CN109486781A

CN109486781A - A kind of alfalfa is overexpressed the method and application of squalene synthase gene

Info

Publication number: CN109486781A
Application number: CN201811387758.XA
Authority: CN
Inventors: 康俊梅; 张铁军; 张俏燕; 王珍; 龙瑞才; 杨青川
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2018-11-21
Filing date: 2018-11-21
Publication date: 2019-03-19

Abstract

The invention discloses methods and application that a kind of alfalfa is overexpressed squalene synthase gene, comprising the following steps: (1) design of primers；(2) cloning vector pEASY-T3 plasmid, plasmid pGEX-4T-1 V are used for prokaryotic expression, and E. coli Trans5 α and E.coli Transetta is used for gene cloning and expression；(3) alfalfa squalene synthase gene MsSQS full-length cDNA is cloned；(4) bioinformatic analysis of MsSQS sequence；(5) expression analysis of squalene synthase gene MsSQS；(6) measurement of squalene synthase content；(7) methyl jasmonate induces the measurement of lower saponin content；(8) Subcellular Localization；(9) prokaryotic expression；(10) building of the overexpression vector of MsSQS gene and the genetic transformation to alfalfa.Squalene synthase gene is transferred in alfalfa by establishing efficient genetic conversion system and is overexpressed by this method, improves the content of the intracorporal Total saponin of transgenic alfalfa, provides breeding material to cultivate the alfalfa variety of high saponin content.

Description

A kind of alfalfa is overexpressed the method and application of squalene synthase gene

Technical field

The present invention relates to plant sapogenin glycosides to synthesize relevant speed limit enzyme gene field, crosses table more particularly to a kind of alfalfa Up to the method and application of squalene synthase gene.

Background technique

Alfalfa saponin (Saponin) also known as saponin, saponin, are made of sapogenin plus one or more sugar chains, are generally existing In a major class glucoside compound of nature, including triterpene compound, sterol, steroids, in mushroom, fern, plant, move It is distributed in object and marine organisms.Although terpenoid is generally existing in plant, the mankind so far can not also be true The complete path of terpenoid synthesis in any one fixed species.

Squalene synthase (Squalene Synthases, SQS) is a kind of membrane bound enzyme that combination is online in endoplasm, and catalysis is double Method ester pyrophosphoric acid (Farnesyl diphosphate, the FPP) condensation of molecule generates squalene, is triterpene and sterol branched metabolic First key enzyme in stream plays important regulating and controlling effect to phytosterol and triterpene biosynthesis.In higher plant, SQS Gene is isolated from tobacco for the first time, is cloned into phase from the various plants such as sweet wormwood, Radix Glycyrrhizae, arabidopsis, ginseng at present The squalene synthase gene cloned in ginseng is found total triterpene saponin(e by the SQS gene answered in ginseng fibrous root's root tissue after overexpression 1.6~3 times are higher by than unconverted control group；Squalene synthetase activity is than control after converting squalene synthase gene in wilsonii 3 times of enhancing or more, the level of total saposins also improves 2~2.5 times.Radix Glycyrrhizae turns squalene synthase gene hairy middle highest glycyrrhizic acid Content is about 3.6 times of wild type hairy.The studies above shows that squalene synthase gene has centainly the synthesis of triterpene and sterol Regulating and controlling effect the squalene synthase gene in arabidopsis be transferred in Withania kansuensis find, terpene substances content is obvious in transgenosis Withania kansuensis It is higher than the content of non-transgenic Withania kansuensis.By importing sweet wormwood antisense squalene synthase gene in tobacco, tobacco is successfully reduced In sterol content.Meanwhile being overexpressed SQS gene can also be such that saponins compound route of synthesis downstream gene expression amount raises, Such as: SE, β-AS, CYP450, it is final to improve sterol and content of ginsenoside in ginseng.

It can be seen that SQS gene may be the key gene of saponin synthesis, there is important regulation to make saponin synthesis With.Correlative study in relation to SQS gene in alfalfa has not been reported.

Alfalfa (Medicago sativa L.) is worldwide widely distributed, is universally acknowledged high yield and high quality Herbage, protein rich in cauline leaf, is known as " King of Pasture ".Clover is not only full of nutrition, secondary metabolite Alfalfa saponin (alfalfa saponins) is considered as the main source of the medicinal function of clover in recent years.

Summary of the invention

The object of the present invention is to provide methods and application that a kind of alfalfa is overexpressed squalene synthase gene.By to purple Clone, expression pattern and the functional analysis of key gene, show squalene synthase gene in lucerne in russian fenugreek herb saponin route of synthesis There is certain adjustment effect in Mu saponin route of synthesis.Squalene synthase gene is turned by establishing efficient genetic conversion system Enter in alfalfa and be overexpressed, improve the content of the intracorporal Total saponin of transgenic alfalfa, for the clover for cultivating high saponin content Kind provides breeding material.

To achieve the above object, The technical solution adopted by the invention is as follows:

A kind of method that alfalfa is overexpressed squalene synthase gene, comprising the following steps:

(1) design of primers；

(2) cloning vector pEASY-T3 plasmid, plasmid pGEX-4T-1V are used for prokaryotic expression, E. coli Trans5 α and E.coli Transetta are used for gene cloning and expression；

(3) alfalfa squalene synthase gene MsSQS full-length cDNA is cloned；

(4) bioinformatic analysis of MsSQS sequence；

(5) expression analysis of squalene synthase gene MsSQS；

(6) measurement of squalene synthase content；

(7) methyl jasmonate induces the measurement of lower saponin content；

(8) Subcellular Localization；

(9) prokaryotic expression；

(10) building of the overexpression vector of MsSQS gene and the genetic transformation to alfalfa.

Wherein, the step (1) is specifically, according to the cds sequence of M. truncatula squalene synthase gene known in NCBI, benefit It is used to expand the cDNA sequence of MsSQS gene with Primer 5.0 design primer MsSQS-f and MsSQS-r.According to Genbank public affairs The alfalfa actin β-actin gene of cloth is as house-keeping gene and the MsSQS gene cloned as target detection Gene, it is each to design pair of primers Actin-f and Actin-r, SQS-qPCR-f and SQS-qPCR-r.Such as SEQ ID NO:1-6 institute Show, sequence is as follows:

MsSQS-f:5 '-GCTTATTCGTAGAAACAAAAGATGG-3 '

MsSQS-r:5 '-ATGCTACCACTGTTCTGTTGCTATC-3 '

Actin-f:5’-CAAAAGATGGCAGATGCTGAGGAT-3’

Actin-r:5’-CATGACACCAGTATGACGAGGTCG-3’

SQS-qPCR-f:5’-CTTCGGTCTTGTTATTCAGCAGC-3’

SQS-qPCR-r:5’-CTTGTATCATCCTCAACGGTGTC-3’。

Wherein, the step (3) specifically, using Trizol method extract alfalfa blade total serum IgE, use spectrophotometric The quality of meter measurement RNA；With the integrality of 1% agar sugar detection RNA；Each sample takes the RNA of 1 μ g for reverse transcription, uses The Prime ScriptTM 1 of Takara^stStrand cDNA synthesis kit carries out PCR amplification, and reaction system is 20 μ L；It using MsSQS-f and MsSQS-r as primer, carries out PCR amplification and obtains MsSQS full-length cDNA, be then cloned into pEASY-T3 load Plasmid pEASY-MsSQS is obtained in body, then is transferred to E. coli DH5 α and in 37 DEG C of dark culturings of LB culture medium；So Plasmid is sequenced assembling afterwards, verifies correct MsSQS insertion point.

Wherein, the step (4) is specifically, analyze target gene MsSQS's using DNAMAN program and NCBI/BLAST Open reading frame sequence, and carry out Multiple sequence alignments and allied species analysis；Utilize SOPMA program analysis SQS protein Molecular weight, amino acid composition and isoelectric point；Egg is encoded by SignalP4.1Server software and SubLoc software prediction MsSQS The signal peptide and subcellular localization of white matter；The transmembrane region of protein and hydrophobic is predicted using TMHMM Server online software Property；Utilize the three-dimensional structure of SWISSMODEL program analysis protein；Utilize Interpro database analysis protein structure domain function Energy；The homologous protein of the plants such as arabidopsis, ginseng is searched in NCBI/GenBank, and is carried out using ClustalX2.0 software Sequence alignment analysis.

