CN108329382A

CN108329382A - A kind of transcription factor MxERF72 and its encoding gene and application

Info

Publication number: CN108329382A
Application number: CN201710030557.3A
Authority: CN
Inventors: 韩振海; 刘伟; 王忆; 李都岳; 吴婷; 张新忠; 许雪峰
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2017-01-17
Filing date: 2017-01-17
Publication date: 2018-07-27
Anticipated expiration: 2037-01-17
Also published as: CN108329382B

Abstract

The invention discloses a kind of transcription factor MxERF72 and its encoding gene and applications.The present invention provides albumen MxERF72, be it is following 1) or 2)：1) protein shown in sequence 1 in sequence table；2) amino acid sequence shown in sequence in sequence table 1 is passed through into the substitution of one or several amino acid residues and/or lacks and ors add and there is identical function protein derived from sequence 1.The experiment proves that, present invention discover that new gene MxERF72 has critically important adjustment effect for the plant of Fe Deficiency, it knocks out its expression and portion gene expression can be improved under the conditions of iron deficiency, show that MxERF72 is of great significance in the resistance to iron deficiency field of plant；In addition, the gene can reduce content of chlorophyll in plant by overexpression, it was demonstrated that it is related with chlorophyll content of plant.

Description

A kind of transcription factor MxERF72 and its encoding gene and application

Technical field

The present invention relates to biotechnology more particularly to a kind of transcription factor MxERF72 and its encoding gene and applications.

Background technology

Fe (iron) is many metabolism participated in including photosynthesis of plant, respiration, nitrogen fixation and nucleic acid synthesis etc. The important composition ingredient of enzyme and albumen in the process is important trace element needed for plant growth.In production of fruit trees, the iron deficiency side of body Compel have strong influence to the yield of fruit tree, quality, tree vigo(u)r and agricultural performance.Study the machine of resistance to iron deficiency of fruit tree crop Reason is the important research direction of gardening fruit tree subject.

Ethylene is semiochemicals important during iron deficiency.Research shows that under iron deficiency processing, arabidopsis, pea, sweet tea After ethylene precursor substance A CC is handled 30 minutes, iron hydrogen reduction enzymatic activity significantly improves (Waters, B.M.and for dish, tomato D.G.Blevins,Ethylene production,cluster root formation,and localization of iron(III)reducing capacity in Fe deficient squash roots.Plant and Soil, 2000.225(1-2):21-31.).Wherein the accumulation of ethylene is earliest in cucumber root, has been improved in Fe Deficiency 1d (ROMERA,F.J.,E.ALCANTARA,and M.D.DE LA GUARDIA,Ethylene production by Fe- deficient roots and its involvement in the regulation of Fe-deficiency stress responses by strategy I plants.Annals of Botany,1999.83(1):51-55.).It is further real It tests and shows in tomato and cushaw root the enhancing of iron hydrogen reduction enzymatic activity under the increase of ethylene contents and Fe Deficiency and root system Consistent (Bacaicoa E.et al., the Relationship between the hormonal balance and of acidization the regulation of iron deficiency stress responses in cucumber.Journal of the American Society for Horticultural Science,2009.134(6):589-601.)。

Ethylene responses factor ERFs is one of the subfamily in AP2/ERF transcription factor families, passes signal in ethylene It is adjusted expression after passing EIN3/EIL1, induces transcription and translation (the Sauter et of ethylene reaction related gene al.Methionine salvage and S-adenosylmethionine:essential links between sulfur,ethylene and polyamine biosynthesis.Biochemical Journal.2013,451:145- 154.).It is in arabidopsis studies have shown that compared with WT lines, AtERF4 is overexpressed plant and is handled through ethylene and Jasmonate The induction of ethylene responses gene PDF1.2 is substantially reduced afterwards, the expression of VSP and CYP79B2 genes improve (Memelink J., Regulation of gene expression by jasmonate hormones.Phytochem, 2009.70:1560- 1570.).RAP2.2 also work as an important adjuster in the response of gray mold and ethylene (Manuela Hinz, Iain W.Wilson,Jun Yang,Katharina Buerstenbinder,Danny Llewellyn,Elizabeth S.Dennis, Margret Sauter, Rudy Dolferus, Arabidopsis RAP2.2:An Ethylene Response Transcription Factor That Is Important for Hypoxia Surviva.Plant Physiology, 2010.153:757-772)。

According to the difference of iron absorpting form, plant is divided into mechanism I (strategy I) and mechanism II (strategy II) plant (Sivitz, A.et al., Proteasome-mediated turnover of the transcriptional activator FIT is required for plant iron‐deficiency responses.The Plant Journal,2011.66(6):1044-1052.).Wherein, mechanism I plants go back Fe (III) under the action of ferric iron back enzyme Originally it was Fe (II), Fe (II) is transported through plasma membrane Fe (II) transport protein into plant again, and I plant of mechanism is also logical under Fe Deficiency Cross the H on activator film⁺- ATPase secretion protons make the soil liquid be acidified, and reduce the solubility that soil liquid pH increases iron.Apple Fruit category malus xiaojinensis is that gardening plant research institute of China Agricultural University screens from the kind or the ecotype of a Malus more than 40 First apple iron efficient fruit genotype (Han Z H et al., the Iron absorption by iron-efficient gone out and-inefficient species of apples.Journal of Plant Nutrition,1998,21:181- 190), it is typical mechanism I plants, originally iron absorbability is strong for root system, and the active Fe content stored in root and spire is higher, It can actively reinforce root iron at low iron processing initial stage to absorb.King is first-class less to be detected in malus xiaojinensis transcript profile sequencing analysis library To some AP2/ERF classes transcription factors under the conditions of iron deficiency notable up-regulated expression, filter out some iron deficiency handle 12h or 2d when There is transcription factor such as RAP2.2, EIN3, ERF72, the ERF4 etc. significantly raised in root, thus it is speculated that these transcription factors may join (Wang, S.et al., Transcriptomic analysis demonstrates the are responded with malus xiaojinensis regulation and control iron deficiency early responses of local ethylene and redox signaling to low iron stress in Malus xiaojinensis.Tree Genetics&Genomes,2014,10:573–584)。

Invention content

It is an object of the present invention to provide a kind of transcription factor and its encoding genes.

