CN108220303B - Soybean BI-1 gene and application - Google Patents

Abstract

The invention discloses a cell apoptosis inhibitor BI-1 gene involved in regulating symbiotic nodulation of leguminous plants, the nucleotide sequence of the gene is shown in a sequence table SEQ ID NO:1, the preparation method is as follows. And provides the application of the gene in regulating the number of leguminous plant nodules. The invention utilizes yeast two-hybrid technology to screen in the soybean nodule AD-cDNA library, finds a new protein interacting with nodulation factor receptor protein, and confirms that the gene is an apoptosis inhibitor gene by methods of sequence comparison, function prediction and the like; the function of the gene in the symbiotic nodulation process of the lotus japonicus of leguminous plants is researched by utilizing an overexpression technology, and the result shows that the protein coded by the gene participates in the symbiotic nodulation process, the number of nodules is obviously increased after overexpression, and the gene has an application prospect in symbiotic nitrogen fixation of leguminous plants.

Description

Soybean BI-1 gene and application

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a cell apoptosis inhibitor BI-1 gene participating in regulating symbiotic nodulation of leguminous plants and application thereof.

Background

The BI-1 gene is an apoptosis inhibitor cloned and identified in 1998, and the protein product thereof can inhibit apoptosis caused by Bax. BI-1 proteins contain 6-7 transmembrane domains, and the C-terminal hydrophilic region may be a key segment for protein interaction, and the protein family members thereof are mainly located in the inner membrane of mammalian and plant cells and have high conservation in higher plants and animals. Studies have proved that the BI-1 gene can enhance the tolerance of plants to abiotic stress such as low temperature, drought, high salt and oxidation stress and biotic stress such as pathogenic bacteria infection. The BI-1 gene participates in various stress signal response channels and is positioned at a key node in a signal channel network.

At present, the research on the function of the inhibitor of apoptosis (BI-1) gene is mainly focused on a model plant Arabidopsis thaliana, the research on the separation and identification of the BI-1 gene in soybean is less, and the research on the function of the BI-1 gene in the symbiotic nodulation process is not reported. The invention screens a potential apoptosis inhibitor BI-1 interacting with the nodulation factor receptor kinase by a yeast two-hybrid technology. After the gene is over-expressed in the lotus japonicus of the leguminous plant, the nodulation number is obviously increased, the rhizobium infection phenotype proves that the gene promotes the organogenesis of the root nodule, and the nodulation process of the leguminous plant is positively regulated, thereby having important significance in agriculture and ecology.

Disclosure of Invention

The invention aims to provide a cell apoptosis inhibitor BI-1 gene involved in regulating symbiotic nodulation of leguminous plants.

Still another object of the present invention is to provide the use of the gene in regulating the number of nodules of leguminous plants (Lotus japonicus, Astragalus sinicus, alfalfa, soybean, peanut, etc.).

The purpose of the invention is realized by the following technical scheme:

the gene provided by the invention is an isolated apoptosis inhibitor gene GmBI-1, and the sequence of the gene is a nucleotide sequence shown in SEQ NO. 1.

The apoptosis inhibitor GmBI-1 gene code is protein, and the sequence is an amino acid sequence shown in SEQ NO. 2.

The invention uses the intramembrane kinase domain of leguminous plant soybean nodulation factor receptor protein GmNFR1 alpha as bait, screens a soybean nodule AD-cDNA library by a yeast double-hybridization method, extracts plasmids of positive transformants and performs sequencing analysis after revolving and verifying colonies growing on a flat plate of a nutrient-deficient yeast culture medium, and obtains the full length of the gene. The gene consists of 741 bases and can code 246 amino acids. The GmBI-1 gene of the invention and any DNA of interest or a DNA homologous thereto may be amplified from the genome, mRNA and cDNA using PCR (polymerase chain reaction) techniques.

In order to prove the expression condition of the gene in leguminous plants, the expression condition of the GmBI-1 gene in roots before and after rhizobium inoculation of soybean is analyzed by Real-Time PCR, and the result shows that the expression level of the GmBI-1 gene is obviously increased 16 days after rhizobium inoculation, so that the induced expression of the GmBI-1 gene by rhizobium is proved. In addition, root nodule tissues (root nodules 12, 30, 42, 64 and 84 days after inoculation) corresponding to 5 important soybean development stages (a branch stage, a flowering stage, a fruit stage, a pod stage and a harvest stage) are selected, and the expression conditions of the GmBI-1 gene in the root nodules at the 5 different stages are analyzed through Real-Time PCR, so that the result shows that the expression level of the GmBI-1 gene is highest in the root nodules at 12 days after inoculation, and the GmBI-1 plays a role in the early development stage of the root nodules.

