CN110468144A - Flavine monooxygenase FMO1 gene order, application and the method for regulating and controlling plant leaf blade ageing process - Google Patents
Flavine monooxygenase FMO1 gene order, application and the method for regulating and controlling plant leaf blade ageing process Download PDFInfo
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- CN110468144A CN110468144A CN201910639848.1A CN201910639848A CN110468144A CN 110468144 A CN110468144 A CN 110468144A CN 201910639848 A CN201910639848 A CN 201910639848A CN 110468144 A CN110468144 A CN 110468144A
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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
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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- C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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- C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
- C12Y114/13008—Flavin-containing monooxygenase (1.14.13.8), i.e. dimethylaniline-monooxygenase
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Abstract
The invention belongs to gene order field of engineering technology, for the aging of Effective Regulation blade, the invention discloses a kind of flavine monooxygenase FMO1 gene order, application and the methods for regulating and controlling plant leaf blade ageing process, and the FMO1 gene order is as shown in SEQ ID NO:1.The method of the present invention being capable of Effective Regulation plant leaf blade aging.
Description
Technical field
The present invention relates to gene order field of engineering technology more particularly to a kind of flavine monooxygenase FMO1 gene order,
Using and regulation plant leaf blade ageing process method.
Background technique
The present invention belongs to the relevant technologies related to the present invention for the description of background technique, be only used for explanation and just
In understanding summary of the invention of the invention, it should not be construed as applicant and be specifically identified to or estimate applicant being considered of the invention for the first time
The prior art for the applying date filed an application.
Blade is the important place that plant utilizes luminous energy anthropogenics, the efficiency and duration of photosynthetic function
It is of great significance to agricultural production.On crop genetic improvement, people very focus on improving the photosynthetic effect of blade for a long time
Rate, and to how by directly adjust the photosynthetic function phase and improve nutriment transfer efficiency come improve crop yield and
Quality concern is less.The adjusting of the latter is substantially exactly the regulation to Leaf senescence development.Plant leaf blade aging course is one
The process of cell death of sequencing, by stringent genetic regulation.Leaf senile is a complicated process, by a variety of inside and outside
The influence of the source factor.
How the aging of Effective Regulation blade is current urgent problem to be solved.
Summary of the invention
The purpose of the embodiment of the present invention is that providing a kind of flavine monooxygenase FMO1 gene order, application and regulation leaves of plants
The method of piece ageing process, the method for the present invention can effectively adjust the aging of plant leaf blade.
The purpose of the embodiment of the present invention is that be achieved through the following technical solutions:
The embodiment of first aspect present invention provides a kind of flavine monooxygenase FMO1 gene order, the FMO1 gene
Sequence is as shown in SEQ ID NO:1.
The embodiment of second aspect of the present invention provides a kind of flavine monooxygenase FMO1 gene order answering in plant
It for regulation plant leaf blade ageing process and/or is cultivated evening with, the described application and declined high-yield transgenic sequence plant variety, it is described
FMO1 gene order is as shown in SEQ ID NO:1.
Further, the plant is arabidopsis.
The embodiment of third aspect present invention provides a kind of method of regulation plant leaf blade ageing process, including regulation
Expression of the FMO1 gene order in plant, the FMO1 gene order is as shown in SEQ ID NO:1.
Further, include the following steps:
(1) recombinant expression carrier is constructed, the particular target sequence of gene order shown in SEQ ID NO:1 is entered thin to eukaryon
In cellular expression carrier；
(2) recombinant expression carrier is transformed into the cell of purpose plant, knocks out FMO1 gene order；
(3) resistance plant is screened, the late plant that declines is obtained.
Further, the eukaryotic expression vector is pCAMBIA1300.
Further, the conversion uses agrobacterium-mediated transformation.
Further, the plant is arabidopsis.
Compared with prior art, flavine monooxygenase FMO1 gene order of the present invention, application and regulation plant leaf blade aging
The method of process at least has the following beneficial effects:
The present invention by using exogenous gene sequence knockout carrier by coded sequence be SEQ ID NO:1 shown in FMO1 base
Because sequence knocks out in plant, plant shows as leaf senile and delays.The present invention is by regulation FMO1 gene order in plant
In expression so that be used to cultivate that leaf senile to be regulatable, high yield new variety of plant, the crop varieties to cultivate new provide
New approaches have great application value on high yield molecular breeding.
