CN105463011B - One plant growth negative regulation cis element SE1 and its regulation plant strain senior middle school application - Google Patents

Abstract

The invention belongs to gene engineering technology fields, specifically disclose a plant growth negative regulation cis elementSE1And its application in regulation plant strain senior middle school.The present invention utilizes the dwarf plant of rice T-DNA insertion, expands flanking sequence discovery by TAIL-PCR, T-DNA insertion is located atEui1At gene intron.eui1It is one of the four big genetic stocks in the rice breeding of heterosis utilization.The present invention, which passes through, utilizes the carrying small missing of introneEui1Genetic transformationeui1The function complementation experiment of mutant,Eui1The sub-segments that include of gene have determined oneEui1The silencer of geneSE1, obtain it is novel withEui1Relevant dwarf plant, this showsSE1Element is used for breeding of hybridized rice in control Plant Height of Rice, to building ideotype, and improving has preferable application potential on the genetic improvement of rice yield.

Description

One plant growth negative regulation cis elementSE1And its in regulation plant strain senior middle school Using

Technical field

The present invention relates to gene engineering technology fields, more particularly, to a plant growth negative regulation cis elementSE1 And its application in regulation plant strain senior middle school.

Background technique

Plant plant height is can to influence one of important agronomic traits of crop yield, for the first time " green revolution " be exactly On the basis of semi-short-stalked breeding, so that the output doubled for rice.Using male-sterile character as the heterosis utilization of core Breeding technique has then started second " green revolution " of tide, and acquirement is made us looking steadily on the crops such as corn, rice, rape Purpose achievement." packet neck " phenomenon that the generally existing tassel of male sterible series of rice can't pump out, brings centainly to hybrid rice breeding Difficulty.Meanwhile to further increase rice yield, breeders propose the concept of ideal strain breeding.For example, plant height is answered Control requires stem hard in 90~100 cm, lower or medium tillering ability, without ineffective tillering etc..And changed by heredity Good suitable control plant height is the importance of ideal strain breeding.Therefore, study and disclose the molecular regulation mechanism of plant height development The understanding built up to plant plant type can not only be enhanced, and effective practice significance can be provided for Instructing manufacture breeding.

Currently, plant plant height is mainly adjusted by relational approaches such as hormone such as gibberellin (GA), brassinosteroids (BR) Control.The relevant gene of two GA that plant type of rice breeding is had an important influence: Semi-dwarf geneSd1With long-neck fringe geneEui1 It has cloned and has delivered.

Rice Semi-dwarf geneSd1It is caused by the afunction of GA20 oxidase gene in GA biosynthesis pathway 's.?sd1The content of plant, GA53 increases, and the decline of the content of GA20 and GA1 causes plant height to become short.Rice long-neck fringe geneEui1Codocyte cytochrome p 450 mainly expresses in small ear, bioactivity GA4 can be made to lose activity.Eui1The mutation of gene Afterwards,eui1Coding albumen cannot inactivate GA4, and the content of GA4 is caused largely to be accumulated in most upper section elongated portion, to promote topmost The cell of internode is grown, and is formedeui1The phenotype of long-neck fringe.The genetic stocks, which becomes in hybrid rice breeding, solves the problems, such as " packet neck " Important means.In the adjustment mechanism of gene, have cis element (cisElement) and trans factors (trans Factor) two kinds.Cis element refers to the DNA sequence dna in nucleotide sequence with regulatory function, and trans factors are mostly Protein transcription factor.Cis-acting elements is usually the binding site of transcription regulaton factor, including promoter, enhancer and silencing Son.However, not yet discovery pair at presentEui1Gene is had negative regulation effect cis element (silencer) and is improved using silencer The method of Plant Height of Rice.

Summary of the invention

The technical problem to be solved by the present invention is to overcome drawbacks described above existing in the prior art, it is raw to provide a kind of plant Long negative regulation cis elementSE1In the application of regulation plant strain senior middle school.

A second object of the present invention is to provide a kind of methods of regulation plant plant height.

The purpose of the present invention is what is be achieved by the following technical programs:

Negative regulation cis element described in SEQ ID NO:1SE1In the application of regulation plant strain senior middle school.

The present invention has determined firstEui1The negative regulation cis element silencer of one 34 bp of geneSE1, pass through conversion The Overexpression vector for lacking the silencer element can control plant height.

Inventor has found a Dwarf Mutant during tissue cultures.It is analyzed by genetic analysis and TAIL-PCR It expands flanking sequence discovery: the mutant is replaced since T-DNA is inserted intoEui1One 135 bp of gene intron front end Segment and the dominant mutant of single site formed, and it is named as dwarf ofEui1(referred to asdEui1), inventor passes through Building is lacked for 2 carrying introne small fragments of the 135 bp sectionEui1Have complementary functions carrier Δ NS1 and Δ NS2, For converting long-neck fringe gene mutation bodyeui1.It was found that the Δ NS1 complementation conversion physical efficiency with small fragment missing willeui1Strain Height is restored to wild-type plant height；And have 34 bp sections (SEQ ID NO:1, sequence ACATCACTAGCTACATGCA TGATGCTTGCTTGCT, underscore section be RY element) missing Δ NS2 complementation transformant then become than wild-type plant more Add short and small, reproducingdEui1Phenotype.As a result, we determined that 34 bp containing a conservative RY element (CATGCATG) Section is that the cis- silencer element with regulatory function is named as Silencer ofEui1,SE1.AlthoughSE1Sequence AsEui1A part of introne has been reported, but its as the cis- silencer element of negative regulation biological function not by It was found that.

