CN102220370A - Application of gene EIN3 and gene EIL1 in promoting growth of plant root hair - Google Patents

Abstract

The invention provides an application of gene EIN3 and gene EIL1 in promoting the growth of plant root hair. The application is that the gene EIN3 and the gene EIL1 are expressed in the plant through a genetic engineering method. Through carrying out a molecular biological mechanism research on the gene EIN3 and the gene EIL1 in the mode plant Arabidopis thaliana, the nutrition absorption efficiency of real crops is improved by over expressing the gene EIN3 and the gene EIL1 in the plant, and the adverse stress resisting capacity of the crops is enhanced, so yield increase in the agriculture is achieved.

Description

Gene EIN3 and EIL1 are promoting the developmental application of plant root hair

Technical field

The invention belongs to the genetically engineered field, specifically, relate to gene EIN3 and EIL1 and promoting the developmental application of plant root hair.

Background technology

Along with population support's growth, how improving crop yield is a problem demanding prompt solution to keep human survival.The Green Revolution first time that the middle of last century proposes, a large amount of agricultural chemicals and chemical fertilizer of using though improved the output of crop, have brought very serious problem thereupon in agriculture production.The excessive use of chemical fertilizer and agricultural chemicals not only causes a large amount of wastes, and be proved to be extremely harmful to human health, the more important thing is the excessive chemical fertilizer and the pesticidal contamination face of land, underground water, and can decomposite a large amount of greenhouse gases that this has caused that the whole world pays close attention to widely.The Green Revolution second time at this problem proposes is intended to utilize molecular biological research and genetic engineering technique, and the efficient that improves dietetic alimentation from crop strengthens resistance, thereby realizes eco-friendly volume increase.

The root hair is the vitals that higher plant absorbs nutrition, and plant root can form many hairs in adverse circumstance, has increased root and the surface-area that soil directly contacts greatly, improves the efficient of plant to dietetic alimentation.Concerning the Green Revolution of future generation, regulating the plant root system development pattern is a brand-new breach, by increasing the length and the density of root hair, can improve the dietetic alimentation efficient of plant.The plasticity-height of plant root hair.With the Arabidopis thaliana is example, and generally the epiblem cell development is divided into two kinds of cells, hair cell and non-hair cell.In the maturation zone of root, most of become hair cell to tend to form projection at outside surface, and and then perpendicular to stem to elongation, grow and be the root hair; Non-hair cell with become hair cell alternately, experience different position signals, do not form the root hair usually.

Known have multiple inside and outside source to stimulate the generation and the elongation that can promote the root hair, increases the mean length of root hair, further improves into the ratio that hair cell grows the root hair, perhaps lures non-hair cell send out roots hair, i.e. dystopy root hair into.Various stimulations comprise nutritive deficiency (such as scarce phosphorus, iron deficiency etc.), the stimulation of hormone (such as growth hormone, ethene etc.).

Summary of the invention

The purpose of this invention is to provide gene EIN3 and EIL1 and promoting the developmental application of plant root hair.

In order to realize the object of the invention, gene EIN3 of the present invention and EIL1 are promoting the developmental application of plant root hair, and it is to cross expressing gene EIN3 and EIL1 by engineered method in plant.Preferably, described plant is an Arabidopis thaliana.

The nucleotide sequence of gene EIN3 and EIN1 is respectively shown in Seq ID No:1 and Seq ID No:2.

Exogenous ethylene can promote generation, the elongation of root hair greatly, and this effect is to realize by transcription factor EIN3 important in the ethylene signaling and EIL1.EIN3 and EIL1 can integrate multiple inside and outside source to be stimulated, and is node important in the plant root hair developmental regulation.The research aspect the molecular biology mechanism in the model plant Arabidopis thaliana by EIN3 and EIL1, find that the arabidopsis mutant body that lacks these two transcription factors presents the phenotype that significant root hair reduces, and induce them in plant, to accumulate, can increase the length and the density of root hair greatly.In crop, cross expression EIN3 and EIL1 by genetic engineering means, improve the dietetic alimentation efficient of actual crop, strengthen crop and resist the ability of environment stress, thereby reach the volume increase purpose on the agricultural.

Description of drawings

Fig. 1 is the result that Col-0 wild-type, ein2-5 mutant, ein3 eil1 mutant, ctr1-1 mutant, EIN3ox transgenic plant vertically grew 6 days on the substratum that add/does not add ACC respectively.

Fig. 2 has integrated the inside and outside source signal of stimulation root hair growth for EIN3 and EIL1.Col-0 wild-type, ein3 eil1 mutant be at the iron deficiency substratum, the growing state on the 3 μ M ABA substratum.