Transcription inhibitory factor provided by the invention is albumen MxERF72, be it is following 1) or 2)：

1) protein shown in sequence 1 in sequence table；

2) by amino acid sequence shown in sequence in sequence table 1 by one or several amino acid residues substitution and/or It lacks and ors add and there is identical function protein derived from sequence 1.

It the substitution of said one or several amino acid residues and/or lacks and ors add residual to be no more than 10 amino acid It the substitution of base and/or lacks and ors add.

The DNA molecular for encoding above-mentioned protein is also the scope of protection of the invention.

Above-mentioned DNA molecular is following 1) -3) in any DNA molecular：

1) code area is DNA molecular shown in sequence 2 in sequence table；

2) hybridize under strict conditions with the DNA sequence dna 1) limited and encode the DNA molecular with identical function protein；

3) with the DNA sequence dna that 1) limits at least with 70%, at least with 75%, at least with 80%, at least have 85%, at least have with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% or at least There is the DNA molecular of 99% homology and coding with identical function protein.

Above-mentioned stringent condition can be to hybridize at 65 DEG C in 6 × SSC, the solution of 0.5%SDS, then with 2 × SSC, It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.

Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing above-mentioned DNA molecular are also that the present invention protects Range.

The primer pair for expanding above-mentioned DNA molecular overall length or its arbitrary segment is also the scope of protection of the invention.

Above-mentioned albumen, above-mentioned DNA molecular or above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium are regulating and controlling Application in plant tissue in Gene Transcription in vitro is also the scope of protection of the invention；Wherein, it is adjusted to inhibit.

Gene is MxHA2.

Above-mentioned albumen, above-mentioned DNA molecular or above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium are regulating and controlling Application in content of chlorophyll in plant or regulation and control plant Chlorophyll Degradation is also the scope of protection of the invention, wherein regulation and control are planted Object Determination of Chlorophyll content is to reduce chlorophyll content of plant, and regulation and control plant chlorophyll, which is degraded to, promotes plant chlorophyll degradation.

Above-mentioned albumen is also the scope of protection of the invention as the application in transcriptional activation repressor.

In above application, the regulation and control carry out under the conditions of iron deficiency.

Second purpose of the invention is to provide a kind of method for cultivating the low genetically modified plants of chlorophyll content.

Method provided by the invention, includes the following steps：Improve expression quantity or the work of the DNA molecular for encoding above-mentioned albumen Property, genetically modified plants are obtained,

The chlorophyll content of the genetically modified plants is less than the purpose plant.

The chlorophyll content of the genetically modified plants is embodied in as follows less than the purpose plant：

The SPAD values of the genetically modified plants are less than the purpose plant；

And/or the PSII Efficiency of primary conversion of light energy of the genetically modified plants is less than the purpose plant.

Among the above, the expression quantity of the DNA molecular for improving above-mentioned albumen or activity are will encode above-mentioned albumen DNA points Son imports purpose plant；

Or the plant is dicotyledon or monocotyledon；

Or the dicotyledon is malus xiaojinensis or tobacco.

The experiment proves that present invention discover that new gene MxERF72 have for the plant of Fe Deficiency it is very heavy The adjustment effect wanted knocks out its expression and portion gene expression can be improved under the conditions of iron deficiency, shows MxERF72 in the resistance to of plant Iron deficiency is of great significance in field；In addition, the gene can reduce content of chlorophyll in plant by overexpression, it was demonstrated that its with Chlorophyll content of plant is related.

Description of the drawings

Fig. 1 is the pcr amplification product result of MxERF72 genes.(1161bp)

Fig. 2 is horizontal lower real-time result of the MxERF72 genes in malus xiaojinensis root of different iron processing.

Fig. 3 is the result of bacterium colony PCR after building pGBKT7-MxERF72 carriers conversion Escherichia coli.

Fig. 4 is the result of bacterium colony PCR after pGBKT7-MxERF72 transformed yeasts.

Fig. 5 is growing state and X-gal colour test of the transgenic yeast on different deficiency culture mediums.

Fig. 6 is the bacterium colony PCR qualification results of yeast list miscellaneous carrier pJG4-5-MxERF72 and pLacZi-proMxHA2.

Fig. 7 is to convert the different miscellaneous carrier yeast X-gal colour tests of list.

Fig. 8 is that Yeast protein interaction carrier pGADT7-MxERF72 and pGBKT7-MxERF4 convert E. coli clones PCR Result.

Fig. 9 is growing state and X-gal colour test of the different plasmid yeast of conversion on different deficiency culture mediums.

Figure 10 is bimolecular fluorescence complementary (BiFC) carrier pSPYNE-MxERF72, pSPYCE-MxERF72, pSPYNE- The result of MxERF4 and pSPYCE-MxERF4 conversion E. coli clones PCR.

Figure 11 is interactions between protein result in plant.

Figure 12 is the result of Tobacco rattle virus carrier pTRV2-MxERF72 3'UTR E. coli clones PCR.

Figure 13 is instantaneous conversion malus xiaojinensis GFP Fluirescence observations.

Figure 14 is to interfere MxERF72 gene expressions and influenced on the expression of downstream functional gene MxHA2.

Figure 15 is the tobacco leaf Phenotype for being overexpressed MxERF72 genes.

Figure 16 is the tobacco leaf chlorophyll fluorescence result for being overexpressed MxERF72 genes.

Figure 17 is the tobacco leaf chlorophyll content result for being overexpressed MxERF72 genes.

Specific implementation mode

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Embodiment 1, the acquisition of MxERF72 and its encoding gene and expression analysis

One, the acquisition of MxERF72 and its encoding gene

(public can obtain malus xiaojinensis (Malus xiaojinensis) from China Agricultural University, record the seas the little Jin The non-patent literature of Chinese bush cherry is：Cheng Minghao, Li Xiaolin, Zhang Yungui, the excellent rootstock resource-malus xiaojinensis of apple, Agricultural University Of Southwest Journal, in October, 2000,22 (5)：383-386.), hereinafter also referred to malus xiaojinensis.

