CN106146633B - The application of EPFL1 albumen and its encoding gene in plant fertility - Google Patents
The application of EPFL1 albumen and its encoding gene in plant fertility Download PDFInfo
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Abstract
The invention discloses the application of EPFL1 albumen and its encoding gene in plant fertility.The present invention utilizes EPFL1 gene expression in antisense nucleic acid silencing arabidopsis body, obtained transgenic arabidopsis stamen becomes smaller, silique shortens, ovule number reduces, it is all with important application prospects that male sterile line, the acceptor material for creating human controllable's male sterility and GMO bio-safety etc. are obtained to proving that EPFL1 gene can regulate and control the fertility of plant, in breeding in agricultural production, seeding procedure.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to EPFL1 albumen and its encoding gene answering in plant fertility
With.
Background technique
The formation of plant male gametophyte pollen includes the generation of microspore and two processes of formation of andro gamete.Microspore
This process occurs all to carry out in immature anther, the formation including microsporocyte, meiosis process and four points
The dispersion process of body.Male gametophyte, that is, pollen grain is that microspore is formed by mitosis twice, wherein comprising being referred to as hero
The spermatid of gamete.It is related to numerous related genes in this serial procedures of pollen development, only they are according to certain
Sequence of time and space normal expression just can guarantee the formation of fertile pollen.
The male sterility in China is studied and is utilized and maintains the leading position in the world.Modern cell biology is applied in recent years
Technology, science of heredity and molecular biology method have done deep grind to the male sterility process of model plant rice and arabidopsis
Study carefully, achieves some new results of study, such as: Ems1, CER1, AtGSL2 gene and rice UDT1, MSP1 in arabidopsis,
GAMYB, UDPG, WDA1 gene etc., the fertility that the mutation of said gene directly results in plant reduce, the complete infertility having.
Ems1 encodes a receptor kinase gene, which is primarily involved in the development of pollen mother cell, tapetum；UDT1 gene is small spore
The key gene of son development, the gene mainly act on Meiosis, it is female for the development of tapetal cell, microspore
The meiosis of cell and the degradation in middle layer are worked；GAMYB gene is close to tapetal cell for microsporocyte and is inhaled
It is required for receiving nutrition and then carrying out normal meiosis；WDA1 gene is required for the formation of paddy pollen wall wax coat
's.Understanding to plants male sterility mechanism has been deepened to their research.
Summary of the invention
It is an object of the present invention to provide the purposes of the substance for the encoding gene expression for inhibiting EPFL1 albumen.
The present invention provides the substances for the encoding gene expression for inhibiting EPFL1 albumen to reduce the application in plant fertility.
In above-mentioned application, the amino acid sequence of the EPFL1 albumen is sequence 5 in sequence table.
In above-mentioned application, the substance of the encoding gene expression for inhibiting EPFL1 albumen is following 1) -3):
1) DNA fragmentation as shown in sequence 2 in sequence table；
2) miRNA encoded as the DNA fragmentation described in 1)；
3) contain 1) described in DNA fragmentation recombinant vector.
In above-mentioned application, the recombinant vector is by the DNA fragmentation and the promoter of its expression to be driven to be inserted into table jointly
Up to carrier obtained in carrier.
In above-mentioned application, the expression vector is pCAMBIA2300；The nucleotides sequence of the promoter is classified as in sequence table
Sequence 3.
In above-mentioned application, the recombinant vector is specially by the Pst I of DNA fragmentation replacement pCAMBIA2300 carrier
DNA fragmentation between Hind III digestion site, and by promoter shown in sequence 3 replacement pCAMBIA2300 Kpn I and
The carrier that DNA fragmentation between Sal I restriction enzyme site obtains.
In above-mentioned application, the reduction plant fertility, which is embodied in, makes plant stamen become smaller and/or silique shortens and/or ovule
Number reduces.
In above-mentioned application, the plant is monocotyledon or dicotyledon；The dicotyledon is specially quasi- south
Mustard.
It is a further object to provide a kind of methods that cultivation fertility reduces or cultivates sterile plants.
