CN105801677A - Compound for regulation and control of plant chlorophyll synthesis and use thereof - Google Patents
Compound for regulation and control of plant chlorophyll synthesis and use thereof Download PDFInfo
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Abstract
The invention provides a compound for regulation and control of plant chlorophyll synthesis and a use thereof. The invention provides a protein-nucleic acid compound. A SCL27 protein is bonded to a specific area of a PORC gene so that the compound is formed. An experiment proves that the compound can regulate and control PORC gene expression, simply and effectively regulate and control plant chlorophyll synthesis and adjust plant chlorophyll content and blade color thereby controlling a leaf color or regulating plant photosynthesis efficiency.
Description
Technical field
The present invention relates to plant genetic engineering field, specifically, the invention provides a kind of regulate and control the complex of plant chlorophyll synthesis, nucleotide sequence and application thereof.
Background technology
The photosynthetic photoreaction of high green plants must complete in chloroplast, the main molecules accepting luminous energy in chloroplast is chlorophyll, and including chlorophyll a and chlorophyll b, chlorophyll can catch photon, luminous energy is changed into chemical energy, thus anabolism and growth for plant provide motive power.
Chlorophyll is encoded by chloroplast DNA, but it is expressed and location is affected by the strict of karyogene and controls.Owing to chlorophyll is the basis of photosynthesis of plant, so research and development participates in the related gene of this approach and controls function, the photosynthetic efficiency and yield improving plant there is important effect.
Research shows, Chlorophyll synthesis Regulation Mechanism is extremely complex.In general, when quick growth of plant, synthesis chlorophyll is also slow.
Chlorophyll is present in protein complexes, participates in two big critical functions, namely catch luminous energy and be passed to photosystem reaction center in photosynthesis.In the process, chlorophyll molecule is inevitably generated high energy singlet oxygen after being excited, thus inhibited photosynthesis and plant growing, can cause cell death when serious.It addition, the many intermediate products in Chlorophyll synthesis process are also potential photosensitizer, under illumination condition, produce active oxygen.Therefore, Chlorophyll synthesis approach must be under precision control.Through the research in century more than one, the check point that now clear and definite Chlorophyll synthesis approach is main be positioned at the synthesis of ALA, Mg branch sites and by the enzymatic protochlorophyllide of POR to chlorophyllous conversion.
In sum, current this area is also knows about less for chlorophyllous synthesis, therefore, and the technology that this area participates in the urgent need to exploitation and/or controlling chlorophyll synthesizes, such as develop the method rapidly promoting chlorophyllous synthesis while plant growing, thus being applied to the field such as agricultural, gardening.
Summary of the invention
It is an object of the invention to provide a kind of can Effective Regulation plant chlorophyll synthesis complex and application.
In a first aspect of the present invention, it is provided that a kind of protein-nucleic acid complex, described complex is SCL27 protein binding being formed selected from the sequence of lower group in PORC gene:
A the nucleic acid fragment II of ()-788nt to-598nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 911-1100 position；
B the nucleic acid fragment III of ()-572nt to-372nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1127-1326 position；
C the nucleic acid fragment W of ()-500nt～-438nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1199-1260 position；
The total length promoter region of (d) POC.
In another preference, described SCL27 protein binding is in the GT cis acting element of described sequence.
In another preference, the sequence of the total length promoter region of described POC is such as shown in SEQIDNO.:1.
In another preference, described complex is that SCL27 protein binding is formed in the nucleic acid fragment of PORC gene-500nt～-438nt.
In a second aspect of the present invention, it is provided that the nucleotide sequence of a kind of separation, described nucleotide sequence is selected from lower group:
A the nucleic acid fragment II of-788nt of () PORC gene to-598nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 911-1100 position；
B the nucleic acid fragment III of-572nt of () PORC gene to-372nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1127-1326 position；
C the nucleic acid fragment W of-500nt～-438nt of () PORC gene, in the sequence of this fragment such as SEQIDNO.:1 shown in 1199-1260 position.
In another preference, for preparing the complex described in first aspect present invention.
In a third aspect of the present invention, it is provided that the purposes of the protein-nucleic acid complex described in a kind of first aspect present invention or the nucleotide sequence described in second aspect present invention, they are used to controlling chlorophyll synthesis, or for preparing the preparation of controlling chlorophyll.
In another preference, described regulation and control include: suppress chlorophyllous synthesis, and/or reduce the content of plant Determination of Chlorophyll.
