CN106811471A - Application of the paddy rice SPL7 genes in plant type is regulated and controled - Google Patents
Application of the paddy rice SPL7 genes in plant type is regulated and controled Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Abstract
The invention belongs to field of plant genetic, and in particular to application of the paddy rice SPL7 genes in plant type is regulated and controled.Described gene energy controlling plant type of rice.The nucleotides sequence of SPL7 genes is classified as the SEQ ID NO in sequence table:1；SEQ ID NO in the protein of its coding such as sequence table:Shown in 3.The whole protein coding region sequence of SPL7 genes is placed under the control of constitutive promoter 35S the overexpression in paddy rice, transfer-gen plant is relative to wild type control, tiller reduction, fringe portion branch are reduced and grain number per spike is reduced, and show that SPL7 genes can be used for the plant type of adjusting and controlling rice.The whole protein coding region sequence of SPL7 genes such as SEQ ID NO:Shown in 2.The present invention not illustrate only biological function of the SPL7 genes in controlling plant type of rice, while also providing a kind of method for improveing plant type of rice.
Description
Technical field
The present invention relates to field of plant genetic.Specifically related to paddy rice SPL7 genes are in plant plant type is adjusted
Using.By transgene method, the protein coding region of overexpression SPL7 genes can reduce dividing for paddy rice in paddy rice
Tiller, reduction fringe portion branch stalk number and reduction grain number per spike, these results show that SPL7 genes have in the genetic improvement of plant type of rice
Certain value.
Background technology
Paddy rice is one of topmost cereal crops in the world, has more than the population of half with it as staple food, therefore paddy rice
Yield height have important influence to social development.With the sustainable growth of world population, the continuous reduction of cultivated area
With the increasingly exacerbation of environmental pollution, cultivate with per unit area yield higher and environment-friendly new rice variety for ensureing China
Grain security has important strategic importance.
The yield of paddy rice is closely related with its plant type, and the Breeding Model with ideotype as Breeding objectives is in agricultural production
Paid attention to by height.The plant type of paddy rice is mainly comprising the big of plant height, tiller, fringe portion branch and grain number, root morphology and organ
Content (Wang and Li, the Molecular basis of plant architecture.Annu Rev such as small and angle
Plant Biol,2008,59:253-279).Wherein plant height, tiller, fringe type and grain weight are all for a long time the weights of genetic improvement
Want objective trait.Plant height be with rice yield, photosynthesis and the proterties closely related with lodging resistance, eighties of last century is dropping
Low plant height greatly improves the yield of global crops for the first time " green revolution " of breeding objective.With science of heredity and point
The development of sub- biology, has illustrated hormone particularly gibberellin and rape element sterol plays crucial in the plant height of regulation plant
Effect (Wang and Li, Molecular basis of plant architecture.Annu Rev Plant Biol,
2008,59:253-279)。
Rice tillering is grown under appropriate conditions by the axillary bud of basal part of stem, is generally comprised the formation of tiller bud and is divided
Two growth courses of elongation of tiller bud.Cloned multiple genes relevant with tiller development at present, such as MOC1, LAX1,
The genes such as LAX2, TAB1 control initial development (Komatsu et al., the LAX and SPA of tiller bud:major
regulators of shoot branching in rice.Proc Natl Acad Sci U S A,2003,100:
11765-11770；Li et al.,Control of tillering in rice.Nature,2003,422:618-621；
Tabuchi et al；LAX PANICLE2of rice encodes a novel nuclear protein and
regulates the formation of axillary meristems.Plant Cell,2011,23:3276-3287；
Tanaka et al.,Axillary meristem formation in rice requires the WUSCHEL
ortholog TILLERS ABSENT1.Plant Cell,2015,27:1173-1184).And the one kind for identifying in recent years is newly
Plant hormone witchweed lactone then control rice tillering bud grow during play core regulating and controlling effect.It is long in paddy rice
The serial Dwarf Mutant that phase genetics research is collected has played important in the synthesis of parsing witchweed lactone and signal transduction
Effect.Witchweed lactone is a type sesquiterpene ene compound, is synthesized by carrotene.D17/HTD1 in paddy rice, D10 and
D27 control witchweed lactone synthesis (Arite et al., DWARF10, an RMS1/MAX4/DAD1ortholog,
controls lateral bud outgrowth in rice.Plant J,2007,51:1019-1029；Lin et al.,
DWARF27,an iron-containing protein required for the biosynthesis of
strigolactones,regulates rice tillers bud outgrowth.Plant Cell,2009,21:1512-
1525；Zou et al.,The rice HIGH-TILLERING DWARF1encoding an ortholog of
Arabidopsis MAX3is required for negative regulation of the outgrowth of
axillary buds.Plant J,2006,48:687-698), the protein of D3, D14 and D53 coding then controls witchweed
Lactone signal transduction (Arite et al., d14, a strigolactone-insensitive mutant of rice,
shows an accelerated outgrowth of tillers.Plant Cell Physiol,2009,50:1416-
1424；Ishikawa et al.,Suppression of tiller bud activity in tillering dwarf of
rice.Plant Cell Physiol,2005,46:79-86；Jiang et al.,DWARF 53acts a repressor
of strigolactone signaling in rice.Nature,2013,504:401-405；Zhou et al.,D14-
SCF(D3)-dependent degradation of D53regulates strigolactone signaling.Nature,
2013,504:406-410)。
The fringe of paddy rice is the purpose organ harvested in agricultural production, and its form and composition directly determine the yield of paddy rice.
