CN110331143A - For the miRNA and coding nucleic acid molecule of the leaf regulation of sweet potato and application - Google Patents
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Abstract
The invention discloses a kind of miRNA for the leaf regulation of sweet potato, belong to plant genetic technical field, with nucleotide sequence shown in SEQ ID NO.1 or SEQ ID No.2.Present invention finds Ib-miR319a and its encoding genes, construct the binary vector of 35S promoter driving target gene analogies low expression IbmiR319a, sweet potato embryonal callus is disseminated using mediated by agriculture bacillus, transgenic positive plant is obtained by screening, breaking up, it is identified by the phenotype to rotaring gene plant blade, show that the expression quantity of miR319a in each transgenic line is significantly lowered, and the width of each transgenic line blade significantly reduces, blade length-width ratio obviously increases.
Description
Technical field
The present invention relates to plant genetic technical field more particularly to a kind of miRNA and coding for the leaf regulation of sweet potato
Nucleic acid molecules and application.
Background technique
MicroRNA (miRNA) is that a kind of endogenic, size generally existing in organism is 21-24 nucleotide
The non-coding tiny RNA of (nucleotides, nt) causes target gene in post-transcriptional level by combine complementary with target-gene sequence
Silencing, to control development, stress reaction and hormone signal or animal and plant metabolism.
In plant, single-stranded RNA molecule (ssRNA) miRNA long in core passes through internal base complementary pairing
The miRNA primary transcription sheet (pri-miRNA) for forming loop-stem structure, through endonuclease DCL1 (Dicer-like RNase
IIIprotein I) cutting generation precursor miRNA (pre-miRNA), it is released by the ring that precursor miRNA is cut in Dicer digestion
Ripe double-strand miRNA (miRNA/miRNA*), 3 ' ends are transported through transport protein again after HEN1 (Hua enchancer1) methylation
It send to cytoplasm.After single-stranded miRNA* is detached from double-strand, silencing complex (RNA- is formed with Argonaute (AGO) and Dicer etc.
Induced Silencing Complex, RISC), said target mrna is acted under the guidance of miRNA chain and shear leads to target base
The silencing of cause.The molecular regulation network that miRNA and its target gene are constituted is located at the upstream core position of gene expression, and miRNA is logical
Cross a series of entire growth and development for controlling transcription factors, stress response albumen and other Regulated Proteins influence plant
Journey, Physiology and biochemistry and the response of a variety of environment stresses.
Theoretical study results show that miRNA has and provide the potential of redundant gene resource for genetic modification of plants, but at present
It relatively lags behind to the research that it is applied in production.Sweet potato (Ipomoea batatas (L.) Lam.), is grain important in the world
Food, vegetables, feed, the raw material of industry and bio-energy crop in China are the baseline crops of China's grain security.In recent years, the guarantor of sweet potato
Health-care function also increasingly causes the concern of people.The improvement of leaf morphology is one of important goal of Sweet Potato Breeding.
Summary of the invention
The object of the present invention is to provide a kind of miRNA for the leaf regulation of sweet potato and coding nucleic acid molecule and applications.
A kind of miRNA for the leaf regulation of sweet potato, with nucleosides shown in SEQ ID NO.1 or SEQ ID No.2
Acid sequence.
In addition, the present invention also provides a kind of expression vector pCAMBIA1301-35S- containing the miRNA
mimicsmiR319a。
In addition, the present invention also provides a kind of Agrobacterium host cell EHA105 containing expression vector:
pCAMBIA1301-35S-mimics miR319a。
The present invention also provides a kind of Agrobacterium host cell LBA4404:pCAMBIA1301-35S-mimics
The construction method of miR319a.
The application in transgenic plant is obtained in conversion plant finally, the present invention also provides a kind of expression vectors.