In a fourth aspect of the present invention, it is provided that a kind of antisense sequences, described antisense sequences is complementary wholly or in part with arbitrary nucleotide sequence in second aspect present invention.
In another preference, described antisense sequences and element (a), (b) or (c) complete complementary.
In a fifth aspect of the present invention, it is provided that the purposes of the antisense sequences described in fourth aspect present invention, they are used to controlling chlorophyll synthesis, or for preparing the preparation of controlling chlorophyll.
In another preference, described regulation and control are to promote chlorophyllous synthesis, or improve the content of plant Determination of Chlorophyll.
In a sixth aspect of the present invention, provide the purposes of a kind of inhibitor or accelerator, described inhibitor or accelerator are inhibitor or the accelerator of the protein-nucleic acid complex described in first aspect present invention, and described inhibitor or accelerator are used to synthesize for controlling chlorophyll, or for preparing the preparation of controlling chlorophyll.
In another preference, the nucleic acid fragment of the described-500nt that inhibitor is PORC gene～-438nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1199-1260 position.
In another preference, described inhibitor is SCL27 albumen, and described regulation and control are to suppress chlorophyllous synthesis or reduce the content of plant Determination of Chlorophyll.
In another preference, described accelerator is miR171, and described regulation and control are to promote chlorophyllous synthesis or improve the content of plant Determination of Chlorophyll.
In a seventh aspect of the present invention, it is provided that a kind of method improveing plant, described improvement includes promoting chlorophyllous synthesis, and wherein, described method includes step: reduce the quantity of complex described in described plant first aspect present invention.
In another preference, described method includes the quantity by improving miR171, thus reducing the quantity of described complex.
In another preference, described plant includes crops (such as Oryza sativa L., Semen Tritici aestivi) and model plant (such as arabidopsis).
In a eighth aspect of the present invention, it is provided that a kind of determine that whether test substances is promote or suppress the accelerator of complex described in first aspect present invention or the method for inhibitor, it is characterised in that described method includes step:
I () is at matched group, when suitably forming in described complex, the specific region of SCL27 albumen or the promoter region of its active fragment and PORC gene is hatched, thus forming the complex of the present invention, measure the quantity of described complex, be designated as C0；And in test group, under the described condition identical with matched group, when with the addition of test substances, measuring the quantity of complex of the present invention in described test group, being designated as C1；
Wherein, described specific region is selected from lower group:
A the nucleic acid fragment II of-788nt of () PORC gene to-598nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 911-1100 position；
B the nucleic acid fragment III of-572nt of () PORC gene to-372nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1127-1326 position；
C the nucleic acid fragment W of-500nt～-438nt of () PORC gene, in the sequence of this fragment such as SEQIDNO.:1 shown in 1199-1260 position；
The total length promoter region of (d) POC；
(ii) compare described C0 and C1, if C1 is significantly higher than C0, then show that described test substances is the accelerator promoting described complex to be formed；If if C1 is substantially less than C0, then show that described test substances is the inhibitor suppressing described complex to be formed.
Preferably, described " being significantly higher than " C1/C0 >=1.5 are referred to, it is preferred that >=2.0, more preferably >=3.0.
Preferably, described " being significantly higher than " C1/C0≤2/3 is referred to, it is preferred that≤1/2, more preferably≤1/3.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus constituting new or preferred technical scheme.As space is limited, tired no longer one by one state at this.
Accompanying drawing explanation
Fig. 1 shows that the SCL albumen of miR171 targeting passes through the synthesis of POR controlling chlorophyll under light.
Wherein, Fig. 1 (a) shows that wild type (Col), MIR171c-OX, scl6scl22scl27 and 35S::LUC-rSCL27 plant grow the phenotype of 25 days under long-day conditions.Bars=1cm.
Fig. 1 (b) shows the chlorophyll content corresponding to each plant leaf in (a) figure.
Fig. 1 (c) shows the activity (Fv/Fm) of plant Photosystem I I after high light (800 μm of olm-2s-1) processes and dark is recovered.
Fig. 1 (d) shows the transcriptional level of HEMA1, GUN4, GUN5, PORC and CAO gene in each mutant of Northern hybridization analysis.
Fig. 1 (e) shows immunoblotting analysis detection POR protein level in each mutant.
Fig. 1 (f) shows the expression lowering POR gene with artificial microRNA technology (por-amiR and por-amiR/MIR171c-OX) respectively in wild type and MIR171c-OX background.Figure is the growth plant phenotype of 22 days.Bars=1cm.
