CN102965392A - Cultivation of sheath blight-resisting transgenic paddy rice and special vector - Google Patents

Abstract

The invention discloses an application of GAFP2 protein in cultivating sheath blight-resisting transgenic paddy rice and a special vector. The invention provides an application of GAFP2 protein or an encoding gene thereof or a recombinant vector in regulating and controlling plant disease resistance. The amino acid sequence of a GAFP2 protein is a sequence 2 inside a sequence table or the (29-171)th site amino acid residue of the sequence 2 inside the sequence table and from an N'tail end. Experiments improve that the GAFP2 is inducted into paddy rice so that transgenic paddy rice is obtained, therefore the resistance of paddy rice sheath blight can be effectively enhanced.

Description

The cultivation of anti-banded sclerotial blight transgenic paddy rice and dedicated carrier

Technical field

The present invention relates to biological technical field, relate in particular to a kind of cultivation and dedicated carrier of anti-banded sclerotial blight transgenic paddy rice.

Background technology

Rice sheath blight disease (sheath blight) is the silborne fungal diseases that is caused by dry thread Pyrenomycetes (Rhizoctonia solani K ü hn).Since the sixties in last century, be accompanied by the using in a large number of popularization, dense planting and chemical fertilizer of short-stalked variety, banded sclerotial blight worldwide becomes one of paddy rice three large diseases, generally causes the underproduction of 15%-20%, but underproduction 60%-70% when serious.

This disease belongs to the leaf sheath disease, starts from the plant base portion.Under the Rice Population condition, generally in 2 onset peaks of later stage to jointing initial stage and heading stage front and back formation in about 20 days of tillering, a rear onset peak particularly, mycelium is not only fast to influences of plant crown expansion (Longitudinal Extension), and laterally spread rapidly, make that healthy plant is sick on every side, endanger very serious.Pathogenic bacteria (R.solani k ü hn) not only endangers leaf sheath, and can cause blade morbidity or dehydration withered, affect Canopy Apparent Photosynthesis, can cause when serious that the stem stalk rots and in flakes lodging, the morbidity of clever shell, tassel is withered even can not extract out fully, and is huge on the yield and quality impact.

Because the resistance level of paddy rice commercial variety is generally undesirable, up to now, the control to this disease in the production mainly relies on chemical prevention.But in current high fertilizer, large group and a large amount of situations about shifting of the light prime of life labor force in rural area, usually because medication is untimely or method is incorrect, cause prevention effect generally not good, banded sclerotial blight is more and more outstanding as the status of Major Diseases, and even a lot of places in south China rice district, this disease have risen to the first disease of paddy rice.

Cultivating anti-banded sclerotial blight new rice variety is the only way that fundamentally solves banded sclerotial blight harm, has also become the task of top priority on China's Rice Production.Along with the fast development of molecular biology, plant pathology and genetic engineering technique, use genetic engineering means to improve the disease resistance of plant, for breeding for disease resistance has been opened up a brand-new approach.

Summary of the invention

Purpose of the present invention provides GAFP ₂Albumen or its encoding gene or contain the application of the recombinant vectors of described encoding gene.

GAFP provided by the invention ₂Albumen or its encoding gene or contain the application of recombinant vectors in the regulating plant disease resistance of described encoding gene; Described GAFP ₂The aminoacid sequence of albumen be in the sequence table in sequence 2 or the sequence table sequence 2 from the terminal 29-171 amino acids of N ' residue.

Sequence 2 in the above-mentioned sequence table is signal peptide by 171 Amino acid profiles from N-terminal 1-28 position, is GAFP from N-terminal 29-171 position ₂Albumen.

In the above-mentioned application, described GAFP ₂The nucleotides sequence of the encoding gene of albumen is classified in sequence 1 in the sequence table or the sequence table sequence 1 as from 5 ' terminal 85-516 position Nucleotide.

Sequence 1 in the above-mentioned sequence table is made of 516 Nucleotide, and ' terminal 1-84 position is the gene of coded signal peptide, and ' terminal 85-516 position is coding GAFP from 5 from 5 ₂The gene of albumen.

