CN105368846A

CN105368846A - Rice lesion mimic mutant gene LIL1 and application

Info

Publication number: CN105368846A
Application number: CN201510923474.8A
Authority: CN
Inventors: 周倩
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2015-12-14
Filing date: 2015-12-14
Publication date: 2016-03-02
Anticipated expiration: 2035-12-14
Also published as: CN105368846B

Abstract

The invention discloses rice lesion mimic mutant gene LIL1; a nucleotide sequence is as shown in SEQ ID No. 1, an amino acid sequence of encoded protein of the mutant gene is as shown in SEQ ID No. 2 and a CDS region of the gene is as shown in SEQ ID No. 3. The LIL1 mutant gene can activate the expression of rice disease resistance-related genes and can significantly enhance resistance to pyticularia oryzae. The gene, with discovery and cloning, provides a new gene resource for the resistance breeding of rice resistant to rice blast.

Description

Paddy rice uneven class sizes mutator gene LIL1 and application

Technical field

The present invention relates to genetically engineered field, be specifically related to a kind of paddy rice uneven class sizes mutator gene, relate to the application of this gene in Pyricularia oryzae resistance simultaneously.

Background technology

Rice blast is global important rice disease, all can work the mischief in the whole breeding time of paddy rice, generalized case underproduction 10%-20%, and heavy reaches 40-50%, and even No kernels or seeds are gathered, as in a year of scarcity.In recent years, rice blast year occurring area present and increase trend year by year, great outburst is quite a few in local sees.Cultivation disease-resistant variety produces the most economical effective approach of upper control rice blast, therefore excavates the breeding for disease resistance of new rice blast resistance gene to paddy rice significant.

Hypersensitive necrosis reaction (hypersensitiveresponse, HR) is a kind of self-protective mechanism that the plant enantiopathy original infects.When hypersensitive necrosis reaction occurs, infected position and host cell energy rapid onset intracellular programmed cell death (ProgrammedCellDeath, PCD) around thereof, form necrotic plaque, restriction pathogen further expands, thus reaches disease-resistant effect.Plant lesion mimics (lesionmimicmutant, LMM) be that a class is under the condition not having apparent adversity, damage or pathogen to infect, can on plant the mutant of spontaneous formation necrotic plaque, the phenotype of these mutant is similar to HR, shows obvious programmed cell death phenomenon.A lot of uneven class sizes mutant material can show the resistance of local or system before or after necrotic plaque is formed.As paddy rice spl11 mutant shows the Double-resistant of Pyricularia oryzae (fungi) and rice leaf spot bacteria (bacterium); Paddy rice spl1 mutant activates the expression of disease-resistant gene PBZ1, all has resistance to the different physiological strains of Pyricularia oryzae.Therefore, discovery as much as possible and cloning rice uneven class sizes mutator gene are the effective ways finding rice blast resistance gene.

Apply the means of molecular genetics in recent years, had been found that and cloned the mutator gene of lesion mimics in part paddy rice, some of them are really relevant to the resistance of rice blast.Such as paddy rice Spl11 and paddy rice Spl18 gene can strengthen the resistance of plant to rice blast, and paddy rice Sekiguchi lesion mimics can improve the resistance of plant to Pyricularia oryzae under illumination condition.

Summary of the invention

The object of the present invention is to provide a kind of new paddy rice uneven class sizes mutator gene, this mutator gene have activated the expression of paddy disease-resistant related gene, and significantly strengthens the resistance of Pyricularia oryzae.

The present inventor finds a lesion mimics from rice variety 93-11EMS mutagenesis colony, called after LIL1 (Light-induced-lesionmimicmutant1).The formation and development of this mutant uneven class sizes is a similar anaphylactoid programmed cell death process.The expression amount versus wild type 93-11 of disease-resistant related gene PR1, PR-10a, POC-1 and POX22.3 gene in LIL1 body obviously increases, and the resistance of mutant to Pyricularia oryzae significantly strengthens.By the method for map based cloning, by LIL1 gene Fine Mapping in the scope of paddy rice No. 7 chromosome 22 2.3kb, at present location or clone paddy rice uneven class sizes mutator gene none be positioned at locating area, therefore this is a new paddy rice uneven class sizes mutator gene.Gene in locating area is checked order, find a kinase whose gene of coding Tyr acceptor class, the variation of a Nucleotide is there is in mutant LIL1, cause this DNA encoding the protein No. 429 amino acid to become Isoleucine by α-amino-isovaleric acid, its aminoacid sequence is as shown in SEQIDNo.2.Adopt the CDS coding region sequence (as shown in SEQIDNo.3) of RT-PCR technology clone LIL1 gene, and construct overexpression vector, verified by transgenosis, determine that the kinase whose gene of Tyr acceptor class of this sudden change is LIL1 gene, its sequence is as shown in SEQIDNo.1.