Fig. 1 (g) shows the chlorophyll content of each genotype corresponding to Fig. 1 (f).
Fig. 1 (h) shows the expression lowering POR gene by artificial microRNA technology in scl6scl22scl27 background, causes that blade turns yellow.Bars=1cm.
Fig. 1 (i) shows the chlorophyll content of each genotype corresponding to Fig. 1 (h).
Fig. 2 shows the SCL27 promoter region directly in conjunction with PORC gene.
Wherein, Fig. 2 (a) shows the relative activity of the PORC promoter region (pPORC-1685, pPORC-861, pPORC-455) of three sections of different lengths.The LUC reporter gene driven by pPORC-1685, pPORC-861, pPORC-455 in tobacco leaf with 6xMYC-rSCL27 or unloaded corotation.The relative activity of LUC and endogenous control 35S::REN weigh after being standardized.
Fig. 2 (b) shows the schematic diagram of PORC promoter and gene thereof.DNA fragmentation I (-1,524bp to-1,324bp), Ι Ι (-778bp arrive-598bp), Ι Ι Ι (-572bp arrives-372bp) test for ChIP, and the DNA fragmentation IV of coding region (1,144bp to 1,246bp) then as negative control.
Fig. 2 (c) shows the relative abundance of each DNA fragmentation in ChIP precipitation.
Fig. 2 (d) shows that EMSA experiment confirms that SCL27 is attached to fragment II and the III of PORC promoter region.
Fig. 2 (e) shows the mutant fragments (M) of DNA fragmentation (W) and the correspondence thereof comprising GT element.
Fig. 2 (f) shows that EMSA experiment confirms that SCL27 can not in conjunction with GT element (M) fragment in conjunction with GT element (W) fragment.
Fig. 2 (g) show when add unlabelled GT element (W) time can competition binding SCL27 albumen, when addition unlabelled GT element (M) time then can not there is competition binding.
Fig. 3 shows the sequence (SEQIDNO.:1) of PORC promoter region, and wherein the region underscore containing 4 GT elements of core indicates.
Detailed description of the invention
The present inventor through extensive and deep research, it was unexpectedly found that, plant exists a kind of SCL27:PORC complex.Utilizing this complex, the overexpression of the effective PORC gene of the present inventor's first passage or silence regulate the character such as content and the leaf color of plant chlorophyll, thus reaching control the dim light of night or regulate the purpose of photosynthesis of plant efficiency.Complete the present invention on this basis.
As used herein, term " complex of the present invention ", " SCL27-PORC complex ", " protein-nucleic acid complex of the present invention " or " SCL27 albumen-PORC gene composite " are used interchangeably, and refer both to SCL27 albumen (or its active fragment) and are incorporated into some specific region of promoter region of PORC gene and the complex that formed.The research of the present invention shows, this complex plays central role in controlling chlorophyll synthesizes.
PORC gene and promoter thereof
In the protein-nucleic acid complex of the present invention, a kind of core parts are the promoter regions of PORC gene.
The sequence of PORC gene can referring to such as public database.
The promoter sequence of a kind of typical PORC gene is such as shown in SEQIDNO.:1.This promoter region is corresponding to the nucleotide sequence of-1698nt to-1nt of PORC upstream region of gene.
SCL27 albumen
In the protein-nucleic acid complex of the present invention, another kind of core parts are SCL27 albumen.
As used herein, term " SCL27 albumen " refers to a distinctive transcription factor of class plant, belongs to GRAS (GAI, RGA, SCR) protein family.The research of the present invention shows, miR171 can targeting SCL27 transcription factor.
