CN105734078A - Genetic establishing body with apple root system development related gene MdMIEL1 and application of genetic establishing body - Google Patents
Genetic establishing body with apple root system development related gene MdMIEL1 and application of genetic establishing body Download PDFInfo
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Abstract
The invention discloses a genetic establishing body with an apple root system development related gene MdMIEL1 and application of the genetic establishing body.The invention relates to the genetic establishing body which has a DNA sequence SEQ ID NO:1 for encoding a ubiquitin ligase E3 of gala apples under the control of a strong promoter.The invention further relates to the application of the genetic establishing body in generation of transgenic arabidopsis.The invention further relates to a method for generating arabidopsis strains with more lateral roots.The method is characterized by including the steps of introducing the genetic establishing body to arabidopsis to obtain transgenic arabidopsis seeds, and screening the offspring of the transgenic arabidopsis seeds multiple times to obtain a purified arabidopsis strain.The transgenic arabidopsis strain with more lateral roots is obtained by means of the genetic establishing body and the method.
Description
Technical field
The present invention relates to genetically engineered plant field, more specifically it relates to use the method that gene engineering obtains the more arabidopsis of lateral root number.
Background technology
Root system of plant moisture absorption, Nutrient Absorption, fixing plant, synthesis plant growth substance and with rhizosphere around microbial interaction etc. in play an important role.The root of plant can be divided into taproot and lateral root from happening part.First the generation surveying root starts from the pericyclic cell of main root, and this cell is by separating, being differentiated to form lateral-root primordia thereafter, then proceedes to grow and break through epidermis and forms side root.
In the lateral root development process of arabidopsis, it is subject to the regulation and control of a series of factor.Auxin is transported to pericyclic cell by Auxin transport gene expression product AuxI, and PIN family member exports auxin so that form auxin concentration gradient in pericyclic cell.When auxin concentration height, the mortifier AUX/IAA in its inducing cell is degraded by Ubiquitin-Proteasome-Dependent approach, occurs to side root thus activating and grows relevant gene pathway.
E3 ubiquitin ligase is the molecule participating in Ubiquitin-Proteasome-Dependent approach, and it can recognise that target site and ubiquitin molecule is connected to target site, plays an important role in the Ubiquitin-Proteasome-Dependent degradation process of protein.Therefore, E3 ubiquitin ligase has likely participated in the process of the root generation of arabidopsis side and growth.
Summary of the invention
It is an object of the invention to obtain the arabidopsis transgenic line that side root increases.Inventor is with the forward shown in SEQIDNO:2 and 3 and reverse primer, with loud, high-pitched sound Fructus Mali pumilae for template, expanded the E3 ubiquitin ligase gene MdMIEL1 obtained in loud, high-pitched sound Fructus Mali pumilae by the mode of PCR, the means then passing through Protocols in Molecular Biology and genetic engineering achieve the purpose of the present invention.
The invention provides a kind of genetic constructs, comprise E3 ubiquitin ligase gene (MdMIEL1) fragment of the loud, high-pitched sound Fructus Mali pumilae being under strong promoter controls, described genetic fragment has the sequence shown in SEQIDNO:1.
Further, described strong promoter be can in arabidopsis the strong promoter of constitutive expression.The promoter of such as viral source, such as CaMV35S, CsVMV promoter, BSV promoter, MMV promoter etc.；The promoter of plant origin, for instance actin2 gene promoter, Ubi.U4 gene promoter etc..
Further, described genetic constructs also comprises the resistant gene of one or more generation antiviral antibiotic, for instance hygromycin gene.By comprising such gene, can be conducive to transgenic arabidopsis cell and the described antibacterial for screening conversion with this genetic constructs is screened.
Further, described genetic constructs comprises the pCAMBIA1300 plasmid of described MdMIEL1 genetic fragment, under the control of the CaMV35S promoter that wherein said MdMIEL1 genetic fragment is on described pCAMBIA1300 plasmid.