The expression of LIL1 gene of the present invention can significantly enhance the resistance of paddy rice to Pyricularia oryzae, and the discovery of this gene and clone are that the resistance breeding of rice anti-rice blast provides new genetic resources.

Accompanying drawing explanation

Fig. 1 is the phenotypic map of mutant LIL1.

Wherein, A: the phenotype of mutant LIL1 under field condition, 1: mutant LIL1 plant, 2: wild-type 93-11; B: mutant LIL1 Lao Ye and young leaves contrast, 1: Lao Ye, 2: young leaves; C: the phenotype of mutant LIL1 under aseptic condition; The phenotype of D: mutant LIL1 shading treatment.

Fig. 2 is the resistance effect diagram of LIL1 gene pairs rice blast.

Fig. 3 is LIL1 gene Fine Mapping figure.

Fig. 4 is the expression of candidate gene in mutant LIL1 and wild-type 93-11.

Fig. 5 is order-checking peak, the mutational site figure of LIL1 gene.

Wherein, A:LIL1; B: wild-type 93-11.

Fig. 6 is LIL1 protein mutation position.

Fig. 7 is the RT-PCR result electrophorogram of LIL1 full length gene cDNA.

Wherein, M:marker500bpDNAladder; 1:LIL1 gene.

Fig. 8 is the structure result electrophorogram of LIL1 gene overexpression vector.

Wherein, M:marker1kbDNAladder; 1,2:LIL1 gene overexpression vector; 3,4: the enzyme of overexpression vector cuts result.

Fig. 9 is transfer-gen plant Totomycin detected result figure.

Wherein, M:markerDL2000; 1-24: turn LIL1 gene plant; 1,3,6,8,10,14,17,19,20,21,23,24 is positive.

Figure 10 is the expression figure of LIL1 resistance related gene.

Wherein, A, C:3 leaf phase; B, D: filling stage.

Figure 11 turns the resistance enhancing figure of LIL1 trans-genetic hybrid rice to rice blast.

Embodiment

The phenotypic evaluation of embodiment 1 mutant LIL1

In conjunction with see Fig. 1, mutant LIL1 plant and wild-type 93-11 are compared, known, mutant LIL1 plant comparatively wild-type is obviously downgraded (Figure 1A), and the uneven class sizes 3 leaf phase begins existing, similar anaphylaxis necrotic plaque.Spot on Lao Ye is many and close (Figure 1B) compared with young leaves, shows uneven class sizes (Fig. 1 C) under gnotobasis equally.Illumination is formed with impact to uneven class sizes, and the position uneven class sizes of masking foil shading treatment obviously few compared with non-shading place (Fig. 1 D).

Mutant LIL1 has obvious resistance (Fig. 2) to for examination Pyricularia oryzae.

The location of embodiment 2LIL1 mutator gene and candidate

Utilize the method for map based cloning by LIL1 gene Fine Mapping in the scope of paddy rice No. 7 chromosome 22 2.3kb (Fig. 3), by Gene expression differential display (Fig. 4) and order-checking, find that a kinase whose gene of coding Tyr acceptor class exists the variation (Fig. 5) of a Nucleotide in mutant LIL1, cause this DNA encoding the protein No. 429 amino acid to become Isoleucine by α-amino-isovaleric acid, and the site of sudden change is just arranged in kinase whose catalyst structure domain (Fig. 6).The sequencing result in multiple variety culture rice and this site of wild-rice illustrates that this site is quite conservative in the middle of the process of natural evolution.Infer that this sports EMS mutagenesis and produces accordingly.

The CDS Clone and sequence of embodiment 3LIL1 mutator gene

According to LIL1 gene order design primer, primer sequence is as follows:

LIL1-F, CA gGTACC| ATGGCCATTTTGACCGTTCTGCC (as shown in SEQIDNo.4)

LIL1-R, CT gGATCC| TTACCTGCCGTTCACAATTGTTATGG (as shown in SEQIDNo.5).