By malus xiaojinensis (Malus xiaojinensis) tissue-cultured seedling in growth medium (MS+0.3mg/L IBA+ 0.3mg/L 6-BA) in culture, after growth to stem lignifying, be transferred to root media (1/2MS+0.5mg/L IBA), After tissue-cultured seedling bears white root, moving to 1/2 pancebrin, (composition is shown in Table 2, overlay film moisturizing 6.0) initial pH value is adjusted to NaOH in Culture 2 weeks, being transferred to pancebrin later, (composition is shown in Table 1, and initial pH value is adjusted to 6.0) cultivate one month with NaOH, changes one weekly Secondary pancebrin, condition of culture are：16 hours (250 μm of olm of photon hypothesis of illumination cultivation^-2·s^-1), temperature 25 ±2℃；Dark culturing 8 hours, temperature are 17 ± 2 DEG C.Iron deficiency processing is carried out later, and method is：By plant from pancebrin Go to that (composition is shown in Table 3, and 6.0) initial pH value is adjusted to NaOH, deionized water is rinsed before shifting containing 0 μM of Fe nutrient solution.

The formula of pancebrin such as the following table 1：

The formula of table 1, pancebrin

The formula of 1/2 pancebrin such as the following table 2：

The formula of table 2,1/2 pancebrin

Formula containing 0 μM of Fe nutrient solution such as the following table 3：

Table 3, the formula containing 0 μM of Fe nutrient solution

Using malus xiaojinensis as experiment material, its iron deficiency is extracted respectively and handles 2 days and 9 days root total serum IgEs, is by its reverse transcription cDNA.Using this cDNA as template, using F1 and R1 as primer, PCR amplification is carried out.

F1：5 '-ATGTGTGGAGGTGCTATTATTT-3 ',

R1：5′-TTAAAAAACTCCCCCAACAA-3′.

Take PCR product into row agarose gel electrophoresis, the results are shown in Figure 1, wherein swimming lane M is the DNA molecular of 2000bp Amount standard, swimming lane 1 are pcr amplification product, obtain the pcr amplification product of 1161bp.Cut purpose band, after purifying recycling according to PCR product is connected to by pEASY-T1-simple Vector Kit support agents box (being purchased from Quan Shijin biotech firms) specification On pEASY-T1-simple, pET-MxERF72 is obtained.

PET-MxERF72 is sequenced, the results showed that, the pcr amplification product sequence on the carrier is 1161bp, In its nucleotide sequence such as sequence table shown in sequence 2, the egg with amino acid residue sequence shown in sequence 1 in sequence table is encoded White matter, sequence 1 is made of 386 amino acid residues in sequence table.It is MxERF72 by the unnamed gene, by the gene code Albumen is named as MxERF72.

Two, the expression analysis of malus xiaojinensis root MxERF72 genes

Extract the malus xiaojinensis root of iron deficiency processing (the Hoagland nutrient solutions for being free of Fe-EDTA) 6 days (d) and 9 days (d) Total serum IgE, ultraviolet specrophotometer measure the rna content of the root of different disposal respectively, the above-mentioned RNA of quality such as take, reverse transcription at Single-stranded cDNA.As a contrast with not iron deficiency processing (pancebrin culture).

MxERF72real-time primers are designed, it is template that iron deficiency malus xiaojinensis cDNA, which is used in combination, with two-step method PCR and standard Curve verifies the feasibility of primer.With the cDNA of the roots malus xiaojinensis iron deficiency 6d, 9d for template, using β-actin as internal reference, analysis The expression of MxERF72, MxIRT1, MxHA2, MxFIT in malus xiaojinensis iron deficiency root and leaf portion.Wherein, real-time expands The primer of increasing is respectively RT-F1 and RT-R1, RT-F2 and RT-R2, RT-F3 and RT-R3, RT-F4 and RT-R4：

RT-F1：5 '-TCACCGTTTCTGGAGGATTC-3 ',

RT-R1：5′-GAAGCATCCCAGCTCTCATC-3′；

RT-F2：5 '-CATGGCACAAGTTCCCTTTT-3 ',

RT-R2：5′-ACCACAGCCATCTCTTGGTC-3′；

RT-F3：5 '-ATCTGCCCTTACTGGGGAGT-3 ',

RT-R3：5′-AAATGCAGAAGTTCCCGATG-3′；

RT-F4：5 '-GGGAAACCATCAAGGAGGTCATA-3 ',

RT-R4：5′-AGCCATTCATCATAAGGTCAGGA-3′；

The primer of β-actin is β-actin-F and β-actin-R：

β-actin-F：5 '-TGGTGAGGCTCTATTCCAAC-3 ',

β-actin-R：5′-TGGCATATACTCTGGAGGCT-3′.

The results are shown in Figure 2, the results showed that, MxERF72 is in the 6th day to the 9th day expression quantity, iron deficiency condition under the conditions of positive iron Lower MxERF72 expression quantity is low in the 6th day calibration iron bar part, is increased between the 6th day to the 9th day and notable in the 9th day expression quantity Higher than the processing of positive iron, high MxIRT1, MxHA2, MxFIT expression quantity of the 6th day expression quantity was fallen after rise at the 9th day, showed that MxERF72 is rung Malus xiaojinensis Fe Deficiency is answered, and expression variation is negatively correlated with MxIRT1, MxHA2, MxFIT.

Embodiment 2, MxERF72 functional verifications

One, MxERF72 genes are the functional verification of Transcription inhibition

This experiment yeast expression system used is the system that a ripe proof albumen is transcription factor.

Yeast strain AH109, yeast vector pGBKT7, deficiency culture medium SD-Trp and deficiency culture medium SD-Trp- His is purchased from the general Jino Science and Technology Ltd. in Beijing, and Yeast Transformation Kit is purchased from Clontech companies.

With NdeI and BamHI double digestions pET-MxERF72 and yeast vector pGBKT7, difference glue recycles purpose digestion piece Section, obtains the carrier segment of the MxERF72 and 7300bp of 1161bp, recombinant expression carrier pGBKT7- is obtained after connection MxERF72.Recombinant expression carrier pGBKT7-MxERF72 is converted into bacillus coli DH 5 alpha competent cell, chooses after spot shakes bacterium and carries out Bacterium colony PCR verifications, after positive colony to be shaken to bacterium upgrading grain respectively, NdeI and BamHI double digestions verify recombinant plasmid.

