CN110396522A - Regulate and control the application technology that lignin improves root crop yield - Google Patents
Regulate and control the application technology that lignin improves root crop yield Download PDFInfo
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Abstract
The present invention relates to the application technologies that regulation lignin improves root crop yield.Present invention discloses a kind of gene--4CL1s closely related with the yield traits of root tuber or tuberous plant, and its coding albumen.The invention discloses the purposes of this 4CL1 gene, in particular for increasing the yield of root tuber or tuberous plant.
Description
Technical field
The invention belongs to botany fields, more particularly it relates to which regulating and controlling lignin improves root crop yield
Application technology.
Background technique
Potato plant refers mainly to have for edible roots or rhizomatous a kind of terrestrial crop.There are root tuber, tubers, such as
Sweet potato (sweet potato, sweet potato), cassava, potato, Chinese yam (Chinese yam), sole potato etc., multirow vegetative propagation only stays potato wedge to plant, and
It can be bred with liana.This kind of general cold-hartliness of plant is weaker, mostly cultivates in frostless season, and low temperature can inhibit tuber crops
Growth, cause the underproduction of block root or tuber, therefore plant tuber crops to avoid prolonged hypothermic phase as far as possible；In addition, dredging
Loose, fertile, deep soil and volume potash fertilizer is conducive to improve tuber crops yield and quality.
It is grain important in the world, feed and insutrial crop, in guarantee using sweet potato as the tuber crops of representative
State's biomass energy and grain security etc. play a significant role.Sweet potato genus Convolvulaceae, sweet potato genus, sweet potato kind, for overgrowing property grass
This plant.Sweet potato plant can be divided into the parts such as root, stem, leaf, flower, fruit, seed.Sweet potato is a kind of important cereal crops,
It is the important source material of food processing industry, nutritive value is abundant.Therefore, sweet potato variety optimization and cultivation, the research of regeneration techniques
Have great importance.The common plant as sweet potato with root tuber structure is also: cassava, purple sweet potato, fleece-flower root etc., and
Potato is tuberous plant.
The storage root of potato plant is important energy storage organ, and the development of storage root is a complicated biological process,
Including expanding initial and subsequent thickening etc..These processes are not only by the regulation of endogenous hormones and gene, also by the shadow of external environment
It rings.However, the molecular mechanism for expanding development for regulation storage root at present is also known little about it.
Therefore, this field is it is necessary to study the excellent genes for the storage root that can regulate and control potato plant in potato plant, from
And new approach is provided for the improvement of plant.
Summary of the invention
The purpose of the present invention is to provide the applications that lignin synthesis pathway gene 4CL1 significantly improves beet yield.
In the first aspect of the present invention, the yield of a kind of raising root crops (crop) or tuberous plant (crop) is provided
Method, which comprises increase the expression of 4- coumaric acid-coacetylase-ligase (4CL1 polypeptide) in root tuber or tuberous plant
Or activity.
In a preferred embodiment, the root crops or tuberous plant are potato plants.
In another preferred example, the root crops or tuberous plant include but is not limited to: sweet potato, purple sweet potato, cassava,
Sweet potato, potato, Chinese yam, taro, pueraria lobata, konjaku, Jerusalem artichoke, yacon.
In another preferred example, 4- coumaric acid-coacetylase-ligase is selected from the group:
(a) such as the polypeptide of SEQ ID NO:2 amino acid sequence；
(b) by SEQ ID NO:2 amino acid sequence by one or more (such as 1-20；Preferably 1-10；More preferably 1-
5) replacing, missing or adding for amino acid residue and formed, and with (a) polypeptide function the polypeptide as derived from (a)；
(c) have 85% or more with (a) polypeptide sequence limited (preferably 90% or more, more preferably 95% or more, such as 98%
More than, 99% or more) homology and with (a) polypeptide function the polypeptide as derived from (a)；Or
(d) protein fragments of the SEQ ID NO:2 with (a) protein function.
In another preferred example, 4- coumaric acid-coacetylase-ligase is 4- coumaric acid-coacetylase-connection of external source
Enzyme introduces root crops or tuberous plant for 4- coumaric acid-coacetylase-ligase polynucleotides are encoded by recombination form
In.
In another preferred example, the method comprising steps of
(i) Agrobacterium for carrying expression vector, expression vector 4- coumaric acid-coacetylase-ligase containing coding are provided
Polynucleotides；
(ii) using Agrobacterium make coding 4- coumaric acid-coacetylase-ligase polynucleotides be transferred to root crops or
Tuberous plant.
In another preferred example, the root crops or tuberous plant are sweet potatoes.
In another preferred example, the yield of the raising root crops or tuberous plant includes:
Increase the storage root of root crops or tuberous plant；And/or
Increase the radix bardanae quantity of root crops or tuberous plant.
In another preferred example, further comprising administering to root crops or tuberous plant HORMONE TREATMENT, the hormone is selected from:
MeJA, GA, IAA, 6-BA or ABA；
Preferably, giving:
MeJA:100~800uM；
GA:50~1500uM；
IAA:80~300uM；
6-BA:10~1500uM；
ABA:10~1500uM.