Wherein, the step (5) is sprouted specifically, No. 3 seeds of lucerne in alfalfa are placed on the filter paper soaked, then It is implanted into Hoagland ' s nutrient solution, is placed in illumination box, 16h daytime/8h night, 24 DEG C of daytime, 20 DEG C of night, relative humidity 40%, it cultivates 4 weeks, changed one time of nutrition liquid every 1 week；It chooses and grows fine after 4 weeks, plant of the same size is used for following place Reason: 1. apart from plant 20cm at, after 15W ultraviolet light 30min, respectively 0,2,4,8,12,24,48h acquire root, Stem, leaf texture's sample；2. be protected from light processing 0,4,8,12,24,48,96,144h acquire root, stem, leaf texture respectively；③200μM MeJA processing 0,4,8,12,24,48,96,144h acquire root, stem, leaf texture respectively；4. 100 μM of ABA processing 0,2,4,8,12, Acquire root, stem, leaf texture respectively for 24 hours；5. 50 μM of GA3 processing 0,2,4,8,12, acquire root, stem, leaf texture respectively for 24 hours；It will receive The sample of collection is respectively used to extract RNA.

Wherein, the step (6) is specifically, different using clover after enzyme linked immunosorbent assay MeJA induction processing The activity of squalene synthase protein in tissue；Various time points root, stem, each 200mg of leaf texture are placed in 2mL centrifugation after taking MeJA to handle In pipe, 10mM phosphate buffer PBS 900mL, PH=7.2-7.4 is added, wears into slurry with grinding rod, stands and draws in a moment Clearly, residue adds 900mL homogenate and grinds again once, is then combined with lapping liquid twice, is turned under the conditions of 4 DEG C with 6000rpm Speed centrifugation 15min, takes supernatant；Enzymatic activity is measured after sample extraction at once；With spectrophotometer, absorbance is read, with standard song Line calculates the actual concentrations of sample.

Wherein, the step (7) is placed in cryogenic vacuum jelly specifically, being chosen at the clover root of MeJA processing, stem, leaf sample It is lyophilized 70 hours for -80 DEG C in dry machine, 60 meshes is crossed after taking-up；It accurately weighs 30mg freeze-drying sample to be placed in 5mL centrifuge tube, add 1mL70% ethyl alcohol Ultrasound Instrument ultrasound 30min, 4 DEG C overnight, again ultrasound 30min；Then 30min is centrifuged with 1000g, taken 100 μ L of clear liquid injects pretreated SPE column, is rinsed respectively with 5mL water and 5mL30% methanol, then with 1mL methanol elution 4 times； Merging eluent, which is placed in the tool plug test tube of 10mL, to be evaporated, and vanillic aldehyde-glacial acetic acid solution of 0.2mL 5% is added after cooling, to 0.8mL perchloric acid is added after completely dissolution；It is placed in 70 DEG C of water-baths after water-bath 15min ice bath immediately, 5mL ice is added after cooling Acetic acid stands 20min；It is compared with blank Duplicate Samples, each sample sets 3 biology and repeats；Existed with spectrophotometric determination The absorbance of sample at 545nm.

Wherein, the step (8) respectively contains I He of Xho specifically, containing upstream and downstream using 5.0 software design of Primer The primer of I restriction enzyme site of Sal, SQS-pA7-f:5 '-CCCTCGAGATGGGAAGTATAAAAGCGATTTTGA-3 ', CTCGAG For I restriction enzyme site of Xho；SQS-pA7-r:5 '-GCGTCGACGTTATTGTAACGGTTGGCAGAGAGA-3, GTCGAC are Sal I Restriction enzyme site；As shown in SEQ ID NO:7-8；Using pEasy-T3-MsSQS plasmid as template, respectively with SQS-pA7-f and SQS- PA7-r is primer, carries out the amplification of target fragment, then target fragment is connected to pEASY-T1 carrier, and names the recon to be pEASY-T1-MsSQS；It is quick with Xho I and Sal I by recombinant plasmid pEASY-T1-MsSQS and transient expression plasmids pA7-GFP Digestion enzyme carries out double digestion, and digestion system is as follows: plasmid volume determines (< 1 μ g) according to plasmid concentration, I 1 I 1 μ of μ L, Sal of Xho L, 10 × Quick Cut Buffer 5 μ L, ddH₂O Up to 50μL；After mixing gently, with 37 DEG C of PCR instrument reaction 5~ 10min；After agarose gel electrophoresis, target fragment is recycled；Then target gene fragment is connected under the action of T4 ligase Carrier pA7-GFP, 16 DEG C of 4~5h of connection are connected to, plasmid pA7-GFP-MsSQS is constructed；MsSQS is integrated into being correctly oriented In transient expression vector pA7-GFP, by the method for biolistic bombardment, by the subcellular wink containing target gene coding region sequence When expression vector pA7-GFP be transferred to onion epidermis, under the driving of 35S strong promoter, fusion protein mistake in onion epidermis cell Amount expression；Green fluorescent protein GFP shows green fluorescence under blue wavelength light, is determined according to the fluorescence signal of fusion protein The positioning of the albumen of SQS gene coding in the cell；

Wherein, the step (9) is specifically, 1. two pairs of primers of design are respectively as follows: the entire code area sequence of MsSQS cDNA Column insertion prokaryotic expression carrier, specific primer are SQS-PE-f:5 '-ATGGGAAGTATAAAAGCGATTTTG-3 ', SQS-PE- r:5'-ATTCCTCAAAA CGTAAGATTTCCTT-3'；2. MsSQS coding region sequence C-terminal to be cut out to the sequence of 30 amino acid Column insertion prokaryotic expression carrier, specific primer are SQS-PE-CTt-f:5 '-ATTCCTCAAAACGTAAGATTTCCTT-3 ', SQS-PE-r:5'-ATTCCTCAAAACGTAAGATTTCCTT-3'；As shown in SEQ ID NO:9-11；Correct MsSQS will be contained The pEASY-T3-MsSQS plasmid of sequence is as template, respectively with SQS-PE-f and SS-PE-r, SQS-PE-CTt-f and SQS- PE-r is amplimer, carries out the amplification of target fragment, then target fragment is connected to pEASY-E2 carrier；PCR response procedures As follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min72 DEG C of extension 5min, 4 DEG C of heat preservations； By above-mentioned PCR product after agarose gel electrophoresis and DNA fragmentation recycling, the target fragment of recycling and prokaryotic expression are carried Body pEASY-E2 connection, and be transferred in Tran-T1 competent cell, the connection and conversion of carrier；

Positive monoclonal is identified with PCR method, to carry out thallus PCR identification for each monoclonal, using bacterium solution as template, Forward primer M13-F, the 5'-CGCCAGGGTTTTCCCAGTCACGAC-3' of carrier, such as SEQ ID NO:12；And target gene Reverse primer SQS-PE-r identifies positive recombinant as primer, and the bacterium solution that will amplify target gene fragment is sent to survey The sequencing of sequence company；Recon is respectively designated as pEASY-E2-MsSQS and pEASY-E2-CTtMsSQS；By two kinds of prokaryotic expressions Recon pEASY-E2-MsSQS and pEASY-E2-CTtMsSQS and empty carrier are transferred to Escherichia coli Transetta respectively； Prokaryotic expression MsSQS albumen and MsSQS-CTt protein SDS-PAGE electrophoresis, take the above-mentioned Escherichia coli containing correct segment Transetta, 37 DEG C of overnight incubations to OD₆₀₀It is 0.4~0.6；Escherichia coli to contain empty carrier are added as control IPTG makes its final concentration of 1mM in bacterium solution, respectively inducing expression 0,30,1,2,3,4,5,6,8h；After IPTG is added, upper It states each time point and takes 2mL bacterium solution, 4 DEG C of 10000rpm are centrifuged 5min, abandon supernatant, and 1mLPBS, 10mM, PH=7.2-7.4 is added Thallus is resuspended, with ultrasonic cell disruption instrument ultrasound 100 times, takes collection precipitating and supernatant, precipitating 100 μ L PBS weight respectively Outstanding, 4 DEG C temporary；After the completion of sampling, takes 50 μ L supernatants to precipitate re-suspension liquid respectively, isometric 2 × SDS loading is added It is immediately placed on cooled on ice after Buffer, 100 DEG C of water-bath 3min, then uses the expression of PAGE gel electroresis appraisal MsSQS.

