CN109971772A - A kind of breeding method of low temperature resistant cotton variety - Google Patents
A kind of breeding method of low temperature resistant cotton variety Download PDFInfo
- Publication number
- CN109971772A CN109971772A CN201910182562.5A CN201910182562A CN109971772A CN 109971772 A CN109971772 A CN 109971772A CN 201910182562 A CN201910182562 A CN 201910182562A CN 109971772 A CN109971772 A CN 109971772A
- Authority
- CN
- China
- Prior art keywords
- ahcmo
- cotton
- gene
- plant
- low temperature
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
- C12Y114/15007—Choline monooxygenase (1.14.15.7)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses prunella asiatica choline single oxygenase genes (referred to as AhCMO) to cultivate the purposes in low temperature resistant plant variety, and the expression vector containing AhCMO gene and plant cell are cultivating the purposes in low temperature resistant plant variety.The invention also discloses a kind of breeding methods of low temperature resistant cotton variety, i.e., by AhCMO genetic transformation into cotton variety, and the material for selecting AhCMO gene expression amount high.AhCMO gene is used for plant frigostabile breeding by the present invention, has opened up a new field for the application of the gene；Secondly, AhCMO transgene cotton, which overcomes cotton, not only avoids the decline of output of cotton reduction and fiber quality to the sensibility of low temperature, but also sown areas of cotton can be expanded, improve the income of peasant.
Description
Technical field
The invention belongs to Cotton Breeding Methods fields, and in particular to prunella asiatica choline single oxygenase gene is low temperature resistant in cultivation
Purposes on cotton variety；And the use of expression vector or plant cell on the low temperature resistant cotton variety of cultivation containing the gene
On the way；Further relate to a kind of breeding method of low temperature resistant cotton variety.
Background technique
Cotton (GossypiumhirsutumL.) originates from subtropical and tropical zones, therefore cotton is very sensitive to cold stress.
Cold stress significantly inhibits cotton seed germination and growth of seedling, also will cause fertilization and fails, shedding even plant death etc.,
It thus will cause output of cotton and fiber quality decline.Cotton in China plantation is distributed mainly on the Northwest inlands such as Xinjiang, Yi Jigao
Latitude and High aititude etc. cool area, and cold stress has become the key constraints of Cotton in China production, cause because of cold stress
Velveteen production loss up to 30%~40%, therefore, it is cold stress have become Cotton in China production in urgent problem to be solved.
Synthesize rapidly and accumulate a large amount of small molecule compounds come maintain the normal function of cell be plant by saline and alkaline or
To a kind of extraneous adaptation reaction when other environment-stress.Glycine betaine just belongs to this kind of important one of small molecule compound,
Under Salt Strees Condition, glycine betaine is largely accumulated in many plants especially Li Ke and graminaceous plant cell, improves cell infiltration
Saturating regulating power stablizes the structure and function of intracellular macromolecules protein and biomembrane.In higher plant, the conjunction of glycine betaine
Chengdu is originated by choline, is reacted and is completed by two-step catalysis, and the enzyme of the first step reaction of catalysis is choline single oxygenase
(Choline Monooxygenase, be abbreviated as CMO), it is the rate-limiting enzyme in glycine betaine biosynthetic process.By improving sweet tea
The salt tolerance or drought resistance of plant can be improved in expression quantity of the dish alkali in plant.Currently, from chenopod spinach
(.PNAS such as Rathinasabapathi, 1997,94 (7): the 3454-3458), beet (.Plant such as Russell
Physiology, 1998,116:859-865), amaranthaceous plant tampala (.Cell such as Meng Research, 2001,11 (3):
187-193), the plants such as gramineae plant rice (the .Plant Cell Reports such as Luo, 2012,31 (9): 1625-1635)
In isolate the gene.
Prunella asiatica (Atriplex hortensis, also known as wild spinach or mountain spinach) belongs to Chenopodiaceae orach, is
A kind of glycine betaine naturally saves plant, can mal-conditions (Tao etc., Scientific such as cold resistant, arid and high salinity
Reports,2018,8(1):2707；The .Theoretical and Applied such as Shen Genetics, 2002).Currently,
Clone obtains the CMO gene (being abbreviated as AhCMO) (the bioengineering journal such as Shen Yiguo, 2001,1:1-6) of prunella asiatica.Pass through structure
Over-express vector (pBI121-AhCMO) is built, by AhCMO genetic transformation tobacco and cotton, obtains and turns AhCMO genetic tobacco
And cotton, test drought tolerance and Saline alkali tolerance (the Zhai Honghong Chinese agriculture section that tobacco is improved after proving to import AhCMO gene
Institute's master thesis, 2015).
Through retrieving, the report applied in the low temperature resistant breeding of cotton in relation to AhCMO gene is not found.
Summary of the invention
In order to solve the problems, such as that plant low temperature damages to plants caused by sudden drop in temperature, it is an object of that present invention to provide prunella asiatica choline single oxygenase genes
(AhCMO) application in low temperature resistant plant variety is being cultivated.
In above-mentioned application, the prunella asiatica choline single oxygenase gene (writing a Chinese character in simplified form are as follows: AhCMO) is by SEQ ID NO:1 institute
The nucleotide sequence composition shown.
In above-mentioned application, the prunella asiatica choline single oxygenase gene (AhCMO), the albumen of coding is by SEQ ID
The composition of amino acid sequence shown in NO:2.
Plant described in above-mentioned application refers to cotton, corn, rice or soybean etc..
Another object of the present invention is that providing the expression vector containing prunella asiatica choline single oxygenase gene (AhCMO) is training
Educate the application in low temperature resistant plant variety.
In above-mentioned application, the expression vector refers to plant expression vector PBI121 etc..
In above-mentioned application, the plant refers to cotton, corn, rice or soybean etc..
Third of the present invention is designed to provide the plant cell containing prunella asiatica choline single oxygenase gene (AhCMO) and is training
Educate the application in low temperature resistant plant variety.
In above-mentioned application, the plant refers to cotton, corn, rice or soybean etc..
The present invention the 4th is designed to provide a kind of method for cultivating low temperature resistant cotton variety, including by prunella asiatica choline list
Monooxygenase gene (AhCMO) is transformed into cotton variety by transgenic method.
