CN106591359A - Construction method of Cannabis sativa L. THCA synthetase gene RNAi vector - Google Patents
Construction method of Cannabis sativa L. THCA synthetase gene RNAi vector Download PDFInfo
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- CN106591359A CN106591359A CN201710076429.2A CN201710076429A CN106591359A CN 106591359 A CN106591359 A CN 106591359A CN 201710076429 A CN201710076429 A CN 201710076429A CN 106591359 A CN106591359 A CN 106591359A
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Abstract
The invention discloses a construction method of a Cannabis sativa L. THCA synthetase gene RNAi vector. The method comprises the following steps: 1, extracting total RNA; 2, carrying out reverse transcription to form cDNA; 3, carrying out a PCR reaction on the above obtained reverse transcription product; 4, linking a THCA synthetase gene forward fragment with a pSKint vector, and carrying out conversion, plasmid extraction and enzyme digestion identification to obtain pSKTHCAihp I; 5, linking the pSKTHCAihp I with a THCA synthetase gene reverse fragment, and carrying out conversion, plasmid extraction and enzyme digestion identification to obtain pSKihpTHCA; and 6, linking a fragment containing forward and reverse synthetase gene and intron with a pCAMBIAsuper1300 + plant expression vector, and carrying out conversion, plasmid extraction and double enzyme digestion identification to obtain the plant expression vector ppCAMBIAsuper1300 + ihpTHCA. The construction method fills the gap in the construction method of Cannabis sativa L. RNAi vectors.
Description
Technical field
The present invention relates to a kind of Fructus Cannabiss RNAi carrier construction method.
Background technology
Fructus Cannabiss (Cannabis Sativa L.) are that Cannabaceae (Cannabinaceae) Cannabis (Cannabis) is annual
Herbaceous plant, due to, containing a kind of active component-tetrahydrocannabinol (THC) of the unreal addiction of cause, easily being used by lawless person in Fructus Cannabiss
Carry out illegal production drugs, cause social danger, many countries that industrial hemp also one is listed as crop of forbidding cultivating.THC in Fructus Cannabiss
Content is determined by the gene (endogenous cause of ill) of Fructus Cannabiss, and is had with growing environment (temperature, humidity, edaphic condition, sunshine etc.) (exopathogenic factor)
Close.Difference due to cultivating subenvironment, THC contents have differences between individuals；As Fructus Cannabiss are planted in there is sufficient space
During the environment of inland, its internal THC content is significantly improved.
Early in nineteen ninety-five, people just have discovered that the key enzyme in THC biosynthesis pathwaies is tetrahydro-cannabinolic acid
(THCA) synzyme, this enzyme is the monomer dehydrogenase that a molecular weight is 74kDa, and it is catalyzed by pentylresorcinol acid to four
The oxidative cyclization reaction of hydrogen cannabinoid acid A.The biological conjunctions of THC are made using the method for knock out mutants (knock-out mutation)
Inactivate into a key enzyme in approach, can be to cultivate the Fructus Cannabiss new lines without THC to lay the foundation.
RNA disturbs (RNA interference, RNAi), and it refers to endogenous or external source double-stranded RNA (double-stranded
RNA, dsRNA) there is selective degradation in the mRNA of endogenous target gene of mediation, and then the expression of suppressor gene makes gene silencing
Phenomenon, produce the phenomenon of corresponding function phenotype disappearance.Some of Caulis et Folium Lini, Boehmeria some crudefiber crops are had reported at present
The structure of gene RNAi carrier, but not yet have the report of Fructus Cannabiss RNAi carrier construction method.
The content of the invention
It is an object of the invention to provide a kind of construction method of Fructus Cannabiss THCA synthase gene RNAi carriers, to fill up Fructus Cannabiss
The blank of RNAi carrier construction method.