Wherein, the step (10) amplifies containing I He of Xba specifically, be amplimer with MsSQS-f and MsSQS-r The MsSQS genetic fragment of I restriction enzyme site of BamH, recovery purifying PCR product, then target fragment is connected to pEASY-T3 carrier； It is sequenced and extracts the recombinant plasmid containing correct sequence, with Xba I and the quick restriction enzyme of BamH I to pBI121 and recombination Sub- pEASY-T3-pMsSQS carries out double digestion, and target gene coding region sequence is inserted into plant under the action of T4 ligase In overexpression vector pBI121, target gene overexpression vector is constructed, and convert Agrobacterium EHA105；Then it is situated between using Agrobacterium Inducing defecation by enema and suppository converts in alfalfa lucerne 3.

The method that any alfalfa of the present invention is overexpressed squalene synthase gene is cultivating high saponin content clover Application in kind.

Compared with the existing technology, protrusion effect of the invention is:

Alfalfa saponin (alfalfa saponins) is considered as the main source of clover medicinal function, is not only had good Norcholesterol, reducing blood lipid the effect of, and there are also the medicinal and bioactivity such as anti-inflammatory, anticancer, antibacterial, desinsection.In order to more preferable Understand alfalfa saponin Biosynthetic pathway in key gene adjustment mechanism and function.The present invention has cloned alfalfa saponin The key gene of synthesis, and functional analysis is carried out, result of study is as follows: obtaining alfalfa shark for the first time by homologous clone method Alkene synthase (MsSQS) cDNA sequence, the gene maximum open reading frame contain 1242bp, codified one containing 413 amino acid, Molecular weight is the albumen of 47kDa.Sequence alignment result shows MsSQS gene and the homology of section shape clover SQS gene is 97.91%, the similarity for encoding albumen is 93.53%.The amino acid sequence of bioinformatic analysis gene coding has 6 guarantors Defending zone, wherein 3 highly conserved, and has 2 transmembrane helical regions, wherein the hydrophobic region of C-terminal is film bond area.In real time Quantitative PCR analysis expression pattern of the MsSQS in alfalfa different tissues, MsSQS can be expressed in root, stem, Ye Zhongjun, but root Middle expression quantity highest, leaf take second place, and expression quantity is minimum in stem.MsSQS gene is also lured by MeJA, ABA, GA3, ultraviolet injury and dark Lead expression.Under MeJa induction, the expression quantity of MsSQS rises in root, stem, leaf, and squalene synthase content and total saposins Content rises accordingly.Prokaryotic expression is the results show that MsSQS cDNA can be about in expression in escherichia coli, molecular weight 45kDa.MsSQS is overexpressed to display in clover, transgenic plant with compare the expression quantity of MsSQS, transcriptional level and Total saposins amount is above wild-type alfalfa.The studies above shows that MsSQS has certain adjusting to the synthesis of alfalfa saponin Effect.

With reference to the accompanying drawing explanation and specific embodiment to alfalfa squalene synthase MsSQS gene of the present invention and It encodes albumen and is described further with application.

Detailed description of the invention

The biological evolution tree that Fig. 1 is MsSQS is analyzed.

Fig. 2 is the Multiple range test of other species squalene synthase protein sequences of alfalfa.

Fig. 3 is Subcellular Localization of the fusion protein MsSQS-GFP in onion epidermis cell；Wherein, a-c: unloaded Body pA7-GFP converts the fluorescence signal distribution situation in onion epidermis cell；The conversion ocean d-f: recombinant plasmid pA7-GFP-MsSQS Fluorescence signal distribution situation in green onion epidermal cell；G-i: the onion epidermis cell of pA7-GFP-MsSQS has been converted in cell dehydration Middle fluorescence signal distribution situation under the conditions of (0.3g/ml sucrose).A, d, g: it is shot under blue wavelength and white light；B, e, h: blue It is shot under color wavelength；C, f, i: it is shot under white light；Scale=100 μm.

Fig. 4 is that the signal peptide (A) of MsSQS albumen and spanning domain predict (B)；Wherein, C-score (raw cleavage Site score): the score value of original shearing site；S-score (signal peptide score): the score value of signal peptide；Y- Score (combined cleavage site score): the score value of comprehensive shearing site；Mean S: signal peptide score value is put down Mean value；D-score (discrimination score): the weighted average of mean S and max.Y.

Fig. 5 is expression analysis of the MsSQS in clover different tissues.

Fig. 6 be Different stress under the conditions of MsSQS different tissues expression analysis.

Fig. 7 is expression analysis of the MsSQS gene under MeJA induction in different tissues.

Fig. 8 is alfalfa squalene synthase coli expression carrier schematic diagram.

Fig. 9 is Bacillus coli expression alfalfa squalene synthase SDS-PAGE analysis；Wherein, A: protein molecular quality mark It is quasi-；B: the e. coli total protein of pEASY-E2-Control has been converted (by IPTG inducing expression 5h)；C: conversion The e. coli total protein of pEASY-E2-CTtMsSQS (without IPTG inducing expression)；D: pEASY-E2-CTtMsSQS has been converted Escherichia coli supernatant protein (passes through IPTG inducing expression 5h)；E: pEASY-E2-CTtMsSQS Escherichia coli precipitating egg has been converted White (passing through IPTG inducing expression 5h)；F: the e. coli total protein for having converted pEASY-E2-MsSQS (induces table without IPTG Up to)；G: pEASY-E2-MsSQS Escherichia coli supernatant protein has been converted (by IPTG inducing expression 5h)；H: pEASY- has been converted E2-CTtMsSQS Escherichia coli protein precipitation (passes through IPTG inducing expression 5h).

Figure 10 is the map of purpose gene overexpression vector.

Figure 11 is the analysis of MsSQS expression quantity and saponin content in transgenic alfalfa strain；Wherein, A: real-time quantitative PCP analyzes MsSQS expression quantity；B: total saponin content measurement；CK1: empty carrier PBI121 transgenic alfalfa；CK2: wild type Alfalfa；Line1~9:MsSQS transgenic alfalfa.

Specific embodiment

1. materials and methods

1.1 vegetable materials are cultivated

Lucerne 1 (Medicago sativa L. ' Zhongmu No.3 ') is salt tolerant alfalfa variety by China in alfalfa Beijing animal and veterinary research institute, Academy of Agricultural Sciences is bred as.It selects and germinates in full alfalfa seed culture dish, then cultivate In hongland nutrient solution, it is placed in artificial intelligence climate box, 25 ± 1 DEG C of temperature, illumination 16h (daytime)/8h (night), humidity 65%, Cultivate the extraction for being used for DNA and RNA in 4 weeks.

1.2 design of primers

According to the cds sequence of M. truncatula squalene synthase gene known in NCBI, 5.0 design primer of Primer is utilized MsSQS-f and MsSQS-r is used to expand the cDNA sequence of MsSQS gene.The alfalfa actin announced according to Genbank β-actin gene, as target detection gene, respectively designs pair of primers as house-keeping gene and the MsSQS gene cloned Actin-f and Actin-r, SQS-qPCR-f and SQS-qPCR-r.As shown in SEQ ID NO:1-6, sequence is as follows:

MsSQS-f:5 '-GCTTATTCGTAGAAACAAAAGATGG-3 '

MsSQS-r:5 '-ATGCTACCACTGTTCTGTTGCTATC-3 '

Actin-f:5’-CAAAAGATGGCAGATGCTGAGGAT-3’

Actin-r:5’-CATGACACCAGTATGACGAGGTCG-3’

SQS-qPCR-f:5’-CTTCGGTCTTGTTATTCAGCAGC-3’

SQS-qPCR-r:5’-CTTGTATCATCCTCAACGGTGTC-3’

1.3 bacterial strains, plasmid and culture medium

Cloning vector pEASY-T3 plasmid, plasmid pGEX-4T-1V are used for prokaryotic expression, plasmid pEASY-T3 and pGEX- The structure map of 4T-1V can be obtained on the net.E. coli Trans5 α and E.coli Transetta (DE3) is used In gene cloning and expression.

The clone of 1.4 alfalfa squalene synthase gene MsSQS full-length cDNAs

The total serum IgE that alfalfa blade is extracted using Trizol method, with the quality of spectrophotometric determination RNA.With 1% The integrality of agar sugar detection RNA.Each sample takes the RNA of 1 μ g for reverse transcription, with the Prime ScriptTM1 of Takara^st Strand cDNA synthesis kit carries out PCR amplification, and reaction system is 20 μ L.Using MsSQS-f and MsSQS-r as primer, It carries out PCR amplification and obtains MsSQS full-length cDNA, be then cloned into pEASY-T3 carrier and obtain plasmid pEASY-MsSQS, then turn Enter E. coli DH5 α and in 37 DEG C of dark culturings of LB culture medium.Then plasmid is sequenced assembling, and verifying is correct MsSQS insertion point.