The present invention is overexpressed transgenic sequence library by screening arabidopsis gene sequence, obtains a leaf presenility mutant
(FMO1ox), FMO1 gene order encodes a kind of flavine monooxygenase.FMO1 gene order has in regulation plant leaf blade aging
It plays an important role and new crop varieties is cultivated by transgenic sequence Delaying Leaf-Senescence process and provide new approaches.
Detailed description of the invention
Fig. 1 be CRISPR carrier psgR-Cas9 (on) and eukaryotic expression vector pCAMBIA1300 carrier (under) figure
Spectrum.Carrier psgR-Cas9 contains two multiple cloning sites of EcoRI and HindIII, and LB indicates the left side in pCAMBIA1300 figure
Boundary, RB indicate right margin, and EcoRI-KpnI-BamHI-XbaI-HindIII is multiple cloning sites；
Fig. 2 is the PCR testing result figure that FMO1 of the present invention knocks out plant (fmo1_cr)；
Fig. 3 is arabidopsis WT lines Col-0, mutant fmo1_cr and 2 FMO1 transgenic sequence strains
The senescent phenotypes figure of (FMO1ox#1 and FMO1ox#2), white line meaning are yellowing leaf region；
Fig. 4 is the leaf senile that arabidopsis Col-0 WT lines and fmo1_cr mutant induce nipecotic acid (Pip)
Phenotypic map.
Specific embodiment
The application is further introduced with reference to the accompanying drawings and examples.
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, it is described below of the invention
Specific embodiment.In the following description, what different " embodiment " or " embodiment " referred to is not necessarily the same embodiment.This
Outside, described below is only some embodiments of the present invention, can replace or merge combination between different embodiments, for
It, without creative efforts, can be with other embodiments for those of ordinary skill in the art.
Experimental method used in the following embodiment is conventional method unless otherwise specified, and the reagent and material used is such as
It is commercial goods without specified otherwise.Wherein, the various mutant used are purchased from ABRC, various medicine and reagents, such as without special
Illustrate to be purchased from Sigma company.
A kind of flavine monooxygenase FMO1 gene order, the FMO1 gene order is as shown in SEQ ID NO:1.
A kind of application of flavine monooxygenase FMO1 gene order in plant, the application decline for regulation plant leaf blade
It old process and/or cultivates evening and declines high-yield transgenic sequence plant variety, the FMO1 gene order is as shown in SEQ ID NO:1.
In some embodiments of the invention, the plant is arabidopsis.
A method of regulation plant leaf blade ageing process, the expression including regulation FMO1 gene order in plant, institute
FMO1 gene order is stated as shown in SEQ ID NO:1.
In some embodiments of the invention, include the following steps:
(1) recombinant expression carrier is constructed, the particular target sequence of gene order shown in SEQ ID NO:1 is entered thin to eukaryon
In cellular expression carrier；
(2) recombinant expression carrier is transformed into the cell of purpose plant, knocks out FMO1 gene order；
(3) resistance plant is screened, the late plant that declines is obtained.
In some embodiments of the invention, the eukaryotic expression vector is pCAMBIA1300.
In some embodiments of the invention, the conversion uses agrobacterium-mediated transformation.
In some embodiments of the invention, the plant is arabidopsis.
Embodiment 1
(1) building knocks out the clone of FMO1
As shown in Figure 1, the present embodiment using the CDS of FMO1 gene order as foundation, designs CRISPR vector construction primer,
Primer are as follows:
Forward primer F:5 '-GATTGGTTCGTGGTTGTGTGTACC-3 ' (as shown in SEQ ID NO:2)
Reverse primer R:5 '-AAACGGTACACACAACCACGAACC-3 ' (as shown in SEQ ID NO:3)
By Fig. 1 (on) shown in the BbsI digestion of psgR-Cas9 carrier, then glue recycle.The above synthetic primer is carried out
Annealing obtains double-stranded segment.By the double-stranded segment in molar ratio 1 after BbsI digestion psgR-Cas9 carrier after the recovery and annealing:
The mixing of 3 ratios, is connected 10 minutes under room temperature with T4 ligase.Escherichia coli are gone to by the method for heat shock, it is mould in ammonia benzyl
Screening positive clone on the LB culture medium of element, and identified through sequencing.
