Oriented evolution method of rice growth-promoting microbiome from top to bottom

Abstract

The invention provides a top-down directional evolution method for growth-promoting microbiome of rice. The top-to-bottom rice growth-promoting microbiome directional evolution method comprises the following steps: step 1, selecting a microbiome with a growth-promoting effect on rice seedlings, combining Bottleneck + Migration into an LB culture medium for iterative culture, and after 15 rounds of non-selective iteration, transferring each round of iteration into a new LB culture solution; 2, respectively inoculating the flora subjected to 15 rounds of non-selective iteration in the step 1 into a Hoagland nutrient solution; 3, the nutrient solution samples with the flora added in the step 2 are respectively and equivalently added into rice seedling culture dish samples; 4, observing the growth vigor of the rice once every five days, and detecting morphological indexes such as leaf length and root length of the rice; 5, repeating the steps 1-4 on the inoculated flora screened in the step 4, and circulating for 5 times; the method has the advantage of screening out the microbiome capable of remarkably promoting the growth of the rice seedlings.

Description

Technical Field

[0001] The present invention relates to the field of rice microbial directed evolution, and in particular to a top-down rice growth-promoting microbiome directed evolution method. Background Art

[0002] Rice is one of the most important and consumed food crops in the world. According to the United Nations, the world population will reach 9.8 billion by 2050. Agricultural productivity is expected to increase by 70% to meet the growing demand for food, feed, fiber and bioenergy. Due to the limited arable land area, the improvement of agricultural productivity relies more on the transformation of crop varieties and the creation of high-efficiency fertilizers and pesticides. Since the 1980s, the input of fertilizers per unit area in rice production has continued to increase. At present, some farmers have applied up to 260 kg hm2 of nitrogen. -2 , which is much higher than the reasonable nitrogen application rate of 150 kg hm -2 level.

[0003] Large amounts of nitrogen fertilizer input not only aggravate the leaching of nitrate nitrogen fertilizer and the volatilization of ammonium nitrogen fertilizer, but also bring about a series of ecological and environmental pollution such as soil compaction, acidification, destruction of microbial community structure, and serious excess of nitrate content in surface water or groundwater, and seriously endanger the sustainable development of agriculture. Plant-related microorganisms have many characteristics that promote plant growth and bioremediation, and have great potential for humans to cope with these challenges. Microorganisms can promote plant growth directly or indirectly. Direct regulation includes regulating the acquisition of essential nutrients such as nitrogen, phosphorus and iron, and regulating hormone levels. Indirect regulation is through antagonistic effects of antibiotics, responding to infection by pathogens, etc. Therefore, the microbial group most suitable for the growth of rice seedlings is selected through a top-down directed evolution method.

[0004] Therefore, it is necessary to provide a new top-down directed evolution method for rice growth-promoting microbiota to solve the above technical problems. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a top-down microbiome directed evolution method for screening microbial flora that has a significant growth-promoting effect on rice seedlings.

[0006] Top-down directed evolution is a form of artificial selection that provides a simple and efficient tool for the development of synthetic biology, that is, through iterative cultivation, based on genotype-phenotype maps to find stable bacterial communities with high biological functions. The early research work of this strategy focused on bacterial community domestication, guiding the ecological selection of existing microbial communities through the design of physical and chemical environments to perform the desired biological processes; in the later stage, metagenomic technology was used for information mining. At present, biotechnology applications often require complex engineering transformation of polygenic traits, but the lack of genetic basis background of complex phenotypes limits people's ability to make rational designs. Using synthetic biology tools, complex phenotypes can be engineered at the system level, and the evolution of the entire biological system can be promoted through directed evolution strategies. The desired phenotype can still be obtained without knowing the genetic basis of the target trait. The engineering of complex traits can be achieved through directed evolution strategies, simulating the natural evolution process through a variety of control settings to achieve the desired phenotype. Adaptive laboratory evolution technology is one example. Microorganisms are cultured under specific selection conditions, and the evolution of phenotypic improvements can be achieved after hundreds of generations of cultivation. Many studies using ALE to engineer Escherichia coli and yeast have successfully improved the production and yield of biofuels or chemicals. Therefore, directed evolution may allow us to find new pathways and mechanisms in hitherto unexplored regions of genotype space. Directed evolution strategies usually rely on artificial selection pressure and multiple generations of amplification to improve mutants and ultimately obtain the desired complex phenotype. Their success depends mainly on two factors, namely the ability to generate functionally diverse mutants and the screening methods to identify truly phenotypically improved mutants. In recent years, the rapid development of the field of synthetic biology has made important contributions to directed evolution strategies. The goal of this field is to design, construct and improve biological components and systems that do not exist in nature. Synthetic biology has developed many tools that can accelerate directed evolution, improve the efficiency of generating diverse progeny, and also expand the scope of screening or selection.

