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Method for improving formation of root nodules of leguminous plants so as to promote their growth

A technology for leguminous plants and higher plants, applied in the field of agricultural biology, can solve the problems of low nitrogen fixation efficiency of leguminous plants, affecting growth and development, etc., and achieve the effects of promoting root cortex cell division, promoting root nodule formation, and increasing leaf area.

Active Publication Date: 2019-04-16
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology

[0004] The purpose of the present invention is to provide a method for improving the root nodule formation of leguminous plants and promoting their growth in view of the low efficiency of nitrogen fixation in leguminous plants and affecting their growth and development

Method used

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  • Method for improving formation of root nodules of leguminous plants so as to promote their growth
  • Method for improving formation of root nodules of leguminous plants so as to promote their growth
  • Method for improving formation of root nodules of leguminous plants so as to promote their growth

Examples

Experimental program
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Effect test

example 1

[0018] Example 1: Construction of SHR expression system

[0019] Soybean root RNA was extracted according to the instructions of OMEGA's E.Z.N.A.® Plant RNA Kit kit. According to the instructions of TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TRANS), it was reverse transcribed to obtain soybean cDNA. Find the cDNA sequence of SHR on the soybean genome website (http: / / www.arabidopsis.org / ), and design primers:

[0020] SEQ ID NO.2 SHR(+): 5'-AAAAAGCAGGCTCCATGGATACCACGTTGTTTAGGG-3' and SEQ ID NO.3 SHR(-): 5'-AGAAAGCTGGGTCCGTCAAGGCCCTCCATGCACT-'3; using soybean cDNA as a template, with primers SEQ ID NO.2 and SEQ ID NO.2 and SEQ ID NO.3 ID NO.3 is a primer to amplify the cDNA target fragment of SHR by PCR, and the obtained target fragment is 1431 bp in size, and its nucleotide sequence is shown in SEQ ID NO.1. The target fragment was recovered, and the recovered fragment was connected to the intermediate vector pDNONR221 by BP reaction. Prepare Escher...

example 2

[0021] Example 2: Establishment of plant expression system

[0022] (1) To prepare GV3101 Agrobacterium competent cells, transfer the 35S:pGWB602-GmSHR expression vector into the GV3101 Agrobacterium competent cells, and spread on the cells containing spectinomycin, 50 mg / L final concentration of 100 mg / L Rifampicin and 15 mg / L gentamicin plate screening positive colonies;

[0023] (2) After soybean seeds are sterilized, prepare for transformation after germination;

[0024] (3) Prepare Agrobacterium carrying the target gene, and use liquid LB medium containing spectinomycin, 50 mg / L rifampicin and 15 mg / L gentamicin at a final concentration of 100 mg / L in large batches at 28 °C Shake, shake to OD 600 =1.2~1.6. The Agrobacterium was centrifuged at 2000 r / min for 10 min, the bacteria were collected, and the Agrobacterium was resuspended in 5 wt% sucrose solution (OD 600 =0.8), to obtain the Agrobacterium suspension.

[0025] (4) Infect soybean cotyledons with the Agrobacte...

example 3

[0027] Example 3: Verification of root structure phenotype after expressing SHR

[0028] The function of SHR in plants is very conserved. In Arabidopsis, it can regulate the division of cortical cells and then affect the development of plant roots. Therefore, we apply the function of SHR to soybean, which can effectively change the structure of soybean roots.

[0029] (1) Soybean seedlings constitutively overexpressing SHR were cultivated in soybean nutrient solution, and after 15 days of cultivation, the roots and transverse shoots of soybean were measured and photographed ( figure 1 A). The results in the figure show that the soybean plants overexpressing SHR have significantly thicker root and transverse stems than the wild-type parents.

[0030] (2) Root cortex structure observation test: Soybean plants cultivated for 15 days were taken from the mature area of ​​the root, and soybean roots were embedded with 5 wt% agarose, and the roots were cut into 100 μm thin slices w...

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Abstract

The invention provides a method for improving formation of root nodules of leguminous plants so as to promote their growth and belongs to the field of agricultural biotechnologies. A constitutively overexpressed soybean transcription factor SHORT-ROOT (SHR) is utilized to promote the division of soybean root cortex cells and thus promote the formation of soybean nodules, accordingly good nitrogenfixation and nutrient transporting of soybeans through roots are and growth and development of the soybeans are promoted. The constitutive expression SHR of a 35S promoter is utilized to remarkably promote the division of soybean root cortex cells, accordingly the number of soybean root nodules is increased, and the biological nitrogen fixation efficiency of the soybeans is promoted. The growth and development of soy plants with constitutive expression SHR are significantly superior than those of control wild type parents. The method has the great significance in improvement of soybean yield and quality and has great potential in the future improvement of various crop biotechnologies.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a method for improving root nodule formation of leguminous plants and promoting their growth. Background technique [0002] The extensive use of chemical fertilizers in our agricultural production and the massive consumption of petroleum and coal have caused serious damage to the nitrogen cycle in the entire earth's element cycle system, and have had a major impact on the natural ecological environment. With the growth of the world population, human demand for agricultural products continues to increase, and the large-scale use of chemical fertilizers in agricultural production has become inevitable, and the natural ecological environment will further deteriorate. Therefore, it is human beings to reduce the use of chemical fertilizers to improve the natural ecological environment and feed the world population. real problems facing society. Reducing the use o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/54
CPCC07K14/415C12N15/8261
Inventor 吴双王春华梁能松
Owner FUJIAN AGRI & FORESTRY UNIV
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