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Application of cotton and rape brassinolide synthetase gene and expression vector containing same

A technology of brassinolide and plant expression vector, which is applied in the field of plant genetic engineering and can solve the problem of low physiological activity

Inactive Publication Date: 2009-09-02
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study found that the substrate of DWF4 accumulates in large quantities in plants but has very low physiological activity, while the direct product of DWF4 has very low content in plants but has high physiological activity

Method used

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  • Application of cotton and rape brassinolide synthetase gene and expression vector containing same
  • Application of cotton and rape brassinolide synthetase gene and expression vector containing same
  • Application of cotton and rape brassinolide synthetase gene and expression vector containing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] [Example 1] Cloning of GhDWF4 Gene Sequence

[0055] 1. Extraction of Cotton RNA

[0056]Select about 3g of fresh cotton material, quickly grind it into a fine powder in liquid nitrogen, put it into a 50mL centrifuge tube, add 15ml of 65°C preheated RNA extraction solution (2% CTAB (W / V), 2% PVP (W / V ), 100mmol / L Tris-HCl (pH8.0), 0.5g / L Spermidine, 2.0mol / L NaCl, 2% mercaptoethanol (V / V, added before use)), and mix well by inversion. Water bath at 65°C for 3-10 minutes, during which time mix 2-3 times. Chloroform:isoamyl alcohol (24:1) extracted twice (10,000r / min, room temperature, 5min). Take the supernatant, add 1 / 4 volume of 10mol / L LiCl solution, place at 4°C for 6h, and extract once each with chloroform:isoamyl alcohol (25:24:1) (10,000r / min, room temperature, 5min). Add 2 times the volume of absolute ethanol, and precipitate in a -70°C refrigerator for more than 30 minutes. Centrifuge at 12,000r / min at 4°C for 20min, discard the supernatant. The precipita...

Embodiment 2

[0075] [Example 2] Construction of Overexpression Vector and Genetic Transformation of Tobacco

[0076] 1. Construction of overexpression vector

[0077] The pUC-GhDWF4 vector was constructed when the GhDWF4 gene was cloned, and the GhDWF4 fragment on it was sequenced. The plant expression vector is a modified pCambia vector, the HPT II gene of the vector is cleaved with XhoI and replaced with the NPT II gene (the designed primers are amplified from the pBI121 vector, and the primers are designed with XhoI sites at both ends), and the restriction enzyme Excision and sequencing results verified the orientation of the NPT II gene. The CaMV35S promoter and NOS terminator were amplified in the pBI121 vector, and the corresponding restriction sites were introduced at both ends of the two elements. Finally, these two elements were respectively introduced into the multiple cloning site of pCambia to form CaMV35S:: MCS::NOS unit. The plant expression vector contains 1 set of 2×Ca...

Embodiment 3

[0096] [Embodiment 3] Detection of the expression level of GhDWF4 gene in transgenic tobacco

[0097] According to the method for extracting cotton RNA in Example 1, the total RNA of transgenic tobacco and wild-type tobacco leaves was extracted, and a cDNA strand was synthesized. Real-Time PCR method was used to analyze the expression of the target gene in transgenic tobacco. PCR was carried out on a quantitative real-time PCR instrument, and 12.5 μL MIX buffer (including PCR buffer, DNA polymerase, dNTPs and MgCl2) was included in the 25 μL reaction system. , provided by quantitative real-time PCR kit, Bio-Rad). The GhDWF4 gene was amplified with primers D4-500 (SEQ ID NO.9) and GhDWF4-2 (SEQ ID NO.6). The internal standard was the tobacco ACTIN gene (accession number: AB158612), and the primer was actin-1 (SEQ ID NO. .7) and actin-2 (SEQ ID NO.8). The amplification program is: pre-denaturation at 94°C for 3min; 94°C, 30sec; 56°C, 30sec; 72°C, 30sec; the preset cycle numb...

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Abstract

The invention discloses a new application of a cotton and rape brassinolide synthetase GhDWF4 gene. The GhDWF4 gene can promote the extension of hypocotyls of plants and can also enlarge areas of plant leaves and increase the yield of fruits so as to promote the growth of the plants if being expressed in the plants, and the GhDWF4 gene can be used for crop breeding and forest tree breeding, thereby enhancing the vegetative growth of transgenic plants and increasing the biological yield of the plants. The invention also provides a plant expression vector which contains the GhDWF4 gene and a transformant thereof and provides a method for cultivating transgenic plants which contain the GhDWF4 gene. The invention has the advantages that the vegetative growth and the reproductive growth of the transgenic plants are promoted, the heights of the transgenic plants are increased, and the yield of fruits is increased.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically relates to the application of the cotton brassinosteroid synthase gene, a protein encoded by the gene and a plant expression vector; the invention also relates to a method for preparing a transgenic plant containing the gene. Background technique [0002] Brassinosteroids (BRs) are a class of hormones widely present in plants with a structure similar to animal sterols. Brassinolides play an important role in many physiological processes of plant growth and development, and the concentration is extremely low (nmol L -1 ) brassinosteroids can show extremely high physiological activity, so brassinosteroids are considered to be the sixth largest plant species after auxin, gibberellin, cytokinin, abscisic acid and ethylene hormone. [0003] Studies on the model plant Arabidopsis thaliana have shown that deletion mutants of BRs synthesis and signal transduction pathways exhib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/10C07J73/00C12N15/52
Inventor 罗明胡明瑜裴炎侯磊李先碧肖月华宋水清李德谋
Owner SOUTHWEST UNIVERSITY
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