The fringe of paddy rice is a kind of panicle, is developed by inflorescence meristem, and it can typically produce Primary branch and Secondary Branch
Stalk, can also produce the branch of higher level to obstruct in a few cases, and branch stalk can further bud into small ear and then produce flower, flower
Rice will be formed after organ chasmogamy.Different from the plant such as arabidopsis, the growth course of rice panicle type is by a series of
The transformation of separate living tissue destiny is determined.Under the conditions of suitable internal and external environment, paddy rice enters reproductive growth by nutrient growth, with this
Meanwhile, apical meristem produces Primary branch separate living tissue also converted into inflorescence meristem by inflorescence meristem,
Produce after a number of Primary branch separate living tissue, inflorescence meristem can degenerate.And Primary branch separate living tissue
The Secondary branch separate living tissue of higher level can be produced, small ear separate living tissue also can be directly differentiated, and Secondary branch separate living tissue
The development models that branch obstructs separate living tissue can be repeated once, final branch stalk separate living tissue is all changed into small ear separate living tissue.And small ear
Separate living tissue can then produce floral meristem, and then produce the differentiation of floral organ.The fringe time of different separate living tissues is very big
Fringe type (Kyozuka et al., the Control of grass inflorescence form by of paddy rice are determined in degree
the fine-tuning of meristem phase change.Curr Opin Plant Biol,2014,17:110-
115).If the time that inflorescence meristem is maintained is more long, more Primary branch will be produced；And if small ear separate living tissue
If differentiation is postponed, it is possible to produce even three times branches of more Secondary branch to obstruct, so as to produce more grain number per spikes.In view of
Significance of the fringe type in agricultural production, scientist has discovered that the gene of substantial amounts of control fringe type development, and some
Gene is applied (Zhang and Yuan, Molecular control of grass in the practices of breeding
inflorescence development.Annu Rev Plant Biol,2014,65:553-578)。
MicroRNA (miRNA) is a class length about in the 20-24 RNA molecule of the endogenous non-coding of nucleotides.
Since having cloned first coding miRNA gene in nematode since 1993, in nearly all higher organism studied
Have been found that the gene of substantial amounts of coding miRNA is present.MiRNA often through regulating mRNA stability or regulation protein
Translation process controls the activity of target gene mRNA, so as to adjust including each including growth, development, metabolism, response environment etc.
Individual vital movement process.Because the regulatory mechanism of its powerful biological function and complexity, miRNA related research has become
One of popular domain of current life science, and it is possible to played an important role in the genetic improvement of crops.
MicroRNA156 (miR156) is the highly conserved tiny RNA in each higher plant species of a class, in paddy rice, there is 11
The transcription factor of SPL families is by its regulation and control (Xie et al., Genomic organization, differential
expression,and interaction of SQUAMOSA promoter-binding-like transcription
factors and microRNA156in rice.Plant Physiol,2006,142:280-293), wherein 2 target genes
The function of SPL14/IPA1 and SPL16 has been parsed (Jiao et al., Regulation of OsSPL14 by
OsmiR156defines ideal plant architecture in rice.Nat Genet,2010,42:541-544；
Miura et al.,OsSPL14 promotes panicle branching and higher grain productivity
in rice.Nat Genet,2010,42:545-549；Wang et al.,Control of grain size,shape and
quality by OsSPL16in rice.Nat Genet,2012,44:950-954), but remaining 9 are adjusted by miR156
The function of the SPL genes of control is still unknown.Can effectively be adjusted disclosure sets forth the SPL7 genes regulated and controled by miR156
The plant type of Water-saving Rice, therefore there is potential value in the genetic improvement of paddy rice.
The content of the invention
It is the application for providing a kind of SPL7 genes in plant type of rice is adjusted that the purpose of the present invention is.It is of the invention in addition
One purpose there is provided a kind of genetic transformation carrier and method that plant type of rice is adjusted by SPL7 genes.SPL7 genes have
Just like SEQ ID NO:Nucleotide sequence shown in 1, also including with SEQ ID NO:DNA sequence dna shown in 1 has more than 90% together
The gene order of source property, also including the mutant allele that is produced because inserting, substitute or lacking one or more bases or
Derivative.Present invention additionally comprises the protein of SPL7 gene codes, its sequence such as SEQ ID NO:Shown in 3, also including with SEQ
ID NO:Protein sequence shown in 3 has the protein sequence of more than 90% homology, also including because of insertion, replacement or missing
One or more amino acid and albumen or albumen analog that the function that produces changes.The core of the protein coding region of SPL7 genes
Nucleotide sequence such as SEQ ID NO:Shown in 2.The present invention is significantly reduced by the protein coding region of overexpression SPL7 genes
The tiller number of paddy rice, fringe portion branch stalk number and grain number per spike, these results show that SPL7 genes may have weight in rice breeding
The value wanted.The SPL7 genes that the present invention is provided can be used for other controlling elements, such as constitutive promoter (such as
CaMV35S promoters) or organ specific promoters construct expression vector；Also can be by transgenic technology, antisense
RNA, RNAi, Zinc finger nuclease (zinc-finger nucleases, ZFN), activating transcription factor sample effector nuclease
(transcription activator-like effector nucleases, TALEN) and CRISPR (Clustered
Regularly Interspersed Short Palindromic Repeats) technology such as/Cas9 manipulates to regulate and control water to it
The plant type of rice.
Realize that particular technique step of the invention is as follows：
1st, the expression pattern of SPL7 genes is detected using microarray data and Real-Time Fluorescent Quantitative PCR Technique；Specific side
Method has a detailed description in embodiment 1.
