阅读说明：本技术 蒺藜苜蓿豆球蛋白基因Mt1g072600启动子PMt1g072600及其应用 (Tribulus medicago conglutin gene Mt1g072600 promoter PMt1g072600 and application thereof ) 是由 魏琦超 关园园 李东霄 宋普文 徐新娟 于 2020-12-18 设计创作，主要内容包括：本发明公开了蒺藜苜蓿豆球蛋白基因Mt1g072600启动子PMt1g072600及其应用,属于生物技术领域。本发明公开了蒺藜苜蓿豆球蛋白基因Mt1g072600启动子PMt1g072600在驱动外源基因在受体植物种子中特异性高水平表达中的应用。转基因拟南芥种子萌发过程及成体植株各组织器官的GUS组织化学染色结果表明,所克隆的编码区上游序列为种子特异性启动子。转基因拟南芥种子的GUS酶活检测结果表明,所克隆启动子驱动GUS的表达量极显著高于CaMV35S启动子,认为其可用于植物基因工程领域,驱动外源基因在转基因植物种子中高水平表达。(The invention discloses a promoter PMt1g072600 of a medicago truncatula legeliin gene Mt1g072600 and application thereof, belonging to the technical field of biology. The invention discloses application of a Tribulus medicaginis conglutin gene Mt1g072600 promoter PMt1g072600 in driving specific high-level expression of an exogenous gene in a receptor plant seed. The germination process of transgenic arabidopsis seeds and GUS histochemical staining results of various tissues and organs of adult plants show that the upstream sequence of the cloned coding region is a seed specific promoter. The GUS enzyme activity detection result of the transgenic arabidopsis seeds shows that the expression quantity of GUS driven by the cloned promoter is remarkably higher than that of a CaMV35S promoter, and the cloned promoter can be used in the field of plant genetic engineering and can drive high-level expression of exogenous genes in transgenic plant seeds.)

1. The promoter PMt1g072600 of the medicago truncatula leguminous globulin gene Mt1g072600 is characterized in that the nucleotide sequence is shown as SEQ ID NO. 15.

2. The use of the Medicago truncatula legelia globulin gene Mt1g072600 promoter PMt1g072600 of claim 1 to drive the specific high level expression of exogenous genes in recipient plant seeds.

Technical Field

The invention relates to the technical field of biology, in particular to a Tribulus terrestris alfalfa legumin gene Mt1g072600 promoter PMt1g072600 and application thereof.

Background

Medicago (Medicago) plants are annual or perennial herbs, among which the vegetal purple alfalfa (m.sativa) has the name "king of pasture" which is a worldwide recognized important legume (Leguminosae) pasture. Medicago truncatula is a diploid selfing plant, has a close relationship with Medicago sativa, and is used as a model plant for researching leguminous plant-rhizobium symbiosis system (legumes-rhizobium symbiosis), and the whole genome sequencing work is completed (Branca et al, 2011; Young et al, 2011).

Seed Storage Proteins (SSPs) refer to storage proteins synthesized by higher plants during the seed maturation process and stored in large quantities, and can provide nitrogen, carbon and sulfur sources for the seed germination process. Because of the high polymorphism, the alfalfa seed storage protein can be used for genetic diversity analysis and variety identification work among varieties or germplasm resources. Alfalfa seeds have no edible value for leguminous plants such as soybean (Glycine max) and kidney bean (Phaseolus vulgaris) from which seeds are derived human proteins, and the types, amounts and genetic information of seed storage proteins are rarely reported in the literature. Stuart et al (1988) extracted alfalfa seed storage proteins using buffers of varying pH and ionic strength and conducted isolation and purification studies, and suggested that the seed storage proteins were predominantly 7S piscine (vicilin) and 11S legumin (legumin) class 2, containing members with apparent molecular weights (apparent molecular weights) of 52, 36, 32, 20kD and 49, 47, 45, 39, 22, 20kD, respectively. Promoters of seed storage protein genes are potential sources of seed-specific promoters.

Therefore, the problem to be solved by the technical personnel in the field is to provide the Tribulus terrestris conglutin gene Mt1g072600 promoter PMt1g072600 and the application thereof.

Disclosure of Invention

In view of the above, the invention provides a promoter PMt1g072600 of a Medicago truncatula legeliin gene Mt1g072600 and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the nucleotide sequence of the promoter PMt1g072600 of the medicago truncatula legumina gene Mt1g072600 is shown in SEQ ID NO. 15.

Furthermore, the application of the Tribulus medicaginis conglutin gene Mt1g072600 promoter PMt1g072600 in driving the specific high-level expression of exogenous genes in the seeds of receptor plants.

According to the technical scheme, compared with the prior art, the invention discloses and provides the Mt1g072600 promoter PMt1g072600 of the medicago truncatula conglutin gene Mt1g and the application thereof, the seed storage protein is a general term of a large amount of storage proteins synthesized and stored in the maturation process of higher plant seeds, and the promoter of the gene is an important source of a seed specific promoter. In order to discover a dicotyledonous plant seed specific promoter, the invention uses Medicago truncatula (Medicago truncatula) as a test material, clones a 2075bp sequence at the upstream of a coding region of a legumin gene Mt1g072600, predicts cis-acting elements by using tool websites PLACE and PlantCARE, constructs a plant expression vector for driving GUS and carries out genetic transformation of arabidopsis thaliana. The result shows that the upstream sequence of the coding region contains a plurality of cis-acting elements related to seed-specific promoters such as E-box, A/T rich element, P-box and the like, and is named as PMt1g 072600; analysis of GUS expression in transgenic Arabidopsis seeds (Arabidopsis thaliana) shows that the expression amount of GUS driven by PMt1g072600 is remarkably higher than PCaMV 35S. Histochemical staining results of each tissue/organ of transgenic arabidopsis thaliana show that the specificity of driving GUS by PMt1g072600 shows tissue/organ expression. The invention successfully clones the seed specific promoter from the medicago truncatula and can be used for driving the expression of exogenous genes in transgenic plant seeds.

The invention obtains the seed specific promoter capable of driving the specific expression of the exogenous gene in the receptor plant seed, and provides a candidate promoter resource for realizing the accurate three-dimensional regulation of the timing, positioning and quantification of the exogenous gene expression and avoiding the transgenic silencing risk caused by the homology with the endogenous promoter of the receptor plant.

The promoter can drive GUS expression amount to be remarkably higher than PCaMV35S, and can be applied to seed bioreactors to drive high-efficiency expression of medical, feed or industrial enzyme genes in dicotyledonous plants.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the result of PCR amplification of the upstream sequence of the coding region Mt1g072600 according to the present invention;

wherein, M is DNA molecular weight standard; mt1g072600 coding region upstream sequence amplification product;

FIG. 2 is a diagram showing the sequence upstream of the coding region of Mt1g072600 according to the invention;

wherein the boxes or underlines indicate cis-acting elements, +1 indicates the transcription start site;

FIG. 3 is a diagram showing a plant expression vector pEXPR (PMt1g072600:: GUS:: T35S) according to the present invention;

FIG. 4 is a diagram showing a part of the result of PCR detection of transgenic Arabidopsis thaliana of the present invention;

wherein, M is DNA molecular weight standard; 1-6, numbering the transgenic arabidopsis strains; CK is wild type arabidopsis negative control;

FIG. 5 is a drawing showing GUS enzyme activity in transgenic Arabidopsis seeds according to the present invention;

FIG. 6 is a drawing showing GUS histochemical staining of transgenic Arabidopsis thaliana of the present invention;

wherein, A: a wild type; b: PCaMV 35S; c: PMt1g 072600; 1: embryo; 2: 3 days old seedlings; 3: 9 days old seedlings; 4: a root; 5: a stem; 6: lotus throne leaves; 7: stem leaves; 8: fruit pieces; 9: flower; 10: inflorescence; a scale: 1 mm.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Medicago truncatula R108, Arabidopsis thaliana (Arabidopsis thaliana) Col0 seeds were maintained by the laboratory.

The entry vector pENTRY (PCaMV35S:: GUS:: T35S) and the target vector pEarleyGate303 are stored in the laboratory.

Wherein the construction process of the pENTRY (PCaMV35S:: GUS:: T35S) vector is as follows:

(1) synthesis of linker-1/2

linker-1：

AATTCGCCCTTGCGATCGCATGCGACTGCGGCCGCTCAGTGCACCCGGGCATGTCATGGCGCGCCAAGGGCG；SEQ ID NO.1；

linker-2：

AATTCGCCCTTGGCGCGCCATGACATGCCCGGGTGCACTGAGCGGCCGCAGTCGCATGCGATCGCAAGGGCG；SEQ ID NO.2；

(2) EcoRI was used to treat Invitrogen corporation products(cat. K250020SC) was subjected to enzyme digestion.

(3) After annealing in linker-1/2 to form a double strand having a cohesive end, ligation is performedThe large fragment of the vector after the enzyme digestion is named pENTR-MCS.

(4) Carrying out PCR amplification on pCAMBIA2300 by using T35S (F)/(R) to obtain a CaMV35S terminator;

T35S(F)：5’-CCCGGGCGGCCATGCTAGAGTCCGCA-3’；SEQ ID NO.3；

T35S(R)：5’-GGCGCGCCATGTCACTGGATTTTGGTTT-3’；SEQ ID NO.4；

(5) carrying out double enzyme digestion on the CaMV35S terminator and pENTR-MCS by using XmaI/AscI, connecting the CaMV35S terminator into the enzyme-digested vector large fragment of the pENTR-MCS, and naming the large fragment as pENTR-MCS-T35S;

(6) carrying out PCR amplification on pMON82053 by using a primer P35S (F)/P35S (R) to obtain P35S;

P35S(F)：5’-GCGATCGCGTCCGATGTGAGACTTTTC-3’；SEQ ID NO.5；

P35S(R)：5’-GCGGCCGCCCTCTCCAAATGAAATGAAC-3’；SEQ ID NO.6；

pMON82053 is described in the following documents: expression of soft words of Arabidopsis thaliana BBX32 gene in soybean innovaties gradinyield.PLoS one.2012; 7(2) e30717.doi: 10.1371/journal.pane.0030717.

(7) The plasmid was digested simultaneously with P35S and pENTR-MCS-T35S using AsiSI/NotI, and the large digested vector fragment containing P35S ligated to pENTR-MCS-T35S was named pENTR-P35S.

(8) Carrying out PCR amplification on pBI121 by using primers GUS (F)/GUS (R) to obtain a GUS fragment;

GUS(F)：5’-GCGGCCGCATGTTACGTCCTGTAGAAAC-3’；SEQ ID NO.7；

GUS(R)：5’-CCCGGGTCATTGTTTGCCTCCCTGCT-3’；SEQ ID NO.8；

(9) the GUS fragment and pENTR-P35S were digested simultaneously with NotI/XmaI, and the GUS fragment was ligated to the digested large fragment of pENTR-P35S, which was named pENTRY (PCaMV35S:: GUS:: T35S).

Escherichia coli (Escherichia coli) DH5 alpha, Agrobacterium tumefaciens (Agrobacterium tumefaciens) GV3101(pMP90) competent cells were purchased from International Biogene technology, Inc., of the Union of Beijing village.

Zero Cloning Kit was purchased from Beijing Quanyu gold Biotechnology Ltd,LR Clonase^TMII Enzyme Mix was purchased from Invitrogen life technology,GXL DNA Polymerase from Baozi physician technology (Beijing) Ltd, Monclone^TMHi-Fusion Cloning Mix V2 was obtained from Morina Biotech, Inc., and restriction endonucleases were obtained from NEB (Beijing) Inc. The polysaccharide polyphenol plant genome DNA extraction kit is purchased from Tiangen Biotechnology (Beijing) Co., Ltd, and the micro-column concentrated DNA gel recovery kit is purchased from Beijing Zhuang Allen internationally biogenetic technology Co., Ltd.

EXAMPLE 1 cloning of sequences upstream of the coding region of the Gene of interest

(1) Extraction of total DNA of medicago truncatula

The leaves of medicago truncatula R108 seedlings are taken as test materials, and total DNA is extracted according to the instruction of a polysaccharide polyphenol plant genome DNA extraction kit.

(2) Target gene discovery and coding region upstream sequence amplification primer design thereof

The UniProt database (https:// www.uniprot.org /) was searched with the search formula name: "seed storage protein" organization: "media truncatula". One gene annotated as "Legumin stored protein" was selected as the subject. Using the corresponding gene name MTR _1g072600 as a keyword, the Tribulus medicago genome sequence information of Ensemblplants (http:// www.plants.ensembl.org /) website was retrieved (Howe et al, 2020). The nucleic acid sequence of 3kb upstream of the coding region of the gene was downloaded as a reference for primer design. The information of the primers used in the present invention is shown in Table 1.

The Latin name of the Medicago truncatula is Medicago truncatula, MTR _1g072600 is the name of a website genome database website for the gene, MTR is the abbreviation of the Latin name, the Latin name is abbreviated as Mt, and the gene is abbreviated as Mt1g 072600.

TABLE 1 primer information

(3) PCR amplification and cloning of amplification products

Reaction system: 5 XPrimeSTAR GXL Buffer 10 u L, dNTP mix 4 u L, 1g072600(F)1 u L, 1g072600(R)1 u L, total DNA template 1 u L, PrimeSTAR GXL DNA Polymerase 1 u L, sterilized ultrapure water 32 u L.

Reaction procedure: 5min at 95 ℃; 15s at 98 ℃,20 s at 60 ℃ and 2min at 68 ℃ for 30 cycles; 5min at 68 ℃. The PCR product was subjected to agarose gel electrophoresis, and the results are shown in FIG. 1, whereby a band of interest having a size corresponding to the expected molecular weight (2075bp) was obtained. Cutting the gel, and recovering the target band by using a microcolumn concentrated DNA gel recovery kit. Reference toZero Cloning Kit instructions attached toA Zero cloning vector. The monoclonal colony is picked, the PCR identification of the bacterial liquid is carried out by adopting a primer 1g072600(F)/1g072600(R), and then the bacterial liquid is sent to a company Limited in Biotechnology engineering (Shanghai) to be sequenced, and the upstream sequence of a coding region is shown in SEQ ID NO. 15.

CTGATTGCTCAAAAGACTTCCTGAACATAGTTGATATGAAAGTTTATAGAAGTTGTGTAACTTGGACTTTCGAATAACCCTAGACGTTGATTTTTGAAAATCTTCGAAATAGCAGTCTTCGAACTTCTGGCTTGTTCTCGATCTTTGAAAAAAATGAACCTAGTTCGCTCGAAACTTTGTTATTAAAAATTTGCATATAACAGACTGAGATTTTCAAACGAGAGGCTACCCCTGCAATGAGATTGCCTCAATAGATAAGGTAAGTTGCACCAAATTAAAAGGACTGGATCTTAGACAATTGATGTACATTAGCTCCTATTAGTTTGTGTCTCTTTCTCTATAATAACTGATACCTTAGTTCGACATACAAATCTATATTTAAATAACTAATTAATCAATGGTTAACAAAAATAAGTTCACAGTAAAAATTGAAATCAACCATTGATATAAGTTGGTAAACTTGATTCAAGTTTATGTATTTATCGGTTGATATTAAGTGATTAAACGTAATTGGTAATTTATTTTGCACTTAAGTTTAGTTAAAACATAAAAGTAGGATGGTACAGATACCATCTAAACTGAAAATCATATGTTAGACTTGATATCTGCTTGAATCATAATTTTTTTGTTTTCAATGTTGTATGCTATCAGCATCACAGGCCATTGCGACCACCACTTGAACTCTGATGATAATACTCTATTAAGAGTGAACTTTGTAAGGGAGGTGGATGAATTGTTGCAAGAGGTTTGTTCGGAACATGACGAAGAAGAGAATGAACATGAGTTTAGTCCTGATACAGTTGCAAATAGATTAAAACAACAGGAGAAGCAACAACAATACACGTAGAAGGTTGTTTTTTTATTTACATATATTTGTTCAGTTGTCTTATAGTTTTGAATAGAAATTAGTATGCAGTGAGTGCAGAAAGGACACAAAAATGTAGACATGGAAATTTTTGGATTATCATGTGTTTTTAAAGGTAAAGGTATGTTAGAAATTGAGTTTTTGAATTTTATTTTATACATCTATGTACATCTGGTGAGTCCACCCAGTATTTTCTAGCATGTTTGGTTTTGTGGTGGTGAAAATGAATTTTAGTTAAAACTTTAAAGTCATGAGTTGAATGTACATTGGTCTATGTTTATATACATTTACATAAATGTGTTCAACAATAATTTGAAGTTAAAAATCTTATAAAGAGGCAAAACATCTAATTACATATTTGAGCTAGAATTTATTTTATAGAACGCTTAAATATATCAAGCAATTTTAATTTATGTGTCTAGAATATTTTTGGATCTTACAAAAGGGAATCCAAATATGCACCAAATGCACTGCATTGATTTAAACATCAATGACAACAATCAACCTGAATTCTCTAGAGCTTACTTGAATTCAAACAACTGGAAGGTAAATACTGAACTTTCAGAGCATAAATTTTAAGAGATTAAGTTATATATGCACTGATGGTGTAAAAAAAATTTTTTACAAACGCATGCAATCAAACTATATCATACTTTTATAAGTTAGATTTTAAAATATTTCTATTTTGATGCATGAGTAAAAATGTTTTACGCTTAAGTTATGCTGTTTCTGTTGCGAAGGCTACAGATTGATAATAACAAAGAGAGGTACTATTAAGCTAGAATTTCTACTTTAATCACTACATACATCTATATATTTGAGTCTGAGCAACACTGTTACTGCTTAATGAAAACTGTAAATGAATTAAGTAAAGTGAAAAAGAAGTAATGATATCAACATGTAAAGAATCGACGCAGAATGCATGGGACCATCCCATGCCATATACATTTGTTCTCATTCATTTAACTGCAGTCATGATTCCATATGTAAAGATACCAACAAATATACACTCTGTGATGTCTCTGTGCACATATATCTTAATGTGATGTGTAGGAAATTAAGAATTCATAGGCATGCATGGTGAAGAATGTCATGAACTAGCAACCTACACTGTGTGACGTGTCCCTTCCTCACTCTTCTCTTCTTACTATAAATCACCACTCCACAGGTTCTTCTCTTCACCAATTCATTCACCAAACTCACAAACACA；SEQ ID NO.15。

(4) Bioinformatic analysis of sequences upstream of coding regions

The sequencing result is compared with the disclosed upstream sequence of the coding region Mt1g072600 of the medicago truncatula genome, the consistency rate of the cloned sequence and the sequence is 100 percent, and the cloned sequence is proved to be the upstream sequence of the coding region Mt1g 072600. Analysis and annotation of promoter-associated cis-acting elements in the cloned sequences was performed using the online tool of the PLACE website (https:// www.dna.affrc.go.jp/PLACE/. Figure 2 results show that: the cloned upstream sequence of the coding region contains a plurality of cis-acting elements related to seed-specific promoters such as E-box, A/T rich element, P-box and the like, and the cloned sequence is preliminarily considered to have the function of the seed-specific promoter and is named as PMt1g 072600.

EXAMPLE 2 construction of GUS expression vector driven by sequence upstream of coding region

Using the correctly sequenced cloning vector as a template, 1g072600(IF)/1g072600(IF) was used for PCR amplification, and the amplification system and procedure were the same as in example 1,after electrophoretic separation, cutting gel and recovering the upstream sequence of the target gene coding region. The entry vector pENTRY (PCaMV35S:: GUS:: T35S) cuts out the PCaMV35S promoter fragment by AsiS I/Not I double enzyme digestion, and after electrophoretic separation, cuts the gel to recover the vector skeleton. Reference MonClone^TMThe Hi-Fusion Cloning Mix V2 kit method, the upstream sequence of the coding region and the vector backbone were ligated. The recombinant entry vector with the correct sequencing was named pENTRY (PMt1g072600:: GUS:: T35S). Reference toLR Clonase^TMII Enzyme Mix kit Specification an LR recombination reaction was performed between the entry vector pENTRY (PCaMV35S:: GUS:: T35S) or pENTRY (PMt1g072600:: GUS:: T35S) and the objective vector pEarleyGate303 to obtain expression vectors pEXPR (PCaMV35S:: GUS:: T35S) and pEXPR (PMt1g072600:: GUS:: T35S) (FIG. 3).

EXAMPLE 3 obtaining of Agrobacterium engineering Strain and genetic transformation of Arabidopsis

1) pEXPR (PCaMV35S:: GUS:: T35S), pEXPR (PMt1g072600:: GUS:: T35S) Agrobacterium GV3101(pMP90) competent cells were transformed by freeze-thaw method to obtain Agrobacterium tumefaciens engineering strains GV3101[ pEXPR (PCaMV35S:: GUS:: T35S) ] and GV3101[ pEXPR (PMt1g072600:: GUS:: T35S) ].

2) Genetic transformation of Arabidopsis thaliana

Wild type Arabidopsis thaliana Col0 was transformed by dipping flower using Agrobacterium tumefaciens engineering strains GV3101[ pEXPR (PCaMV35S:: GUS:: T35S) ] and GV3101[ pEXPR (PMt1g072600:: GUS:: T35S) ] respectively, as follows:

(1) taking the day of transformation as T day; at night of the day of T-2, selecting a monoclonal of the agrobacterium engineering bacteria, and inoculating the monoclonal to 10mL of YEB, Rif50 mg/L, Kan 50mg/L and Gent 50 mg/L; culturing at 28 deg.C and 220rpm for 24 hr; in the night of "T-1" day, the culture was expanded at a ratio of 1: 100.

(2) Preparing a dip dyeing solution (1L): MSB₅2.2g of dry powder (product number M404 from Phytotech), 50g of sucrose, 0.044. mu. mol/L of 6-BA, 0.77200. mu.L of Silwet L, and pH 5.7.

(3) At noon of T, await Agrobacterium OD₆₀₀When the strain is equal to 1, stopping shaking the strain; centrifuge at 4,000rpm for 15min, enriching the bacteria.

(4) Resuspending the bacterial pellet with a staining solution, and adjusting the OD of the bacterial solution₆₀₀＝0.8-1.0。

(5) Wild type arabidopsis thaliana was pod cut and inflorescences were immersed in the dip for exactly 30 s.

(6) The stained Arabidopsis thaliana was placed on its side on the bottom of a large plastic tray. Normally culturing after being protected from light for about 14 h.

3) Harvesting, screening and PCR detection of Arabidopsis seeds

(1) After the fruit pods are mature, dry and yellow, the latex gloves are worn by both hands, the fruit branches are cut off, and the seeds fall on clean newspaper through proper rubbing. Removing fruit pods by adopting a proper mode, pouring clean seeds into a centrifuge tube filled with a proper amount of dry silica gel, and preserving at 4 ℃. In the operation process, the gloves should be changed frequently, so that the cross contamination of the seeds is avoided.

(2)T₁The seeds can be directly sowed in nutrient soil, and after four true leaves are grown, the seeds are sprayed on the surfaces of the leaves at intervals of 3-4 days by 20 percent (W/V) Basta diluted by 5,000 times. T is₂/T₃The seeds can be sowed on a glufosinate-ammonium plate containing 8-10mg/L for screening.

Collecting warpT positive for resistance selection₁The Arabidopsis seedlings were bred, and genomic DNA was extracted and PCR detection of GUS sequence was carried out using primers GUS (F)/GUS (R).

And (3) PCR reaction system: 5 XPrimeSTAR GXL Buffer 10. mu.L, dNTP mix 4. mu.L, GUS (F) 1. mu.L, GUS (R) 1. mu.L, total DNA template 1. mu.L, PrimeSTAR GXL DNA Polymerase 1. mu.L, sterilized ultrapure water 32. mu.L.

PCR reaction procedure: 5min at 95 ℃; 15s at 98 ℃,20 s at 60 ℃ and 2min at 68 ℃ for 30 cycles; 5min at 68 ℃.

The results show that 13 and 15 transgenic positive materials are obtained by transforming the agrobacterium, and the PCR detection results of partial plants are shown in figure 4.

Example 4 detection of GUS Activity

Activity analysis of upstream sequence of Mt1g072600 coding region for driving GUS expression in transgenic arabidopsis mature seed

(1) Reagent preparation

A. 0.2M sodium phosphate buffer (pH7.0)

0.2MNa₂HPO₄305ml of 0.2M NaH was taken₂PO₄195ml was taken.

0.2M NaH₂PO₄Solution: 11.998g NaH₂PO₄(CAS:7558-80-7, MW 119.98) was dissolved in 500ml water.

0.2M Na₂HPO₄Solution: 14.196g Na₂HPO₄(CAS:7558-79-4, MW 141.96) was dissolved in 500ml water.

B. 10% SDS solution

90ml of water was heated slightly, 10g of SDS was added, the mixture was dissolved with stirring, NaOH was added to adjust the pH to 7.2, and then water was added to make a volume of 100 ml.

C、0.5M EDTA(pH8.0)

18.61g of Na was added to 80ml of water₂EDTA·2H₂O (disodium ethylenediamine tetraacetic acid, CAS:6381-92-6, MW:372.24), adjusting pH to 8.0 with NaOH (about 2g solid NaOH is needed), dissolving and fixing to 100 ml.

D. Gus extraction buffer

50mM sodium phosphate buffer (pH7.0), 10mM EDTA, 0.1% Triton X-100, 0.1% SDS, 10mM beta-mercaptoethanol, stored at 4 ℃.

The preparation method comprises the following steps: 250ml of 0.2M sodium phosphate buffer (pH7.0) is taken; taking 10ml of 10% SDS; taking 20ml of 0.5M EDTA (pH8.0); 1ml of TritonX-100 is taken; 1ml of beta-mercaptoethanol; the volume is adjusted to 1L by water.

E. Reaction buffer (1mM MUG)

0.1219g of MUG (4-Methylumbelliferyl-. beta. -D-Glucuronide, C) were weighed out₁₆H₁₆O₉·3H₂O, CAS:6160-80-1, MW:406.4), dissolved in 300ml of Gus extraction buffer and stored for 2 weeks at 4 ℃.

F. Reaction terminating solution (0.2 mol/LNa)₂CO₃)

Weighing 21.198gNa₂CO₃(CAS:497-19-8, MW: 105.99) by diluting with water to a constant volume1L。

G. Coomassie brilliant blue G₂₅₀Solutions of

Coomassie brilliant blue G₂₅₀0.01g, 5ml of 95% ethanol, H₃PO₄10ml, constant volume to 100ml, filtering and storing at 4 ℃.

H. 0.25mg/ml BSA stock solution

0.0125g BSA, made up to 50ml with Gus extraction buffer.

I、1mM 4-MU

0.008808g of 4-MU (4-methylumbelliferone, CAS:90-33-5, MW 176.17) was weighed out and made up to 50ml with the reaction-terminating solution. Can be stored for 1 month at 4 ℃ in the dark, and can be crystallized during the storage process.

(2) Experimental protocol

Extraction of Gus

Taking 2mg of arabidopsis seeds, quickly freezing the arabidopsis seeds by liquid nitrogen, and smashing the arabidopsis seeds; adding 500 μ l Gus extraction buffer solution, and extracting under shaking for 5 min; centrifuge at 13,000rpm at 4 ℃ for 10-15min, transfer the supernatant to another clean Ep tube, and place on ice until ready for use.

② protein quantification (Bradford method) purchasing plastic micro cuvette

A. Making a standard curve

Quantitative requirement (kang Shiji BCA protein quantitative kit)

When the BCA method is used for measuring the protein concentration, the light absorption value continuously increases along with the increase of time. Therefore all sample assays need to be completed within 3-5 minutes, otherwise the accuracy of protein quantification is affected. 2-3 parallel reactions are recommended for each sample assayed.

6 Ep tubes were taken and the solutions were prepared according to the following table.

Adding Coomassie brilliant blue G250 solution, mixing, and standing at room temperature for 5 min. The absorbance at 595nm was measured with an ultraviolet spectrophotometer. A standard curve was plotted as absorbance A595 against protein concentration (. mu.g/ml).

B. Taking 5 mul of protein sample to be detected, adding 245 mul of Gus extraction buffer solution, adding 1.25ml of Coomassie brilliant blue G250 solution, fully mixing, and standing for 5min at room temperature. The absorbance at 595nm was measured with an ultraviolet spectrophotometer. Substituting the formula to obtain the concentration of the protein sample.

Note: the volume of the reagent related to the method comprehensively considers the extraction efficiency of the sample protein and the sample loading amount of the cuvette, and refers to the rose sage hydronium hydrochloride laboratory doctor thesis. BSA and samples were all treated with Gus extraction buffer to pre-assay results. The concentration of BSA mother liquor is adjusted in consideration of accurate liquor taking. A micro cuvette is required.

③ enzyme reaction

Pre-heating Gus reaction buffer solution at 37 ℃; adding 1.96ml of reaction termination solution into each EP tube, numbering according to reaction time, and standing by; mu.l of the protein supernatant was taken, and 995. mu.l of the reaction buffer preheated at 37 ℃ was added thereto, and the mixture was incubated at 37 ℃. 40 μ l of the mixed reaction mixture was added to 1.96ml of the reaction termination solution at 0min, 20min, 40min and 60min, respectively, and stored at room temperature in the dark.

Fluorescence measurement

A. Making a standard curve

Preparing a solution according to the table above, measuring the fluorescence of each sample by using a fluorescence spectrophotometer under the excitation wavelength of 365nm, the emission wavelength of 455nm and the slit of 10nm, drawing a standard curve by using the reaction termination solution as a blank solution and plotting the fluorescence value to the concentration of 4-MU.

B. And measuring the fluorescence intensity values of the samples at different time points by using a fluorescence spectrophotometer under the conditions of an excitation wavelength of 365nm, an emission wavelength of 455nm and a slit of 10nm and taking a 0min tube as a blank.

(3) Calculation of enzyme Activity

A. The fluorescence intensity values of the samples were measured for different reaction times. The quantitative concentration of the 4-MU substance was calculated from the 4-MU standard curve. The amount concentration of 4-MU was plotted against the reaction time, and the slope was the amount of 4-MU produced (amount concentration of the substance) per unit time.

B. The amount of 4-MU produced per unit time (the amount concentration of the substance) multiplied by the amount of the substance at the time of measurement, which is 4-MU in volume.

C. This value is divided by the mass of crude protein to obtain the specific activity of the final enzyme.

D. Such as nmol 4-MU. mg protein^-1·min^-1

T was determined using the constitutive promoter PCaMV35S as a control₂GUS enzyme activity of transgenic Arabidopsis seeds. The result (figure 5) shows that the upstream sequence of the cloned Mt1g072600 coding region has the function of a promoter and can drive GUS to be expressed in the seeds of transgenic receptor materials by taking the amount of substances which catalyze and generate 4-MU (4-methylumbelliferone) in unit time of total soluble protein of each mg of seed sources of the transgenic strains as an index. And (2) taking the type of the promoter as a processing factor, taking the enzyme activity of the transgenic arabidopsis seeds of GUS driven by PCaMV35S (13 independent transformation events) and PMt1g072600(15 independent transformation events) in different lines as a dependent variable, and performing one-factor analysis of variance on the enzyme activity data: p value (P-value) 9.27 × 10^-10<0.01 and F (test statistic) 81.05>F crit (test cut-off value) is 7.64, which shows that the transcriptional activity of PMt1g072600 driving GUS in Arabidopsis seeds is remarkably higher than PCaMV35S commonly used in plant genetic engineering.

Secondly, driving GUS to be stained in the germination process of transgenic arabidopsis seeds by PMt1g072600 to observe histochemical staining and decoloring of GUS activity

Plant material to be stained was immersed in GUS stain in an appropriate container, vacuumed for 1h, and stained at 37 ℃ for 30min to overnight. After the plant material was sufficiently colored, a decolorizing solution [ acetic acid: the decolorization was performed with 3:1(V/V) ethanol pair. The decolorization liquid needs to be changed frequently in the decolorization process, and heating is avoided.

GUS dye solution formula

(1) Mother liquor

A. 0.2M sodium phosphate buffer (pH7.0)

0.2MNa₂HPO₄305ml of 0.2M NaH was taken₂PO₄195ml was taken.

0.2M NaH₂PO₄Solution: 11.998g NaH₂PO₄(CAS:7558-80-7, MW 119.98) was dissolved in 500ml water.

0.2M Na₂HPO₄Solution: 14.196g Na₂HPO₄(CAS:7558-79-4, MW 141.96) was dissolved in 500ml water.

B、0.5M EDTA(pH8.0)

C. 0.1M Potassium ferricyanide (MW 329.24)

1.647g of potassium ferricyanide is taken and the volume is adjusted to 50 ml.

D. 0.1M Potassium ferrocyanide (MW 422.39)

2.112g of potassium ferrocyanide is taken and the volume is determined to 50 ml.

E、X-Gluc 10mg/ml

DMSO (approximately 4ml DMSO for 10ml solution)

F、Triton X-10020％

(2) Working fluid

The Mt1g072600 genome is annotated as a seed storage protein gene, the promoter sequence is presumed to be a seed-specific promoter, in order to observe the expression mode of the promoter driving GUS in the seed germination process, wild type Arabidopsis seeds are used as a negative control, PCaMV35S driving GUS transgenic seeds are used as a positive control, the seeds are sown on MS plates after being sterilized, and the seeds are transplanted into soil at a proper time period for conventional management. The day of exposure of the seeds to the white is recorded as 1 day of age, the sampling and GUS histochemical staining work of the seeds in different periods of germination and each organ of the adult plants is carried out, and the result is shown in figure 6.

The results in FIG. 6 show that: pEXPR (PMt1g072600:: GUS:: T35S) transgenic Arabidopsis 3 days old seedlings had GUS expression in their stems and cotyledons, but no expression in their roots; a weaker blue color was observed in the cotyledons only for 9-day-old seedlings; in each organ of the adult plant, except the end of the fruit flap, GUS expression is expressed, and the other organs are GUS staining negative. The Mt1g072600 promoter PMt1g072600 is considered to be a seed-specific promoter, the organ (cotyledon and stem) with positive GUS staining in the seedling is a product for driving GUS transcription and expression by the PMt1g072600 in the seed development stage, and because the PMt1g072600 cannot drive GUS transcription in other organs except the seed, the GUS in the cotyledon and stem part is gradually degraded or diluted along with the growth of the seedling, and cannot be detected in each organ of the adult plant.

Based on the sequencing and annotation result of the medicago truncatula genome, the upstream 2075bp sequence of the Mt1g072600 coding region annotated as seed storage protein is cloned, a GUS-driving plant expression vector is constructed, and the genetic transformation of arabidopsis is carried out. The germination process of transgenic arabidopsis seeds and GUS histochemical staining results of various tissues and organs of adult plants show that the upstream sequence of the cloned coding region is a seed specific promoter. The GUS enzyme activity detection result of the transgenic arabidopsis seeds shows that the expression quantity of GUS driven by the cloned promoter is remarkably higher than that of a CaMV35S promoter, and the cloned promoter can be used in the field of plant genetic engineering and can drive high-level expression of exogenous genes in transgenic plant seeds.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> institute of science and technology of Henan

<120> Medicago truncatula legeliin gene Mt1g072600 promoter PMt1g072600 and application thereof

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 72

<212> DNA

<213> Artificial Sequence

<400> 1

aattcgccct tgcgatcgca tgcgactgcg gccgctcagt gcacccgggc atgtcatggc 60

gcgccaaggg cg 72

<210> 2

<211> 72

<212> DNA

<213> Artificial Sequence

<400> 2

aattcgccct tggcgcgcca tgacatgccc gggtgcactg agcggccgca gtcgcatgcg 60

atcgcaaggg cg 72

<210> 3

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 3

cccgggcggc catgctagag tccgca 26

<210> 4

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 4

ggcgcgccat gtcactggat tttggttt 28

<210> 5

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 5

gcgatcgcgt ccgatgtgag acttttc 27

<210> 6

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 6

gcggccgccc tctccaaatg aaatgaac 28

<210> 7

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 7

gcggccgcat gttacgtcct gtagaaac 28

<210> 8

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 8

cccgggtcat tgtttgcctc cctgct 26

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 9

ctgattgctc aaaagacttc ctg 23

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 10

tgtgtttgtg agtttggtga at 22

<210> 11

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 11

cgaattcgcc cttgcgatcg cctgattgct caaaagactt cctg 44

<210> 12

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 12

ctacaggacg taacatgcgg ccgctgtgtt tgtgagtttg gtgaat 46

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 13

cgccgatgca gatattcgta a 21

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 14

aaagtcccgc tagtgccttg t 21

<210> 15

<211> 2075

<212> DNA

<213> Artificial Sequence

<400> 15

ctgattgctc aaaagacttc ctgaacatag ttgatatgaa agtttataga agttgtgtaa 60

cttggacttt cgaataaccc tagacgttga tttttgaaaa tcttcgaaat agcagtcttc 120

gaacttctgg cttgttctcg atctttgaaa aaaatgaacc tagttcgctc gaaactttgt 180

tattaaaaat ttgcatataa cagactgaga ttttcaaacg agaggctacc cctgcaatga 240

gattgcctca atagataagg taagttgcac caaattaaaa ggactggatc ttagacaatt 300

gatgtacatt agctcctatt agtttgtgtc tctttctcta taataactga taccttagtt 360

cgacatacaa atctatattt aaataactaa ttaatcaatg gttaacaaaa ataagttcac 420

agtaaaaatt gaaatcaacc attgatataa gttggtaaac ttgattcaag tttatgtatt 480

tatcggttga tattaagtga ttaaacgtaa ttggtaattt attttgcact taagtttagt 540

taaaacataa aagtaggatg gtacagatac catctaaact gaaaatcata tgttagactt 600

gatatctgct tgaatcataa tttttttgtt ttcaatgttg tatgctatca gcatcacagg 660

ccattgcgac caccacttga actctgatga taatactcta ttaagagtga actttgtaag 720

ggaggtggat gaattgttgc aagaggtttg ttcggaacat gacgaagaag agaatgaaca 780

tgagtttagt cctgatacag ttgcaaatag attaaaacaa caggagaagc aacaacaata 840

cacgtagaag gttgtttttt tatttacata tatttgttca gttgtcttat agttttgaat 900

agaaattagt atgcagtgag tgcagaaagg acacaaaaat gtagacatgg aaatttttgg 960

attatcatgt gtttttaaag gtaaaggtat gttagaaatt gagtttttga attttatttt 1020

atacatctat gtacatctgg tgagtccacc cagtattttc tagcatgttt ggttttgtgg 1080

tggtgaaaat gaattttagt taaaacttta aagtcatgag ttgaatgtac attggtctat 1140

gtttatatac atttacataa atgtgttcaa caataatttg aagttaaaaa tcttataaag 1200

aggcaaaaca tctaattaca tatttgagct agaatttatt ttatagaacg cttaaatata 1260

tcaagcaatt ttaatttatg tgtctagaat atttttggat cttacaaaag ggaatccaaa 1320

tatgcaccaa atgcactgca ttgatttaaa catcaatgac aacaatcaac ctgaattctc 1380

tagagcttac ttgaattcaa acaactggaa ggtaaatact gaactttcag agcataaatt 1440

ttaagagatt aagttatata tgcactgatg gtgtaaaaaa aattttttac aaacgcatgc 1500

aatcaaacta tatcatactt ttataagtta gattttaaaa tatttctatt ttgatgcatg 1560

agtaaaaatg ttttacgctt aagttatgct gtttctgttg cgaaggctac agattgataa 1620

taacaaagag aggtactatt aagctagaat ttctacttta atcactacat acatctatat 1680

atttgagtct gagcaacact gttactgctt aatgaaaact gtaaatgaat taagtaaagt 1740

gaaaaagaag taatgatatc aacatgtaaa gaatcgacgc agaatgcatg ggaccatccc 1800

atgccatata catttgttct cattcattta actgcagtca tgattccata tgtaaagata 1860

ccaacaaata tacactctgt gatgtctctg tgcacatata tcttaatgtg atgtgtagga 1920

aattaagaat tcataggcat gcatggtgaa gaatgtcatg aactagcaac ctacactgtg 1980

tgacgtgtcc cttcctcact cttctcttct tactataaat caccactcca caggttcttc 2040

tcttcaccaa ttcattcacc aaactcacaa acaca 2075