The results are shown in Figure 3 for agarose gel electrophoresis detection, and wherein swimming lane M is the DNA molecular amount standard of 15000bp, Swimming lane 1 is to turn to have in the recombinant bacterium of DH5 α/pGBKT7-MxERF72 amplification MxERF72's as a result, swimming lane 2 is to expand by template of water Increase the negative control of MxERF72.To the bacterium that shakes that qualification result is positive bacteria, extraction plasmid carries out sequence verification.Sequencing result table Bright pGBKT7-MxERF72 is the NdeI and BamHI that MxERF72 shown in sequence in sequence table 2 is inserted into pGBKT7 expression vectors The carrier obtained between double enzyme site.

With yeast growth type culture medium (YPDA) and deficiency culture medium (SD-Trp and SD-Trp-His), utilize Clontech Yeast Transformation Kits by recombinant vector pGBKT7-MxERF72 and negative control pGBKT7, have transcription self-activation (pGBKT7 carriers are purchased from Clontech to active positive control pGBKT7-HB2, which expresses HB2 genes as skeleton.) point Recombinant bacterium AH109/pGBKT7-MxERF72, AH109/pGBKT7 and AH109/ Dao Ru not be obtained in yeast strain AH109 Recombinant bacterium is coated on SD-Trp and SD-Trp-His culture mediums by pGBKT7-HB2, and 30 DEG C are inverted culture.

The result shows that AH109/pGBKT7-MxERF72 has bacterium colony to grow in SD-Trp, grown on SD-Trp-His bright Aobvious to be suppressed, negative control AH109/pGBKT7 has bacterium colony to grow and sterile on SD-Trp-His culture mediums on SD-Trp It is born length.Positive control AH109/pGBKT7-HB2 has bacterium colony growth on SD-Trp and SD-Trp-His culture mediums.

Picking single bacterium colony carries out the positive colony that bacterium colony PCR identifies AH109/pGBKT7-MxERF72 with primers F 1 and R1, As shown in Figure 4：Wherein, M is the DNA molecular amount standard of DL2000bp, and 1 is the recombinant bacterium for turning to have AH109/pGBKT7-MxERF72 Middle amplification MxERF72's as a result, 2 for using water as the negative control of template amplification MxERF72.The result shows that carrier pGBKT7- MxERF72 is successfully transferred in yeast strain AH109.

By AH109/pGBKT7-MxERF72 (BD-MxERF72), AH109/pGBKT7 (BD) and AH109/pGBKT7-HB2 (BD-HB2) positive bacteria of picking is added in 50ml SD-Trp/Kan fluid nutrient mediums, 30 DEG C, 200rpm shake cultures 16- For 24 hours, with UV spectrophotometer measuring OD₆₀₀Value, is all higher than 0.8 and bacterial concentration is close, illustrate bait plasmid to yeast strain It is non-toxic.Bacterium solution is taken to cross on SD-Trp and SD-Trp-His culture mediums respectively, 30 DEG C are inverted culture, and the bacterium colony grown is done X-gal colour tests, the results are shown in Figure 5.X-gal colour tests prove to turn have the yeast strain of pGBKT7-MxERF72 not show Blue, and turn have the aobvious blue of the yeast strain of pGBKT7-HB2, negative control cannot develop the color, and illustrate pGBKT7-MxERF72 to ferment Female AH109 does not have self-excitation viability.

Two, yeast one-hybrid verifies MxERF72 and proMxHA2 interactions

With EcoRI and XhoI double digestions pET-MxERF72, pET-proMxHA2 and carrier pJG4-5 (AD), pLacZi (BD), respectively glue recycle purpose endonuclease bamhi, obtained after connection recombinant expression carrier pJG4-5-MxERF72 (AD-MxERF72), pLacZi-proMxHA2(BD-proMxHA2).Recombinant expression carrier AD-MxERF72 and BD-proMxHA2 are converted into large intestine bar Bacterium DH5 α competent cells are chosen after spot shakes bacterium and carry out bacterium colony PCR verifications, and the results are shown in Figure 6 for agarose gel electrophoresis detection, Wherein Fig. 6 (A) swimming lanes M is the DNA molecular amount standard of 2000bp, and swimming lane 1 is to be expanded in the recombinant bacterium for turning to have AD-MxERF72 MxERF72's as a result, swimming lane 2 is using water as the negative control of template amplification MxERF72；Fig. 6 (B) swimming lanes M is the DNA of 2000bp Molecular weight standard, swimming lane 1 are to turn to have in the recombinant bacterium of BD-proMxHA2 amplification proMxHA2's as a result, swimming lane 2 is using water as mould Plate expands the negative control of proMxHA2.It is that positive bacterial plaque shakes bacterium to qualification result, extraction plasmid carries out sequence verification.Sequencing The result shows that AD-MxERF72 is the expression vector containing MxERF72, BD-proMxHA2 is that the expression containing proMxHA2 carries Body.

Using Yeast Transformation Kit, by carrier AD-MxERF72/BD-proMxHA2, AD-MxERF72/ of various combination BD, AD/BD-proMxHA2, AD/BD are transformed into respectively in yeast strain E GY48, after growing single bacterium colony, picking positive bacteria drop point In carrying out chromogenic reaction 2-3 days on SD/-Trp-Ura solid mediums X-gal colour developing tablets, the results are shown in Figure 7, AD- The aobvious blue of MxERF72/BD-proMxHA2 single crosses, and the not aobvious blue of other three kinds of single crosses, illustrate transcription factor MxERF72 It can be combined with each other with MxHA2 gene promoters.

Three, yeast two-hybrid verifies MxERF72 and MxERF4 interactions

With BamHI and XhoI double digestions pET-MxERF72 and yeast vector pGADT7, difference glue recycles purpose digestion piece Section, obtains the carrier segment of the MxERF72 and 7987bp of 1161bp, recombinant expression carrier pGADT7- is obtained after connection MxERF72.Recombinant expression carrier pGADT7-MxERF72 is converted into bacillus coli DH 5 alpha competent cell, chooses after spot shakes bacterium and carries out Bacterium colony PCR verifications, as a result as shown in Fig. 8 (A), wherein swimming lane M is the DNA molecular amount standard of 2000bp, and swimming lane 1 is pGADT7- The positive bacterial plaque PCR of MxERF72 is as a result, swimming lane 2 is using water as the negative control of template amplification MxERF72.It is to qualification result Positive bacterial plaque shakes bacterium, and extraction plasmid carries out sequence verification.Sequencing result shows that pGADT7-MxERF72 is containing MxERF72's Expression vector.