The bioinformatic analysis of 1.5MsSQS sequence

The open reading frame sequence of target gene MsSQS is analyzed using DNAMAN program and NCBI/BLAST, and is carried out Multiple sequence alignments and allied species analysis.Using the molecular weight of SOPMA program analysis SQS protein, amino acid composition and wait The physicochemical properties such as electricity point.By SignalP4.1Server software and SubLoc the software prediction letter of MsSQS coding protein Number peptide and subcellular localization.The transmembrane region and hydrophobicity of protein are predicted using TMHMM Server online software.It utilizes SWISSMODEL program analyzes the three-dimensional structure of protein.Utilize Interpro database analysis protein structure domain-functionalities. The homologous protein of the plants such as arabidopsis, ginseng is searched in NCBI/GenBank, and carries out sequence using ClustalX2.0 software Compare analysis.

The expression analysis of 1.6 squalene synthase gene MsSQS

No. 3 seeds of lucerne in alfalfa are placed on the filter paper soaked and are sprouted, Hoagland ' s nutrient solution is then implanted into In, it is placed in illumination box (16h daytime/8h night, 24 DEG C of daytime, 20 DEG C of night, relative humidity 40%) cultivation 4 weeks, was changed every 1 week One time of nutrition liquid.It chooses and grows fine after 4 weeks, plant of the same size is used for following processing: 1. at plant 20cm, using After 15W ultraviolet light 30min, root, stem, leaf texture's sample are acquired in 0,2,4,8,12,24,48h respectively；2. be protected from light processing 0, 4,8,12,24,48,96,144h acquires root, stem, leaf texture respectively；3. 200 μM of MeJA processing 0,4,8,12,24,48,96, 144h acquires root, stem, leaf texture respectively；4. 100 μM of ABA processing 0,2,4,8,12, acquire root, stem, leaf texture respectively for 24 hours；⑤ 50 μM of GA3 processing 0,2,4,8,12, acquire root, stem, leaf texture respectively for 24 hours.The sample of collection is respectively used to extract RNA.

The measurement (enzyme-linked immunosorbent assay) of 1.7 squalene synthase contents

MeJA is measured using enzyme-linked immunosorbent assay (ELISA:enzyme linked immunosorbant assay) After induction processing in clover different tissues squalene synthase protein activity.Various time points root, stem, leaf texture after taking MeJA to handle Each 200mg is placed in 2mL centrifuge tube, 10mM phosphate buffer PBS 900mL is added, (PH=7.2-7.4) is worn into grinding rod Slurry, standing draw supernatant in a moment, and residue adds 900mL homogenate and grinds again once, lapping liquid twice is then combined with, 4 15min is centrifuged with 6000rpm revolving speed under the conditions of DEG C, takes supernatant.Enzymatic activity is measured after sample extraction at once.With spectrophotometer, It reads absorbance (OD) and calculates the actual concentrations of sample with standard curve.

1.8 methyl jasmonates induce the measurement of lower saponin content

The clover root, stem, leaf sample for being chosen at MeJA processing, which are placed in cryogenic vacuum freeze dryer, to be lyophilized 70 hours for -80 DEG C, 60 meshes are crossed after taking-up.It accurately weighs 30mg freeze-drying sample to be placed in 5mL centrifuge tube, adds 1mL70% ethyl alcohol Ultrasound Instrument ultrasonic 30min, 4 DEG C overnight, again ultrasound 30min.Then 30min is centrifuged with 1000g, takes 100 μ L of supernatant injection pretreated SPE column is rinsed with 5mL water and 5mL30% methanol respectively, then with 1mL methanol elution 4 times.Merge the tool that eluent is placed in 10mL It is evaporated in plug test tube, vanillic aldehyde-glacial acetic acid solution of 0.2mL 5% is added after cooling, to which 0.8mL high chlorine is added after completely dissolution Acid.It is placed in 70 DEG C of water-baths after water-bath 15min ice bath immediately, 5mL glacial acetic acid is added after cooling, stands 20min.It is flat with blank Row sample compares, and each sample sets 3 biology and repeats.With the absorbance of spectrophotometric determination sample at 545nm.

1.9 Subcellular Localization

Contain primer of the upstream and downstream respectively containing I restriction enzyme site of Xho I and Sal, SQS- using 5.0 software design of Primer pA7-f:5’-CCCTCGAGATGGGAAGTATAAAAGCGATTTTGA-3 ' (CTCGAG is I restriction enzyme site of Xho), SQS-pA7- R:5 '-GCGTCGACGTTATTGTAACGGTTGGCAGAGAGA-3 ' (GTCGAC is I restriction enzyme site of Sal).Such as SEQ ID NO: Shown in 7-8.Using pEasy-T3-MsSQS plasmid as template, respectively using SQS-pA7-f and SQS-pA7-r as primer, purpose is carried out The amplification of segment, then target fragment is connected to pEASY-T1 carrier, and naming recon is pEASY-T1-MsSQS.It will recombination Plasmid pEASY-T1-MsSQS and transient expression plasmids pA7-GFP carries out double digestion, enzyme with Xho I and the quick digestion enzyme of Sal I It is as follows to cut system: plasmid volume determines (< 1 μ g) according to plasmid concentration, I 1 I 1 μ L, 10 × Quick Cut of μ L, Sal of Xho Buffer 5 μ L, ddH₂O Up to 50μL.After mixing gently, with 37 DEG C of 5~10min of reaction of PCR instrument.Through Ago-Gel electricity After swimming, target fragment is recycled.Then target gene fragment is connected to (16 DEG C of carrier pA7-GFP under the action of T4 ligase Connect 4~5h), construct plasmid pA7-GFP-MsSQS.MsSQS is integrated into transient expression vector pA7-GFP to be correctly oriented In, by the method for biolistic bombardment, the subcellular transient expression vector pA7-GFP containing target gene coding region sequence is turned Enter onion epidermis, under the driving of 35S strong promoter, fusion protein overexpression in onion epidermis cell.Green fluorescent protein GFP shows green fluorescence under blue wavelength light, determines the albumen of SQS gene coding thin according to the fluorescence signal of fusion protein Positioning intracellular.(Fig. 3)

1.10 prokaryotic expression

Simultaneously signal peptide is not present in the albumen n end of MsSQS gene coding, but since there may be a films for its C-terminal hydrophobic region Bond area, it is thus possible to influence its expression in Escherichia coli.Therefore 1. two pairs of primers of design are respectively as follows: MsSQS cDNA Entire coding region sequence is inserted into prokaryotic expression carrier, and specific primer is SQS-PE-f:5 '- ATGGGAAGTATAAAAGCGATTTTG-3 ', SQS-PE-r:5 '-ATTCCTCAAAACGTAAGATTTCCTT-3 '；2. will MsSQS coding region sequence C-terminal cuts out the sequence insertion prokaryotic expression carrier of 30 amino acid, and specific primer is SQS-PE- CTt-f:5 '-ATTCCTCAAAACGTAAG ATTTCCTT-3 ', SQS-PE-r:5 '-ATTCCTCAAAACGTAA GATTTCCTT-3'.As shown in SEQ ID NO:9-11.To contain the pEASY-T3-MsSQS plasmid of correct MsSQS sequence as Template carries out target fragment respectively using SQS-PE-f and SS-PE-r, SQS-PE-CTt-f and SQS-PE-r as amplimer Amplification, then target fragment is connected to pEASY-E2 carrier.PCR response procedures are as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min72 DEG C of extension 5min, 4 DEG C of heat preservations.Above-mentioned PCR product is coagulated by agarose After gel electrophoresis and DNA fragmentation recycling, the target fragment of recycling is connect with prokaryotic expression carrier pEASY-E2, and be transferred to Tran- In T1 competent cell, the connection and conversion of carrier.

