gus reporter gene; and (2) using the expression carrier for plant callus transgenosis research induced by agrobacterium tumefacien and obtaining the transgenosis plant by inducing, dip dyeing, coincubating and taking root of the plant callus. The gene promoter comes from coconut, can specially start expression of target gene in endosperm histiocyte, provides new promoter type in the future crop genetic improvement and has very high stability and operability without transgenic silencing.
Description
Technical field
The present invention relates to field of plant breeding, be specifically related to utilize genetic engineering technique to obtain a kind of glycerol phosphate acyltransferase (LAPPT) gene promoter, and be applied to paddy endosperm transgeneic procedure and genetic improvement.
Background technology
Endosperm is starch important in the crop seed and the types of organization that stores with protein, and 60% energy and protein all derive from the endosperm of crop in the world food production.In crop transgenic research process; Express organ and types of organization with respect to other; The endosperm of using crop as the platform of heterologous gene or protein expression have cost low, be easy to many advantages (Takaiwa et al, 2007, Endosperm tissue is a good production platform for artificial recombinant proteins intransgenic rice.Plant Biotechnol J such as control, output are big, production process safety; 5,84-92.; Wu et al; 2007; Oral immunization withtransgenic rice seeds expressing VP2 protein of infectious bursal disease virusinduces protective immune responses in chickens.Plant Biotechnol J.5,570-578).At present; Through the mode of genetic modification the composition of the product in the crop endosperm being modified is present important research direction; Certain achievement in research (Bajaj and Mohanty, 2005, Recent advances inrice biotechnology--towards genetically superior transgenic rice.Plant Biotechnol J have also been obtained; 3 (3), 275-307).Yet, improve in the research of crop endosperm in applying transgene mode in the past, make the correlative study progress very slowly owing to lack effective endosperm specific promotor.Therefore, screen with clone new endosperm specific promotor type and with the foreign gene specifically expressing in the endosperm of crop with being one of focus of relevant from now on crop genetic improvement research.
In crop genetic improvement process in the past; It is major cause (the Bhullar et al that causes transgene silencing that multiple uses same promotor type; 2003; Strategies for development of functionallyequivalent promoters with minimum sequence homology for transgene expression inplants:cis-elements in a novel DNA context versus domain swapping.Plant Physiol; 132,988-998).In order to reach the purpose that polygene transforms, need in the genetic improvement of crop, develop a series of have organizing specific type and the promotor types that efficiently express.In the research process in early stage; The investigator has screened some endosperm specific promotors (Rasmussen and Donaldson successively from paddy rice, wheat, corn and barley; 2006; Investigation of the endosperm-specific sucrosesynthase promoter from rice using transient expression of reporter genes in guar seedtissue.Plant Cell Rep, 25,1035-1042; Qu and Takaiwa, 2004, Evaluation of tissuespecificity and expression strength of rice seed component gene promoters intransgenic rice; Plant Biotechnol J, 2,113-125; Lamacchia et al, 200l, Endosperm-specific activity of a storage protein gene promoter in transgenic wheatseed.J Exp Bot, 52,243-250; Wiley et al; 2007; Promoter analysis andimmunolocalisation show that puroindoline genes are exclusively expressed instarchy endosperm cells of wheat grain.Plant Mol Biol; 64,125-136; Russell andFromm 1997, Tissue-specific expression in transgenic maize of four endospermpromoters from maize and rice.Transgenic Research, 6,157-168).Yet from the effect of reality, promotor type that at present can the successful Application crop genetic improvement is still very limited, and can't effectively be directed against the important storage organ of crop---seed carries out the modification and the improvement of orientation.