The method provided by the invention cultivated fertility reduction or cultivate sterile plants includes the following steps: to inhibit
The substance of the encoding gene expression of EPFL1 albumen imports in purpose plant, obtains genetically modified plants, the genetically modified plants educate
Property be lower than the purpose plant.
In the above method, the substance of the encoding gene expression for inhibiting EPFL1 albumen is EPFL1 RNAi segment；
The nucleotides sequence of the EPFL1 RNAi segment is classified as sequence 2 in sequence table；
The EPFL1 RNAi segment is by importing purpose plant in recombinant vector；
The recombinant vector is that will obtain in the EPFL1 RNAi fragment inserting expressioning carrier as shown in sequence 2 in sequence table
Carrier.
In the above method, the purpose plant is monocotyledon or dicotyledon；The dicotyledon is specially
Arabidopsis.
Final object of the present invention is to provide a kind of DNA molecular.
DNA molecular provided by the invention be it is following 1) or 2):
1) sequence is the DNA molecular of sequence 2 in sequence table；
2) under strict conditions with the DNA molecular that 1) hybridizes and encode same protein.
The present invention is using EPFL1 gene expression in antisense nucleic acid silencing arabidopsis body, obtained transgenic arabidopsis stamen
Become smaller, silique shortens, ovule number reduce, to prove that EPFL1 gene can regulate and control the fertility of plant, in agricultural production
The receptor for obtaining male sterile line in breeding, seeding procedure, creating human controllable's male sterility and GMO bio-safety
Material etc. is all with important application prospects.
Detailed description of the invention
Fig. 1 is that RNAi segment building primer matches schematic diagram.
Fig. 2 is pEPFL1::EPFL1 RNAi recombinant plasmid map.
Fig. 3 is that the expression quantity of transgenic line 2#, 3#, 4#, 8#, 9# are identified.
Fig. 4 is the stamen fertility that Alexandria decoration method observes transgenic line 2#, 3#, 4#, 8#, 9#.
Fig. 5 is that the silique of transgenic plant 3#, 4# observes result.
Fig. 6 is the ovule number of transgenic plant 3#, 4#.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
PRS300 plasmid (5ng/ul) is in document " Highly specific gene silencing by artificial
microRNAs in Arabidopsis.Schwab R,Ossowski S,Riester M,Warthmann N,Weigel
D.Plant Cell.2006 May.18 (5): being disclosed in 1121-33. ", and the public can obtain from Peking University's Life Science College
?.
PQGIE110 plasmid is in document " Expression and functional analysis of the rice
plasma-membrane intrinsic protein gene family.Lei Guo,Zi Yi Wang,Hong Lin,Wei
Er Cui,Jun Chen,Meihua Liu,Zhang Liang Chen,Li Jia Qu and Hongya Gu.Cell
It is disclosed in Research.2006.March.16 (16) 277-286. ", the public can obtain from Peking University's Life Science College.
PCAMBIA2300 plasmid is Beijing DingGuo ChangSheng Biology Technology Co., Ltd, catalog number MCV036.
The acquisition of embodiment 1, pEPFL1::EPFL1 RNAi transgenic arabidopsis
One, the acquisition of pEPFL1::EPFL1 RNAi recombinant plasmid
1, EPFL1 RNAi segment designs
Website WMD3 (http://wmd3.weigelworld.org/cgi-bin/ is designed using online RNAi segment
Webapp.cgi the EPFL1 RNAi segment that following primer is used to construct EPFL1 (At5g10310)) is designed, primer sequence is as follows:
A:5′-CTGCAAGGCGATTAAGTTGGGTAAC-3′；
B:5′-CTGTTTCCTGTGTGAAATTGTTATCCGC-3′；
I (miR-s): 5 '-GACGACCACTTTTATTATCCTTTTCTCTCTTTTGTATTCC-3 '；
II (miR-a): 5 '-GAAAAGGATAATAAAAGTGGTCGTCAAAGAGAATCAATGA-3 '；
III (miR*s): 5 '-GAAACGGATAATAAATGTGGTCGTCACAGGTCGTGATATG-3 '；
IV (miR*a): 5 '-GACGACCACATTTATTATCCGTTTCTACATATATATTCCT-3 '.
