CN103109740A - New strain breeding method of high-quality purple perilla - Google Patents
New strain breeding method of high-quality purple perilla Download PDFInfo
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Abstract
The invention discloses a new strain breeding method of high-quality purple perilla for effectively overcoming the defects of long period, low mutation rate, narrow mutation spectrum and the like of the conventional breeding method. The new strain breeding method has the beneficial effects that the purple perilla new strain which is high in yield, strong in stress resistance and high in alpha-linolenic acid can be bred, and the purple perilla bioindustry is promoted to be continuously, healthily and rapidly developed in Gansu province even in China, and necessary technical conditions and experimental basis are provided for breed improvement and development and utilization of the purple perilla as one precious Chinese medicinal herb.
Description
Technical field
The present invention relates to the selection of plant new lines, be specifically related to the selection and breeding method for new strain of high-quality purple perilla.
Background technology
Purple perilla
(Perilla frutescens L.)Belonging to Labiatae Perilla annual herb plant, is one of 60 kinds of first batch of food and medicament dual-purpose article of the Ministry of Public Health, is also one of the highest plant resources of alpha-linolenic acid content, is a kind of good health vegetables, is also a kind of important herbal medicine.Perilla herb oil (Perilla Seed Oil), also be perilla oil, to extract the natural oil of a kind of high degree of unsaturation that obtains in the ripe seed of purple perilla, Main Ingredients and Appearance is alpha-linolenic acid, it is the highest health care nutritional edible oil of alpha-linolenic acid content in all crude vegetals of finding at present, do not contain cholesterol, human body had significant health care and medical efficacy, can be converted into the vital activity factor D HA(DHA of metabolism necessity in human body, docosahexaenoic acid, DHA) and the EPA(eicosapentaenoic acid
,Eieosapentaenoieaeid, EPA), so alpha-linolenic acid is enjoyed the good reputation of " plant gold " in nutrition educational circles.
Purple perilla has the cultivation history of more than 2000 year in China, be distributed in more than 20 province of China, resource distribution is extensive, but the research of current relevant purple perilla breed breeding is less, improved seeds are deficient, cause the purple perilla cultivated area little, yield poorly, quality is unstable, and these factors have seriously restricted the development of purple perilla industry.Change this unfavorable situation, must rely on scientific and technological advances and strengthen the research dynamics of breeding technique and method, make breeding level that larger breakthrough be arranged.In order to improve breeding efficiency, shortening the breeding cycle, the breeding brainstrust is constantly being explored new breeding method and approach.
Present breeding technique mainly contains traditional conventional cross-breeding, mutation breeding, ploidy breeding, tissue cultivation, somatic hybridization, protoplast fusion and genetic engineering breeding etc.Wherein, the radiaction mutation method is easy, safety, economy, mutation rate are high, variation is stable fast, but brings out new varieties within a short period of time.The heavy ion beam that the CAS Institute of Modern Physics's Lanzhou Heavy Ion Cyclotron produces is having high, the unique ionization peak of linear energy transfer (Bragg peak), relative biological effectiveness high (RBE) aspect mutagenic organism, and the radioactive breeding mutation spectrum is wide, mutant character is easily stablized, the high unique advantage of forward mutation rate, has critical role in the nuclear agricultural science field, heavy ion irradiation mutation breeding has developed into a kind of new method and the new tool in current radiation breeding field, has extremely wide prospect.At present, heavy ion irradiation mutation breeding technology is sugar grass, Radix Angelicae Sinensis, and wheat, and obtained great successes in the flower plant such as wandering jew, petunia, dahlia.
Proteomics is utilized two dimensional gel electrophore-sis (two-dimensional protein electrophoresis exactly, 2DE) technology, detect the situation of protein expression on whole genomic level, it is the feature of Study on Protein on extensive level, comprise the protein expression level, modification after translation, protein-protein interaction etc., and mass spectrum
(Mass Spectrometry, MS
)It is one of the strongest instrument of proteomics research.Dielectrophoresis-mass spectrometry
(2DE-MS
)Be used in mutant and detect, can unite mutation type surface and mutant in the expression of protein level, further confirm mutant at protein level.
Gas-chromatography (GC) technology due to its uniqueness, efficiently, stalling characteristic fast, become physics, the indispensable important tool of chemical analysis, at present, this technology has had extensive use at aspects such as oil, chemical industry, environmental protection, medicines.The high resolution gas chromatography technology can be applied to by measuring certain main Secondary Metabolite Contents the quality-screening of mutant.
