Application of FoSyn1 gene in regulation and control of cadmium tolerance of endophytic fungi or rice symbiotic system

文档序号：1811101 发布日期：2021-11-09 浏览：40次中文

阅读说明：本技术 FoSyn1基因在调控内生真菌或水稻共生体系耐镉性中的用途 (Application of FoSyn1 gene in regulation and control of cadmium tolerance of endophytic fungi or rice symbiotic system ) 是由林福呈苏珍珠代梦迪于 2021-07-23 设计创作，主要内容包括：本发明公开了FoSyn1基因在调控内生真菌或水稻共生体系耐镉性中的用途,属于基因工程技术领域。所述FoSyn1基因的核苷酸序列如SEQ IDNO.1所示。本发明首次公开水稻内生真菌中调控对镉胁迫耐受性的基因FoSyn1,通过对FoSyn1基因的的克隆和分析,并利用同源重组的技术对该基因进行功能验证,发现敲除FoSyn1基因导致菌株自身以及与之共生的水稻对于镉胁迫的耐受性显著降低,该研究结果在可持续农业中具有巨大的应用潜力,为提高水稻耐受性和保证粮食安全提供了研究思路。(The invention discloses an application of a FoSyn1 gene in regulation and control of cadmium tolerance of endophytic fungi or rice symbiotic systems, belonging to the technical field of genetic engineering. The nucleotide sequence of the FoSyn1 gene is shown in SEQ ID NO. 1. The invention discloses a gene FoSyn1 for regulating cadmium stress tolerance in rice endophytic fungi for the first time, and by cloning and analyzing the FoSyn1 gene and carrying out functional verification on the gene by utilizing a homologous recombination technology, the fact that the knockout of the FoSyn1 gene causes the cadmium stress tolerance of a strain and rice symbiotic with the strain to be obviously reduced is found.)

1. The FoSyn1 gene with a nucleotide sequence shown as SEQ ID NO.1 is used for regulating and controlling cadmium resistance of endophytic fungi of the genus bottled mould or endophytic fungi-rice symbiotic system.

2. The use according to claim 1, wherein the FoSyn1 gene encodes a protein having an amino acid sequence as set forth in SEQ ID No. 2.

3. The use according to claim 1, wherein the knockout of the endophytic fungus FoSyn1 of the genus Calycota results in a decrease in the tolerance of the endophytic fungus of the genus Calycota or the symbiotic system of endophytic fungus of the genus Calycota-rice to cadmium stress.

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 genetic engineering, in particular to application of a FoSyn1 gene in regulation and control of cadmium tolerance of endophytic fungi or rice symbiotic systems.

Background

With the rapid development of industrialized, urbanized and agricultural intensive production in China, the problem of heavy metal pollution of farmlands is increasingly serious, and the food quality safety and human health are seriously threatened. As an important food crop in China, the problem that cadmium in rice exceeds the standard is very serious at present, and the problem of how to reduce cadmium accumulation in rice is of great concern. With the improvement of environmental protection consciousness of people, the position of green prevention and control in agricultural prevention and control is increased year by year. Therefore, it is well recognized in the art to seek an environmentally friendly, ecologically safe, efficient, economical, and generalizable strategy for solving the problem of cadmium accumulation in rice.

In recent years, there has been increasing interest among bacteriologists and ecologists in the application of endophytic fungi to plant stress resistance, particularly to heavy metal stress. In 1989, Buttrey studies found that endophytic fungi (Acremonium coenophialium) were able to reduce copper levels in Festuca arundinacea (Buttrey,1989), and thus the effect of endophytic fungi on host heavy metal tolerance was discovered. Endophytic fungi are beneficial fungi which are colonized in healthy plants, biological functions are abundant, many fungi can live in a heavy metal environment with high concentration, and benefit from many unique tolerance mechanisms, such as isolation and chelation of extracellular heavy metals, binding of heavy metals on cell walls, intracellular isolation and complexation, compartmentalization and the like (Fomina et al, 2005). Endophytic fungi possess a metal sequestering and chelating system that can increase the tolerance of host plants to heavy metals (Aly et al, 2011). Therefore, the biological technology for improving quality and increasing efficiency of plants by using endophytic fungi can also become a new means which is matched with the traditional adversity-resistant breeding and transgenic cultivation technology, and has very wide application prospect.

