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Application of gene GmMDH12 in promotion of nodulation and nitrogen fixation capacity of soybeans

A gene and soybean technology, applied in the application field of promoting soybean nodulation and nitrogen fixation, to achieve the effects of improving the efficient use of crop nutrients, promoting soybean nodulation and nitrogen fixation, and increasing the synthesis of malic acid

Active Publication Date: 2018-11-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the important genes that regulate malic acid synthesis in root nodules, and whether increasing malic acid synthesis can promote root nodulation and improve nitrogen nutrition in legumes are still unclear

Method used

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  • Application of gene GmMDH12 in promotion of nodulation and nitrogen fixation capacity of soybeans
  • Application of gene GmMDH12 in promotion of nodulation and nitrogen fixation capacity of soybeans
  • Application of gene GmMDH12 in promotion of nodulation and nitrogen fixation capacity of soybeans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 GmMDH12 Gene cloning and expression analysis

[0046] 1. GmMDH12 protein expression analysis

[0047] Experimental design of root nodule proteomics analysis: 5 days after seed germination of soybean variety "HN66", the seedlings were inoculated with Rhizobium BXYD3 for 1 hour, and then transferred to low phosphorus (LP, 5 μM KH 2 PO 4 ) or high phosphorus (HP, 250 μM KH 2 PO 4 ) low nitrogen nutrient solution (50 μM NH 4 NO 3 ), after 25 days of culture, the shoots, roots and nodule samples were harvested and stored at -80 ℃ for the extraction of nodulin and RNA. Experimental design of gene expression analysis at different time points: 5 days after the germination of soybean HN66 seeds, the seedlings were inoculated with Rhizobium BXYD3 for 1 hour, and then transferred to low phosphorus (LP, 25 μM KH 2 PO 4 ) or high phosphorus (HP, 500 μM KH 2 PO 4 ) low nitrogen nutrient solution (250 μM NH 4 NO 3 ) in culture, on the 14th, 21st, 30th and 40th da...

Embodiment 2

[0058] Example 2 GmMDH12 Gene promoter cloning, vector construction and tissue localization analysis

[0059] 1. Experimental method

[0060] GmMDH12 Gene promoter cloning and GUS expression vector construction: extract soybean root genomic DNA, use soybean genomic DNA as a template, use upstream specific primer 5'-GGGATCCCCGTGCATGTGTTGA-3' (SEQ ID NO:9) and downstream specific primer 5'-ATGAATTCAGTGGCGATTAGTGAG- 3' (SEQ ID NO:10) amplification GmMDH12 Promoter 2182 bp (SEQ ID NO: 11), after the PCR fragment was recovered and sequenced, passed Bam H I and EcoR I carried out double enzyme digestion to the recovered fragment and the target vector pCAMBIA1391, connected the promoter fragment to pCAMBIA1391, and then transformed the constructed GUS expression vector into Agrobacterium rhizogenes K599 by the freeze-thaw method (the K599 bacterial strain was obtained from Queensland, Australia Donated by the Leguminous Comprehensive Research Center of the University, it is pre...

Embodiment 3

[0065] Example 3 GmMDH12 enzymatic property analysis

[0066] 1. Experimental method

[0067] (1) Construction of prokaryotic expression vector: using soybean root nodule cDNA as template, using upstream specific primer 5'-GGATCCATGATGAAGCCATCGA-3' (SEQ ID NO:12) and downstream specific primer 5'-ACATGAATTCTTACTGGTTGGCAAATT-3' (SEQ ID NO: 13) Amplification GmMDH12 The full length of the gene, after the PCR fragments were recovered and sequenced correctly, passed Bam H I and EcoR After carrying out double digestion of the fragment and the prokaryotic expression vector pGEX6P-3 (GE-Healthcare, USA), the GmMDH12 The gene was connected to the target vector pGEX6P-3, and then the constructed expression vector was transformed into BL21 Escherichia coli by freeze-thaw method.

[0068] (2) Take an appropriate amount of the BL21 bacterial solution containing the target gene and culture it in 200 mL of fresh LB liquid medium at 28°C until the OD600 is 0.4, then add 0.2 mM IPTG t...

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Abstract

The invention discloses an application of a gene GmMDH12 in promotion of nodulation and nitrogen fixation capacity of soybeans, and particularly relates to an application of a malate dehydrogenase gene GmMDH12 in control for synthesis of malic acid of root nodules and in promotion of nodulation of leguminous crop root system. The research indicates that GmMDH12 has a function of regulating and controlling synthesis of malic acid of the soybean root nodules, concentration of endogenous malic acid of the root nodules of transgenic composite soybean plants is remarkably increased by over-expression of GmMDH12, the number and the biomass of the root nodules are increased, and accordingly, nitrogen content and biomass of the plants are increased. According to the invention, the gene GmMDH12 haspotential of improving biological nitrogen fixation function of the leguminous crops with a transgenic technology and has great theoretical and practical significance for efficient utilization of crop nutrients and sustainable development of environment-friendly agriculture.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. More specifically, it involves GmMDH12 Application of genes in promoting soybean nodulation and nitrogen fixation. Background technique [0002] The roots of leguminous plants can form a mutually beneficial and complementary symbiotic system with rhizobia -- root nodules. The host plant can provide carbon source and suitable growth environment for the growth of rhizobia, and the rhizobia can carry out biological nitrogen fixation through the action of nitrogenase to provide nitrogen source for the growth of the host. Applying rhizobia inoculation technology to agricultural production to improve crop nitrogen nutrition is an effective measure to save fertilizer and increase yield of leguminous crops, and plays an important role in agricultural production (Qin et al., 2012). The growth of rhizobia, root nodule formation and biological nitrogen fixation in leguminous plants is a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/82A01H5/00A01H6/54
CPCC12N9/0006C12N15/8243C12Y101/01037
Inventor 田江陈志坚廖红梁翠月白振龙
Owner SOUTH CHINA AGRI UNIV
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