2, the acquisition of EPFL1 RNAi segment
(1) with pRS300 plasmid (5ng/ul) for template, PCR is carried out according to primer matching method shown in FIG. 1, is obtained
(a), (b), (c) three intermediate PCR products.
PCR reaction system (50ul): 2xKOD Mix 25ul, pRS300 (1:100) 2ul, upstream primer 2ul, downstream are drawn
Object 2ul, KODase 1ul, ddH2O supplies 50ul.
PCR reaction condition: 95 DEG C 5 '；94 DEG C 30 ", 58 DEG C 30 ", 68 DEG C of 40 ", 24cycles；68℃10'.
(2) add 5ul (10X) loading into three intermediate PCR products of (a), (b), (c) that above-mentioned steps (1) obtains
Buffer, with the separation of 2% agarose gel electrophoresis method for detecting, purifying, recycling, adjustment concentration is 5ng/ul；With (a), (b), (c) three
A intermediate PCR product is template, carries out PCR reaction, obtains PCR product.
PCR reaction system (50ul): 2xKOD Mix 25ul, OligoA-SmaI 2ul, oligoB-SacI 2ul,
KODase 1ul、(a)0.5ul、(b)0.5ul、(c)0.5ul、ddH2O supplies 50ul.
PCR reaction condition: 95 DEG C 5 '；94 DEG C 30 ", 58 DEG C 30 ", 68 DEG C of 40 ", 24cycles；68℃10'.
(3) add 5ul (10X) loading buffer in the PCR product obtained to step (2), with 2% Ago-Gel electricity
The separation of swimming method, purifying, recycling obtain the Human disturbance that the EPFL1 RNAi template (d) that size is 701bp is EPFL1
MiR-96 gene segment (as shown in sequence 1 in sequence table).
(4) double enzymes are carried out to the EPFL1 RNAi template that above-mentioned steps (3) obtain with Restriction enzyme Sma I and Sac I
It cuts, obtains the DNA fragmentation that size is 701bp；Double enzymes are carried out to pQGIE110 plasmid with Restriction enzyme Sma I and Sac I
It cuts, obtains skeleton carrier large fragment.
(5) DNA fragmentation obtained above is connected with T4 ligase with skeleton carrier large fragment, obtains recombinant vector.Even
It is as follows to connect reaction system: 10x T4ase Buffer 1ul, T4ase 1ul, DNA fragmentation 2ul, skeleton carrier 1ul, ddH2O
5ul.16 DEG C of constant temperature connection reaction 16h.
(7) DH5 α bacterial strain, screening are converted using the recombinant vector that Escherichia coli thermal shock conversion method obtains above-mentioned steps (6)
Obtain positive colony.
(8) Oligo A and EPFL1-RNAi-a-Hind III primer are used, the positive colony obtained with above-mentioned steps (7)
PCR amplification is carried out for template, obtaining the DNA fragmentation as shown in sequence 2 in sequence table is EPFL1 RNAi segment.
Oligo A:5 '-CCCTGCAAGGCGATTAAGTTGGGTAAC-3 '；
EPFL1-RNAi-a-Hind III:5 '-TCCCCAAGCTTGGCCGCTCTAGAACTAGT-3 '.
3, the acquisition of pEPFL1::EPFL1 RNAi recombinant plasmid
(1) with restriction enzyme Pst I and Hind III to the EPFL1 RNAi segment of above-mentioned acquisition and
PCAMBIA2300 plasmid carries out double digestion, connects (the same above-mentioned steps of reaction system (5)) with T4 ligase, obtains containing EPFL1
The recombinant plasmid of RNAi segment.