In another aspect of this invention, a kind of 4- coumaric acid-coacetylase-ligase is provided or encodes the multicore glycosides of the polypeptide
The purposes of acid, for improving the yield of root crops or tuberous plant.
In a preferred embodiment, the yield of the raising root crops or tuberous plant include: increase root crops or
The storage root of tuberous plant.
In another aspect of this invention, a kind of 4- coumaric acid-coacetylase-ligase is provided or encodes the multicore glycosides of the polypeptide
The purposes of acid, the molecular marked compound for the yield as plant identification.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure
's.
Detailed description of the invention
Fig. 1, sweet potato 4CL gene family member 4792 (4CL1), 5346 (4CL2), 380 (4CL3), 675 (4CL4) are out of office
Tissue specific expression in raw type sweet potato.
Lf is indicated: leaf color (leaf color)；St indicates stem；
Ft1 is indicated: white fibrous root (white fibrous root)；
Ft2 is indicated: red fibrous root (red fibrous root)；
Dt is indicated: development root (development root)；
Mt is indicated: matured root (mature root).
The building and Transformation of sweet potato callus of Fig. 2, Ib4CL1 overexpression vector.
A~J, the preparation of callus and the acquisition of genetically modified plants.
K, the schematic diagram of the construction of Ib4CL1 overexpression vector is constructed.
Fig. 3, Real-time PCR detect the expression quantity of Ib4CL1 in transgenic plant.Wherein, OE4CL1-1, OE4CL1-
2, OE4CL1-7 respectively indicates several plants of specific transgenic plants obtained after transgenic culturing.
The phenotype and yield of Fig. 4, Ib4CL1 overexpression.
A, the phenotype of Ib4CL1 overexpression；
B, dyeing of the phloroglucin to Sweet Potato；
C, the storage root of two months plant and the statistics of radix bardanae quantity are grown；
D, the statistics of two months plant yields is grown；
Wherein, OE4CL1-1, OE4CL1-2, OE4CL1-3 respectively indicate that several plants obtained after transgenic culturing are specific to be turned
Gene plant.
Fig. 5, Ib4CL1 overexpress plant root tuber in the phenotype and output statistics in crop field.
A, Ib4CL1 overexpresses plant root tuber in the phenotype in crop field；
B, Ib4CL1 overexpresses plant beet yield statistics.
Fig. 6, various concentration MeJA, GA, IAA, 6-BA, ABA for Ib4CL1 expression influence column diagram.
Specific embodiment
The present inventor passes through in-depth study, discloses a kind of closely related with the yield traits of root tuber or tuberous plant
Gene--4- coumaric acid-coacetylase-ligase (4CL1), and its coding albumen.The invention also discloses this 4CL1 genes
Purposes there are extremely significant excellent properties in particular for increasing the yield of root tuber or tuberous plant.
4CL1 is the gene in lignin synthesis access, and the expression of the gene can cause mentioning for content of lignin in Sweet Potato
Height, the present inventor has found that Ib4CL1 can cause storage root to become larger for the first time, to significantly improve yield.
4CL1 albumen (polypeptide) of the invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide.Polypeptide of the invention can
To be native purified product or chemically synthesized product, or use recombinant technique from protokaryon or eucaryon host (for example, thin
Bacterium, yeast, higher plant, insect and mammalian cell) in generate.
The invention also includes the segments of 4CL1 albumen, derivative and analogue.As used herein, term " segment ", " derivative
Object " and " analog ", which refer to, is kept substantially the identical biological function of 4CL1 albumen or active polypeptide of the invention.This hair
Bright polypeptide fragment, derivative or the like, which can be (i), has one or more conservative or non-conservative amino acid residues (preferably
Conservative amino acid) substituted polypeptide, and such substituted amino acid residue can be and may not be by heredity
Cipher coding, or (ii) in one or more amino acid residues with the polypeptide of substituent group, or (iii) additional amino
Acid sequence is fused to this polypeptide sequence and the polypeptide (such as leader sequence or secretion sequence or for purifying the sequence of this polypeptide that are formed
Or proprotein sequence or fusion protein).According to the definition of this paper these segments, that derivative and analogue belong to this field is skilled
Range well known to technical staff.
The bioactive fragment of any 4CL1 albumen can be applied in the present invention.Herein, 4CL1 albumen
The meaning of bioactive fragment refers to all or part of function that the 4CL1 albumen of overall length is still able to maintain as a kind of polypeptide
Energy.Under normal conditions, the bioactive fragment at least keeps the activity of 50% overall length 4CL1 albumen.In preferred item
Under part, the active fragment is able to maintain 60%, 70%, 80%, 90%, 95%, 99% or the 100% of overall length 4CL1 albumen
Activity.
