CN108424920A - The resistance to inversely related transcription factor ZmNAC33 genes of corn and its application - Google Patents

The resistance to inversely related transcription factor ZmNAC33 genes of corn and its application Download PDF

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Publication number
CN108424920A
CN108424920A CN201810374938.8A CN201810374938A CN108424920A CN 108424920 A CN108424920 A CN 108424920A CN 201810374938 A CN201810374938 A CN 201810374938A CN 108424920 A CN108424920 A CN 108424920A
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zmnac33
resistance
corn
arabidopsis
genes
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CN108424920B (en
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李晓辉
刘文平
张春宵
李淑芳
李万军
金峰学
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Jilin Academy of Agricultural Sciences
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    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

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Abstract

The invention discloses a kind of resistance to inversely related transcription factor ZmNAC33 genes of corn, and nucleotide sequence is as shown in SEQ ID No.1.The transcription regulatory factor ZmNAC33 gene that the present invention is cloned into from resistance to inverse corn inbred line, self-mating system is under drought stress, ZmNAC33 gene expressions up-regulation.ZmNAC33 dicotyledon binary vectors are built, by dipping in colored method by ZmNAC33 channel genes arabidopsis, the seed of harvest is identified by hygromycin selection and PCR, obtains homozygous lines.Transgenic seedling has obtained the transgenic line that can significantly increase arabidopsis drought-resistant ability by means such as drought stress, seed germination rates.This illustrates that ZmNAC33 can enhance plant to the tolerance of resistance to inverse stress, is of great significance.