Summary of the invention
Purpose of the present invention is exactly for above-mentioned defective of the prior art, provides a kind of and has overcome effectively that the conventional hybridization breeding technique cycle is long, mutation rate is low and the selection and breeding method for new strain of the high-quality purple perilla of the defective such as mutation spectrum is narrow.
To achieve these goals, technical scheme provided by the invention is: the selection and breeding method for new strain of high-quality purple perilla comprises the following steps:
1) choose full seed, size evenly, without damaged, without the purple perilla dry seeds of mould contamination;
2) the purple perilla dry seeds that step 1) is obtained can carbon ion beam carry out irradiation with middle, obtains purple perilla dry seeds after irradiation;
3) with step 2) obtain irradiation after the purple perilla dry seeds carry out the land for growing field crops primary dcreening operation, obtain purple perilla primary dcreening operation mutant;
4) the purple perilla primary dcreening operation mutant that step 3) is obtained, by Phenotypic Observation, record, mark, RAPD molecular markers for identification, protein science analysis confirmation and high resolution gas chromatography technology for detection quality, screening obtains a plant height quality purple perilla new lines.
Further, the selection and breeding method for new strain of above-mentioned high-quality purple perilla, described step 2) in middle can carbon ion beam be that the middle of 100MeV/u can carbon ion beam; Described radiation mode is for vertically running through.
Further, the selection and breeding method for new strain of above-mentioned high-quality purple perilla, described step 2) irradiance method in is specially: the purple perilla dry seeds is contained in a thick 0.9cm at random, diameter is in the Tissue Culture Dish of 3.5cm, irradiation dose is respectively 0,40,80 and 120 Gy, the Ping Qu irradiation, dose rate is 4Gy/min.
Further, the selection and breeding method for new strain of above-mentioned high-quality purple perilla, in described step 3), the method for land for growing field crops primary dcreening operation is specially: adopt the method for single factors randomized blocks design, each dosage triplicate; Residential quarter area 15m
2(5m * 3m), sowing depth is 4cm, and line-spacing is 30cm, and spacing in the rows is 15m, and 4～5 leaf phase final singlings according to land for growing field crops phenotype Preliminary screening mutant, are carried out mark, and when ripe, individual plant is gathered.
Further, the selection and breeding method for new strain of above-mentioned high-quality purple perilla, in described step 4), a plant height quality purple perilla new lines that screens shows as the top shrinkage, tillers many, and inflorescence is long, and output is high, and plant is tall and big, and alpha-linolenic acid content is high.
The new lines Breeding Scheme flow process of high-quality purple perilla as shown in Figure 1.
Beneficial effect of the present invention is: the selection and breeding method for new strain of high-quality purple perilla provided by the invention, can breeding high-yield, the purple perilla new lines of strong stress resistance and high alpha-linolenic acid content, promote the purple perilla biological industry in Gansu Province and even the whole nation continues, healthy, development fast, be the breed improvement of this precious Chinese herbal medicine of purple perilla and exploitation provide the necessary technical condition and experimental basis.
Each demonstration test step:
One, RAPD molecular markers for identification experimental procedure:
Take the young leaflet tablet of purple perilla new lines (KJ0801) and contrast as material, adopt the CTAB method to extract genomic DNA, the pcr template in analyzing take this DNA as RAPD, RAPD-PCR as shown in table 1 analyzes primer, all available from Shanghai biotechnology Co., Ltd.
Adopt the PCR reaction system of 20ul, include: 2ul 2mmol/L MgCl
2The dNTP of 2ul 100umol/L; 1ul 0.2 umol/L primer; 20 ng template DNAs; 0.15ul 0.5U Taq enzyme, 13.85 μ l ddH
2O。
Table 1
。
The pcr amplification program: 94 ℃ of 5 min, 94 ℃ of 1 min, 36 ℃ of 1 min, 72 ℃ of 1 min, after 45 circulations, 72 ℃ of 10 min.Amplified production separates through 1.5% agarose gel electrophoresis, and observes with gel imaging system and take a picture, as shown in Figure 2.
Two, protein science is analyzed experimental procedure:
1.TCA-acetone method extracts albumen:
(1) protein extraction: get 0.5g purple perilla Tender leaf, put into rapidly the mortar of precooling, be ground to superfine after, the medication spoon is transferred to rapidly in the centrifuge tube of precooling, add 1mL 10%TCA-acetone solution (containing 0.07% beta-mercaptoethanol), mixing, lie in-20 ℃ of refrigerator overnight fast.