The invention still further relates to a kind of method for producing the arabidopsis strain that side root increases, including the genetic constructs described in claim 1 is imported in arabidopsis, obtain transgenic arabidopsis seed, then pass through the filial generation to described transgenic arabidopsis seed and carry out multi-turns screen and obtain arabidopsis strain of isozygotying..
Further, said method comprising the steps of:
1) convert Agrobacterium competent cell with genetic constructs of the present invention, and screening obtains monoclonal transformant；
2) incubation step 1) in the monoclonal transformant that obtains, collect thalline, and make and infect liquid；
3) by step 2) in the liquid that infects that obtains infect arabidopsis inflorescence, by infecting the arabidopsis obtaining transgenic；
4) collect the arabidopsis seed that described inflorescence produces, carry out antibiotic (hygromycin) and the PCR screening in many generations, to obtain the transgenic arabidopsis strain isozygotied.Described screening technique is to it known in the art, such as first to be cultivated in the culture medium containing 50mg/l hygromycin by the seed infecting inflorescence generation, the Arabidopsis plant that can grow wherein is carried out PCR, to confirm to there is institute's transgenic in its genome.Then, collecting the seed that described plant produces, carry out next round screening, in later screening process, hygromycin concentration is 100mg/l.This screening can carry out three-wheel and at most take turns, till guaranteeing the transgenic arabidopsis strain obtaining isozygotying.
Accompanying drawing explanation
Fig. 1 is the plasmid figure of the genetic constructs of the present invention；
Fig. 2 illustrates the expression of MdMIEL1 in the transgenic arabidopsis (#1, #7 and #8) and wildtype Arabidopsis thaliana (col) that the method by the present invention obtains；
Fig. 3 is the transgenic arabidopsis (#1, #7 and #8) that obtained by the method for the present invention of the present invention and wildtype Arabidopsis thaliana (col) sprout after plant and the photo of root system；
Fig. 4 illustrates the main root length of transgenic arabidopsis (#1, #7 and #8) that the method by the present invention obtains and wildtype Arabidopsis thaliana (col)；
Fig. 5 illustrates the lateral root number of transgenic arabidopsis (#1, #7 and #8) that the method by the present invention obtains and wildtype Arabidopsis thaliana (col).
Detailed description of the invention
Inventor finds its E3 ubiquitin ligase gene (MdMIEL1) up-regulated expression in process is in adverse circumstances by loud, high-pitched sound Fructus Mali pumilae in the research process to loud, high-pitched sound Fructus Mali pumilae, implies that it is likely to play certain effect in the degeneration-resistant border process of loud, high-pitched sound Fructus Mali pumilae.By this gene clone out, when studying in model organism arabidopsis, surprisingly it was found that the process LAN that this gene is in arabidopsis can increase the lateral root number of arabidopsis.Inventor, according to this characteristic, completes the present invention.
Below in conjunction with accompanying drawing, principles of the invention and feature being described, example is served only for explaining the present invention, is not intended to limit the scope of the present invention.