Preferably, a height of plant for making normal plant height of regulation plant strain generates Dwarfing phenotypes.

Preferably, the plant is rice.

It is building to the negative regulation cis element the present invention also provides a kind of method of regulation plant plant heightSE1It carries out The carrier of inside missing, and convert long-neck fringe mutanteui1Plant or normal plants obtain genetically modified plants, from transgenosis It is filtered out in plant and long-neck fringe mutanteui1Plant or normal plants are compared, and generate the genetically modified plants of Dwarfing phenotypes i.e. It can.

Specifically, the above method the following steps are included:

S1. cis element containing negative regulation is constructedSE1MissingEui1Complementing vector, and convert and (turned by mediated by agriculture bacillus Change) long-neck fringe mutanteui1Plant obtains genetically modified plants；

S2. it screens and obtains and long-neck fringe mutant from the obtained genetically modified plants of S1eui1Plant is compared, and generates short Change the genetically modified plants of phenotype.

In addition, the above method can with the following steps are included:

S1. it constructs without introneEui1The coding Sequence Transformed carrier of CDS (Eui1- cDNA), and convert and (pass through agriculture Bacillus mediated transformation) normal plants obtain genetically modified plants；

S2. it screens and is obtained compared with normal plants from the obtained genetically modified plants of S1, generate Dwarfing phenotypes turns base Because of plant.

It should be noted that internal missing negative regulation cis element of the present inventionSE1Carrier can be equally loaded into Deleted carrier is imported purpose plant with any method for transformation, to obtain short by other any plant conversion carriers The transformed variety of change.

Preferably, in the above method, the plant is rice.

Compared with prior art, the invention has the following advantages:

The present invention provides a plant growth negative regulation cis elementsSE1It is benefit in the application of regulation plant strain senior middle school The dwarf plant being inserted into a rice T-DNA expands flanking sequence discovery by TAIL-PCR, and T-DNA insertion is located atEui1At gene intron.By rightEui1Inside deleting genetic conversion and function complementation experiment,Eui1Gene includes Sub-segments have determined oneEui1The silencer of geneSE1 (silencer ofEui1), obtain it is novel withEui1It is related Dwarf plant, this showsSE1Element is used for breeding of hybridized rice in control Plant Height of Rice, to building ideotype, improves water There is preferable application potential on the genetic improvement of rice yield.

Detailed description of the invention

Fig. 1 is Dwarf MutantdEui1Phenotype and T-DNA be inserted into schematic diagram；Wherein, Tu1AZhongEui1It is that nonfunctional is prominent Varianteui1, WT is wild type ZH11；dEui1It is the plant phenotype of ZH11 T-DNA insertion Dwarf Mutant；Figure 1B is T-DNA Position in genome andEui1Structural schematic diagram, wherein P1, P2, P3 are the primers for genotype identification；Fig. 1 C is short Change the T of mutant₁The genotyping of generation individual, w indicate wild type, and h indicates heterozygous, and d indicates to downgrade pure and mild type；Fig. 1 D isEui1Expression of the gene in mutant different tissues organ, L indicate leaf；S indicates stem；R indicates root；Scale represents 25 in figure Cm height.

Fig. 2 is genetic transformation and the functional verification for carrying the complementing vector of introne small fragment missing；Wherein Fig. 2A is to take Complementing vector structure with introne small fragment missing, Δ NS1 areEui1The complementation of genomic deletion introne 3 3-bp small fragment Carrier；Δ NS2 isEui1The complementing vector of genomic deletion introne 3 4-bp small fragment；Underscore is shown containing conservative RY element (CATGCATG)；Fig. 2 B, Fig. 2 C are Δ NS1 and Δ NS2 conversion long-neck fringe mutant respectivelyeui1T afterwards₀Generation conversion is planted Object hygromycin geneHptQualification result；Fig. 2 D, Fig. 2 E are Δ NS1 and Δ NS2 conversion long-neck fringe mutant respectivelyeui1Afterwards T₀The phenotype of generation conversion plant.Δ NS1 conversioneui1Plant recovery is normal plant height afterwards；Δ NS2 conversioneui1Plant performance afterwards To downgrade, reappeardEui1Plant phenotype；Fig. 2 F, Fig. 2 G are Δ NS1 or Δ NS2 conversionseui1Afterwards, T₁For in genetically modified plantsEui1The qualification result of expression (Fig. 2 F is semi-quantitative results, and Fig. 2 G is quantitative result)；It can be seen thatEui1Gene expression Level in Δ NS2 anddEui1In significantly improve.

