CN107360970A - Promote the method for Momordica grosvenori UGT74AC1 gene expressions - Google Patents
Promote the method for Momordica grosvenori UGT74AC1 gene expressions Download PDFInfo
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- CN107360970A CN107360970A CN201710629292.9A CN201710629292A CN107360970A CN 107360970 A CN107360970 A CN 107360970A CN 201710629292 A CN201710629292 A CN 201710629292A CN 107360970 A CN107360970 A CN 107360970A
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- momordica grosvenori
- ugt74ac1
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- methyl jasmonate
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- 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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- 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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a kind of method for promoting Momordica grosvenori UGT74AC1 gene expressions, including：Cultivated Step 1: Luohanguo With Plantlets of Tissue Culture is seeded in the solid medium containing methyl jasmonate, concentration of the methyl jasmonate in solid medium is 50 400 μm of ol/L；Luohanguo With Plantlets of Tissue Culture is taken out from the solid medium every 3 days, the root of Luohanguo With Plantlets of Tissue Culture is soaked in temperature as in the clear water of 5 DEG C and room temperature successively, then Momordica grosvenori is trained into seedling renewed vaccination and returned in the solid medium；Step 2: cultivating Momordica grosvenori plant, the methyl jasmonate that concentration is 50 400 μm of ol/L is sprayed on to the Momordica grosvenori surface of 20 30d after pollination.The present invention induces the high expression of key gene UGT74AC1 in momordica glycoside V biosynthesis pathway by applying methyl jasmonate in Luohanguo With Plantlets of Tissue Culture and cultivation Momordica grosvenori.
Description
Technical field
The present invention relates to gene technology, more particularly to a kind of method for promoting Momordica grosvenori UGT74AC1 gene expressions.
Background technology
Momordica glycoside V belongs to cucurbitane type tetracyclic triterpenes material, and the precursor substance of momordica glycoside V biosynthesis is
Base pyrophosphoric acid (DMAPP) in isopentenyl diphosphate (IPP) and 3,3- dimethyl alkene, the two is by mevalonic acid (MVA) and first
Two approach of base antierythrite phosphorylation (MEP) are formed, and MVA approach occurs in kytoplasm, and MEP approach occurs in plastid.Come
The IPP or DMAPP for coming from above-mentioned two approach form Mang ox base pyrophosphoric acid through Mang ox base pyrophosphate synthase (GPS) catalysis
(GPP), IPP and GPP under farnesyl pyrophosphate synthase (FPS) catalytic action and then forms farnesyl pyrophosphate (FPP), so
2,3- oxidosqualenes, cucurbit dienol synthase are formed by the catalysis of squalene synthase (SS), squalene epoxidase (SE)
(CS) further catalysis forms cucurbit dienol, finally in the presence of CYP450 enzymes and glucosyltransferase (UGT), is formed
Momordica glycoside V.
The generation of isoprenoid material is considered as on its biosynthesis way always by the strict regulation and control of speed limit enzymatic activity
Important regulating and controlling effect is played in footpath.As the rate-limiting enzyme in isoprene approach, the expression of UGT genes is to momordica glycoside V
Biosynthesis play decisive role.UGT genes are overexpressed, the accumulation of momordica glycoside V can be promoted；If on the contrary, suppression
The expression of UGT genes processed, the yield of momordica glycoside V will significantly reduce.Promotion Momordica grosvenori is there are no in the prior art
The report of UGT74AC1 gene expressions.
The content of the invention
For above-mentioned technical problem, the present invention has designed and developed a kind of side for promoting Momordica grosvenori UGT74AC1 gene expressions
Method.
Technical scheme provided by the invention is：
A kind of method for promoting Momordica grosvenori UGT74AC1 gene expressions, including：
Cultivated Step 1: Luohanguo With Plantlets of Tissue Culture is seeded in the solid medium containing methyl jasmonate, wherein, jasmonic
Concentration of the methyl esters in solid medium is 50-400 μm of ol/L；During culture, every 3 days by Luohanguo With Plantlets of Tissue Culture from described solid
Taken out in body culture medium, the root of Luohanguo With Plantlets of Tissue Culture is soaked in the clear water that temperature is 5 DEG C, soaked 15 minutes, Zhi Houzai
The root of Luohanguo With Plantlets of Tissue Culture is soaked in the clear water that temperature is room temperature, soaked 5 minutes, afterwards again by Luohanguo With Plantlets of Tissue Culture weight
Newly it is inoculated with back in the solid medium；
It is 50-400 μ by concentration during cultivation Step 2: cultivating the Momordica grosvenori plant that the step 1 culture obtains
Mol/L methyl jasmonate is sprayed on the Momordica grosvenori surface of 20-30d after pollination, untill surface is dripped, early, middle and late each sprinkling
Once, 5d is continuously sprayed.