(2) washing protein: take out the protein liquid that spends the night, at 4 ℃, 12000r/min, then centrifugal 40min removes supernatant with rifle, adds 100% acetone solution, shakes even, keeps flat 1h in-20 ℃ of refrigerators, then centrifugal 40min.The above step of reference uses 100%, 80%, 100% acetone washing protein until the powder pure white successively.At vacuum drying 30min on ice.
(3) cracking: add a certain amount of lysate in dried powder, this process is preferably on ice carries out.After 2h, centrifugal 1h gets supernatant.
(4) protein quantification: carry out protein quantification with Coomassie brilliant blue with reference to the Brandford method.Final applied sample amount is 500ug/gel (Bradford 1976).
Dielectrophoresis:
(1) point focusing (IEF) such as: protein liquid and the hydrating fluid of certain volume are mixed, join in adhesive tape groove after centrifugal.Take out adhesive tape, room temperature was placed 10 minutes, write down numbering and sample number.The glue face of adhesive tape is put in adhesive tape groove gently, bubble do not occur.Add 500uL after aquation 1h and cover oil.Then seetting program carries out isoelectric focusing.Program is as follows: aquation 1h, 30v 11h, 200v 1h, 500v 1h, 1000v 2h, 8000v 5h.
(2) balance: take out adhesive tape and rushed 5 seconds facing to the adhesive tape seating surface with ultra-clean water, then be sidelong on moistening filter paper 5 minutes.Be put in equilibrium liquid I balance 15 minutes, every adhesive tape adds 10mL equilibrium liquid (now adding 0.1 gram DTT).Then change the balance 15min that (now adds 0.25 gram iodoacetamide) in equilibrium liquid II over to.Then on moistening filter paper, be sidelong 5 minutes.
(3) SDS-PAGE: take out the separation gel for preparing, adhesive tape is put on the glue face of separation gel, the glue surface plaster of noting avoiding adhesive tape is to glass, and the glue face of separation gel is close in the bottom surface of adhesive tape.Then add rapidly sealing liquid, notice that temperature can not too lowly cause too early cohesion or bubble occur, temperature can not too highly cause the PD sex change.Install electrophoretic apparatus and add electrophoresis liquid to carry out electrophoresis, first 10mA/gel, 1h, rear 30mA/gel is until bromophenol blue is run extremely apart from 1cm place, glue bottom.
(4) dyeing and decolouring: after bromophenol blue is run out of, take out the electrophoresis glass plate, note not damaged the glue face in the time of stripping glue.First dye in coomassie brilliant blue staining liquid, and then dye with destainer.
Analysis result as shown in Figure 3.
Three, mass spectral analysis experimental procedure:
1) enzymolysis and Ziptip desalination in glue:
Put into the Eppendorf pipe after each micelle chopping, every pipe adds 200-400 μ L 100mmol/L NH
4HCO
3/ 30% ACN decolouring (if silver dyes, is got 30-50 μ L 30mmol/L K
3Fe (CN)
6: 100mmol/L Na
2S
2O
3The volume ratio decolouring of=1:1), after freeze-drying, (enzyme is generally 1 with analyzed protein quality ratio: 20-1: 100), 37 ℃ of reactions are spent the night, about 20 hours to add 5 μ L 2.5-10 ng/ μ L order-checking level Trypsin (Promega) solution; The sucking-off enzymolysis liquid is transferred in new EP pipe, and former pipe adds 100 μ L 60% ACN/0.1% TFA, ultrasonic 15 minutes, merges last time solution, freeze-drying; If salt is arranged, use Ziptip(millipore) carry out desalination.
) mass spectral analysis:
Enzymolysis sample after freeze-drying is got 2 ml 20% acetonitriles and is redissolved.Get the 1ml sample, directly put on the sample target, allow the solvent natural seasoning, then get 0.5 μ L supersaturation CHCA matrix solution (solvent is 50% ACN, 0.1% TFA) point to corresponding target position and natural seasoning.the sample target is put into instrument and is advanced the target groove also with 4800 time-of-flight mass spectrometry instrument (4800 Plus MALDI TOF/TOFTM Analyzer) (Applied Biosystems after nitrogen blows off, USA) carry out mass spectral analysis, lasing light emitter is the Nd:YAG laser instrument of 355 nm wavelength, accelerating potential is 2 kV, adopt the type collection data of positive ion mode and automatic acquisition data, PMF mass scanning scope is 800-4000Da, the selection signal to noise ratio is carried out second order ms (MS/MS) analysis greater than 50 parent ion, select 8 parent ions on each sample spot, secondary MS/MS laser excitation 2500 times, collision energy 2 kV, CID closes.