The method of the above-mentioned low temperature resistant cotton variety of cultivation, the specific steps are as follows:
(1) gene cloning: using AhCMO-F and AhCMO-R as primer, PCR amplification is carried out by substrate of prunella asiatica cDNA, is obtained
Pcr amplification product, as AhCMO genetic fragment；The wherein primer are as follows:
AhCMO-F:5 '-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3 ',
AhCMO-R:3 '-GTGTTGCTTTTGATGCTTTCGTAAGCGATCCATTC-5 '；
(2) intermediate vector is constructed: the AhCMO genetic fragment obtained by PstI and XhoI double digestion step (1), Ago-Gel
Genetic fragment is recycled after electrophoresis；With PstI and XhoI double digestion carrier pUC19, linear pUC19 segment is recycled, AhCMO is connected into
In the pUC19 of Enhanced expressing sequence containing Cozk, Ω and p1oyA, and gene is made to connect CaMV35 promoter and Nos termination
Son is built into intermediate vector pUC19-AhCMO；
(3) expression vector establishment: the III resulting intermediate vector pUC19- of double digestion step (2) of EcoRI and Hihd is used
AhCMO recycles the segment containing AhCMO gene；Carrier pBI121 after being connected into III double digestion of EcoRI and Hihd respectively, building
At over-express vector pBI121-AhCMO；
(4) it converts: taking the 100 μ L of Agrobacterium GV3101 competent cell prepared, 10 μ g pBI121-AhCMO matter are added
Grain DNA is mixed, and is placed in 5min on ice；Then 2mm electric shock cup is added in competence mixed liquor, 2500V shocks by electricity, adds rapidly after electric shock
Enter 800 μ L of YEB fluid nutrient medium, Fiber differentiation 5h under the conditions of 28 DEG C, 180rpm；It takes out the bacterium solution induced and is coated in contain and block that
On the YEB plate of mycin and each 50 μ g/mL of rifampin, cultivated 48 hours at 28 DEG C；Picking monoclonal, which is inoculated in, to be blocked containing for 5mL
In the YEB fluid nutrient medium of that mycin and rifampin (concentration is 50 μ g/mL), bacterium is shaken under the conditions of 180rpm 36 hours, it is small
Amount extracts plasmid enzyme restriction and PCR identification；The AhCMO carrier converting cotton that will be built using Agrobacterium infestation method is obtained AhCMO and turned
The cotton regenerated seedling of gene；
(5) RT-PCR identification evaluation and screening: is carried out to the resulting regenerated transgenic seedling of step (4)；Harvest is accredited as the positive
The T0 of plant is for seed；The transgenosis T0 of harvest is sowed for seed, the T1 for taking growing way good extracts DNA for leaf tissue and carries out
RT-PCR detection, the strain that screening positive material continues to cultivate, and selects AhCMO gene expression amount high, is bred as AhCMO transgenosis
Cotton variety.
The present invention the 5th, which is designed to provide, cultivates low temperature resistant cotton variety using the transgene cotton containing AhCMO gene
Breeding method, the breeding method be back cross breeding method or cross breeding method；
The back cross breeding method, includes the following steps:
(1) using the transgene cotton containing AhCMO gene as nonrecurrent parent, using the excellent cotton variety of character as circulation
Parent hybridizes to obtain F1Generation；
(2), with F1On behalf of female parent, it is returned using cotton variety described in step (1) as male parent, obtains BC1F1Generation；With list
Seed is unit, extracts BC1F1For single grain genomic DNA, and using the genomic DNA as substrate, with AhCMO-F and AhCMO-R
PCR identification is carried out for primer, selects pcr amplification product size for the offspring of 1314bp；Positive backcross progeny is accredited as with PCR
For male parent, it is maternal backcrossing with cotton variety described in step (1), obtains BC2F1Generation；The wherein primer sequence are as follows:
AhCMO-F:5 '-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3 ',
AhCMO-R:3 '-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5 '；
(3), it repeats described in step (2) backcrossing 3~6 times；It was finally selfed for 1 generation, and the seed of self progeny is walked
Suddenly PCR described in (2) is identified, it is unseparated to retain AhCMO gene in the single plant offspring of identification, is as bred as containing AhCMO
The new varieties of gene.
Transgene cotton containing AhCMO gene described in above-mentioned back cross breeding method and step (1) refer to CMO24 or
The Derivative line of CMO24 containing AhCMO gene.
Character described in above-mentioned back cross breeding method and step (1) excellent cotton variety refers to be promoted and applied in production
Cotton variety；Or the cotton line being just bred as.Wherein the character refers to economical character and fiber quality etc..
The reaction system (20ul) of PCR described in above-mentioned back cross breeding method and step (2): transgene cotton gene to be measured
Group 1 μ l of DNA, each 1 μ l, Mix10 μ l of primer add sterile water to 20 μ l；The reaction condition of the PCR are as follows: 94 DEG C, 3min；94
℃,30Sec；56℃,30Sec；72℃,30Sec；Circulation 30 times；72 DEG C, 10min.
The cross breeding method, includes the following steps:
(1), it is one of parent with the transgene cotton containing AhCMO gene, is that another parent is miscellaneous with elite cotton kind
It hands over, obtains F1Generation；
(2), by gained F1Generation selfing, harvests to obtain F2Generation；
(3), identification and selection F are assisted by molecular labeling2The single grain of AhCMO gene is had in generation, field plot test is
Positive offspring, and roguing is selfed；It repeats above-mentioned PCR identification and roguing was selfed for 4~6 generations, is i.e. cotton of the incubation containing AhCMO gene
Flower new varieties；Wherein the molecular labeling refers to using the genomic DNA of cotton seed to be measured as substrate, with AhCMO-F and
AhCMO-R is that primer carries out PCR amplification, selects amplified production size for the progeny material of 1314bp.
Cotton containing AhCMO gene described in above-mentioned cross breeding method step (1) refers to CMO24.The guarantor of CMO24
Hiding number: CGMCC No.17381.
The source of CMO24: Biological Technology institute, Chinese Academy of Agricultural Sciences is by AhCMO genetic transformation to cotton line R15
(R15 is the cotton that institute, the Chinese Academy of Agricultural Sciences biotechnology research institute is selected from cotton variety Ke word 312 (or being Coker312)
Flower strain) in, AhCMO gene expression amount highest and the strong strain of lower temperature resistance are then selected in transformant, are named as
CMO24。
The present invention also provides for identifying the molecular labeling of AhCMO transgene cotton, the molecular labeling is by SEQ
The composition of nucleotide sequence shown in ID NO:1.