Positive monoclonal is identified with PCR method, to carry out thallus PCR identification for each monoclonal, using bacterium solution as template, Forward primer M13-F, the 5'-CGCCAGGGTTTTCCCAGTCACGAC-3' of carrier, such as SEQ ID NO:12；And target gene Reverse primer SQS-PE-r identifies positive recombinant as primer, and the bacterium solution that will amplify target gene fragment is sent to survey The sequencing of sequence company.Recon is respectively designated as pEASY-E2-MsSQS and pEASY-E2-CTtMsSQS.By two kinds of prokaryotic expressions Recon pEASY-E2-MsSQS and pEASY-E2-CTtMsSQS and empty carrier are transferred to Escherichia coli Transetta respectively (DE3).Prokaryotic expression MsSQS albumen and MsSQS-CTt protein SDS-PAGE electrophoresis take the above-mentioned large intestine bar containing correct segment Bacterium Transetta (DE3), 37 DEG C of overnight incubations to OD₆₀₀It is 0.4~0.6.To contain the Escherichia coli of empty carrier as control, IPTG, which is added, makes its final concentration of 1mM in bacterium solution, respectively inducing expression 0,30,1,2,3,4,5,6,8h.After IPTG is added, 2mL bacterium solution is taken in above-mentioned each time point, 4 DEG C of 10000rpm are centrifuged 5min, abandon supernatant, and 1mLPBS (10mM, PH=7.2- is added 7.4) thallus is resuspended, with ultrasonic cell disruption instrument ultrasound 100 times, takes collection precipitating and supernatant, 100 μ L PBS of precipitating respectively It is resuspended, 4 DEG C temporary.After the completion of sampling, takes 50 μ L supernatants to precipitate re-suspension liquid respectively, isometric 2 × SDS loading is added It is immediately placed on cooled on ice after Buffer, 100 DEG C of water-bath 3min, then uses the expression of PAGE gel electroresis appraisal MsSQS.

The building of the overexpression vector of 1.11MsSQS gene and genetic transformation to alfalfa

It is amplimer with MsSQS-f and MsSQS-r, amplifies the MsSQS gene containing I restriction enzyme site of Xba I and BamH Segment, recovery purifying PCR product, then target fragment is connected to pEASY-T3 carrier.It is sequenced and extracts containing correct sequence Recombinant plasmid carries out double enzymes to pBI121 and recon pEASY-T3-pMsSQS with Xba I and the quick restriction enzyme of BamH I It cuts, target gene coding region sequence is inserted into plant overexpression vector pBI121 under the action of T4 ligase, construct mesh Gene overexpression vector (Figure 10), and convert Agrobacterium EHA105.Then using in Agrobacterium-mediated transformation alfalfa Lucerne 3.

The identification of 1.12 transgenic alfalfas

Using alfalfa kalamycin resistance plant genomic DNA as template, with the pale reddish brown lucerne of empty carrier PBI121 transgenosis Mu and wild-type alfalfa DNA as negative control and PBI121-SQS plasmid as positive control, with the 35S on pBI121 The downstream primer SQS-r of upstream primer 35S-f and the SQS gene of promoter carries out PCR amplification as primer, to detect transgenosis Clover.

The measurement of MsSQS expression analysis and saponin content in 1.13 transgenic alfalfas

It extracts transgenic alfalfa aerial part total serum IgE and carries out reverse transcription, respectively with SQS-qPCR-f and SQS- QPCR-r and Actin-f and Actin-r be primer, the transcriptional level of testing goal gene M sSE1 and house-keeping gene β-actin, To turn empty carrier and wild-type alfalfa as control, the transcriptional level variation of MsSQS is detected.The positive for turning MsSQS is taken to turn Gene alfalfa and the transgenic alfalfa for turning empty carrier PBI121 and wild-type alfalfa aerial part crush de- Rouge, -80 DEG C are lyophilized 70 hours, cross 60 meshes and utilize colorimetric method for determining saponin content after procyanidin.

2 results and analysis

The sequence of 2.1 alfalfa squalene synthase gene MsSQS is analyzed

According to the squalene synthase nucleic acid sequence of known M. truncatula, homologous clone is pale reddish brown out from alfalfa lucerne 1 The full length cDNA sequence of clover squalene synthase gene.Sequence analyzes the opening code-reading frame that MsSQS cDNA includes a 1242bp, Encode 413 amino acid.DNAMAN software sequences comparison result is shown: the homology of itself and M. truncatula SQS gene is 97.91%, while also height is similar for the albumen encoded, is 93.53%.

Homology analysis discovery, the MsSQS amino acid sequence and M. truncatula, chick-pea, soybean, Radix Glycyrrhizae, hundred arteries and veins derived Root, ginseng SQS Amino acid sequence identity be respectively 98%, 95%, 94%, 93%, 92%, 92%.12 kinds are chosen simultaneously The SQS amino acid sequence of different plant species carries out multiple sequence comparison together, the results showed that the SQS protein sequence in each species Homology is higher, and global homology is up to 80%, this illustrate plant SQS albumen during evolution than more conservative, different plant species SQS albumen should belong to the same family.(Fig. 1)

Pass through the MsSQS cDNA alfalfa SQS amino acid sequence derived and the mankind, mouse, yeast, arabidopsis, fragmentation ferment The SS amino acid sequence of the different plant species such as mother is made comparisons, and discovery SQS amino acid sequence has 6 regions than more conservative, each conservative The amino acid residue in region is generally 14~23 (Fig. 2).It is highly conserved for wherein having 3 regions (IIIth area, IVth area and Vth area) , in animal, plant as being almost in fungi, these three regions are considered related with enzyme active center；IIth area and IV Respectively containing 1 rich aspartic acid conservative region (DXXXD) in area, it is considered to be Mg²⁺The activated centre of binding site is terpene The typical structure of synthase；Although VI conservative of region is relatively low, contain 1 section of very hydrophobic sequence, this with it is all other The squalene synthase of species is similar, also consistent with amino acid sequence hydrophobic region prediction result

2.2 secondary structure predictions and analysis of physical and chemical property

Secondary structure is predicted with SOPMA online software, shows alfalfa SS secondary structure with alpha-helix and random volume Qu Weizhu, wherein random coil is 20.19%, alpha-helix 64.48%, and β-corner is 6.57%, extended chain 8.76%. By the analysis of hydrophilic and hydrophobic to MsSQS, overall average hydrophilicity is -0.134 (negative value is bigger, and expressions hydrophily is better, on the occasion of getting over It is big to indicate that hydrophobicity is stronger).Analysis of physical and chemical property (table 1) is carried out to MsSQS protein sequence by ProtParam program.

The physicochemical property of table 1MsSQS albumen

2.3 subcellular localizations and signal peptide prediction

Utilize the cellular localization of TargetP 1.1 and PSORT prediction analysis alfalfa squalene synthase.As a result Show (table 2): MsSQS albumen seldom, should belong to non-secretory albumen in secretory pathway, chloroplaset, mitochondria.Main distribution In cytoplasma membrane, Thylakoid membrane, microbody, minority is distributed on endoplasmic reticulum.

By the protein sequence of biological software SignalP4.1Server software analysis MsSQS coding, detecting it is It is no that there is N-terminal signal peptide (Fig. 4).As the result is shown: the 51st phenylalanine residue of MsSQS protein sequence has highest original cut Enzyme site score value 0.113 and the 1st methionine residues have highest signal peptide score value 0.167, the 11st proline residue Score value 0.121 with the comprehensive shearing site of highest.Since the weighted average of last counted amino acid residue is smaller by 0.122 (< 0.5), thus, thus it is speculated that signal peptide is not present in the protein sequence N-terminal of MsSQS coded by said gene, is non-secretory albumen.As a result It is consistent with subcellular localization conclusion, illustrate that alfalfa SQS after cytoplasm synthesis, not can be carried out Protein transport.

The subcellular localization of table 2MsSQS is predicted

Analyzed by TMHMM Server online software: MsSQS encodes albumen, and there are two may transmembrane helical region (TM Helix): one is from the 288th amino acid residue to the 310th amino acid residue, and direction is ecto-entad.Second be from For 385th amino acid residue to the 407th amino acid residue, direction is from inside to outside.Trans-membrane region prediction result shows Respectively there is 1 hydrophobic region for being about 22 bases with C-terminal between amino acid residue from 288 to 310 in the amino acid sequence of MsSQS Domain may be the region that MsSQS albumen is combined with film anchoring in conjunction with wherein C-terminal hydrophobic region.

The expression analysis of 2.4 squalene synthase gene MsSQS

2.4.1 the expression analysis in different tissues

Using leaf, stem, each tissue sample of root reverse transcription cDNA as template, with SQS-qPCR-f and SQS-qPCR-r be draw Object, that detects MsSQS respectively organizes the variation of transfer record level in alfalfa.As a result (Fig. 5) is shown: MsSQS is expressed in root Highest, followed by leaf are measured, expression quantity is minimum in stem.Wherein 3 times higher than the expression quantity of stem of root or more.