Preferably, in the method for described promotion Momordica grosvenori UGT74AC1 gene expressions, in the step 1, the training
Foster condition is：Relative humidity 60-66%, intensity of illumination 1400lux, light application time 8h/d, cultivate under the conditions of 21-25 DEG C of temperature
30d。
Preferably, in the method for described promotion Momordica grosvenori UGT74AC1 gene expressions, described solid culture basigamy
Than for MS+6-BA 1.5mg/L, IBA 0.3mg/L, agar 3.5g/L, sucrose 30g/l, activated carbon 1.0g/L.
Preferably, it is described to contain in the step 1 in the method for described promotion Momordica grosvenori UGT74AC1 gene expressions
The solid medium for having methyl jasmonate is prepared in the following manner：It is water-soluble with the ethanol that concentration of volume percent is 2%
Liquid dissolves methyl jasmonate, is configured to 10000 μm of ol/L methyl jasmonate mother liquor, and is sterilized with 0.22 μm of miillpore filter,
Methyl jasmonate mother liquor after sterilizing is added in solid medium, the ultimate density to methyl jasmonate is 50-400 μ
Mol/L, solid medium is sterilized and is cooled to 24-26 DEG C.
Preferably, the method for described promotion Momordica grosvenori UGT74AC1 gene expressions, in addition to：
Step 3: detecting the step 2 using qRT-PCR cultivates UGT74AC1 genes in obtained Lo Han Guo fruit
Expression.
Preferably, in the method for described promotion Momordica grosvenori UGT74AC1 gene expressions, in the step 3, using changing
Good Trizol methods extract Lo Han Guo fruit total serum IgE.
The present invention in Luohanguo With Plantlets of Tissue Culture and cultivation Momordica grosvenori by applying methyl jasmonate, in a short time rapid induction arhat
Key gene UGT74AC1 high expression in fruit sweet tea glycosides V biosynthesis pathways, so as to promote the notable of momordica glycoside V content
Improve.The present invention is easy to operate, and cost is low, environmentally friendly, suitable for large-scale production, has stronger practicality and promotion price
Value.
Embodiment
The present invention is described in further detail below, to make those skilled in the art being capable of evidence with reference to specification word
To implement.
Embodiment one
A kind of method for promoting Momordica grosvenori UGT74AC1 gene expressions, including：
Cultivated Step 1: Luohanguo With Plantlets of Tissue Culture is seeded in the solid medium containing methyl jasmonate, wherein, jasmonic
Concentration of the methyl esters in solid medium is 50 μm of ol/L.The condition of culture is：Relative humidity 60-66%, intensity of illumination
1400lux, light application time 8h/d, 30d is cultivated under the conditions of 21 DEG C of temperature.Described solid medium proportioning is MS+6-BA
1.5mg/L, IBA 0.3mg/L, agar 3.5g/L, sucrose 30g/l, activated carbon 1.0g/L.During culture, every 3 days by arhat
Fruit tissue-cultured seedling takes out from the solid medium, the root of Luohanguo With Plantlets of Tissue Culture is soaked in the clear water that temperature is 5 DEG C, leaching
Bubble 15 minutes, the root of Luohanguo With Plantlets of Tissue Culture is soaked in the clear water that temperature is room temperature again afterwards, soaked 5 minutes, Zhi Houzai
Luohanguo With Plantlets of Tissue Culture renewed vaccination is returned in the solid medium.
The solid medium containing methyl jasmonate is prepared in the following manner：It is with concentration of volume percent
2% ethanol water dissolving methyl jasmonate, 10000 μm of ol/L methyl jasmonate mother liquor is configured to, and with 0.22 μm
Miillpore filter is sterilized, and the methyl jasmonate mother liquor after sterilizing is added in solid medium, final dense to methyl jasmonate
Spend for 50 μm of ol/L, solid medium is sterilized and is cooled to 24-26 DEG C.