The present invention also provides the primer pair for expanding above-mentioned molecular labeling, the primer pair by AhCMO-F and
AhCMO-R composition；
AhCMO-F:5 '-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3 ',
AhCMO-R:3 '-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5 '.
Primer pair the present invention also provides the detection kit of AhCMO transgene cotton, in the detection kit
It is made of AhCMO-F and AhCMO-R；
AhCMO-F:5 '-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3 ',
AhCMO-R:3 '-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5 '.
Compared with prior art, the present invention have the advantage that and the utility model has the advantages that (1), the present invention for the first time by prunella asiatica choline list
Oxygenase (AhCMO) gene is used for plant frigostabile breeding, provides a good GENE SOURCES for the low temperature resistant breeding of plant,
A new field has been opened up for the application of AhCMO gene；(2) AhCMO base in present invention gained AhCMO transgene cotton strain
Because of expression quantity height, and lower temperature resistance is strong, provides a new approach for the low temperature resistant breeding of plant；(3), the method for the present invention is educated
At cotton variety lower temperature resistance it is strong, overcome cotton to the sensibility of low temperature, damaged to plants caused by sudden drop in temperature caused by can not only overcoming because of low temperature,
The reduction of yield and fiber quality is avoided, but also planting area can be expanded, expand cultivated area, or can will be mentioned in sowing time
Before, extension breeding time increases farmers' income to improve yield and fiber quality.
Biological deposits: upland cotton (GossypiumhirsutumLinn) CMO24 of the present invention is the applicant's Chinese agriculture section
Prunella asiatica choline single oxygenase gene (AhCMO) is transformed into cotton variety by transgenic method by institute's biotechnology research institute
(R15 is that institute, the Chinese Academy of Agricultural Sciences biotechnology research institute is selected from cotton variety Ke word 312 (or being Coker312) to R15
Cotton line) in, and select AhCMO gene expression amount high and the strong material of lower temperature resistance in gained transformant and be bred as
Transgene cotton strain.CMO24 was preserved in Chinese culture presevation administration committee common micro-organisms on 2 25th, 2019
Center, deposit number are as follows: CGMCC No.17381.
Detailed description of the invention
Fig. 1 is the schematic diagram of AhCMO expression vector pBI121-AhCMO.
Fig. 2 is that the PCR of AhCMO gene identifies electrophorogram；Wherein CK is wild type R15, and 1~23, which is respectively 23 plants, turns base
Because of plant positive identification result.
Fig. 3 is the expression quantity of AhCMO gene in 6 week old Transgenic cotton plants；Wherein 1 is wild type R15, and 2 are
CMO20,3 be CMO24.
Fig. 4 is wild type R15 and transgene cotton strain CMO24 seedling photo before and after low-temperature treatment；Wherein 1 is before handling
Wild type R15,2 be the CMO24 before processing；3 be wild type R15 after processing, and 4 be treated CMO24.
Fig. 5 is wild type R15 and transgene cotton strain CMO24 blade desk tray indigo plant stained photographs before and after low-temperature treatment；Its
In 1 be wild type R15 before handling, 2 be the CMO24 before processing, and 3 be wild type R15 after processing, and 4 be CMO24 after handling.
Fig. 6 is wild type R15 and transgene cotton strain CMO24 chlorophyll content in leaf blades column diagram before and after low-temperature treatment；
Wherein 1 is wild type R15, and 2 be CMO24.
Fig. 7 is wild type R15 and the free nitrogen content cylindricality of transgene cotton strain CMO24 cotton leaf before and after low-temperature treatment
Figure；Wherein 1 is wild type R15, and 2 be CMO24.
Fig. 8 is beet alkali content cylindricality in low-temperature treatment front and back wild type R15 and transgene cotton strain CMO24 blade
Figure；Wherein 1 is wild type R15, and 2 be CMO24.
Specific embodiment
The present invention will be further explained by the following examples and explanation, but does not constitute to the scope of the present invention
Any restrictions.Unless otherwise specified, agents useful for same is conventional reagent in following embodiments, and method therefor is this field routine
Method, or carried out according to the specification of purchased reagent.
The building of embodiment 1AhCMO gene cloning and expression carrier
(1) materials and methods
1) cotton material: cotton line R15 is the self-fertile strain of Biological Technology institute, Chinese Academy of Agricultural Sciences, and in
Academy of Agricultural Sciences, state biotechnology research institute crop molecular breeding research room saves.
2) strain: Escherichia coli Transl-T1, Agrobacterium GV3101.
3) carrier: pEASY-Blunt, pEASY-T5, pUC19, pBI121.
4) toolenzyme and modification enzyme: various restriction enzymes and modification enzyme purchased from TaKaRa company, NEB company and
TIANGEN company.
5) chemical reagent: chemicals is that domestic outer analysis is pure.
6) primer synthesizes: being synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
7) it is sequenced: being completed by Huada gene company.
(2) test procedure
AhCMO gene is connected into pBI121 carrier, is built into single-gene expression vector, operating procedure is as follows:
1) according to degenerate codon design primer, the code area AhCMO cDNA full length gene is expanded, with PstI and XhoI digestion
Genetic fragment, recycle after agarose gel electrophoresis Gene A hCMO (referring to Zhai Honghong, Chinese Academy of Agricultural Sciences's master thesis,
2015), sequencing is it is found that AhCMO gene nucleotide sequence shown in SEQ ID NO:1 forms.The albumen of AhCMO gene coding
The amino acid sequence shown in SEQ ID NO:2 forms.
2) PstI and XhoI double digestion carrier pUC19 is used, linear carrier segments are recycled, by AhCMO gene obtained by step 1)
It is connected into the pUC19 containing the Enhanced expressings sequence such as Cozk, Ω and p1oyA, and AhCMO gene is made to connect CaMV35s promoter
With Nos terminator, it is built into intermediate vector pUC19-AhCMO.
3) the intermediate vector pUC19-AhCMO obtained in EcoRI and HindIII double digestion step 2), recycling contain
The expression nuclear fragmentation of AhCMO gene, the carrier pBI121 after being connected into EcoRI and HindIII double digestion respectively, is built into overexpression
Carrier pBI121-AhCMO (see Fig. 1).
