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Mutant of rhizobium japonicum SMH12 and application of mutant

A technology of SMH12, mutant, applied in applications, microorganism-based methods, chemicals for biological control, etc., to achieve good nodule nitrogen fixation ability, enhance nodule nitrogen fixation effect, and improve the effect of matching

Active Publication Date: 2020-06-16
华创佳农生物科技(武汉)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] After searching, there is no report on rhizobia inoculants based on the mutation or knockout of specific effector proteins to improve the nodulation and nitrogen fixation ability of legumes

Method used

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  • Mutant of rhizobium japonicum SMH12 and application of mutant
  • Mutant of rhizobium japonicum SMH12 and application of mutant
  • Mutant of rhizobium japonicum SMH12 and application of mutant

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Soybean Rhizobium SMH12ΔnopP Mutant

[0039] Using the CreloxP system double exchange replacement method, the nopP gene deletion mutant ( figure 1 ). Using SMH12 total genomic DNA as a template, the homologous upper and lower arms (692bp and 691bp) were amplified, respectively ( figure 2 A), the homologous upper arm was connected to the vector pCM351 via KpnI and Nde I, and then the homologous lower arm was connected to the vector pCM351::up via Apa I and Age I. The constructed recombinant plasmid was introduced into the fast-growing soybean rhizobia SMH12, and the exchange fragment on the recombinant plasmid underwent homologous recombination with the upstream and downstream fragments of nopP in the rhizobium genome, and integrated into the chromosome, thereby replacing the gene. With the primer Gm-F of vector pCM351 and the screening primer nopP-MAP-R of the target gene corresponding to the restriction site, carry out PCR verification and amplify about 16...

Embodiment 2

[0040] Example 2 Symbiotic Nitrogen Fixation Phenotype Investigation

[0041] In order to identify the effect of the SMH12(ΔnopP) mutant on the symbiotic nitrogen fixation ability, nopP overexpression OE-nopP and complementary CM-nopP were also constructed, and the symbiotic phenotypes of each strain with Jidou 17 and Zhonghuang 13 were investigated.

[0042] The results showed that there were differences in the growth of the aboveground parts of different strains inoculated with Jidou 17 and Zhonghuang 13. The growth of the mutants inoculated with Jidou 17 was tall and the leaves were green, and the growth of the mutants inoculated with overexpression was weaker than that of the wild type and showed yellow. The phenotype of the plants inoculated with the complementary strain was between the wild type and the mutant, and could partially recover to the wild type phenotype ( image 3 A). Similarly, the aboveground plants inoculated with mutants and inoculated with overexpressi...

Embodiment 3

[0043]Example 3 Quantitative analysis of symbiotic nitrogen fixation phenotype

[0044] The nitrogenase activity of root nodules, the number of nodules, the weight of nodules and the fresh weight of above-ground parts were further statistically analyzed. The results showed that the nitrogenase activity of the mutant plants inoculated with Jidou 17 was 24.3 μmol / g / h, and the symbiotic nitrogen fixation of wild-type plants was 24.3 μmol / g / h. The enzyme activity was 11.1 μmol / g / h, and the nitrogenase activity of the mutant was significantly higher than that of the wild type (P<0.05), and the number of root nodules was also significantly higher than that of the wild type. The nodule enzyme activity, nodule number, nodule weight and fresh weight of above-ground parts of plants inoculated with the overexpression strain were significantly lower than those of the wild type. The nodule nitrogenase activity of the Zhonghuang 13 plants inoculated with the mutant was significantly higher ...

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Abstract

The invention discloses a mutant of rhizobium japonicum SMH12 and application of the mutant. The nopP gene deletion mutant is constructed by utilizing a CreloxP system double-exchange replacement method. By taking fast-growing rhizobium SMH12 genome DNA as a template and adopting a nopP-up-F / R primer pair and a nopP-down-F / R primer pair, a nopP homologous exchange upper arm and a nopP homologous exchange lower arm are respectively amplified, a product is connected with a digested pCM351 vector in two steps after being subjected to double enzyme digestion, plasmid transformation is obtained, and the mutant can be obtained. Therefore, the rhizobium target gene mutant is constructed and obtained. After the nopP gene is mutated or deleted, the defense reaction of leguminous plants is inhibitedor eliminated, so that rhizobium infection and nitrogen fixation are promoted. The improved rhizobium can improve the growth and nitrogen fixation capacity of leguminous crops, reduce nitrogen fertilizer application in agricultural production and generate huge economic and ecological benefits.

Description

technical field [0001] The invention belongs to the technical field of agricultural microbial bacterial agents, and in particular relates to a mutant of soybean rapid-growing rhizobium SMH12 and an application thereof. Background technique [0002] There are many factors that affect the performance of rhizobia agents, mainly including host leguminous plants, rhizobia, and environment. From the perspective of bacteria-plant interaction, legume-rhizobia symbiotic nitrogen fixation is related to the differences of host plant species. The types and structures of flavonoids secreted by different legumes are different; the genetics and genotypes of different legumes are also different, all of which affect the application effect of rhizobia inoculation. This phenomenon is also known as rhizobia-legume symbiotic compatibility. The characteristics of nodulation and nitrogen fixation of the same rhizobia on different soybean varieties, or different rhizobia on the same soybean varie...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/90C12N15/31C12N1/21A01N63/20A01P21/00C12R1/41
CPCA01N63/00C07K14/195C12N15/743C12N15/902Y02P60/21
Inventor 李友国孙轶芳
Owner 华创佳农生物科技(武汉)有限公司
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