In the above-mentioned application, described regulating plant disease resistance is for improving the plant sharp eyespot resistance;

Described plant is monocotyledons or dicotyledons, and described monocotyledons is paddy rice.The kind name of paddy rice is specially paddy rice " Xu rice No. 3 ".

In the above-mentioned application, described raising plant sharp eyespot resistance is for improving resisting rice sheath blight;

Described rice sheath blight disease is specifically caused by dry thread Pyrenomycetes (Rhizoctonia solani K ü hn).

Another object of the present invention provides a kind of method of cultivating transgenic plant.

Method provided by the invention is for GAFP ₂The encoding gene of albumen imports the purpose plant and obtains transgenic plant, and the sharp eyespot resistance of described transgenic plant is higher than described purpose plant; Described GAFP ₂The aminoacid sequence of albumen be in the sequence table in sequence 2 or the sequence table sequence 2 from the terminal 29-171 amino acids of N ' residue.

In the aforesaid method, described GAFP ₂The nucleotides sequence of the encoding gene of albumen is classified in sequence 1 in the sequence table or the sequence table sequence 1 as from 5 ' terminal 85-516 position Nucleotide.

In the aforesaid method, described purpose plant is monocotyledons or dicotyledons, and described monocotyledons is paddy rice; The kind name of paddy rice is specially paddy rice " Xu rice No. 3 ".

Described banded sclerotial blight is rice sheath blight disease, and described rice sheath blight disease is caused by dry thread Pyrenomycetes (Rhizoctonia solani K ü hn);

Described GAFP ₂The encoding gene of albumen imports in the described purpose plant by the recombinant vectors in following the 3rd purpose.

The 3rd purpose of the present invention provides a kind of recombinant vectors.

Recombinant vectors provided by the invention is for GAFP ₂The promotor of the encoding gene of albumen and the described encoding gene of driving is inserted expression vector and is obtained; Described GAFP ₂The aminoacid sequence of albumen be in the sequence table in sequence 2 or the sequence table sequence 2 from the terminal 29-171 amino acids of N ' residue.

Above-mentioned promotor can be composing type, vascular tissue specifically expressing or fungal induction type promotor, and described constitutive promoter is preferably 35S promoter.Above-mentioned recombinant vectors is with GAFP ₂The promotor 35S of the encoding gene of albumen and the described encoding gene of driving inserts the carrier that obtains among the expression vector pCambia2300;

Described GAFP ₂The nucleotides sequence of the encoding gene of albumen is classified in sequence 1 in the sequence table or the sequence table sequence 1 as from 5 ' terminal 85-516 position Nucleotide.

Above-mentioned recombinant vectors specifically is prepared as follows:

1) with described GAFP ₂The encoding gene of albumen is replaced the GUS fragment in the pBI221 carrier, the intermediate carrier pBI 35S-GAFP2 that obtains;

With described GAFP ₂GUS fragment in the encoding gene replacement pBI221 carrier of albumen further is specially cuts the recombinant vectors that the small segment (GUS fragment) between recognition site obtains with XbaI and the SacI enzyme of the replacement of the Nucleotide shown in the sequence 1 pBI221 carrier in the above-mentioned sequence table.

2) obtain the 1300bp35S-GAFP2 fragment with a HindIII and SacI enzyme carrier pBI35S-GAFP2 that hits, with described 35S-GAFP ₂Fragment is inserted between pCambia2300 carrier HindIII and SacI restriction enzyme site and is obtained recombinant vectors.

The recombinant bacterium or the transgenic cell line that contain above-mentioned expression vector also are the scope of protection of the invention.

Of the present invention experimental results show that: the GAFP that will contain signal peptide ₂Obtain transgenic paddy rice in the gene Introduced into Rice, can resist efficiently infecting of sheath blight fungus, compare with the non-transgenic paddy rice, turn GAFP ₂Paddy rice has anti-banded sclerotial blight ability stable, that obviously strengthen, and its sick index significantly reduces, and illustrates to turn GAFP ₂The GAFP that paddy rice is expressed ₂Albumen can be with mature peptide GAFP under the guiding of signal peptide ₂Accurately be positioned outside the born of the same parents, thereby effectively strengthened the resistance that transgenic plant are infected fungi pathogeny thing.