The purpose of the present invention is achieved through the following technical solutions：
A kind of construction method of Fructus Cannabiss THCA synthase gene RNAi carriers, comprises the steps：
First, with Fructus Cannabiss florescence blade, male flower, female flower aggregate sample as material, total serum IgE is extracted；
2nd, reverse transcription is cDNA；
3rd, with reverse transcription product as template, specific primer is designed, performing PCR is entered to reverse transcription product with high-fidelity Taq enzyme
Reaction, obtains PCR primer and reclaims；
4th, by the THCA synthase genes forward direction fragment and the double digestions of I/Hind of Sal III of the double digestions of I/Hind of Jing Sal III
PSKint carriers connect, and convert bacillus coli DH 5 alpha competent cell, and screening positive clone extracts the identification of bacterium solution PCR correct
Recombiant plasmid, by the double digestions of I/Hind of Sal III, further identification obtains pSKTHCAihp I；
5th, the THCA synthase genes of the double digestions of I I/Sac of Jing BamH of pSKTHCAihp I and the double digestions of I/Sac of Jing BamH I
Reversely fragment is attached, and converts bacillus coli DH 5 alpha competent cell, and screening positive clone extracts bacterium solution PCR and identifies just
True recombiant plasmid, by the double digestions of I/Sac of Sal I, further identification obtains pSKihpTHCA；
6th, glue reclaim obtains the fragment containing forward and reverse THCA synthase genes and intron, with I/Sac of Jing Sal, I pair of enzyme
Cut pCAMBIAsuper1300+ plant expression vectors to be attached, convert bacillus coli DH 5 alpha competent cell, positive gram of screening
Longzi, extracts the identification of bacterium solution PCR, and the double digestions of I/Sac of Sal I are further identified, plant expression vector pCAMBIAsuper1300+
IhpTHCA is successfully constructed.
The present invention builds the gene of THCA synthase gene RNAi carriers, extracts from Fructus Cannabiss florescence blade, male flower, female flower and mixes
In closing sample, its nucleotide sequence is as shown in SEQ ID NO.1.
The THCA synthase genes RNAi carrier that the present invention builds can be to cultivate the Fructus Cannabiss without tetrahydrocannabinol (THC)
New lines lay the foundation, and create more excellent new germ plasms, serve industrial hemp breeding and theoretical research.
Description of the drawings
Fig. 1 is, by the intron two ends of the forward and reverse gene clonings of THCA to intermediate carrier pSKint, to build
PSKintihpTHCA, restriction enzyme site is Sal I, Hind III, BamH I, Sac I；
Fig. 2 is plant expression vector pCAMBIASuper1300+, and it is Jing pCAMBIA1300 transformations, will
The multiple clone site of pCAMBIA1300 is entirely scaled off, and adds super promoter and multiple clone site, and restriction enzyme site is successively
For Xba I, Hind III, Pst I, Apa I, Sma I, Sal I, Swa I, Spe I, Kpn I, Sac I；
Fig. 3 is the structure flow chart of RNAi carrier pCAMBIASuper1300+ihpTHCA；
Fig. 4 is Fructus Cannabiss florescence blade, male flower, the picture of female flower aggregate sample Total RNAs extraction；
Fig. 5 is CsTHCAihp I and the pcr amplification product figures of ihp II, and base number is 504bp；
Fig. 6 is the plasmid figures of pSK THCAihp I, and 1 swimming lane is pSKint plasmids；
Fig. 7 is the enzyme action qualification figures of I/Sac of pSKihpTHCA plasmid Sal I, and intron (163bp)+forward and reverse gene order is about
For 1171bp；
Fig. 8 is the enzyme action qualification figures of I/Sac of RNAi carrier pCAMBIASuper1300+ihpTHCA plasmid Sal I；
Fig. 9 is plasmid PCR identification after the conversion of pCAMBIASuper1300+ihpTHCA RNAi carrier Agrobacteriums freeze-thaw method
Figure.
Specific embodiment
Technical scheme is further described below in conjunction with the accompanying drawings, but is not limited thereto, it is every to this
Inventive technique scheme is modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention, all should be covered
In protection scope of the present invention.