Description

The resistance to inversely related transcription factor ZmNAC33 genes of corn and its application
Technical field
The invention belongs to genetic engineering fields, and in particular to a kind of resistance to inversely related transcription factor ZmNAC33 genes of corn and It is applied.
Background technology
Plant often suffers from various adverse circumstance impact factors, including arid, with high salt, low temperature and disease pest in growth course. Since such environmental effects the growth of plant, plant evolution goes out various mechanism to cope with these challenges.Plant reply arid and Complicated regulation process with high salt is related to the various aspects such as the synthesis of osmotic protection substance, ion-select electrode and plasma membrane protection, and these The expression of stress-related genes be unable to do without the Effective Regulation of all kinds of transcription factors in plant.Transcription factor by with gene promoter The DNA cis elements of son and enhancer interact, and directly or indirectly adjust the transcript and expression of target gene.Plant it is degeneration-resistant Character is the quantitative character by controlled by multiple genes, and transcription factor can regulate and control the multiple and relevant gene expression of degeneration-resistant character, and Promote these adversity genes to play corresponding effect by enhancing the effect of certain key regulators, makes the resistance of plant Improved.The study found that NAC albumen participates in the stress response of the abiotic stress in plant, such as freeze injury, the arid side of body A variety of abiotic stress such as urgent, with high salt, NAC regulate and control plant pair environment stress induction of the expression of many stress-related genes Reaction.
Studies have shown that the environment stresses such as arid, with high salt or abscisic acid can induce rice SNAC1, OsNAC6/SNAC2, OsNAC5, OsNAC10, OsNAC45, corn ZmSNAC1, ZmNAC111, ZmNAC55, arabidopsis ATAF1, ANAC019, The expression of the NAC transcription factors gene such as ANAC055 and ANAC072, and functional study also turn out these NAC genes it is resistance to it is inverse, There is effect in resistance to reactant salt.
Invention content
The technical problems to be solved by the invention are：How a kind of new corn transcription factor gene of resistance to inversely related is provided, Enhance the drought-resistance ability of plant.
The technical scheme is that：The resistance to inversely related transcription factor ZmNAC33 genes of corn, nucleotide sequence such as SEQ Shown in ID No.1.
The albumen of the resistance to inversely related transcription factor ZmNAC33 codings of corn, amino acid sequence is as shown in SEQ ID No.2.
The egg of the resistance to inversely related transcription factor ZmNAC33 genes of corn or the resistance to inversely related transcription factor ZmNAC33 codings of corn Application in the enhancing resistance to inverse ability of plant in vain.
Further, the enhancing resistance to inverse ability of plant refers to enhancing drought tolerance in plants ability.
Further, the plant is dicotyledon.
Transcription regulatory factor a ZmNAC33, ZmNAC33 that the present invention is cloned into from resistance to inverse corn inbred line with There is NAC structural domains in ZmNAC55 and ZmNAC111, they belong to the same NAC families, and ZmNAC33 and ZmNAC55 is in albumen Similarity is 28.71%, ZmNAC33 in sequence and ZmNAC111 similarities on protein sequence are 24.75%.Drought stress Under, ZmNAC33 gene expressions up-regulation.ZmNAC33 dicotyledon binary vectors are built, by dipping in colored method by ZmNAC33 genes It imports in arabidopsis, the seed of harvest is identified by hygromycin selection and PCR, obtains homozygous lines.Transgenic seedling is by arid The means such as stress, seed germination rate have obtained the transgenic line that can significantly increase arabidopsis drought-resistant ability.This explanation ZmNAC33 can enhance plant to the tolerance of resistance to inverse stress, be of great significance.
Description of the drawings
Fig. 1 is the gene constructed schematic diagrames of ZmNAC33 on dicotyledon binary vector ZmNAC33-pGreen carriers (ZmNAC33：Target gene；pCAMBIA1300 super-GFP：Dicotyledon binary vector)；
Fig. 2 is the agarose gel electrophoresis figure that PCR identifies transgenic arabidopsis ZmNAC33 genes, wherein there is about 880bp ZmNAC33 gene bands, the genomic DNA of wildtype Arabidopsis thaliana is that negative object carries out PCR identifications, does not occur 880bp's ZmNAC33 gene bands；wt：Wildtype Arabidopsis thaliana pcr amplification product；L1-L12：Transgenosis ZmNAC33 arabidopsis PCR amplifications Figure；
Fig. 3 measures for transgenic arabidopsis seed germination rate, Wt：Wildtype Arabidopsis thaliana；L2：The quasi- south of ZmNAC33 transgenosis Mustard strain L2；Wildtype Arabidopsis thaliana seed and the L2 sowings of ZmNAC33 transgenic arabidopsis strains are in 1/ containing 350mM mannitol On 2MS culture mediums, the germination rate of seed is measured；