The application of an apoptosis inhibitor GmBI-1 gene participating in symbiotic nodulation of leguminous plants in crowtoe, astragalus sinicus, alfalfa, soybean and peanut is realized through genetic transformation. According to the prediction of the GmBI-1 gene structure, an over-expression vector is constructed, the constructed vector is transferred into agrobacterium LBA1334 through a freeze-thaw method, then the transformed agrobacterium tumefaciens LBA1334 is transferred into the Lotus corniculatus of leguminous plants through hairy root transformation, after a positive plant is obtained through GUS identification, the positive plant is planted in a mixed pot culture of sand and vermiculite 1:1, and rhizobium is inoculated. The result is shown in table 1, compared with a control plant, the root nodule number of the crowtoe is obviously increased after overexpression, and the gene is found in the crowtoe for the first time, confirms the apoptosis inhibitor gene for positively regulating and controlling the nodulation, and has application prospect in symbiotic nitrogen fixation of leguminous plants.

TABLE 1 comparison of nodulation numbers of Lotus corniculatus normal plants and plants with overexpression of GmBI-1 Gene

Compared with the prior art, the invention has the following beneficial effects:

1. the invention utilizes yeast two-hybrid technology to screen in the soybean nodule AD-cDNA library, finds a new protein interacting with nodulation factor receptor protein, and confirms that the gene is an apoptosis inhibitor gene by methods of sequence comparison, function prediction and the like; detecting the expression of the gene in different tissues at different times by using Real-Time PCR (polymerase chain reaction), and determining that the expression of the gene in the soybean of the leguminous plant is induced by rhizobia; meanwhile, the gene is proved to participate in and positively regulate the nodulation of leguminous plants by an overexpression genetic transformation method, and the gene is found and proved to positively regulate the symbiotic nodulation of the leguminous plants by the apoptosis inhibitor in the soybeans for the first time.

2. Leguminous plants increase the fertility of soil by symbiotic nitrogen fixation of root nodules, but the number of the root nodules in the leguminous plants is limited under the natural condition, so that the fertility of the soil is increased by increasing the number of the root nodules and further increasing the nitrogen fixation capacity of the leguminous plants, the use of chemical fertilizers and the like is effectively reduced, and the leguminous plants have great significance in agriculture and ecology; the invention proves that the gene positively regulates nodulation by an overexpression technology, so that the gene can be overexpressed in leguminous plants by a transgenic means, the number of nodules can be greatly increased, and the nitrogen fixation capability is enhanced, thereby having a certain application prospect in the research of nitrogen fixation mechanisms of leguminous crops and agricultural production.

Drawings

FIG. 1 is a schematic diagram of alignment of a protein sequence predicted by a GmBI-1 gene and a GmBI-1 homologous protein sequence by using DNAMAN software, wherein:

Gs:accession KHN09861.1Sequence source:Glycine soja

Ad:accession XP_015960981.1Sequence source:Arachis duranensis

Lj:accession AFK35071.1Sequence source:Lotus japonicus

La:accession XP_019422063.1Sequence source:Lupinus angustifolius

Cc:accession XP_020231227.1Sequence source:Cajanus cajan

Ha:accession XP_022002312.1Sequence source:Helianthus annuus

Ai:accession XP_020976213.1Sequence source:Arachis ipaensis

Pv:accession AGV54668.1Sequence source:Phaseolus vulgaris

Va:accession XP_017439409.1Sequence source:Vigna angularis。

FIG. 2 is a schematic diagram of the expression of the GmBI-1 gene in different tissues at different times by using Real-Time PCR.

FIG. 3 is a schematic diagram of detecting the overexpression efficiency of an overexpressed transgenic plant and the nodulation of an overexpressed GmBI-1 gene promoted plant by using Real Time-PCR, wherein 3A is the nodulation comparison of the overexpressed transgenic plant and a control normal plant; and 3B is the relative expression quantity comparison of the over-expression transgenic plant and the control normal plant.