2nd, using PCR method from rice young panicle RNA reverse transcriptions and come cDNA in expand SPL7 genes protein compile
Code area；Specific method has a detailed description in example 2.
3rd, the protein coding region of SPL7 genes is connected on pCAMBIA1301S carriers, builds overexpression carrier
35S:SPL7OE；Specific method has a detailed description in embodiment 3.
4th, using agriculture bacillus mediated transgenic method (Lin and Zhang, the Optimising for optimizing
tissue culture conditions for high efficiency transformation of indica
rice.Plant Cell Rep,2005,23:540-548) by expression vector 35S:11 are spent in SPL7OE Introduced into Rice acceptors, is obtained
Obtain transformed plant；Specific method has a detailed description in example 4.
5th, analyze and identify positive transgenic plant by the method for PCR and isolate detection, and table is carried out for individual plant to T1
Type is identified and statistical analysis；Specific method has a detailed description in embodiment 5.
6th, 35S is detected using the method for real-time fluorescence quantitative PCR:The expression of the SPL7 genes in SPL7OE transfer-gen plants
Amount；Specific method has a detailed description in embodiment 6.
Compared with prior art, advantages of the present invention is as follows：
1st, the function disclosure sets forth paddy rice SPL7 genes in controlling plant type of rice.
2nd, the protein coding region Introduced into Rice of SPL7 genes is realized the genetic improvement to plant type of rice by the present invention.
3rd, a kind of method that genetic improvement is carried out to paddy rice using SPL7 genes of the invention.
Brief description of the drawings
Sequence table SEQ ID NO:1 is the nucleotide sequence of SPL7 genes.
Sequence table SEQ ID NO:2 is the nucleotide sequence of the protein coding region of SPL7 genes.
Sequence table SEQ ID NO:3 is the protein sequence of SPL7 gene codes.
Fig. 1：SPL7 genes are with microRNA156 oligonucleotide ligands to schematic diagram.
Fig. 2：The expression pattern of SPL7 genes.Description of reference numerals：
A figures in Fig. 2：In 25 tissues for coming self-evident extensive 63, the 10 SPL genes regulated and controled by microRNA156
The dendrogram of genetic chip signal value.Chip data comes from disclosed database CREP (http://crep.ncpgr.cn).
B figures in Fig. 2：The expression pattern of SPL7 genes in spending 16 of 11 to organize in from rice varieties.Callus takes
Sample is in 20d after subculture；Plumule and radicle are sampled in 48h after sprouting；Seedling top is separately sampled in 20d after sprouting and 40d；Root, leaf
Piece, leaf sheath, internode, section, cob and flower were sampled in heading stage；Seed sampling is in after fertilization 7d；Young fringe is separately sampled in 1mm, 4-
The length of 5mm and 10mm.Data display is 3 average value ± standard errors of repetition.SA：Seedling top；YP：Young fringe；DAG：Sprout
Number of days after hair.
Fig. 3：Expression vector 35S:SPL7OE building process schematic diagrames.Description of reference numerals：
A figures in Fig. 3：Insertion expression vector 35S:The DNA fragmentation for including SPL7 protein coding regions of SPL7OE shows
It is intended to
B figures in Fig. 3：For construction of expression vector 35S:The structural representation of the empty carrier pCAMBIA1301S of SPL7OE
Figure.
Carrier shown in the B that fragment shown in A figures in Fig. 3 is inserted in Fig. 3 by KpnI and BamHI double digestions is schemed
The expression vector 35S of conversion is formed in the middle of KpnI the and BamHI restriction enzyme sites of pCAMBIA1301S:SPL7OE.
Fig. 4：The expression vector 35S that the present invention builds:SPL7OE collection of illustrative plates.
Fig. 5：11 (WT) and 35S are spent in wild type:The phenotype of the plant of SPL7OE overexpressions (SPL7OE) compares.Accompanying drawing
Description of symbols：
A figures in Fig. 5：11 (WT) and 35S are spent in wild type:The plant height of the plant of SPL7OE overexpressions (SPL7OE) and
The form of tiller compares.
B figures in Fig. 5：11 (WT) and 35S are spent in wild type:The fringe type ratio of the plant of SPL7OE overexpressions (SPL7OE)
Compared with.
Fig. 6：11 (WT) and 35S are spent in detecting wild type using real-time fluorescence quantitative PCR:SPL7OE overexpressions
(SPL7OE) in plant SPL7 genes expression quantity.Data display is 3 average value ± standard errors of repetition.
Specific embodiment
Embodiment 1：The spatial and temporal expression profile of SPL7 genes
The result of the rice at whole growth periods gene expression spectrum analysis based on applicant laboratory early stage, has 3 by miR156
The special high expression in young fringe of the SPL genes of regulation and control, and their expression quantity gradually lowered with the process of Young spike development
(Wang et al.,A dynamic gene expression atlas covering the entire life cycle
of rice.Plant J,2010,61:752-766), therefore applicant envisages that this 3 genes participate in the young fringe hair of adjusting and controlling rices
Educate process.The function of one of member SPL7 genes be it is unknown, the present invention make a concrete analysis of the gene biological function and
The potential value in rice genetic improvement.
SPL7 genes are regulated and controled by miR156, and its nucleotides pair relationhip between miR156 is shown in Fig. 1.According to paddy gene
Result (the http of group annotation://rice.plantbiology.msu.edu/), the accession number of SPL7 genes is LOC_
Os04g46580, the SEQ ID NO in its nucleotide sequence such as sequence table:Shown in 1, the sequence such as sequence of the protein of its coding
SEQ ID NO in table:Shown in 3.In order to analyze the function of SPL7 genes, applicant analyzes the expression mould of the gene first
Formula.