The invention provides a kind of construction method of Fructus Cannabiss THCA synthase gene RNAi carriers, concrete construction step is such as
Under：
First, with Fructus Cannabiss florescence blade, male flower, female flower aggregate sample as material, using TRIzol extracting solution extracting RNAs, specifically
Step is as follows：
(1) take vegetable material about 0.1g and fine powder is ground in liquid nitrogen, proceed to the 1.5mL centrifuge tubes of pre-cooling；Add 1mL
The concussion of TRIzol extracting solution shakes up；
(2) 5min on ice is placed in after mixing, 4 DEG C, 12000rpm is centrifuged 15min；Supernatant is taken, order adds 100 μ L 3M
NaOAC (pH 5.2), mixes, the phenol/chloroform/isoamyl alcohol (25 of 200 μ L:24:1), fully mix.It is placed in and stands on ice
10min；
(3) 4 DEG C, 12000rpm centrifugation 15min；Add 0.5mL isopropanols under the conditions of -20 DEG C, precipitate more than 20min；
(4) and then under the conditions of 2～8 DEG C, 12000rpm is centrifuged 20min, RNA precipitate tube wall and bottom；
(5) remove liquid portion, add 1mL75% ethanol；Under the conditions of 2～8 DEG C, 12000rpm centrifugation 10min；
(6) remove liquid portion, after air-drying, add 30 μ L DNase/RNase-free deionized water dissolving RNA, storage
It is standby in -80 DEG C of refrigerators, obtain Fructus Cannabiss florescence blade, male flower, female flower aggregate sample total serum IgE (Fig. 4).
Note：Wear masks and glove, and will normal hand-off set.
2nd, reverse transcription is cDNA, wherein：Each component is as follows in the synthesis of the chains of cDNA first：
Reaction condition：50 DEG C, 45min；85 DEG C, 5min, cDNA product is in -20 DEG C of storages, its nucleotide sequence such as SEQ
Shown in ID NO.1.
3rd, with reverse transcription product as template, specific primer is designed, performing PCR is entered to reverse transcription product with high-fidelity Taq enzyme
Reaction, obtains PCR primer and reclaims, wherein：
(1) primer sequence is as follows：
Positive sequence：Restriction enzyme site Sal I and Hind III
THCAihpⅠSalF：5’-CTAGTCGACCTCAGCATTTTCCTTTTG-3’；
THCAihpⅠHinR：5’-GCCCCAAGCTTAAACTAAGATTCTCATTC-3’；Gene size：504bp.
Reverse sequence：Restriction enzyme site Sac I and BamH I
THCAihpⅡSacF：5’-GTAGAGCTCCTCAGCATTTTCCTTTTG-3’；
THCAihpⅡBamR：5’-GGCGGATCCAAACTAAGATTCTCATTC-3’；Gene size：504bp.
(2) each component is as follows in PCR reactions：
Reaction condition is as follows：
CsTHCAihp I and the pcr amplification products of ihp II are as shown in Figure 5.
4th, as shown in figure 1, by the THCA synthase genes of the double digestions of I/Hind of Jing Sal III forward direction I/Hind of fragment and Sal
III double digestion pSKint carriers connect, and convert bacillus coli DH 5 alpha competent cell, and screening positive clone extracts bacterium solution PCR
Correct recombiant plasmid is identified, further identification obtains pSKTHCAihp I, pSK THCAihp I by the double digestions of I/Hind of Sal III
Plasmid figure is as shown in fig. 6, wherein：
(1) reaction system of the enzyme action CsTHCAihp I and pSKint of Sal I is as follows：
In 37 DEG C of water-bath 8h or so, digestion products recovery is carried out.
(2) reaction system of the enzyme action previous step recovery products of Hind III is as follows：
In 37 DEG C of water-bath 8h or so, digestion products recovery is carried out.
(3) reaction system of forward direction gene cloning to carrier pSKint is as follows:
In 16 DEG C, about 10h.
(4) competent escherichia coli cell thermal shock method for transformation is as follows：
A () is placed in a competent cell (100uL is stored in -80 DEG C) on ice.