Description of drawings

Fig. 1 is for turning GAFP ₂The Molecular Identification result of rice strain

Fig. 2 is cultivation and the paddy rice inoculation picture of Rhizoctonia solani Kuhn

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Embodiment 1, contain GAFP ₂Efficient expression vector pCambia2300-35S-GAFP ₂Structure

Synthetic GAFP ₂Gene, the nucleotides sequence of GAFP2 are classified the sequence 1 in the sequence table as, and the aminoacid sequence of this genes encoding is the sequence 2 in the sequence table.Sequence 1 in the above-mentioned sequence table is made of 516 Nucleotide, and ' terminal 1-84 position is the gene of coded signal peptide, and ' terminal 85-516 position is coding GAFP from 5 from 5 ₂The gene of albumen.Sequence 2 in the above-mentioned sequence table is signal peptide by 171 Amino acid profiles from N-terminal 1-28 position, is GAFP from N-terminal 29-171 position ₂Albumen.

With GAFP ₂Gene (sequence 1 in the sequence table) is connected to pUCm-T(and gives birth to worker's biotechnology company limited available from Shanghai) on, carrier construction pUCm-GAFP ₂

Cut pUCm-GAFP with XbaI and SacI enzyme ₂, obtain the GAFP after the 516bp enzyme is cut ₂Fragment connects through this fragment and pBI221 carrier (available from Clontech Laboratories, the Inc.) skeleton that cuts 1.9Kb GUS fragment with same enzyme, obtains intermediate carrier pBI 35S-GAFP ₂

With a HindIII and SacI enzyme carrier pBI35S-GAFP that hits ₂Obtain 1300bp35S-GAFP ₂Fragment, this fragment with through the pCambia2300(of the 8740bp of same enzyme after cutting available from Cambia Laboratories company) carrier framework is connected, and obtains expression vector pCambia2300-35S-GAFP ₂(the process enzyme is cut and is verified as the positive); The bacterium of this expression vector and resistant gene in plant are kalamycin resistance gene Kan r+ and NPTII selection markers gene, this expression vector pCambia2300-35S-GAFP ₂For with 35S-GAFP ₂The carrier that obtains between the HindIII of (35S+ sequence table 1) insertion vector pCambia2300 and SacI restriction enzyme site.

Embodiment 2, turn GAFP ₂The acquisition of paddy rice and functional study

One, turns GAFP ₂The acquisition of paddy rice

1, turns GAFP ₂The acquisition of paddy rice

1) with the expression vector pCambia2300-35S-GAFP that obtains among the embodiment 1 ₂Be transformed in the agrobacterium tumefaciens lba4404 (available from Invitrogen company), obtain agrobacterium tumefaciens lba4404/pCambia2300-35S-GAFP ₂

2) difference picking agrobacterium tumefaciens lba4404/pCambia2300-35S-GAFP ₂Single bacterium colony is inoculated in the YEP substratum that 20ml contains 50mg/L kantlex, 50mg/L Rifampin and 30mg/L Streptomycin sulphate, 28 ° of C, 280rpm incubated overnight to OD600 be 0.7～0.8.Then 5000rpm is centrifugal, abandons supernatant liquor, precipitation NB-liquid nutrient medium Eddy diffusion.At last with paddy rice " Xu rice No. 3 " (Xu Jun, Duan Xiangmao, Liu Qiang, Yang Bo, Xu Zongjin, Zhou Wei keeps No. 3 Super-high-yielding Cultivation in Rice experimental studies of root and Zhong Wei merit .2008. Xu rice. and the Jiangsu agricultural sciences .3:29-31. public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Being designated hereinafter simply as the wild-type paddy rice) callus of rataria soaked in suspension 30 minutes.