In the present invention, term " 4CL1 albumen " refers to the polypeptide of the SEQ ID NO:2 sequence with 4CL1 protein active.It should
Term further includes having and 4CL1 albumen identical function, SEQ ID NO:2 sequence variant form.These variant forms include
(but being not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, also more
It is good such as 1-8,1-5) missing, insertion and/or the substitution of amino acid, and in C-terminal and/or N-terminal (especially N-terminal)
Add or lack it is one or several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, also more
Good such as 1-8,1-5) amino acid.For example, in the art, when being substituted with similar nature or similar amino acid, leading to
The function of Chang Buhui change protein.For another example, one or several ammonia are added in C-terminal and/or N-terminal (especially N-terminal)
Base acid will not generally also change the function of protein.The term further includes the active fragment and reactive derivative of 4CL1 albumen.
It is any high with the described 4CL1 albumen homology (for example to be with the homology of sequence shown in SEQ ID NO:2
50% or higher；Preferably, homology is 60% or higher；Preferably, homology is 70% or higher；Preferably, homology
It is 80% or higher；It is furthermore preferred that homology be 90% or higher, such as homology 95%, 98% or 99%) and have
The albumen of 4CL1 albumen identical function is also included in the present invention.
The invention further relates to the polynucleotide sequences for encoding 4CL1 albumen of the present invention or its conservative variation's polypeptides.Described
Polynucleotides can be DNA form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can
To be single-stranded or double-strand.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be with
Coding region sequence shown in SEQ ID NO:1 is identical or the variant of degeneracy.As used herein, " variant of degeneracy " In
Referring to coding in the present invention has the protein of SEQ ID NO:2, but has difference with coding region sequence shown in SEQ ID NO:1
Nucleic acid sequence.
The polynucleotides for encoding the mature polypeptide of SEQ ID NO:2 include: the coded sequence of an encoding mature polypeptide；It is mature
The coded sequence of polypeptide and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide and
Non-coding sequence.
Term " polynucleotides of coding polypeptide " can be the polynucleotides including coding said polypeptide, be also possible to also wrap
Include the polynucleotides of additional code and/or non-coding sequence.
The invention further relates to the variant of above-mentioned polynucleotides, coding has the more of identical amino acid sequence with the present invention
The segment of peptide or polypeptide, analogs and derivatives.The variant of this polynucleotides can be the allelic variant naturally occurred or
The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insertion variant.Such as this
Known to field, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide substitution,
Missing or insertion, but not from substantially change its encode polypeptide function.
The invention further relates to hybridize with above-mentioned sequence and between two sequences with the multicore glycosides of at least 80% phase same sex
Acid.The present invention is more particularly directed under strict conditions with the interfertile polynucleotides of polynucleotides of the present invention.In the present invention,
" stringent condition " refers to: (1) compared with hybridization and elution low ionic strength and higher temperature under, such as 0.2 × SSC, 0.1%SDS,
60℃；Or (2) when hybridizing added with denaturant, such as 50% (v/v) formamide, 0.1% calf serum/0.1%Ficoll, 42 DEG C
Deng；Or (3) only the phase same sex between two sequences at least just hybridizes at 90% or more, more preferably 95% or more.And
And mature polypeptide shown in the polypeptide of interfertile polynucleotide encoding and SEQ ID NO:2 have identical biological function and
Activity.
4CL1 protein nucleotides full length sequence or its segment of the invention can usually use PCR amplification method, recombination method or people
Work synthetic method obtains.For PCR amplification method, can disclosed related nucleotide sequence according to the present invention, it is especially open
Reading frame sequence carrys out design primer, and with the commercially available library cDNA or by prepared by conventional method well known by persons skilled in the art
The library cDNA expands as template and obtains related sequence.
The present invention also relates to the carriers comprising the polynucleotides, and with the carrier or 4CL1 encoding histone sequence
Arrange genetically engineered host cell.
In the present invention, 4CL1 protein polynucleotide be can be plugged into recombinant expression carrier." recombinant expression carries term
Body " refer to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other
Carrier.As long as any plasmid and carrier can be used in short, can replicate and stablize in host.One weight of expression vector
It is characterized in usually containing replication orgin, promoter, marker gene and translation control element.
Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, can be used for converting suitable
When host cell, allow it to expression protein.Host cell can be prokaryotic cell, such as bacterial cell；Or it is low
Eukaryocyte, such as yeast cells；Or higher eucaryotic cells, such as plant cell.Representative example has: Escherichia coli, streptomycete
Belong to, Agrobacterium；Fungal cell's such as yeast；Plant cell etc..
When the polynucleotides are expressed in higher eucaryotic cells, if will when being inserted into enhancer sequence in the carrier
Enhance transcription.Enhancer is the cis-acting factors of DNA, generally about there is 10 to 300 base-pairs, acts on starting
Son is to enhance the transcription of gene.
Persons skilled in the art are aware that how to select carrier, promoter, enhancer and host cell appropriate.
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.Converting plant can
Use the methods of Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as spraying, leaf disk method, Rice Young Embryo conversion method etc..
In a preferred embodiment of the invention, the present inventor has cloned the 4CL gene of lignin access and has constructed overexpression
Carrier, Transformation of sweet potato obtain transgenic plant of the 35S::Ib4CL1 in sweet potato.Later, in the field on two ground of Shandong and Shanghai
Experimental data discovery, the beet yield of transgenic plant sweet potato significantly improve.The pilot experiment of different zones is it has been shown that turn base
Because the yield of plant sweet potato is all improved.Experiment in vivo shows that Ib4CL1 may cause the change of methyl jasmonate (MeJA) content
Change the development of de-regulation sweet potato storage root.
