Application of FoCcw14 gene in regulation and control of endophytic fungi-rice symbiotic system to rice blast resistance

文档序号：1811100 发布日期：2021-11-09 浏览：45次中文

阅读说明：本技术 FoCcw14基因在调控内生真菌-水稻共生体系抗稻瘟病中的用途 (Application of FoCcw14 gene in regulation and control of endophytic fungi-rice symbiotic system to rice blast resistance ) 是由林福呈代梦迪苏珍珠于 2021-07-23 设计创作，主要内容包括：本发明公开了FoCcw14基因在调控瓶霉属内生真菌-水稻共生体系抗稻瘟病中的用途,属于植物病害防治技术领域。所述FoCcw14基因的核苷酸序列如SEQ ID NO.1所示。本发明通过对FoCcw14基因的克隆和分析,并利用同源重组的技术对该基因进行功能验证,发现敲除FoCcw14基因导致内生真菌-水稻共生体系抗稻瘟病的性能显著降低。本发明为后续水稻育种提供了新的思路。(The invention discloses an application of a FoCcw14 gene in regulating and controlling rice blast resistance of a pitcher fungus endophytic fungus-rice symbiotic system, and belongs to the technical field of plant disease control. The nucleotide sequence of the FoCcw14 gene is shown in SEQ ID NO. 1. According to the invention, by cloning and analyzing the gene FoCcw14 and carrying out functional verification on the gene by using a homologous recombination technology, the fact that the knockout of the gene FoCcw14 leads to the remarkable reduction of the rice blast resistance of an endophytic fungus-rice symbiotic system is found. The invention provides a new idea for subsequent rice breeding.)

1. The application of the FoCcw14 gene with the nucleotide sequence shown as SEQ ID NO.1 in regulating and controlling the resistance of endophytic fungi of the botrytis to rice blast of a rice symbiotic system.

2. The use according to claim 1, wherein the amino acid sequence of the protein encoded by the FoCcw14 gene is shown in SEQ ID No. 2.

3. The use according to claim 1, wherein the knockout of the endophytic fungus FoCcw14 of the genus Calycota results in a reduction in the resistance of the symbiotic system of endophytic fungi of the genus Calycota to Pyricularia oryzae diseases.

4. The use according to claim 1, wherein the endophytic fungus belonging to the genus Botrytis is Falciphora oryzae FO-R20 with a collection number of CCTCC NO: M2021505.

5. The use according to claim 4, wherein the construction method of the symbiotic system of endophytic fungi of the genus Calycota-rice comprises: the endophytic fungus Falciphora oryzae FO-R20 was co-cultured with rice to colonize the root tissue of rice.

6. The use according to claim 5, wherein the endophytic fungus Falciphora oryzae FO-R20 strain is inoculated to PDA medium for activation culture and dark culture at 25 ℃ for 7 days before co-cultivation.

7. The use of claim 5, wherein co-culturing comprises inoculating germinated rice seeds and a clump of the endophytic fungus Falciphora oryzae FO-R20 on sterile 1/2MS medium for culturing.

8. Use according to claim 7, wherein the co-cultivation conditions are: culturing at 22-25 deg.C for 20-25 days under illumination for 16 hr per day, and culturing in dark for 8 hr.

Technical Field

The invention relates to the technical field of plant disease control, in particular to application of a FoCcw14 gene in regulation and control of rice blast resistance of endophytic fungi-rice symbiotic system of Chlamydomonas.

Background

The rice blast is one of important diseases of rice, can cause great yield reduction, and can reduce the yield by 40 to 50 percent even if the yield is serious, and even no grains are harvested. The disease is an epidemic disease spread by airflow, has great threat to rice production, has different harm degrees due to different varieties, cultivation techniques and climatic conditions, and occurs in all rice regions in the world, wherein the number of the leaves and the nodes is large, so that the yield can be reduced to different degrees after the occurrence, particularly, the panicle neck blast or the node blast occurs early and seriously, and the white spike can be caused to cause dead production.