To achieve the above object, applicant is using disclosed database CREP (http://crep.ncpgr.cn；Wang
et al.,A dynamic gene expression atlas covering the entire life cycle of
rice.Plant J,2010,61:752-766) analyze the expression pattern of SPL7 genes.The database has been included from two
The chip data of multiple tissues in rice variety treasure's Shan 97 and the bright extensive 63 covering time of infertility.The present invention has made a concrete analysis of and has come self-evident
The expression mould of SPL7 and other the 9 SPL genes by miR156 regulation and control in extensive 63 25 tissues (details are shown in Table 1)
Formula, is analyzed using the method for hierarchical clustering to chip data, and as a result as shown in the A figures in Fig. 2, it shows that SPL7 genes exist
(panicle1-panicle4) special expression high in the young fringe of early development.
Table 1 is used for the chip data source-information of bright extensive the 63 of SPL Gene Expression Profile Analysis
Applicant have detected the expression pattern of SPL7 genes further with the method for real-time fluorescence quantitative PCR.To realize
The purpose, devises following primer first：
SPL7QRTF (forward primer)：5'-CTGCTGCTGTGTAACGACGCTA-3',
SPL7QRTR (reverse primer)：5'-GCACACGGACGGACCTATGTGTAA-3'；
UbiF (forward primer):5'-AACCAGCTGAGGCCCAAGA-3',
UbiR (reverse primer):5'-ACGATTGATTTAACCAGTCCATGA-3'；
Wherein primer combination SPL7QRTF and SPL7QRTR amplifications is SPL7 genes, and primer combination UbiF and UbiR expands
Increase be ubiquitin genes, and by the use of ubiquitin genes expression quantity as real-time fluorescence quantitative PCR equilibrating
Internal reference.
Applicant acquire respectively from spent in rice varieties 11 (Institute of Crop Science, Chinese Academy of Agricultural Science) after
Callus for 20d, sprout after 48h plumule and seedling top, the root at heading stage, blade, the leaf of 20d and 40d after radicle, sprouting
The seed of sheath, internode, section, cob and flower, Post flowering 7d, and length is respectively in the young fringe of 1mm, 4-5mm and 10mm, and take out
Carrying this 16 RNA of sample carries out reverse transcription to carry out real-time fluorescence quantitative PCR analysis.Specific operating procedure is as follows：
(1) RNA is extracted
Extracting is above-mentioned from 16 RNA for organizing that 11 are spent in rice varieties, and the reagent of RNA extractings is to buy certainly
Trizol extraction agents box (concrete operation step is shown in kit specification) of Invitrogen companies.
(2) the reverse transcription synthesis chains of cDNA first
Step is as follows：1) the μ g of total serum IgE 2 of extracting are taken, the μ l of 1 μ l, 10xDNaseI buffer of DNaseI 1 are added, plus
DEPC (pyrocarbonic acid diethyl ester, the strong inhibition agent of RNase, working concentration be 0.01%) treated water to 10 μ l, after mixing
Place 15min to remove the genomic DNA of residual in room temperature；2) the μ l of 0.2M EDTA 1 are added after 15min, and in 65 DEG C of water-baths
Middle incubation 10min is removing the activity of DNaseI；3) 1 μ l primers oligo (dT) is added15, and it is incubated 10min in 65 DEG C of water-baths
To destroy the secondary structure of RNA, 5min is then placed on ice；4) 5x first strand buffer 4 μ l, 0.1M are added
2 μ l, 10mM dNTP mixture of DTT (mercaptoethanol) 1 μ l, the μ l of reverse transcriptase 1, are placed in warm bath in 42 DEG C of water-baths after mixing
1.5h；5) reverse transcription product is placed in 90 DEG C of dry bath 3min to inactivate reverse transcriptase by reaction after terminating；6) in reverse transcription product
120 μ l water are added, -20 DEG C preserve reaction final product after mixing.The reagent used in reaction is all public purchased from Invitrogen
Department.
(3) real-time fluorescence quantitative PCR amplification
With step (2) reverse transcription come cDNA as template, be utilized respectively primer combine SPL7QRTF and SPL7QRTR,
UbiF and UbiR carries out real-time fluorescence quantitative PCR amplification.Real-time fluorescence quantitative PCR related reagent is purchased from precious bioengineering and is connected with greatly
Limit company, reaction system is referring to specification.PCR instrument is 95 DEG C of predegeneration 10s for 7500, the PCR parameters of American AB I companies, is entered
Enter 95 DEG C of denaturation 5s after circulation, 60 DEG C of annealing extend 40s, 45 circulations.It is with the result that primer combines UbiF and UbiR amplifications
The result of internal reference equilibrating each sample, and the expression quantity of the SPL7 genes in callus is set to 1 calculating remaining each group
Knit the relative expression quantity of middle SPL7 genes.As shown in the B figures in Fig. 2, it shows SPL7 genes to the result of real-time fluorescence quantitative PCR
The expression high in the young fringe of development, the result is identical with the above-mentioned result from chip detection.
Embodiment 2：The clone of the protein coding region of paddy rice SPL7 genes
In order to analyze the function of SPL7 genes, the present invention use the RNA of rice young panicle by cDNA that reverse transcription is obtained for
Template, using the special primer of SPL7 genes, the protein coding region of clone's SPL7 genes, its core is separated by the method for PCR
SEQ ID NO in nucleotide sequence such as sequence table:Shown in 2.To realize the purpose, the present invention is public from Invitrogen using purchase
The Trizol extraction agents box extracting of department is from spending the RNA of 11 length in the young fringe of 1mm or so, the tool of extracting in rice varieties
The specification of related reagent is shown in body operating process.After RNA extractings are completed, inverted using the identical method of same embodiment 1
The record synthesis chains of cDNA first.