Fig. 3 directly causes the growth of root hair vigorous for the proteic accumulation of EIN3.Growing state after β-oestrogenic hormon of transgenic plant induction type EIN3 (ein3 eil1 ebf1 ebf2 quadruple mutant background) interpolation different concns is handled.

Fig. 4 is for after adding β-oestrogenic hormon, and EIN3 albumen is in the confirmatory experiment result of plant interior accumulation.

Fig. 5 is the result that the EIL1ox transgenic plant vertically grew 6 days on the MS substratum.

Embodiment

Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.

The various mutant that use in following examples are all available from ABRC, and various medicine and reagents are if no special instructions all available from Sigma company.

Embodiment 1 EIN3 and EIL1 are the important transcription factors of regulation and control root hair growth

CTR1 is the negative regulatory factor in the Ethylene Signal path, and CTR1 has not closed the ethylene reaction in downstream when having ethene, and Arabidopis thaliana can be closed the inhibit feature of CTR1 to the downstream after experiencing Ethylene Signal.EIN2 is the positive regulatory factor in the Ethylene Signal approach, and it can promote the accumulation of transcription factor EIN3 and EIL1, and EIN3 and EIL1 transcribe various ethylene responses genes.With insensitive mutant ein2-5 of ethene and ein3 eil1, constitutive mutation body ctr1-1, hypersensitization mutant EIN3ox sows respectively that (ethene synthesizes precursor at MS (available from PTL company) and ACC, 10 μ M) on the substratum, 4 ℃ of imbibitions were placed in 22 ℃ of long day greenhouses in 3 days vertically cultivated 6 days, observed phenotype.The mutant ctr1-1 that the discovery composing type is opened the Ethylene Signal path has very many root hairs, and the root hair of blocking the non-sensitive type mutant ein2-5 of Ethylene Signal path and ein3 eil1 double mutant fully seldom, and only hair is also very short, is difficult to observe.Ein3 eil1 mutant is applied the growth that exogenous ethylene can not promote the root hair.Yet crossing the seedling root of expressing EIN3, transgenosis have significantly a large amount of root hairs to form.A pair of transcription factor EIN3 and EIL1 are necessary to the growth of Arabidopis thaliana root hair in this explanation Ethylene Signal path.(Fig. 1)

Embodiment 2 EIN3 and EIL1 have integrated the inside and outside source signal that stimulates the growth of root hair

Known iron deficiency can lure that the root hair increases into, and the hormone ABA that simulating drought is coerced handles the growth that also can promote the root hair.A. ein3 eil1 is sowed on the MS substratum, light was vertically cultivated 3 days down after the imbibition, then seedling was transferred on the MS substratum that has added 3 μ M ABA, continued to cultivate after 3 days under the light and observed phenotype.B. with same planting seed (5mM KNO on MS ' substratum ₃, 2mM MgSO ₄, 2mM Ca (NO ₃) ₂, 2.5mM K ₂PO ₄, 70 μ MH ₃BO ₃, 14 μ M MnCl ₂, 1 μ M ZnSO ₄, 0.5 μ M CuSO ₄, 10uM NaCl, 0.2 μ MNa ₂MoO ₄40 μ M FeEDTA, 0.8% agar, pH=5.7), same method cultivate and transfer to after 3 days on the iron deficiency substratum (MS ' do not add 40 μ M FeEDTA in the substratum, add 100 μ M 5,6-phenylbenzene-3-(2-pyridyl)-1,2,4-triazine-4,4 '-disulfonic acid disodium salt) under light, cultivate after 3 days and observe phenotype.The seedling root of contrast wild-type Arabidopis thaliana and ein3 eil1 mutant can find that the root hair that iron deficiency causes increases the living EIN3 and the EIL1 of all depending on of root hair of growth and ABA stimulation.The mutant that lacks EIN3 and EIL1 can not respond these exogenous stimulations, remains root hair phenotype seldom.Illustrate that EIN3 and EIL1 are the important node of integrating the stimulus that these root hairs educate.(Fig. 2)

The proteic accumulation of embodiment 3 EIN3 directly causes the growth of root hair vigorous