B () adds 10uL connection products in competent cell, be placed in 30min on ice.
C () is placed in 42 DEG C of thermal shock 1min30s, be subsequently placed at least 5min on ice.
D () adds LB fluid mediums of the 400uL without any antibiotic, 37 DEG C in shaking table, 200rpm shakes 60min.
E above-mentioned culture is divided equally two parts by (), coat on the LB solid mediums containing Amp, and 37 DEG C just put culture 1h
Afterwards, put upside down culture.
(5) each component is as follows in bacterium solution PCR：
Reaction condition is as follows：
The good extraction plasmid of electrophoresis result, plasmid electrophoresis result is as shown in Figure 6.
(6) reaction system of the enzyme action CsTHCAihp II of Sac I and the plasmids of pSKTHCAihp I is as follows：
In 37 DEG C of water-bath 8h or so, digestion products recovery is carried out.
(7) reaction system of the enzyme action previous step recovery products of BamH I is as follows：
In 30 DEG C of water-bath 8h or so, digestion products recovery is carried out.
5th, as shown in figure 1, the THCA of the double digestions of I I/Sac of Jing BamH of pSKTHCAihp I and the double digestions of I/Sac of Jing BamH I
The reverse fragment of synthase gene is attached, and converts bacillus coli DH 5 alpha competent cell, and screening positive clone extracts bacterium solution
PCR identifies correct recombiant plasmid, and by the double digestions of I/Sac of Sal I, further identification obtains pSKihpTHCA (Fig. 7), wherein：
(1) cdna reverse be cloned into carrier pSKTHCAihp I reaction system it is as follows:
In 16 DEG C, about 10h.
(2) reaction system of the enzyme action pSKihpTHCA of Sal I is as follows:
In 37 DEG C of water-bath 4h or so, digestion products recovery is carried out.
(3) reaction system of the enzyme action previous step recovery products of Sac I is as follows：
In 37 DEG C of water-bath 8h or so, electrophoresis checking is carried out.That verifies successful connection carries out glue reclaim.
6th, as shown in figure 3, glue reclaim obtains fragment and Jing Sal containing forward and reverse THCA synthase genes and intron
Double digestions pCAMBIAsuper1300+ plant expression vectors (Fig. 2) of I/Sac I are attached, and convert bacillus coli DH 5 alpha competence
Cell, screening positive clone extracts the identification of bacterium solution PCR, and the double digestions of I/Sac of Sal I are further identified (Fig. 8), and plant expression is carried
Body pCAMBIAsuper1300+ihpTHCA is successfully constructed, wherein：
(1) reaction system of the enzyme action pCAMBIA super1300+ of Sal I is as follows:
In 37 DEG C of water-bath 8h or so, digestion products recovery is carried out.
(2) reaction system of the enzyme action previous step recovery products of Sac I is as follows：
(3) reaction system of forward and reverse gene cloning to expression vector pCAMBIASuper1300+plasmid is as follows：
In 16 DEG C, about 10h.
(4) preparation of Agrobacterium competent cell：
A () goes bail for the Agrobacterium tumefaciens EHA105/LBA4404 strains deposited in the flat lining outs of the LB containing Rif, 28 DEG C
Culture 2d；
B the fresh Agrobacterium EHA105/LBA4404 single bacterium colonies of () picking (contain rifampicin in the LB fluid mediums of 3mL
Rif in), 28 DEG C of 180rpm shaken cultivation are overnight；
C () takes incubated overnight bacterium solution and takes 1mL bacterium solutions, in adding LB containing 100mL (Rif containing rifampicin) fluid medium, 28
DEG C shaken cultivation about 4～8h to OD600For 0.5 or so；
D () ice-water bath 30min, in 4 DEG C, 5,000rpm, is centrifuged 10min, abandon supernatant；
E () plus 10mL 0.15mol/L NaCl suspension agrobatcerium cells, in 4 DEG C 5,000rpm, are centrifuged 10min；
F () abandons supernatant, add the 20mmol/L CaCl of l mL pre-coolings2(DMSO containing dimethyl sulfoxide) suspension cell, ice
Bath, is distributed into every pipe 200uL, and quick-freezing l min in liquid nitrogen put -80 DEG C and save backup.