Embodiment
The clone of embodiment 1 loud, high-pitched sound Fructus Mali pumilae MdMIEL1 gene
1. loud, high-pitched sound Fructus Mali pumilae group training blade RNA extracts
Extracted the total serum IgE of loud, high-pitched sound Fructus Mali pumilae group training blade by CTAB method, comprise the following steps:
1) take 1.5g through the 200mMNaCl salt treatment loud, high-pitched sound Tissue-cultured apple seedling of 24 hours, put into the mortar of pre-cooling, add liquid nitrogen and grind, proceed in the 50ml centrifuge tube of pre-cooling；
2) it is rapidly added 10ml and is preheating to the Extraction buffer (CTAB20%w/v of 65 DEG C, Tris-HCl0.1mol/l, EDTA25mol/l, NaCl2mol/l, mercaptoethanol 2%w/v, PVP2%w/v, distilled water constant volume without RNase, wherein PVP and mercaptoethanol are now with now adding), mix gently, in 65 DEG C of water-baths 0.5 hour；
3) adding and the isopyknic water-saturated phenol of upper step centrifuge tube liquid/chloroform/isoamyl alcohol (25:24:1) mixture, ice bath vibrates 0.5 hour, 4 DEG C, 12,000rpm centrifugal 20 minutes；Supernatant is transferred in new 50ml centrifuge tube；
4) adding the 10mol/L pre-cooling LiCl of 1/3 supernatant volume, place 3 hours for-20 DEG C, 12,000rpm are centrifuged 30 minutes, abandon supernatant；
5), after adding 500 μ lSSTE buffer (NaCl1mol/l, SDS0.5%w/v, EDTA10mol/l, without the distilled water constant volume of RNase) fully suspension precipitation, average mark installs in 2 1.5ml centrifuge tubes；
6) being separately added into water-saturated phenol isopyknic with suspension/chloroform/isoamyl alcohol (25:24:1) mixture, ice bath vibrates 10 minutes, 4 DEG C, 12,000rpm centrifugal 10 minutes；Supernatant is transferred to new 1.5ml centrifuge tube；
7) being separately added into chloroform isopyknic with supernatant/isoamyl alcohol (24:1) mixture, ice bath vibrates 10 minutes, 4 DEG C, 12,000rpm centrifugal 10 minutes；Supernatant is transferred to new 1.5ml centrifuge tube；
8) add the pre-cooling dehydrated alcohol of 2.5 times of supernatant volume, place 1-2 hour for-20 DEG C；
9) in 4 DEG C, 12,000rpm centrifugal 20 minutes, 70% ethanol washes 2 times；
10) in 4 DEG C, 14,000rpm centrifugal 10 minutes, air-dry precipitation on super-clean bench；Add 20 μ lDEPC water dissolution RNA.
11) put-80 DEG C to be in store for, or carry out following reverse transcription experiment immediately.
2. total serum IgE reverse transcription is obtained cDNA
The reverse transcription of total serum IgE uses commercially available Reverse Transcription box, and the description according to manufacturer carries out.
The amplification of 3.MdMIEL1 full length DNA sequence
Use primer pair MdMIEL1-F/MdMIEL1-R, carry out pcr amplification with the cDNA that above-mentioned reverse transcription synthesizes for template.
MdMIEL1-F:5 '-ATGGAAGGCTCAGCCAATGAAC-3 ' (SEQIDNO:3)；
MdMIEL1-R:5 '-TTGAGGAAGAACTGGAGGGGC-3 ' (SEQIDNO:4).
Amplification system: cDNA product 25 μ l, 5 × TdT buffer 10 μ l, 0.1%BSA5 μ l, 10mMdCTP2.5 μ l, the TdT15U of purification, distilled water is settled to 50 μ l.
Amplification program: 94 DEG C of denaturations 5 minutes；Loop parameter is 94 DEG C of degeneration 30 seconds, 30 seconds, 72 DEG C extensions of 56 DEG C of annealing 60 seconds, carries out 32 circulations；72 DEG C of abundant extensions 10 minutes.
PCR reaction is reclaimed PCR primer and is connected on pMD-18T carrier after terminating, and sequencing result shows, its nucleotide sequence is such as shown in SEQIDNO:1；Its aminoacid sequence is such as shown in SEQIDNO:2.
Embodiment 2. is for the structure of the plasmid of transgenic
1. using primer pair EMdMIEL1-F/EMdMIEL1-R, with the pMD18-T plasmid with MdMIEL1 obtained above for template, pcr amplification is with the MdMIEL1 sequence of EcoRI and PstI restriction enzyme site.
EMdMIEL1-F:5 '-GAATTCATGGAAGGCTCAGCCAATGAAC-3 ' (SEQIDNO:5), drawing horizontal line part is EcoRI restriction enzyme site；
EMdMIEL1-R:5 '-CTGCAGTTGAGGAAGAACTGGAGGGGC-3 ' (SEQIDNO:6), drawing horizontal line part is PstI restriction enzyme site.