3.4.2 influence of the different inductive conditions to MsSQS gene transcription level

In order to study the table that different inductive condition ultraviolet irradiations, dark, 100 μM of ABA and 50 μM of GA3 induce lower MsSQS Expression patterns, we determine the expression of different tissues root, stem, Ye Zhong MsSQS.In 15w ultraviolet light irradiation 30min, Ye Zhong The transcriptional level of MsSQS significantly increases, and highest is reached at 24 hours, and the transcriptional level in root is on a declining curve, at 32 hours Minimum (Fig. 6 A).Dark condition processing, the transcriptional level of Ye Zhong MsSQS are declined slightly, being basically unchanged in stem, and in root Transcriptional level is in significant ascendant trend (Fig. 6 B).It is handled with 100 μM of ABA, the transcriptional level of MsSQS does not obviously become in root Change, and the small time expression quantity of 4-12 significantly increases in cauline leaf.It is handled with 50 μM of GA3, the transcriptional level of Ye Zhong MsSQS is obvious It increases, it is as a result similar to ABA processing.Transcriptional level improves 15 times or more compared with the control when handling 8 hours.

3.5MeJA induces the expression to MsSQS gene, the influence of enzyme content and saponin content

The adjustment effect synthesized to saponin(e with sterol substance is expressed to understand MsSQS.We have studied methyl jasmonates Relationship under induction, between the expression and squalene synthase content and the variation of total saponin content of MsSQS.With 200 μM MeJA handles alfalfa, takes leaf, stem, root tissue to extract total serum IgE by time gradient after processing.Using reverse transcription cDNA as template, Using SQS-qPCR-f and SQS-qPCR-r as primer, MsSQS transcriptional level (Fig. 7 A) in alfalfa different tissues is detected.? In the 144h of MeJA processing, expression quantity of the MsSQS in leaf, stem, root is increased, and shows that MeJA can induce MsSQS expression.Root The expression quantity presentation of MsSQS first increases the trend reduced afterwards, and expression quantity highest, then gradually declines when for 24 hours, but general trend All it is higher than control；MsSQS transcriptional level shows two peak values different from root in leaf, and first peak value transcribes water at 8 hours Flat is 15 times of control, and second of lift-off value is 24 times of control at 144 hours.In stem although the transcriptional level of MsSQS has It is increased, but amplitude of variation is little.

Under MeJA induction, clover respectively organize in the overall raising of squalene synthase content, but with the length of stress time Certain fluctuation is presented, squalene synthase content content highest when coercing 24 hours in root, is 2.26 times of control；Leaf and stem The transcriptional level of middle MsSQS is consistent, i.e. mentioning with MsSQS gene transcription level with the variation tendency of squalene synthase content Height, the content of squalene synthase also increase (Fig. 7 B).

Under MeJA induction, the total saponin content in clover different tissues changes such as Fig. 7 C, with MeJA stress time Extend, it is 1.2 times of control that total saponin content is in rising trend in root, and content reaches maximum at 24 hours.Total saposins in leaf Content changes greatly, and general trend is that saponin content constantly rises, and saponin content reaches maximum value at 144 hours, is control 1.78 times.Total saponin content variation is not significant in stem.

2.6 prokaryotic expression results

Success in Experiment constructs MsSQS full length gene code area cDNA and C-terminal truncates (carboxy-terminal Truncated) the MsSQS prokaryotic expression recon of 90 bases, and its recon is respectively designated as pEASY-E2-MsSQS With pEASY-E2-CTtMsSQS (Fig. 8).It constructs simultaneously and inserts one section of crt gene sequence in pEASY-E2 expression vector Column, as blank control, are named as pEASY-E2-Control.And above-mentioned recon is transferred to Escherichia coli Transetta (DE3) in competent cell, under the action of T7 promoter, recombinant protein big scale in colibacillus is induced with 1mM IPTG It reaches.

SDS-PAGE electrophoresis result is shown: compared with before induction, the large intestine of pEASY-E2-MsSQS has been converted after induction 2h There is obvious protein band, the MsSQS-HIS of recombinant protein relative molecular mass and prediction in 45~50kDa or so in bacillus sample Molecular weight of albumen theoretical value (49kDa) is close, and expression quantity reaches maximum value after 5h；Meanwhile pEASY-E2- is converted There is obvious protein band in 40~45kDa or so in the E. coli SampLes of CTtMsSQS, recombinate egg after IPTG inducing expression 5h White expression quantity reaches highest, the recombinant protein molecular size range of expression and desired CTtMsSQS-HIS recombinant protein theoretical value (46kDa) is consistent.

To have converted the Escherichia coli of pEASY-E2-Control as blank control, after IPTG inducing expression 5h, respectively The broken liquid of Bacillus coli cells for having converted pEASY-E2-MsSQS and pEASY-E2-CTtMsSQS is extracted, is taken respectively after centrifugation Supernatant precipitating carries out SDS-PAGE electrophoretic analysis.As the result is shown (Fig. 9): after IPTG is induced, having converted pEASY-E2- In the supernatant total protein and sediment total protein of CTtMsSQS Escherichia coli target weight can be detected in 40~45kDa or so Histone band, and converted pEASY-E2-MsSQS Escherichia coli can only detect target weight in its sediment total protein Histone band.Therefore, MsSQS albumen can in Escherichia coli great expression, but be inclusion body protein, be not detected solvable Property albumen.And after C-terminal truncates 30 amino acid, MsSQS albumen still can be in expression in escherichia coli, and can give expression to solvable Property albumen.

The variation of 2.7 turns of MsSQS gene alfalfa saponins contents

The concentration of the total saposins of the aerial part of different strains is acquired, according to oleanolic acid standard curve with empty carrier PBI121 transgenic alfalfa and wild-type alfalfa are control, detect MsSQS transgenic alfalfa aerial part soap Salidroside content shows that total saponin content is apparently higher than control in transgenic alfalfa, and the plant than turning empty carrier is 1.5~2 times high, 1~1.6 times (Figure 11) higher than wild type.Illustrate, MsSQS is overexpressed in alfalfa, and saponin in alfalfa can be improved Content.It can be seen that MsSQS gene plays a key effect in alfalfa saponin route of synthesis.

The present invention has found that alfalfa SQS gene is compiled by the amino acid alignment encoded to different plant species SQS gene The protein and the SQS protein sequence homologies in other species of code are up to 80%, illustrate plant SQS albumen during evolution Than more conservative, the SQS albumen of different plant species should belong to the same family.And the coding protein structure phase one of different plant species SQS It causes, have 6 conservative regions: wherein 3 highly conserved regions (IIIth area, IVth area and Vth area) are related with enzyme active center；Other 2 A conservative region (IIth area and IVth area) is considered as Mg²⁺The activated centre of binding site is the terpene rich in aspartic acid (DXXXD) The typical structure of class synthase；Although VI conservative of region is lower, dredged containing 1 section of height as the sequence in other species Water sequence, prediction may be membrane anchor domain (Robinson et al., 1993).Subcellular localization shows its coding Albumen may be mainly distributed on cytoplasma membrane, Thylakoid membrane, microbody, and minority is distributed on endoplasmic reticulum.Cross-film simultaneously Structure domain analysis show that MsSQS albumen has 2 possible transmembrane helical regions, and wherein C-terminal hydrophobic region may be film combined area Domain.Blast search is carried out to MsSQS albumen using SWISSMODEL, the results showed that the albumen and birds method Buddhist nun ester pyrophosphoric acid close Enzyme, people's squalene synthase activity division center are quite similar, form a hydrophobic space reaction substrate in center, show originally to grind Study carefully the albumen squalene synthase catalytic activity having the same of the MsSQS sequential coding of clone.

Using real-time fluorescence quantitative PCR detection MsSQS in the differential expression of clover different tissues, MsSQS exists as the result is shown Expression quantity highest in root, followed by leaf, expression quantity is minimum in stem.By containing to squalene synthase activity in different tissues and saponin(e The analysis of amount, MsSQS gene become in the expression quantity of different tissues with the variation having the same of squalene synthase activity and saponin content Gesture, i.e. MsSQS gene are positively correlated with squalene synthase content and saponin content, illustrate that squalene synthase gene may be regulation saponin(e The key gene of synthesis.

