Stress tolerance related 14-3-3 protein GRF9 and application thereof

A technology of stress tolerance and plant stress tolerance, applied in the field of plant genetic engineering, can solve problems such as soil secondary salinization, crop yield decline, degradation, etc., and achieve the effect of drought resistance and low temperature resistance

Inactive Publication Date: 2012-03-28
INST OF SOIL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Falling into the dilemma that if no fertilization is applied, the crop yield will decrease, and fertilizati

Method used

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  • Stress tolerance related 14-3-3 protein GRF9 and application thereof
  • Stress tolerance related 14-3-3 protein GRF9 and application thereof
  • Stress tolerance related 14-3-3 protein GRF9 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Cloning of Arabidopsis 14-3-3 protein grf9 gene and construction of plant expression vector

[0019] The specific primers were designed according to the DNA information (NM_001036456) of the grf9 gene of Arabidopsis thaliana published by NCBI. The 5'end of the upstream primer was added with the XbaI restriction site, and the 5'end of the downstream primer was added with the SacI restriction site. The PCR product is 1332bp. Upstream primer F1: 5′-GC TCTAGA GTAGTAATTACCCCGTTTAACCCG-3' (adding XbaI restriction site), downstream primer R1: 5'-GCGAGCTCCTCAGCAATTCAGAAATGAAGGAAT-3' (adding SacI restriction site). Extract the total RNA of Arabidopsis thaliana grown in hydroponic culture for 15 days and use it as a template. Oligo(dT) 18 For primer reverse transcription, the reverse transcription product cDNA is obtained. Using this cDNA as a template, PCR amplification was performed with specific primers F1 and R1. Then the pMD18 simple vector was used for TA cloning...

Embodiment 2

[0021] Example 2: Preparation of transgenic tomatoes

[0022] The steps to transform tomato with LBA4404::GRF9-pBI121 are as follows:

[0023] (1) Cultivation of Agrobacterium: Pick a single colony of LBA4404::GRF9-pBI121 from the plate and inoculate it with 100mgL -1 kanamycin(Kan), 25mg L -1 rifampicin (Rif) and 50mg L -1 Streptomycin (Strep) in Luria-Bertani (LB) liquid medium, 28℃, 250rpm shaking culture to OD 600nm =1.0, dilute the bacterial solution 10 times with LB liquid medium, continue shaking and culture for 4h; pour the bacterial solution into a sterile screw-top centrifuge tube with a lid, cover the tube, and centrifuge at 4000 rpm for 10 minutes; discard the supernatant and invert the centrifuge tube 1min, drain off the remaining liquid; add an appropriate amount of co-culture medium-I (MS, 30g L -1 Sucrose, pH 5.8), suspend the bacteria, transfer to a sterile container, use co-culture liquid medium-I, and dilute to OD again 600nm = 0.1.

[0024] (2) Pre-cultivati...

Embodiment 3

[0028] Example 3: Detection of tolerance to low phosphorus and / or drought and / or low temperature tolerance of transgenic grf9 tomato

[0029] (1) Determination of phosphorus content in genetically modified tomatoes: T 2 Generation transgenic tomatoes were planted in the greenhouse of Nanjing Institute of Soil Science, Chinese Academy of Sciences. The soil used for greenhouse potting was collected from the vegetable growing area (greenhouse soil) in Yixing City, Jiangsu Province. T 2 One month after the generation of transgenic tomatoes were transplanted into the soil, harvest the above-ground samples and control samples of the transgenic tomatoes, wash them with deionized water, divide them into different parts, kill them at 105°C for 30 minutes, and dry them to constant weight at 65°C; crush; ; Weigh. The results showed that under the same phosphorus supply conditions, the biomass of genetically modified tomatoes increased significantly (Table 1). It shows that the transgenic ...

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Abstract

The invention relates to a stress tolerance related 14-3-3 protein GRF9 and an application thereof. The amino acid sequence of the 14-3-3 protein GRF9 is represented by SEQ ID NO.1. According to a technical scheme in the invention, a genetic technology is adopted and the 14-3-3 protein GRF9 is utilized to screen out transgenetic tomato with substantially improved low phosphor resistance, drought resistance and low temperature resistance. The obtaining of a new transgenetic crop species which can effectively utilize the soil phosphor resource and has the drought resistance and the low temperature resistance has an important meaning to the reduction of the production cost of agricultural plantation and the reduction of water and soil pollution caused by applying a large amount of phosphate fertilizers.

Description

Technical field [0001] The invention belongs to the field of plant genetic engineering. Specifically, it is a 14-3-3 protein GRF9 related to stress tolerance and its application in cultivating plants tolerant to soil low phosphorus and soil stress. Background technique [0002] my country's facility agriculture is developing rapidly. At the same time, more serious problems have emerged in facility agricultural production. Compared with the open field soil, the biggest feature of the facility soil is its airtightness, controllability, lack of rainwater washing and high multiple crop index. In addition, facility cultivation is mostly carried out in the opposite seasons of winter and spring, and low temperature and low light conditions inhibit the absorption of soil nutrients by vegetable roots. In addition, facility horticulture has the characteristics of "high input, high output, and high efficiency". Under the pressure of the market economy, facility agriculture in various plac...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/79C12N15/84A01H5/00
Inventor 高南施卫明许卫峰闵炬强晓敏
Owner INST OF SOIL SCI CHINESE ACAD OF SCI
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