(MxERF4 shown in sequence 3 is inserted into pEASY-T1-simple with EcoRI and BamHI double digestions pET-MxERF4 In, obtained carrier.) and yeast vector pGBKT7, respectively glue recycle purpose endonuclease bamhi, obtain 693bp MxERF72 and The carrier segment of 7300bp, obtains recombinant expression carrier pGBKT7-MxERF4 after connection.By recombinant expression carrier pGBKT7- MxERF4 converts bacillus coli DH 5 alpha competent cell, chooses after spot shakes bacterium and carries out bacterium colony PCR verifications, as a result as shown in Fig. 8 (B), Wherein swimming lane M is the DNA molecular amount standard of 2000bp, and swimming lane 1 is the positive bacterial plaque PCR of pGBKT7-MxERF4 as a result, swimming lane 2 For using water as the negative control of template amplification MxERF4.It is that positive bacterial plaque shakes bacterium to qualification result, extraction plasmid is sequenced Verification.Sequencing result shows that pGBKT7-MxERF4 is the expression vector containing MxERF4.

Using Clontech Yeast Transformation Kits, by carrier pGADT7/pGBKT7, pGADT7- of various combination MxERF72/pGBKT7, pGADT7/pGBKT7-MxERF4, pGADT7-MxERF72/pGBKT7-MxERF4 are transformed into ferment respectively In mother strains AH109, and upgrading grain is verified, positive colony is lined respectively two lack and four scarce deficiency culture mediums on, And make colour developing experiment, the results are shown in Figure 9, and the yeast strain containing pGADT7-MxER72/pGBKT7-MxERF4 can lack four Grown on culture medium, and develop the color au bleu, and other three kinds conversion yeast can only on two scarce culture mediums normal growth, four It is apparent suppressed to lack growth on culture medium, does not develop the color.Illustrate that there are interactions between MxERF72 and MxERF4.

Four, bimolecular fluorescence complementary verifies MxERF72 and MxERF4 interactions

It is carried with XbaI and BamHI double digestions pET-MxERF72, pET-MxERF4 and pSPYNE-35S and pSPYCE-35S Body, glue recycling purpose endonuclease bamhi, obtains recombinant expression carrier MxERF72-YFP respectively after connection^N、MxERF72-YFP^C、 MxERF4-YFP^NWith MxERF4-YFP^C.Recombinant expression carrier is converted into bacillus coli DH 5 alpha competent cell, choosing spot, to shake bacterium laggard Positive colony, is shaken bacterium upgrading grain and carries out bacterium colony PCR, the results are shown in Figure 10, wherein Figure 10 (A) by the PCR verifications of row bacterium colony respectively Swimming lane M is the DNA molecular amount standard of 2000bp, and swimming lane 1 is MxERF72-YFP^NPositive bacterial plaque PCR is as a result, swimming lane 2 is to be with water The negative control of template amplification MxERF72；Figure 10 (B) swimming lanes M is the DNA molecular amount standard of 2000bp, and swimming lane 1 is MxERF72- YFP^CPositive bacterial plaque PCR is as a result, swimming lane 2 is using water as the negative control of template amplification MxERF72；Figure 10 (C) swimming lanes M is The DNA molecular amount standard of 2000bp, swimming lane 1 are MxERF4-YFP^NPositive bacterial plaque PCR is as a result, swimming lane 2 is using water as template amplification The negative control of MxERF4；Figure 10 (D) swimming lanes M is the DNA molecular amount standard of 2000bp, and swimming lane 1 is MxERF4-YFP^CPositive bacteria Spot PCR is as a result, swimming lane 2 is using water as the negative control of template amplification MxERF4.It is that positive bacterial plaque shakes bacterium to qualification result, carries Plasmid is taken to carry out sequence verification.Sequencing result shows MxERF72-YFP^N、MxERF72-YFP^CFor the carrier containing MxERF72, MxERF4-YFP^N、MxERF4-YFP^CFor the carrier containing MxERF4.

Carrier bacterium solution is pressed into MxERF72-YFP^N/MxERF4-YFP^C、MxERF72-YFP^N/YFP^C、YFP^N/MxERF4- YFP^C、YFP^N/YFP^CCombo, combination of two and mixed bacteria liquid, co-injection tobacco (university degree gave birth to cigarette in 5 weeks) blade, after injecting 2-4 days It is observed using Nikon D-ECLIPSE C1 Spectral Confocal laser scanning systems, uses 488nm exciting lights.Use ECLIPSE TE2000-E inverted fluorescence microscopes carry out fluorescence analysis.

As a result such as Figure 11, MxERF72-YFP^N/MxERF4-YFP^CCombination has green fluorescence, table on Tobacco Epidermis core Bright MxERF72 and MxERF4 is positioned in nucleus, and there are physics interactions.

Five, transgenosis verifies MxERF72 functions

1, the transgenosis verification of silence MxERF72 gene expressions

1), the structure of the recombinant expression carrier of silence MxERF72 gene expressions

With EcoRI and XbaI double digestion pET-MxERF72 3'UTR sequences (sequence 4) and with GFP carriers pTRV2 (notes It carries in the following literature, Tian, J.et al., TRV-GFP:a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function.Journal of Experimental Botany.2014,65:311-322. or less carried pTRV2 carriers all carry GFP labels), glue returns respectively Purpose endonuclease bamhi is received, the MxERF72 3'UTR sequences of 237bp and the carrier segment of 9663bp is obtained, is recombinated after connection Expression vector MxERF72-pTRV2.

Recombinant expression carrier MxERF72-TRV2 is converted into bacillus coli DH 5 alpha competent cell, chooses after spot shakes bacterium and carries out bacterium PCR verifications are fallen, positive colony progress bacterium colony PCR results are as shown in figure 12, and wherein swimming lane M is the DNA molecular amount standard of 2000bp, Swimming lane 1 is positive colony PCR as a result, swimming lane 2 is using water as the negative control of template amplification MxERF72.It is sun to qualification result Property bacterial plaque shake bacterium, extraction plasmid carries out sequence verification.