It is 50 μm of ol/L by concentration during cultivation Step 2: cultivating the Momordica grosvenori plant that the step 1 culture obtains
Methyl jasmonate be sprayed on the Momordica grosvenori surface of 21d after pollination, untill surface is dripped, early, middle and late each sprinkling once, even
It is continuous to spray 5d.
Step 3: detecting the step 2 using qRT-PCR cultivates UGT74AC1 genes in obtained Lo Han Guo fruit
Expression.
First Lo Han Guo fruit is handled, picking pulp, be cut into 2-4mm fritters and wrapped respectively with masking foil, immediately
Be placed in it is quick-frozen in liquid nitrogen, be stored in -80 DEG C it is standby.
Lo Han Guo fruit total serum IgE is extracted using improved Trizol method.
Using ABI7500 real-time fluorescence quantitative PCR instrument.
It is μ L, the PrimeScript RT Enzyme Mix I of RNA 10.0 by the reaction system that RNA reverse transcriptions are cDNA
μ L, the RNase Free dH of 1.0 μ L, RT Primer Mix, 1.0 μ L, 5 × PrimeScript Buffer 24.02O 4.0μL；
Reaction condition is 37 DEG C of (15min) → 85 DEG C (5s) → 4 DEG C.
Using the software Design primers of Primer Premier 5.0, closed by Sangon Biotech (Shanghai) Co., Ltd.
Into.Wherein, UGT74AC1 primer sequences are (FP：
CGCCATATGCACCACCACCACCACCACATGGTGGTTTCCACTTCCAGCG(SEQ ID NO:1), RP：
CCCTCGAGTTATCTGATTAAATTAGTTGATT(SEQ ID NO:2)), reference gene UBQ5, sequence are (FP：
ATAAAAGACCCAGCACCACATTC(SEQ ID NO:3), RP：CCCTTGCCGACTACAACATCC(SEQ ID NO:4)).
QRT-PCR reaction systems (20 μ L) are SYBR Premix Ex Taq II (Tli RNaseH Plus) (2 ×)
10.0 μ L, PCR Forward Primer (10 μM) 0.8 μ L, PCR Reverse Primer (10 μM) 0.8 μ L, ROX
Reference Dye of Dye II (50 ×) 0.4 μ L, Template 2.0 μ L, dH2O6.0μL.Reaction condition is 95 DEG C
(30s), then carry out 40 cycles, [95 DEG C (5s), 95 DEG C (34s)].
Using the content of HPLC methods measure momordica glycoside V.Chromatographic condition is：Chromatographic column：DIKMA Diamomsil(TM)
C18 (4.6mm × 250mm, 5 μm)；Mobile phase：Acetonitrile-water (gradient 0-40min；10% linearly rises to 40%；40.01-45mm；
It is back to 10%)；Flow velocity：1.0ml/min, Detection wavelength：203nm.Test sample sample liquid is formulated as：Grosvenor Momordica Fruit 2g is taken, essence
It is close weighed, it is placed in 100ml tool grinding port plug conical flasks, precision adds methanol 25ml, shakes up, weighed weight.1h is ultrasonically treated, is put
It is cold, then weighed weight, and the weight of less loss is supplied with methanol, shake up, with 0.45 μm of filtrate filtering with microporous membrane.
Comparative example one
Cultivated Step 1: Luohanguo With Plantlets of Tissue Culture is inoculated in the solid medium for do not contain methyl jasmonate；Step 2:
Methyl jasmonate is not sprayed to Momordica grosvenori plant；Other conditions are consistent with embodiment one.
QRT-PCR testing results are shown, in comparative example one, the expression quantity of Momordica grosvenori UGT73AF1 genes is 1, and in reality
Apply in example one, the expression quantity of Momordica grosvenori UGT73AF1 genes is up to 8.9, improves 790% compared with comparative example one, it is seen then that embodiment
One can remarkably promote the high expression of Momordica grosvenori UGT73AF1 genes.HPLC testing results are shown, in comparative example one, Momordica grosvenori
Sweet tea glycosides V content is 0.75%, and in embodiment one, momordica glycoside V content is up to 1.89%, is improved compared with comparative example one
152.00%, it is seen then that embodiment one can also remarkably promote the raising of momordica glycoside V yield.