(5) conversion (Agrobacterium freeze-thaw method conversion) of the plant expression vector to Agrobacterium competent cell：
A () takes the Agrobacterium EHA105/LBA4404 competent cell 200uL for preparing and melts on ice, in super-clean bench
20uL expression vector pCAMBIASuper1300+ihpTHCA plasmid DNA is added, is gently mixed, 10min is stood in ice-water bath.
B quick-freezing 5min in () liquid nitrogen, then centrifuge tube is quickly placed in 37 DEG C of water-baths keep 5min, should not rock water
Face；
C () puts back to centrifuge tube in ice-water bath, keeping 5min.
D () adds the LB fluid mediums without any antibiotic of 800uL after taking out, 28 DEG C vibrate (150rmp) at a slow speed
Culture 4h；
E () 5,000rpm centrifugation 1min receive bacterium, leave and take 100uL or so supernatants, gently blow and beat resuspended bacterium solution, then coat
On LB solid medium flat boards (containing Kna and Rif), about 1h is just being put, after fully absorbing, be inverted flat board, 28 DEG C of culture 48h-
72h。
(6) identification of positive colony：
The single bacterium colony grown on picking flat board, in being inoculated in LB liquid mediums (containing Kan and Rif), 28 DEG C of vibration trainings
Support overnight；Plasmid identification is extracted, with plasmid (50 times of dilution) as the identification of template PCR.pCAMBIASuper1300+ihpTHCA
Plasmid PCR qualification figure is as shown in Figure 9 after the conversion of RNAi carrier Agrobacterium freeze-thaw method.
<110>Daqing Branch Institute of Heilongjiang Academy of Sciences
<120>A kind of construction method of Fructus Cannabiss THCA synthase gene RNAi carriers
<160>1
<210>1
<211>504
<212>DNA
<400>1
1 CTCAGCATTT TCCTTTTGGT TTGTTTGCAA AATAATATTT TTCTTTCTCT CATTCCATAT
61 CCAAATTTCA ATAGCTAATC CTCGAGAAAA CTTCCTTAAA TGCTTCTCAA AACATATTCC
121 CAACAATGTA GCAAATCCAA AACTCGTATA CACTCAACAC GACCAATTGT ATATGTCTAT
181 CCTGAATTCG ACAATACAAA ATCTTAGATT CATCTCTGAT ACAACCCCAA AACCACTCGT
241 TATTGTCACT CCTTCAAATA ACTCCCATAT CCAAGCAACT ATTTTATGCT CTAAGAAAGT
301 TGGCTTGCAG ATTCGAACTC GAAGCGGTGG CCATGATGCT GAGGGTATGT CCTACATATC
361 TCAAGTCCCA TTTGTTGTAG TAGACTTGAG AAACATGCAT TCGATCAAAA TAGATGTTCA
421 TAGCCAAACT GCGTGGGTTG AAGCCGGAGC TACCCTTGGA GAAGTTTATT ATTGGATCAA
481 TGAGAAGAAT GAGAATCTTA GTTT
Claims (4)
1. a kind of construction method of Fructus Cannabiss THCA synthase gene RNAi carriers, it is characterised in that the construction method step is such as
Under：
First, with Fructus Cannabiss florescence blade, male flower, female flower aggregate sample as material, total serum IgE is extracted；
2nd, reverse transcription is cDNA；
3rd, with reverse transcription product as template, specific primer is designed, with Taq enzyme performing PCR reaction is entered to reverse transcription product, obtained
PCR primer is simultaneously reclaimed；
4th, by the THCA synthase genes forward direction fragment and the double digestions of I/Hind of Sal III of the double digestions of I/Hind of Jing Sal III
PSKint carriers connect, and convert bacillus coli DH 5 alpha competent cell, and screening positive clone extracts the identification of bacterium solution PCR correct
Recombiant plasmid, by the double digestions of I/Hind of Sal III, further identification obtains pSKTHCAihp I；
5th, the THCA synthase genes of the double digestions of I I/Sac of Jing BamH of pSKTHCAihp I and the double digestions of I/Sac of Jing BamH I
Reversely fragment is attached, and converts bacillus coli DH 5 alpha competent cell, and screening positive clone extracts bacterium solution PCR and identifies just
True recombiant plasmid, by the double digestions of I/Sac of Sal I, further identification obtains pSKihpTHCA；
6th, glue reclaim obtains the fragment containing forward and reverse THCA synthase genes and intron, with the double digestions of I/Sac of Jing Sal I
PCAMBIAsuper1300+ plant expression vectors are attached, and convert bacillus coli DH 5 alpha competent cell, screening positive clone
Son, extracts the identification of bacterium solution PCR, and the double digestions of I/Sac of Sal I are further identified, plant expression vector pCAMBIAsuper1300+
IhpTHCA is successfully constructed.
