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A cloning method and microbial technology, applied in the field of genetics, can solve the problems of complicated workload, low exchange rate, strong subjectivity, etc., and achieve the effects of reducing risks, clear results, and broadening the scope of cloning and utilization.
Inactive Publication Date: 2013-01-30
JIANGHAN UNIVERSITY
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Problems solved by technology
[0005] (1) Microorganisms are small, so observation and identification are very difficult and highly subjective
However, linkage marker mapping requires identifying the phenotype of each strain in the segregating population, which is very complicated and difficult
[0006] (2) At present, the molecular markers developed for microorganisms are limited, which greatly restricts their application; in fine positioning, the available markers are less
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Embodiment 1
[0024] A method for microbial gene cloning, comprising the following steps (see figure 1 ):
[0025] (1) Construction of segregation population: use parent 1 and parent 2 with differences in target traits (such as toxicity, drug resistance, temperature sensitivity, etc.) to cross. If the microorganism is haploid, then F 1 It is a segregation group, if it is diploid, then go to F 2 Generations become a segregating population, which is the pre-screening population.
[0026] (2) Automatic screening of isolated populations: according to the characteristics of the target traits, select the selection pressure for the target traits (such as resistant plants, drugs, high temperature, etc.), automatically screen the isolated populations, and obtain the screened populations;
[0027] (3) DNA extraction: extract the genomic DNA of 2 parents, 2 mixed populations before and after screening;
[0028] (4) Determination of the target site: According to the high-throughput sequencing proce...
Embodiment 2
[0031] A kind of rice blast avirulent gene cloning method (see figure 2 ):
[0032](1) Identification of virulent and avirulent rice blast races. The physiological races of rice blast were isolated and cultured from the diseased leaves of Lijiang Xintuan Heigu rice in Enshi, Hubei Province, and the rice material IRBL 12 was inoculated with these physiological races. It was found that after inoculation with race S223, the typical rice blast fusiform appeared Lesions, indicating that the S223 race is virulent to IRBL 12. After inoculation with S12, IRBL 12 had almost no lesions, indicating that the S12 race was not toxic to IRBL 12.
[0033] (2)F 1 Obtaining and culturing hybrid ascospores. The parent strains of Magnaporthe grisea S223 and S12 were cultured on oatmeal medium (30g of oatmeal, 10g of sucrose, 18g of agar, 1000ml of water) at 25°C until the mycelium met, and continued to cultivate at 20°C under light until the junction of the mycelium appeared Ascus shell bla...
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Abstract
The invention discloses a method for microbe gene clone. The method comprises the steps of constructing segregation populations by hybridizing parents having target character differences; automatically screening the segregation populations by setting a selection pressure to obtain screened populations; high-throughput genome sequencing of the two parents, populations before screening and populations after screening; obtaining specific loci of the parents by preliminary direct assembly and comparison; comparing the specific loci of the parents with the genomes of the populations before screening and the populations after screening in a completely matching mode; looking for loci which exists in the parents and the populations before screening but disappears in the populations after screening, wherein the loci are candidate gene loci for controlling target characters; obtain full-length sequence of the genes by comparing with the genomes of the parents; cloning target genes via PCR amplification; and verifying functions of the cloned target genes via genetic complementary experiments. The method directly clones genes themselves, and reduces a lot of microspecies isolation and identification work of the microbes. The method has little workload, fast speed and low risk.
Description
technical field [0001] The invention belongs to the field of genetics and discloses a microbial gene cloning method. Background technique [0002] The types of microorganisms in nature are extremely complex, including bacteria, fungi, and viruses, and their hosts also include humans, animals, and plants. For humans, the functions of microbial genes are rich and diverse, both good and bad. For example, rhizobia and leguminous plants form root nodules symbiotically, and with the help of nitrogenase genes, nitrogen in the air can be fixed for plant nutrition, which can reduce the application of chemical fertilizers, protect environment. However, the rice blast caused by the virulence gene in Magnaporthe grisea is one of the most serious fungal diseases on rice, which can cause about 30% of rice yield reduction, or even no harvest, seriously threatening national food security. Therefore, cloning the toxicity genes and drug resistance genes of microorganisms is very important t...
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