(2) transgenic sequence plant pCAMBIA1300-psgR-Cas9-FMO1 (fmo1_cr) is constructed
The present embodiment constructs transgenic sequence plant pCAMBIA1300-psgR-Cas9-FMO1 (fmo1_cr), building
Method includes the following steps:
By Fig. 1 (under) shown in the positive plasmid EcoRI and HindIII that obtain in pCAMBIA1300 carrier and example 1
Enzymes double zyme cutting, then glue recycles the segment for obtaining an about 9000bp and 5000bp respectively.By two segment massages of size
You mix than 1:3 ratio, are connected under room temperature 10 minutes with T4 ligase.Escherichia coli, In are gone to by the method for heat shock
Screening positive clone on the LB culture medium of kanamycins, and plasmid pCAMBIA1300-psgR-Cas9- is obtained through sequencing identification
FMO1。
It takes 2 μ l plasmids to be added in 100 μ l Agrobacterium GV3101, is rapidly added 800 μ l LB culture mediums after 2200V electric shock, 28
DEG C, 220rpm cultivate 1.5 hours, be evenly coated on the LB culture medium containing 50 μ g/ml kanamycins and 50 μ g/ml rifampins, 28
DEG C be inverted culture 2 days；
Single bacterium colony is inoculated in 5ml LB culture medium (50 μ g/ml+ kanamycins of rifampin, 50 μ g/ml), 28 DEG C of 220rpm
Overnight incubation；
LB (rifampin 50 μ g/ml+ kanamycins of the 500mL containing appropriate antibiotic is incubated at volume ratio 1:100 expansion
50 μ g/ml) in, continue to shake training 5-8hr or so to stand density be OD600 value 1.0-1.2；
Room temperature, 5000g are centrifuged 10min, collect thallus；
With conversion medium, (mass percentage is 5% sucrose, 2.033g/L MgCl2) suspension thalline；Silwet L-77
(200 μ l/L) (efficient organic silicon surfactant is one of necessary reagent of the conversions such as arabidopsis, rape, can reduce the table of water
Face tension, effective component are polycyclic ether modified dimethyl polysiloxane) there is injury to bacterium solution, it should be added before converting plant；
Conversion medium containing Agrobacterium is poured into beaker, the arabidopsis just bloomed is inverted thereon, so that entirely
Inflorescence all immerses in conversion medium (inflorescence of lotus throne base portion can pour leaching with rifle), 30s~120s；
Arabidopsis is taken out, is lain on one's side and is placed on clean plastic pallet, and is protected from light moisturizing r for 24 hours with film covering；
Arabidopsis is propped up, is cultivated under light, when siliqua is completely withered and yellow, to be cracked after about 3-4 weeks, seed can be harvested；
Seed sowing is used for screening transgenic sequence plant on the MS culture medium containing hygromycin, not without resistant seeds
It can sprout, if normal seed germination and growth as transgenic sequence resistance plant；
Using round pcr, FMO1 specific fragment can be amplified by detecting in arabidopsis Col-0 wild-type plant, and transgenosis
Amplification shows that FMO1 is knocked out successfully, as fmo1_cr (Fig. 2) less than specific fragment in sequence resistance plant.
Embodiment 3 knocks out FMO1 gene order and regulates and controls plant leaf blade anti-aging process
By wildtype Arabidopsis thaliana Col-0, the clone (fmo1_cr) of the knockout FMO1 built and FMO1 in the present embodiment
Vegetable seeds sowing is overexpressed on MS culture medium, is placed on culture in artificial incubator, cultivation temperature is 22 DEG C, and humidity is
60%, illumination in 16 hours, 8 hours dark long-day (use incandescent lamp to carry out illumination, continuous action light intensity as light source in light
Degree is 200 μm of olm-2s-1) under grow 5 days after shift soil in, plant culturing room cultivate, cultivation temperature be 22 DEG C, humidity
It is 60%, illumination in 16 hours, dark long-day culture in 8 hours.Leaf senile situation is looked first at, when the 40th day,
Individual blades of wild-type plant have begun flavescence aging, and the blade of fmo1_cr all also keeps green, and FMO1ox two
The blade of strain has the blade of nearly half withered death (Fig. 3).Then wildtype Arabidopsis thaliana Col-0, fmo1_cr are taken respectively
It is measured with FMO1 the 4th leaf for being overexpressed plant for chlorophyll content in leaf blades, as the result is shown fmo1_cr leaf chlorophyll
Content is higher compared to Col-0, and FMO1 transgenic sequence plant leaf chlorophyll content is lower compared to Col-0, explanation
Fmo1_cr leaf senile delays.It compares for wild-type plant the above result shows that FMO1 is knocked plant, blade is just
Being frequently grown the aging time is postponed, i.e. knockout plant FMO1 gene order can be with Delaying Leaf-Senescence process.