The aminoacid sequence of a kind of typical SCL27 is as follows:
MPLSFERFQGEGVFGLSSSSFYSDSQKIWSNQDKTEAKQEDLGYVVGGFLPEPTSVLDALRSPSPLASYSSTTTTLSSSHGGGGTTVTNTTVTAGDDNNNNKCSQMGLDDLDGVLSASSPGQEQSILRLIMDPGSAFGVFDPGFGFGSGSGPVSAPVSDNSNLLCNFPFQEITNPAEALINPSNHCLFYNPPLSPPAKRFNSGSLHQPVFPLSDPDPGHDPVRRQHQFQFPFYHNNQQQQFPSSSSSTAVAMVPVPSPGMAGDDQSVIIEQLFNAAELIGTTGNNNGDHTVLAQGILARLNHHLNTSSNHKSPFQRAASHIAEALLSLIHNESSPPLITPENLILRIAAYRSFSETSPFLQFVNFTANQSILESCNESGFDRIHIIDFDVGYGGQWSSLMQELASGVGGRRRNRASSLKLTVFAPPPSTVSDEFELRFTEENLKTFAGEVKIPFEIELLSVELLLNPAYWPLSLRSSEKEAIAVNLPVNSVASGYLPLILRFLKQLSPNIVVCSDRGCDRNDAPFPNAVIHSLQYHTSLLESLDANQNQDDSSIERFWVQPSIEKLLMKRHRWIERSPPWRILFTQCGFSPASLSQMAEAQAECLLQRNPVRGFHVEKRQSSLVMCWQRKELVTVSAWKC(SEQIDNO.:2)。
Should be understood that in the present invention, SCL27 albumen not only includes the SCL27 albumen from arabidopsis or Oryza sativa L., also includes deriving from other different homologous proteins with chlorophyllous plant.In addition, this SCL27 albumen not only includes wild-type protein, but also include its mutein or active fragment, as long as described mutein or active fragment still retain promoter region (especially fragment II, fragment III and the fragment A) binding ability with PORC gene and then the regulation and control function of PORC gene expression or ability.
As having chlorophyllous plant, representational example includes: monocotyledon, dicotyledon and other rudimentary plants.Preferred plant example (but being not limited to): crops (such as Oryza sativa L., Semen Tritici aestivi, Semen Maydis), flowers, trees and model plant (such as arabidopsis) etc..
Protein-nucleic acid complex
In the present invention, provide a kind of SCL27-PORC complex (also referred to as " complex of the present invention "), this complex is that SCL27 albumen (or its active fragment) is incorporated into some specific region of the promoter region of PORC gene and is formed, and these specific regions include (but being not limited to):
A the nucleic acid fragment II of ()-788nt to-598nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 911-1100 position；
B the nucleic acid fragment III of ()-572nt to-372nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1127-1326 position；
C the nucleic acid fragment of ()-500nt～-438nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1199-1260 position；
The total length promoter region of (d) POC.
Utilizing the complex of the present invention, reversibly regulation and control in being possible not only in plant (include promoting or suppressing) chlorophyllous synthesis, it is also possible to screening (a) efficiently promotes the accelerator that described complex is formed in vitro；(b) inhibitor that described complex is formed is suppressed.
Typically, a kind of typically determine that whether test substances is promote or suppress the accelerator of complex of the present invention or the method for inhibitor to include step:
A () is at matched group, when suitably forming in complex of the present invention, the above-mentioned specific region of SCL27 albumen (or its active fragment) Yu the promoter region of PORC gene is hatched, thus forming the complex of the present invention, measure the quantity of described complex, be designated as C0；And in test group, under the described condition identical with matched group, when with the addition of test substances, measuring the quantity of complex of the present invention in described test group, being designated as C1；
B () be described C0 and C1 relatively, if C1 is significantly higher than C0, then show that described test substances is the accelerator promoting described complex to form (or preventing described complex dissociation)；If if C1 is substantially less than C0, then show that described test substances is the inhibitor suppressing described complex to form (or promoting described complex dissociation).
Generally, described " being significantly higher than " C1/C0 >=1.5 are referred to, it is preferred that >=2.0, more preferably >=3.0.
Generally, described " being significantly higher than " C1/C0≤2/3 is referred to, it is preferred that≤1/2, more preferably≤1/3.
Preferably, described test substances includes (but being not limited to): micromolecular compound, hormone, albumen, saccharide, nucleic acid etc..
Polynucleotide construction
According to miRNA sequence provided by the present invention (i.e. miR171), the polynucleotide construction that can be processed to affect the miRNA of corresponding mrna expression after being imported into can be designed, namely described polynucleotide construction can raise the amount of corresponding miRNA in vivo.Therefore, the invention provides the polynucleotide of a kind of separation (construction), described polynucleotide (construction) can be become precursor miRNA by people's cell transcription, and described precursor miRNA can be sheared and be expressed as described miRNA by host cell.
As a kind of optimal way of the present invention, described polynucleotide construction contains the structure shown in Formula II:
SeqForward-X-SeqReversely
Formula II
In Formula II,
SeqForwardFor the nucleotide sequence of described miR171, Seq can be expressed as in cellReverselyFor with SeqForwardThe nucleotide sequence being substantially complementary；Or, SeqReverselyFor the nucleotide sequence of described miRNA, Seq can be expressed as in cellForwardFor with SeqForwardThe nucleotide sequence being substantially complementary；
X is for being positioned at SeqForwardAnd SeqReverselyBetween intervening sequence, and described intervening sequence and SeqForwardAnd SeqReverselyNot complementary；
Structure shown in Formulas I, after proceeding to host cell, forms the secondary structure shown in formula III:
Formula III
In formula III, SeqForward、SeqReverselyAs defined above with X is stated；
| | represent at SeqForwardAnd SeqReverselyBetween formed base pair complementarity relation.