In order to expand the protein coding region of SPL7 genes, we devise following primer：
SPL7OEF (forward primer):5'-GGTACCATGGAAGGAAACGGCTGCG-3'(underlined sequences are known for KpnI
Other site)；
SPL7OER (reverse primer):5'-GGATCCTCAGACCACGCGGGCGCCCT-3'(underlined sequences are BamHI
Recognition site)；
This pair of primer can amplify the initiation codon of SPL7 genes to the protein coding region between terminator codon
Section, its sequence such as sequence table SEQ ID NO:Shown in 2.
The cDNA come with reverse transcription is entered performing PCR and expanded as template, with primer SPL7OEF and SPL7OER.PCR reactions are total
Volume is 20 μ l, comprising μ l, 10mM the primer SPL7OEF of 1 μ l, 10xPCR buffer of cDNA templates, 2 μ l, 10mM dNTP 2 and
Each μ l of 0.3 μ l, ExTaq enzyme 0.2 of SPL7OER, plus deionized water is to 20 μ l (used PCR buffer, dNTP, rTaq enzymes etc.
It is purchased from precious bioengineering Dalian Co., Ltd).PCR reaction conditions are as follows：1. 94 DEG C of 4min, 2. 94 DEG C of 40s, 3. 58 DEG C of 40s,
4. 72 DEG C of 2min, 5. from 2. -4. circulate 38 times, 6. 72 DEG C of 7min, 7. 4 DEG C of preservations.PCR primer is in 1% (mass/volume)
Electrophoresis detection on TBE Ago-Gels, recovery length is that (the target dna section of 1083bp is added 1095bp plus on primer
Two restriction enzyme site 12bp) DNA fragmentation.The PCR primer of recovery is connected to T-A cloning vectors pGEMT-vector
Upper (being purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd, i.e. U.S. Promega companies), is drawn using T7 on carrier and SP6
Thing carries out sequence verification to the PCR primer, and the recombinant plasmid comprising the PCR primer is named as into TA-SPL7.
Embodiment 3：The overexpression vector construction of SPL7 genes
With KpnI and BamHI double digestion plasmids TA-SPL7, (described KpnI and BamHI is bought from precious bioengineering Dalian
Co., Ltd, detailed directions refer to the specification of the said firm's corresponding product with consumption, and plasmid TA-SPL7 comes from embodiment 2, such as schemes
In 3 A figure shown in), reclaim comprising SPL7 genes protein coding region 1095bp DNA fragmentation, then with by KpnI
With the vector plasmid pCAMBIA1301S of BamHI double digestions using T4DNA ligases (be purchased from Promega companies, detailed directions with
Specification of the consumption with reference to the said firm's product) it is attached.Carrier pCAMBIA1301S is to be disclosed to report (Zhou et
al.,Over-expression of aspartate aminotransferase genes in rice resulted in
altered nitrogen metabolism and increased amino acid content in seeds.Theor
Appl Genet,2009,118:Carrier 1381-1390), its basic framework derives from Australia CAMBIA laboratories
(http://www.cambia.org/daisy/cambia/materials/overview.html) pCAMBIA1301, lead to
Cross and add 35S promoter to realize the expression regulation to transformed gene, its structure is as shown in the B figures in Fig. 3.Connection product is by electricity
(electric conversion instrument is eppendorf Products to the method for conversion, and applied voltage of the present invention is 1800V, and concrete operations refer to the instrument
The operation instructions of device) import in Escherichia coli DH10B (being purchased from Promega companies), plus the recovery of 400 μ l LB culture mediums
45min, is applied on the LA culture medium flat plates of the kanamycins containing 50mg/L, and (LA and LB is formulated ginseng to 37 DEG C of incubator culture 14-16h
Examine：Pehanorm Brooker,《Molecular Cloning:A Laboratory guide》The third edition, Science Press, 2002).Picking monoclonal, Amplification Culture
And plasmid is extracted, by KpnI and BamHI double digestion screening positive clones, and the expression vector of gained is named as 35S:
SPL7OE, its structure is as shown in Figure 4.
Embodiment 4：The acquisition of transgenic paddy rice
By expression vector 35S:The genetic transforming method that SPL7OE (coming from embodiment 3) is mediated by Agrobacterium EHA105 is led
Enter the callus that 11 are spent in rice varieties, by preculture, infect, co-culture, screen with hygromycin (for screening the positive
Transgenosis callus a kind of antibiotic, purchase is from the Co., Ltd of Roche Group of Denmark) callus of resistance, differentiation, give birth to
Root and acclimatization and transplantses crop field, obtain transfer-gen plant.The method of Agrobacterium genetic transformation and reagent used and formula are bases
Report optimization (Hiei et al., the Efficient transformation of rice (Oryza of Hiei et al.
sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries
of the T-DNA.Plant J,1994,6:271-282；Lin and Zhang,Optimising the tissue
culture conditions for high efficiency transformation of indica rice.Plant
Cell Rep,2005,23:540-548)。
The Agrobacterium-mediated genetic transformation reagent and formula that the present invention relates to are as follows：
(1) reagent and solution are abridged
6-BA (6-BenzylaminoPurine, 6-benzyladenine)；KT (Kinetin, kinetin)；NAA
(Napthalene acetic acid, methyl α-naphthyl acetate)；IAA (Indole-3-acetic acid, heteroauxin)；2,4-D(2,4-
Dichlorophenoxyacetic acid, 2,4- dichlorphenoxyacetic acids)；AS (Acetosringone, acetosyringone)；CH
(Casein Enzymatic Hydrolysate, caseinhydrolysate)；HN (Hygromycin B, hygromycin)；DMSO
(Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO))；N6max (a large amount of ingredient solutions of N6)；N6min (N6 a small amount of ingredient solution)；
MSmax (a large amount of ingredient solutions of MS)；MSmin (MS a small amount of ingredient solution)
(2) main solution formula
1) N6max mother liquors [10 times of concentrates (10X)]
Dissolve one by one, 1000ml is then settled at room temperature.