Embodiment 2AhCMO genetic transformation and the identification of transgenic line and screening
1) AhCMO expression vector converts Agrobacterium GV3101
Agrobacterium culture: taking the Agrobacterium GV3101 competent cell 100uL prepared, and 10ug pBI121- is added
AhCMO Plasmid DNA mixes, is placed in 5min on ice, obtains competence mixed liquor.2mm electric shock cup is added in competence mixed liquor (to add
When to avoid as far as possible generate bubble), 2500V electric shock；YEB fluid nutrient medium 800uL is rapidly added after electric shock, 28 DEG C,
Fiber differentiation 5h under the conditions of 180rpm；The bacterium solution induced is taken out, is coated in containing kanamycins and each 5Oug/mL of rifampin
On YEB plate, cultivated 48 hours at 28 DEG C.Picking monoclonal is inoculated in that (concentration is 50ug/ containing kanamycins and rifampin
ML in 5mL YEB fluid nutrient medium), bacterium is shaken at 180rpm 36 hours, it is a small amount of to extract plasmid enzyme restriction and PCR identification.It will test
It demonstrate,proves correct monoclonal and is inoculated in 5OmL and contain in the YEB fluid nutrient medium that concentration is 50ug/mL kanamycins and rifampin,
It is incubated overnight until OD600=1.0, is added 50% glycerol, saves backup in -80 DEG C.
2) Cotton Transformation of AhCMO gene
1. the culture of cotton: the seed of cotton line R15 is tested by Chinese Academy of Agricultural Sciences's biology institute crop molecular breeding
Room provides, and uses 98%H2SO4Unsticking is to prepare original material.By seed with 70% ethyl alcohol surface sterilizing 30 seconds, it is then immersed in
15%H2O2In 2 hours, then with sterile distilled water rinse 3 times.Then, by seed in 28 DEG C of sterile water soaked overnight.
After sprouting starts, kind of a skin is removed, kernel is then implanted into MS culture medium, is recycled 28 DEG C, illumination 16 hours and dark 8 hours
Lower germination, luminous intensity are 70 μm of ol m-2s-1.Cotyledon is cut when 5-6 days ages seedling and hypocotyl is used as the outer of callus induction
Implant.
2. Cotton Transformation: explant being impregnated 30 minutes in agrobacterium suspension, is gently shaken 3~5 times.Then, will
The explant of infection is dry on aseptic filter paper, and is transferred to callus inducing medium (CIM) upper 2 day, in 24 DEG C, dark
Under the conditions of co-culture.After co-cultivation, it is mould that the explant of infection is transferred to the tide containing 500mg/L cefotaxime and various concentration
It on the CIM of element, and is cultivated under circulation 28 DEG C, illumination 16 hours and dark 8 hours, is used for callus induction, acquisition turns base
Because of 23 plants of cotton.
3. the positive identification of transgene cotton: the genomic DNA for extracting transgenic cotton plant is detected for PCR.With
AhCMO-F and AhCMO-R is that primer carries out PCR amplification,
AhCMO-F:5 '-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3 ',
AhCMO-R:3 '-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5 '；
PCR reaction system (20ul): 1 μ l of template (transgene cotton genomic DNA), each 1 μ l, Mix10 μ l of primer add nothing
Bacterium water is to 20 μ l.
PCR reaction condition: 94 DEG C, 3min；94℃,30Sec；56℃,30Sec；72℃,30Sec；30 circulations；72 DEG C,
10min。
As a result 23 plants of transgene cotton are identified (see Fig. 2), wherein 8 plants of positive plant.
3) AhCMO gene qRT-PCR is detected
It takes T2 generation to grow the blade of two weeks transgenosis AhCMO strains, extracts RNA.The RNA of extraction is used for reverse transcription, and
QRT-PCR is as template using the cDNA of reverse transcription.
Forward primer: 5 '-ACCCAACTACTGTTTGTGGAATACC-3 '
Reverse primer: 3 '-GTTAACGGGTTTATTAGGAGTACGG-5 '
QRT-PCR reaction system (20ul): 0.8 μ l of template (cDNA), each 1 μ l, KOD-Mix10 μ l of primer add sterile water
To 20 μ l.
QRT-PCR reaction condition: 98 DEG C, 2min；98℃,10Sec；60 DEG C, 30Sec；68℃,30Sec；
40cycle；65 DEG C -99 DEG C primary every 0.5 DEG C of reading.
As a result (see Fig. 3), qRT-PCR detection shows have in a transgenic plant compared to other 7 positive plants
The expression quantity highest of AhCMO gene is named as CMO24 and (entrusted on 2 25th, 2019 in Chinese microorganism strain preservation management
Member's meeting common micro-organisms center preservation, deposit number are as follows: CGMCC No.17381)；There are one AhCMO gene expression quantity compared with
Height is only second to CMO24, is named as CMO20.
Embodiment 3AhCMO transgenic plant lower temperature resistance qualification test
1) transgene cotton CMO24 and wild type cotton R15 are placed under normal growth conditions (28 DEG C) and continuously cultivate 3
In week, as a result (see Fig. 4-1 and Fig. 4-2), the blade of transgenosis and wild type cotton plant is all acted normally.Then by transgenosis and
Wild type cotton plant is transferred to 12 DEG C and cultivates 24 hours, as a result, it has been found that transgene cotton CMO24 leaf morphology it is still normal (see
Fig. 4-4), rather than there is serious even death of wilting in rotaring gene plant blade (see Fig. 4-3).Illustrate AhCMO transgene cotton
CMO24 is insensitive to low temperature, i.e., lower temperature resistance is strong.
2) placenta indigo plant dyes: selection plant type size is similar, wild type R15 and transgenosis CMO24 cotton before and after low-temperature treatment
It floral leaf piece each 3, is put into 50ml centrifuge tube and writes number.Pour into prepared placenta indigo plant dye liquor, vacuum filtration make dye liquor into
Enter space between cells, boiling water boils 3-5min, is stored at room temperature dyeing 6-8h.It is fully transparent up to organizing with 95% ethanol decolorization
Until.The blade to have decolourized, which is placed on filter paper, to be observed and takes pictures.As a result (see Fig. 5-1 and Fig. 5-2) before low-temperature treatment, wild type
R15 and transgene cotton CMO24 blade do not dye substantially, and non-transgenic cotton R15 leaf tissue is serious after low-temperature treatment
It colours (see Fig. 5-3), the coloring of transgene cotton CMO24 leaf tissue is relatively light (see Fig. 5-4), illustrates low-temperature treatment to wild
Type cotton causes to damage, and low temperature is smaller on the influence of AhCMO transgene cotton, and lower temperature resistance is strong.