Generally, described polynucleotide construction is positioned on expression vector.Therefore, present invention additionally comprises a kind of carrier, it contains described miRNA or described polynucleotide construction.Described expression vector is generally possibly together with promoter, origin of replication and/or marker gene etc..Method well-known to those having ordinary skill in the art can be used for building expression vector required for the present invention.These methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..Described expression vector preferably comprises one or more selected marker, to provide the phenotypic character of the host cell for selecting conversion, such as kalamycin, gentamycin, hygromycin, amicillin resistance.
The antisense nucleic acid molecule of miR171
Present invention also offers a kind of antisense nucleic acid molecule for miR171, described antisense nucleic acid molecule contestable ground suppresses the effect of miR171, by building the expression vector containing this antisense nucleic acid molecule, antisense nucleic acid molecule is carried out overexpression, thus the effect of miR171 is disturbed.
Those of ordinary skill in the art can use general method to build the overexpression vector of described antisense nucleic acid molecule.
The present invention has a major advantage in that:
A () provides the means of a kind of effectively and easily controlling chlorophyll synthesis first.
B () utilizes " protein-nucleic acid complex " that present invention firstly discovers that, be possible not only to conveniently and effectively just regulate and control (promotion) chlorophyllous synthesis, and can conveniently and effectively negative regulation (suppression) chlorophyllous synthesis.
C () utilizes " protein-nucleic acid complex " that find first of the present invention, it is possible to the reversibly synthesis of controlling chlorophyll.
D () utilizes " protein-nucleic acid complex " that find first of the present invention, it is convenient to screening promotes or suppresses compound or other materials of Chlorophyll synthesis in vitro.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally conventionally condition, such as Sambrook et al., molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to manufacturer it is proposed that condition.Unless otherwise indicated, otherwise percentage ratio and number are percentage by weight and parts by weight.
Material
In embodiment, the primer is as shown in table 1 below:
Table 1
Method:
Vegetable material and growth conditions
MIR171c-OX and 35S::LUC-rSCL27 is the environmental background of arabidopsis Colombia (Columbia)；Scl6, scl22, scl27 Trimutant is obtained by scl6 (Ler background), scl27 (Ws background) and scl22 (Col background) hybridizing method；Trimutant and Col backcrossed for four generations afterwards；Por-amiR/MIR171c-OX plant is by obtaining por-amiR and MIR171c-OX hybridization；All of seed all germinates in the 1/2MS culture medium containing 1% sucrose, and plant grows under long-day conditions, and temperature is 21 DEG C, and intensity of illumination is 110 μm of ol m-2S-1。
Plasmid construction and Plant Transformation
PORC promoter region 1.7kb fragment expands from wild type gene group DNA, then passes through enzyme action connection and is cloned into pGREEN0800LUC carrier, builds and produce pPORC-1685::LUC carrier.
PORC upstream from start codon 861bp and 455bp fragment are also building up to pGREEN0800LUC carrier by same method.In order to build PORamiRNA, the amiRNA targeting sequence of PORC gene is by WMD3 website (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi) prediction, amiRNA precursor fragment passes through round pcr from pRS300 (purchased from T ü bingenUniversity, Germany) template amplification obtains, and with gateway (invitrogen) system constructing to plant expression vector pGWB2 (invitrogen).In order to carry out external protein-DNA binding analysis, SCL27 is cloned into pET28b carrier and converts expression strain BL21.
Quantitative fluorescent PCR
Quantitative PCR analysis SYBR-GreenPCRMastermix (Takara) carries out, and its amplification procedure is monitored PCR system record (AppliedBiosystems) by fluorescent quantitation.
ChIP analyzes
The 6xMYC-rSCL27-OX transfer-gen plant about 3 grams growing three weeks is fixed, ultrasonic and be resuspended in extracting solution and be divided into three parts.A copy of it compares as applied sample amount, and second part adds MYC-agarose (Sigma) and precipitate, and the 3rd part adds HA-agarose as negative control.The DNA sample PCR purification kit (Qiagen) obtained is purified, and the concentration of corresponding DNA fragments is analyzed by quantitative PCR.