3) will be through above-mentioned 2) process the callus that obtains and blot with aseptic thieving paper, be positioned on the NB substratum, the dark cultivation after 4～6 days, with sterilized water explant is cleaned, carry out a sieve in the upper cultured calli of semi-solid NB substratum (adding Pyocianil or the cefotaxime of 250ug/ml), cultivated 16 days for 28 ℃ under cold luminescent lamp, kanamycin-resistant callus tissue chooses and carries out two sieves.The transformant of Fast Growth is chosen, forward in the division culture medium, illumination in 12 hours, under 12 hours dark conditions, continue to cultivate about about two weeks until differentiate budlet.

4) seedling is transferred in the box that contains 30ml 1/2MS substratum, under cold luminescent lamp, cultivated seedling 10-14 days for 28 ℃.At last seedling is transferred in the basin, in the greenhouse, cultivated, obtain T ₀In generation, turn GAFP ₂Paddy rice.

The prescription of NB substratum is: contain following substances: N6 (Chu (N6) Basal Salt Mixture in every 1000mL water, Sigma-Aldrich, C1416) a large amount of (20 *) 50ml, B5 (B5 minimum medium, the Shanghai striking biological reagent of perseverance company limited, BS1304) organic (100 *) 10ml, B5 trace (100 *) 10ml, molysite (100 *) 10ml, 2,4-D (0.2mg/ml) 10ml, proline(Pro) 500mg, caseinhydrolysate 300mg, sucrose 30g, agar 8g, pH 5.8.

The prescription of root media is: MS (Murashige﹠amp; The Skoog minimum medium, Sigma-Aldrich, M5519) pulvis 2.17g, B5 (B5 minimum medium, the Shanghai striking biological reagent of perseverance company limited, BS1304) organic (100 *) 10ml, NAA (1-naphthylacetic acid, Sigma-Aldrich, N0640,0.2mg/ml), 2.5ml MET (methionine(Met), Sigma-Aldrich, 459240,0.2mg/ml) 5ml, sucrose 20g, agar 8g, pH5.8.

Adopt and use the same method, empty carrier pCambia2300 is changed in the paddy rice " Xu rice No. 3 " obtain T ₀In generation, turn the empty carrier paddy rice.

With T ₀In generation, turn GAFP ₂Paddy rice and T ₀In generation, turn the empty carrier rice growing, and sowing obtains respectively T ₁In generation, turn GAFP ₂Paddy rice and T ₁In generation, turn the empty carrier paddy rice.

2, turn GAFP ₂The Molecular Identification of paddy rice

1), extracts total DNA

Extract T ₁In generation, turn GAFP ₂The genomic dna of the fresh young leaflet tablet of paddy rice individual plant adopts conventional CTAB method to extract.

2), PCR detects

The genomic dna of each single-strain blade that obtains take step 1) is as template, with GAFP ₂-F primer: 5 '-AGC CAT GGC AGC ATC CGC AAG-3 ' and GAFP ₂-R primer: 5 '-GCG TCG ACC TAT TCT CTT AGA CCG T-3 ' carries out pcr amplification as the upstream and downstream primer, obtains the PCR product.

The cumulative volume of PCR reaction is 20 μ l, and program is: 94 ° of C 3min of elder generation; Then 94 ° of C 40s, 55 ° of C 30s, 72 ° of C40s are after 30 circulations; 72 ° of C extend 10min.

With the plasmid pCambia2300-35S-GAFP that obtains among the embodiment 1 ₂Positive contrast (CK2) is with the negative contrast of the genomic dna of wild-type paddy rice (CK1).

After reaction finished, the PCR product was through 1.5% agarose gel electrophoresis detected result, the result as shown in Figure 1,1-16 turns GAFP at T1 generation ₂Paddy rice; The negative contrast of CK1; The positive contrast of CK2 can be found out, obtains the positive T1 of 516bp purpose fragment for turning GAFP ₂Paddy rice, swimming lane is T1 generation of 1-5,8-10,12-16 to turn GAFP among the figure ₂The positive T1 of paddy rice is for turning GAFP ₂Paddy rice, and negative control wild-type paddy rice does not have the purpose fragment.