As an improvement, the plant is the embryo callus of sweet potato stem tip induction, detailed process is as follows:
Step 1: positive colony is inoculated into the 50mlYEB fluid nutrient medium containing 100 μ g/ml Kan, 28 DEG C of 200rpm
Continue culture to OD600Then=0.5-1.0 is centrifuged 10 minutes for 4 DEG C of 6000rpm, abandons culture solution, collects thallus；
Step 2: 4 DEG C of 6000rpm are centrifuged 10 minutes, then use after cleaning thallus with the 50ml MS culture solution that pH is 5.3
The 20ml MS culture solution that pH is 5.3 adds 200 μM of acetosyringone suspension thallines to OD600=1.0, it is prepared into infected liquid, with
Sweet potato embryonal callus suspension cell co-cultures 3 days at 22 DEG C；
Step 3: the embryo callus after co-culturing is transferred to additional 20mg/L hygromycin with aseptic water washing 3 times
It with the screening and culturing medium of 200mg/L cephalosporin, is cultivated in 25 DEG C of illumination (16L/8D), obtains transgenic plant.
The present invention is based on IbmiR319a and its coding nucleic acid molecules, can utilize the core by the method for nucleic acid molecules engineering
The width of acid molecule effectively regulation and control Sweet Potato Leaf changes the length-width ratio of blade, and then changes the leaf morphology of sweet potato,
To the new variety of plant that cultivation width of blade is reduced, length-width ratio increases flesh, shape changes and to the improvement of plant plant type with important meaning
Justice.
Detailed description of the invention
Fig. 1 is the mature body sequence of sweet potato IbmiR319；
(wherein red lines marking part is divided into mature body to the secondary structure schematic diagram that Fig. 2 is sweet potato pre-miR319a
MiR319 is 3 ' flank miRNA)；
Fig. 3 is the expression pattern of quantitative RT PCR analysis IbmiR319 gene (IbActin gene is reference gene)；
Fig. 4 is that miRNA is checked by target gene analogies (Target mimicry) to the shearing of target gene to inhibit
The active method of miRNA, the mimics miR319 carrier structure schematic diagram of building；
Fig. 5 is quantitative RT PCR analysis IbmiR319 gene in mimics miR319 Transgenic Sweet Potato and wild type control
In expression；
Fig. 6 is mimics miR319 Transgenic Sweet Potato compared with the blade of wild type control；
Fig. 7 is mimics miR319 Transgenic Sweet Potato compared with the blade length and width of wild type control.
Specific embodiment
In order to make the objectives, technical solutions and advantages of the present invention clearer, below by accompanying drawings and embodiments, to this
Invention is further elaborated.However, it should be understood that described herein, specific examples are only used to explain the present invention, and
It is not used in and limits the scope of the invention.
Unless otherwise defined, all technical terms and scientific terms used herein are led with technology of the invention is belonged to
The normally understood meaning of the technical staff in domain is identical, and term as used herein in the specification of the present invention is intended merely to retouch
State the purpose of specific embodiment, it is not intended that in the limitation present invention.
A kind of RNA provided by the invention comes with nucleotide sequence shown in SEQ ID NO.1 or SEQ ID No.2
From sweet potato cultivation kind Xu-shu No.22 number (Ipomoea batatas (L.) Lam.cv Xu22), the recombination containing above-mentioned nucleic acid molecules
Carrier, recombinant bacterium are also the scope of the present invention.
Above-mentioned recombinant vector are as follows:
Overexpression target gene analog (target mimic) coding nucleic acid molecule of above-mentioned RNA is inserted into pCABIA1301
Between XbaI the and SalI restriction enzyme site of carrier, the recombinant vector pCAMBIA1301-35S-mimics for expressing above-mentioned RNA is obtained
miR319a；
The application of above-mentioned RNA or above-mentioned recombinant vector or recombinant bacterium in adjusting plant phenotype is also what the present invention protected
Range；In above-mentioned application, the phenotype is embodied in width of blade reduction, and blade length-width ratio increases.
It is a further object to provide a kind of methods for cultivating transglutaminase nucleic acid molecule plant.