Embodiment two
A kind of method for promoting Momordica grosvenori UGT74AC1 gene expressions, including：
Cultivated Step 1: Luohanguo With Plantlets of Tissue Culture is inoculated in the solid medium containing methyl jasmonate, wherein, jasmonic
Concentration of the methyl esters in solid medium is 100 μm of ol/L.The condition of culture is：Relative humidity 60-66%, intensity of illumination
1400lux, light application time 8h/d, 30d is cultivated under the conditions of 25 DEG C of temperature.Described solid medium proportioning is MS+6-BA
1.5mg/L, IBA 0.3mg/L, agar 3.5g/L, sucrose 30g/l, activated carbon 1.0g/L.During culture, every 3 days by arhat
Fruit tissue-cultured seedling takes out from the solid medium, the root of Luohanguo With Plantlets of Tissue Culture is soaked in the clear water that temperature is 5 DEG C, leaching
Bubble 15 minutes, the root of Luohanguo With Plantlets of Tissue Culture is soaked in the clear water that temperature is room temperature again afterwards, soaked 5 minutes, Zhi Houzai
The solid medium is returned into Luohanguo With Plantlets of Tissue Culture renewed vaccination.
It is 300 μm of ol/ by concentration during cultivation Step 2: cultivating the Momordica grosvenori plant that the step 1 culture obtains
L methyl jasmonate is sprayed on the Momordica grosvenori surface of 25d after pollination, and untill surface is dripped, early, middle and late each sprinkling once, connects
It is continuous to spray 5d.
Other conditions are consistent with embodiment one.
Comparative example two
Cultivated Step 1: Luohanguo With Plantlets of Tissue Culture inoculation is not contained in the solid medium of methyl jasmonate；Step 2: not
Methyl jasmonate is sprayed to Momordica grosvenori plant；Other conditions are consistent with embodiment two.
QRT-PCR testing results are shown, in comparative example two, the expression quantity of Momordica grosvenori UGT73AF1 genes is 1, and in reality
Apply in example two, the expression quantity of Momordica grosvenori UGT73AF1 genes is up to 16.7, and 1570% is improved compared with comparative example two, it is seen then that implement
Example two can remarkably promote the high expression of Momordica grosvenori UGT73AF1 genes.HPLC testing results are shown, in comparative example two, arhat
Fruit sweet tea glycosides V content is 0.76%, and in embodiment two, momordica glycoside V content is up to 2.05%, is improved compared with comparative example two
169.74%, it is seen then that embodiment two can also remarkably promote the raising of momordica glycoside V yield.
Embodiment three
A kind of method for promoting Momordica grosvenori UGT74AC1 gene expressions, including：
Cultivated Step 1: Luohanguo With Plantlets of Tissue Culture is inoculated in the solid medium containing methyl jasmonate, wherein, jasmonic
Concentration of the methyl esters in solid medium is 200 μm of ol/L.The condition of culture is：Relative humidity 60-66%, intensity of illumination
1400lux, light application time 8h/d, 30d is cultivated under the conditions of 25 DEG C of temperature.Described solid medium proportioning is MS+6-BA
1.5mg/L, IBA 0.3mg/L, agar 3.5g/L, sucrose 30g/l, activated carbon 1.0g/L.During culture, every 3 days by arhat
Fruit tissue-cultured seedling takes out from the solid medium, the root of Luohanguo With Plantlets of Tissue Culture is soaked in the clear water that temperature is 5 DEG C, leaching
Bubble 15 minutes, the root of Luohanguo With Plantlets of Tissue Culture is soaked in the clear water that temperature is room temperature again afterwards, soaked 5 minutes, Zhi Houzai
The solid medium is returned into Luohanguo With Plantlets of Tissue Culture renewed vaccination.
It is 100 μm of ol/ by concentration during cultivation Step 2: cultivating the Momordica grosvenori plant that the step 1 culture obtains
L methyl jasmonate is sprayed on the Momordica grosvenori surface of 27d after pollination, and untill surface is dripped, early, middle and late each sprinkling once, connects
It is continuous to spray 5d.
Other conditions are consistent with embodiment one.
Comparative example three
Cultivated Step 1: Luohanguo With Plantlets of Tissue Culture inoculation is not contained in the solid medium of methyl jasmonate；Step 2: not
Methyl jasmonate is sprayed to Momordica grosvenori plant；Other conditions are consistent with embodiment three.