Therefore, the present invention provides the purposes of the 4CL1 albumen or its encoding gene, for improving block root or tuber
The yield of plant；It include: the storage root for increasing root crops or tuberous plant and/or the ox for increasing root crops or tuberous plant
Burdock radical amount.
The invention further relates to a kind of method for improveing plant, this method includes improving 4CL1 in the root tuber or tuberous plant
The expression of albumen.After the purposes for knowing the 4CL1 albumen, can using a variety of methods well known in the art come
Improve the expression of the 4CL1 albumen.For example the expression list of 4CL1 gene can will be carried by approach known to those skilled in the art
Position (such as expression vector or virus etc.) is delivered on target spot, and is allowed to the 4CL1 albumen of expression activity.
Preferably, a kind of method of prepare transgenosis plant is provided, comprising:
(1) coded polynucleotide of the 4CL1 albumen of external source is transferred to plant tissue, organ or tissue, is transformed into
Plant tissue, organ or the seed of the coded polynucleotide of 4CL1 albumen；With
(2) plant tissue, organ or the kind for the coded polynucleotide for being transferred to external source 4CL1 albumen for obtaining step (1)
Son regeneration plant.
Other 4CL1 genes or the method for its homologous gene expression of increasing are well known in the art.For example, can be by with strong
Promoter driving is to enhance 4CL1 gene or the expression of its homologous gene.Or pass through enhancer (such as rice waxy gene the
One introne, Actin gene First Intron etc.) enhance the expression of the 4CL1 gene.Suitable for opening by force for the method for the present invention
Mover includes but is not limited to: 35s promoter, rice, Ubi promoter of corn etc..
Any conventional means appropriate can be used, implement the method including reagent, temperature, pressure condition etc..
Moreover, it relates to the chasing after as a kind of genetic transformation progeny of plants using 4CL1 albumen or its encoding gene
Track label.The invention further relates to using 4CL1 albumen or its encoding gene as a kind of molecular labeling, by detection plant
The expression of 4CL1 albumen, the yield characteristics of plant identification, storage root feature.And utilize 4CL1 albumen or its encoding gene
The screening improved the breed.
Present invention firstly discovers that 4CL1 gene expands in development in the storage root of root tuber or tuberous plant in lignin access
Play an important role, overexpress the gene, can cause root tuber or tuberous plant expand in advance and can significantly improve root tuber or
The yield of tuberous plant, to generate more economic benefits.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or
According to the normal condition proposed by manufacturer.
The Cloning and sequence analysis of embodiment 1, Ib4CL
The 4CL gene of the function of lignin metabolism's access is cloned into using the method for homologous clone, the present inventor will
The unnamed gene is Ib4CL1.
The code area the Ib4CL1 long 1710bp of nucleotide, encodes the polypeptide of 569 amino acid.
The nucleotide sequence of Ib4CL1 is following (SEQ ID NO:1):
ATGCTATCTGTGGAAACTCAGAAACCAGAGCTTGAAGTTGCAGATTCTGGGAAGACACAAGCACAGTCT
TCCCAGATTCATATTTTCCGATCGAGGTTGCCGGATATACCCATCCCCAATCAAATCCCACTCCATACTTATTGTTT
CCAGAATCTGGCCGAGTACCGGGACCGGACATGTCTCATCGTGGGTTCCACCGGAAAAACATATTCCTTCGCCGAAA
CCCATTTGATTTGCCGGAAAGTCGCGTCGGGATTGGCCAAACTCGGGGTTAAGAAGGGGGATGTGATTATGACCCTA