For the coding nucleic acid molecule of above-mentioned RNA is imported purpose plant, transglutaminase nucleic acid molecule plant is obtained, the nucleic acid that turns divides
Sub- plant has following at least one feature: 1) width of blade of the transglutaminase nucleic acid molecule plant is less than the purpose plant；2)
The transglutaminase nucleic acid molecule plant leaf blade length-width ratio is greater than the purpose plant.
In the above method, the coding nucleic acid molecule of above-mentioned RNA imports purpose plant by above-mentioned recombinant vector.
In the above method, above-mentioned blade is first climax leaves.
In the above method, the purpose plant is specially the sweet potato of dicotyledon.
The primer pair for expanding the RNA or its any segment also belongs to protection scope of the present invention.
The expression vector for carrying the nucleic acid molecules can be by using standard biologics such as mediated by agriculture bacillus, nucleic acid molecules rifles
Method Transformation of sweet potato embryo callus, and the embryo callus of conversion is cultivated into plant.The purpose plant is double
Cotyledon plant.The dicotyledon concretely sweet potato, such as sweet potato cultivation kind Xu-shu No.22 number.
Embodiment 1
Sweet potato blade profile regulates and controls discovery and the expression analysis of relevant miRNA and its encoding gene.
1) sweet potato blade profile width regulates and controls the discovery of relevant miRNA and its encoding gene, specifically includes the following steps:
According to this laboratory library sweet potato microRNA sequencing result, one obtained, 20 alkali as shown in sequence 1
The nucleotide sequence of base, compared online by miRBase database with other plant miR319a gene very high homology, belong to one
A-miRNA319 family, miRNA family guarded in plant.By degradation group sequencing result obtain as shown in sequence 2
Pre-miRNA sequence compares (http://public-genomes-ngs.molgen.mpg.de/ by online database
SweetPotato/), on No. 15 chromosome of sweet potato genome, homology 100%.
MiRNA319a primary transcript (that is: pri-miRNA319a), is processed by DCL1 etc. containing stem-loop (stem-
Loop) the miRNA precursor pre-IbmiR319a (sequence 2) of structure (their stem-loop structure is as shown in Figure 2)；Using into one
Step processing finally obtains mature miR319a (Fig. 1, nucleotide sequence are sequence 1).
MiRNA is checked to the shearing of target gene by target gene analogies (Target mimic) to inhibit miRNA living
Property method, building closing miR319 function mimic miR319a recombinant plasmid pass through agrobacterium mediation converted sweet potato embryo
Property callus, the width of Transgenic Sweet Potato blade of the inhibition miR319a expression of acquisition is obviously reduced, and blade length-width ratio increases
Add.
2) expression analysis of IbmiR319a gene
In order to study the expression pattern of IbmiR319a gene, it is analyzed in sweet potato cultivation kind Xu's potato by qRT-PCR technology
No. 22 spires, first expansion leaf, climax leaves, tender stem, fibrous root, radix bardanae, the expression in the tissue such as expand root.
The amplimer of IbmiR319a are as follows:
Forward primer: 5 '-ACACTCCAGCTGGGTTGGACTGAAGGGA-3 '；
Reverse primer: 5 '-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGGAGCTC-3 '；
URP:5 '-TGGTGTCGTGGAGTCG-3 '；
The amplimer of reference gene IbActin are as follows:
F:5 '-AGCAGCATGAAGATTAAGGTTGTAGCAC-3 '；
R:5 '-TGGAAAATTAGAAGCACTTCCTGTGAAC-3 '
The fluorescence signal (being indicated with Ct value) of IbmiR319a is believed with the fluorescence of IbActin gene (reference gene) respectively
Number (being indicated with Ct value), according to formula (relative expression quantity=2-ΔCt, wherein Δ Ct=CtTarget gene-CtReference gene) calculated value makees
For the relative expression quantity of IbmiR319a gene.
As a result as shown in Figure 3, show that IbmiR319a includes spire, young stem, expanding at the tender vigorous growth position of children
Expressed in root it is relatively high, have in climax leaves and fibrous root, radix bardanae expression it is lower.
Embodiment 2
The acquisition of the Transgenic Sweet Potato of the mimic IbmiR319a of low expression miR319 and phenotypic analysis.