Conveniently be cloned into expression vector, add the sequence of corresponding restriction enzyme site (underscore) before primer sequence, RT-PCR clones the CDS coding region (Fig. 7) of LIL1 gene.

Specific operation process is as follows:

(1) extraction of plant RNA

Getting mutant LIL13 leaf phase complete stool is material extraction RNA, and step is as follows:

1) 100-200mg tissue is collected, with liquid nitrogen by abundant for organization material grind into powder;

2) after liquid nitrogen volatilization, powder is transferred to rapidly Rnasefree centrifuge tube, adds the Trizol extracting solution of 1ml immediately, cover tightly pipe lid, vortex concussion 30s, sample is fully mixed, and room temperature places 5min;

3) add 0.2ml chloroform thermal agitation and mixed for 15 seconds, room temperature places 10min;

4) 4 DEG C, the centrifugal 10min of 12000rpm, transfer supernatant, to the centrifuge tube of new 2ml, adds 0.5 times of volume dehydrated alcohol, fully mixes, precipitation at room temperature 10min;

5) supernatant liquor is transferred to centrifugal adsorbing column, 4 DEG C, and the centrifugal 30s of 12000rpm, discards waste liquid;

6) add 500 μ l protein liquid removals, 4 DEG C, the centrifugal 30s of 12000rpm, discards waste liquid;

7) add 700 μ l and remove rinsing liquid, 4 DEG C, the centrifugal 30s of 12000rpm, discards waste liquid;

8) add 500 μ l and remove rinsing liquid, 4 DEG C, the centrifugal 30s of 12000rpm, discards waste liquid;

9) 4 DEG C, the centrifugal 2min of 12000rpm, uncap, dries 2min;

10) in the middle of adsorption column, 30 μ lRnase-FreeddH are added ₂o leaves standstill a moment, 4 DEG C, the centrifugal 2min of 12000rpm;

11) RNA concentration and quality is detected with ultraviolet spectrophotometer and 1%Agrose running gel.

(2) RT-PCR amplification

Reverse transcription: get OligodT (500 μ g/ml) 1 μ l, dNTPMixture (each 10mM) 1 μ l, RNA template 1 μ l and RNAfreeddH ₂o9 μ l mixes, in 65 DEG C reaction 5min, cool rapidly on ice, of short duration centrifugal after add 5 × Firststrandbuffer4 μ l, RNaseOUT again ^tM(40U/ μ l) 1 μ l and 0.1MDTT2 μ l mixes gently, hatches 2min for 37 DEG C, finally adds M-MLV (200U/ μ l) 1 μ l, blows and beats mixing lightly, hatch 50 minutes for 37 DEG C.

The reverse transcription product of gained is directly used as pcr amplification, reaction system: 10 × PCRBuffer2 μ l, LIL1-F (10 μMs) 1 μ l, LIL1-R (10 μMs) 1 μ l, cDNA2 μ l, Taq enzyme (2.5U/ μ l) 0.5 μ l, dNTPMixture (each 10mM) 0.5 μ l and ddH ₂o13 μ l mixes, and increases by following condition: 94 DEG C of denaturations 5 minutes, 94 DEG C of sex change 30 seconds, and 55 DEG C of annealing 30 seconds, 72 DEG C extend 90 seconds, carry out 30 circulations, last 72 DEG C of extension 10min.

The amplified production agarose gel electrophoresis of 1%, then reclaims test kit with gel and reclaims fragment, send order-checking company sequence verification, and the coding region of display LIL1 gene is 2064bp.

The structure of embodiment 4LIL1 gene overexpression vector

The LIL1 gene C DS total length respectively RT-PCR increased and pCAMBIA1301 carrier KpnI and BamHI double digestion (RT-PCR product or pCAMBIA1301 carrier 10 μ l, 10 × KBuffer5 μ l, KpnI1 μ l, BamHI1 μ l, ddH ₂o33 μ l was in 37 DEG C of reactions 1 hour), then cross post and reclaim digestion products, connect (linked system: PCR recovery product 8 μ l, pCAMBIA1301 carrier 2 μ l, T4DNA ligase enzyme (350U/ μ l) 1 μ l, 10 × T4DNALigationBuffer2 μ l and ddH ₂o7 μ l, spends the night in 16 DEG C), connect product and cross post recovery, heat-shock transformed E.coliDH5 ɑ, build and obtain LIL1 gene overexpression vector.Positive strain extracts plasmid, and whether digestion verification LIL1 gene successfully inserts expression vector (Fig. 8).

