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Application of rice OsMADS27 gene and protein in improving nitrogen fertilizer use efficiency of rice

A utilization rate and rice technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of nitrogen fertilizer utilization resource waste, water eutrophication pollution, and lower nitrogen use efficiency, so as to promote expression, transport and The effect of absorbing and improving nitrogen utilization efficiency

Inactive Publication Date: 2019-02-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to meet the growing population demand, it is necessary to increase the yield of rice. The input of nitrogen fertilizer in rice production is becoming one of the powerful measures to increase yield. Irrational use has also brought about a series of environmental pollution problems such as waste of resources and eutrophication of water bodies.

Method used

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  • Application of rice OsMADS27 gene and protein in improving nitrogen fertilizer use efficiency of rice
  • Application of rice OsMADS27 gene and protein in improving nitrogen fertilizer use efficiency of rice
  • Application of rice OsMADS27 gene and protein in improving nitrogen fertilizer use efficiency of rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Cloning of the rice OsMADS27 gene.

[0026] (1) Extraction of total RNA. From rice (Nipponbare) seedlings to the 3-leaf stage, take the roots of the rice seedlings and put them in liquid nitrogen for quick storage, weigh about 0.1g of roots, grind them with liquid nitrogen, put them into a 1.5ml centrifuge tube after grinding, and quickly add TRIzol reagent (purchased from Invitrogen Company) was shaken thoroughly, and the total RNA was extracted.

[0027] (2) Synthesis of cDNA.

[0028] The first strand of cDNA was synthesized using the M-MLV reverse transcription system (Takara code: D2639A), the steps are as follows:

[0029] RNA 1-2μg

[0030] Oligo d(T)18 Primer (50μM) 2μl

[0031] RNase-free H 2 O make up to 10 μl

[0032] 70 ℃ water bath for 10 minutes, placed on ice for 2 minutes to cool. Centrifuge for a few seconds to pool the template RNA / primer / water denaturing solution at the bottom, then add:

[0033]

[0034] Incubate at 42°C for 1h. Incubate ...

Embodiment 2

[0047] OsMADS27 gene interference vector construction and genetic transformation.

[0048] (1) Construction of RNAi expression vector

[0049] The cDNA sequence of the OsMADS27 gene was compared with the rice full-length cDNA database, the 200-300bp fragment of the non-conserved region was selected as the interference fragment, and the primers were designed with the interference fragment as the target interference sequence. The vector used was pTCK303, and primers were designed using Primer Primer 5.0 software as follows:

[0050] RiOsMADS27-FP:

[0051] 5'-GCGACTAGTGGTACCGAATCGGAAGGGAAGTCTAG-3';

[0052] RiOsMADS27-RP:

[0053] 5'-GCGGAGCTCGGATCCTTGTTCGGCGTCACTATGT-3'.

[0054] Using the cDNA of the rice root system in Example 1 as a template, carry out PCR amplification with the above-mentioned primers, and use the high-fidelity enzyme Prime HS DNA Polymerase (Takara Code: DR010A) system. Take 50 μl of PCR product and 5 μl of DL2000 DNA Marker (Takara Code: D501A), an...

Embodiment 3

[0064] OsMADS27 gene overexpression vector construction and genetic transformation.

[0065] (1) Construction of overexpression vector

[0066] With the cDNA of the rice root system in Example 1 as a template, the carrier used is pENTR 1A, and the Primer Primer5.0 software is utilized to design primers as follows:

[0067] OE-OsMADS27-FP:

[0068]5'-GCGGTCGACATGGGGAGGGGGAAGATTGT-3';

[0069] OE-OsMADS27-RP:

[0070] 5'-GCGCCATGGTCATGGATTCAACTGTAACC-3'.

[0071] After digestion and purification, the PCR product was connected to the vector pENTR 1A, and the sequenced correct primary vector was exchanged with the Gateway overexpression vector pH2GW7.0, using Gateway LR ClonaseTM Enzyme Mix (Invitrogen, Cat.No.11791-019), the system as follows:

[0072] 1) Add the following reagents to a 1.5ml centrifuge tube and mix well at room temperature:

[0073] Entry clone 50-150ng

[0074] Destination vector 150ng

[0075] TE buffer (pH=8.0) make up to 8μl

[0076] 2) Take LR Clon...

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Abstract

The invention discloses application of rice OsMADS27 gene and protein in improving the nitrogen fertilizer use efficiency of rice. According to the application, it is found according to research thatthe rice OsMADS27 protein can interact with nitrate translocator cofactors OsNAR2.1, overexpression OsMADS27 gene can significantly promote the expression of nitrate translocator, thereby promoting the transfer and absorption of the rice to nitrate, and improving the nitrogen fertilizer use efficiency. The application of the rice OsMADS27 gene and protein in improving the nitrogen fertilizer use efficiency of the rice is helpful for researching key regulatory genes related to the nitrogen absorption of the rice, is of great significance for screening and breeding of nutrient efficient plant varieties, can also provide a theoretical basis and technical support for explaining a physiological mechanism of nitrogen utilization of the rice, improving the nitrogen utilization efficiency of the rice and genetic improvement of rice varieties, and has important theoretical significance and potential application value.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the application of rice OsMADS27 gene and protein in improving rice utilization rate of nitrogen fertilizer. Background technique [0002] Rice is one of the most important food crops in the world, accounting for 1 / 3 of the world's cereal crop planting area. my country is the country with the largest rice planting area in the world. In order to meet the growing population demand, it is necessary to increase the yield of rice. The input of nitrogen fertilizer in rice production is becoming one of the powerful measures to increase yield. Irrational use has also brought about a series of environmental pollution problems such as waste of resources and eutrophication of water bodies. Therefore, studying the mechanism of rice nitrogen uptake and fully understanding the potential of rice to absorb and utilize nitrogen to improve rice nitrogen use efficiency is of great importanc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/46
CPCC07K14/415C12N15/8243
Inventor 甘银波吴君宇于春燕黄琳丽吴敏洁姜蕲宁
Owner ZHEJIANG UNIV
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