In seed endosperm specific promotor clone process, mainly be to study to glucide and protein metabolism relevant enzymes, be basic cloning promoter and ignored with lipid metabolism enzyme in the past.(1-acyl-sn-glycerol-3-P acyltransferase LPAAT) is a kind of key enzyme that acts on fatty acyl phosphatidic acid (LPA) sn-2 position acetylize generation phosphatidic acid PA (Phosphatidicacid) in the Kennedy approach to the glycerol phosphate acyltransferase.Phosphatidic acid is under the phosphatidic acid phosphatase effect; Hydrolysis discharges inorganic phosphate; And change triglyceride into, generate triglyceride level (Okazaki et al.2006, Thesignificance of C16 fatty acids in the sn-2 positions of glycerolipids in thephotosynthetic growth of Synechocystis sp.PCC6803.Plant Physiol through further esterification again; 141,546-556).At present; Glycerol phosphate acyltransferase gene is proved to be in the distribution of glycerine fatty acid play an important role (Coleman and Lee, 2004, Enzymes of triacylglycerol synthesis and their regulation.Prog Lipid Res; 43,134-176; Nath et al; 2009; Increasing erucic acid content throughcombination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT+Bn-fael transgenes in rapeseed (Brassica napus L.) .Theor Appl Genet; 118,765-773).
Summary of the invention
The objective of the invention is to provides a kind of glycerol phosphate acyltransferase (LPAAT) gene promoter region sequence that from coconut, extracts to existing deficiency in existing albumen Idiotype promotor and the application thereof.
Another object of the present invention is to provide the application method of above-mentioned glycerol phosphate acyltransferase gene promoter in crop endosperm transgeneic procedure and genetic improvement.
A further object of the invention is to provide a kind of preparation method who prepares the relevant glycerol phosphate acyltransferase gene promoter of fatty acid metabolism approach that from coconut, extracts.
Glycerophosphate acyltransferase gene promoter region sequence of the present invention derives from coconut, and after methylating on original sequence basis, gene adds or disappearance etc. modifies, the action effect of promotor changes to some extent.Particularly, above-mentioned purpose of the present invention is achieved through following scheme:
At first, the present invention extracts from coconut and has prepared glycerol phosphate acyltransferase gene promoter, and concrete steps are following:
(1) be the basis with coconut genomic dna and coconut fatty acid metabolic enzyme, the design special primer, primer sequence is shown in SEQ ID NO:2 and SEQ ID NO:3;
(2) through the chromosome walking method amplimer, obtain glycerophosphate acyltransferase gene promoter region sequence.
The clone of endosperm promotor was based on and carried out on the basis of storage protein and glucide genes involved in the crop in the past; The promotor type that obtains also is that proteinoid is relevant with carbohydrate metabolism therewith; And the present invention is the basis with the enzyme in the fatty acid metabolism approach; The endosperm specificity promoter that the clone obtains will all have on control, expressive site and the expression amount of pathways metabolism to be had obviously differently with the promotor type of discovery in the past, on use range and effect, also has different.Therefore, promotor of the present invention is the basic design Auele Specific Primer with coconut genomic dna and glycerol phosphate acyltransferase.
In the above-mentioned preparation process, used chromosome walking method is the common technology in this area.
Acquisition glycerol phosphate acyltransferase gene promoter region sequence process plant promoter analysis software PlantCARE (
Http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/); This sequence of analytical proof has typical plant tissue specificity promoter characteristic, like 1 TATA box (being positioned at-4), 6 CAAT box (lay respectively at-878 ,-877 ,-842 ,-356 ,-335 and+18) and 45 ' UTR cytosine(Cyt) content rich region (laying respectively at-170 ,-216 ,-276 and-326); In addition, the cis-acting elements Skn-1 motif (being positioned at-127) that also contains an important endosperm specific expression.This promoter sequence is shown in SEQ IDNO:1.
Glycerol phosphate acyltransferase gene promoter region sequence of the present invention is specific expressed in the crop endosperm, can be used for preparing genetically modified crops.
Further, glycerol phosphate acyltransferase gene promoter region sequence of the present invention prepares transfer-gen plant through starting the expression of gus gene in paddy endosperm.