2. the construction method of Fructus Cannabiss THCA synthase gene RNAi carriers according to claim 1, it is characterised in that described
In step one, using TRIzol extracting solution extracting RNAs.
3. the construction method of Fructus Cannabiss THCA synthase gene RNAi carriers according to claim 1, it is characterised in that described
In step 2, the nucleotide sequence of cDNA is as shown in SEQ ID NO.1.
4. the construction method of Fructus Cannabiss THCA synthase gene RNAi carriers according to claim 1, it is characterised in that described
In step 3, primer sequence is as follows：
Positive sequence：Restriction enzyme site Sal I and Hind III
THCAihp Ⅰ SalF：5’-CTAGTCGACCTCAGCATTTTCCTTTTG-3’；
THCAihp Ⅰ HinR：5’-GCCCCAAGCTTAAACTAAGATTCTCATTC-3’；
Reverse sequence：Restriction enzyme site Sac I and BamH I
THCAihp Ⅱ SacF：5’-GTAGAGCTCCTCAGCATTTTCCTTTTG-3’；
THCAihp Ⅱ BamR：5’-GGCGGATCCAAACTAAGATTCTCATTC-3’.
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| CN110923242A (en) * | 2019-12-14 | 2020-03-27 | 厦门梓蔓生物科技有限公司 | Transcription factor CsAPL1 separated from hemp glandular hair and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039394A (en) * | 2015-09-08 | 2015-11-11 | 内蒙古民族大学 | Method for obtaining new castor-oil plant materials with improved ricinoleic acid content |
| WO2016189384A1 (en) * | 2015-05-28 | 2016-12-01 | Tweed Inc. | Cannabis plants having modified expression of thca synthase |
| CN107405314A (en) * | 2015-02-27 | 2017-11-28 | 埃布公司 | Cannboid comprising purifying and the composition of the combination of at least one flavonoids, terpene or mineral matter |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107405314A (en) * | 2015-02-27 | 2017-11-28 | 埃布公司 | Cannboid comprising purifying and the composition of the combination of at least one flavonoids, terpene or mineral matter |
| WO2016189384A1 (en) * | 2015-05-28 | 2016-12-01 | Tweed Inc. | Cannabis plants having modified expression of thca synthase |
| CN107846861A (en) * | 2015-05-28 | 2018-03-27 | 推德有限公司 | The cannabis plants that THCA synthesis expression of enzymes changes |
| CN105039394A (en) * | 2015-09-08 | 2015-11-11 | 内蒙古民族大学 | Method for obtaining new castor-oil plant materials with improved ricinoleic acid content |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923242A (en) * | 2019-12-14 | 2020-03-27 | 厦门梓蔓生物科技有限公司 | Transcription factor CsAPL1 separated from hemp glandular hair and application thereof |
| CN110923242B (en) * | 2019-12-14 | 2021-04-13 | 厦门梓蔓生物科技有限公司 | Transcription factor CsAPL1 separated from hemp glandular hair and application thereof |
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