2) N6min mother liquors [100 times of concentrates (100X)]
1000ml is dissolved and is settled at room temperature.
3)Fe2- EDTA stores liquid (100X)
300ml distilled water and ferric sulfate (FeSO are added in a big triangular flask4·7H2O)2.78g
300ml distilled water is added in another big triangular flask and 70 DEG C are heated to, b diammonium edta two is subsequently adding
Sodium (Na2EDTA·2H2O)3.73g
Mixed after they all dissolve, 2h is kept in 70 DEG C of water-baths, be settled to 1000ml, 4 DEG C save backup.
4) vitamins stock liquid (100X)
Add water and be settled to 1000ml, 4 DEG C save backup.
5) MSmax mother liquors (10X)
1000ml is dissolved and is settled at room temperature.
6) MSmin mother liquors (100X)
1000ml is dissolved and is settled at room temperature.
7) 2,4-D stores liquid (1mg/ml)
2,4-D 100mg.
1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, and room temperature is protected
Deposit.
8) 6-BA stores liquid (1mg/ml)
6-BA 100mg.
1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, and room temperature is protected
Deposit.
9) NAA stores liquid (1mg/ml)
NAA 100mg.
1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, 4 DEG C of preservations
It is standby.
10) IAA stores liquid (1mg/ml)
IAA 100mg.
1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, 4 DEG C of preservations
It is standby.
11) glucose storage liquid (0.5g/ml)
Glucose 125g
Distillation water dissolves are settled to 250ml, and 4 DEG C save backup after sterilizing.
12) AS storages liquid
AS 0.392g
DMSO 10ml
In packing to 1.5ml centrifuge tubes, 4 DEG C save backup.
13) 1N potassium hydroxide storage liquid
Potassium hydroxide (KOH) 5.6g
Distillation water dissolves are settled to 100ml, and room temperature preservation is standby.
14) KT stores liquid (1mg/ml)
KT 100mg.
1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, and room temperature is protected
Deposit.
(3) culture medium prescription
1) inducing culture
Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 5.9, boils and be settled to 1000ml, is dispensed into 50ml
Triangular flask (25ml/ bottles), sealing sterilizing.
2) subculture medium
Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 5.9, boils and be settled to 1000ml, is dispensed into 50ml
Triangular flask (25ml/ bottles), sealing sterilizing.
3) pre-culture medium
Plus distilled water is to 250ml, 1N potassium hydroxide adjusts pH value to 5.6, sealing sterilizing.
Using preceding heating for dissolving culture medium and 5ml glucose storages liquid and 250 μ l AS are added to store liquid, training is poured into packing
Support in ware (25ml/ wares).
4) base is co-cultured
Plus distilled water is to 250ml, 1N potassium hydroxide adjusts pH value to 5.6, sealing sterilizing.
Using preceding heating for dissolving culture medium and 5ml glucose storages liquid and 250 μ l AS are added to store liquid, training is poured into packing
Support in ware (25ml/ wares).
5) suspension medium
Plus distilled water, to 100ml, regulation pH value is dispensed into two triangular flasks of 100ml to 5.4, sealing sterilizing.
Liquid is stored using preceding addition 1ml glucose storages liquid and 100 μ lAS.
6) Selective agar medium
Plus distilled water, to 250ml, regulation pH value to 6.0, sealing sterilizes.
Using preceding dissolving culture medium, the hygromycin and 400ppm cephalosporins of 250 μ l 50mg/ml are added, training is poured into packing
Support in ware (25ml/ wares).
7) pre- differential medium
Plus distilled water is to 250ml, 1N potassium hydroxide adjusts pH value to 5.9, sealing sterilizing.
Using preceding dissolving culture medium, the hygromycin and 400ppm cephalosporins of 250 μ l 50mg/ml are added, training is poured into packing
Support in ware (25ml/ wares).
8) differential medium
Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 6.0.
1000ml is boiled and be settled to, 100ml triangular flasks (50ml/ bottles), sealing sterilizing is dispensed into.
9) root media
Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 5.8.
1000ml is boiled and be settled to, is dispensed into pipe of taking root (25ml/ pipes), sealing sterilizing.
10) LA culture mediums (LB culture mediums are free of agar powder)
Distillation water dissolves are settled to 250ml, and loaded on 500ml triangular flasks, room temperature is saved backup after sterilizing.
The step of Agrobacterium-mediated genetic transformation that the present invention relates to, is as follows：
(1) callus induction
1) ripe rice paddy seed is shelled, then successively with 70% Ethanol Treatment 1min, 0.15% mercury chloride
(HgCl2)15min
2) sterilizing washing seed 4-5 times；
3) seed is placed on inducing culture；
4) it is placed at dark and cultivates 5 weeks, 26 ± 1 DEG C of temperature.