3) chlorophyll and free nitrogen analysis: before and after low-temperature treatment, to AhCMO transgene cotton strain CMO24 and open country
Raw type cotton R15 blade Determination of Chlorophyll and free nitrogen analysis, as a result, it has been found that before (see Fig. 6 and Fig. 7) low-temperature treatment, wild type
The chlorophyll of R15 and transgene cotton CMO24 is identical with free nitrogen content；And wild type R15 and transgenic cotton after low-temperature treatment
The chlorophyll and free nitrogen content of flower CMO24 reduces, and transgene cotton CMO24 is small compared to wild type R15 reduction amplitude.Explanation
The expression of AhCMO gene reduces the damage that low temperature stress acts on cotton photosynthetic.
4) beet alkali content measures: before and after low-temperature treatment, respectively to wild type R15 and AhCMO transgene cotton CMO24
Beet alkali content be measured.Measuring method are as follows: the sample of wild type R15 and transgene cotton CMO24 are dried respectively, filled
0.1g is weighed after dividing grinding, is added 1ml extracting solution (80% methanol), 60 DEG C of extraction 30min, is during which shaken 1 time every 5min.25
DEG C, be centrifuged 10min under the conditions of 10000rpm, take supernatant.Measurement pipe is+350 μ l (reagent one) of 250 μ l (supernatant), sufficiently
It mixes, 4 DEG C of reaction 2h, 10min is centrifuged under the conditions of 25 DEG C, 10000rpm, abandon supernatant；99% ether, 300 μ l is added, 25
DEG C, be centrifuged 10min under the conditions of 10000rpm, being placed in draught cupboard makes ether volatilize；70% acetone 1ml is added, concussion fills precipitating
Divide dissolution, 1ml cuvette, 70% acetone returns to zero, and light absorption value is measured at 525nm.
As a result (see Fig. 8) after low-temperature treatment 24 hours, beet alkali content is significantly larger than in AhCMO transgene cotton CMO24
Wild type R15 illustrates that the overexpression of AhCMO gene improves the content of glycine betaine.
The above result shows that AhCMO can significantly improve the resistance to low temperature of cotton plants.
Embodiment 4 is tested using the back cross breeding that the transgene cotton containing AhCMO gene cultivates low temperature resistant cotton variety
Include the following steps:
(1), 2016 in Langfang in Hebei Province proving ground using transgene cotton CMO24 as nonrecurrent parent, with North SinKiang morning
Ripe cotton (this laboratory from material selection) is that recurrent parent is hybridized, and harvests to obtain F1For seed；
(2), winter in 2016 plants F to Hainan1In generation, extracts F in Seedling Stage1For the genomic DNA of cotton plants, and with
It is substrate, carries out PCR amplification by primer of AhCMO-F and AhCMO-R；Selecting transgene cotton positive strain, (amplified fragments are long
Spend about 1300bp) it is returned with North SinKiang Shine Early cotton, harvest to obtain BC1F1For seed；The wherein primer are as follows:
AhCMO-F:5 '-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3 ',
AhCMO-R:3 '-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5 '；
PCR reaction system (20ul): 1 μ l of template (cotton genomic dna to be measured), each 10 μ l of 1 μ l, Mix of primer add nothing
Bacterium water is to 20 μ l.PCR reaction condition: 94 DEG C, 3min；94℃,30Sec；56℃,30Sec；72℃,30Sec；30cycles；72
DEG C, 10min.
(3), BC is planted in Langfang in Hebei Province proving ground within 20171F1In generation, is identified by PCR method described in step (2)
BC1F1For plant, it is returned using selected positive single plant as male parent and North SinKiang Shine Early cotton, is harvested by single plant, obtain BC2F1For seed；
(4), as unit of single grain, according to step (2) described PCR method to BC2F1PCR identification is carried out for seed, is selected and remain
Positive plant；Winter in 2017 plants BC in Hainan2F1In generation, is returned, by single using selected positive single plant as male parent and North SinKiang Shine Early cotton
Strain harvest, obtains BC3F1For seed；
(5), as unit of single grain, according to step (2) described PCR method to BC3F1In generation, carries out PCR identification, and selection is positive
Plant plants BC in Langfang in Hebei Province proving ground in 20183F1Generation, and be returned by male parent and North SinKiang Shine Early cotton of selected single plant, it presses
Single plant harvest, obtains BC4F1For seed；
(6), as unit of single grain, BC is identified according to step (2) PCR method4F1For plant, positive strain is selected and remain,
Winter in 2018 plants in Hainan, and roguing selfing is harvested by single plant, obtains BC4F2For seed；
(7), to BC as unit of single grain4F2PCR identification (method is shown in step (2)) is carried out for seed, selects and remain the corresponding positive
Strain, and the unseparated offspring of AhCMO, the transgene cotton new varieties for the gene containing AhCMO being as bred as are named as CMO24bc.
CMO24bc is identified under 12 DEG C of low temperature, as a result CMO24bc has lower temperature resistance, illustrates backcrossing of the present invention
Breeding method has been bred as low temperature resistant new varieties.
Embodiment 5 is tested using the crossbreeding that the transgene cotton containing AhCMO gene cultivates low temperature resistant cotton variety
Include the following steps:
(1), 2016 Langfang in Hebei Province proving ground with transgene cotton CMO24 and Soviet Union (the applicant laboratory of cotton 12
From material selection) hybridization, harvest to obtain F1For seed；
(2), winter in 2016 plants F in Hainan1For seed, selfing, single plant harvest obtains F2For seed；
(3), as unit of single grain, to F2It for seed and carries out PCR identification (referring to 4 step of embodiment (2)), selects and remain sun
Property strain (expanding fragment length about 1300bp) seed, 2017 Langfang in Hebei Province proving ground plant, select economical character it is good
Individual plant selfing, by single plant harvest, obtain F3For seed；
(4), step (3) are repeated, continues roguing in Hainan and Langfang base within 2017 and was selfed for 2 generations, it is auxiliary in addition to being identified with PCR
It helps outside selection AhCMO gene, the same conventional breeding of the character determinations such as economical character obtains F5Generation；
(5), as unit of single grain, to F5For seed and PCR identification is carried out, AhCMO gene is unseparated in offspring, i.e.,
For the new cotton variety of the gene containing AhCMO of incubation, it is named as CMO24f.
