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Leghemoglobin GmLbc2 gene involved in symbiotic nodulation of leguminous plants and its application

A leguminous plant, symbiotic nodulation technology, applied in the field of plant genetic engineering, can solve problems such as affecting early root nodule formation, and achieve the effects of increasing the number of root nodules, enhancing nitrogen fixation ability, and reducing chemical fertilizers

Inactive Publication Date: 2019-08-06
XINYANG NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report about whether soybean leghemoglobin affects the formation of early nodules

Method used

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  • Leghemoglobin GmLbc2 gene involved in symbiotic nodulation of leguminous plants and its application
  • Leghemoglobin GmLbc2 gene involved in symbiotic nodulation of leguminous plants and its application
  • Leghemoglobin GmLbc2 gene involved in symbiotic nodulation of leguminous plants and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] A preparation method of the leghemoglobin gene GmLbc2 participating in the symbiotic nodulation of leguminous plants, the steps of which are:

[0022] After surface sterilization of W82 soybean seeds, spread them on sterile wet filter paper with the hilum facing down, and cultivate them in the dark at 28°C until they germinate. Fresh soybean root tissues were collected and quick-frozen in liquid nitrogen. According to the instructions of the RNA extraction kit (Invitrogen, USA), the total RNA of soybean root tip tissue was extracted. The first strand of cDNA was obtained according to the instructions of the reverse transcription kit of TIANGEN Company. Primers F-GmLbc2 and R-GmLbc2 (Table 1) were designed according to the soybean GmLbc2 gene (Glyma.20g191200) sequence published on https: / / www.soybase.org / search / website, and the GmLbc2 target gene was amplified by PCR. The target fragment was recovered, connected to the T vector, and sent to Nanjing GenScript Company ...

Embodiment 2

[0031] A kind of apoptosis suppressor gene that participates in leguminous plant symbiotic nodulation in the application of lotus root, milk vetch, alfalfa, soybean and peanut (legume plant) root nodule number, its steps are:

[0032] In order to study the biological function of GmLbc2 in the leguminous plant Lotus japonicus, the applicant explored the biological function of GmLbc2 by the method of overexpression ( image 3 ). It mainly involves the construction and genetic transformation of the overexpression vector, and the specific implementation steps are as follows:

[0033] Construction of the overexpression fusion vector: design primers F-OX and R-OX (Table 1) that introduce restriction sites Kpn I and BamH I, and insert the sequenced correct target gene into the plant expression vector p1301U (Gus gene as a screening marker gene ), the p1301U-GmLbc2 recombinant plasmid was constructed and transformed into Agrobacterium rhizogenes A. rhizogenes LBA1334 (this strain is ...

Embodiment 3

[0060] Embodiment 3 is basically the same as Embodiment 2, the difference is that

[0061] In the specific implementation step (1), the plant culture material is to germinate alfalfa seeds 5 days in advance; in the step (7), the rhizobia is inoculated as the alfalfa rhizobium Sinorhizobium meliloti 1021 (this bacterial strain is an internationally announced bacterial strain, Juan etc., Comparison of Developmental and Stress-Induced Nodule Senescence in Medicagot runcatula. Plant Physiol, 152:1574-1584, 2010).

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Abstract

The invention discloses a leghemoglobin GmLbc2 gene involved in symbiotic nodulation of leguminous plants, which has a length of 438 bp and is located on chromosome 20 of soybean genome, and the invention also discloses an application of the gene in regulating the number of nodules of leguminous plants (Lotus corniculatus, Chinese milk vetch, clover, soybeans and peanuts). A reverse transcriptionPCR technology is used to isolate a soybean hemoglobin gene GmLbc2 in soybean root, and confirms that the gene is a leghemoglobin gene by homologous protein multi-sequence alignment and cladogram analysis; the application confirms for the first time that a soybean nodulation factor receptor protein GmNFR1a and the leghemoglobin GmLbc2 can interact in yeast and tobacco; further, a method of overexpression of genetic transformation confirms that the gene participates in and regulates the nodulation of legumes. The application confirms for the first time that the soybean leghemoglobin gene GmLbc2is regulating the symbiotic nodulation of legumes.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a leghemoglobin GmLbc2 gene involved in symbiotic nodulation of leguminous plants and its application. Background technique [0002] A large amount of evidence shows that soybean leghemoglobin is closely related to nitrogen fixation, and it regulates free O in root nodules. 2 concentration, protects the susceptible O produced by bacteroids 2 Destroyed nitrogenase. The content of leghemoglobin in root nodules is positively correlated with nitrogenase activity. After rhizobia infect soybean roots to induce leghemoglobin expression, root nodules can normally fix nitrogen and provide the host plant with the nitrogen source needed for growth. However, there is no report about whether soybean leghemoglobin affects the formation of early nodules. [0003] The present invention connects soybean nodulation factor receptor protein GmNFR1a with leghemoglobin GmLbc2 for ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/54A01H6/82
CPCC07K14/415C12N15/8261
Inventor 柯丹霞舒勇贺月丽樊柳霄牛若鑫
Owner XINYANG NORMAL UNIVERSITY
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