(2) callus subculture
The embryo callus subculture of glassy yellow, consolidation and relatively dry is selected, dark lower culture 2 weeks, temperature is put on subculture medium
26 ± 1 DEG C of degree.
(3) preculture
The embryo callus subculture of consolidation and relatively dry is selected, dark lower culture 4d, 26 ± 1 DEG C of temperature is put on pre-culture medium.
(4) Agrobacterium culture
1) Agrobacterium EHA1052d of the preculture containing structure good vector, temperature 28 on the LA culture mediums with kanamycins
℃；
2) Agrobacterium is transferred in suspension medium, 2-3h is cultivated on 28 DEG C of shaking tables.
(5) Agrobacterium is infected
1) callus of preculture is transferred in the bottle for having sterilized；
2) suspension of regulation Agrobacterium is to OD6000.8-1.0；
3) callus is soaked into 30min in agrobacterium suspension；
4) blotted on transfer callus to the filter paper for having sterilized；It is then placed within co-culturing on base and cultivates 2d, temperature 19-20
℃。
(6) callus washing and selection culture
1) sterilize water washing callus to invisible Agrobacterium；
2) it is immersed in 30min in the aqua sterilisa containing 400ppm cephalosporins；
3) blotted on transfer callus to the filter paper for having sterilized；
4) selected 2-3 times, every time 2 weeks on transfer callus to Selective agar medium.(first time cephalosporin screening concentration is
400ppm, is 250ppm after second)
(7) break up
1) kanamycin-resistant callus tissue is transferred to and cultivates 5-7d on pre- differential medium at dark；
2) shift on callus to the differential medium of pre- differentiation culture, cultivated under illumination, 26 DEG C, 5-7 weeks of temperature.
(8) take root
1) young plant for having broken up is extracted, the root produced during differentiation is cut；
2) it is then transferred to be cultivated 2-3 weeks under illumination in root media, 26 DEG C of temperature.
(9) transplant
Wash the remaining medium on root off, the seedling with good root system is transferred to greenhouse, while being protected at initial several days
Water holding point moistening.After greenhouse hardening about 2 weeks or so, then transfer load to crop field.
Embodiment 5：The identification of transgenic paddy rice and Phenotypic Observation
T0 is for 35S for extracting:The STb gene of SPL7OE transformed plants (coming from embodiment 4) blade, then with PCR method to super
The T0 for expressing SPL7 carries out positive detection for transformed plant.
PCR positive detections are carried out using the primer of beta-glucosiduronatase gene (GUS), and the sequence of GUS primers is as follows：
GUS-F (forward primer):5'-CCAGGCAGTTTTAACGATCAGTTCGC-3',
GUS-R (reverse primer):5'-GAGTGAAGATCCCTTTCTTGTTACC-3'；
DNA method for extracting is CTAB methods (Zhang et al., Genetic diversity and
differentiation of indica an japonica rice detected by RFLP analysis.Theor
Appl Genet,1992,83:495-499).With paddy rice 35S:SPL7OE transformed plant blades STb gene is template, uses primer
GUS-F and GUS-R enter performing PCR amplification.PCR reaction cumulative volumes are 20 μ l, comprising DNA profiling 100ng, the μ of 10xPCR buffer 2
Each μ l of 0.3 μ l, rTaq enzyme 0.2 of μ l, 10mM primers GUS-F and GUS-R of l, 10mM dNTP 2, plus deionized water is (used to 20 μ l
To PCR buffer, dNTP, rTaq enzymes etc. be purchased from precious bioengineering Dalian Co., Ltd).PCR reaction conditions are as follows：①
94 DEG C of 4min, 2. 94 DEG C of 40s, 3. 58 DEG C of 40s, 4. 72 DEG C of 1min, 5. from 2. -4. circulate 38 times, 6. 72 DEG C of 7min, 7. 4 DEG C of guarantors
Deposit.PCR primer electrophoresis detection on the TBE Ago-Gels of 1% (mass/volume).
PCR testing results show that GUS positive transfer-gen plant all shows as tiller for negative plant
Reduce, fringe portion branch stalk is reduced and grain number per spike is reduced, its phenotype is shown in Fig. 5.
In order to carry out phenotype statistical analysis, applicant is sub for positive plant point individual plant sowing to T0, and continues to plant T1
The transgenic line in generation, the PCR amplifications that gus gene is also carried out to the plant in T1 generations separate the yin and yang attribute of individual plant to detect, carry out
Transgenic event and character mutation isolate detection, and statistics phenotypic data.What applicant carried out 3 independent transformations
35S:The seed of the T0 plant of SPL7OE is transplanted to crop field after 20d and obtains T1 generations turn by being seeded in rice seedling bed after seed soaking, vernalization
Gene plant.Planting density is 24 centimetres of 15 cm x, and plantation place is that Wuhan City of Hubei China province Hongshan District Central China agricultural is big
Experimental plot, the paddy rice planting method under the conditions of having safety protection facility at routinely carries out field management.Using PCR
Method detection gus gene carry out yin and yang attribute identification, and analyze the corresponding relation of PCR testing results and phenotype.Analysis result
Show that all GUS positive individual plants separated in T1 generations show as tiller reduction, fringe portion for negative individual plant, all
Branch stalk is reduced and grain number per spike is reduced, and shows the transgenic event of SPL7 with the present invention signified plant type change (tiller reduction, fringe portion
Branch stalk is reduced and grain number per spike is reduced) isolate.The data to the character mutation of record T1 generation related plant are investigated, including is divided
Tiller number, every fringe Primary branch number, every fringe Secondary branch number and grains per panicle, and the GUS the moon separated with each family
Property individual plant for control, statistical analysis are carried out respectively, it the results are shown in Table 2.