Sequencing result shows that MxERF72-TRV2 is that MxERF72 3'UTR shown in sequence 4 are replaced carrier pTRV2's The carrier that segment between EcoRI and XbaI double digestions obtains.

Bacterium containing the carrier is named as DH5 α/MxERF72-TRV2.

2), the acquisition of the malus xiaojinensis of silence MxERF72

MxERF72-TRV2 Agrobacteriums bacterium solution, pTRV2 Agrobacteriums bacterium solution and pTRV1 Agrobacterium bacterium solutions prepare as follows：First By MxERF72-TRV2, pTRV1 and pTRV2 vector plasmid (pTRV1 and pTRV2 carriers record in the following literature, Tian, J.et al.,TRV–GFP:a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function.Journal of Experimental Botany.2014, 65:311-322) it converts in Agrobacterium GV1301 bacterial strains, culture is containing 50 μ g/ml cards that and the 50 dual anti-trainings of μ g/ml rifampins Support and cultivate 2-3 days in base, choose single bacterium colony progress bacterium colony PCR verifications, by positive colony in containing 50 μ g/ml cards that with 50 μ g/ml 28 DEG C of 200rmp are incubated overnight in the LB culture mediums of rifampin, i.e., prepared by vector Agrobacterium liquid completes.

Using vacuum infiltration method, by DH5 α/MxERF72-pTRV2 and pTRV1 Agrobacterium bacterium solutions and pTRV2 Agrobacterium bacterium Liquid and pTRV1 vector Agrobacteriums bacterium solution press volume 1 respectively:1 mixing (MxERF4-pTRV2:PTRV1=1:1, pTRV2:pTRV1 =1:1) wild type malus xiaojinensis tissue-cultured seedling Rooted Cuttings, are transferred to two-by-two respectively, obtain turning MxERF72-pTRV2:The seas pTRV1 little Jin Chinese bush cherry and turn pTRV2:PTRV1 malus xiaojinensis.

Using ZEISS Stereo fluorescence microscopes to turning MxERF72-pTRV2:PTRV1 malus xiaojinensis (TRV-GFP- MxERF4) and turn pTRV2:PTRV1 malus xiaojinensis (TRV-GFP) carries out GFP Fluirescence observations (Figure 13), shows that carrier successfully turns Enter, turns MxERF72-pTRV2:PTRV1 malus xiaojinensis (TRV-GFP-MxERF72) is denoted as the malus xiaojinensis of silence MxERF72.

With the total serum IgE of the malus xiaojinensis root for the silence MxERF72 for cultivating 12 days (d) after vacuumizing, ultraviolet specrophotometer The content for measuring the RNA of different disposal in root such as takes at the above-mentioned RNA of quality, and reverse transcription is at single-stranded cDNA.Design MxERF72, The real-time primers of MxHA2, the cDNA of the malus xiaojinensis root of silence MxERF72 is template after being used in combination iron deficiency to handle, with two steps The feasibility of method PCR and standard curve verification primer, it is specific as follows：

The cDNA of malus xiaojinensis root (TRV-MxERF72) #2, #3, #4 of silence MxERF72 is template after being handled using iron deficiency, Using β-actin as internal reference, expressions of analysis MxERF72, the MxHA2 in malus xiaojinensis root.Wild type after being handled with iron deficiency Malus xiaojinensis is control (CK), turns pTRV2 after being handled with iron deficiency:PTRV1 malus xiaojinensis is control (TRV#2, #3).

Wherein, the real-time amplimers of MxERF72 are RT-F1 and RT-R1, and the real-time amplifications of MxHA2 are drawn Object is RT-F2 and RT-R2：

RT-F1：5 '-TTGTTGGGGGAGTTTTTTAAGC-3 ',

RT-R1：5′-ATGACATTAGCTACGGCTT-3′；

RT-F2：5 '-ATCTGCCCTTACTGGGGAGT-3 ',

RT-R2：5′-AAATGCAGAAGTTCCCGATG-3′；

The primer of β-actin is β-actin-F and β-actin-R：

β-actin-F：5 '-TGGTGAGGCTCTATTCCAAC-3 ',

β-actin-R：5′-TGGCATATACTCTGGAGGCT-3′.

As a result as shown in figure 14,3 strains (TRV-MxERF72) #2, #3, #4 of the malus xiaojinensis of silence MxERF72 are lacked The expression quantity of root MxERF72 is less than wild type malus xiaojinensis under the conditions of iron, it was demonstrated that in the malus xiaojinensis of silence MxERF72 MxERF72 is silenced (left figure).In addition, compared with wild type malus xiaojinensis, under the conditions of the malus xiaojinensis iron deficiency of silence MxERF72 The expression quantity of root MxHA2 expression quantity significantly raises (right figure).Speculate that MxERF72 is inhibited to the transcription of MxHA2, card Bright MxERF72 inhibits target gene to transcribe, and after silence, target gene transcriptional level improves.

Turn pTRV2 after wild type malus xiaojinensis and iron deficiency processing:PTRV1 malus xiaojinensis is without significant difference.

2, it is overexpressed the transgenosis verification of MxERF72

1) acquisition of expression vector

With XbaI and BamHI double digestions pET-MxERF72 and carrier pBI121 (Tian, J.et al., McMYB10 regulates coloration via activating McF30H and later structural genes in ever-red leaf crabapple.Plant Biotechnology Journal,2015,13:948-961.), difference glue Purpose endonuclease bamhi is recycled, the MxERF72CDS sequences of 1161bp and the carrier segment of 14752bp is obtained, is recombinated after connection Expression vector MxERF72-pBI121.

Recombinant expression carrier MxERF72-pBI121 is converted into bacillus coli DH 5 alpha competent cell, chooses after spot shakes bacterium and carries out Positive colony is shaken bacterium extraction plasmid order-checking verification by bacterium colony PCR verifications, and sequencing result shows that MxERF72-pBI121 is by sequence MxERF72 shown in sequence 2 replaces the plasmid between XbaI the and BamHI double digestions of pBI121 carriers in table.

Bacterium containing the carrier is named as DH5 α/MxERF72-pBI121.