Mass spectrometry results as shown in Figure 4.
) database retrieval:
Database retrieval uses following database analysis-by-synthesis search: Database: NCBI; Taxonomy: Viridiplantae(900091); Type of search: Peptide Mass Fingerprint (MS/MS Ion Search); Enzyme: Trypsin; Fixed modifications: Carbamidomethyl (C); Mass values:Monoisotopic; Protein Mass:Unrestricted; Peptide Mass Tolerance: ± 100 ppm; Fragment Mass Tolerance: ± 0.4 Da; Peptide Charge State: 1+Max Missed Cleavages: 1.Identify successful standard: protein score c.i.% is greater than 90.The albumen score has some differences because length protein is different with sequence, greatly more than 50-55 divides.
) data statistics
All indexs of measuring all arrange the repetition more than three, as the standard of significant difference, the mean parameter of measuring are carried out the t check with P≤0.05 level.
Four, high resolution gas chromatography is analyzed experimental procedure:
Collect the purple perilla seed, drying is ground, and extracts refining, and urea clathrate is with purifying alpha-linolenic acid, then adopts the high resolution gas chromatography technology to carry out qualitative and quantitative analysis to the alpha-linolenic acid content in mutant and adjoining tree seed.Adopt gas chromatography determination, the peak area normalizing calculates, and represents with relative percentage.Wherein chromatogram (GC) analysis condition is: the hollow quartz capillary column of Rtx-5 (WCOT), 30 m * 0.25 mm * 0.25 μ m; Split sampling, split ratio 30: 1; Carrier gas is N
2, press 112 kPa before post; Detector is FID, air 39 kPa, hydrogen 30 mL/min; 270 ℃ of injection port and detector temperatures; Adopt programmed temperature method: 130 ℃ of initial temperatures, after being warmed up to 185 ℃ with 10 ℃/min speed, keep 20 min, be warming up to 220 ℃ with 5 ℃/min speed, then rise to 285 ℃ with 10 ℃/min, and keep this temperature 5 min.The GC/MS condition: carrier gas is helium, presses 49 kPa before post, and other condition is identical with GC.250 ℃ of transmission line temperature, 200 ℃ of ion source temperatures.The high resolution gas chromatography analysis result as shown in Figure 5.
Description of drawings
Fig. 1 is the new lines Breeding Scheme of high-quality purple perilla.
Fig. 2 is the RAPD molecular markers for identification of purple perilla new lines and contrast.
Wherein, 1 road is purple perilla contrast, and the 2-7 road is the purple perilla new lines.
Fig. 3 is the protein science analysis confirmation of purple perilla new lines.
Fig. 4 is the protein science-mass spectral analysis figure of purple perilla new lines.
Fig. 5 is the high resolution gas chromatography analysis of alpha-linolenic acid content in purple perilla new lines and contrast.
Embodiment
Embodiment 1:
the selection and breeding method for new strain of high-quality purple perilla, in conjunction with agronomy, genetics, radiation biology, the means such as plant physiology and molecular biology, take the CAS Institute of Modern Physics's HIRFL (HIRFL) provide middle can carbon ion beam (100MeV/u) as mutation source, adopt the radiation mode that vertically runs through, the range of the carbon ion beam of this energy in the purple perilla dry seeds be 2.0cm approximately, LET is about 20 keV/ μ m, the purple perilla dry seeds is through irradiation mutagenesis and a series of subsequent treatment, screen good mutant, subsequent treatment comprises: through the plantation in 6 seasons, phenotypic screen and mark, the RAPD molecular marking technique is identified, after protein science analysis and high resolution gas chromatography technology are measured alpha-linolenic acid content, be defined as high-quality purple perilla new lines.
Test material and irradiation: the purple perilla seed source is in From Shijiazhuang City of Hebei Province agricultural and forest science institute's medicinal plants study center, choose full seed, size evenly, without damaged, without the purple perilla dry seeds of mould contamination.Seed is contained in a thick 0.9cm at random, and diameter is in the Tissue Culture Dish of 3.5cm, and irradiation dose is respectively 0,40,80 and 120 Gy, the Ping Qu irradiation, and dose rate is 4Gy/min, compares with the seed without irradiation
First carry out primary election and mark large Tanaka by phenotype, adopt again the RAPD molecular marking technique to identify from gene level, with bidirectional electrophoresis technique-mass spectral analysis (2DE-MS), new lines and contrast are carried out the analysis and research of protein science afterwards, measured alpha-linolenic acid content in new lines and normal control purple perilla seed to determine the quality of new lines by the high resolution gas chromatography technology at last.