FIG. 4 is a schematic diagram of a vector structure of a overexpression vector pU1301 in plants.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the following detailed description is given with reference to the accompanying drawings and preferred embodiments of the present invention.

Example 1

A preparation method of an apoptosis inhibitor GmBI-1 gene participating in symbiotic nodulation of leguminous plants comprises the following steps:

cloning of candidate gene GmBI-1: an intramembrane kinase structural domain GmNFR1 alpha-pk of nodulation factor receptor protein GmNFR1 alpha of leguminous plant soybean is fused with a yeast protein expression vector pGBKT7 to be used as a bait plasmid, a soybean AD-cDNA library is screened by a yeast two-hybrid method, a colony growing on an SD/-Leu-Trp-Ade-His (nutrient-deficient yeast culture medium) plate is subjected to rotary verification, a plasmid is extracted from a positive transformant and is subjected to sequencing analysis, and a GmBI-1 gene is obtained, wherein the sequence of the gene is a nucleotide sequence shown as SEQ NO: 1. The implementation steps comprise the steps of preparing yeast competent cells by a LiAc method, converting yeast and screening a library, and specifically comprise the following steps:

(1) frozen yeast Y187 (from clontech) was streaked onto YPDA plates from-70 ℃. Inverting the culture dish, and culturing for 2-3 days at 30 ℃ until clones appear;

(2) under aseptic condition, 3mL of 2YPDA liquid culture medium is bottled by PA, a yeast with the diameter of 2-3mm is inoculated, and shaking culture is carried out at 30 ℃ and 200r/min for 8 h;

(3) transferring, transferring 5 μ L of the culture into a triangular flask with a volume of 250mL containing 50mL LYPDA, and culturing at 30 deg.C and 230-;

(4) centrifuging at room temperature (20-25 deg.C, the same below) for 5min at 700 g;

(5) discarding the supernatant, and resuspending the cells with 100ml LYPDA;

(6) culturing at 30 ℃ and 230 ℃ for 3-5h at 250r/min until the OD600 is 0.5-0.6;

(7) centrifuging at room temperature for 5min at 700 g;

(8) discard the supernatant and resuspend the cells with 3mL of 1.1 XTE/LiAc;

(9) the resuspension solution was dispensed into two sterile 1.5mL centrifuge tubes (1.5 mL per tube);

(10) centrifuging at high speed for 15 s;

(11) discard the supernatant and resuspend the cells in 600. mu.L of 1.1 XTE/LiAc for each tube; the prepared cells should be used for transformation immediately, and if necessary, the cells can be stored at room temperature for 1-3 hours without obvious change of transformation capacity;

(12) samples were added to a pre-cooled 1.5ml EP tube and mixed as follows:

the positive control plasmid pGBKT7-53 (from clotech Co.);

the negative control plasmid pGBKT7-lam (from clotech Co.);

(13) culturing at 30 deg.C for 30min, and shaking every 10 min;

(14) adding 20 μ L DMSO into each tube, mixing, placing the centrifuge tube in 42 deg.C water bath for 15min, and shaking once every 5 min;

(15) centrifuging at high speed for 15s, discarding supernatant, and resuspending cells with 1 mLYPDA;

(16) culturing at 30 deg.C for 90 min;

(17) centrifuging at high speed for 15 s;

(18) discarding the supernatant, resuspending the cells with 1ml LYPDA, and mixing well;

(19) plates coated with SD/-Trp in a volume of 100. mu.L;

(20) performing inverted culture at 30 ℃ for 2-4d until a clone appears;

(21) selecting transformed clones, culturing in 50ml SD/-Trp at 30 deg.C for 20-24hrs until OD reaches 0.8;

(22) centrifuging at 2500rpm for 5min, removing supernatant, and suspending with SD/-Trp (4-5 ml);

(23) thawing the library plasmid in room temperature water bath, mixing 1ml library plasmid with 5ml bait strain, adding 45ml into 2L triangular flask, and culturing at 30 deg.C under light suspension (30-50rpm) for 20-24 hr;

(24) centrifuging at 2500rpm for 10min, and resuspending the cells with 10ml of 0.5 × YPDA;

(25) 350ul of the coated SD/-Leu-Trp-Ade-His plate was incubated at 30 ℃ for 3-8 days.