(2) pEPFL1-Kpn-lp and pEPFL1-Sal-rp primer is used, EPFL1 coding is cloned from arabidopsis gene group
The DNA fragmentation (as shown in sequence 3 in sequence table) in area, the region 2kb, upstream carries out base in situ as driving EPFL1 RNAi segment
Because of the promoter of silencing.Primer sequence is following (sequence of underscore represents restriction enzyme site):
PEPFL1-Kpn-lp:5 '-CCGGGGTACCTGTGAAAGTCTTCTCTTTCTG-3'；
PEPFL1-Sal-rp:5 '-TCGCGTCGAC TAAAGGAGGAGCTTCATGTGG-3’。
(3) promoter and step (1) acquisition that above-mentioned steps (2) are obtained using restriction enzyme Kpn I and Sal I
The segment of RNAi containing EPFL1 recombinant plasmid carry out double digestion, with T4 ligase connect, obtain pEPFL1: as shown in Figure 2:
EPFL1 RNAi recombinant plasmid, and sequence verification is carried out to it.
Sequencing result shows: pEPFL1::EPFL1 RNAi recombinant plasmid is by EPFL1 RNAi segment shown in sequence 2
The DNA fragmentation between the Pst I and Hind III digestion site of pCAMBIA2300 carrier is replaced, and by promoter shown in sequence 3
Replace the carrier that the DNA fragmentation between Kpn I and Sal the I restriction enzyme site of pCAMBIA2300 obtains.
Two, the acquisition of transgenic plant
The pEPFL1::EPFL1 RNAi recombinant plasmid that step 1 is obtained, it is electroporated to arrive Agrobacterium GV3101 bacterial strain
In, the Agrobacterium containing pEPFL1::EPFL1 RNAi recombinant plasmid is obtained, then will be contained using titbit infusion method
The Agrobacterium infection wildtype Arabidopsis thaliana (Columbia) of pEPFL1::EPFL1 RNAi recombinant plasmid.Using containing 100ug/mL
The solidified MS media screening transgenic T of kanamycin sulfate0The seed in generation obtains 18 plants of transgenosis T1For plant, and use RT-
PCR method is to 18 plants of transgenosis T1It is detected for the EPFL1 expression quantity of plant.
Testing result is as shown in Figure 3: comparing with control, the EPFL1 expression quantity of transgenic line 2#, 3#, 4#, 8#, 9# are bright
Aobvious decline, chooses transgenic line 2#, 3#, 4#, 8#, and 9# strain is used for further phenotypic analysis.
1, Agrobacterium electric shock transformation method is as follows:
(1) the electroporated competence preparation of Agrobacterium
A, recovery agrobacterium strains, from -80 DEG C of picking agrobacterium strains GV3101 in 5ml sulfur acid gentamicin and Li Fu
In the LB culture medium of flat resistance, 28 DEG C, 220rpm activation is overnight；
B, expand culture, the GV3101 of activation is accessed into 800ml sulfur acid gentamicin and rifampin in 1:1000 ratio
In the LB culture medium of resistance, 28 DEG C, 220rpm is cultivated to OD550 ≈ 1.0；
C, bacterium solution is divided into two bottles, every bottle of 400ml, 4 DEG C by sterile working, and 4000 × g is centrifuged 10min, abandons supernatant, and every bottle
10% glycerol is pre-chilled with 200ml, thallus is resuspended；
D, sterile working, 4 DEG C, 4000 × g is centrifuged 10min, abandons supernatant, and every bottle is pre-chilled 10% glycerol with 15ml and bacterium is resuspended
Body；
E, sterile working, 4 DEG C, 4000 × g is centrifuged 10min, abandons supernatant；10% glycerol is pre-chilled with 0.5ml, thallus is resuspended, eventually
Volume 0.75ml or so, immediately using or be packed as freezing for -80 DEG C after the every pipe of 50ul.