(6), CMO24f is identified under 12 DEG C of low temperature, as a result its lower temperature resistance is strong；Illustrate crossbreeding of the present invention
The new varieties for the gene containing AhCMO that method is bred as have lower temperature resistance.
Sequence table
<110>Biological Technology institute, Chinese Academy of Agricultural Sciences；Three heap of Guo
<120>a kind of breeding method of low temperature resistant cotton variety
<130> 2019S1471IHCY
<141> 2019-03-12
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1314
<212> DNA
<213> Atriplex hortensis
<400> 1
atggcagcaa gtgcaacaac aatgttgcta aaatacccaa ctactgtttg tggaatacca 60
aattcatccg caaacaattc tactgatcct tcaaataaca tcgtccaaat tccacaaact 120
actactacta atagcccgct acttaagttc cgtactccta ataaacccgt taacgccgtc 180
gctgccccgg cttttccgtc cgtaaccacc actacaacca ccactccgtc gtccatccaa 240
tcacttgtca aggatttcga tcctcttgtt ccggccgagg atgctcttac tcctcctagc 300
tcttggtata ccgaacctgc cttctatgct catgaacttg accgtatctt ttacaaagga 360
tggcaagtcg cagggtacag tgatcaagtt aaggaggcta accaatattt caccggaacg 420
ttaggaaatg ttgaatattt ggtgtgtcga gatggagaag gaaaagttca tgcatttcac 480
aatgtttgca cccatcgtgc atctatcctt gcttgtggaa gtggcaaaaa gtcatgtttc 540
gtatgccctt atcatggatg ggtatatggc atgaatggat cgcttacgaa agcttcaaaa 600
gcaacaccag aacaatcact aaatcccgat gaacttgggc ttgtaccact aaaagttgca 660
gtatggggcc catttatact catcagtttg gacagatcaa gccgtgaagt aggtgacgtt 720
ggatctgaat ggcttggtag ttgtgctgaa gatgttaagg cccatgcttt tgacccgaat 780
cttcagttca ttaataggag tgaatttcca attgaatcta attggaagat tttcagtgac 840
aactatttgg atagctctta ccatgttcct tatgcacaca aatactatgc aactgagctc 900
gactttgata cttaccaaac cgatatggtt ggaaatgtca cgattcaaag ggtggctggg 960
acttcaaaca atggttttaa tagacttgga actcaagcct tctatgcttt tgcataccct 1020
aactttgctg tggaaaggta tggcccttgg atgactacaa tgcatattgt tccattagga 1080
ccaaggaaat gcaaactagt ggtggactac tatattgaaa aatcaaagct ggacgacaag 1140
gattacatcg aaaagggcat agcaatcaat gataatgtgc agaaagaaga tgtggtgttg 1200
tgtgaaagcg tccaaaaagg gttggagaca ccagcatatc gtagtggaag atatgtgatg 1260
ccaattgaga aaggaattca ccatttccac tgctggttac accaagtgtt gaag 1314
<210> 2
<211> 438
<212> PRT
<213> Atriplex hortensis
<400> 2
Met Ala Ala Ser Ala Thr Thr Met Leu Leu Lys Tyr Pro Thr Thr Val
1 5 10 15
Cys Gly Ile Pro Asn Ser Ser Ala Asn Asn Ser Thr Asp Pro Ser Asn
20 25 30
Asn Ile Val Gln Ile Pro Gln Thr Thr Thr Thr Asn Ser Pro Leu Leu
35 40 45
Lys Phe Arg Thr Pro Asn Lys Pro Val Asn Ala Val Ala Ala Pro Ala
50 55 60
Phe Pro Ser Val Thr Thr Thr Thr Thr Thr Thr Pro Ser Ser Ile Gln
65 70 75 80
Ser Leu Val Lys Asp Phe Asp Pro Leu Val Pro Ala Glu Asp Ala Leu
85 90 95
Thr Pro Pro Ser Ser Trp Tyr Thr Glu Pro Ala Phe Tyr Ala His Glu
100 105 110
Leu Asp Arg Ile Phe Tyr Lys Gly Trp Gln Val Ala Gly Tyr Ser Asp
115 120 125
Gln Val Lys Glu Ala Asn Gln Tyr Phe Thr Gly Thr Leu Gly Asn Val
130 135 140
Glu Tyr Leu Val Cys Arg Asp Gly Glu Gly Lys Val His Ala Phe His
145 150 155 160
Asn Val Cys Thr His Arg Ala Ser Ile Leu Ala Cys Gly Ser Gly Lys
165 170 175
Lys Ser Cys Phe Val Cys Pro Tyr His Gly Trp Val Tyr Gly Met Asn
180 185 190
Gly Ser Leu Thr Lys Ala Ser Lys Ala Thr Pro Glu Gln Ser Leu Asn
195 200 205
Pro Asp Glu Leu Gly Leu Val Pro Leu Lys Val Ala Val Trp Gly Pro
210 215 220
Phe Ile Leu Ile Ser Leu Asp Arg Ser Ser Arg Glu Val Gly Asp Val
225 230 235 240
Gly Ser Glu Trp Leu Gly Ser Cys Ala Glu Asp Val Lys Ala His Ala
245 250 255
Phe Asp Pro Asn Leu Gln Phe Ile Asn Arg Ser Glu Phe Pro Ile Glu
260 265 270
Ser Asn Trp Lys Ile Phe Ser Asp Asn Tyr Leu Asp Ser Ser Tyr His
275 280 285
Val Pro Tyr Ala His Lys Tyr Tyr Ala Thr Glu Leu Asp Phe Asp Thr
290 295 300
Tyr Gln Thr Asp Met Val Gly Asn Val Thr Ile Gln Arg Val Ala Gly
305 310 315 320
Thr Ser Asn Asn Gly Phe Asn Arg Leu Gly Thr Gln Ala Phe Tyr Ala
325 330 335
Phe Ala Tyr Pro Asn Phe Ala Val Glu Arg Tyr Gly Pro Trp Met Thr
340 345 350
Thr Met His Ile Val Pro Leu Gly Pro Arg Lys Cys Lys Leu Val Val
355 360 365
Asp Tyr Tyr Ile Glu Lys Ser Lys Leu Asp Asp Lys Asp Tyr Ile Glu
370 375 380
Lys Gly Ile Ala Ile Asn Asp Asn Val Gln Lys Glu Asp Val Val Leu
385 390 395 400
Cys Glu Ser Val Gln Lys Gly Leu Glu Thr Pro Ala Tyr Arg Ser Gly
405 410 415
Arg Tyr Val Met Pro Ile Glu Lys Gly Ile His His Phe His Cys Trp
420 425 430
Leu His Gln Val Leu Lys
435
Claims (10)
1. prunella asiatica choline single oxygenase gene (AhCMO) is cultivating the application in low temperature resistant plant variety；It is characterized in that institute
Prunella asiatica choline single oxygenase gene (writing a Chinese character in simplified form are as follows: the AhCMO) nucleotide sequence shown in SEQ ID NO:1 stated forms.