The brief introduction and the formula of the steps are as follows:

salmon sperm (Salmon) DNA is a Sigma product and has the concentration of 2 mg/mL;

adding 20mg of salmon sperm DNA into 10mL of sterilized TE buffer (10mmol/L Tris-HCl pH 8.0, 1.0mmol/L EDTA), repeatedly blowing and sucking by Tip for dispersion, and fully mixing for 2-3 h on a magnetic stirrer until the salmon sperm DNA is completely dissolved; placing the mixed solution in a refrigerator at 4 ℃ for overnight; then subpackaging in a 1.5mL centrifuge tube, and storing at-20 ℃; before use, a tube was boiled in a water bath for 5min, taken out and immediately placed on ice. The tube can be used 3-4 times, and if the conversion efficiency is reduced, the tube needs to be boiled for 1 time, or new salmon sperm DNA is used.

Polyethylene glycol 3350(PEG 3350) is a Sigma product. Concentration 50% w/v: PEG 335050 mg was added to 35mL sterile ddH₂In O, fully stirring on a magnetic stirrer until the mixture is completely dissolved, and adding sterilized ddH₂And (4) metering the volume of O to 100mL, filtering and sterilizing by using a 0.45-micron filter membrane, and storing in a reagent bottle with good sealing property. The concentration fluctuation thereof lowers the conversion efficiency.

10 XTE 0.1M Tris-HCl, 10mM EDTA, pH7.5, autoclaved

10 XLiAc 1M LiAc, pH7.5, autoclaved

1.1 × TE/LiAc Solution (ready for use):

10×TE 1.1mL

10×LiAc 1.1mL

add ddH₂The volume of O is up to 10 mL.

PEG/LiAc solution:

8mL 50％PEG 3350

1mL 10×TE

1mL 10×LiAc

add ddH₂The volume of O is up to 10 mL.

YPDA medium formula (100ml) with pH of 5.5-6.5

SD medium formulation (1L): PH 5.8-6.5

Yeast nitrogeo 1.7g

(NH₄)₂SO₄ 5.0g

Glucose 20g

Amino acid (-Trp): L-Arg 200 mg; L-Iso 300 mg; 300mg of L-lys; l-me +200 mg; l-phe500 mg; L-Thr 2000 mg; L-Tyr 300 mg; L-Val 1500 mg; L-Ura 200 mg; 20mg of His; leu 100 mg; ade 30mg

Amino acid (-Trp-Leu-His-Ade): L-Arg 200 mg; L-Iso 300 mg; 300mg of L-lys; l-me +200 mg; 500mg of L-phe; L-Thr 2000 mg; L-Tyr 300 mg; L-Val 1500 mg; L-Ura 200mg

Example 2

The application of an apoptosis inhibitor GmBI-1 gene participating in symbiotic nodulation of leguminous plants in regulating and controlling the number of root nodules of leguminous plants (Lotus japonicus, Astragalus sinicus, alfalfa, soybean and peanut) comprises the following steps:

A. detecting the tissue specific expression condition of GmBI-1 in soybean:

the root tissues of soybeans at different times before and after inoculation and nodules at 12, 30, 42, 64 and 84 days after inoculation were harvested from cultivated soybean W82 as a material, total RNA (Trizol reagent, available from Invitrogen corporation) was extracted from the tissues at different times, and the expression thereof was detected by the fluorescent quantitative PCR technique (shown in FIG. 2). The specific implementation steps are as follows: the extracted Total RNA is reversely transcribed into cDNA, the first strand of the cDNA is synthesized by using a Kit PrimeScriptTM RT reagent Kit (Perfect RealTime), a 20 mu l system reversely transcribes 500ng Total RNA, the primer uses Oligo-dT, and the operation is carried out according to the use instruction. PCR reaction (25. mu.L): 12.5 μ L SYBR O R Premix Ex TaqTM II (2 ×); 1. mu. mol/L PCR, Forward Primer; 1 mu mol/L PCR Reverse Primer; 2 μ L of cDNA template; add ddH2O to 25. mu.L. PCR reaction procedure: 10s at 95 ℃; 5s at 95 ℃,12 s at 60 ℃,15 s at 72 ℃ and 40 cycles; dissolution curve analysis was then performed. The quantitative method adopts a relative quantitative delta-delta Ct method.

