Use of boea crassifolia BcBCP1 gene for breeding drought-salt-tolerant plants

A technology of spinocystis and genes, applied in the field of drought-related blue copper protein-like genes, can solve the problem of still few

Inactive Publication Date: 2006-12-20
林忠平
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still little information about higher plant azuretin at home and abroad (Turner, 2002)

Method used

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  • Use of boea crassifolia BcBCP1 gene for breeding drought-salt-tolerant plants
  • Use of boea crassifolia BcBCP1 gene for breeding drought-salt-tolerant plants
  • Use of boea crassifolia BcBCP1 gene for breeding drought-salt-tolerant plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Obtaining the blue copper protein-like gene BcBCP1:

[0053] 1. Obtaining the BcBCP1 gene fragment

[0054] In order to obtain the sequence tags differentially expressed under drought stress in Cymbidium crassicarpa, we adopted the method of mRNA different display combined with reverse Northern blotting. Extract the total RNA of Cymbidium sphaeroides that was induced by drought (about 43% of water loss) and grown in natural environments without drought treatment. According to the instructions of the fluroDD kit, three anchor primers (AP1, AP6 and AP10) and Four random primers (APR7, APR9, APR18 and APR20) were combined for mRNA differential display analysis, and the cDNA fragments obtained were separated by 5.6% denaturing polyacrylamide gel electrophoresis. The difference display was repeated 3 times, and 26 fragments that appeared in the 3 experiments were selected for further analysis. Put the excavated fragments into 30ulTE, incubate at 37°C for 1-2 days, tak...

Embodiment 2

[0074] Example 2: Construction of plant expression vector:

[0075] See the structure of plant expression vector p3300-BCP1 figure 1 .

[0076] Construction of plant expression vector p3300-BCP1: pBI121 was digested with HindIII and EcoRI to recover a 2.8 kb fragment, pCAMBIA3301 was digested with HindIII and EcoRI, and ligated with the recovered 2.8 kb fragment, named p3301-121. At the same time, p BI121 was digested with EcoRI and Sac I to recover small 0.2kb fragments, pCAMBIA3300 was digested with EcoRI and Sac I, and ligated with the above 0.2kb small fragments to obtain a plasmid named p3300-Tnos. The p3300-Tnos was digested with HindIII and SacI to recover a small 0.2kb fragment, and p3301-121 was digested with HindIII and SacI to connect the 0.2kb fragment to obtain a plasmid named p35S-3300-Tnos. The BcBCP1 gene was ligated to the pGEM-Teasy vector and named pT-BcBCP1. pT-BcBCP1 was digested with EcoRI, the 0.6kb fragment was recovered and the vector pGEM-7Z was digested...

Embodiment 3

[0081] Example 3: Transformation of Agrobacterium:

[0082] (1) Preparation of Agrobacterium Competent

[0083] 1. Pick a single colony of Agrobacterium and inoculate it in 5ml of LB / YEB liquid medium containing appropriate antibiotics, and cultivate overnight at 28°C and 250rpm with shaking;

[0084] 2. Inoculate 1:100 in 40ml LB / YEB liquid medium containing appropriate antibiotics and continue to culture for 4-6h;

[0085] 3. Centrifuge at 5,000 rpm for 10 min at 4°C and remove the supernatant;

[0086] 4. Pre-cooled 0.05M CaCL with 600μl ice 2 Solution washing bacteria;

[0087] 5. Centrifuge at 5,000 rpm for 10 min at 4°C and remove the supernatant;

[0088] 6. Pre-cool 0.05M CaCL with 200μl ice-pre-cooled ice 2 Resuspend the bacteria and store at 4°C.

[0089] (2) Conversion

[0090] 1. Add about 0.5-1.0 μg plasmid DNA to competent Agrobacterium, mix gently, and place on ice for 5 minutes;

[0091] 2. Place in liquid nitrogen for 3 minutes;

[0092] 3. Incubate at 37°C for 5 ...

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Abstract

The invention relates to the application of Boea crassifolia Hemsl. ex Dunn BccBcp1 gene 606bp arid correlation in cultivation drought resistance and salt-resistant plants, this gene codes 201 amino acids and compositions the multi- peptides, which has the protoplasmic membrane guide signal peptide in the amino terminal, in the middle of which has the Cu2 + union the structure territory, the carboxyl group terminal includes the plant cell wall and extends the protein unique PPR structure, which has the typical small molecular weight of the blue copper protein structure characteristic. Inducts the plant this gene obviously and enhance the plant's drought resistance, salt-resistant, height temperature-resistant forces. The construction constructed suits and expresses the carrier to the single seed leaf plant and the double seed leaf plant's BccBcp1 gene plant, which involved the start included the CacMv35s start, Ubiquitin started sub- and induction start subBcDh2, which lay between the method and through the gene gun and the agricultural bacillus which led to the tobacco, petunidin, the lawn grass and the forage grass carried on the heredity transforms, and obtained the drought resistance, salt-resistant.

Description

1. Technical Field: [0001] The present invention relates to the use of a drought-related blue copper protein-like gene (BcBCP1) with a coding region of 606 bp cloned from a plant of the genus Cymbidium spp., which encodes a polypeptide composed of 201 amino acids and has a quality at the amino terminal. Membrane targeting signal peptide with Cu in the middle 2+ The carboxyl end of the binding domain contains the PPR structure unique to plant cell wall extensin, which has the typical structure of small molecular weight blue copper protein. Methods to improve the drought resistance of plants through transgenic technology and to study their possible functions. 2. Background technology: [0002] Drought stress is one of the main adversity factors affecting plant growth and development, and it is a major obstacle to agricultural development in many regions. The loss of crops caused by drought ranks first among all abiotic stresses. Therefore, the use of transgenic methods to obtain dr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N15/65C12Q1/68
Inventor 林忠平吴韩英胡鸢雷申业
Owner 林忠平
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