TTTCAGAACTGCCCGGAGTTTGTTTTCTCCTTCATGGGGGCTTCTATGATCGGCGCCGTCACCACCACGGCCAACCC
TTTCTACACCAAAGCCGAGATTTTTAAGCAGATGAGTGCTTCCAAGGCGAAGGTTGTCATTACCCAATCTGTGTACG
TAGAAAAGCTCAAAGATGCCGGCGAGGAGAACCCTAAAATCGGGGAGGATTTCTCGGTGGTCACCATCGATGACCCG
CCGGAAAATTGCTTGCATTTCTCCGTGCTGTCGGAGGCCGACGAGGAGGATATGCCGGCGACGGTGGAAATCGCCCC
TGACGACGCCGTGGCTCTGCCGTTCTCTTCCGGGACTACCGGGCTGCCGAAAGGAGTGGTGTTGACCCACAAGAGCT
TGATCACCAGCGTGGCCCAGCAAGTCGACGGCGAGAACCCGAACTTATACCTCAAGGAAGACGACGTCGTTCTGTGC
GTGCTACCATTGTTCCACATATTTTCCCTCAACTCCGTTCTCCTCTGCTCCCTCCGAGCAGGGGCGGCGGTGCTGCT
GATGCAGAAATTCGAAATCAAATCGTTGCTGGAGCTGATAAAGAAGCACCGGGTGTCCGTGGCGGCGGTGGTGCCGC
CGCTGGTCCTGGCGCTGGCCAAGAATCCGATCGTGGATTCCTACGATTTGAGCTCGATTCGGGTGGTGCTCTCCGGC
GCGGCGCCGCTGGGGAAGGAGCTGGAGGAGGCCCTCCATCAGAGAGTCCCTCAGGCCATTTTTGGTCAGGGTTATGG
AATGACTGAGGCAGGGCCAGTATTGTCAATGTGTCCGGCTTTCGCGAAGCAGGCGCTGCCTGCTAAATCCGGCTCAT
GCGGCTCAGTAGTGAGGAACGCGGAGCTGATGGTGGTAGATCCGGAAACCGGCTGCTCCCTTGGCCGCAACCAACCC
GGAGAGATTTGCATACGCGGCTCTCAAATCATGAAAGAGTATTTGAATGATCCGGCGGCCACCGCTCGGACTATTGA
CGTGGATGGCTGGCTCCATACCGGCGACATTGGCTATGTAGACGATGACGATGAAGTTTTCATCGTCGATAGAGTGA
AAGAACTCATCAAATTTAAAGGCTTCCAGGTGCCACCAGCTGAGCTTGAGGCTCTGCTTCTGAGCCACCCGATGATT
GCAGATGCTGCTGTCGTACCGCAAAAAGATGATGCAGCCGGAGAAGTCCCTGTTGCGTTTGTGGTTCGATCCGCGGA
TGGATTTGATCTCACTGAAGAAGCTGTAAAGGAATTTATTGCCAAACAGGTGGTATTTTATAAGAAGTTACACAAGG
TATACTTTATTCATGCAATTCCAAAATCAGCATCCGGAAAAATATTGAGAAAAGAGCTCCGGGAAAAACTACAAGCT
GCGCCACCTTCCACGCCGCAATAA
The amino acid sequence of Ib4CL1 is following (SEQ ID NO:2):
MLSVETQKPELEVADSGKTQAQSSQIHIFRSRLPDIPIPNQIPLHTYCFQNLAEYRDRTCLIVGSTGKTYSFAETHL
ICRKVASGLAKLGVKKGDVIMTLFQNCPEFVFSFMGASMIGAVTTTANPFYTKAEIFKQMSASKAKVVITQSVYVEK
LKDAGEENPKIGEDFSVVTIDDPPENCLHFSVLSEADEEDMPATVEIAPDDAVALPFSSGTTGLPKGVVLTHKSLIT
SVAQQVDGENPNLYLKEDDVVLCVLPLFHIFSLNSVLLCSLRAGAAVLLMQKFEIKSLLELIKKHRVSVAAVVPPLV
LALAKNPIVDSYDLSSIRVVLSGAAPLGKELEEALHQRVPQAIFGQGYGMTEAGPVLSMCPAFAKQALPAKSGSCGS
VVRNAELMVVDPETGCSLGRNQPGEICIRGSQIMKEYLNDPAATARTIDVDGWLHTGDIGYVDDDDEVFIVDRVKEL
IKFKGFQVPPAELEALLLSHPMIADAAVVPQKDDAAGEVPVAFVVRSADGFDLTEEAVKEFIAKQVVFYKKLHKVYF
IHAIPKSASGKILRKELREKLQAAPPSTPQ*
Different sweet potato 4CL gene family member 4792 (4CL1), 5346 (4CL2), 380 (4CL3), 675 (4CL4) In
The specific expressed situation of tissue (root, stem, leaf, root) in wild type sweet potato is as shown in Figure 1.
Embodiment 2, the building of Ib4CL1 overexpression vector and Transformation of sweet potato
Using sweet potato cDNA as template, with primer 5 '-cggggtaccatgctatctgtggaaactca-3 ' (SEQ ID NO:
3) it is expanded with 5 '-aactgcagttattgcggcgtggaaggtg-3 ' (SEQ ID NO:4), obtains amplified production.
PCambia1301 is expression vector, is inserted into Ib4CL1 coded sequence in the site KPNI/PstI of the expression vector
(amplified production i.e. above-mentioned).The recombinant expression carrier of the acquisition, structural schematic diagram such as Fig. 2 K, can be with tobacco mosaic virus (TMV)
35S promoter starting Ib4CL1 expression.
By the callus of the recombinant expression carrier Transformation of sweet potato of above-mentioned building, use hygromycin as the anti-of the positive seedling of conversion
Property selection markers.Wherein, the preparation of callus and the acquisition of genetically modified plants such as Fig. 2A~J.
After about 6 months time, the transgenic plant of Ib4CL1 overexpression is obtained.Simultaneously with Southern Blot and
Real-time PCR detects the transgenic plant of acquisition.
Real-time PCR detects result such as Fig. 3 of the expression quantity of Ib4CL1 in transgenic plant.The result shows that turning base
Because the expression quantity versus wild type of Ib4CL1 in plant significantly increases.
