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Application of miR169 or target genes NFYA5 thereof in nitrogen stress adaptation of plants

1. The technology of mir169 and target gene is applied in the field of genetic engineering to achieve the effect of enhancing ability and increasing sensitivity

Inactive Publication Date: 2011-01-26
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Combier et al. found that miR169 was involved in the development of root nodules of Medicago truncatula (Combier et al., 2006, Gene Dev20: 3084-3088); previous research results showed that AtNFYA5 (a target gene of miR169) in Arabidopsis resistance to water stress It plays an important role in the process, and its expression is regulated by both transcriptional and post-transcriptional levels, and the post-transcriptional level is mainly regulated by miR169. Further research results show that although miR169 is a relatively large family, only miR169a is involved in It plays an important role. After overexpressing miR169a, the mutant strain becomes sensitive to drought (Li et al., 2008, Plant Cell 20: 2238-2251)
So far, no studies have shown that miR169 and its target genes are associated with nitrogen stress

Method used

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  • Application of miR169 or target genes NFYA5 thereof in nitrogen stress adaptation of plants
  • Application of miR169 or target genes NFYA5 thereof in nitrogen stress adaptation of plants
  • Application of miR169 or target genes NFYA5 thereof in nitrogen stress adaptation of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation of embodiment 1 material reagent

[0022] 1. Strains and tool plasmids

[0023] Materials used in the examples of the present invention include: Escherichia coli DH10B, DB3.1 and Agrobacterium tumefaciens GV3101, pGEM T-Easy cloning vector purchased from Promega Company; pENTR TM The / D-TOPO vector was purchased from Invitrogen; the plant expression vector pMDC32 was purchased from TAIR.

[0024] 2. Tool enzymes and biochemical reagents

[0025] Ordinary Taq enzymes were prepared by this experiment; high-fidelity Taq enzymes and SYBR were purchased from Takara; dNTPs mixture was purchased from Shanghai Sangong; Trizol and LR Clonase were purchased from Invitrogen; T4 DNA kinase was purchased from New England Biolab; Propylcarbodiimide was purchased from Promega; ampicillin (Amp), kanamycin (Kan), spectinomycin (Spe), and rifampicin (Rif) were purchased from Xinjingke.

Embodiment 2

[0026] Example 2 Small RNA Northern analysis of the response of miR169 to nitrogen stress

[0027] 1. Total RNA extraction

[0028] Arabidopsis was cultured in hydroponics, and the composition of the nutrient solution was as follows: 1mM NH 4 NO 3 , 1mM NaH 2 PO 4 , 1.5 mM MgSO 4 , 1.5mM CaCl 2 , 100 μM Fe-EDTA, 50 μM HO 3 BO 3 , 12 μM MnCl 2 , 2 μM ZnSO 4 , 1 μM CuSO 4 , and 0.2 μM Na 2 MoO 4 . Change the nutrient solution every 2 days. Twenty-eight days after germination, they were transferred into nitrogen-free nutrient solution for 0, 1, 2, 3, and 4 days of nitrogen deficiency, and total RNA was extracted from the roots and shoots of the corresponding nitrogen treatments.

[0029]Add 1ml Trizol to the DEPC-treated Eppendorf tube; grind 0.15g sample into powder in liquid nitrogen, transfer the powder to the above-mentioned centrifuge tube with a small medicine spoon before the liquid nitrogen is volatile, and mix it upside down; add 200μl Chloroform, vigorous...

Embodiment 3

[0048] Example 3Real-Time PCR analysis of the response of miR169 precursor and NFYA5 to nitrogen stress

[0049] Using the total RNA extracted in Example 2 as a template, and oligo(dT) as a primer to reverse transcribe the first strand of cDNA, the method is as follows:

[0050] (1) Mix 2μg total RNA, 2μl Oligo(dT)18nt, 1μl 10mmol L -1 Add dNTPs into a PCR tube and mix well;

[0051] (2) Insulate at 65°C for 5 minutes, then quench on ice;

[0052] (3) Add the following components in order on ice:

[0053] 5×M-MLV buffer 4μl

[0054] 0.1×DTT 2μl

[0055] RNase inhibitor (40U / μl) 1μl

[0056] M-MLV reverse transcriptase 1μl

[0057] DEPC water make up to 20μl

[0058] (4) Gently mix and keep warm at 37°C for 50 minutes;

[0059] (5) Incubate at 70°C for 15 minutes to terminate the reaction, and cool on ice.

[0060] The following primers were used to detect the expression of miR169 precursor and its NFYA5 gene by Real-Time PCR.

[0061] Primer miR169a F 5′-TGGGTATAGCTA...

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Abstract

The invention discloses application of small RNA miR169 or target genes NFYA5 thereof related to nitrogen metabolism in the nitrogen stress adaptation of plants, which is characterized in that miR169 is excessively expressed in Arabidopsis thaliana (L.) Heynh to increase the sensitivity of the Arabidopsis thaliana (L.) Heynh on nitrogen deficiency stress; and the target genes NFYA5 of miR169 is correspondingly excessively expressed to reduce the sensitivity of the Arabidopsis thaliana (L.) Heynh on the nitrogen deficiency stress and enhance the capability of the Arabidopsis thaliana (L.) Heynh on low nitrogen stress resistance. The invention can ensure that the analysis and the deeply knowledge of the complicated regulate and control network can be further applied to the cultivation of crops of high-efficiency nitrogen.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of Arabidopsis miR169 and its target gene NFYA5 in plant adaptation to nitrogen stress. Background technique [0002] Nitrogen, the most demanded element of plants, is closely related to the yield and quality of plants. my country's agricultural production consumes 26.21 million tons of synthetic nitrogen fertilizers every year, but the current utilization rate of nitrogen fertilizers in my country is only about 30%, far below the world average of 45%. On the one hand, it increases the cost of agricultural production, and on the other hand, it is very easy Causes pollution of groundwater and its surface water (Ju et al., 2009, Proc Natl Acad Sci USA 96:3041-3046). In order to ensure food security, it is impossible to simply adopt the method of some developed countries to reduce the amount of nitrogen fertilizer used by reducing the yield, thereby incre...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/113C12N15/29A01H5/00
Inventor 李文学赵勐丁红张福锁
Owner CHINA AGRI UNIV
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