EMSA.
The SCL27 albumen of fluorescently-labeled for Cy5 DNA Yu purification is hatched 20 minutes in 30 DEG C in binding soln, then with 6% acrylamide native glue electrophoresis 1-2h (electrophoresis liquid is 5xTris-borate-EDTA) when 4 DEG C of 100V. after electrophoresis, the DNA fluorescence signal StarionFLA-9000 on glue detects.
Result
The SCL albumen of miR171 targeting regulates chlorophyllous synthesis by POR under light
Consistent with report before, MIR171c is excessive is all bottle-green with scl6scl22scl27 Trimutant blade, and its chlorophyll content is higher than about wild type 40%.And the overexpression not rotaring gene plant blade of LUC-rSCL27 luciferase fusion protein by miR171 regulation and control turns yellow, chlorophyll content significantly reduces.The SCL albumen of these results prompting miR171 targeting is the negative growth factor of Chlorophyll synthesis.Then the present inventor is by detecting the maximal photochemistry efficiency Fv/Fm of reaction Photosystem I I activity, have evaluated the SCL albumen function when plant strong ligh stress.Compared to wild type, the Photosystem I I activity decrease of MIR171c-OX and scl6scl22scl27 plant slow, the Photosystem I I activity of LUC-rSCL27-OX plant then declines faster, the therefore SCL albumen involved in plant of the miR171 targeting adaptability to high light.Because Chlorophyll synthesis approach is subject to different regulation and control under dark and illumination condition, first the present inventor have detected the function at dark lower SCL albumen.Ironically, wild type compared to MIR171c-OX, scl6, scl22, scl27 or LUC-rSCL27-OX plant on the content of protochlorophyllide and the structure of prolamellar body all without significant difference.To sum up, inventor thinks that SCL albumen mainly participates in the suppression of Chlorophyll synthesis under light.
In order to prove whether SCL albumen directly participates in the regulation and control of Chlorophyll synthesis approach, the present inventor have detected the transcriptional level that some genes participating in Chlorophyll synthesis committed step include HEMA1, GUN4, GUN5, PORC and CAO.Northern hybridization and quantitative PCR analysis show, in these genes, only the expression of PORC and CAO raises in MIR171c-OX and scl6scl22scl27 plant and reduces in LUC-rSCL27-OX plant.This expression pattern of PORC and CAO and the chlorophyll content in MIR171c-OX, scl6, scl22, scl27 and LUC-rSCL27-OX plant are identical, and the expression of prompting PORC and CAO is affected by SCL protein regulation.And the expression pattern of GUN4, GUN5 and HEMA1 is uncorrelated with the transcriptional level of SCL.Immunoblot experiment display MIR171c-OX and scl6scl22scl27 accumulates higher levels of PORC and PORB albumen compared to wild type and LUC-rSCL27-OX plant.Because CAO is a key enzyme controlling chlorophyll b synthesis, if CAO is also a major target of SCL, then the value of chlorophyll a/b should be able to change along with the expression of SCL.But, not having significant difference at wild type, MIR171c-OX, scl6scl22scl27 and LUC-rSCL27-OX plant Determination of Chlorophyll a/b, prompting CAO activity is not exposed to the transcriptional control of SCL.Therefore test result indicate that of the present invention, in Chlorophyll synthesis approach, POR is a key enzyme by SCL protein regulation.
Whether the Chlorophyll synthesis in order to determine SCL protein regulation is mediated by POR, utilizes artificial microRNA technology to lower the expression of POR in wild type, MIR171c-OX and scl6scl22scl27.In these plant, the downward of POR expression causes the phenotype of young leaves pale green and being remarkably decreased of chlorophyll content.These results all confirm that POR plays an important role in the SCL Chlorophyll synthesis approach regulated and controled.
SCL27 is incorporated into PORC promoter
The result of the test of the present invention shows that expressing of POR is closely related with the expression of SCL, and this promotes to it is considered as desirable by the inventor to whether SCL can directly control the promoter activity of POR gene.
In order to detect whether this hypothesis is set up, LUC and the 6xMYC-rSCL27 that POR promoter merges is used Transient Expression System cotransformation by the present inventor in Nicotiana tabacum L..The expression of LUC declines a lot when with 6xMYC-rSCL27 corotation, and prompting PORC promoter region is the direct action target of SCL27.