The application of glycerol phosphate acyltransferase gene promoter region sequence of the present invention in crop genetic improvement comprises the steps:
(1) glycerol phosphate acyltransferase gene promoter is inserted promotor checking expression vector pBI101.3, start the expression of gus reporter gene;
(2) above-mentioned expression vector is used for Agrobacterium tumefaciens mediated plant callus transgenic research, through the plant callus acquisition transfer-gen plant of inducing, contaminate, cultivate altogether, screen, take root.
In order to verify the expression control effect of glycerol phosphate acyltransferase gene promoter in the crop endosperm; The present invention has obtained 3 kind of 5 ' glycerol phosphate acyltransferase gene promoter sequence that end lacks through design of primers and pcr amplification, and the disappearance position is respectively-896 ,-817 and-453.Insert the expression that promotor checking expression vector pBI101.3 starts the gus reporter gene with above-mentioned three kinds of deletion fragments, with not inserting the control vector of any segmental pBI101.3 empty carrier as later stage research.
The above-mentioned plant expression vector that builds is used for Agrobacterium tumefaciens mediated rice callus organizes transgenic research.Change the transgenic paddy rice plant that obtains over to expression checking that the seed of gathering in the crops after the hot-house culture is used for promoter function.GUS expression analysis through to the results seed proves: in the seed of transgenic paddy rice; The glycerol phosphate acyltransferase gene promoter sequence startup in various degree of 3 kinds of deletion fragments the expression of gus gene in the rice paddy seed endosperm, but in blade, inflorescence, root, stem and leaf sheath, do not detect the gus expression of gene.Simultaneously, 3 kinds of different deletion fragments also show different expression amounts, are indicating that special role element Skn-1 motif has played vital role on final expression effect in the process of 5 ' end disappearance.
Compared with prior art, the present invention has following beneficial effect:
(1) expression of startup goal gene in the endosperm tissue cell that glycerol phosphate acyltransferase gene promoter of the present invention can be special for new promotor type is provided in the crop genetic improvement, has very high stability and operability;
(2) homology between glycerol phosphate acyltransferase gene promoter of the present invention and other the existing promotors is very little, can not cause the transgene silencing phenomenon;
(3) glycerol phosphate acyltransferase gene promoter of the present invention is the basic design primer with the glycerol phosphate acyltransferase in coconut genomic dna and the fatty acid metabolism approach, and the promotor of acquisition can be used in the regulation and control of fatty acid metabolism approach.
Description of drawings
Fig. 1 is a glycerol phosphate acyltransferase gene promoter expression vector establishment mode, and wherein A is different 5 ' end deletion fragment and gus gene mode of connection; B is the coded message of connection site;
Fig. 2 changes the gus Color of rice paddy seed and other types of organization over to for glycerol phosphate acyltransferase transgenic promotor.Wherein, 1 is that promoterless empty carrier pBI101.3 (contrast) transforms; 2 is that deletion fragment 1 carrier construction (pBI101.3-L1) transforms; 3 is that deletion fragment 2 carrier constructions (pBI101.3-L2) transform; 4 is that deletion fragment 3 carrier constructions (pBI101.3-L3) transform; 5 is transfer-gen plant root dyeing effect; 6 is transfer-gen plant leaf sheath Color; 7 are transfer-gen plant flower Color; 8 is the rotaring gene plant blade Color; 9 is transfer-gen plant stem Color.
Embodiment
Below in conjunction with specific embodiment the present invention is done further description, but specific embodiment is not done any qualification to the present invention.