The overexpression 35S of table 2:The phenotype statistical value of the T1 generations positive and negative individual plant of SPL7OE (SPL7OE)
The explanation of table 2：(+) and (-) represents transgenic positive and negative individual plant respectively.Statistics credit is carried out using t tests
Analysis,a,b,cP is represented respectively<0.05,0.01 the level of signifiance with 0.001.
Embodiment 6：The expression analysis of SPL7 genes in transgenic paddy rice
Except entering performing PCR augmentation detection 35S using GUS primers:Outside the yin and yang attribute of SPL7OE transfer-gen plants, applicant is also
The expression quantity of SPL7 genes in transfer-gen plant is have detected further with the method for real-time fluorescence quantitative PCR.It is specifically grasped
Make method with described in embodiment 1.
11 are spent in coming from wild type from the Trizol extraction agents box extracting of Invitrogen companies using purchase and turn base
Because of the RNA (concrete operation step is shown in kit specification) of positive plant blade, using the method for the reverse transcription described in embodiment 1
Synthesize first chain of cDNA, then carry out real-time fluorescence quantitative PCR detection, the side of real-time fluorescence quantitative PCR using following primer
Method is also as described in Example 1.
Used primer：
SPL7QRTF (forward primer)：5'-CTGCTGCTGTGTAACGACGCTA-3',
SPL7QRTR (reverse primer)：5'-GCACACGGACGGACCTATGTGTAA-3'；
UbiF (forward primer):5'-AACCAGCTGAGGCCCAAGA-3',
UbiR (reverse primer):5'-ACGATTGATTTAACCAGTCCATGA-3'；
Wherein primer combination SPL7QRTF and SPL7QRTR amplifications is SPL7 genes, and primer combination UbiF and UbiR expands
What is increased is ubiquitin genes.By the use of ubiquitin genes expression quantity as real-time fluorescence quantitative PCR equilibrating internal reference,
And SPL7 genes are spent in wild type the relative expression quantity in 11 be set to 1.The result of real-time fluorescence quantitative PCR is shown in Fig. 6,
It shows SPL7 genes in 35S:The transfer-gen plant up-regulated expression of SPL7OE.
Claims (4)
1. a kind of application of SPL7 genes in controlling plant type of rice, it is characterised in that the nucleotide sequence of the gene such as SEQ
ID NO:Shown in 1.
2. application of a kind of SPL7 genes in controlling plant type of rice, it is characterised in that the protein sequence of the gene code is such as
SEQ ID NO:Shown in 3.
3. a kind of plant expression vector 35S:SPL7OE, it is characterised in that the carrier includes the SPL7 bases described in claim 1
The CDS sequences of cause, its nucleotide sequence such as sequence table SEQ ID NO:Shown in 2.
4. a kind of method for improveing paddy rice, including structure plant expression vector and Agrobacterium-mediated genetic transformation, its feature exist
In described method is to include by overexpression SPL7 genes, and improvement includes reducing tiller number, reduces fringe portion branch stalk number and subtract
Few grain number per spike, or with using the gene with SPL7 genes with more than 90% homology, by insertion, substitute or missing one
Or multiple bases and mutant allele for producing or derivatives thereof carrys out rice transformation.
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| CN107299102A (en) * | 2017-07-20 | 2017-10-27 | 中国科学院东北地理与农业生态研究所 | The positive regulatory factor OsWRKY53 genes of paddy rice BR signals and its encoding proteins |
| CN107460204A (en) * | 2016-06-03 | 2017-12-12 | 中国科学院上海生命科学研究院 | The upstream and downstream action pathway of OsSPL7 controlling plant type of rice and its application |
| CN109112138A (en) * | 2017-12-01 | 2019-01-01 | 华中农业大学 | A kind of gene OsVAS1 of adjusting and controlling rice ideotype |
| CN113501868A (en) * | 2021-08-09 | 2021-10-15 | 北京大学 | Application of SPL7 in regulation and control of plant drought resistance |
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2015
- 2015-12-01 CN CN201510872393.XA patent/CN106811471A/en active Pending
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460204A (en) * | 2016-06-03 | 2017-12-12 | 中国科学院上海生命科学研究院 | The upstream and downstream action pathway of OsSPL7 controlling plant type of rice and its application |
| CN107460204B (en) * | 2016-06-03 | 2021-07-16 | 中国科学院分子植物科学卓越创新中心 | Upstream and downstream action way for regulating rice plant type through OsSPL7 and application of upstream and downstream action way |
| CN107299102A (en) * | 2017-07-20 | 2017-10-27 | 中国科学院东北地理与农业生态研究所 | The positive regulatory factor OsWRKY53 genes of paddy rice BR signals and its encoding proteins |
| CN107299102B (en) * | 2017-07-20 | 2019-11-08 | 中国科学院东北地理与农业生态研究所 | The positive regulatory factor OsWRKY53 gene of rice BR signal and its coding albumen |
| CN109112138A (en) * | 2017-12-01 | 2019-01-01 | 华中农业大学 | A kind of gene OsVAS1 of adjusting and controlling rice ideotype |
| CN109112138B (en) * | 2017-12-01 | 2020-12-08 | 华中农业大学 | Gene OsVAS1 for regulating and controlling ideal plant type of rice |
| CN113501868A (en) * | 2021-08-09 | 2021-10-15 | 北京大学 | Application of SPL7 in regulation and control of plant drought resistance |
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