In the present invention after ultraviolet processing, part rises the mRNA level in-site of squalene synthase gene transcription on the ground, root The transcriptional level in portion is declined slightly.It may be to cause directly to injure to cauline leaf due to ultraviolet irradiation, by increasing aerial part shark The expression of alkene synthase gene is to adjust the synthesis of saponin(e, to reduce damage caused by ultraviolet radiation, thus illustrates that saponin(e is joined With the self-defensive system of plant.At present for the oxidation resistant research of alfalfa saponin it has been reported that still closing In it whether there is certain anti-ultraviolet radiation effect not yet to see relevant report.

Under after being protected from light processing, root MsSQS expression quantity obviously rises the present invention, but the expression quantity of aerial part slightly has Drop.May be in the different tissues of different plants secondary metabolite synthesis have certain difference to lighting requirements, be that plant is logical Cross a kind of adaptation reaction of the secondary metabolism to poor environment (shading).Studies have shown that the conditions such as intensity of illumination, time, light quality Change can influence the synthesis of secondary metabolite in plant.

The present invention adds ABA and GA3 respectively in alfalfa culture solution, and interior for 24 hours after treatment, MsSQS is in blade Expression quantity significantly raise, and root expression quantity is not significant in each period difference.Gibberellin and abscisic acid can be in blades Middle induction MsSQS expression quantity up-regulation, it may be possible to because when gibberellin exogenous in plant or abscisic acid increase, from first hydroxyl Valeric acid (mevalonate, MVA), which begins to flow to gibberellin or the carbon flow of abscisic acid synthesis, to be reduced, and accordingly flows to steroidal synthesis Carbon flow but increase, thus the expression of positive regulation squalene synthase gene.In addition, plant hormone and triterpene compound synthesis tool There is common intermediate, the variation of plant hormone in vivo content may will affect the synthesis of triterpene compound, and then influence squalene The change of synthase expression amount.

The present invention analyzes under 200mM MeJA induction, the transcriptional level of MsSQS gene, the expression for encoding albumen And the variation of final product-saponin content, the expression quantity of the MsSQS in different tissues have rising, while squalene is closed in clover Fluctuation status is also presented in enzyme content and activity accordingly；It is measured by Solid Phase Extraction-ultraviolet spectrophotometer method total in each tissue The content of saponin(e also accordingly increases, and the variation tendency of total saponin content is consistent with the variation tendency of MsSQS expression.Study table Bright, under MeJa induction, MsSQS gene expression amount and saponin content ascensional range are maximum in alfalfa-leaves, illustrate MeJA to leaf In MsSQS induction it is the most significant.Preliminary proof is tested, methyl jasmonate can pass through induction alfalfa MsSQS gene Transcription, to influence the activity and the synthesis of saponins active material of squalene synthase.

By carrying out the detection of transcriptional level and total saponin content, as a result table to the clover transgenic positive seedling for turning MsSQS MsSQS expression quantity and total saposins amount are above wild-type alfalfa and turn empty carrier alfalfa in bright transgenic plant.Into One step card, MsSQS gene have certain adjustment effect in total saponin of alfalfa route of synthesis.The present invention not only establishes Clover turns the Efficient Conversion system of MsSQS gene, and expression pattern to MsSQS and function have preliminary understanding；It obtains Transgenic alfalfa strain can be used as the germplasm materials for cultivating high saponin content alfalfa new varieties, be clover heredity Improvement has certain meaning and application value.

Embodiment described above only describe the preferred embodiments of the invention, not to model of the invention It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention The various changes and improvements that case is made should all be fallen into the protection scope that claims of the present invention determines.

Sequence table

<110>Institute of Animal Sciences, Chinese Academy of Agricultural Sciences

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Claims

1. a kind of method that alfalfa is overexpressed squalene synthase gene, which comprises the following steps:

(1) design of primers；

(2) cloning vector pEASY-T3 plasmid, plasmid pGEX-4T-1V are used for prokaryotic expression, E. coli Trans5 α Gene cloning and expression is used for E.coli Transetta；

(3) alfalfa squalene synthase gene MsSQS full-length cDNA is cloned；

(4) bioinformatic analysis of MsSQS sequence；

(5) expression analysis of squalene synthase gene MsSQS；

(6) measurement of squalene synthase content；

(7) methyl jasmonate induces the measurement of lower saponin content；

(8) Subcellular Localization；

(9) prokaryotic expression；

2. the method that alfalfa according to claim 1 is overexpressed squalene synthase gene, it is characterised in that: the step (1) specifically, according to the cds sequence of M. truncatula squalene synthase gene known in NCBI, 5.0 design primer of Primer is utilized MsSQS-f and MsSQS-r is used to expand the cDNA sequence of MsSQS gene.The alfalfa actin announced according to Genbank β-actin gene, as target detection gene, respectively designs pair of primers as house-keeping gene and the MsSQS gene cloned Actin-f and Actin-r, SQS-qPCR-f and SQS-qPCR-r.As shown in SEQ ID NO:1-6, sequence is as follows:

MsSQS-f:5 '-GCTTATTCGTAGAAACAAAAGATGG-3 '

MsSQS-r:5 '-ATGCTACCACTGTTCTGTTGCTATC-3 '

Actin-f:5’-CAAAAGATGGCAGATGCTGAGGAT-3’

Actin-r:5’-CATGACACCAGTATGACGAGGTCG-3’

SQS-qPCR-f:5’-CTTCGGTCTTGTTATTCAGCAGC-3’

SQS-qPCR-r:5’-CTTGTATCATCCTCAACGGTGTC-3’。

3. the method that alfalfa according to claim 2 is overexpressed squalene synthase gene, it is characterised in that: the step (3) specifically, the total serum IgE of alfalfa blade is extracted using Trizol method, with the quality of spectrophotometric determination RNA；With 1% Agar sugar detection RNA integrality；Each sample takes the RNA of 1 μ g for reverse transcription, with the Prime ScriptTM of Takara 1^stStrand cDNA synthesis kit carries out PCR amplification, and reaction system is 20 μ L；It is to draw with MsSQS-f and MsSQS-r Object carries out PCR amplification and obtains MsSQS full-length cDNA, is then cloned into pEASY-T3 carrier and obtains plasmid pEASY-MsSQS, It is transferred to E. coli DH5 α again and in 37 DEG C of dark culturings of LB culture medium；Then plasmid is sequenced assembling, and verifying is correct MsSQS insertion point.

4. the method that alfalfa according to claim 3 is overexpressed squalene synthase gene, it is characterised in that: the step (4) it specifically, analyzing the open reading frame sequence of target gene MsSQS using DNAMAN program and NCBI/BLAST, and carries out Multiple sequence alignments and allied species analysis；Using the molecular weight of SOPMA program analysis SQS protein, amino acid composition and wait Electric point；By the signal peptide and subcellular of SignalP4.1Server software and SubLoc software prediction MsSQS coding protein Positioning；The transmembrane region and hydrophobicity of protein are predicted using TMHMM Server online software；Utilize SWISSMODEL program Analyze the three-dimensional structure of protein；Utilize Interpro database analysis protein structure domain-functionalities；It is looked into NCBI/GenBank The homologous protein of the plants such as arabidopsis, ginseng is looked for, and carries out sequence alignment analysis using ClustalX2.0 software.

5. the method that alfalfa according to claim 4 is overexpressed squalene synthase gene, it is characterised in that: the step (5) it is sprouted specifically, No. 3 seeds of lucerne in alfalfa are placed on the filter paper soaked, is then implanted into Hoagland ' s nutrition In liquid, it is placed in illumination box, in 16h daytime/8h night, on 24 DEG C of daytime, 20 DEG C of night, relative humidity 40% was cultivated 4 weeks, every 1 week Change one time of nutrition liquid；It chooses and grows fine after 4 weeks, plant of the same size is used for following processing: 1. at plant 20cm, After 15W ultraviolet light 30min, root, stem, leaf texture's sample are acquired in 0,2,4,8,12,24,48h respectively；2. being protected from light processing 0,4,8,12,24,48,96,144h acquires root, stem, leaf texture respectively；3. 200 μM of MeJA processing 0,4,8,12,24,48,96, 144h acquires root, stem, leaf texture respectively；4. 100 μM of ABA processing 0,2,4,8,12, acquire root, stem, leaf texture respectively for 24 hours；⑤ 50 μM of GA3 processing 0,2,4,8,12, acquire root, stem, leaf texture respectively for 24 hours；The sample of collection is respectively used to extract RNA.