The invention patent with application number 201410068912.2 discloses a fusarium oryzicola separated from roots of wild rice grain warts, the preservation number is CGMCC No.2737, and the strain can enhance the tolerance of plants to heavy metals and achieve the reciprocal symbiotic relationship with the plants.

At present, the research on regulation genes of endophytic fungi responding to heavy metal stress is less, related genes are excavated by researching the molecular mechanism of endophytic fungi interaction, and then the related performance of endophytic fungi is modified by utilizing the molecular biology means, so that the cadmium resistance of an interaction object is improved, and the interaction object is developed into a biological prevention and control preparation applied to agricultural production, so that the biological prevention and control preparation has an important application value.

Disclosure of Invention

The invention aims to provide a gene for regulating and controlling cadmium stress resistance of a strain and a rice symbiotic system obtained by cloning from rice endophytic fungi, and provides a research idea for improving rice tolerance and ensuring grain safety.

In order to achieve the purpose, the invention adopts the following technical scheme:

the gene FoSyn1 with cadmium stress resistance is identified and cloned from endophytic fungus Falciphora oryzae FO-R20 of the bottle mold with the preservation number of CCTCC NO: M2021505. Under the heavy metal cadmium stress condition, the expression of the gene is obviously up-regulated.

The full length of the FoSyn1 gene is 1842bp, and the nucleotide sequence is shown in SEQ ID NO. 1. FoSyn1 encodes a 505aa protein sequence, and the amino acid sequence is shown in SEQ ID No. 2.

The invention utilizes homologous recombination technology to knock out FoSyn1 gene in the genome of endophytic fungus Falciphara oryzae FO-R20 to obtain a mutant delta FoSyn1, and compared with a wild strain, the minimum inhibitory concentration of the mutant to cadmium is reduced to 12.5mg/L from 20 mg/L. Under the condition of cadmium stress, compared with a wild type rice symbiotic system of endophytic fungi, the plant height of rice symbiotic with the mutant at the seedling stage is obviously reduced by 34.69%, and the result shows that the FoSyn1 gene is a key gene for regulating and controlling the cadmium stress resistance of the endophytic fungi and influencing the cadmium resistance of a host.

Therefore, the invention provides the application of the FoSyn1 gene with the nucleotide sequence shown as SEQ ID NO.1 in regulating and controlling the cadmium resistance of the endophytic fungi of the bottled mold or the endophytic fungi-rice symbiotic system of the bottled mold. The gene enhances the tolerance of endophytic fungi and an endophytic fungi-rice symbiotic system to heavy metal cadmium stress. After the FoSyn1 gene is silenced, the tolerance of the endophytic fungi of the genus Botrytis or the endophytic fungi of the genus Botrytis-rice symbiotic system to cadmium stress 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 improving the cadmium stress tolerance of rice, which comprises the following steps: the FoSyn1 gene 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 FoSyn1 for regulating cadmium stress tolerance in rice endophytic fungi for the first time, and by cloning and analyzing the FoSyn1 gene and carrying out functional verification on the gene by utilizing a homologous recombination technology, the fact that the knockout of the FoSyn1 gene causes the cadmium stress tolerance of a strain and rice symbiotic with the strain to be obviously reduced is found.

Drawings

FIG. 1 shows the colony morphology of an endophytic fungi gene knockout mutant delta FoSyn1 strain.

FIG. 2 shows the content of endophytic fungi at different concentrations (0-25.0 mg. L)^-1) Growth conditions on PDA medium of cadmium, wherein WT is wild type strain, delta FoSyn1 is FoSyn1 gene knockout mutant, and FoSyn1c is anaplerosis strain.

FIG. 3 shows the growth of rice symbiotic with endophytic fungi under cadmium stress (cadmium concentration of 5 μ M), (a) shows photographs of rice plants, (b) shows statistical results of rice plant height, and (c) shows statistical results of green leaf content of rice leaves. The significance is as follows: denotes P < 0.01.

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 previous work of a subject group as a material, knocks out the FoSyn1 gene, and has important significance for explaining the molecular mechanism of the endophytic fungus FO-R20-rice symbiotic system responding to heavy metal cadmium stress.

Example 1 FoSyn1 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 Wuhan, Wuhan university at 2021, 5 months and 8 days, with the preservation number: CCTCC NO: M2021505.