Biological control is a research hotspot for controlling plant diseases, and biological control is to reduce the number or pathogenicity of pathogenic bacteria by utilizing beneficial microorganisms to generate various adverse effects (such as antibiosis, bacteriolysis, competition, parasitism and the like) on the pathogenic bacteria; meanwhile, the beneficial microorganisms for biological control can also induce the enhancement of plant disease resistance, improve plant immunity and delay, alleviate or inhibit the occurrence of diseases.

The plant endophytic fungi is one of beneficial microorganisms existing in nature, at least part of life history of the endophytic fungi is in healthy plant tissues, and the endophytic fungi colonize and absorb nutrition in a host, but different from pathogenic fungi, the endophytic fungi can not obviously harm the host and can keep a balance relationship with the host in the host.

Endophytic fungi exhibit many beneficial biological relationships during their interaction with plants, such as promoting growth of the host, increasing yield of the host; producing toxins to protect plants from feeding by insects, herbivores; when plants face abiotic stress adversities such as high and low temperature and heavy metal stress, physiological and biochemical reactions of the plants can be regulated and controlled, so that the plants are helped to live through adverse environments; in addition, colonization by endophytic fungi can induce the host to produce systemic disease resistance, thereby protecting against fungi, bacteria, nematodes, viruses, and the like.

In the aspect of resisting rice blast stress, patent document with application number 201911150894.1 discloses that the sugarcane endophytic bacillus amyloliquefaciens CGB15 strain has strong inhibiting effect on sugarcane smut, rice blast, litchi rinderpest fungus, litchi colletotrichum gloeosporioides and banana vascular wilt. Patent document No. 201610064924.7 discloses that antagonistic bacterial strain HDXY isolated from the bulb of laughing neglecta has a very significant antagonistic effect against rice blast. At present, no report indicates the function of the wild rice endophytic fungi strain in preventing and controlling the neck blast of rice.

At present, the research on the molecular mechanism of how endophytic fungi induce a host to resist rice blast stress is few, so that the related genes are excavated by researching the molecular mechanism of endophytic fungi interaction, the related performance of the endophytic fungi is improved by using a molecular biology means, the disease resistance of an interaction object is improved, and the endophytic fungi is developed into a biological prevention and control preparation to be applied to agricultural production, and the prospect is wide.

Disclosure of Invention

The invention aims to provide a gene which is cloned from rice endophytic fungi and has the characteristic of promoting the rice blast disease stress resistance of host rice, and provides a theoretical basis and related genes for the improvement and production of endophytic fungi-rice symbiotic system.

In order to achieve the aim, the gene FoCcw14 with the characteristic of promoting the host rice blast disease stress resistance is identified and cloned from endophytic fungus Falciphara oryzae FO-R20 with the preservation number of CCTCC NO: M2021505, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1. The CDS region of the gene has the full length of 453bp and codes a protein sequence of 150 aa. The amino acid sequence of the protein coded by the FoCcw14 gene is shown as SEQ ID No. 2.

According to the invention, a homologous recombination technology is utilized to knock out FoCcw14 gene in the genome of endophytic fungus Falciphora oryzae FO-R20 to obtain a mutant delta FoCcw14, and compared with a wild type-rice symbiotic system of the endophytic fungus, the rice blast resistance of the rice system is remarkably reduced by 15% after the mutant is co-cultured with the root of rice, and the result shows that the FoCcw14 gene is a key gene for regulating and controlling the endophytic fungus to influence the disease resistance of a host.

Therefore, the invention provides the application of the FoCcw14 gene with the nucleotide sequence shown as SEQ ID NO.1 in regulating and controlling the resistance of endophytic fungi of the botrytis to rice blast of a rice symbiotic system. The gene enhances the resistance of a symbiotic system to rice blast germs. After the gene FoCcw14 is knocked out, the function of resisting rice blast disease of a pitcher fungus endophytic fungus-rice symbiotic system is reduced.