The extracting of embodiment 1 coconut genomic dna
High-quality coconut genomic dna extracts from young leaflet tablet through the CTAB method.Get 0.1g after the material liquid nitrogen grinding and add 600 μ L CTAB extracting solutions (containing 4% beta-mercaptoethanol), continue grinding it is suspended evenly, put 65 ℃ of water-bath 45min; Add 700 μ L phenol: chloroform: primary isoamyl alcohol, 37 ℃ of water-bath temperature are bathed 10min.The centrifugal 10min of 12000rpm; Get supernatant, add 60 μ L 5M NaCl, the freezing absolute ethyl alcohol of 1mL, freezing 30min deposit D NA; The centrifugal 5min of 10000rpm abandons behind the alcohol air-dry.DNA after air-dry adds TE, and 37 ℃ of water-bath 15-30min to DNA dissolve fully, add 700 μ L chloroforms: primary isoamyl alcohol (24: 1), put upside down 15min, the centrifugal 5min of 12000rpm; Get supernatant, add 30 μ L 5M NaCl, 1mL freezing absolute ethyl alcohol-20 ℃ deposition 30min, the centrifugal 3min of 10000rpm abandons supernatant; Add 70% alcohol, abandon supernatant behind the centrifugal 3min of 10000rpm, air-dry; Add 100 μ L, after TE fully dissolves, add 5 μ L Rnase, 37 ℃ of water-bath 1h.Add 200 μ L chloroforms again: primary isoamyl alcohol (24: 1), put upside down 15min, the centrifugal 5min of 12000rmp; Get supernatant, add 30 μ L 5M NaCl, the freezing absolute ethyl alcohol of lmL is put upside down back-20 ℃ of depositions 30min, the centrifugal 3min of 10000rpm gently; Abandon alcohol, add 70% alcohol-pickledly, 2h changes once, back soaked overnight; Abandon alcohol behind the centrifugal 3min of 8000rpm, air-dry; Adding 100 μ L TE fully dissolves.Measure 260nm and 280nm place light absorption value with behind 1000 times of the DNA diluted samples that obtains respectively with spectrophotometer.Use the purity of ratio calculation sample of the light absorption value at two places, and through the dna content in the 260nm place light absorption value calculation sample.
The clone and the analysis of embodiment 2 LPAAT upstream region of gene promoter sequences
The clone of glycerol phosphate acyltransferase upstream region of gene promoter sequence carries out with reference to the operational manual of Universal GenomeWalker Kit (Clontech).Genomic dna earlier with 4 kinds of flush end restriction endonucleases (EcoRI, Pvu II, Dra I, Stu I) respectively enzyme cut.Enzyme is cut the purified back of product and is connected with given joint; Connect the nest-type PRC reaction that product is used for the first round; Glycerol phosphate acyltransferase gene specific primer nucleotide sequence is shown in SEQ ID NO:2, and joint special primer nucleotide sequence is shown in SEQ ID NO:3.The PCR response procedures is: 94 ℃ of 25s, 72 ℃ of 3min, 7 circulations: 94 ℃ of 25s, 67 ℃ of 3min, 32 circulations; 67 ℃ of 7min.Second PCR that take turns is a substrate with the product of first round PCR reaction, uses identical amplification program, and the primer nucleotide sequence is respectively shown in SEQ ID NO:4 and SEQ ID NO:5.Be connected after amplified production reclaims pMD18-T (TaKaRa) go up and carry out sequential analysis (TaKaRaBiotechnology Company, Dalian, China).Sequencing result is analyzed with plant promoter dedicated analysis software PlantCARE, carries out further functional verification on the basis of analyzing.
The structure and the genetic transformation of embodiment 3 plant expression vectors
Obtain the effect of promoter region sequence in tissue specific expression control in order to verify, the promoter fragment type of three kinds of difference 5 ' end disappearances is used to the structure of plant expression vector.Deletion fragment inserts promotor checking expression vector pBI101.3, difference called after pBI101.3-L3, pBI101.3-L2, pBI101.3-L1 respectively.The site of 3 fragment deletions is respectively-896, and-817 and-453.The restriction enzyme site HindIII and the BamHI of 8 bases added at pcr amplification primer two ends respectively.The relevant primer sequence is respectively:
The p101-1L nucleotide sequence is shown in SEQ ID NO:6;
The p101-2L nucleotide sequence is shown in SEQ ID NO:7;
The p101-3L nucleotide sequence is shown in SEQ ID NO:8;
The p101-R nucleotide sequence is shown in SEQ ID NO:9.