Earlier researches show that the endophytic fungus Falciphora oryzae FO-R20 is colonized in root tissues of rice plants, and the cadmium tolerance of rice can be remarkably improved. The mutualistic symbiosis relationship provides a foundation for researching the molecular mechanism of rice stress response and improving the quality.

The proteomics of Falciphora oryzae FO-R20 under heavy metal cadmium stress and in a natural state is analyzed, and 88 proteins with obvious differential expression are identified. Of these, 50 proteins up-regulate expression and 38 down-regulate expression. We found that a Syntaxin1 protein FoSyn1 was significantly up-regulated under cadmium stress. We selected this gene as a candidate gene and investigated its role in cadmium resistance in Falciphora oryzae FO-R20.

Specifically, the nucleotide sequence of the FoSyn1 gene is shown in SEQ ID NO.1, and the gene sequence contains an intron. The coded amino acid sequence is shown in SEQ ID NO. 2.

Example 2 validation of FoSyn1 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 Delta FoSyn1

Designing upstream and downstream primers of the target gene, and amplifying upstream and downstream fragments and SUR fragments. Specific primers are shown in Table 1.

TABLE 1

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, a mutant delta FoSyn1 is obtained by an agrobacterium-mediated transformation method, DNA of a transformant is extracted for long-short fragment verification and copy number verification, the transformant which is verified to be correct is inoculated to a PDA culture medium for subsequent experiments, and is inoculated to a PDA inclined plane for storage. The growth of mutant Δ FoSyn1 on PDA medium is shown in figure 1.

In addition, the FoSyn1 gene is complemented back into a FoSyn1 knockout mutant to obtain a complemented strain FoSyn1 c.

The results show that the knockout mutant strain delta FoSyn1 and the replenisher strain FoSyn1c have no difference from the wild strain in colony morphology, hyphae are creeping, aerial hyphae are not flourishing, and the hyphae are black brown.

3. FO-R20 wild strain, mutant strain delta FoSyn1 and anaplerosis strain FoSyn1c are inoculated to different concentrations (0-25.0 mg. L)^-1) PDA culture medium of cadmium

Inoculating wild type fungus cake and fungus cake (diameter is 0.5cm) of gene knockout mutant delta FoSyn1 in different concentrations (0-25.0 mg. L)^-1) Culturing in the dark at 25 deg.C for 5-7 days in PDA culture medium containing cadmium, and observing its growth condition.

As shown in FIG. 2, the minimal inhibitory concentration of Δ FoSyn1 was 12.5 mg.L, which was lower than that of the wild type strain^-1It was shown that the deletion of FoSyn1 resulted in a decrease in the resistance of Fusarium oryzae to heavy cadmium metals. The complementation mutant strain FoSyn1c has recovered cadmium tolerance and is consistent with the wild type.

4. Co-culture of FO-R20 wild strain, mutant strain delta FoSyn1, anaplerotic strain FoSyn1c 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 were transferred into tissue culture flasks containing 1/2MS medium with 5. mu.M cadmium, 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.

5. Counting the plant height and chlorophyll content of rice seedlings

Plant height measurement: after the rice is symbiotically cultured with wild strains, mutant strains and anaplerotic strain FoSyn1c for 15d, three strains are selected from each tissue culture tube to measure the plant height, and each experiment is repeated three times.

Measuring the chlorophyll content: after the rice is symbiotically cultured with the wild strain and the mutant strain for 15 days, the chlorophyll content of the leaf of three rice plants in each culture tube is measured by a chlorophyll content measuring instrument SPAD-502 (Japan), and the three measurements are repeated for three times each time and three times each experiment.

As shown in FIG. 3, the rice inoculated with Δ FoSyn1 showed dysplasia under heavy metal stress (FIG. 3a), and the plant height was significantly reduced by 34.69% (FIG. 3b) as compared with the wild-type rice inoculated. The chlorophyll content of the leaves did not change significantly compared to wild type inoculated seedlings (fig. 3 c). The rice inoculated with the anaplerotic strain FoSyn1c recovers the characteristic of improving the cadmium resistance of the rice and has the same function with the wild strain. These results indicate that deletion of FoSyn1 results in reduced cadmium tolerance of the Falciphora oryzae FO-R20, thereby affecting cadmium tolerance of symbiotic systems.

Sequence listing

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