In order to identify the SCL27 PORC promoter region combined, the present inventor is by the fragment of three different lengths of PORC promoter region (-1685bp ,-861bp and-455bp) from promoter to upstream and LUC reporter gene fusion, the expression of the LUC that pPORC-1685 and pPORC-861 drives can significantly inhibit when 6xMYC-rSCL27 corotation, and the expression of the LUC that pPORC-455 drives is relatively low, and not by the impact of 6xMYC-rSCL27.The cis acting element of SCL27 is contained in these results prompting region between POR promoter region-861 to-455.
The present inventor has carried out chromatin immune co-precipitation (ChIP) experiment further and quantitative PCR analysis reduces the binding site of SCL27 further.
Experimental result shows, in 6xMYC-rSCL27 transfer-gen plant, fragment II (-788bp arrives-598bp) and fragment III (-572bp arrives-372bp) is enriched with in co-immunoprecipitation, and the fragment IV (1 of fragment I (-1524bp arrives-1324bp) and coding region, 144bp to 1,246bp) not enrichment, prompting fragment II and III all contains the SCL cis acting element combined.The present inventor in be by EMSA experiment detect whether that SCL27 can be bonded directly to the fragment II and fragment III of PORC promoter region.Consistent with CHIP-qPCR result, when SCL27 recombiant protein and DNA fragmentation II and III can detect the band of migration after hatching, and the intensity being with strengthens gradually along with the increase of SCL protein concentration, when SCL27 and fragment I is then not detected by after hatching migrating.In a word, the experiment of the vivo and vitro of the present invention all shows that SCL27 can directly in conjunction with PORC promoter, and the expression of suppressor gene.
Bioinformatic analysis shows, fragment II and fragment III comprises cis acting element G (A/G) (A/T) AA (A/T) consistent with GT1 land, is called GT element.GT element permitted polygenic promoter region widely distributed.In order to detect whether that SCL27 is directly in conjunction with GT element, the present inventor have selected GT element mutant fragments (M) of the DNA fragmentation (W) and correspondence that are positioned at the 62bp length that-500bp to-438bp region comprises three GT element repetitive structure territories and carries out EMSA experiment, result display SCL recombiant protein can be attached to W fragment, but not can be incorporated into M fragment, and the combination of SCL27-DNA can be suppressed by excessive unmarked W fragment, and M fragment does not then act on.
The above results shows, SCL27 regulates and controls the expression of PORC by being directly combined to the specific GT element (especially the GT element in nucleic acid fragment A) of PORC gene promoter.
In addition, test shows, one GT element is not enough to be effectively formed the complex of the present invention, and 4 the GT elements being numbered 8-11 in following table are maximally effective SCL27 calmodulin binding domain CaMs (i.e. 1199-1260 position in SEQIDNO.:1) in PORC gene promoter region, it also it is the region to PORC expression regulation best results.
GT element in table PORC promoter
Discuss
Research shows, transcribing of POR gene largely take part in Chlorophyll synthesis.There is three POR gene: PORA (expression is the highest in etiolated seedling, degrades under light), PORB (light and all expression under dark) and PORC (expressing under illumination condition) in arabidopsis gene group.Research shows, PORA and PORB transcribes the regulation and control being limited primarily by EIN3 and its homologous protein EIL1, and the two transcription factor directly in conjunction with the promoter of PORA and PORB, can be referred to as EBS site.And the forward being subject to transcription factor PIF1 of transcribing of PORC regulates.But, based on these interaction mechanisms, Chlorophyll synthesis is regulated and controled, be but difficult to obtain gratifying regulating effect.
Although existing research display: miR171 action target SCL6/22/27 participates in the many important biological process of regulation and control plant, but the molecular mechanism of its effect is still known little about it.For this, the research of the present inventor proves: the expression of miR171 controllable SCL, and SCL plays central role in the molecular mechanism of controlling chlorophyll route of synthesis.SCL sudden change causes that under light, growing plants chlorophyll content raises (Fig. 1).Further study showed that, SCL albumen is by G (A/G) (A/T) AA (A/T) the GT cis element in conjunction with photoresponse gene PORC promoter region, thus the expression (Fig. 2) of the key gene of Effective Regulation coding protochlorophyllide oxidoreductase.
The all documents mentioned in the present invention are incorporated as reference all in this application, are individually recited as reference such just as each section of document.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally.
Claims (10)
1. a protein-nucleic acid complex, it is characterised in that described complex is SCL27 protein binding being formed selected from the sequence of lower group in PORC gene:
A the nucleic acid fragment II of ()-788nt to-598nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 911-1100 position；
B the nucleic acid fragment III of ()-572nt to-372nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1127-1326 position；
C the nucleic acid fragment W of ()-500nt～-438nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1199-1260 position；
The total length promoter region of (d) POC.