Amplified production reclaim the back with two kinds of enzymes respectively enzyme cut (HindIII and BamHI) and be connected in the pBI101.3 carrier.Relating operation all carries out according to the scheme that the molecular cloning experiment guide provides.Do not insert the pulsating pBI101.3 carrier of any promotor and be used as negative control, the carrier after the connection verifies that with the mode of pcr amplification the primer nucleotide sequence is respectively shown in SEQ ID NO:10 and SEQ ID NO:11.
Embodiment 4 transgeneic procedures and expression checking
4 kinds have the pulsating expression vector pBI101.3-L1 of different glycerol phosphate acyltransferase gene promoters, pBI101.3-L2, pBI101.3-L3 and pBI101.3-L0 and change rice varieties Zhonghua11 among the agrobacterium tumefaciens Agrobacterium tumefaciens EHA105 (Oryza sativa L ssp.Japonica) over to through the mode that electric shock transforms; The operation of callus induction and genetic transformation is carried out according to the schedule of operation that pertinent literature provides; The transgenic rice plant that obtains is transplanted and is cultivated to the greenhouse, until the seed fully matured.
Seed, blade, leaf sheath, stem, inflorescence and the root of collecting the transgenic rice plant that different expression vectors transform respectively carry out GUS dyeing, and dyeing process carries out with reference to pertinent literature, coloration result under Stereo microscope, observe and take pictures (Fig. 2).
The application sequence table of glycerol phosphate acyltransferase gene promoter region sequence in crop genetic improvement
SEQUENCE?LISTING
< 110>University Of Hainan
< 120>application of glycerol phosphate acyltransferase gene promoter region sequence in crop genetic improvement
<130>
<160>11
<170>PatentIn?version?3.2
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gattacgaat?tcgagctcgg?tacccgggga?tcctctagag?attactatag?ggcacgcgtg 60
gtcgacggcc?cgggctggta?aattaaacca?attaaattat?ggcagtgatt?ttggaaccac 120
tacaataggc?ctgatctatt?acggtgattt?tcagtaatca?ctgccatagg?ttaggtcatt 180
ctcactacac?gcccagccca?ccttattttg?gttttcctct?ttcgagcact?ctctctccct 240
cctctccacc?gccagtaagc?tcctcctcct?ctctccctcc?tctccaccat?cagagcgctc 300
atccccggca?tccctcgacc?ggccctgcct?cctctttctt?cctagccagc?gctgcctcca 360
cccctgcctc?ctccttcttc?cttgactgac?cccacctcct?acttcttcct?cggtcggccc 420
tacattctcc?ttcttcctcg?actagcctca?cctcctgctt?cttttttgat?cggccctacc 480
accttcccca?cctccttctt?tcttgaccga?cctcgcctcc?ctctttttcg?actggccttg 540
cctccagtct?tgcctccttt?tttctcgacc?agccctacct?cctccctcac?tcctttttcc 600
ccaatcggcc?ccatcttctc?tctctttctc?tttctctcct?ctcttccatt?ggccagaccc 660
actgcagccc?cctcagcctt?tcttctctct?cactccttct?gtctctctct?tcctgctttc 720
tcttatctat?cactctctct?cttctctctt?tctctatttg?gtctgcactc?tagaatcttc 780
tctttcttct?ctctccacca?agaacccata?gaatttgttc?gttgctggat?tccgattccg 840
acctattcgc?cagttcccta?ctcggaaccc?tcaacccttt?acgtagtcct?cgtttgcctt 900
tcttgctcgt?ggtattggtg?gtgggaagtg?ggggatatat?agtcct 946
<210>2
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ttcggttggg?acatcccgaa?gctcgctt 28
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<211>22
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<400>3
gtaatacgac?tcactatagg?gc 22
<210>4
<211>28
<212>DNA
< 213>artificial sequence
<400>4
gaacttgccc?ctgaagcatc?cataggac 28
<210>5
<211>19
The application sequence table of glycerol phosphate acyltransferase gene promoter region sequence in crop genetic improvement