Primer and template as described above, are only replaced by amplification system and amplification program.
2. reclaiming PCR primer and be connected on pMD-18T carrier, and checking order, the sequence to guarantee gained fragment is correct.
3. respectively above-mentioned plasmid and pCAMBIA1300 carrier are used EcoRI and PstI double digestion, reclaim MdMIEL1 fragment and carrier, with T4DNA ligase, the two is coupled together, and be used for converting escherichia coli, select the escherichia coli with the plasmid being correctly inserted into direction, that is, the transcriptional orientation that MdMIEL1 fragment starts with the CaMV35S promoter on pCAMBIA1300 carrier inserts on pCAMBIA1300 (Fig. 1).
4. convert Agrobacterium LB4404 competent cell with the recon pCAMBIA1300-MdMIEL1 built.Carrying out PCR qualification, picking positive bacteria drops into row order-checking.The recon pCAMBIA1300-MdMIEL1 monoclonal the checking order correct conversion of arabidopsis later.
Embodiment 3. obtains transgenic arabidopsis
1. the arabidopsis seed that will obtain, respectively with 70% alcohol disinfecting 3min, 4% hypochlorite disinfectant 8-10min (period repeatedly rocks), aquesterilisa rinses 5 times, blots water.Be seeded on seed germination medium (being directly laid on surface), light cultivate (25-28 DEG C, 16h long-day/8h short-day, 10d), grow to seedling.It is transplanted to substrate to cultivate and bloom.
2. picking Agrobacterium monoclonal colony inoculation is in 10mLYEP fluid medium (containing 50mg/L hygromycin), 28 DEG C, 200rpm, and shaken cultivation is to OD600For 06-0.8 (about 48h)；Taking wherein lmL bacterium solution to add in 20mLYEP fluid medium, 28 DEG C, 200rpm, shaken cultivation is to OD600For 06-0.8 (about 5h).Centrifugal collection thalline, with infecting liquid (containing 0.05g/ml sucrose, 0.03-0.05%Silweet) suspension dilution 20 times, standby；
3. arabidopsis inflorescence is dipped into and infects 15-20s in liquid, collect fruit pod, 50mg L-1After hygromycin resistance screening, PCR detection obtains positive transgenic plant, obtains T3 for homozygote through continuous 3 generations screening, collects seed, carry out phenotype analytical.
4. utilize screening culture medium (containing 100mg/L hygromycin) to screen candidate's transgenic line that hygromycin is positive, obtain 3 35S:MdMIEL1 positive strain (#1, #7 and #8) altogether；For identifying transgenic line further, when carrying out successive transfer culture, every strain takes the tissue cultured seedling of about 0.1g, extracts corresponding RNA, and reverse transcription also carries out quantitative PCR detection, to determine the expression of MdMIEL1 in these strains.Result shows that such as Fig. 2 shows, the MdMIEL1 in #1, #7 and #8 expresses far above wild type.
Embodiment 4: the root system phenotype of transgenic arabidopsis
In order to further determine that the function of transfer-gen plant, arabidopsis is sowed in vertical MS culture medium, observe arabidopsis root system development phenotype.
(1) seed treatment.By the arabidopsis seed of the wild type (col) obtained and transgenic line (#1, #7, #8) respectively with 70% alcohol disinfecting 3min, 4% hypochlorite disinfectant 8-10min (period repeatedly rocks), aquesterilisa flushing 5 times, blot water.It is seeded on seed germination medium.4 DEG C of Stratificated treatment cultivate to light after 4 days (21-24 DEG C, 16h long-day/8h short-day).
(2) select to sprout latter about 4 days, the consistent wildtype Arabidopsis thaliana (col) of root system development and transgenic arabidopsis (#1, #7, #8), transfer in MS culture medium and vertically to cultivate (21-24 DEG C, 16h long-day/8h short-day).After continuing cultivation 8 days, observe arabidopsis root system phenotype and also take pictures.