[0007] The top-down directed evolution method of rice growth-promoting microbiome provided by the present invention comprises the following steps:

[0008] Step 1: Select 12 rice soil environmental samples, take out 1.0g of one of the environmental samples and place it in a 15mL sterile test tube, resuspend it in 10mL phosphate buffered saline to inhibit the growth of eukaryotic cells, phosphate buffered saline is referred to as PBS; the sample is homogenized by vortexing, and the supernatant is used as the inoculum of the experiment; similarly, the initial bacterial supernatant of the 12 environmental samples is inoculated into LB culture medium as the initial inoculum pool according to the above method, and 8 parallel samples are set for each sample to form 96 groups of samples; 4μL subsamples from each of the 96 inoculum pools are taken and diluted in PBS at a ratio of 1:10 in a new 96-well plate. 3After dilution at a ratio of , the microbial flora combination is formed as diverse as possible through bottleneck perturbation and random addition of exogenous flora. The bottleneck perturbation is referred to as Bottleneck, and the exogenous random addition of flora is referred to as Migration. After 15 rounds of non-selective iterations, each round of iteration is transferred to a new LB culture medium.

[0009] Step 2: Inoculate the bacterial colonies that have undergone 15 rounds of non-selective iterations in step 1 into Hoagland nutrient solution respectively;

[0010] Step 3: adding equal amounts of the nutrient solution samples after adding the bacterial flora in step 2 into the rice seedling culture dish samples;

[0011] Step 4: Observe the growth of rice every five days, test the morphological indicators such as leaf length and root length of rice, and select the inoculated bacterial colonies with better growth as the mother generation of the new round of subculture based on the morphological indicators such as leaf length and root length;

[0012] Step 5: Repeat steps 1 to 4 with the inoculated bacterial community selected in step 4, and cycle for 5 times to select the microbial community with the highest biological function. The microbial community is called directed evolution microbial group 1.

[0013] Preferably, in step 1 Bottleneck: This method involves subjecting each colony unit to a dilution step.

[0014] Preferably, in step 1, Migration (random addition of exogenous bacteria): this method involves disturbing the community by invading a group of random species sampled from different environmental microbiomes.

[0015] Preferably, the number of nutrient solution samples in step three is the same as the number of water source seedling culture medium samples.

[0016] Compared with the related art, the top-down directed evolution method of rice growth-promoting microbiome provided by the present invention has the following beneficial effects:

[0017] Through a method of artificially selecting a growth-promoting microbiome, the microbiome has a significant growth-promoting effect on the growth period of rice seedlings and is universal for different rice varieties. In a controlled greenhouse environment, the microbiome was differentially propagated between non-evolved indica conventional rice 9311; therefore, only the microbiome evolved in the experiment, but the plants did not evolve in parallel. In simple terms, the rice soil microbiome from different sources was used as a bacterial library, and the microbial flora combinations were formed as diverse as possible through dilution (Bottleneck) and random addition of exogenous flora (Migration). After 15 rounds of unselected iterations, they were inoculated into Hoagland nutrient solution for rice seedling growth. Based on rice physiological indicators (leaf length, root length and corresponding fresh-dry weight) and microbial physiological indicators (gibberellins, indoleacetic acid, and siderophore production ability), the flora with significant growth-promoting effects on rice seedlings was selected as the parent generation for the next generation of cultivation. The bottleneck and migration combination strategy was used to form a new mother generation for a new round of unselected iterative cultivation to screen the rice growth-promoting microbiome with the highest biological function. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A flow chart of the directed evolution of the rice growth-promoting microbiome provided by the present invention;

[0019] Figure 2 Schematic diagram of the directed evolution microbial group 1 provided by the present invention promoting the growth of rice seedlings. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and implementation modes.

[0021] Please refer to Figure 1 and Figure 2 ;

[0022] Step 1: Select 12 rice soil environmental samples, take out 1.0 g of one of the environmental samples and place it in a 15 mL sterile test tube, resuspend it in 10 mL phosphate buffered saline (PBS) to inhibit the growth of eukaryotic cells, and vortex the sample to homogenize it. The supernatant is used as the inoculum of the experiment; Similarly, the initial bacterial supernatant of the 12 environmental samples is inoculated into LB culture medium as the initial inoculum pool according to the above method, and 8 parallel samples are set for each sample to form 96 groups of samples; Take 4 μL subsamples from each of the 96 inoculum pools and dilute them in a new 96-well plate with PBS at a ratio of 1:10 3 After dilution at a ratio of , the most diverse microbial flora combinations were formed through bottleneck disturbance (Bottleneck) and random addition of exogenous flora (Migration), and 15 rounds of non-selective iterations (each round of iteration was transferred to a new LB culture medium);

[0023] Step 2: Inoculate the bacterial colonies that have undergone 15 rounds of non-selective iterations in step 1 into Hoagland nutrient solution respectively;

[0024] Step 3: adding equal amounts of the nutrient solution samples after adding the bacterial flora in step 2 into the rice seedling culture dish samples;