<212>DNA
< 213>artificial sequence
<400>5
actatagggc?acgcgtggt 19
<210>6
<211>26
<212>DNA
< 213>artificial sequence
<400>6
ataagcttgg?cacgcgtggt?cgacgg 26
<210>7
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<400>7
ataagcttcc?tgatctatta?cggtga 26
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<400>8
ataagcttcc?ttctttcttg?accgac 26
<210>9
<211>32
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<400>9
atggatccag?gactatatat?cccccacttc?cc 32
<210>10
<211>22
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< 213>artificial sequence
<400>10
ggacacttta?tgcttccggc?tc 22
<210>11
<211>21
<212>DNA
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attccacagt?tttcgcgatc?c 21
Claims (3)
1. the application of glycerol phosphate acyltransferase gene promoter region sequence in crop genetic improvement; It is characterized in that said glycerol phosphate acyltransferase gene promoter region sequence is shown in SEQ ID NO:1; This promotor is specific expressed in the crop endosperm, is used to prepare crop genetic improvement.
2. the application of glycerol phosphate acyltransferase gene promoter region sequence according to claim 1 in crop genetic improvement is characterized in that comprising the steps:
(1) glycerol phosphate acyltransferase gene promoter is inserted promotor checking expression vector pBI101.3, start the expression of gus reporter gene;
(2) above-mentioned expression vector is used for Agrobacterium tumefaciens mediated plant callus transgenic research, through the plant callus acquisition transfer-gen plant of inducing, contaminate, cultivate altogether, screen, take root.
3. the application of glycerol phosphate acyltransferase gene promoter region sequence according to claim 2 in crop genetic improvement is characterized in that the preparation method of said glycerol phosphate acyltransferase gene promoter region sequence comprises the steps:
(1) be the basis with coconut genomic dna and coconut fatty acid metabolic enzyme, the design special primer, primer sequence is shown in SEQ ID NO:2 and SEQ ID NO:3;
(2) through the chromosome walking method amplimer, obtain glycerophosphate acyltransferase gene promoter region sequence.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2146712C (en) * | 1995-04-10 | 2002-06-25 | Jas Singh | Cold induced promoter from winter brassica napus |
| WO2008022208A2 (en) * | 2006-08-16 | 2008-02-21 | Yorktown Technologies, L.P. | Recombinant constructs and transgenic fluorescent ornamental fish therefrom |
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2009
- 2009-11-13 CN CN200910193929XA patent/CN101705242B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2146712C (en) * | 1995-04-10 | 2002-06-25 | Jas Singh | Cold induced promoter from winter brassica napus |
| WO2008022208A2 (en) * | 2006-08-16 | 2008-02-21 | Yorktown Technologies, L.P. | Recombinant constructs and transgenic fluorescent ornamental fish therefrom |
Non-Patent Citations (2)
| Title |
|---|
| Deborah S. Knutzon，et al.Cloning of a Coconut Endosperm cDNA Encoding a 1 -Acyl-sn-Clycerol-3-Phosphate Acyltransferase That Accepts Medium-Chain-Length Substrates.《Plant Physiol》.1995,999-1006. |
| Deborah S. Knutzon,et al.Cloning of a Coconut Endosperm cDNA Encoding a 1-Acyl-sn-Clycerol-3-Phosphate Acyltransferase That Accepts Medium-Chain-Length Substrates.《Plant Physiol》.1995,999-1006. * |
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