Further, the construction method of the pitcher endophytic fungus-rice symbiotic system comprises the following steps: the endophytic fungus Falciphora oryzae FO-R20 was co-cultured with rice to colonize the root tissue of rice.

Preferably, before co-cultivation, the endophytic fungus Falciphora oryzae FO-R20 strain was inoculated into PDA medium for activation culture and dark culture at 25 ℃ for 7 days.

Preferably, the co-culture comprises inoculating germinated rice seeds and a hypha block of an endophytic fungus Falciphora oryzae FO-R20 on a sterile 1/2MS culture medium for culture.

Preferably, the co-cultivation conditions are: culturing at 22-25 deg.C for 20-25 days under illumination for 16 hr per day, and culturing in dark for 8 hr.

The invention also provides a method for preventing and treating rice blast, which comprises the following steps: the gene FoCcw14 with the nucleotide sequence shown as SEQ ID NO.1 is introduced into endophytic fungi of the genus Botrytis to obtain recombinant bacteria, and the recombinant bacteria and rice are co-cultured to obtain a recombinant bacteria-rice symbiotic system.

The invention has the following beneficial effects:

the invention discloses a gene FoCcw14 influencing the performance of host rice blast resistance in rice endophytic fungi for the first time, and discovers that the performance of endophytic fungi-rice symbiotic system rice blast resistance is obviously reduced by knocking out the FoCcw14 gene through cloning and analyzing the FoCcw14 gene and carrying out functional verification on the gene by utilizing a homologous recombination technology. The invention provides a new idea for subsequent rice breeding.

Drawings

FIG. 1 shows the bioinformatics analysis of FoCcw14 in the endophytic fungus Falciphora oryzae FO-R20, panel A shows the multiple sequence alignment, panel B shows the SMART prediction result, and panel C shows the phylogenetic analysis.

FIG. 2 shows the strain of the endophytic fungal gene mutant Δ FoCcw 14.

FIG. 3 is a cross section of rice roots after the endophytic fungal gene mutant strain delta FoCcw14 is colonized on the rice roots, the left figure is a view under white light, and the right figure is a view under 488nm laser.

FIG. 4 shows the lesion area of the endophytic fungal gene mutant strain Δ FoCcw14 after the colonization of the rice roots and the spraying of Magnaporthe grisea.

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.

In the present invention, the equipment and materials used are commercially available or commonly used in the art, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.

The invention separates a new endophytic fungus Falciphara oryzae FO-R20 of the genus Chlophora from a wild rice root system of Yunnan verrucous granules by the prophase work of a subject group as a material, knocks out a FoCcw14 gene, and has important significance for explaining the molecular mechanism of responding to rice blast germ stress of an endophytic fungus FO-R20-rice symbiotic system.

Example 1 FoCcw14 Gene analysis

The endophytic fungus Falciphora oryzae FO-R20 is isolated from root system of Oryza sativa Miquel, and is preserved in China center for type culture Collection of Wuhan university in 5 months and 8 days in 2021, with the preservation numbers: CCTCC NO: M2021505.

Earlier studies show that the endophytic fungus Falciphora oryzae FO-R20 is colonized in root tissues of rice plants, and the resistance of rice to rice blast stress can be remarkably improved.

The complete genome sequence analysis was performed on Falciphora oryzae FO-R20, and the signal peptide and domain of the protein were predicted by the online software SMART. The FoCcw14 was determined to be the subject of study by multiple sequence alignment and phylogenetic analysis. The nucleotide sequence of the FoCcw14 gene is shown in SEQ ID NO.1, the coded amino acid sequence is shown in SEQ ID NO.2, and the bioinformatics analysis result is shown in FIG. 1.

Example 2 verification of FoCcw14 Gene function

Test endophytic fungi: falciphora oryzae FO-R20;

the test plants: rice Oryza sativa l., conventional variety, CO 39.