The land for growing field crops prescreening method is:
Adopt the method for single factors randomized blocks design, each dosage triplicate.Residential quarter area 15m
2(5m * 3m), sowing depth is the 4cm left and right.Line-spacing 30cm, spacing in the rows 15m, 4～5 leaf phase final singlings.Experimental field fertility is even, and landform is smooth, and every residential quarter is except irradiation dose, and other each measure keeps strict conformance.According to land for growing field crops phenotype Preliminary screening mutant, carry out mark again, when ripe, individual plant is gathered.
Selection is as follows:
(1) at first sow the purple perilla dry seeds of irradiation large Tanaka, change according to phenotype and select from individual plant, and carry out listing mark and record, according to the record of listing, individual plant results seed stays after selected and does seed of lower season when results;
(2) second season selected single-strain seed seedling-cultivating tray is grown seedlings, move into the land for growing field crops during seven one-tenth leaf a slice young leaves, and with the same period first season growing state compare, each strain to the phenotype vary stable is done the RAPD molecular markers for identification, protein science is analyzed and the high resolution gas chromatography technology is measured alpha-linolenic acid content, relatively quality, screen good strain seed list receipts, stays after selected and does seed of lower season;
(3) five seasons of the third quarter to the were all the seeds that a upper season is chosen, grow seedlings with seedling-cultivating tray according to individual plant, be transplanted into the land for growing field crops during seven one-tenth leaf a slice young leaves, transplant according to recording the single-strain seed subregion, and with former same period in season growing state compare, observe stability, select single district of stable, the consistent strain of growth traits to gather, do kind;
The type of seeding of (4) the 6th seasons is with (3), and then subregion carries out the RAPD molecular markers for identification to each district's strain, and protein science-mass spectrum (2DE-MS) is analyzed, and the high resolution gas chromatography technology is measured alpha-linolenic acid content.With compare quality, be defined as high-quality purple perilla new lines with when results output strain high, that alpha-linolenic acid content is high.
It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment, the present invention is had been described in detail, for a person skilled in the art, it still can be modified to the technical scheme that aforementioned each embodiment puts down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (5)
1. the selection and breeding method for new strain of high-quality purple perilla, is characterized in that, comprises the following steps:
1) choose full seed, size evenly, without damaged, without the purple perilla dry seeds of mould contamination;
2) the purple perilla dry seeds that step 1) is obtained can carbon ion beam carry out irradiation with middle, obtains purple perilla dry seeds after irradiation;
3) with step 2) obtain irradiation after the purple perilla dry seeds carry out the land for growing field crops primary dcreening operation, obtain purple perilla primary dcreening operation mutant;
4) the purple perilla primary dcreening operation mutant that step 3) is obtained, by Phenotypic Observation, record, mark, RAPD molecular markers for identification, protein science analysis confirmation and high resolution gas chromatography technology for detection quality, screening obtains a plant height quality purple perilla new lines.
2. the selection and breeding method for new strain of high-quality purple perilla according to claim 1, is characterized in that, described step 2) in middle can carbon ion beam be that the middle of 100MeV/u can carbon ion beam; Described radiation mode is for vertically running through.
3. the selection and breeding method for new strain of high-quality purple perilla according to claim 2, it is characterized in that, described step 2) irradiance method in is specially: the purple perilla dry seeds is contained in a thick 0.9cm at random, diameter is in the Tissue Culture Dish of 3.5cm, irradiation dose is respectively 0,40,80 and 120 Gy, the Ping Qu irradiation, dose rate is 4Gy/min.
4. the selection and breeding method for new strain of high-quality purple perilla according to claim 3, is characterized in that, in described step 3), the method for land for growing field crops primary dcreening operation is specially: adopt the method for single factors randomized blocks design, each dosage triplicate; Residential quarter area 15m
2, sowing depth is 4cm, and line-spacing is 30cm, and spacing in the rows is 15m, and 4～5 leaf phase final singlings according to land for growing field crops phenotype Preliminary screening mutant, are carried out mark, and when ripe, individual plant is gathered.
5. the selection and breeding method for new strain of high-quality purple perilla according to claim 4, is characterized in that, in described step 4), a plant height quality purple perilla new lines that screens shows as the top shrinkage, tillers many, and inflorescence is long, output is high, and plant is tall and big, and alpha-linolenic acid content is high.
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Application publication date: 20130522 |