Embodiment 4fmo1_cr mutant plant blade is insensitive to nipecotic acid
In the present embodiment, by the sowing of wildtype Arabidopsis thaliana Col-0 and fmo1_cr mutant seeds on MS culture medium, put
It sets and is cultivated in artificial incubator, cultivation temperature is 22 DEG C, humidity 60%, illumination in 16 hours, 8 hours dark long-day,
(incandescent lamp is used to carry out illumination as light source, continuous action luminous intensity is 200 μm of olm in light-2s-1) under grow 5 days after shift
It in soil, is cultivated in plant culturing room, cultivation temperature is 22 DEG C, humidity 60%, illumination in 16 hours, 8 hours dark long-day
Culture.When the 21st day, the 4th blade is taken, is handled with the nipecotic acid (Pip) of 1mM, MES buffer is used as control, the
Three days observation leaf senile phenotypes.The blade of wild type Col-0 and fmo1_cr do not occur senescent phenotypes under MES processing,
But the blade of wild type Col-0 shows obvious aging in Pip processing third day, and the blade of fmo1_cr almost with MES condition
Lower consistent (Fig. 4).Above data shows that fmo1_cr mutant is insensitive to nipecotic acid.
It should be noted that above-described embodiment can be freely combined as needed.Described above is only of the invention preferred
Embodiment is not intended to restrict the invention, for those skilled in the art, the present invention can have it is various change and
Variation.All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in this hair
Within bright protection scope.
SEQUENCE LISTING
<110>Beijing Forestry University
<120>flavine monooxygenase FMO1 gene order, application and the method for regulating and controlling plant leaf blade ageing process
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1593
<212> DNA
<213>arabidopsis (Arabidopsis thaliana)
<400> 1
atggcttcta actatgataa gcttacttct tcgagagtag ccatcatcgg tgctggtgtt 60
agcggattag cagccgctaa gaacttagtt catcacaacc cgaccgtatt cgaagcctcg 120
gattcagtcg gaggtgtttg gaggagctgc acttacgaga cgactaaatt acaatcggct 180
cgagtcgatt acgagttctc cgactttcca tggcccaata atagagatga cacaactttc 240
ccaccttacc ttgagatatt agattacttg gaatcttatg ccaaacattt tgatcttctc 300
aaattcatga agtttggctc taaggtcatc gaagtaaggt tcatcggtga tggcgaaact 360
cctcagatgg ttgaccttgg tgcttacggc aacttgttgc ccggaaaacc tgtatgggaa 420
gttgctgttc agatcggaga ttctggagat attcagtggc atgcatttga gttcgtggtt 480
gtgtgtaccg ggaaatacgg cgacgttcca agaataccag cttttccggc aaagaaaggg 540
ccggagatgt ttcaaggaaa agtaatgcat tcgatggatt actgcaagtt agagaaagaa 600
gaagcttcta ctctcctcag tggcaaaaaa gtagccgtca tcggcttcaa gaaatccgcc 660
attgatttgg ctttagagtc tgctttagct aatcaagggg aaggaggaaa agcttgcaca 720
atggtggtga gaacaactca ttgggggatc ccacattatt gggtgtgggg cttaccattc 780
ttcttgttct actcttcgag agcttctcag ttcctccatg ataggcctaa ccaaagcttc 840
cttagaactc tcttttgcct cctcttctct cttctgcgtg ccgtagtttc caagttcatc 900
gaatcatatg ttttatggaa gctacctctt gagaaatatg gtctaaaacc aaaccattct 960
ttcgaggaag actatgcttc ttgtcaaatg gcgatcatac cggagaattt ctttgaggaa 1020
gcggataaag ggatgatccg gtttaagaaa tcatcaaaat ggtggtttta tgaagaaggg 1080
attgtgtttg aagatgggac gacattagaa gctgatgttg tgatacttgc tactggttat 1140
gatggcaaga agaagctcaa agctattgtt cctgaacctt tccgaacttg gcttgagttt 1200
ccaagcggtg tcatgccttt atacagggga acaatccatc cattgatacc aaacatgggt 1260
ttcgtaggat acgtccaaag cagctcgaac ttacacacat cagagctacg ttcaatgtgg 1320
ctaagccggc tagtggatga gaaattcaga ttaccaagca aagagaagat gttggatcaa 1380
ttcttaaaag aaatggaagt gacgagaaac tcaagcagat tctacaaacg tcattgcatt 1440
tccacgttca gcatccaaca tgctgatgat atgtgcaacg acatgggact caatccttgg 1500
cgtaaatcca acttcctcct tgaagctttt agtccttatg gttctcaaga ttaccgactc 1560
ggtcaagaag aaaaagaaga tatgactgct taa 1593
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gattggttcg tggttgtgtg tacc 24
<210> 3
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aaacggtaca cacaaccacg aacc 24
Claims (8)
1. a kind of flavine monooxygenase FMO1 gene order, which is characterized in that the FMO1 gene order such as SEQ ID NO:1 institute
Show.