Adopting uses the same method detects T ₁Generation turns the empty carrier paddy rice does not have the purpose fragment yet.

Two, turn GAFP ₂The sharp eyespot resistance of paddy rice detects (field connects the disease experiment)

1, bacterial classification and cultural method thereof

The pathogenic bacteria of rice sheath blight disease is that dry thread Pyrenomycetes (Rhizoctonia solani K ü hn) bacterial strain RH-9(is documented in as in the Publication about Document: Zuo Shimin, Zhang Yafang, Yin Yuejun, the foundation of the happy and auspicious Pan Xue young tiger .2006. field water sheath and culm blight of rice Resistance Identification system of old ancestor and perfect, Yangzhou University's journal agricultural and life science version .27 (4): 57-61; The public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Also can obtain from academy of agricultural sciences, Jiangsu Province Plant Protection Institute.

The spawn culture method: flat wooden stub (10 * 2 * 2) mm, place culture dish (only spreading 1 layer), add PDA substratum (not covering inoculum), after the sterilization, access mycelia piece, the dark cultivation after 3-4 days under the room temperature, when treating that mycelia spreads all over media surface, can be used for Inoculated Rice.

2, expert evidence field planting

Adopt randomized block design, 3 repetitions, every residential quarter rice cultivation 3 row, every row 12 strains.Seeding row spacing is 22.5cm * 12.0cm.Medium Fertilization Level keeps shallow irrigation not dewater.Expert evidence is planted by normal sowing time of paddy rice and farming method in Tanaka's stochastic distribution, keeps the suitable humidity in field, is beneficial to the generation of banded sclerotial blight.Above-mentioned paddy rice is for being numbered the positive T1 of SG2 for turning GAFP ₂Paddy rice, paddy rice " Xu rice No. 3 " and T ₁In generation, turn the empty carrier paddy rice.

3, Field inoculation method

In the positive T1 of the SG2 generation of being numbered for the treatment of that 2 kinds of above-mentioned steps plant, turn GAFP2 paddy rice, paddy rice " Xu rice No. 3 " and T ₁In generation, turn the empty carrier paddy growth to the land for growing field crops jointing initial stage (rice shoot of normal half non-irrigated seedling seedling, about transplanting rear about 35 days) from up to down the 3rd leaf sheath is inboard will to lack the match stick embedding with tweezers, make leaf sheath phimosis state constant, and carry out mark at corresponding blade.3 stem stalks of every strain inoculation see Fig. 2 for details.

With non-transgenic wild-type paddy rice " Xu rice No. 3 " in contrast (CK), T ₁In generation, turn the empty carrier paddy rice also in contrast.

4, morbidity detects

Susceptible contrast is fully fallen ill, and the state of an illness is carried out the state of an illness investigation when basicly stable (greatly about rear about 30 days of heading).8 strains (being that 2 strains are respectively removed on both sides) in the middle of the investigation middle row of cells, the sick level of the 0-9 level investigation standard based on the leaf sheath position that adopts the Pan Xue of Agricultural College Affiliated to Yangzhou Univ. young tiger study group to formulate: wherein 0 grade is that plant is anosis; 9 grades is due to illness unusual death of plant; 0～3 grade is disease-resistant; 4～6 grades is medium disease-resistant to medium susceptible; 7～9 grades is susceptible, and (it is quick to see left scholar for details, Zhang Yafang, Yin Yuejun, the foundation of the happy and auspicious Pan Xue young tiger .2006. field water sheath and culm blight of rice Resistance Identification system of old ancestor and perfect, Yangzhou University's journal agricultural and life science version .27 (4): 57-61 as the grading index; Wang Zibin, a left side shows quick, Li Gang, Chen Xijun, Chen Zongxiang, the comparative studies of the sick level investigation in several fields of Zhang Yafang and Pan Xue young tiger .2009. rice sheath blight disease standard. Yangzhou University's journal agricultural and life science version .3 (4): 9-14).