Make up β-estrogen-induced and express the clone of EIN3, be transformed in the ein3 eil1 ebf1 ebf2 quadruple mutant, EIN3 and the proteic EBF1 of EIL1 or EBF2 exist owing to degrade, the external source EIN3 albumen of β-estrogen-induced can be in plant stable existence.On the MS substratum, light growth down shifts seedling to the MS substratum that has added β-oestrogenic hormon (10 μ M) after 3 days, vertically cultivate after 3 days under the light again and observe phenotype with transgenosis homozygote planting seed.Do not adding estrogen-induced, promptly do not having to be difficult to observe the root hair in the proteic control group experiment of EIN3 and EIL1; And when having added the protein product stable existence of β-estrogen-induced EIN3 genetic expression and translation, can see that the plant seedlings root has grown a large amount of root hairs.As long as this explanation induces EIN3 in the intravital accumulation of plant, just can make plant grow more root hair, EIN3 is the sufficient condition (Fig. 3 and Fig. 4) that the root hair is educated.Fig. 5 is the result that the EIL1ox transgenic plant vertically grew 6 days on the MS substratum, same as the important transcription factor in the Ethylene Signal path, also the root hair than wild-type is many to cross the transgenic plant EIL1ox of expression EIL1 (EIN3-LIKE1), and the same growth that can promote plant root hair with EIN3 of EIL1 is described.

Owing to can constantly be degraded by F-box albumen EBF1/2 after endogenous EIN3 albumen is synthetic, for can manually operated stable EIN3 albumen at plant interior expression, the present invention has made up transgenic plant pER8:EIN3-FLAG/ein3 eil1 ebf1 ebf2, but the proteic accumulation of EIN3 in the inducing plant body when applying the β of external source-oestrogenic hormon, and, realize the variation of protein content by applying different concns or different treatment time.The method that makes up transgenic plant pER8:EIN3-FLAG/ein3 eil1ebf1 ebf2 is:

1.PCR amplification target fragment EIN3 gene coding region, cut target fragment and plasmid pER8 (Jianru Zuo etc. by the endonuclease enzyme, An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants, The Plant Journal, 2000, building the scholar laboratory by heredity institute an of Chinese Academy of Sciences left side is so kind as to give) the generation cohesive terminus, fragment is connected with ligase enzyme with plasmid, (building process is seen Chen etc. to obtain plasmid pER8:EIN3-FLAG by the order-checking evaluation, ETHYLENEINSENSITIVE3 and ETHYLENE INSENSITIVE3-LIKE1 re-press SALICYLIC ACID INDUCTION DEFICIENT2 expression to neg-atively regulate plant innate immunity in Arabidopsis, Plant Cell, 2009).

2. get 2 μ l plasmids and add among the 100 μ l Agrobacterium GV3101,2200V electric shock back adds 500-800 μ l LB substratum rapidly, and 28 ℃ of 220rpm cultivated 1.5 hours, were evenly coated on the 40 μ g/ml spectinomycin LB substratum, were inverted for 28 ℃ and cultivated 1-2 days;

3. inoculate single bacterium colony in 5ml LB substratum (Rifampin 50 μ g/ml+ spectinomycins 40 μ g/ml; Also can only add the transgenosis resistance), 28 ℃ of 220rpm overnight incubation;

4. contain among the suitable antibiotic LB in 400mL (adjusting) with 1: 100 enlarged culturing according to plant quantity, continue to shake about training 5-8hr to stand density be OD600 value 1.0-1.2;

5. room temperature, 4000g, centrifugal 10min collects thalline;

6. with conversion medium (5% sucrose, 2.033g/L MgCl ₂) the suspension thalline; Silwet L-77 (200 μ L/L) has injury to bacterium liquid, should add before transforming plant;

7. the conversion medium that will contain Agrobacterium is poured in the beaker, and the Arabidopis thaliana of just having bloomed is inverted on it, makes whole inflorescence all immerse (inflorescence of lotus throne base portion can water pouring with rifle) in the conversion medium, 30s～120s;

8. taking-up Arabidopis thaliana, it is lain on one's side is placed on the clean plastic pallet, and covers the lucifuge 24hr that preserves moisture with film;

9. Arabidopis thaliana is propped up, normal light is cultivated down, about 3-4 after week silique withered and yellow fully, when desire ftractures, can gather in the crops seed;

10. the MS substratum with 50 μ g/ml Totomycin screens transgenic plant.

In sum, discover that through the present invention two transcription factor EIN3 of Arabidopis thaliana and EIL1 are the abundant necessary factors that plant root hair is grown, and they having integrated the factor of some exogenous stimulation root hairs growths, is the important node that regulation and control root hair is educated.Studies show that in the model plant Arabidopis thaliana, the proteic accumulation of EIN3 can impel the growth of more hairs, and this provides new thinking for improve root by genetic engineering means in crop to the assimilated efficiency of nutrition.

Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims (2)

1. gene EIN3 and EIL1 are promoting the developmental application of plant root hair.

2. application according to claim 1 is characterized in that, described plant is an Arabidopis thaliana.

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