Fig. 4 is transgenic arabidopsis seedling Stress treatment, Wt：Wildtype Arabidopsis thaliana；L2、L3、L13：Respectively ZmNAC33 Transgenic arabidopsis strain L2, L3, L13；The wildtype Arabidopsis thaliana and ZmNAC33 transgenic arabidopsis strains L2, L3 of growth 5 days It is transplanted seedlings with L13 to measuring growing state on the 1/2MS culture mediums containing 400mM mannitol；
Fig. 5 is the processing of transgenic arabidopsis drought stress, Wt：Wildtype Arabidopsis thaliana；L2、L3：Respectively ZmNAC33 turns base Because of arabidopsis strain L2, L3.Wildtype Arabidopsis thaliana and ZmNAC33 transgenic arabidopsis strain L2, L3 kinds are planted in Nutrition Soil, into The processing of row drought stress, measures the drought-resistance ability of transfer-gen plant.
Specific implementation mode
Embodiment 1：Corn anti-reverse transcripfactor ZmNAC33 gene clonings and dicotyledon binary vector con- struction
According to positioning of the early period to Maize QTL is tested, the transcription of one and resistance to inversely related are found in the candidate regions QTL Factor Z mNAC33；With reference to the gene order of Maize genome database, designed for drawing for structure dicotyledon binary vector Object sequence.Sense primer F1:5 '-CCAAGCTTATGAGCGGCGCCGGTCCGGATC-3 ' (SEQ ID No.3), downstream primer R1:5 '-TCCCCCGGGTGAACGGCTTGCCCCAGTACATGAG-3 ' (SEQ ID No.4), PCR product 897bp.Extraction It is that primer carries out reverse transcription with Oligo (dT) 18 with the total serum IgE of the resistance to inverse corn inbred line of vermiculite culture to one heart stage of three leaves, Obtain the cDNA of corn inbred line.Use this cDNA as template.Primers F 1 and R1 carry out PCR amplification gene.Ago-Gel electricity Swimming recycling PCR product, the target fragment recycled with restriction enzyme HindIII and XmaI digestion and dicotyledon double base carry Then body recycles digestion products with QIAquick Gel Extraction Kit.
With the target fragment and binary vector of the connection recycling of T4DNA ligases, 25 DEG C connect 1 hour.Connection product converts E.coli DH5 α carry out the bacterium colony on PCR identification conversion tablets with primers F 1 and R1, obtain the band of about 897bp.Picking contains Clone's sample presentation sequencing of purposeful band.The correct bacterium colony of sequencing result is selected, plasmid is extracted, completes ZmNAC33 genes (ZmNAC33-pGreen, T-DNA schematic diagram are shown in Fig. 1 to the structure of dicotyledon binary plasmid.With chemical transformation the double base Plasmid converts Agrobacterium GV3101, obtains engineering Agrobacterium GV3101：：ZmNAC33-pGreen can be used for Plant Transformation.
Embodiment 2：The transformation of Arabidopsis thaliana of ZmNAC33 genes
Use GV3101：：It is as follows that ZmNAC33-pGreen infects the step of arabidopsis：
(1) culture of Agrobacterium：Picking contains the Agrobacterium GV3101 of ZmNAC33 target gene from tablet, is inoculated in Containing 50mg/L rifampins, 50mg/L kanamycins YEB fluid nutrient mediums in, 28 DEG C, 200rpm shaken cultivations stay overnight.By 1: 100 ratios take the Agrobacterium 1ml being incubated overnight to be inoculated in new 100ml liquid YEB culture mediums, continue shaken cultivation 9h.With Thalline were collected by centrifugation for centrifuge tube.Room temperature, 4000g centrifuge 10min, abandon supernatant, are inverted centrifuge tube, residual liquid is allowed to flow to end.
(2) Agrobacterium infects arabidopsis：When bud occurs in arabidopsis, the top of arabidopsis is cut off with scissors, to promote Break up more side shoots into arabidopsis, increases more buds.After cutting off top 1 week, prepares to infect arabidopsis with Agrobacterium, use Scissors cut off the siliqua grown up to.Agrobacterium is resuspended with the 1/2MS fluid nutrient mediums containing 5% sucrose, is diluted to OD600=0.8, final concentration of 0.04% Sliwet L-77 surfactants are added in suspension.Suspension is poured into small burning In cup, arabidopsis is inverted, so that inflorescence is immersed in the suspension containing Agrobacterium, during which about 1min constantly stirs bacterium solution.Leaching After dye, arabidopsis is covered inside with black plastic bag, dark culturing is for 24 hours.After light culture, arabidopsis is placed in normal light It is cultivated according under the conditions of.It was infected again every 4 days once, it is total to infect 3 times.After seed maturity, seed is collected.