Each residential quarter is got three individual plants and is identified, the highest sick level is that the sick level of the heaviest stem stalk of morbidity is index in each stem stalk of tillering of a strain plant, average sick level is take stem and the mean number of the disease level of tillering greatly is index, the results are shown in shown in following table 1 and table 2 with the identification and analysis of these two kinds of indexs (disease grade index).T ₁In generation, turns the empty carrier paddy rice and contrasts disease-resistant result and non-transgenic contrast indifference.

Table 1 is take the highest sick level as index, the disease-resistant as a result appraisal that carries out

The disease-resistant as a result appraisal of table 2 for carrying out as index take average sick level

Can find out that from the disease-resistant qualification result in above-mentioned field CK compares with non-transgenic wild-type paddy rice, be numbered the positive T1 of SG2 for turning GAFP ₂Paddy rice has anti-banded sclerotial blight ability more stable, that strengthen, and its sick level index significantly reduces, and illustrates to turn GAFP ₂The rhizoma Gastrodiae antifungal protein GAFP2 that expresses in the paddy rice can accurately be positioned outside the born of the same parents under the guiding of signal peptide (SP), thereby has effectively strengthened the resistance that transgenic plant are infected fungi pathogeny thing.

Claims (10)

1.GAFP ₂Albumen or its encoding gene or contain the application of recombinant vectors in the regulating plant disease resistance of described encoding gene; Described GAFP ₂The aminoacid sequence of albumen be in the sequence table in sequence 2 or the sequence table sequence 2 from the terminal 29-171 amino acids of N ' residue.

2. application according to claim 1 is characterized in that:

The nucleotides sequence of the encoding gene of described GAFP2 albumen is classified in sequence 1 in the sequence table or the sequence table sequence 1 as from 5 ' terminal 85-516 position Nucleotide.

3. application according to claim 1 and 2 is characterized in that:

Described regulating plant disease resistance is for improving the plant sharp eyespot resistance;

Described plant is monocotyledons or dicotyledons, and described monocotyledons is paddy rice.

4. application according to claim 3 is characterized in that:

Described raising plant sharp eyespot resistance is for improving resisting rice sheath blight;

Described rice sheath blight disease is specifically caused by dry thread Pyrenomycetes (Rhizoctonia solani K ü hn).

5. a method of cultivating transgenic plant obtains transgenic plant for the encoding gene with GAFP2 albumen imports the purpose plant, and the sharp eyespot resistance of described transgenic plant is higher than described purpose plant; The aminoacid sequence of described GAFP2 albumen be in the sequence table in sequence 2 or the sequence table sequence 2 from the terminal 29-171 amino acids of N ' residue.

6. method according to claim 5 is characterized in that: the nucleotides sequence of the encoding gene of described GAFP2 albumen is classified in sequence 1 in the sequence table or the sequence table sequence 1 as from 5 ' terminal 85-516 position Nucleotide.

7. according to claim 5 or 6 described methods, it is characterized in that: described purpose plant is monocotyledons or dicotyledons, and described monocotyledons is paddy rice;

Described banded sclerotial blight is rice sheath blight disease, and described rice sheath blight disease is caused by dry thread Pyrenomycetes (Rhizoctonia solani K ü hn);

The encoding gene of described GAFP2 albumen imports in the described purpose plant by following claim 8 or 9 described recombinant vectorss.

8. a recombinant vectors obtains for the encoding gene with GAFP2 albumen inserts expression vector with the promotor that drives described encoding gene; The aminoacid sequence of described GAFP2 albumen be in the sequence table in sequence 2 or the sequence table sequence 2 from the terminal 29-171 amino acids of N ' residue.

9. recombinant vectors according to claim 8 is characterized in that: described recombinant vectors is for inserting the carrier that obtains among the expression vector pCambia2300 with the encoding gene of GAFP2 albumen and the promotor 35S that drives described encoding gene;

The nucleotides sequence of the encoding gene of described GAFP2 albumen is classified in sequence 1 in the sequence table or the sequence table sequence 1 as from 5 ' terminal 85-516 position Nucleotide.

10. the recombinant bacterium or the transgenic cell line that contain claim 8 or 9 described expression vectors.

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