In order to study the biological function of sweet potato IbmiR319a, checked by target gene analogies (Target mimic)
MiRNA inhibits the active method of miRNA, the mimic miR319a of the low expression miR319 of building to the shearing of target gene
Recombinant plasmid (Fig. 4), and convert into sweet potato cultivation kind Xu-shu No.22 embryo callus.
1) preparation of recombinant plasmid
With arabidopsis IPS1 gene (AF236376.1) for skeleton, the simulation of miR319 target gene is driven under 35S promoter
The binary vector of object low expression miR319.
2) acquisition of Transgenic Sweet Potato
2.1) the mimic miR319a recombinant plasmid transformed Agrobacterium (Agrobactericum for constructing step 1)
Tumefaciens) bacterial strain LBA4404 (Cat.No.18313-015, Invitrogen company) obtains recombinational agrobacterium
LBA4404:pCAMBIA1301-35S-mimics miR319a.Sequencing result is correct.
2.2) the recombinational agrobacterium LBA4404:pCAMBIA1301-35S-mimics miR319a for obtaining step 2.1)
In the embryo callus for converting wild type sweet potato (WT), 7 plants of Transgenic Sweet Potatos are respectively obtained after hygromycin selection culture.
2.3) positive transgenic rice is identified
Above-mentioned Transgenic Sweet Potato blade total DNA is extracted respectively, and the IPS gene in Transgenic Sweet Potato plant is detected by PCR
Whether it is positive that (having on pCAMBIA1301-35S-mimics miR319a carrier) and GUS histochemical staining method primarily determine
Plant.The primer is
IPSF2(5'-GAGGTTATTCATCTTTTAGGG-3')；
IPSR2(5’-GGTCTGACTATTCTCCAAACT-3’)。
Wherein 5 plants of transgenic plants can amplify the IPS genetic fragment that expected length is 500bp, and WT lines are (negative
Property control) do not amplify corresponding band.PCR is analyzed the result shows that 5 plants of positives, which are obtained, turns LBA4404:pCAMBIA1301-
35S-mimics miR319a sweet potato.GUS histochemical staining method result is consistent with PCR result.
3) the qRT-PCR identification of the mimics IbmiR319a Transgenic Sweet Potato of low expression miR319
Respectively extract number be 2,3,8,9 and 10 turn mimics miR319a sweet potato (m3-8, m3-9) and wild type is sweet
The total serum IgE of potato (WT) carries out qRT-PCR identification using stem-loop method, and the primer is specific as follows:
319aF:5 '-ACACTCCAGCTGGGTTGGACTGAAGGGA-3 '；
319aRT:5 '-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGGAGCTC-3 '；
URP:5 '-TGGTGTCGTGGAGTCG-3 '；
The amplimer of reference gene IbActin are as follows:
F:5 '-AGCAGCATGAAGATTAAGGTTGTAGCAC-3 '；
R:5 '-TGGAAAATTAGAAGCACTTCCTGTGAAC-3 '
For identifying that the primer pair of IbmiR319a is the primer pair that 319aF and 319aRT is formed, target sequence about 170bp.
For identifying that the primer pair of IbActin gene is the primer pair that F and R is formed, target sequence about 135bp.
The fluorescence signal (being indicated with Ct value) of IbmiR319a is believed with the fluorescence of IbActin gene (reference gene) respectively
Number (being indicated with Ct value) is according to formula (relative expression quantity=2-ΔCt, wherein Δ Ct=CtTarget gene-CtReference gene) calculated value makees
For the relative expression quantity of IbmiR319a gene.
As a result as shown in figure 5, being the opposite table of 1, m3-2, m3-3, m3-8, m3-9 and m3-10 with wild type sweet potato Δ Ct
Up to amount 0.582,0.722,0.188,0.104,0.158, the results showed that, number 2,3,8,9 and 10 turn
The expression quantity of IbmiR319a gene is substantially reduced in mimicsmiR319a sweet potato, it is believed that the reduction of Transgenic Sweet Potato width of blade,
The increased phenotype of length-width ratio is caused by driving the low expression of lower IbmiR319a as 35S promoter.