1. Culturing of bacterial strains

The FO-R20 strain preserved on the filter paper sheet is inoculated on a Potato Dextrose Agar (PDA) solid medium for activation culture at 25 ℃ and cultured in the dark for 7 days for standby.

PDA culture medium: the product contains glucose 20g, potato 200g, and agar 15 g. Weighing the required potato according to the volume of the culture medium to be prepared, boiling in water, mashing, dissolving, filtering, adding glucose and agar, and sterilizing with high pressure steam at 121 deg.C for 20 min.

2. Acquisition of Gene mutant Strain Δ FoCcw14

Designing upstream and downstream primers of the target gene, and amplifying upstream and downstream fragments and SUR fragments. The specific primer sequences are as follows:

FoCcw14-UP-F：

5’-GGTACCCGGGGATCCTCTAGATTACCTGCCTACCTACCTAG-3’；

FoCcw14-UP-R：

5’-GGCACTGTGGCGTTGGCACGCGTCTGCTGTGTCTTGAC-3’；

FoCcw14-DOWN-F：

5’-GGGAATTGCATGCTCTCACTCATTCCTTCTAGTGCTTCA-3’；

FoCcw14-DOWN-R：

5’-ACGACGGCCAGTGCCAAGCTTGAATACTACAAGAACCGTGAC-3’；

SUR-F：5’-GTGCCAACGCCACAGTGCCCCAC-3’；

SUR-R：5’-GATTGTGAATCGTGAGAGCATGCAATTC-3’。

the vector pKO1B was digested with the restriction enzymes XbaI and Hind III. According to the instructions of the Clonexpress II One Step clone Kit (Vazyme, China), the recovered fragment and the digested pKO1B vector were ligated together to construct a knockout vector and transformed into E.coli competent DH5 α. The grown transformant is picked out and put into 1mL LB liquid medium (containing 50 ug/mL kanamycin), shaken at the speed of 200rpm and the temperature of 37 ℃ for 4-6h, and 1-2 mul of bacterial liquid is sucked and added into a verification system for PCR amplification and gel electrophoresis.

Plasmid extraction was performed according to the AxyPrep-96(Axygen, US) protocol. And the plasmid is transferred into agrobacterium-induced AGL1, mutant transformants are obtained by an agrobacterium-mediated transformation method, DNA of the transformants is extracted for long-short fragment verification and copy number verification, the transformants which are verified to be correct are inoculated to a PDA culture medium for subsequent experiments, and are inoculated to a PDA slant of a freezing storage tube for storage.

Colony morphology of mutant strain Δ FoCcw14 is shown in fig. 2, and it was experimentally found that mutant strain Δ FoCcw14 substantially coincides with wild type in colony morphology and diameter.

3. Co-culture of FO-R20 wild strain, mutant strain delta FoCcw14 and rice root

Removing hull of rice seed, placing in triangular flask, sterilizing with 1% NaClO surface for 15min, and cleaning with sterile water for 3 times. The sterilized seeds were spread evenly on 1/2MS (Murashige and Skoog) medium, sealed with Parafilm sealing film, and placed in a 25 ℃ plant incubator (16h light/8 h dark). After 3 days, the seeds appeared white, and they were transferred into tissue culture flasks containing 1/2MS medium, 10 seeds per flask. 3 pieces of the cake (0.5 cm in diameter) were inoculated simultaneously. The control group was sterile PDA agar blocks. Let 3 replicates.

As shown in FIG. 3, the mutant GFP-labeled hyphae spread only on the epidermal layer and did not enter the rice cells.

4. Spraying of conidia of rice blast germs

The rice blast fungus strain Guy11 is inoculated in CM solid culture medium and cultured for 10-12 days at 25 ℃ under illumination. Washing Guy11 conidia with sterile water, filtering with three-layer filter paper, and making into spore suspension with concentration of 1 × 10⁵. Subsequently, 0.4% gelatin solution was prepared and mixed with the spore suspension at a volume ratio of 1:1 to give a final concentration of 5X 10 of Magnaporthe grisea spore suspension⁴. The spore suspension was uniformly sprayed onto the rice seedling leaves with a sprayer at 1mL per bottle. Then, the tissue culture bottle was placed in a plant incubator at 25 ℃ and cultured in the dark for 2 days. After 2 days, supplementing light, 16h light/8 h dark, culturing for 4-5 days, and recording the disease condition of the leaves.