(3) transgenic molecules detect：By the arabidopsis seed of harvest after the surface of the seed sterilizes, sowing containing On the 1/2MS culture medium flat plates of 50mg/L hygromycin, first handle 2 days for 4 DEG C, then illumination cultivation 7 days.Selecting being capable of normal growth Arabidopsis seedling, the arabidopsis of survival is transplanted in matrix and is cultivated.PCR identifications are carried out to the arabidopsis of survival, cut quasi- south The leaf of mustard；Genomic DNA is extracted with CTAB methods, PCR identifications are carried out with primers F 1 and R1, with the base of wildtype Arabidopsis thaliana Because group DNA is as negative control, it is carried out at the same time PCR amplification.PCR product is after agarose gel electrophoresis, and wildtype Arabidopsis thaliana is not There is target gene band, target gene band occurs in transgenic line (see Fig. 2).After the seed maturity of positive seedling, single plant Collect seed.The seed of collection is seeded on the 1/2MS culture mediums containing hygromycin and is screened again, again to the plant of survival Strain carries out PCR identifications.Finally, ripe seed is collected.
Embodiment 3：Turn the drought tolerance identification of ZmNAC33 gene arabidopsis
Seed germination rate：Wildtype Arabidopsis thaliana and transgenic arabidopsis sowing in the 1/2MS trainings containing 350mM mannitol It supports on base tablet, every part of strain sows 50 seeds, is repeated 3 times.Tablet is placed on 4 DEG C of low-temperature treatments 2 days, is transferred to culturing room's training It supports 7 days, takes a picture (see Fig. 3).Compared with wildtype Arabidopsis thaliana, the seed number that transfer-gen plant is sprouted is more than wild type, shows ZmNAC33 transfer-gen plant seeds are drought-enduring when sprouting.
Seedling Stress treatment：5 days Arabidopsis thaliana Seedlings are cultivated in selection, move on to the culture of the 1/2MS containing 400mM mannitol It is vertical to cultivate 10 days (Fig. 4) on base tablet.Compared with wildtype Arabidopsis thaliana, transgenic arabidopsis root long is longer than wild type, shows ZmNAC33, which turns Arabidopsis plant, can enhance the drought-resistance ability of arabidopsis.
Osmotic treatment：By T3For transgenic arabidopsis kind in matrix, normal growth stops watering after 4 weeks, wild after 3 weeks Raw type Arabidopsis leaf is wilted,；Transgenic Arabidopsis plants blade still maintains green, remains to continued growth (Fig. 5), table Bright ZmNAC33 can enhance the drought-resistance ability of arabidopsis.
Sequence table
<110>Applicant's title:Jilin Academy of Agricultural Science
<120>The resistance to inversely related transcription factor ZmNAC33 genes of corn and its application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 882
<212> DNA
<213>Corn (Zea mays)
<400> 1
atgagcggcg ccggtccgga tctgcagctg ccaccggggt tccggttcca cccgacggac 60
gaggagctgg tgatgcacta cctctgccgc cgctgcgccg gcctgcccat cgccgtcccc 120
atcatcgccg agatcgacct ctacaagttc gacccatggc agctccccag gatggcgctg 180
tacggcgaga aggagtggta cttcttctcc ccgcgggacc gcaagtaccc gaacgggtcc 240
aggcccaacc gcgccgccgg ggctgggtac tggaaggcca ccggcgctga caagcccgtg 300
ggcacgccca agccgctggc catcaagaag gcgctcgtct tctacgccgg caaggcgccc 360
aagggcgaga agaccaactg gatcatgcac gagtaccgcc tcgccgacgt cgaccgctcg 420
gcgcgcaaga agaacagcct caggttggat gactgggtcc tgtgccgcat ctacaacaag 480
aagggcggcg ggctggagaa ggcgccggcg gccggcggcg accacaagcc tgtgttcgcc 540
acggcggcgg tgagctcccc gccggagcag aagccgttcg tggcggcggc gggcgggctg 600
cccccggcgt tcccggagct ggcggcgtac tacgaccggc cgtcggactc gatgccgcgg 660
ctgcacgcgg actactccag ctgctcggag caggtgctgt ccccggagca gctggcgtgc 720
gaccgggagg tgcagagcca gcccaagatc agcgagtggg agcggacctt cgcctccgac 780
cccgtgaacc ccgcgggctc catgctcgac cccgtcctcg gccacgccgg cggcgacccg 840
ctgctgcagg acatcctcat gtactggggc aagccgttct ag 882
<210> 2
<211> 293
<212> PRT
<213>Corn (Zea mays)
<400> 2
Met Ser Gly Ala Gly Pro Asp Leu Gln Leu Pro Pro Gly Phe Arg Phe
1 5 10 15
His Pro Thr Asp Glu Glu Leu Val Met His Tyr Leu Cys Arg Arg Cys
20 25 30
Ala Gly Leu Pro Ile Ala Val Pro Ile Ile Ala Glu Ile Asp Leu Tyr
35 40 45
Lys Phe Asp Pro Trp Gln Leu Pro Arg Met Ala Leu Tyr Gly Glu Lys
50 55 60
Glu Trp Tyr Phe Phe Ser Pro Arg Asp Arg Lys Tyr Pro Asn Gly Ser
65 70 75 80
Arg Pro Asn Arg Ala Ala Gly Ala Gly Tyr Trp Lys Ala Thr Gly Ala
85 90 95
Asp Lys Pro Val Gly Thr Pro Lys Pro Leu Ala Ile Lys Lys Ala Leu
100 105 110
Val Phe Tyr Ala Gly Lys Ala Pro Lys Gly Glu Lys Thr Asn Trp Ile
115 120 125
Met His Glu Tyr Arg Leu Ala Asp Val Asp Arg Ser Ala Arg Lys Lys
130 135 140
Asn Ser Leu Arg Leu Asp Asp Trp Val Leu Cys Arg Ile Tyr Asn Lys
145 150 155 160
Lys Gly Gly Gly Leu Glu Lys Ala Pro Ala Ala Gly Gly Asp His Lys
165 170 175
Pro Val Phe Ala Thr Ala Ala Val Ser Ser Pro Pro Glu Gln Lys Pro
180 185 190
Phe Val Ala Ala Ala Gly Gly Leu Pro Pro Ala Phe Pro Glu Leu Ala
195 200 205
Ala Tyr Tyr Asp Arg Pro Ser Asp Ser Met Pro Arg Leu His Ala Asp
210 215 220
Tyr Ser Ser Cys Ser Glu Gln Val Leu Ser Pro Glu Gln Leu Ala Cys
225 230 235 240
Asp Arg Glu Val Gln Ser Gln Pro Lys Ile Ser Glu Trp Glu Arg Thr
245 250 255
Phe Ala Ser Asp Pro Val Asn Pro Ala Gly Ser Met Leu Asp Pro Val
260 265 270
Leu Gly His Ala Gly Gly Asp Pro Leu Leu Gln Asp Ile Leu Met Tyr
275 280 285
Trp Gly Lys Pro Phe
290
<210> 3
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ccaagcttat gagcggcgcc ggtccggatc 30
<210> 4
<211> 34
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tcccccgggt gaacggcttg ccccagtaca tgag 34