2. application according to claim 1, it is characterised in that the prunella asiatica choline single oxygenase gene (AhCMO),
Its albumen amino acid sequence shown in SEQ ID NO:2 encoded forms.
3. application according to claim 1 or 2, it is characterised in that the plant refers to cotton, corn, rice or soybean etc..
4. the expression vector containing prunella asiatica choline single oxygenase gene (AhCMO) is cultivating answering in low temperature resistant plant variety
With；It is characterized in that the expression vector refers to plant expression vector pBI121 etc.；The plant refer to cotton, corn,
Rice or soybean etc..
5. the plant cell containing prunella asiatica choline single oxygenase gene (AhCMO) is cultivating answering in low temperature resistant plant variety
With；It is characterized in that the plant refers to cotton, corn, rice or soybean etc..
6. a kind of method for cultivating low temperature resistant cotton variety, including by prunella asiatica choline single oxygenase gene (AhCMO) by turning
Genetic method is transformed into cotton variety；Wherein the prunella asiatica choline single oxygenase gene is as shown in SEQ ID NO:1
Nucleotide sequence composition.
7. a kind of method for cultivating low temperature resistant cotton variety, it is characterised in that specific step is as follows:
(1) gene cloning: using AhCMO-F and AhCMO-R as primer, PCR amplification is carried out by substrate of prunella asiatica cDNA, obtains PCR
Amplified production, as AhCMO genetic fragment；The wherein primer are as follows:
AhCMO-F:5 '-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3 ',
AhCMO-R:3 '-GTGTTGCTTTTGATGCTTTCGTAAGCGATCCATTC-5 '；
(2) intermediate vector is constructed: the AhCMO genetic fragment obtained by PstI and XhoI double digestion step (1), agarose gel electrophoresis
After recycle genetic fragment；With PstI and XhoI double digestion carrier pUC19, recycle linear pUC19 segment, by AhCMO be connected into containing
In the pUC19 of the Enhanced expressing sequence of Cozk, Ω and p1oyA, and gene is made to connect CaMV35 promoter and Nos terminator, structure
Build up intermediate vector pUC19-AhCMO；
(3) expression vector establishment: using the III resulting intermediate vector pUC19-AhCMO of double digestion step (2) of EcoRI and Hihd, returns
Receive the segment containing AhCMO gene；Carrier pBI121 after being connected into III double digestion of EcoRI and Hihd respectively, is built into overexpression
Carrier pBI121-AhCMO；
(4) it converts: taking the 100 μ L of Agrobacterium GV3101 competent cell prepared, 10 μ g pBI121-AhCMO plasmids are added
DNA is mixed, and is placed in 5min on ice；Then 2mm electric shock cup is added in competence mixed liquor, 2500V shocks by electricity, is rapidly added after electric shock
800 μ L of YEB fluid nutrient medium, Fiber differentiation 5h under the conditions of 28 DEG C, 180rpm；The bacterium solution that taking-up has induced is coated in mould containing that is blocked
On the YEB plate of element and each 50 μ g/mL of rifampin, cultivated 48 hours at 28 DEG C；Picking monoclonal, which is inoculated in, blocks that containing for 5mL
In the YEB fluid nutrient medium of mycin and rifampin (concentration is 50 μ g/mL), bacterium is shaken under the conditions of 180rpm 36 hours, in a small amount
Extract plasmid enzyme restriction and PCR identification；The AhCMO carrier converting cotton that will be built using Agrobacterium infestation method, is obtained AhCMO and turns base
Because of cotton regenerated seedling；
(5) RT-PCR identification evaluation and screening: is carried out to the resulting regenerated transgenic seedling of step (4)；Harvest is accredited as positive plant
T0 for seed；The transgenosis T0 of harvest is sowed for seed, the T1 for taking growing way good extracts DNA for leaf tissue and carries out RT-
PCR detection, the strain that screening positive material continues to cultivate, and selects AhCMO gene expression amount high, is bred as AhCMO transgenic cotton
Flower variety.
8. cultivating the breeding method of low temperature resistant cotton variety, the breeding side using the transgene cotton containing AhCMO gene
Method is back cross breeding method or cross breeding method；
The back cross breeding method, includes the following steps:
(1) using the transgene cotton containing AhCMO gene as nonrecurrent parent, using the excellent cotton variety of character as circulation parent
This, hybridizes to obtain F1Generation；
(2), with F1On behalf of female parent, it is returned using cotton variety described in step (1) as male parent, obtains BC1F1Generation；With single grain
For unit, BC is extracted1F1It is to draw with AhCMO-F and AhCMO-R for single grain genomic DNA, and using the genomic DNA as substrate
Object carries out PCR identification, selects pcr amplification product size for the offspring of 1314bp；Positive backcross progeny is accredited as with PCR as father
This, is maternal backcrossing with cotton variety described in step (1), obtains BC2F1Generation；The wherein described primer sequence are as follows:
AhCMO-F:5 '-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3 ',
AhCMO-R:3 '-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5 '；
(3), it repeats described in step (2) backcrossing 3~6 times；It was finally selfed for 1 generation, and step (2) is carried out to the seed of self progeny
Described in PCR identification, it is unseparated to retain AhCMO gene in the single plant offspring of identification, is as bred as containing AhCMO gene
New varieties.