[0025] Step 4: Observe the growth of rice every five days, test the morphological indicators such as leaf length and root length of rice, and select the inoculated bacterial colonies with better growth as the mother generation of the new round of subculture based on the morphological indicators such as leaf length and root length;

[0026] Step 5: Repeat steps 1 to 4 with the inoculated bacterial community selected in step 4, and cycle 5 times to select the microbial group with the highest biological function (directed evolution microbial group 1); In order to verify whether the directed evolution microbial group 1 obtained by artificial selection has universal applicability to different rice varieties; three rice varieties, namely, indica conventional rice 9311, hybrid rice Yongyou 1540 and Zhongzu 18, were selected, and it was found that the directed evolution microbial group 1 has the same function of promoting the growth of rice seedlings for different rice varieties (such as attached Figure 2 shown).

[0027] Bottleneck perturbation in step 1: This method involves subjecting each community unit to a dilution step; Migration in step 1: This method involves perturbing the community by invading a set of random species sampled from different environmental microbiomes. A perturbation strategy combining bottleneck perturbation (Bottleneck) and random addition of exogenous bacteria (Migration) can find bacterial communities with higher biological functions. The inherent randomness of sampling will ensure that each variant has a different community composition; at the same time, a set of species randomly sampled from different bacterial communities will be combined to invade to obtain different species combinations.

[0028] The number of nutrient solution samples in step three is the same as the number of water source seedling culture medium samples, in order to ensure that each nutrient solution sample corresponds to a water source seedling sample and improve the accuracy of the experimental results.

[0029] Through a method of artificially selecting a growth-promoting microbiome, the microbiome has a significant growth-promoting effect on the growth period of rice seedlings and is universal for different rice varieties. In a controlled greenhouse environment, the microbiome was differentially propagated between non-evolved indica conventional rice 9311; therefore, only the microbiome evolved in the experiment, but the plants did not evolve in parallel. In simple terms, the rice soil microbiome from different sources was used as a bacterial library, and the microbial flora combinations were formed as diverse as possible through dilution (Bottleneck) and random addition of exogenous flora (Migration). After 15 rounds of non-selective iterations, they were inoculated into Hoagland nutrient solution for rice seedling growth. Based on rice physiological indicators (leaf length, root length and corresponding fresh-dry weight) and microbial physiological indicators (gibberellic acid, indoleacetic acid, and siderophore production ability), the flora with significant growth-promoting effects on rice seedlings was selected as the parent generation for the next generation of cultivation. The bottleneck and migration combination strategy was used to form a new mother generation for a new round of non-selective iterative cultivation to screen the microbial flora most suitable for rice growth.

[0030] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A top-down directed evolution method for rice growth-promoting microbiome, characterized in that: The following steps are involved: Step 1: Select 12 rice soil environmental samples, take out 1.0g of one of the environmental samples and place it in a 15mL sterile test tube, resuspend it in 10mL phosphate buffered saline to inhibit the growth of eukaryotic cells, phosphate buffered saline is referred to as PBS; the sample is homogenized by vortexing, and the supernatant is used as the inoculum of the experiment; similarly, the initial bacterial supernatant of the 12 environmental samples is inoculated into LB culture medium as the initial inoculum pool according to the above method, and 8 parallel samples are set for each sample to form 96 groups of samples; 4μL subsamples from each of the 96 inoculum pools are taken and diluted in PBS at a ratio of 1:10 in a new 96-well plate. 3 After dilution at a ratio of , the microbial flora combination is formed as diverse as possible through bottleneck perturbation and random addition of exogenous flora. The bottleneck perturbation is referred to as Bottleneck, and the exogenous random addition of flora is referred to as Migration. After 15 rounds of non-selective iterations, each round of iteration is transferred to a new LB culture medium. Step 2: Inoculate the bacterial colonies that have undergone 15 rounds of non-selective iterations in step 1 into Hoagland nutrient solution respectively; Step 3: adding equal amounts of the nutrient solution samples after adding the bacterial colonies in step 2 into the rice seedling culture dish samples; Step 4: Observe the growth of rice every five days, test the morphological indicators such as leaf length and root length of rice, and select the inoculated bacterial colonies with better growth as the mother generation of the new round of subculture based on the morphological indicators such as leaf length and root length; Step 5: Repeat steps 1 to 4 with the inoculated bacterial community selected in step 4, and cycle for 5 times to select the microbial community with the highest biological function. The microbial community is called directed evolution microbial group 1.

2. The top-down directed evolution method of rice growth-promoting microbiome according to claim 1, characterized in that: Bottleneck in step 1: This method involves subjecting each community unit to a dilution step.

3. The top-down directed evolution method for rice growth-promoting microbiota according to claim 1, characterized in that: Migration in step 1: This method involves perturbing the community by invading a set of random species sampled from different environmental microbiomes.

4. The top-down directed evolution method for rice growth-promoting microbiome according to claim 1, characterized in that: The number of nutrient solution samples in step 3 is the same as the number of water source seedling culture medium samples.

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