B. Construction and genetic transformation of a GmBI-1 gene overexpression vector:

in order to study the biological function of GmBI-1 in Lotus corniculatus of Leguminosae, the applicants explored the biological function of GmBI-1 by overexpression (FIG. 3). Mainly relates to the construction and genetic transformation of an overexpression vector, and comprises the following specific implementation steps:

construction of the overexpression fusion vector: taking the library plasmid as a template, and amplifying a GmBI-1 gene sequence by using a primer containing KpnI and BamHI enzyme cutting sites, wherein the sequence is a nucleotide sequence shown as SEQ ID NO. 1; the GmBI-1 gene sequence was cleaved with KpnI and BamHI and ligated into the Mazie-ubquitin reporter gene downstream of the international commonly used plant overexpression vector pU1301 (FIG. 4, from Wangshiping laboratories, university of agriculture, Huazhong).

The constructed overexpression fusion vector is transformed into agrobacterium rhizogenes A. rhizogenes LBA1334 (the strain is a strain published internationally and is from professor hongzongzi in the United states) by a freeze-thaw method for hairy root transformation of crowtoe. The transformation method of the hairy roots of the Lotus japonicus refers to Lotus japonica Handbook.

The specific implementation steps are as follows:

(1) plant material cultivation: lotus japonicus MG20 seeds were germinated 5 days earlier. Sanding the seeds (lighter than the ordinary germinated seeds) with sand paper, freezing in liquid nitrogen for 1 minute, soaking in 75% (by mass/volume) ethanol for 1 minute, soaking in 5% (effective chlorine concentration) NaClO for 15 minutes, washing with sterile water for 5-6 times, and removing residual NaClO. After the surface of the seeds is disinfected, a little sterile water is left to just submerge the seeds, the seeds are vernalized for 1 day at 4 ℃ under the dark condition, then the seeds are transferred to a solid culture medium without sucrose for MS and cultured for 2 days at 23 ℃ in the dark, and then the seeds are placed in an illumination incubator (16h illumination and 8h darkness) for continuous culture for 2 days at 23 ℃ for standby application.

(2) Strain and plasmid preparation: the strain is A.rhizogenes LBA1334 (spectinomycin resistance), and plasmid containing target gene is introduced into Agrobacterium A.rhizogenes LBA1334 by electrotransformation, and the strain is preserved at-70 deg.C after identification. The preserved strain is taken out for plating (containing plasmid resistance plate) for activation 2 days in advance.

(3) Infection transformation: the first day: in the afternoon, a single colony was selected and inoculated into 5ml of LB medium (containing plasmid resistance), and shake-cultured at 28-30 ℃ for 16-24 hours. The next day: and (3) carrying out amplification culture on the small amount of bacteria inoculated on the previous day, wherein the mass ratio of 1: inoculating at a ratio of 100, and culturing at 28-30 deg.C for about 8 hr under shaking. The mass-cultured bacterial solution was centrifuged at 6000 rpm for 10 minutes, and the cells were collected and resuspended in sterile water so that the OD600 became about 0.8. Cutting off hypocotyl base of seedling, soaking the part with cotyledon with the re-suspended bacteria liquid for 30min, taking out explant, drying with filter paper, and co-culturing in MS culture medium without sucrose.

(4) Co-culturing: co-culturing in dark for 3-5 days. The explants were then transferred to HRE and 300. mu.g mL-1cefotaxime medium for another 10 days during which time hair roots grew out of the hypocotyl incisions.

(5) Positive transgenic root identification: a root segment about 0.5cm long at the root tip is cut and put into GUS dye solution, dark culture is carried out at 37 ℃ overnight, and the blue root is a positive transgenic root.

(6) Hardening and transplanting seedlings: after identification, non-transformed roots were cut off, and the seedlings were put in a dish filled with water (without being covered) to be acclimatized for 1 day, and then transplanted into a flowerpot to be cultured in a light incubator (16h light, 8h dark) at 23 ℃. The base material mixed by vermiculite (vermiculite) and sand (sand) according to the proportion of 1:1 is added in the flowerpot in advance. The nitrogen-free nutrient solution is poured once every 3 days.