Fig. 3 isEui1The phenotype of cDNA conversion wild-type plant；Fig. 3 A is removal introneEui1- cDNA conversion Wild type ZH11 obtains the transgene positive plant downgraded；Fig. 3 B, Fig. 3 C are conversionsEui1After-cDNA, T₁For genetically modified plants InEui1The qualification result of expression (Fig. 3 B is semi-quantitative results, and Fig. 3 C is quantitative result)；It can be seen thatEui1Gene table Exist up to levelEui1It is significantly improved in-cDNA genetically modified plants；Scale represents 25 cm height in figure.

Specific embodiment

The contents of the present invention are further illustrated below in conjunction with Figure of description and specific embodiment, but should not be construed as pair Limitation of the invention.Without departing from the spirit and substance of the case in the present invention, it is repaired to made by the method for the present invention, step, condition Change or replace, all belongs to the scope of the present invention.Unless otherwise noted, experimental method used in embodiment is art technology Conventional method known to personnel and technology, used reagent or material are to be obtained by commercial sources.

1 rice dwarf mutant of embodimentdEui1Related gene identification and clone

Inventor obtains the Dwarf Mutant of a long-neck fringe gene function acquisition type in tissue culture procedures, name FordEui1(Figure 1A).dEui1In T₀In generation, shows typical Dwarfing phenotypes, and in T₁Dai Zhong, only 1/4 plant performance are Normal plant height, this explanationdEui1It is a dominant Dwarf Mutant.It is found through analysisdEui1It is the prominent of a T-DNA insertion Variant.Other adjacent sequence is obtained by TAIL-PCR, the site T-DNA insertion is located at one of No. 5 chromosome in Codocyte color The long-neck fringe gene of plain P450 monooxygenase (CYP450)Eui1(LOC_Os05g40384).Eui1By two exons and one Introne composition, analyzed by further sequence, T-DNA instead ofEui1135 alkali of the introne close to First Exon Basic sequence (Figure 1B).In order to further confirm thatdEui1It is as caused by T-DNA insertion, by primer P1/P3, P2/P3 two is right Primer (table 1) is to T₁Linkage analysis is carried out for plant, as long as it is short to find that transgenosis heterozygous individual and homozygous individual are shown as Change, other plant then shows as normal (Fig. 1 C).This is sufficiently proveddEui1It is to have a unit as caused by T-DNA insertion The dominant Dwarf Mutant of point.In addition,dEui1Root, stem, the Ye Zhong of mutantEui1The expression of gene is significantly improved (Fig. 1 D).

The building of embodiment 2 carriesEui1Introne small fragment missing Δ NS1 or Δ NS2 have complementary functions carrier heredity turn Changeeui1Mutant obtains the genetically modified plants of different plant heights

To spend 11 genomic DNAs template in japonica rice, to be segmented the strategy of amplification, using in pCAMBIA1300 and table 2 Primer, construct containingEui1The conversion carrier Δ NS1 and Δ NS2(Fig. 2A that has complementary functions of the small missing of introne), then by this A little plasmids convert Agrobacterium, are conducted into respectivelyeui1In mutant.T is carried out using hygromycin gene primer (table 1)₀Generation Detection GMOs (Fig. 2 B, 2C).Inventor's discovery is converted with pCAMBIA1300- Δ NS1 expression vectoreui1The conversion of mutant It is horizontal (Fig. 2 D) that body plant height reverts to wild type plant height.And it is converted with pCAMBIA1300- Δ NS2 expression vectoreui1Mutant Transformant plant height then show as compared with wild type downgrade plant height phenotype (Fig. 2 E).Further transcriptional expression analysis shows, Δ NS2 T₁For in genetically modified plantsEui1Expression withdEui1It is similar, (Fig. 2 F and 2G) is significantly improved compared with wild type.

3 rice conversion intronless of embodimentEui1Code cDNA obtain dwarf transgenic plants

Using the primer in pCAMBIA1300 and table 2, removal introneEui1Code area cDNA converts wild type water 11 are spent in rice, it is same to obtain the transgene positive plant (Fig. 3 A) downgraded.Transcriptional expression analysis shows,Eui1- cDNA transgenosis In plantEui1Expression withdEui1It is similar, (Fig. 3 B and 3C) is significantly improved compared with wild type in root, stem, Ye Zhongjun.

SEQUENCE LISTING

<110>Agricultural University Of South China

<120>one plant growth negative regulation cis element SE1 and its regulation plant strain senior middle school application

<130>

<160> 20

<170> PatentIn version 3.3

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<213>negative regulation cis-regulating element SE1 sequence

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acatcactag ctacatgcat gatgcttgct tgct 34

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ggtggctagc catatatcgt ggtcattgac 30

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aggctgccat ggagagcttc ttcgtc 26

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Claims (1)

1. Application of the negative regulation cis element SE1 shown in 1.SEQ ID NO:1 in regulation plant strain senior middle school, the regulation plant strain It is a height of that plant is made to generate Dwarfing phenotypes；The plant is rice.