Embodiment 3, the phenotype and yield of Ib4CL1 overexpression plant
The phenotype discovery for observing Ib4CL1 overexpression sweet potato plant, in greenhouse-grown 2 months transgenic plants compared to open country
Raw type stem is short and sturdy, and anthocyanidin (Fig. 4 A) is assembled on top.
It to being found after the stem phloroglucinol stain of transgenic plant, compares wild type color burn (Fig. 4 B), illustrate to gather
More lignin is collected, i.e. Ib4CL1 overexpresses the content of lignin in plants stems and improves, and stem elongation growth is impacted, and stem becomes
It obtains short and thick.
Meanwhile statistics discovery, the storage root and fructus arctii of Transgenic Sweet Potato plant also are carried out to the yield of 2 months sweet potato plant
Radical amount is significantly higher than wild type (Fig. 4 C)；The yield of Transgenic Sweet Potato plant is apparently higher than wild type, illustrates transgenic plant
Root tuber expand and shift to an earlier date (Fig. 4 D) compared to wild type.
Embodiment 4, Ib4CL1 overexpress plant root tuber in the phenotype and output statistics in crop field
By Transgenic Sweet Potato plant in July, 2017 and May, field planting is carried out in the field on two ground of Shandong and Shanghai,
Wild type sweet potato is planted simultaneously as control.After planting 4 months and 6 months respectively, sweet potato is harvested, observes phenotype, and
Carry out output statistics comparison.
The results show that yield of the Transgenic Sweet Potato plant in crop field is significantly improved than wild type sweet potato, such as OE4CL1-2
Yield reaches 6kg/ plants, is wild type more than 4 times, and the yield of whole Transgenic Sweet Potato strain has compared to wild type significantly to be mentioned
Height, such as Fig. 5 A-B.This provides more economic values and guarantee for the safety of industrial application and food.
The expression of embodiment 5, different exogenous hormone processing sweet potatoes and Ib4CL1 gene
The overexpression of Ib4CL1 gene causes expanding and increasing production for root tuber, and the present inventor's guess may have with the adjusting of hormone
It closes, specifically what hormone plays regulatory role not clear, therefore at hormone of the present inventor with various concentration (0~1000uM)
Reason.The hormone for choosing optimum concentration detects Ib4CL1 in the wild type for going processing sweet potato with the different time of optimum concentration hormone
Expression, thus speculate it is possible regulation Ib4CL1 expression hormone.
As a result it is influenced as Fig. 6, methyl jasmonate (MeJA), GA, IAA, 6-BA, ABA have the expression of Ib4CL1, and
And there are optimum concentration for the treatment of.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>Shanghai Inst. of Life Science, CAS
<120>regulation lignin improves the application technology of root crop yield
<130> 179649
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1710
<212> DNA
<213>sweet potato (Ipomoea batatas)
<400> 1
atgctatctg tggaaactca gaaaccagag cttgaagttg cagattctgg gaagacacaa 60
gcacagtctt cccagattca tattttccga tcgaggttgc cggatatacc catccccaat 120
caaatcccac tccatactta ttgtttccag aatctggccg agtaccggga ccggacatgt 180
ctcatcgtgg gttccaccgg aaaaacatat tccttcgccg aaacccattt gatttgccgg 240
aaagtcgcgt cgggattggc caaactcggg gttaagaagg gggatgtgat tatgacccta 300
tttcagaact gcccggagtt tgttttctcc ttcatggggg cttctatgat cggcgccgtc 360
accaccacgg ccaacccttt ctacaccaaa gccgagattt ttaagcagat gagtgcttcc 420
aaggcgaagg ttgtcattac ccaatctgtg tacgtagaaa agctcaaaga tgccggcgag 480
gagaacccta aaatcgggga ggatttctcg gtggtcacca tcgatgaccc gccggaaaat 540
tgcttgcatt tctccgtgct gtcggaggcc gacgaggagg atatgccggc gacggtggaa 600
atcgcccctg acgacgccgt ggctctgccg ttctcttccg ggactaccgg gctgccgaaa 660
ggagtggtgt tgacccacaa gagcttgatc accagcgtgg cccagcaagt cgacggcgag 720
aacccgaact tatacctcaa ggaagacgac gtcgttctgt gcgtgctacc attgttccac 780
atattttccc tcaactccgt tctcctctgc tccctccgag caggggcggc ggtgctgctg 840
atgcagaaat tcgaaatcaa atcgttgctg gagctgataa agaagcaccg ggtgtccgtg 900