Claims (6)

1. the resistance to inversely related transcription factor ZmNAC33 genes of corn, nucleotide sequence is as shown in SEQ ID No.1.
2. the albumen of the resistance to inversely related transcription factor ZmNAC33 codings of corn, amino acid sequence is as shown in SEQ ID No.2.
3. the resistance to inversely related transcription factor ZmNAC33 genes or according to claim 2 of corn according to claim Application of the albumen of the resistance to inversely related transcription factor ZmNAC33 codings of corn in the enhancing resistance to inverse ability of plant.
4. application according to claim 3, which is characterized in that the enhancing resistance to inverse ability of plant refers to enhancing drought tolerance in plants energy Power.
5. application according to claim 3, which is characterized in that the plant is dicotyledon.
6. application according to claim 3, which is characterized in that the plant is corn.
CN201810374938.8A 2018-04-24 2018-04-24 Corn stress tolerance related transcription factor ZmNAC33 gene and application thereof Active CN108424920B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109837296A (en) * 2019-03-22 2019-06-04 济南大学 The drought-enduring new function of a salt tolerant of corn gene ZmNAC77 and its application
CN111172175A (en) * 2020-03-12 2020-05-19 北京农学院 Application of Zma006493 gene, transgenic arabidopsis thaliana plant expressing Zma006493 and preparation method
CN115161330A (en) * 2022-06-07 2022-10-11 青岛农业大学 Application of corn ZmGAPB gene in improving stress resistance of plants

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CN103626858A (en) * 2007-10-19 2014-03-12 先锋高级育种国际公司 Maize stress-responsive nac transcription factors and promoter and methods of use

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WO2008034648A1 (en) * 2006-04-05 2008-03-27 Metanomics Gmbh Process for the production of a fine chemical
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109837296A (en) * 2019-03-22 2019-06-04 济南大学 The drought-enduring new function of a salt tolerant of corn gene ZmNAC77 and its application
CN109837296B (en) * 2019-03-22 2022-06-24 济南大学 New salt-tolerant drought-tolerant function of corn gene ZmNAC77 and application thereof
CN111172175A (en) * 2020-03-12 2020-05-19 北京农学院 Application of Zma006493 gene, transgenic arabidopsis thaliana plant expressing Zma006493 and preparation method
CN115161330A (en) * 2022-06-07 2022-10-11 青岛农业大学 Application of corn ZmGAPB gene in improving stress resistance of plants

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