Four, the research of Transgenic Sweet Potato phenotype
The positive is turned into the plantation of pCAMBIA1301-35S-mimics miR319a sweet potato in Jiangsu Normal University's life science
Institute planting base, Routine Management.Implantation time is June to November.It is control with wild type sweet potato (WT).
The length and width of the climax leaves of each strain is observed in early July, as a result as shown in Figure 6.
It counts the width of above-mentioned transgenic plant climax leaves, wild type sweet potato and turns pCAMBIA1301-35S-
The width of mimicsmiR319a sweet potato first climax leaves of strain m3-8, m3-9 be respectively 15.97mm, 11.81mm,
10.34mm illustrates that the width of the blade of above-mentioned genetically modified plants significantly reduces (Fig. 7 A) relative to the generation of wild type；
It counts the length of above-mentioned transgenic plant climax leaves, wild type sweet potato and turns pCAMBIA1301-35S-
The length of mimicsmiR319a sweet potato first climax leaves of strain m3-8, m3-9 is respectively 15.66mm, 15.34mm,
14.01mm, illustrate the length of the blade of above-mentioned genetically modified plants relative to wild type length of blade without significant change (Fig. 7 B)；
The length-width ratio of above-mentioned transgenic plant climax leaves is counted, with the length-width ratio of first climax leaves of wild type sweet potato for 1,
The length-width ratio of first climax leaves of transgenic line m3-8, m3-9 is 1.36；Illustrate the length of the blade of above-mentioned genetically modified plants
Width significantly increases (Fig. 7 C) than the generation relative to wild type.
Present invention finds Ib-miR319a and its encoding genes, and it is low to construct 35S promoter driving target gene analogies
The binary vector for expressing IbmiR319a is disseminated sweet potato embryonal callus using mediated by agriculture bacillus, is obtained by screening, breaking up
Transgenic positive plant is identified by the phenotype to rotaring gene plant blade, shows miR319a in each transgenic line
Expression quantity is significantly lowered, and the width of each transgenic line blade significantly reduces, and blade length-width ratio obviously increases.
Sequence table
<110>Jiangsu Normal University
<120>for the miRNA of the leaf regulation of sweet potato and coding nucleic acid molecule and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> RNA
<213>sweet potato cultivation kind Xu-shu No.22 number (sweet potato cv. Xushu 22)
<400> 1
uuggacugaa gggugcuccc 20
<210> 2
<211> 177
<212> RNA
<213>sweet potato cultivation kind Xu-shu No.22 number (sweet potato cv. Xushu 22)
<400> 2
aagagagctt tcttcagtcc actcatgggt agaagtagga ttcaattagc ttctggctca 60
ttcatccaaa tgctgagatg aactgcatgg aaaagtctct gtaactgagt gaatgatatg 120
ggagacaagt tggatcctaa tcttcctgta cttggactga agggagctcc cttttct 177
Claims (6)
1. a kind of miRNA for the leaf regulation of sweet potato, which is characterized in that it is with SEQ ID NO.1 or SEQ ID No.2 institute
The nucleotide sequence shown.
2. a kind of expression vector pCAMBIA1301-35S-mimics miR319a containing miRNA described in claim 1.
3. a kind of Agrobacterium host cell EHA105:pCAMBIA1301-35S- containing expression vector described in claim 2
mimics miR319a。
4. Agrobacterium host cell LBA4404:pCAMBIA1301-35S-mimics miR319a described in a kind of claim 3
Construction method.
5. expression vector described in a kind of claim 2 obtains the application in transgenic plant in conversion plant.