2), turn the acquisition of MxERF72 tobaccos (overexpression)

Using tobacco injection method, MxERF72-pBI121 carriers and zero load pBI121 are converted into Agrobacterium GV3101, name For GV3101/MxERF72-pBI121 and GV3101/pBI121.By positive colony GV3101/MxERF72-pBI121 and GV3101/pBI121 shakes bacterium overnight, then by GV3101/pBI121 bacterium solutions (empty vector) and GV3101/MxERF72- PBI121 bacterium solutions (MxERF72) equivalent injects same this life Tobacco Leaves (Tian, J.et al., TRV-GFP:a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function.Journal of Experimental Botany.2014,65:311-322.), the left half vane of vein and the right side Half vane, 16 hours (250 μm of olm of photon hypothesis of illumination cultivation after culturing room is protected from light for 24 hours^-2·s^-1), temperature 25 ±2℃；Dark culturing 8 hours, temperature are 17 ± 2 DEG C and cultivate 10 days.Camera is taken pictures, phenotypic map such as Figure 15, GV3101/ Chlorophyll content in leaf blades after the injection of MxERF72-pBI121 bacterium solutions is less than the blade after the injection of GV3101/pBI121 bacterium solutions.

By GV1301/pBI121 bacterium solutions inject after blade and GV1301/MxERF72-pBI121 injection after blade in It is placed at room temperature for 20min in darkroom, chlorophyll fluorescence detection (Karel is carried out with chlorophyll fluorescence instrumentK.et al.Relaxation of the non-photochemical chlorophyll fluorescence quenching in diatoms:kinetics,components and mechanisms.Phil.Trans.R.Soc.B 2013.369:1-7.), Three samples of each processing detection, as a result as shown in figure 16.

The blade after blade and GV1301/MxERF72-pBI121 injections after GV1301/pBI121 bacterium solutions are injected is adopted Chlorophyll content detection is carried out with Chlorophyll meter, three samples of each processing detection, as a result as shown in figure 17.

The above results show that the blade Determination of Chlorophyll content after GV1301/MxERF72-pBI121 injections is less than Blade after the injection of GV1301/pBI121 bacterium solutions illustrates that being overexpressed MxERF72 promotes chlorophyll degradation, makes chlorophyll content drop It is low.

Sequence table

<110>China Agricultural University

<120>A kind of transcription factor MxERF72 and its encoding gene and application

<160>4

<210> 1

<211> 386

<212> PRT

<213>Malus xiaojinensis (Malus xiaojinensis)

<400> 1

Met Cys Gly Gly Ala Ile Ile Ser Asp Phe Ile Ala Pro Val Arg Ser

1 5 10 15

Arg Arg Leu Thr Ala Asp Tyr Leu Trp Pro Asp Leu Lys Lys Pro Ser

20 25 30

Ser Gly Lys Arg Leu Ser Lys Pro Leu Lys Pro Glu Ile Val Asp Leu

35 40 45

Asp Asp Asp Phe Glu Ala Asp Phe Gln Glu Phe Lys Asp Glu Ser Asp

50 55 60

Val Asp Glu Asp Asp Glu Met Val Asp Ser Lys Pro Ser Ala Phe Ser

65 70 75 80

Ala Gly Asn Pro Ser Phe Ala Arg Gly Ser Thr Ala Val Lys Ser Val

85 90 95

Glu Phe Asp Gly Arg Ala Glu Lys Ser Ala Lys Arg Lys Arg Lys Asn

100 105 110

Gln Tyr Arg Gly Ile Arg Gln Arg Pro Gly Gly Lys Trp Ala Ala Glu

115 120 125

Ile Arg Asp Pro Arg Lys Gly Val Arg Val Trp Leu Gly Thr Phe Asn

130 135 140

Thr Ala Glu Glu Ala Ala Arg Ala Tyr Asp Ala Glu Ala Arg Arg Ile

145 150 155 160

Arg Gly Lys Lys Ala Lys Val Asn Phe Pro Glu Glu Thr Pro Cys Ala

165 170 175

Ser Ala Lys Arg Ser Ile Lys Glu Asn Pro Gln Lys Leu Ile Ala Lys

180 185 190

Thr Asn Leu Asn Gly Thr Gln Ser Asn Pro Asn Gln Lys Phe Asn Phe

195 200 205

Val Asn Asp Ser Ser Glu Asp Tyr Tyr Ser Ala Leu Gly Phe Leu Asp

210 215 220

Glu Lys Pro Thr Met Asn Asn Phe Arg Tyr Met Ser Thr Phe Pro Ala

225 230 235 240

Asn Glu Asp Val Ala Leu Lys Ser Ser Val Pro Ser Glu Asn Ala Pro

245 250 255

Phe Tyr Phe Ser Ser Asp Gln Gly Ser Asn Ser Phe Asp Cys Ser Asp

260 265 270

Phe Gly Trp Gly Glu Gln Gly Ser Lys Thr Pro Glu Ile Ser Ser Val

275 280 285

Ile Ser Ser Val Met Glu Glu Ser Asp Asn Ser Pro Phe Leu Glu Asp

290 295 300

Ser Asn Pro Thr Lys Lys Met Lys Pro Asn Ser Gln Asp Leu Glu Pro

305 310 315 320

Pro Glu Asp Asn Ser Gly Lys Thr Leu Ser Asp Glu Leu Ser Ala Phe

325 330 335

Glu Met Lys Tyr Phe Gln Thr Pro Tyr Leu Asp Glu Ser Trp Asp Ala

340 345 350

Ser Val Asp Ala Phe Leu Asn Gly Asp Ala Thr Gln Asp Gly Gly Asn

355 360 365

Pro Met Gly Leu Trp Ser Phe Asp Asp Leu Pro Ala Ile Val Gly Gly

370 375 380

Val Phe

385

<210> 2

<211> 1161

<212> DNA

<213>Malus xiaojinensis (Malus xiaojinensis)