6. the method that alfalfa according to claim 5 is overexpressed squalene synthase gene, it is characterised in that: the step (6) specifically, using squalene synthase protein in clover different tissues after enzyme linked immunosorbent assay MeJA induction processing Activity；Various time points root, stem, each 200mg of leaf texture are placed in 2mL centrifuge tube after taking MeJA to handle, and it is slow that 10mM phosphoric acid is added Fliud flushing PBS 900mL, PH=7.2-7.4 wear into slurry with grinding rod, and standing draws supernatant in a moment, and it is even that residue adds 900mL Slurries are ground once again, are then combined with lapping liquid twice, are centrifuged 15min under the conditions of 4 DEG C with 6000rpm revolving speed, are taken supernatant；Sample Product measure enzymatic activity after extracting at once；With spectrophotometer, absorbance is read with standard curve and calculates the actual concentrations of sample.

7. the method that alfalfa according to claim 6 is overexpressed squalene synthase gene, it is characterised in that: the step (7) specifically, being chosen at the clover root of MeJA processing, that stem, leaf sample are placed in -80 DEG C of freeze-dryings 70 in cryogenic vacuum freeze dryer is small When, 60 meshes are crossed after taking-up；It accurately weighs 30mg freeze-drying sample to be placed in 5mL centrifuge tube, adds 1mL70% ethyl alcohol Ultrasound Instrument ultrasonic 30min, 4 DEG C overnight, again ultrasound 30min；Then 30min is centrifuged with 1000g, takes 100 μ L of supernatant injection pretreated SPE column is rinsed with 5mL water and 5mL30% methanol respectively, then with 1mL methanol elution 4 times；Merge the tool that eluent is placed in 10mL It is evaporated in plug test tube, vanillic aldehyde-glacial acetic acid solution of 0.2mL 5% is added after cooling, to which 0.8mL high chlorine is added after completely dissolution Acid；It is placed in 70 DEG C of water-baths after water-bath 15min ice bath immediately, 5mL glacial acetic acid is added after cooling, stands 20min；It is flat with blank Row sample compares, and each sample sets 3 biology and repeats；With the absorbance of spectrophotometric determination sample at 545nm.

8. the method that alfalfa according to claim 7 is overexpressed squalene synthase gene, it is characterised in that: the step (8) specifically, containing primer of the upstream and downstream respectively containing I restriction enzyme site of Xho I and Sal, SQS- using 5.0 software design of Primer pA7-f:5’-CCCTCGAGATGGGAAGTATAAAAGCGATTTTGA-3 ', CTCGAG are I restriction enzyme site of Xho；SQS-pA7-r: 5’-GCGTCGACGTTATTGTAACGGTTGGCAGAGAGA-3, GTCGAC are I restriction enzyme site of Sal；Such as SEQ ID NO:7-8 institute Show；Using pEasy-T3-MsSQS plasmid as template, respectively using SQS-pA7-f and SQS-pA7-r as primer, target fragment is carried out Amplification, then target fragment is connected to pEASY-T1 carrier, and naming recon is pEASY-T1-MsSQS；By recombinant plasmid PEASY-T1-MsSQS and transient expression plasmids pA7-GFP carries out double digestion, digestion system with Xho I and the quick digestion enzyme of Sal I As follows: plasmid volume determines (< 1 μ g) according to plasmid concentration, I 1 I 1 μ L, 10 × Quick Cut Buffer of μ L, Sal of Xho, 5 μ L, ddH₂O Up to 50μL；After mixing gently, with 37 DEG C of 5~10min of reaction of PCR instrument；After agarose gel electrophoresis, recycling Target fragment；Then target gene fragment is connected to carrier pA7-GFP, 16 DEG C of 4~5h of connection under the action of T4 ligase, Construct plasmid pA7-GFP-MsSQS；MsSQS is integrated into transient expression vector pA7-GFP with being correctly oriented, passes through gene The method of rifle bombardment, is transferred to onion epidermis for the subcellular transient expression vector pA7-GFP containing target gene coding region sequence, Under the driving of 35S strong promoter, fusion protein overexpression in onion epidermis cell；Green fluorescent protein GFP is in blue wave Green fluorescence is shown under long light, and the positioning of the albumen of SQS gene coding in the cell is determined according to the fluorescence signal of fusion protein；

1. the entire coding region sequence of MsSQS cDNA is inserted into protokaryon specifically, two pairs of primers of design are respectively as follows: by the step (9) Expression vector, specific primer are SQS-PE-f:5 '-ATGGGAAGTATAAAAGCGATTTTG-3 ', SQS-PE-r:5 '- ATTCCTCAAAA CGTAAGATTTCCTT-3'；2. the sequence that MsSQS coding region sequence C-terminal cuts out 30 amino acid is inserted Enter prokaryotic expression carrier, specific primer is SQS-PE-CTt-f:5 '-ATTCCTCAAAACGTAAGATTTCCTT-3 ', SQS-PE- r:5'-ATTCCTCAAAACGTAAGATTTCCTT-3'；As shown in SEQ ID NO:9-11；Correct MsSQS sequence will be contained PEASY-T3-MsSQS plasmid is respectively to expand with SQS-PE-f and SS-PE-r, SQS-PE-CTt-f and SQS-PE-r as template Increase primer, carries out the amplification of target fragment, then target fragment is connected to pEASY-E2 carrier；PCR response procedures are as follows: 94 DEG C Initial denaturation 5min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min72 DEG C of extension 5min, 4 DEG C of heat preservations；It will be above-mentioned PCR product is after agarose gel electrophoresis and DNA fragmentation recycling, by the target fragment of recycling and prokaryotic expression carrier pEASY- E2 connection, and be transferred in Tran-T1 competent cell, the connection and conversion of carrier；

Positive monoclonal is identified with PCR method, to carry out thallus PCR identification, using bacterium solution as template, carrier for each monoclonal Forward primer M13-F, 5'-CGCCAGGGTTTTCCCAGTCACGAC-3', as shown in SEQ ID NO:12；And target gene Reverse primer SQS-PE-r identifies positive recombinant as primer, and the bacterium solution that will amplify target gene fragment is sent to survey The sequencing of sequence company；Recon is respectively designated as pEASY-E2-MsSQS and pEASY-E2-CTtMsSQS；By two kinds of prokaryotic expressions Recon pEASY-E2-MsSQS and pEASY-E2-CTtMsSQS and empty carrier are transferred to Escherichia coli Transetta respectively； Prokaryotic expression MsSQS albumen and MsSQS-CTt protein SDS-PAGE electrophoresis, take the above-mentioned Escherichia coli containing correct segment Transetta, 37 DEG C of overnight incubations to OD₆₀₀It is 0.4~0.6；Escherichia coli to contain empty carrier are added as control IPTG makes its final concentration of 1mM in bacterium solution, respectively inducing expression 0,30,1,2,3,4,5,6,8h；After IPTG is added, upper It states each time point and takes 2mL bacterium solution, 4 DEG C of 10000rpm are centrifuged 5min, abandon supernatant, and 1mLPBS, 10mM, PH=7.2-7.4 is added Thallus is resuspended, with ultrasonic cell disruption instrument ultrasound 100 times, takes collection precipitating and supernatant, precipitating 100 μ L PBS weight respectively Outstanding, 4 DEG C temporary；After the completion of sampling, takes 50 μ L supernatants to precipitate re-suspension liquid respectively, isometric 2 × SDS loading is added It is immediately placed on cooled on ice after Buffer, 100 DEG C of water-bath 3min, then uses the expression of PAGE gel electroresis appraisal MsSQS.

9. the method that alfalfa according to claim 8 is overexpressed squalene synthase gene, it is characterised in that: the step (10) specifically, being amplimer with MsSQS-f and MsSQS-r, the MsSQS containing I restriction enzyme site of Xba I and BamH is amplified Genetic fragment, recovery purifying PCR product, then target fragment is connected to pEASY-T3 carrier；It is sequenced and extracts containing correct sequence The recombinant plasmid of column carries out pBI121 and recon pEASY-T3-pMsSQS with Xba I and the quick restriction enzyme of BamH I Target gene coding region sequence is inserted into plant overexpression vector pBI121, structure by double digestion under the action of T4 ligase Target gene overexpression vector is built, and converts Agrobacterium EHA105；Then using lucerne 3 in Agrobacterium-mediated transformation alfalfa Number.

10. the method that any alfalfa of claim 1-9 is overexpressed squalene synthase gene is cultivating high saponin content Application in alfalfa variety.