(7) Rhizobium inoculation: rhizobium baenii MAFF303099 (from the laboratory of professor Wu national river, south plant Garden of China, see the genetic resource table) of Chinese academy of sciences is activated on a YMA plate, inoculated into a liquid TY culture medium, and subjected to shaking culture at 28 ℃ for 24-36 h; transferring the cultured rhizobia into a sterile centrifuge tube, centrifuging at 4 ℃ at 7000r/min for 5min, and collecting thalli; washing with sterile Fahraeus nitrogen-free nutrient solution and centrifuging for 2 times; adding nitrogen-free nutrient solution to re-suspend the thallus, and inoculating the thallus to the roots of the crowtoe seedlings.

The formula of the culture medium and the reagent in the steps is as follows:

1) MS culture medium: 4.3g of MS basal salt mix (Sigma), 0.103g of MS vitamin powder, 0.7-0.8% (mass to volume) of agar powder and ddH₂And O is metered to 1000mL and is adjusted to pH 5.8.

2) YMA medium: 10.0g mannitol (or sucrose), 0.4g Yeast Extract, 0.5g K₂HPO₄，0.2g MgSO₄·7H₂O，0.1g CaCl₂·6H₂O, 0.1g NaCl, 4mL Rh microelement liquid, ddH₂And (4) metering the volume of O to 1000mL, adjusting the pH value to 6.8-7.0, and sterilizing at 115 ℃ for 20 min.

3) Rh trace element liquid: 5.0g H₃BO₃，5.0g Na₂MoO₄，ddH₂And O is metered to 1000 mL.

4) Fahraeus nitrogen-free nutrient solution: 0.10g of CaCl₂·2H₂O，0.12g MgSO₄·7H₂O，0.10g KH₂PO₄，0.15g Na₂HPO₄·12H₂O, 1mL of Gibson's microelement liquid, 5mg of ferric citrate, ddH₂And O is metered to 1000 mL.

5) Gibson trace element liquid: 2.86g H₃BO₃，0.22g ZnSO₄·7H₂O，2.03g MnSO₄·4H₂O，0.13g Na₂MoO₄·2H₂O，0.08g CuSO₄·5H₂O，ddH₂And O is metered to 1000 mL.

6) GUS dye solution: 100mM sodium phosphate buffer, pH 7.0, 0.1% Triton X-100, 0.1% N-laurylsarcosine, 10mM Na₂EDTA, 1mM potassium ferricyanide (K)₃Fe(CN)₆) 1mM potassium ferrocyanide (K)₄Fe(CN)₆) And 0.5mg/mL X-GluC。

7) HRE medium: 20X SH-A salt, 20X UM-C vitamin, 10g sucrose, 3mL 1M MES, 0.7-0.8% (mass/volume ratio) agar powder, ddH₂And (4) metering the volume of O to 1000mL, adjusting the pH value to 5.8, and sterilizing at 110 ℃ for 30 min.

20X SH-A salt:

dissolving FeSO in 100mL of sterile water respectively₄.7H₂Dissolving O and NaEDTA and other salts in 700mL of sterile water, mixing to a constant volume of 1000mL, and subpackaging into 50mL tubes for preservation at-20 ℃.

20X UM-C vitamin:

ddH₂and O is metered to 1000mL, and each tube is subpackaged with 50mL and stored at-20 ℃.

MES (2- [ N-Morpholino ] ethane-sulfonic acid),1M stock solution:

weigh 29.28g MES (Sigma) in ddH₂And (4) metering the volume of O to 150mL, adjusting the volume to pH 5.8, subpackaging into 6mL tubes, and storing at-20 ℃.

Example 3

Example 3 is substantially the same as example 2, except that,

B. construction and genetic transformation of a GmBI-1 gene overexpression vector:

the specific implementation of the plant material culture in the step (1) is that alfalfa is germinated 5 days in advance respectively; the Rhizobium in step (7) was inoculated with Sinorhizobium meliloti 1021 (a strain that has been published internationally, Juan et al, company of development and Stress-Induced Nodule Senesence in medical procedure apparatus, plant physiology, 152:1574-, (2010)).

Example 4

Example 4 is substantially the same as example 2, except that,

B. construction and genetic transformation of a GmBI-1 gene overexpression vector:

specifically, the Rhizobium japonicum strain inoculated in the step (7) is Rhizobium huakuii (the strain is a strain published internationally, and A novel plant protein protease such as Li, AsNODF32, is infected in novel sensory activity of the green human spleen arsenic bacterium. New phytologist 180: 185-1922008).