QRT-PCR testing results are shown, in comparative example three, the expression quantity of Momordica grosvenori UGT73AF1 genes is 1, and in reality
Apply in example three, the expression quantity of Momordica grosvenori UGT73AF1 genes is up to 12.9, and 1190% is improved compared with comparative example three, it is seen then that implement
Example three can remarkably promote the high expression of Momordica grosvenori UGT73AF1 genes.HPLC testing results are shown, in comparative example three, arhat
Fruit sweet tea glycosides V content is 0.74%, and in embodiment three, momordica glycoside V content is up to 2.13%, is improved compared with comparative example three
187.84%, it is seen then that embodiment three can also remarkably promote the raising of momordica glycoside V yield.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, it is of the invention and unlimited
In specific details.
SEQUENCE LISTING
<110>Li Huazheng
<120>Promote the method for Momordica grosvenori UGT74AC1 gene expressions
<130> 2016
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 49
<212> DNA
<213>Artificial sequence
<400> 1
cgccatatgc accaccacca ccaccacatg gtggtttcca cttccagcg 49
<210> 2
<211> 31
<212> DNA
<213>Artificial sequence
<400> 2
ccctcgagtt atctgattaa attagttgat t 31
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<400> 3
ataaaagacc cagcaccaca ttc 23
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
cccttgccga ctacaacatc c 21
Claims (6)
- A kind of 1. method for promoting Momordica grosvenori UGT74AC1 gene expressions, it is characterised in that including：Cultivated Step 1: Luohanguo With Plantlets of Tissue Culture is seeded in the solid medium containing methyl jasmonate, wherein, methyl jasmonate Concentration in solid medium is 50-400 μm of ol/L；During culture, Luohanguo With Plantlets of Tissue Culture is trained from the solid every 3 days Support and taken out in base, the root of Luohanguo With Plantlets of Tissue Culture is soaked in the clear water that temperature is 5 DEG C, soaked 15 minutes, afterwards again by sieve The root of Chinese fruit tissue-cultured seedling is soaked in the clear water that temperature is room temperature, is soaked 5 minutes, is afterwards again connect Luohanguo With Plantlets of Tissue Culture again Plant back in the solid medium；It is 50-400 μm of ol/L by concentration during cultivation Step 2: cultivating the Momordica grosvenori plant that the step 1 culture obtains Methyl jasmonate be sprayed on the Momordica grosvenori surface of 20-30d after pollination, untill surface is dripped, early, middle and late each sprinkling once, Continuously spray 5d.
- 2. promote the method for Momordica grosvenori UGT74AC1 gene expressions as claimed in claim 1, it is characterised in that the step 1 In, the condition of culture is：Relative humidity 60-66%, intensity of illumination 1400lux, light application time 8h/d, 21-25 DEG C of bar of temperature 30d is cultivated under part.
- 3. promote the method for Momordica grosvenori UGT74AC1 gene expressions as claimed in claim 2, it is characterised in that described solid Medium Proportion is MS+6-BA 1.5mg/L, IBA 0.3mg/L, agar 3.5g/L, sucrose 30g/l, activated carbon 1.0g/L.
- 4. promote the method for Momordica grosvenori UGT74AC1 gene expressions as claimed in claim 3, it is characterised in that the step 1 In, the solid medium containing methyl jasmonate is prepared in the following manner：It is 2% with concentration of volume percent Ethanol water dissolves methyl jasmonate, is configured to 10000 μm of ol/L methyl jasmonate mother liquor, and filtered with 0.22 μm of micropore Film sterilizes, and the methyl jasmonate mother liquor after sterilizing is added in solid medium, the ultimate density to methyl jasmonate is 50- 400 μm of ol/L, solid medium is sterilized and is cooled to 24-26 DEG C.
- 5. promote the method for Momordica grosvenori UGT74AC1 gene expressions as claimed in claim 1, it is characterised in that also include：Step 3: detecting the step 2 using qRT-PCR cultivates UGT74AC1 gene expressions in obtained Lo Han Guo fruit.
- 6. promote the method for Momordica grosvenori UGT74AC1 gene expressions as claimed in claim 5, it is characterised in that the step 3 In, Lo Han Guo fruit total serum IgE is extracted using improved Trizol method.
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| CN114752577A (en) * | 2021-01-08 | 2022-07-15 | 桂林莱茵生物科技股份有限公司 | Momordica grosvenori-derived glycosyltransferase mutant and application thereof |
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