Embodiment 5LIL1 gene genetic transforms and detects

(1) evoked callus: select full grains, be material without the rice paddy seed of spot, by the rice paddy seed that shells through 75% alcohol immersion 5min, 0.01% HgCl ₂soak 15min, aseptic water washing 4-5 time, be then inoculated in inducing culture (MS+2,4-D1-2mg/L), under 25 DEG C of low light levels, cultivate evoked callus formed.Select subculture callus once as experiment material.

(2) via Particle Bombardment Transformation: adopt PDS-1000/He particle gun (Bio-Rad).Get bronze suspension (60mg bronze dehydrated alcohol is sterilized, and is suspended in 1ml sterilized water) 50 μ l, add 5 μ gDNA, 50 μ l2.5mol/LCaCl ₂, 20 μ l0.1mol/L spermidines, abundant mixing, centrifugal, wash precipitation with dehydrated alcohol, Eddy diffusion, in 60 μ l dehydrated alcohols, evenly drips on 6 bombardment films, according to program described in this particle gun handbook, target material is bombarded, the pressure that wherein can split film is 1100psi, and target material distance can split film 6cm, every batch of material bombardment twice.

(3) detection of transient expression: the material after conversion is cultivated two days later on subculture medium, immerses in X-GluC dye liquor and cultivates 8-24h in 37 DEG C, dissects Microscopic observation, record Bluepoint number.

(4) screening and regeneration: the final concentration using Totomycin is 50mg/L, initial screening pressure is Totomycin 30mg/L, after subculture 2 times, proceed to regeneration culture medium illumination cultivation, regenerate plantlet, plantlet is proceeded to (Totomycin 50mg/L) in 1/2MS substratum, treat that plantlet is grown up, be transplanted into greenhouse.

(5) Molecular Detection of transfer-gen plant: utilize plant DNA extraction kit to extract the DNA of plant to be detected, with primers F: GGACTTCGGGGCAGTCCT (SEQIDNo.6), primer R:CGATGTAGGAGGGCGTGG (SEQIDNo.7), PCR detect hygromycin gene.

Reaction system: 10 × PCRBuffer2 μ l, LIL1-F (10 μMs) 1 μ l, LIL1-R (10 μMs) 1 μ l, cDNA2 μ l, Taq enzyme (2.5U/ μ l) 0.5 μ l, dNTPMixture (each 10mM) 0.5 μ l, ddH ₂o13 μ l, amplification condition: 94 DEG C of denaturations 5 minutes, 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C extend 90 seconds, carry out 30 circulations, last 72 DEG C of extension 10min.The amplified production agarose gel electrophoresis of 1% detects (Fig. 9), and 24 strain paddy rice of detection have 12 strains to be transgenic positive.

The preliminary study of embodiment 6LIL1 gene function

The method of Semiquatitative RT-PCR assay is adopted to have detected the expression of transgenic paddy rice and wild-type 93-11 disease-resistant related gene PR-1, PR-10a, POC-1 and POX22.3 in 3 leaf phases and filling stage respectively.Result shows, and in 3 leaf phase LIL1 bodies, POC-1 gene expression amount is apparently higher than wild-type 93-11, and the difference of the expression amount of PR-1, PR-10a and POX22.3 gene is not obvious; The expression amount of filling stage four genes in LIL1 body all obviously increases (Figure 10), and the expression of resistance related gene of mutant LIL1 vivo activation is described.

Observe morbidity result (Figure 11) by after LIL1 transgenic paddy rice spray inoculation magnaporthe grisea spore suspension, result shows that the paddy rice turning LIL1 gene strengthens the resistance of Pyricularia oryzae.

Claims

1. a paddy rice uneven class sizes mutator gene LIL1, its nucleotide sequence is as shown in SEQIDNo.1.

2. a paddy rice uneven class sizes mutator gene LIL1 proteins encoded, its aminoacid sequence is as shown in SEQIDNo.2.

3. a paddy rice uneven class sizes mutator gene LIL1CDS coding region, its sequence is as shown in SEQIDNo.3.

4. gene described in claim 1 in paddy rice to the application in Pyricularia oryzae resistance.

5. the application of gene described in claim 1 in the resistance breeding of rice anti-rice blast.

6. gene described in claim 1 is cultivating the application in blast resisting transgenic paddy rice.