gcggcggtgg tgccgccgct ggtcctggcg ctggccaaga atccgatcgt ggattcctac 960
gatttgagct cgattcgggt ggtgctctcc ggcgcggcgc cgctggggaa ggagctggag 1020
gaggccctcc atcagagagt ccctcaggcc atttttggtc agggttatgg aatgactgag 1080
gcagggccag tattgtcaat gtgtccggct ttcgcgaagc aggcgctgcc tgctaaatcc 1140
ggctcatgcg gctcagtagt gaggaacgcg gagctgatgg tggtagatcc ggaaaccggc 1200
tgctcccttg gccgcaacca acccggagag atttgcatac gcggctctca aatcatgaaa 1260
gagtatttga atgatccggc ggccaccgct cggactattg acgtggatgg ctggctccat 1320
accggcgaca ttggctatgt agacgatgac gatgaagttt tcatcgtcga tagagtgaaa 1380
gaactcatca aatttaaagg cttccaggtg ccaccagctg agcttgaggc tctgcttctg 1440
agccacccga tgattgcaga tgctgctgtc gtaccgcaaa aagatgatgc agccggagaa 1500
gtccctgttg cgtttgtggt tcgatccgcg gatggatttg atctcactga agaagctgta 1560
aaggaattta ttgccaaaca ggtggtattt tataagaagt tacacaaggt atactttatt 1620
catgcaattc caaaatcagc atccggaaaa atattgagaa aagagctccg ggaaaaacta 1680
caagctgcgc caccttccac gccgcaataa 1710
<210> 2
<211> 569
<212> PRT
<213>sweet potato (Ipomoea batatas)
<400> 2
Met Leu Ser Val Glu Thr Gln Lys Pro Glu Leu Glu Val Ala Asp Ser
1 5 10 15
Gly Lys Thr Gln Ala Gln Ser Ser Gln Ile His Ile Phe Arg Ser Arg
20 25 30
Leu Pro Asp Ile Pro Ile Pro Asn Gln Ile Pro Leu His Thr Tyr Cys
35 40 45
Phe Gln Asn Leu Ala Glu Tyr Arg Asp Arg Thr Cys Leu Ile Val Gly
50 55 60
Ser Thr Gly Lys Thr Tyr Ser Phe Ala Glu Thr His Leu Ile Cys Arg
65 70 75 80
Lys Val Ala Ser Gly Leu Ala Lys Leu Gly Val Lys Lys Gly Asp Val
85 90 95
Ile Met Thr Leu Phe Gln Asn Cys Pro Glu Phe Val Phe Ser Phe Met
100 105 110
Gly Ala Ser Met Ile Gly Ala Val Thr Thr Thr Ala Asn Pro Phe Tyr
115 120 125
Thr Lys Ala Glu Ile Phe Lys Gln Met Ser Ala Ser Lys Ala Lys Val
130 135 140
Val Ile Thr Gln Ser Val Tyr Val Glu Lys Leu Lys Asp Ala Gly Glu
145 150 155 160
Glu Asn Pro Lys Ile Gly Glu Asp Phe Ser Val Val Thr Ile Asp Asp
165 170 175
Pro Pro Glu Asn Cys Leu His Phe Ser Val Leu Ser Glu Ala Asp Glu
180 185 190
Glu Asp Met Pro Ala Thr Val Glu Ile Ala Pro Asp Asp Ala Val Ala
195 200 205
Leu Pro Phe Ser Ser Gly Thr Thr Gly Leu Pro Lys Gly Val Val Leu
210 215 220
Thr His Lys Ser Leu Ile Thr Ser Val Ala Gln Gln Val Asp Gly Glu
225 230 235 240
Asn Pro Asn Leu Tyr Leu Lys Glu Asp Asp Val Val Leu Cys Val Leu
245 250 255
Pro Leu Phe His Ile Phe Ser Leu Asn Ser Val Leu Leu Cys Ser Leu
260 265 270
Arg Ala Gly Ala Ala Val Leu Leu Met Gln Lys Phe Glu Ile Lys Ser
275 280 285
Leu Leu Glu Leu Ile Lys Lys His Arg Val Ser Val Ala Ala Val Val
290 295 300
Pro Pro Leu Val Leu Ala Leu Ala Lys Asn Pro Ile Val Asp Ser Tyr
305 310 315 320
Asp Leu Ser Ser Ile Arg Val Val Leu Ser Gly Ala Ala Pro Leu Gly
325 330 335
Lys Glu Leu Glu Glu Ala Leu His Gln Arg Val Pro Gln Ala Ile Phe
340 345 350
Gly Gln Gly Tyr Gly Met Thr Glu Ala Gly Pro Val Leu Ser Met Cys
355 360 365
Pro Ala Phe Ala Lys Gln Ala Leu Pro Ala Lys Ser Gly Ser Cys Gly
370 375 380
Ser Val Val Arg Asn Ala Glu Leu Met Val Val Asp Pro Glu Thr Gly
385 390 395 400
Cys Ser Leu Gly Arg Asn Gln Pro Gly Glu Ile Cys Ile Arg Gly Ser
405 410 415
Gln Ile Met Lys Glu Tyr Leu Asn Asp Pro Ala Ala Thr Ala Arg Thr
420 425 430
Ile Asp Val Asp Gly Trp Leu His Thr Gly Asp Ile Gly Tyr Val Asp
435 440 445
Asp Asp Asp Glu Val Phe Ile Val Asp Arg Val Lys Glu Leu Ile Lys
450 455 460
Phe Lys Gly Phe Gln Val Pro Pro Ala Glu Leu Glu Ala Leu Leu Leu
465 470 475 480
Ser His Pro Met Ile Ala Asp Ala Ala Val Val Pro Gln Lys Asp Asp
485 490 495
Ala Ala Gly Glu Val Pro Val Ala Phe Val Val Arg Ser Ala Asp Gly
500 505 510
Phe Asp Leu Thr Glu Glu Ala Val Lys Glu Phe Ile Ala Lys Gln Val
515 520 525
Val Phe Tyr Lys Lys Leu His Lys Val Tyr Phe Ile His Ala Ile Pro
530 535 540
Lys Ser Ala Ser Gly Lys Ile Leu Arg Lys Glu Leu Arg Glu Lys Leu
545 550 555 560
Gln Ala Ala Pro Pro Ser Thr Pro Gln
565
<210> 3
<211> 29
<212> DNA
<213>primer (Primer)
<400> 3
cggggtacca tgctatctgt ggaaactca 29
<210> 4
<211> 28
<212> DNA
<213>primer (Primer)
<400> 4
aactgcagtt attgcggcgt ggaaggtg 28
Claims (12)
1. a kind of method for the yield for improving root crops or tuberous plant, which is characterized in that the described method includes: in root tuber or
Increase 4- coumaric acid-coacetylase-ligase expression or activity in tuberous plant.