9. breeding method according to claim 8, it is characterised in that turn described in step (1) containing AhCMO gene
Gene cotton refers to the Derivative line of CMO24 or the CMO24 containing AhCMO gene；The deposit number of CMO24: CGMCC
No.17381；The excellent cotton variety of the character refers to the cotton variety promoted and applied in production；Or the cotton being just bred as
Strain；Wherein the character refers to economical character and fiber quality etc.；The reaction system (20ul) of the PCR: to be measured turn
1 μ l of gene cotton genomic dna, each 1 μ l, Mix10 μ l of primer, adds sterile water to 20 μ l；The reaction condition of the PCR are as follows:
94 DEG C, 3min；94℃,30Sec；56℃,30Sec；72℃,30Sec；Circulation 30 times；72 DEG C, 10min.
10. cultivating the breeding method of low temperature resistant cotton variety, the breeding side using the transgene cotton containing AhCMO gene
Method is back cross breeding method or cross breeding method；It is characterized in that the cross breeding method, includes the following steps:
(1), it is one of parent with the transgene cotton containing AhCMO gene, with elite cotton kind for another parents, obtains
F1Generation；
(2), by gained F1Generation selfing, harvests to obtain F2Generation；
(3), identification and selection F are assisted by molecular labeling2The single grain of AhCMO gene is had in generation, field plot test is positive
Offspring, and roguing is selfed；It repeats above-mentioned PCR identification and roguing was selfed for 4~6 generations, is i.e. cotton new product of the incubation containing AhCMO gene
Kind；Wherein the molecular labeling refers to using the genomic DNA of cotton seed to be measured as substrate, is with AhCMO-F and AhCMO-R
Primer carries out PCR amplification, selects amplified production size for the progeny material of 1314bp；
The cotton containing AhCMO gene refers to CMO24；The deposit number of CMO24: CGMCC No.17381.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910182562.5A CN109971772B (en) | 2019-03-12 | 2019-03-12 | Breeding method of low-temperature-resistant cotton variety |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910182562.5A CN109971772B (en) | 2019-03-12 | 2019-03-12 | Breeding method of low-temperature-resistant cotton variety |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109971772A true CN109971772A (en) | 2019-07-05 |
| CN109971772B CN109971772B (en) | 2021-05-28 |
Family
ID=67078401
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910182562.5A Active CN109971772B (en) | 2019-03-12 | 2019-03-12 | Breeding method of low-temperature-resistant cotton variety |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109971772B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643629A (en) * | 2019-09-19 | 2020-01-03 | 湖北省农业科学院经济作物研究所 | Method for creating high-quality cotton material based on wild germplasm |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1364905A (en) * | 2001-01-12 | 2002-08-21 | 大连理工大学 | Suaeda liaotungensis kitag chloine monoxygenase gene and its cloning |
-
2019
- 2019-03-12 CN CN201910182562.5A patent/CN109971772B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1364905A (en) * | 2001-01-12 | 2002-08-21 | 大连理工大学 | Suaeda liaotungensis kitag chloine monoxygenase gene and its cloning |
Non-Patent Citations (3)
| Title |
|---|
| JITENDER GIRI: "Glycinebetaine and abiotic stress tolerance in plants", 《PLANT SIGNALING & BEHAVIOR》 * |
| KENTA SHIRASAWA ET AL.: "Accumulation of Glycinebetaine in Rice Plants that Overexpress Choline Monooxygenase from Spinach and Evaluation of their Tolerance to Abiotic Stress", 《ANNALS OF BOTANY》 * |
| SHEN,Y.G. ET AL.: "Atriplex hortensis choline monooxygenase (CMO) mRNA, complete cds", 《GENBANK: AF270651.1》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643629A (en) * | 2019-09-19 | 2020-01-03 | 湖北省农业科学院经济作物研究所 | Method for creating high-quality cotton material based on wild germplasm |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109971772B (en) | 2021-05-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102174560B (en) | The delay senility of plant and adverse circumstance endurance | |
| CN108948164B (en) | Salt-tolerant drought-resistant sweet potato related protein IbbZIP1 as well as encoding gene and application thereof | |
| CN1884518B (en) | Directional gene transfer method of cabbage type rape C chromosome set | |
| CN103484436B (en) | Corn (zea mays) derived growth period related protein ZmHUB2 and coding gene and application thereof | |
| CN111926097B (en) | Insect-resistant herbicide-resistant corn transformation event and creation method and detection method thereof | |
| CN104004781A (en) | Preparation method of glyphosate resistant transgenic rice | |
| CN113789334B (en) | Application of HY5 gene in regulation and control of plant resistance to pest and disease damage | |
| CN110358772A (en) | The OsEBP89 gene and preparation method of raising rice abiotic stress resistance and application | |
| CN109468333A (en) | Citrus laccase family gene CsiLAC4 and its application | |
| CN109971772A (en) | A kind of breeding method of low temperature resistant cotton variety | |
| CN100413965C (en) | Phosphorus starvation induced gene promoter and its application | |
| CN102732553B (en) | Improve the gene engineering method and material of plant products | |
| CN103602688B (en) | Helianthus tuberosus L. Na<+>/H<+> reverse transport protein genes HtNHX1 and HtNHX2 and use thereof | |
| CN104278053B (en) | A kind of method for improving drought tolerance in plants ability | |
| CN106995492B (en) | Sucrose transporter and application thereof in regulation and control of plant male sterility | |
| CN109504687A (en) | A kind of soybean lipid transport protein gene GmABCA9 and its application | |
| CN102676576B (en) | Method for improving moisture resistance of cabbage type rape | |
| CN112724213B (en) | Sweet potato anthocyanin synthesis and stress resistance related protein IbMYB4, and coding gene and application thereof | |
| CN106868038A (en) | The method that cotton transgenic formulates cytoplasmic male sterile line | |
| CN107630021A (en) | The albumen and application of asparagus resistant gene of salt AoSOS2 and its coding | |
| CN106929518A (en) | A kind of rubber tree HbAG genes and its application | |
| CN106701783A (en) | Rice gene OsDF1 and application of disease control functions | |
| CN108642066B (en) | Protein from wild soybean and application of coding gene thereof in improving salt tolerance of plants | |
| CN107602694B (en) | Trichoderma reesei metallothionein TrCRD2S, encoding gene and its application in heavy metal resistance plant is cultivated | |
| CN110484545A (en) | A kind of mosaic disease resisting poison GsCAD1 gene separated from wild soybean, coding albumen and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |