CN106434659A - Soybean low-temperature inducing promoter and recombinant expression vector containing same and application of soybean low-temperature inducing promoter - Google Patents

Soybean low-temperature inducing promoter and recombinant expression vector containing same and application of soybean low-temperature inducing promoter Download PDF

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CN106434659A
CN106434659A CN201610847705.6A CN201610847705A CN106434659A CN 106434659 A CN106434659 A CN 106434659A CN 201610847705 A CN201610847705 A CN 201610847705A CN 106434659 A CN106434659 A CN 106434659A
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翟莹
邵淑丽
张军
赵艳
任巍巍
张闯
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Qiqihar University
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Abstract

The invention discloses a soybean low-temperature inducing promoter, a recombinant expression vector containing the same, an application of the soybean low-temperature inducing promoter and particularly relates to the field of plant genetic engineering, in particular to expression of a soybean gene promoter sequence can serve as the promoter to control gene under low-temperature stress with an aim to effectively solve the problem that the soybean low-temperature inducing promoter is not developed yet. The promoter is derived from a promoter sequence of soybean ethylene response factor gene GmERF9, the promoter sequence is named as GmERF0P with a base sequence indicated as SEQ ID No: 1; the sequence contains multiple stress-related cis-acting elements; the cis-elements are respectively: GT-1, BIHD10S, WRKY, MYB, MYC and G-box. The soybean low-temperature inducing promoter is used for inducing cold-resistance gene expression under low temperature.

Description

Soybean cold-inducible promoter, comprise this promoter recombinant expression carrier and Application
Technical field
The present invention relates to plant genetic engineering field is and in particular to soybean gene promoter sequence is permissible under low temperature stress Expression as promoter regulation gene.
Background technology
Soybean is important industrial crops, worldwide generally plants, and is not only the main of human protein and lipid Source, is also important feed stripped and the raw material of industry simultaneously, medically also has very big value.But low temperature, arid, high salt Deng adverse environment condition, the growth and development of soybean being produced with impact, thus leading to soybean yields to reduce, causing serious warp Ji loss.Except traditional breeding method, recently as the development of molecular biology and becoming of plant genetic engineering breeding technique Ripe, application gene engineering method improvement soybean resistance is possibly realized.
Promoter is to be located at structural gene upstream on DNA molecular, in conjunction with RNA polymerase thus startup structure genetic transcription Section of DNA sequence.Gene expression efficiency depends primarily on the activity of promoter, and high-caliber expression is endogenous or foreign gene needs There is efficient promoter.In genetic engineering, commonly use some constitutive promoters such as CaMV35S at present to control genes of interest Expression.Although these promoters can make genes of interest overexpression, they are inorganization, no environment, no temporal Constitutive expression.Although plant can be improved under adverse environmental factor using the expression that constitutive promoter controls anti contravariance related gene Anti-adversity ability, even in related degeneration-resistant albumen under plant positive reason growing state also can overexpression, this is to plant metabolism For be a kind of waste.And the long-time excess accumulation of degeneration-resistant albumen also can be normal to plant in plant body under home Growth has a negative impact, and causes genetically modified plants aberrant morphogenesis, and delayed growth is even dead.And adverse circumstance induction starts Son just starts downstream gene only when plant is by environment stress in a large number is expressed, and can avoid foreign gene in genetically modified plants Plant growth defect caused by middle continuous expression.Therefore clone and apply adverse circumstance inducible promoter, it is possible to achieve efficiently, can The expression of the regulation and control adversity gene of control and special (pinpointing, regularly, quantitatively), has greatly in terms of the resistance of improvement plant Superior, become the focus of current research.
There are some reports about low temperature induction promoter at present both at home and abroad.For example arabidopsis rd29A promoter is permissible Induced by abiotic stress such as low temperature, arid and high salt, be that in current adversity gene engineering, most widely used induction type starts Son.The blt101.1 promoter by low temperature induction is obtained from winter wheat.Clone and have low temperature induction expression from wild rice The cold induced promoter p-LTT1 of characteristic.Obtain low temperature strong evoked promoter POscold6 from rice varieties Japan is fine, this opens Mover can be special driving foreign gene under low warm/cold stress conditions in plant express.But generally speaking, at present can be In genetic engineering, the cold-inducible promoter of application is still few, therefore, to effectively new cold-inducible promoter Clone and research remain the emphasis studied from now on.
Content of the invention
The invention aims to solving the problems, such as that effective soybean cold-inducible promoter is not developed, and The recombinant expression carrier provide soybean cold-inducible promoter, comprising this promoter and application..
The promoter of the present invention derives from the promoter sequence of soybean ethylene response factor gene GmERF9, is named as GmERF9P, its base sequence such as SEQ ID NO：Shown in 1.
Multiple cis-acting elements related to stress are contained in described sequence.
Comprise the recombinant expression carrier of soybean cold-inducible promoter, the initial carrier of its recombinant expression carrier is pCAMBIA1301.
The soybean cold-inducible promoter of the present invention induces the application in Cold resistant genes expression at low temperature.
The present invention utilize Real-Time Fluorescent Quantitative PCR Technique detect soybean leaves in GmERF9 gene 4 DEG C process 2h when Expression, now the expression of GmERF9 significantly raise it was demonstrated that GmERF9 gene has low temperature induction expression characterization.Therefore, exist Search for GmERF9 upstream region of gene general 2000bp sequence in soybean gene group database, design primer, from soybean leaves genome In clone the GmERF9 promoter sequence of 1885bp, be named as GmERF9P.GmERF9P is building up to plant expression vector On pCAMBIA1301, by leaf disk method transformation of tobacco.By hygromycin selection and PCR identification, obtain T1For positive transgenic cigarette Grass.K cryogenic treatment 2h is carried out to transgene tobacco.Untreated tobacco and K cryogenic treatment tobacco are carried out GUS histochemical stain and Real-time fluorescence quantitative PCR detects the expression of gus gene, shows that GmERF9P has low temperature induction under low temperature stress and starts spy Property, it is cold-inducible promoter.
The present invention is with respect to the advantage of prior art：
Although plant can be improved under adverse environmental factor using the expression that constitutive promoter controls anti contravariance related gene Anti-adversity ability, even in related degeneration-resistant albumen under plant positive reason growing state also can overexpression, this to plant metabolism come Say it is a kind of waste.And the long-time excess accumulation of degeneration-resistant albumen also can be to the normal life of plant in plant body under home Length has a negative impact, and causes genetically modified plants aberrant morphogenesis, delayed growth is even dead.And adverse circumstance evoked promoter Just start downstream gene in a large number to be expressed only when plant is by environment stress, foreign gene can be avoided in genetically modified plants Plant growth defect caused by continuous expression.The present invention has cloned a soybean cold-inducible promoter, this promoter Obtain to study for genetic engineering of cold-resistance in plants and tool is provided, the efficient table of cold-resistant related gene can be started under cryogenic Reach, thus cultivating the cold resistant plant kind of practicability and effectiveness.
Brief description
The real-time fluorescence quantitative PCR testing result of Fig. 1 GmERF9 gene expression in K cryogenic treatment 0h and 2h；
The PCR amplification of Fig. 2 GmERF9 gene promoter sequence, wherein M is DL2000Marker, and 1 is amplification bar Band；
Fig. 3 GmERF9P promoter sequence cis-acting elements forecast analysis；
The plasmid double digestion result of Fig. 4 recombinant vector pCAMBIA1301-GmERF9P, wherein M is DL2000Marker, 1- 3 is double digestion band；
Fig. 5 GmERF9P T1For positive transgenic tobacco PCR qualification result, wherein M is DL2000Marker, and 1-6 is T1 For positive transgenic tobacco amplified band, 7 is wild-type tobacco negative control, and 8 is pCAMBIA1301-GmERF9P plasmid positive Comparison；
Fig. 6 untreated wild-type tobacco blade GUS histochemical stain result；
Fig. 7 K cryogenic treatment 2h wild-type tobacco blade GUS histochemical stain result；
The untreated T of Fig. 81For transgenic tobacco leaf GUS histochemical stain result；
Fig. 9 K cryogenic treatment 2h T1For transgenic tobacco leaf GUS histochemical stain result；
The real-time fluorescence quantitative PCR detection knot of Figure 10 gus gene expression during K cryogenic treatment 2h in transgene tobacco Really, wherein 1 is untreated T1For gus gene expression in transgene tobacco, 2 is T during K cryogenic treatment 2h1For in transgene tobacco Gus gene expression, 3 is gus gene expression in positive control pCAMBIA1301 transgene tobacco.
Specific embodiment
Technical solution of the present invention is not limited to act specific embodiment set forth below, also includes between each specific embodiment Arbitrarily reasonable combination.
Specific embodiment one：SEQ in the sequence of the soybean cold-inducible promoter of present embodiment such as sequence table IDNO：Shown in 1.
Specific embodiment two：Present embodiment from unlike specific embodiment one, in described sequence contain with The related cis-acting elements of stress.Other steps are identical with specific embodiment one with parameter.
Specific embodiment three：From unlike specific embodiment two, described cis-acting elements divides present embodiment Wei not GT-1, BIHD10S, WRKY, MYB, MYC and G-box.Other steps are identical with specific embodiment one with parameter.
Specific embodiment four：The recombinant expression carrier comprising soybean cold-inducible promoter of present embodiment.
Specific embodiment five：Present embodiment from unlike specific embodiment four, described recombinant expression carrier Initial carrier is pCAMBIA1301.Other steps are identical with specific embodiment one with parameter.
Specific embodiment six：Present embodiment soybean cold-inducible promoter induces Cold resistant genes to express at low temperature In application.
Embodiment 1：The present embodiment will be carried out to the sequence of the soybean cold-inducible promoter of the present invention from following experiment Checking：
(1) under K cryogenic treatment, the real-time fluorescence quantitative PCR of GmERF9 gene expression amount detects
Potted plant four leaf stage soybean seedling under normal condition is placed in 4 DEG C of incubators and carries out K cryogenic treatment, processing 0h During with 2h, clip 0.1g blade is immediately placed in liquid nitrogen respectively, and -80 DEG C save backup.(it is purchased from using Plant RNAzol reagent Beijing Ding Guo biotech company) extract soybean K cryogenic treatment and untreated when blade total serum IgE, according to cDNA first chain synthesize examination The specification of agent box (purchased from Beijing Ding Guo biotech company) synthesizes the first chain cDNA.
According to GmERF9 gene (GenBank accession number：AK245092) cDNA sequence design real-time fluorescence quantitative PCR primer (F:5′-CATACCAACCTTCAAATGCCTC-3′；R:5′-TTTCTATTAGGGTCACGGATTTC-3′).In BIO-RAD In CFX96Real-Time PCR instrument, with soybean constitutive expression gene β-Tubuin (GenBank accession number：GMU12286) make For reference gene (F:5′-GGAAGGCTTTCTTGCATTGGTA-3′；R:5 '-AGTGGCATCCTGGTACTGC-3 '), with soybean Leaf cDNA is template.Reaction system is 2 × SYBR Premix Ex Taq (purchased from TaKaRa company) 10 μ L, ROX Reference Dye II 0.2 μ L, cDNA 2 μ L, Primer F 0.4 μ L, Primer R 0.4 μ L, moisturizing to cumulative volume 20 μ L.Response procedures：95 DEG C of denaturations 10s；95 DEG C of denaturation 20s, 58 DEG C of annealing 20s, 72 DEG C of extension 30s, 40 circulations.Each process All do the relative expression quantity computing repeatedly gene 3 times.Result of the test carries out data using BIO-RAD CFX Manager software and divides Analysis.As shown in table 1 and Fig. 1, result shows that GmERF9 gene expression in K cryogenic treatment 2h significantly raises.
The relative expression quantity (numerical value is three repetition mean values) of GmERF9 gene under table 1 K cryogenic treatment
(2) clone of GmERF9 gene promoter sequence and cis-acting elements analysis
Soybean gene group database GmGDB (http is searched for according to soybean GmERF9 gene cDNA sequence：// Www.plantgdb.org/GmGDB/), obtain the promoter sequence of the general 2000bp in its upstream.Using primer-design software Primer5 designs primer, and sequence is as follows, F：5'-ACGCGTCGACCCGTGCAACTTGATATTCGT-3'(underscore represents Sal I restriction enzyme site)；R：5'-GGCCATGGTTTTTGGTTGTGAAATTGAGG-3'(underscore represents Nco I restriction enzyme site).Using Plant genome DNA extracts kit (purchased from Beijing Ding Guo biotech company) extracts soybean leaves genomic DNA, with gene Group DNA is template, enters performing PCR amplification.PCR program：94℃8min；94 DEG C of 40s, 56 DEG C of 40s, 72 DEG C of 1min, carry out 30 and follow Ring；72 DEG C of extension 8min.The amplified band obtaining such as Fig. 2.With cloning vector pMD18-T (purchase after above-mentioned amplified fragments are reclaimed From TaKaRa company) it is attached, screening is obtained positive plasmid and is named as pMD18-T-GmERF9P, and it is public to serve the raw work in sea Department is sequenced, and checking sequence is correct.PLACE(http：//www.dna.affrc.go.jp/PLACE/) database is used for opening The forecast analysis of mover cis-acting elements.
Obtain GmERF9 gene promoter sequence GmERF9P through PCR amplification, length 1885bp is (as SEQ ID in sequence table NO：Shown in 1).Predicted GmERF9P sequence contains multiple cis-acting elements related to adverse circumstance, as seen from Figure 3 with inverse The related cis-acting elements in border includes 3 GT1 elements, 1 G-box element, 2 BIHD10S binding sites, 5 WRKY knots Close site, 3 MYB binding sites and 1 MYC binding site.GmERF9P sequence is carried out in ncbi database homology Blast, does not obtain thering is the promoter sequence of homology with it, shows that GmERF9P is a new promoter sequence.
(3) Transformation of tobacco of GmERF9P and transgene tobacco identification
Plant expression vector construction method：
With Sal I and Nco I two kinds of restriction enzymes (various restriction enzymes are purchased from TaKaRa company) double digestion PMD18-T-GmERF9 plasmid, digestion products are purified QIAquick Gel Extraction Kit (purchased from Beijing ancient cooking vessel state biology skill through Ago-Gel DNA Art company) after purification, (purpose is that excision CaMV35S starts with the same pCAMBIA1301 carrier with Sal I and Nco I double digestion Son) connect, obtain pCAMBIA1301-GmERF9P.Connection product conversion bacillus coli DH 5 alpha is (purchased from Beijing ancient cooking vessel state biotechnology Company), plasmid, after double digestion checking (result is as shown in Figure 4), converts Agrobacterium EHA105 (purchased from Biovector company).
Infect tobacco leaf disc method using Agrobacterium by pCAMBIA1301-GmERF9P transformation of tobacco NC89 (purchased from middle cigarette kind Sub- Co., Ltd), conversion pCAMBIA1301 empty carrier is as positive control simultaneously.Concrete grammar is as follows：
1 tobacco seed is placed in 1.5mL centrifuge tube, with 10% sodium hypochlorite soaking disinfection 3min, aseptic water washing 4-5 time；
2 tobacco seed is laid on MS culture medium, condition of culture：(22 DEG C) of 16h (28 DEG C) illumination/8h is dark；
3 take the growth aseptic tobacco leaf of 1-2 month after sprouting, are cut into 0.5cm2Fritter (removing master pulse), and It is inoculated on MS differential medium (MS adds 3mg/L 6-BA and 0.2mg/L NAA), preculture 2 days；
4 picking pCAMBIA1301-GmERF9P monoclonals add 50 μ g/mL rifampins and 50mg/L kanamycins in 5mL YEP (peptone 10g/L, dusty yeast 10g/L, NaCl 5g/L) in, 28 DEG C, 120rpm shaken cultivation 24h；
5 press 1：Above-mentioned culture is proceeded to 50mL and adds 50 μ g/mL rifampins and 50 μ g/mL kanamycins by 100 ratio YEP in, 28 DEG C, 120rpm shaken cultivation to OD600=0.4-0.5；
6 5000rpm, are centrifuged 15min under room temperature, remove supernatant, thalline is resuspended in MS Liquid Culture and concentrates, to OD600 =0.5 about；
Tobacco leaf after preculture is infected 20min in re-suspension liquid by 7, blots the bacterium solution of blade surface, is placed in common training On foster base (PH to be adjusted to 5.4 about), co-culture 3 days；
Tobacco leaf after co-culturing is cleaned by 8 with the MS fluid nutrient medium containing 500mg/L carbenicillin, turns after drying Move on on screening and culturing medium that (it is mould that MS adds 3mg/L 6-BA, 0.2mg/L NAA, 500mg/L carbenicillin and 8mg/L tide Element), every 15 days subcultures are once；
9 when resistant budses grow to 1cm, move into culture of rootage and concentrate (MS interpolation 200mg/L carbenicillin and 5mg/L tide Mycin), promote its long root；
10 after tobacco seedling root system development is good, moves in soil, Routine Management.
The screening of transgene tobacco and authentication method：
1 takes T0For tobacco leaf, (public purchased from TaKaRa according to Universal Genomic DNA Extraction Kit Department) method in specification extracts blade STb gene；
After the DNA extracting is diluted 50 times by 2,1 μ L is taken to be template, with primer (F：5'-CCGTGCAACTTGATATTCGT- 3'；R：5'-TTTTTGGTTGTGAAATTGAGG-3') carry out Standard PCR checking.Wherein with unconverted Nicotiana gossei as feminine gender Comparison, plasmid pCAMBIA1301-GmERF9P is positive control；
3 seeds harvesting positive tobacco plant, i.e. T1For seed；
4 by T1Bury middle acquisition T for seed kind1For tobacco seedling, it is continued with extraction DNA and enters performing PCR detection, take T1Dai Yang Property tobacco plant carries out follow-up Resistance Identification test.
Fig. 5 is GmERF9P in part T1For the PCR qualification result in transgene tobacco, show GmERF9P promoter Successfully it is incorporated in tobacco gene group.
(4) transgene tobacco K cryogenic treatment lower blade GUS histochemical stain
By 6 weeks of normally potted plant culture big T1It is placed in 4 DEG C of incubators for transgene tobacco and carry out K cryogenic treatment.Clip is not The T processing1T for transgenic tobacco leaf and K cryogenic treatment 2h1For transgenic tobacco leaf, clip is untreated simultaneously and low temperature The wild-type tobacco blade processing 2h, as negative control, carries out GUS histochemical stain：(soybean is stearic to add GUS dyeing liquor The clone of acid-ACP desaturase gene promoter and its expression activity analysis, Zhang Qinglin etc., 2011), it is incubated overnight in 37 DEG C, It is wholly absent to background color with 75% ethanol decolorization.
As Figure 6-9, untreated and K cryogenic treatment 2h wild-type tobacco blade does not have GUS histochemical stain result It is dyed to blueness；Untreated T1It is dyed to for transgenic tobacco leaf light blue, but coloring is shallower, shows that GmERF9P has The startup activity of promoter；The T of K cryogenic treatment 2h1It is dyed to blueness for transgenic tobacco leaf and colours relatively deep, show The startup activity of GmERF9P is induced in K cryogenic treatment 2h, so that the expression of downstream Reporter gene GUS is increased.
(5) under transgene tobacco K cryogenic treatment, the real-time fluorescence quantitative PCR of gus gene expression detects
By 6 weeks of normally potted plant culture big T1It is placed in K cryogenic treatment 2h in 4 DEG C of incubators for transgene tobacco.Clip low temperature Process 2h and untreated T1For transgenic tobacco leaf 0.1g, clip pCAMBIA1301T simultaneously1Make for transgenic tobacco leaf For positive control, it is immediately placed in liquid nitrogen, -80 DEG C save backup.(biological purchased from Beijing ancient cooking vessel state using Plant RNAzol reagent Technology company) extract tobacco leaf total serum IgE, according to cDNA the first chain synthetic agent box (purchased from Beijing Ding Guo biotech company) Specification synthesizes the first chain cDNA.
In BIO-RAD CFX96Real-Time PCR instrument, with tobacco constitutive expression gene α-tubulin (GenBank accession number：AB052822) as reference gene (F：5′-ATGAGAGAGTGCATATCGAT-3′；R：5′- TTCACTGAAGAAGGTGTTGAA-3 '), gus gene real-time fluorescence quantitative PCR primer is as follows, F：5′- GATCGCGAAAACTGTGGAAT-3′；R：5′-TAATGAGTGACCGCATCGAA-3′.With tobacco leaf cDNA as template.Real When quantitative fluorescent PCR reaction system and response procedures ibid, annealing temperature is changed to 55 DEG C.As shown in Figure 10 and Biao 2, real-time fluorescence Quantitative PCR result shows that GmERF9P can significantly improve the expression of downstream Reporter gene GUS in K cryogenic treatment 2h.
The relative expression quantity (numerical value is three repetition mean values) of gus gene under table 2 transgene tobacco K cryogenic treatment

Claims (6)

1. soybean cold-inducible promoter it is characterised in that：SEQ ID in the nucleotide sequence of this promoter such as sequence table NO：Shown in 1.
2. soybean cold-inducible promoter according to claim 1 it is characterised in that：Contain and the side of body in described sequence Compel related cis-acting elements.
3. soybean cold-inducible promoter according to claim 1 it is characterised in that：Described cis-acting elements is GT-1, BIHD10S, WRKY, MYB, MYC and G-box.
4. comprise the recombinant expression carrier of soybean cold-inducible promoter described in claim 1.
5. recombinant expression carrier according to claim 4 it is characterised in that：The initial carrier of described recombinant expression carrier is pCAMBIA1301.
6. soybean cold-inducible promoter as claimed in claim 1 induces the application in Cold resistant genes expression at low temperature.
CN201610847705.6A 2016-09-23 2016-09-23 Soybean low-temperature inducible promoter, recombinant expression vector containing promoter and application Expired - Fee Related CN106434659B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111763672A (en) * 2020-06-30 2020-10-13 安徽省农业科学院水稻研究所 Rice low-temperature inducible expression promoter Poscold10 and application thereof
CN113151305A (en) * 2021-06-01 2021-07-23 安徽农业大学 Tea tree WRKY29 gene and application thereof in improving cold resistance of plants

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CARLOS M HERNANDEZ-GARCIA ET AL: "High level transgenic expression of soybean (Glycine max) GmERF and Gmubi gene promoters isolated by a novel promoter analysis pipeline", 《BMC PLANT BIOLOGY》 *
CARLOS MANUEL HERNANDEZ GARCIA: "ISOLATION AND CHARACTERIZATION OF SOYBEAN PROMOTERS", 《THE OHIO STATE UNIVERSITY DISSERTATION》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111763672A (en) * 2020-06-30 2020-10-13 安徽省农业科学院水稻研究所 Rice low-temperature inducible expression promoter Poscold10 and application thereof
CN111763672B (en) * 2020-06-30 2022-03-25 安徽省农业科学院水稻研究所 Rice low-temperature inducible expression promoter Poscold10 and application thereof
CN113151305A (en) * 2021-06-01 2021-07-23 安徽农业大学 Tea tree WRKY29 gene and application thereof in improving cold resistance of plants

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