2. the nucleotide sequence separated, it is characterised in that described nucleotide sequence is selected from lower group:
A the nucleic acid fragment II of-788nt of () PORC gene to-598nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 911-1100 position；
B the nucleic acid fragment III of-572nt of () PORC gene to-372nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1127-1326 position；
C the nucleic acid fragment W of-500nt～-438nt of () PORC gene, in the sequence of this fragment such as SEQIDNO.:1 shown in 1199-1260 position.
3. the purposes of nucleotide sequence as claimed in claim 2, it is characterised in that for preparing the complex described in claim 1.
4. the purposes of the protein-nucleic acid complex described in a claim 1 or the nucleotide sequence described in claim 2, it is characterised in that synthesize for controlling chlorophyll, or for preparing the preparation of controlling chlorophyll.
5. an antisense sequences, it is characterised in that described antisense sequences is complementary wholly or in part with arbitrary nucleotide sequence in claim 2.
6. the purposes of the antisense sequences described in a claim 5, it is characterised in that synthesize for controlling chlorophyll, or for preparing the preparation of controlling chlorophyll.
7. the purposes of an inhibitor or accelerator, it is characterized in that, described inhibitor or accelerator are inhibitor or the accelerator of the protein-nucleic acid complex described in claim 1, and described inhibitor or accelerator are used to synthesize for controlling chlorophyll, or for preparing the preparation of controlling chlorophyll；
It is preferred that the nucleic acid fragment of the described-500nt that inhibitor is PORC gene～-438nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1199-1260 position.
8. purposes as claimed in claim 7, it is characterised in that described inhibitor is SCL27 albumen, and described regulation and control are to suppress chlorophyllous synthesis or reduce the content of plant Determination of Chlorophyll；Or
Described accelerator is miR171, and described regulation and control are to promote chlorophyllous synthesis or improve the content of plant Determination of Chlorophyll.
9. the method improveing plant, it is characterised in that described improvement includes promoting chlorophyllous synthesis, wherein, described method includes step: reduce the quantity of complex described in claim 1 in described plant.
10. determine whether test substances is promote or suppress the accelerator of complex described in claim 1 or the method for inhibitor for one kind, it is characterised in that described method includes step:
I () is at matched group, when suitably forming in described complex, the specific region of SCL27 albumen or the promoter region of its active fragment and PORC gene is hatched, thus forming the complex of the present invention, measure the quantity of described complex, be designated as C0；And in test group, under the described condition identical with matched group, when with the addition of test substances, measuring the quantity of complex of the present invention in described test group, being designated as C1；
Wherein, described specific region is selected from lower group:
A the nucleic acid fragment II of-788nt of () PORC gene to-598nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 911-1100 position；
B the nucleic acid fragment III of-572nt of () PORC gene to-372nt, in the sequence of this fragment such as SEQIDNO.:1 shown in 1127-1326 position；
C the nucleic acid fragment W of-500nt～-438nt of () PORC gene, in the sequence of this fragment such as SEQIDNO.:1 shown in 1199-1260 position；
The total length promoter region of (d) POC；
(ii) compare described C0 and C1, if C1 is significantly higher than C0, then show that described test substances is the accelerator promoting described complex to be formed；If if C1 is substantially less than C0, then show that described test substances is the inhibitor suppressing described complex to be formed.
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| WO2020108620A1 (en) * | 2018-11-29 | 2020-06-04 | 中国科学院分子植物科学卓越创新中心 | Target gene for improving regeneration ability of plants, regulatory molecule and application thereof |
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| CN103160541A (en) * | 2011-12-09 | 2013-06-19 | 中国科学院上海生命科学研究院 | Transcription factor for regulating physical and chemical properties of plant seed starch |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020108620A1 (en) * | 2018-11-29 | 2020-06-04 | 中国科学院分子植物科学卓越创新中心 | Target gene for improving regeneration ability of plants, regulatory molecule and application thereof |
| CN112522281A (en) * | 2020-12-07 | 2021-03-19 | 上海师范大学 | Gene PeGRAS sequence for regulating and controlling petal development of phalaenopsis miniata and application thereof |
| CN112522281B (en) * | 2020-12-07 | 2022-11-11 | 上海师范大学 | Gene PeGRAS sequence for regulating and controlling petal development of phalaenopsis miniata and application thereof |
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