Example 5

Example 5 is substantially the same as example 2, except that,

B. construction and genetic transformation of a GmBI-1 gene overexpression vector:

specifically, the inoculation of Rhizobium japonicum with HN01lux in step (7) is carried out (this strain is a strain published internationally, Chenchan et al, and the promotion of nitrogen fixation efficiency of Rhizobium japonicum HN01lux by constitutive nifA, scientific report 44(5) 1999).

Example 6

Example 6 is substantially the same as example 2, except that,

B. construction and genetic transformation of a GmBI-1 gene overexpression vector:

specifically, the rhizobium inoculation in the step (7) is peanut rhizobium Spr (the strain is a strain published internationally, in Jingli and the like, the celB gene marking method is used for researching the inoculation and molybdenum application effects of acid soil peanuts, and the report on plant nutrition and fertilizer, 12(2): 250-.

The leguminous plant (Leguminosae sp) is a dicotyledonous plant, such as an arbor, a shrub, a subshrubb or a herb, and is erected or climbed, and root nodule plants capable of fixing nitrogen are frequently used. The mode belongs to: faba p. About 650 genus, 18000 species, widely distributed all over the world. Chinese 172 genus, 1485 species, 13 subspecies, 153 variety, 16 variant; all provinces are distributed. The family has important economic significance and is one of the important sources of starch, protein, oil and vegetables in human food. The leguminous plant is specifically Lotus japonicus (Lotus japonicus), alfalfa (Medicago truncatula), Astragalus sinicus (Astragalius sinicus), Soybean (Soybean), Peanut (Peanout) and the like.

The above description is only for the specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto, and other modifications or equivalent substitutions made by the technical solution of the present invention by the ordinary skilled in the art should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Sequence listing

<110> Xinyang college of teachers and schools

<120> soybean BI-1 gene and application

<141> 2018-03-28

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Met Ala Thr Pro Pro Leu Ser Pro Ser Ser Ser Ser Ser Ala Ser Ser

1 5 10 15

Thr Ser Thr Ala Thr Leu Leu Ala Pro Ala Gly Ile Ser Pro Leu Val

20 25 30

Gly Ala His Ile Leu Leu Val Thr Pro Thr Leu Cys Cys Ala Val Val

35 40 45

Ala Ala Ala Val Gly Ala Pro Leu His Val Leu Thr Ala Ile Gly Gly

50 55 60

Pro Leu Thr Thr Val Ala Ser Ile Gly Ser Met Pro Thr Leu Leu Ser

65 70 75 80

Thr Pro Pro Pro Gly Gly Gly Leu Ala Leu Ser Leu Leu Met Ala Ser

85 90 95

Ala Leu Pro Gly Gly Ala Ser Ile Gly Pro Leu Ile Gly Leu Ala Pro

100 105 110

Ala Ile Ala Pro Gly Leu Ile Ile Gly Ala Pro Val Ala Thr Ser Leu

115 120 125

Ala Pro Ala Cys Pro Ser Ala Val Ala Leu Val Ala Ala Ala Ala Gly

130 135 140

Thr Leu Thr Leu Gly Gly Leu Leu Ser Ser Thr Leu Ser Ile Leu Met

145 150 155 160

Thr Leu His Ser Ala Ser Ser Leu Pro Gly Gly Ser Ile Ala Leu Pro

165 170 175

Leu Pro Gly Leu Thr Pro Gly Leu Leu Val Pro Val Gly Thr Val Ile

180 185 190

Val Ala Thr Gly Val Ile Ile Gly Ala Ala His Pro Gly Ala Leu Ala

195 200 205

Thr Val Leu His Ala Leu Thr Leu Pro Thr Ala Leu Ala Ala Ile Pro

210 215 220

Val Ala Ile Leu Ala Ile Met Leu Ala Ala Ser Ser Leu Ala Ala Gly

225 230 235 240

Leu Leu Ala Ala Ala Ala

245

Claims (1)

1. The application of the cell apoptosis inhibitor BI-1 gene participating in regulating symbiotic nodulation of leguminous plants in regulating the number of roots of crowtoe plants; the sequence of the BI-1 gene is a nucleotide sequence shown as SEQ ID NO. 1; the sequence of the protein coded by the BI-1 gene is an amino acid sequence shown in SEQ ID NO. 2.

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