Gene GmSPX5 for regulating and controlling growth of leguminosae root nodules, and application of gene GmSPX5

A gene and root nodule technology is applied to the gene GmSPX5 that regulates the growth of leguminous root nodules and its application fields, which can solve the problems of few reports, few studies, and no gene function.

Active Publication Date: 2018-10-09
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on the molecular mechanism of nodule adaptation to phosphorus deficiency, especially the key regulators of nodule phosphorus signaling.
Previous studies have shown that soy

Method used

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  • Gene GmSPX5 for regulating and controlling growth of leguminosae root nodules, and application of gene GmSPX5
  • Gene GmSPX5 for regulating and controlling growth of leguminosae root nodules, and application of gene GmSPX5
  • Gene GmSPX5 for regulating and controlling growth of leguminosae root nodules, and application of gene GmSPX5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 GmSPX5 Gene cloning and vector construction

[0052] 1. Excess (OX -GmSPX5- pTF) expression vector construction

[0053] (1) design GmSPX5 Gene-specific primer OX- GmSPX5 -pTF-F and OX- GmSPX5 -pTF-R:

[0054] Primer OX- GmSPX5 -pTF-F (SEQ ID NO. 3):

[0055] 5'-GCTCTAGAATGAAGTTTGAGAAGATCCTGAAG-3'

[0056] Primer OX- GmSPX5 -pTF-R (SEQ ID NO.4):

[0057] 5'-TCCCCCGGGCTAGTTGTGTGGAGAAGATGGG-3'.

[0058] (2) PCR amplification: using soybean genotype YC03-3 phosphorous deficiency treated root nodule cDNA as a template, using gene-specific primers OX- GmSPX5 -pTF-F (SEQ ID NO.3) and primer OX- GmSPX5 - pTF-R (SEQ ID NO.4) was amplified GmSPX5 Coding region fragment. The PCR reaction system is 50 microliters, including 5 microliters of 10×Ex Taq Buffer, 4 microliters of 2.5 mmol / L dNTP, 3 microliters of cDNA template, and 1 microliter of 10 micromol / liter forward and reverse primers. liters, 0.5 microliters of ExTaq enzyme, and finally make up to 50 ...

Embodiment 2

[0068] Example 2 GmSPX5 Analysis of gene expression patterns and protein subcellular localization

[0069] 1, GmSPX5 Gene expression pattern analysis

[0070] (1) Experimental method

[0071] Experimental design: Set up two phosphorus concentration treatments, high phosphorus (+P) is 250 micromol / L KH 2 PO 4 , phosphorus (-P) deficiency of 5 μmol / L KH 2 PO 4 ; Nitrogen level is 500 micromole / L of total nitrogen; The cultivation device is a 15-liter blue light-proof bread box, which is cultivated in a solar greenhouse, and each treatment is set to replicate four times.

[0072] Seedlings: use roll paper for seedlings. Pick seeds of the same size and dry them with chlorine gas (100 ml of sodium hypochlorite + 4.2 ml of hydrochloric acid) for 12 hours, then place them side by side on one side of the wet seedling paper with an interval of about 1 cm, roll up the seedling paper, and fix the seeds One end of the plant is facing up (the hilum is facing down), put it verticall...

Embodiment 3

[0088] Example 3 Research on transgenic materials

[0089] 1. Obtaining genetically modified materials

[0090] Agrobacterium tumefaciens mediated whole plant transformation of cotyledon nodes. The main steps include:

[0091] (1) Seed germination. Select soybean seeds with undamaged seed coats for surface disinfection in chlorine for 12-14 hours, then place the seeds in an ultra-clean workbench for 30 minutes to remove excess chlorine; Cultured for 4 days.

[0092] (2) Preparation of bacterial solution. Overexpress the constructed OX -GmSPX5- The pTF vector plasmid was transformed into Agrobacterium tumefaciens EHA101 by freeze-thaw method, and the positive clones were picked and inoculated in YEP culture medium containing corresponding resistance, cultured at 28°C and 200 rpm until the OD650 was 1.0, and then the colonies were collected by centrifugation and used Suspend the bacteria in CM liquid until the OD650 is 1.0.

[0093](3) Cotyledon node infection and co-cul...

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Abstract

The invention discloses a gene GmSPX5 for regulating and controlling growth of leguminosae root nodules, and the application of gene GmSPX5. The nucleotide sequence of the gene is as shown in SEQ ID NO.1 and the coding amino acid sequence is as shown in SEQ ID NO.2. The research shows that excessive expression of the GmSPX5 can increase the number of the leguminosae root nodules and the density ofroot nodules infecting cells under high phosphorus treatment; furthermore, the nitrogen phosphorus content and the yield of soybeans can be remarkably increased. Therefore, the GmSPX5 can regulae andcontrol the growth and development of the root nodules and has important effects of improving the nitrogen phosphorus nitration of plants and increasing the yield; and the gene GmSPX5 can be used forregulating and controlling the adaptability on phosphorus deficiency and nitrogen deficiency in soil by the plants through a transgenic technology, also can be applied to leguminous crop nitrogen phosphorus synergistic and efficient genetic improvement and has a very important market prospect.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. More specifically, a gene that regulates legume root nodule growth GmSPX5 and its application. Background technique [0002] In recent years, there have been more and more research reports on SPX proteins, and the progress is very fast. Many studies have proved that proteins containing SPX domains are involved in phosphorus signaling and phosphorus balance regulation (Duan et al., 2008; Wang et al., 2009; Gu et al., 2012). Arabidopsis SPX There are 4 genes, namely AtSPX1 ~ AtSPX4 (Duan et al., 2008), where the overexpressed AtSPX1 Increased phosphorus starvation-induced genes such as ACP5 , PAP2 as well as RNS1 The expression of , which increases the phosphorus concentration, indicates that AtSPX1 Involved in the regulation of phosphorus starvation signaling at the transcriptional level (Duan et al., 2008). Instead, the mutation Atspx3 able to increase AtP...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415A01H5/00A01H6/54
CPCC07K14/415C12N15/8243
Inventor 田江薛迎斌廖红庄庆礼梁翠月
Owner SOUTH CHINA AGRI UNIV
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