(2) electroporated
A, plasmid to be transformed is extracted, concentration is at least up to 20ng/ μ l；
B, -80 DEG C of 50 μ l of the Agrobacterium competence frozen are dissolved on ice, while Bio-Rad 0.2cm electric shock cup is pre-chilled；
C, it operates on ice, takes 100ng Plasmid DNA in Agrobacterium competence, flick tube wall mixing；
D, Bio-Rad GenePulser XcellTM PC Module electric shock instrument V=2.4kv, C=25 μ F, PC=are set
200ohm, shock by electricity cup parameter 0.2cm；
E, electric shock bottom of a cup portion is added in the plasmid competence mixed liquor in step 3；
F, electric shock cup is put into electric shock tank, is shocked by electricity；
G, it is rapidly added 1ml room temperature LB culture medium after electric shock, is transferred in 1.5ml EP pipe；
H, 28 DEG C, 220rpm recovery 3h takes 200ul to be applied to sulfur acid gentamicin, rifampin, kanamycin sulfate resistance
LB solid medium on, 28 DEG C of back-off plate screenings grind positive bacterium colony.
2, transfection method is as follows:
(1) agrobacterium strains containing target gene of -80 DEG C of picking preservations, transfer into 5ml sulfur acid gentamicin, Li Fu
Flat, the LB culture medium of kanamycin sulfate, 28 DEG C of 220rpm activation are overnight；
(2) bacterium solution activated is transferred into the LB culture medium of the corresponding resistance of 500ml with the ratio of 1:100,28 DEG C,
220rpm is cultivated to OD600=1.2-1.6；
(3) room temperature is centrifuged 4000rpm, 10min, abandons supernatant；
(4) with conversion buffer (conversion buffer formulation (1L): MS powder (Sigma of equal volume (500ml)
M5519) 2.2g, sucrose 50g, use KOH tune pH=5.7,1mg/mL 6-benzyl aminopurine (6-BA) 10 μ L, Silwet L-77
200 μ L) thallus is resuspended；
(5) it takes bolting to reach wild type (Columbia) arabidopsis of 10-15cm, watering to saturation, while wiping out completion and awarding
The flower and silique of powder only retain the titbit of non-flowering parts；
(6) above-ground plant parts is inverted in conversion buffer.In the Agrobacterium of resuspension, guarantee plant basal leaf with top
Divide and be all immersed in bacterium solution, infects 8min (can continuously infect 3 basin plant with portion conversion fluid)；
(7) plant is taken out, flowerpot side is put and is protected from light culture for 24 hours；
(8) according to regular culture conditions culture (attention can not water to above-ground plant parts)；
T is collected after silique is mature0For seed；
(9) to T0It after carrying out sterilization treatment for seed, plants on resistant MS plate, screens T1For positive plant.
Embodiment 2, the phenotypic evaluation of transgenic line
One, stamen
Using Alexandria decoration method to T1 for transgenic line 2#, 3#, 4#, the stamen of 8#, 9# carry out staining analysis, with
Wild-type Arabidopsis plants (Col) are control.Alexandria colouring method is as follows:
The Alexandria 200uL dyeing liquor is drawn in 1.5mL EP pipe, the little Hua of suitable time is taken to be completely immersed in dyeing,
Room temperature dyes 8h or more.The little Hua in dye liquor is taken out, dissects out by stamen under stereoscope, be placed on glass slide, is fitted in drop
Measure transparent liquid, coverslip mounting, transparent 8~for 24 hours after, microscopically observation is taken pictures.
Alexandria dyeing liquor be by 95% ethyl alcohol 10mL, 1% peacock green (preparation of 95% ethyl alcohol) 1mL, distilled water 50mL,
Glycerol 25mL, phenol 5g, chloral hydrate 5g, 1% acid fuchsin 5mL, 1% orange G (Orange G) 0.5mL and glacial acetic acid
1mL (pollen of loose powder need to use 4mL not from anther) is uniformly mixed so as to obtain.
Transparent liquid is to be uniformly mixed so as to obtain trichloroacetaldehyde, glycerol, water according to the volume ratio of 8:1:2.
As a result as shown in Figure 4: it is compared as a whole with wild-type Arabidopsis plants (Col), transgenic line 2#, 3#,
The stamen of 4#, 8#, 9# become smaller, and the development of some coyote holes is abnormal.
Two, silique
Transgenic line 3# and 4# is chosen to be further analyzed silique.It is with wild-type Arabidopsis plants (Col)
Control.