6. a kind of expression vector according to claim 5 obtains the application in transgenic plant, feature in conversion plant
It is, the plant is the embryo callus of sweet potato stem tip induction, and detailed process is as follows:
Step 1: being inoculated into positive colony in the 50mlYEB fluid nutrient medium containing 100 μ g/ml Kan, 28 DEG C of 200rpm continue
It cultivates to OD600Then=0.5-1.0 is centrifuged 10 minutes for 6000rpm4 DEG C, abandons culture solution, collects thallus；
Step 2: 6000rpm4 DEG C is centrifuged 10 minutes, then is with pH after cleaning thallus with the 50ml MS culture solution that pH is 5.3
5.3 20ml MS culture solution adds 200 μM of acetosyringone suspension thallines to OD600=1.0, it is prepared into infected liquid, with sweet potato
Embryo callus suspension cell co-cultures 3 days at 22 DEG C；
Step 3: co-culture after embryo callus, with aseptic water washing 3 times, be transferred to additional 20mg/L hygromycin and
The screening and culturing medium of 200mg/L cephalosporin is cultivated in 25 DEG C of illumination (16L/8D), obtains transgenic plant.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114958845A (en) * | 2022-04-24 | 2022-08-30 | 中国农业科学院作物科学研究所 | Application of miR319-TaGAMYB3 module in regulating wheat plant type and increasing yield |
| NL2032335B1 (en) * | 2021-12-03 | 2023-06-21 | Xuzhou Institute Of Agricultural Sciences In Jiangsu Xuhuai District Jiangsu Xuzhou Sweetpotato Res | Use of sweet potato ibsap15 gene in regulating leaf shape and flower shape of sweet potato |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361348A (en) * | 2012-03-29 | 2013-10-23 | 中国科学院遗传与发育生物学研究所 | Rice leaf width adjustment and control related microRNA (ribonucleic acid) and encoded nucleic acid molecule thereof as well as application |
| CN104004071A (en) * | 2014-05-20 | 2014-08-27 | 中国农业大学 | Plant salt tolerance-related protein IbERD3 as well as encoding gene and application of plant salt tolerance-related protein IbERD3 |
| CN106701756A (en) * | 2015-11-16 | 2017-05-24 | 华中农业大学 | Application of microRNA172 to regulation of plant height, ear type and leaf size of rice |
-
2019
- 2019-07-10 CN CN201910620077.1A patent/CN110331143B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361348A (en) * | 2012-03-29 | 2013-10-23 | 中国科学院遗传与发育生物学研究所 | Rice leaf width adjustment and control related microRNA (ribonucleic acid) and encoded nucleic acid molecule thereof as well as application |
| CN104004071A (en) * | 2014-05-20 | 2014-08-27 | 中国农业大学 | Plant salt tolerance-related protein IbERD3 as well as encoding gene and application of plant salt tolerance-related protein IbERD3 |
| CN106701756A (en) * | 2015-11-16 | 2017-05-24 | 华中农业大学 | Application of microRNA172 to regulation of plant height, ear type and leaf size of rice |
Non-Patent Citations (3)
| Title |
|---|
| FRANCO-ZORRILLA ET AL.: "Target mimicry provides a new mechanism for regulation of microRNA activity", 《NATURE GENETICS》 * |
| MARLENE REICHEL ET AL.: "Specificity of plant microRNA target MIMICs: Cross-targeting of miR159 and miR319", 《JOURNAL OF PLANT PHYSIOLOGY》 * |
| TOMOTSUGU KOYAMA ET AL.: "Roles of miR319 and TCP Transcription Factors in Leaf Development", 《PLANT PHYSIOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL2032335B1 (en) * | 2021-12-03 | 2023-06-21 | Xuzhou Institute Of Agricultural Sciences In Jiangsu Xuhuai District Jiangsu Xuzhou Sweetpotato Res | Use of sweet potato ibsap15 gene in regulating leaf shape and flower shape of sweet potato |
| CN114958845A (en) * | 2022-04-24 | 2022-08-30 | 中国农业科学院作物科学研究所 | Application of miR319-TaGAMYB3 module in regulating wheat plant type and increasing yield |
| CN114958845B (en) * | 2022-04-24 | 2023-07-21 | 中国农业科学院作物科学研究所 | Application of miR319-TaGAMYB3 module in regulation and control of wheat plant types and increase of yield |
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