As in Figure 3-5, compared with wildtype Arabidopsis thaliana, the transgenic arabidopsis main root length of process LAN MdMIEL1 does not have notable difference to result, but side radical order significantly increases.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (10)
1. a genetic constructs, it is characterised in that comprising the E3 ubiquitin ligase gene fragment of the coding loud, high-pitched sound Fructus Mali pumilae being under strong promoter controls, described genetic fragment has the DNA sequence shown in SEQIDNO:1.
2. genetic constructs according to claim 1, it is characterised in that described strong promoter be can in arabidopsis the strong promoter of constitutive expression.
3. genetic constructs according to claim 2, it is characterised in that described strong promoter is CaMV35S promoter.
4. genetic constructs according to claim 1, it is characterised in that also comprise the resistant gene of one or more generation antiviral antibiotic.
5. genetic constructs according to claim 4, it is characterised in that the one or more resistant gene includes hygromycin gene.
6. genetic constructs according to claim 1, it is characterized in that, described structure body is the pCAMBIA1300 plasmid of the DNA sequence of the E3 ubiquitin ligase comprising described coding loud, high-pitched sound Fructus Mali pumilae, under the control of the CaMV35S promoter that the DNA sequence of the E3 ubiquitin ligase of wherein said coding loud, high-pitched sound Fructus Mali pumilae is on described pCAMBIA1300 plasmid.
7. the genetic constructs according to any one of claim 1-6 is for producing the purposes of transgenic arabidopsis.
8. the method for producing the arabidopsis strain that side root increases, it is characterized in that, genetic constructs according to any one of claim 1-6 is imported in arabidopsis, obtain transgenic arabidopsis seed, then pass through the filial generation to described transgenic arabidopsis seed and carry out multi-turns screen and obtain arabidopsis strain of isozygotying.
9. method according to claim 8, it is characterised in that comprise the following steps:
1) convert Agrobacterium competent cell with described genetic constructs, and screening obtains monoclonal transformant；
2) incubation step 1) in the monoclonal transformant that obtains, collect thalline, and make and infect liquid；
3) by step 2) in the liquid that infects that obtains infect arabidopsis inflorescence；
4) the transgenic arabidopsis seed that described inflorescence produces is collected, by the filial generation of described transgenic arabidopsis seed being carried out antibiotic and the PCR screening in many generations, to obtain the transgenic arabidopsis strain isozygotied.
10. method according to claim 9, it is characterised in that described antibiotic is hygromycin.
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Cited By (2)
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| CN106566835A (en) * | 2016-11-08 | 2017-04-19 | 山东农业大学 | Application of MdMIEL1 gene to improvement of sensitivity of plant to abiotic stress |
| CN111793636A (en) * | 2020-07-29 | 2020-10-20 | 山东农业大学 | Apple gene MdBT2 for regulating and controlling adventitious root development and application thereof |
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| US20040123349A1 (en) * | 2002-12-20 | 2004-06-24 | Qi Xie | SINAT5, an Arabidopsis thaliana gene promotes ubiquitin related degradation |
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| US20040123349A1 (en) * | 2002-12-20 | 2004-06-24 | Qi Xie | SINAT5, an Arabidopsis thaliana gene promotes ubiquitin related degradation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106566835A (en) * | 2016-11-08 | 2017-04-19 | 山东农业大学 | Application of MdMIEL1 gene to improvement of sensitivity of plant to abiotic stress |
| CN111793636A (en) * | 2020-07-29 | 2020-10-20 | 山东农业大学 | Apple gene MdBT2 for regulating and controlling adventitious root development and application thereof |
| CN111793636B (en) * | 2020-07-29 | 2021-09-28 | 山东农业大学 | Apple gene MdBT2 for regulating and controlling adventitious root development and application thereof |
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