As a result as shown in Figure 5: the fertility of transgenic plant is decreased obviously, wherein all there is the silique of abortion, 3# is more bright
It is aobvious, but be not complete stool stamen abortion, it can also bear some shorter siliques.
These siliques are further looked at, the silique of transgenic plant obviously shortens for wild type,
In, transgenic line 3# silique is most short, and the silique of 4# is slightly long, but still shorter than wild type.Fertility is remarkably decreased.
Three, ovule
The silique for dissecting transgenic line 3# and 4#, observes the ovule in silique.With wild-type Arabidopsis plants
It (Col) is control.
As a result as shown in Figure 6: transgenic plant only have part ovule can normal development, there are abortion feelings in a large amount of ovules
Condition counts the ovule number of these plant, and the ovule number of discovery transgenic line 3# and 4# are remarkably decreased, transfer
The 50% or less of wild type is downgraded under ovule number contained by each silique of gene strain 3#.
Claims (6)
1. inhibiting the substance of the encoding gene expression of EPFL1 albumen is reducing the application in plant fertility；The EPFL1 albumen
Amino acid sequence is sequence 5 in sequence table；The reduction plant fertility, which is embodied in, makes that plant stamen becomes smaller and/or silique shortens
And/or ovule number reduces；The plant is arabidopsis.
2. application according to claim 1, it is characterised in that: the object of the encoding gene expression for inhibiting EPFL1 albumen
Matter is following 1) -3):
1) DNA fragmentation as shown in sequence 2 in sequence table；
2) miRNA encoded as the DNA fragmentation described in 1)；
3) contain 1) described in DNA fragmentation recombinant vector.
3. application according to claim 2, it is characterised in that: the recombinant vector is by the DNA fragmentation and to drive it
The promoter of expression is inserted into carrier obtained in expression vector jointly.
4. application according to claim 3, it is characterised in that: the expression vector is pCAMBIA2300；The promoter
Nucleotides sequence be classified as sequence 3 in sequence table.
5. a kind of method for cultivating fertility and reducing or cultivating sterile plants, includes the following steps: that the volume of EPFL1 albumen will be inhibited
The substance of code gene expression imports in purpose plant, obtains genetically modified plants, and the fertility of the genetically modified plants is lower than the mesh
Plant；The fertility reduction, which is embodied in, makes plant stamen become smaller and/or silique shortens and/or ovule number reduces；The plant
Object is arabidopsis；The amino acid sequence of the EPFL1 albumen is sequence 5 in sequence table.
6. according to the method described in claim 5, it is characterized by: the object of the encoding gene expression for inhibiting EPFL1 albumen
Matter is EPFL1 RNAi segment；
The nucleotides sequence of the EPFL1 RNAi segment is classified as sequence 2 in sequence table；
The EPFL1 RNAi segment is by importing purpose plant in recombinant vector；
The recombinant vector is that will carry obtained in the EPFL1 RNAi fragment inserting expressioning carrier as shown in sequence 2 in sequence table
Body.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103304653A (en) * | 2013-07-11 | 2013-09-18 | 北京大学 | Application of arabidopsis ERF protein and coding gene of arabidopsis ERF protein for regulating and controlling plant pollen fertility |
| CN104292319A (en) * | 2014-09-18 | 2015-01-21 | 中国农业科学院生物技术研究所 | Application of OsGSL5 protein in controlling plant fertility |
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| CN103304653A (en) * | 2013-07-11 | 2013-09-18 | 北京大学 | Application of arabidopsis ERF protein and coding gene of arabidopsis ERF protein for regulating and controlling plant pollen fertility |
| CN104292319A (en) * | 2014-09-18 | 2015-01-21 | 中国农业科学院生物技术研究所 | Application of OsGSL5 protein in controlling plant fertility |
Non-Patent Citations (1)
| Title |
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| Generation of Signaling Specificity in Arabidopsis by Spatially Restricted Buffering of Ligand–Receptor Interactions;Emily B. Abrash et al.;《Plant Cell》;20110823;1-16 |
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