CM medium (1L): yeast Extract (1g), Casamino acid (1g), D-glucose (10g), KH₂PO₄(1.52g)，NaNO₃(6g)，Peptone140(2g)，KCl(0.52g)， MgSO₄·7H₂O (0.52g), 0.1% (v/v) Vitamin solution, 0.1% (v/v) Trace Element. The pH was adjusted to 6.5 with NaOH and 15g/L agar was added to the solid medium. Sterilizing with high pressure steam at 121 deg.C for 15 min.

5. Statistics of rice blast scab rate

The ill rice leaves are pasted on white paper, and a scale is placed for photographing. Randomly selecting 5 cm-long leaves in Photoshop, counting the whole pixels of the selected leaves, adjusting tolerance, selecting green non-scab areas, and counting the pixels.

The plaque rate (%) is calculated by: (capture leaf full pixels-green non-lesion area pixels)/capture leaf full pixels.

As shown in fig. 4, the typical spindle-shaped lesions appeared in the blank control and rice leaves symbiotic with mutant Δ FoCcw 14. The rice leaves symbiotic with the wild strains have small and few scabs. Therefore, the gene FoCcw14 plays an important role in regulating and controlling the endophytic fungus Falciphora oryzae FO-R20-rice symbiotic system to respond to the rice blast fungus stress.

Sequence listing

<110> Zhejiang province academy of agricultural sciences

Application of FoCcw14 gene in regulation and control of endophytic fungi-rice symbiotic system to rice blast resistance

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<212> DNA

<213> Falciphora oryzae

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gccggcgacc tgcccaagtg cgctcaagac tgcgcgaacc agttcctcct gaacggcatc 120

ggcaactgcg gccgcgaccc aaagtgcatc tgctccaaca agtccttcat cggagacatc 180

tcgtgctgcc tggccaagcc gggcggctgc agcgccgacg accagaagaa ggccatcgcc 240

tacgccgccc agctgtgcca ggccaacggc gtcaccgtcc ccgaccaggt cgtctgcaac 300

agcaacagca acaacggcac cgcggccacc tctggcgccg gcggcgccgc ggccaccacc 360

tcgtcgtcca gggcctgggc cccgcaccag accggcgccc ctgctgctgt cggcctcctc 420

ggcggcctgg ctgctgtcgc cgtgctgctg tga 453

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<213> Falciphora oryzae

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Met Lys Thr Ser Ala Val Leu Ala Ala Leu Ala Leu Ala Val Ala Thr

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Asn Ala Gln Thr Ala Gly Asp Leu Pro Lys Cys Ala Gln Asp Cys Ala

20 25 30

Asn Gln Phe Leu Leu Asn Gly Ile Gly Asn Cys Gly Arg Asp Pro Lys

35 40 45

Cys Ile Cys Ser Asn Lys Ser Phe Ile Gly Asp Ile Ser Cys Cys Leu

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Ala Lys Pro Gly Gly Cys Ser Ala Asp Asp Gln Lys Lys Ala Ile Ala

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Tyr Ala Ala Gln Leu Cys Gln Ala Asn Gly Val Thr Val Pro Asp Gln

85 90 95

Val Val Cys Asn Ser Asn Ser Asn Asn Gly Thr Ala Ala Thr Ser Gly

100 105 110

Ala Gly Gly Ala Ala Ala Thr Thr Ser Ser Ser Arg Ala Trp Ala Pro

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His Gln Thr Gly Ala Pro Ala Ala Val Gly Leu Leu Gly Gly Leu Ala

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Ala Val Ala Val Leu Leu

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