2. a kind of application of flavine monooxygenase FMO1 gene order in plant, which is characterized in that the application is regulation
It plant leaf blade ageing process and/or cultivates evening and declines high-yield transgenic sequence plant variety, the FMO1 gene order such as SEQ ID
Shown in NO:1.
3. application according to claim 2, which is characterized in that the plant is arabidopsis.
4. a kind of method of regulation plant leaf blade ageing process, which is characterized in that including regulation FMO1 gene order in plant
Expression, the FMO1 gene order is as shown in SEQ ID NO:1.
5. according to the method described in claim 4, it is characterized by comprising the following steps:
(1) recombinant expression carrier is constructed, the particular target sequence of gene order shown in SEQ ID NO:1 is entered to eukaryocyte table
Up in carrier；
(2) recombinant expression carrier is transformed into the cell of purpose plant, knocks out FMO1 gene order；
(3) resistance plant is screened, the late plant that declines is obtained.
6. according to the method described in claim 5, it is characterized in that, the eukaryotic expression vector is pCAMBIA1300.
7. according to the method described in claim 5, it is characterized in that, the conversion uses agrobacterium-mediated transformation.
8. according to the method described in claim 4, it is characterized in that, the plant is arabidopsis.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008060555A2 (en) * | 2006-11-14 | 2008-05-22 | Purdue Research Foundation | Activation of the arabidopsis hypertall (hyt1/yucca6) locus affects several auxin mediated responses |
| CN109200276A (en) * | 2018-08-09 | 2019-01-15 | 浙江大学 | A kind of application of flavin containing monooxygenase 2 in preparation treatment heart disease drug |
-
2019
- 2019-07-16 CN CN201910639848.1A patent/CN110468144A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008060555A2 (en) * | 2006-11-14 | 2008-05-22 | Purdue Research Foundation | Activation of the arabidopsis hypertall (hyt1/yucca6) locus affects several auxin mediated responses |
| CN109200276A (en) * | 2018-08-09 | 2019-01-15 | 浙江大学 | A kind of application of flavin containing monooxygenase 2 in preparation treatment heart disease drug |
Non-Patent Citations (5)
| Title |
|---|
| MICHAEL BARTSCH ET AL.: ""Accumulation of Isochorismate-derived 2,3-Dihydroxybenzoic 3-O-β-D-Xyloside in Arabidopsis Resistance to Pathogens and Ageing of Leaves"", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| SALMA BALAZADEH ET AL.: ""ORS1, an H2O2-Responsive NAC Transcription Factor, Controls Senescence in Arabidopsis thaliana"", 《MOLECULAR PLANT》 * |
| THEOLOGIS, A. ET AL.: ""Arabidopsis thaliana flavin-dependent monooxygenase 1 (FMO1), mRNA，Genbank: NM_101783.4"", 《GENBANK》 * |
| YIPING WANG ET AL.: ""ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis"", 《THE PLANT JOURNAL》 * |
| ZHONGHAI LI ET AL.: ""Genetic redundancy of senescence-associated transcription factors in Arabidopsis"", 《JOURNAL OF EXPERIMENTAL BOTANY》 * |
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