<400> 2

atgtgtggag gtgctattat ttccgatttc atagcgccgg tacggtcccg ccggctcacg 60

gccgactacc tctggccgga tctcaaaaaa cccagttctg ggaagcggct ctcgaagcct 120

ctgaagcccg aaatcgttga cttggacgac gacttcgagg ctgatttcca ggagttcaag 180

gacgagtccg atgtggacga ggacgatgaa atggttgatt ccaagccctc tgctttctct 240

gccggaaacc cctcttttgc tcgtggttct accgctgtga aatctgtgga gtttgatggg 300

cgagctgaga aatctgcaaa gagaaaaagg aagaaccagt acaggggaat tcgccagcgc 360

ccagggggta agtgggctgc agagatccga gacccaagga aaggggttcg ggtttggctt 420

ggaacattca acactgcaga agaagctgca agggcgtatg atgccgaggc acgtagaatt 480

cgcggcaaga aagcgaaggt taatttccct gaagaaactc cttgtgcttc tgcaaagcgt 540

tccatcaagg aaaatcctca gaaattgata gccaagacaa acttgaatgg cactcagtct 600

aatccgaacc agaagttcaa ttttgtgaac gactcaagtg aggactacta cagtgctctg 660

ggttttctgg atgaaaagcc aacaatgaat aactttcgat atatgtccac cttccctgcc 720

aatgaagatg ttgcactgaa atcctctgtt ccatcagaaa atgccccttt ttacttcagt 780

tccgatcagg gaagcaactc ctttgattgt tctgactttg gctggggaga acaaggctcg 840

aagactccag aaatctcatc cgttatttca tctgttatgg aagaaagtga taactcaccg 900

tttctggagg attctaaccc aacaaagaag atgaagccta actcacagga tctggagcct 960

cctgaggata attcgggaaa gacactgtct gatgagctct cagcttttga gatgaagtac 1020

tttcagaccc catatctcga tgagagctgg gatgcttcag tggacgcgtt cctcaacggt 1080

gacgctactc aggatggtgg taatccaatg ggcctttgga gctttgatga tctgcccgca 1140

attgttgggg gagtttttta a 1161

<210> 3

<211> 693

<212> DNA

<213>Malus xiaojinensis (Malus xiaojinensis)

<400> 3

atggctccga cgacggcgaa gtccctaccc aaatccggct ctgaggacca gaatccgaca 60

tccaacgaga tccggtaccg gggtgtccgg aagaggcctt ggggacgcta cgccgccgag 120

atcagagatc ccgggaagaa gacccgcgtc tggcttggga ccttcgacac cgccgaggag 180

gcagcgcgtg cgtacgacaa ggccgcgcgt gagtttcgcg gaggcaaggc gaagactaac 240

ttccccaccc cctctgagct ccagctcgac gccgtcaaca tcgtcaacat gaacaacgcc 300

gccaaatcag cggcggcgac gaacgtcagc aacagtccca gcagccagag cagcaccgtg 360

gagtcctcct cgccgccgcc tccgccgcca ctcgacctca ctctcaaaaa cccccgtttc 420

tccaccggcg gcggctactt caccgctgcg agcggcttcc gcccgctccc cgtgccccgt 480

cccgtgttct tcttcgacgc cttcgcacgg gccgacggcg ccgccgccct ccagctcgcc 540

cgcgaaactt gcaggttcga ccgccccgct cccatatccg gctcggccca cagcgactcc 600

tccacgtcct cggtcgtcga cttcgagcgt agctcccgca acgtacggct cgatctcgac 660

ctgaacctcc cagccccctc ggaagtcgcc taa 693

<210> 4

<211> 237

<212> DNA

<213>Artificial sequence

<220>

<223>

<400>4

atgacattag ctacggctta aaatactccc cgcacaggcc ttatgcatgt tcaacagcct 60

acacagttgc actccaaccc gacaactctt tctggataaa atgggagcga acacgagacc 120

aataatgaaa caatgtagaa gctcacagag gactgaaaaa ctcactaaca tgtagcttta 180

tttacataaa ctagaaaggt ggggaaaggt tcatcgctta aaaaactccc ccaacaa 237

Claims

1. a kind of albumen, be it is following 1) or 2)：

1) protein shown in sequence 1 in sequence table；

2) substitution by amino acid sequence shown in sequence in sequence table 1 by one or several amino acid residues and/or missing And/or it adds and there is identical function protein derived from sequence 1.

2. encoding the DNA molecular of protein described in claim 1.

3. DNA molecular as claimed in claim 2, it is characterised in that：The DNA molecular is following 1) -3) in any DNA Molecule：

1) code area is DNA molecular shown in sequence 2 in sequence table；

3) with the DNA sequence dna that 1) limits at least with 70%, at least with 75%, at least with 80%, at least with 85%, extremely Less with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% or at least with 99% The DNA molecular of homology and coding with identical function protein.

4. the recombinant vector, expression cassette, transgenic cell line containing DNA molecular described in Claims 2 or 3 or recombinant bacterium.

Or, the primer pair of DNA molecular overall length or its arbitrary segment described in amplification Claims 2 or 3.

5. recombinant vector described in DNA molecular or claim 4 described in albumen, Claims 2 or 3 described in claim 1, expression The application of box, transgenic cell line or recombinant bacterium in regulating and controlling plant tissue in Gene Transcription in vitro；

Or recombinant vector described in DNA molecular or claim 4 described in albumen, Claims 2 or 3 described in claim 1, expression The application of box, transgenic cell line or recombinant bacterium in regulation and control content of chlorophyll in plant or in regulating and controlling plant chlorophyll degradation；

Or albumen described in claim 1 is as the application in transcriptional activation repressor.

6. application according to claim 5, it is characterised in that：The regulation and control under the conditions of iron deficiency carrying out.

7. a method of the low genetically modified plants of chlorophyll content are cultivated, are included the following steps：Improve coding claim 1 institute It states the expression quantity of the DNA molecular of albumen or activity obtains genetically modified plants, the chlorophyll content of the genetically modified plants is less than institute State purpose plant.

8. application according to claim 5 or 6 or the method described in 7, it is characterised in that：

It is described to improve the expression quantity of the DNA molecular of albumen or activity described in coding claim 1 as that encode described in claim 1 The DNA molecular of albumen imports purpose plant.

9. application according to claim 5 or 6 or the method described in 7, it is characterised in that：The plant is dicotyledon Or monocotyledon.

10. application according to claim 5 or 6 or the method described in 7, it is characterised in that：

The plant is dicotyledon, and the dicotyledon is malus xiaojinensis or tobacco.