2. the method as described in claim 1, which is characterized in that the root crops or tuberous plant is potato plant.
3. the method as described in claim 1, which is characterized in that the root crops or tuberous plant includes: sweet potato, purple
Potato, cassava, potato, Chinese yam, taro, pueraria lobata, konjaku, Jerusalem artichoke, yacon, the fleece-flower root.
4. the method as described in claim 1, which is characterized in that the 4- coumaric acid-coacetylase-ligase is selected from the group:
(a) such as the polypeptide of SEQ ID NO:2 amino acid sequence；
(b) SEQ ID NO:2 amino acid sequence is formed by one or more replacing, missing or adding for amino acid residue
, and the polypeptide as derived from (a) with (a) polypeptide function；
(c) there is 85% or more homology with (a) polypeptide sequence limited and there is the polypeptide as derived from (a) of (a) polypeptide function；
Or
(d) protein fragments of the SEQ ID NO:2 with (a) protein function.
5. the method as described in claim 1, which is characterized in that the 4- coumaric acid-coacetylase-ligase is the 4- of external source
Coumaric acid-coacetylase-ligase introduces block for 4- coumaric acid-coacetylase-ligase polynucleotides are encoded by recombination form
It takes root in object or tuberous plant.
6. method as claimed in claim 5, which is characterized in that the method comprising steps of
(i) Agrobacterium for carrying expression vector is provided, expression vector 4- coumaric acid-coacetylase-ligase containing coding is more
Nucleotide；
(ii) coding 4- coumaric acid-coacetylase-ligase polynucleotides are made to be transferred to root crops or stem tuber using Agrobacterium
Plant.
7. the method as described in claim 1, which is characterized in that the root crops or tuberous plant is sweet potato.
8. the method as described in claim 1, which is characterized in that the yield packet for improving root crops or tuberous plant
It includes:
Increase the storage root of root crops or tuberous plant；And/or
Increase the radix bardanae quantity of root crops or tuberous plant.
9. the method as described in claim 1, which is characterized in that further comprising administering to root crops or tuberous plant HORMONE TREATMENT,
The hormone is selected from: MeJA, GA, IAA, 6-BA or ABA；
Preferably, giving:
MeJA:100~800uM；
GA:50~1500uM；
IAA:80~300uM；
6-BA:10~1500uM；
ABA:10~1500uM.
10. the purposes of a kind of 4- coumaric acid-coacetylase-ligase or the polynucleotides for encoding the polypeptide, for improving root crops
Or the yield of tuberous plant.
11. purposes as claimed in claim 10, which is characterized in that the yield packet for improving root crops or tuberous plant
It includes: increasing the storage root of root crops or tuberous plant.
12. the purposes of a kind of 4- coumaric acid-coacetylase-ligase or the polynucleotides for encoding the polypeptide, for being used as plant identification
Yield molecular marked compound.
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Cited By (3)
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| CN113331051A (en) * | 2020-03-02 | 2021-09-03 | 中国科学院分子植物科学卓越创新中心 | Culture medium and method for inducing plants to form storage roots |
| CN114262745A (en) * | 2021-12-22 | 2022-04-01 | 山东省农业科学院作物研究所 | Sweet potato anthocyanin related SNP molecular marker, identification and application thereof |
| CN116377108A (en) * | 2023-01-10 | 2023-07-04 | 江西农业大学 | Molecular marker, primer pair and kit for detecting purple sweet potato seeds and Chinese yam and application of molecular marker |
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| CN114262745B (en) * | 2021-12-22 | 2022-07-15 | 山东省农业科学院作物研究所 | Sweet potato anthocyanin related SNP molecular marker, identification and application thereof |
| CN116377108A (en) * | 2023-01-10 | 2023-07-04 | 江西农业大学 | Molecular marker, primer pair and kit for detecting purple sweet potato seeds and Chinese yam and application of molecular marker |
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| CN110396522B (en) | 2023-03-28 |
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