Method for promoting plant seed augmentation and cotton fibre growth by using RDL1 gene

A gene and seed technology, applied in the fields of plant bioengineering and plant improvement genetic engineering, can solve problems such as less functional research

Inactive Publication Date: 2009-08-12
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The C-terminus of GhRDL1 protein contains a plant-specific BURP domain protein, and its function is less studied

Method used

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  • Method for promoting plant seed augmentation and cotton fibre growth by using RDL1 gene
  • Method for promoting plant seed augmentation and cotton fibre growth by using RDL1 gene
  • Method for promoting plant seed augmentation and cotton fibre growth by using RDL1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1. Isolation of RDL1 cDNA

[0096] Cotton RNA was extracted by cold phenol method:

[0097] 2g of material (fibers on the ovule surface of upland cotton 9 days after flowering) was ground into powder in liquid nitrogen, transferred to a 50ml centrifuge tube, and 8ml of extraction buffer (1M Tris HCl, 50mM EDTA, 1% SDS, pH9.0) and an equal volume of water-saturated phenol:chloroform:isoamyl alcohol (25:24:1), vortexed and mixed, placed on ice for 1h, and mixed every 10 minutes. Centrifuge at 13000g for 20 minutes at 4°C. Repeat phenol: chloroform: isoamyl alcohol extraction 2 to 4 times, and finally extract once with chloroform: isoamyl alcohol (24:1). Take the supernatant, add 1 / 2 volume of high salt solution (0.8M sodium citrate, 1.2M NaCl) and 1 / 2 volume of isopropanol, mix well, and place at -70°C for 1h. Centrifuge at 13,000g for 20 minutes at 4°C, remove the supernatant, dissolve the pellet in 1ml of DEPC-treated water, and centrifuge at 13,000g for 10 m...

Embodiment 2

[0103] Construction and Agrobacterium transformation of embodiment 2.RDL1 and GFP fusion vector

[0104] The GFP gene used was derived from pEGFP-1 (Clontech, Palo Alto, USA). Appropriate enzyme cleavage sites were introduced by PCR. The stop codon of GFP was removed, and an unfolded sequence of 6 amino acids (GPGGGG) was added.

[0105] Using GS1: 5'-CTAGTCTAGAATGGTGAGCAAGGGCGAGGAG-3' (SEQ ID NO: 9) and GA1: 5'-GTCCCCCGGGCGTCCTCCTCCTCCTGGTCCCTGGTACAGCTCGTCCATGCC-3' (SEQ ID NO: 10) as primers, pEGFP-1 vector as template, and using Pyrobest DNA polymerase (purchased from TAKARA company) amplified the GFP coding region, recovered from rubber tapping, double-digested with SmaI and NcoI, and connected between the EcoRV and NcoI sites of pET32c to construct the GFP-32c intermediate vector.

[0106] The target fragment was amplified with Pyrobest DNA polymerase, and the corresponding restriction sites BamHI and SacI were introduced at the same time (see Example 1). After checking ...

Embodiment 3

[0109] Example 3. Plant Transformation and Screening of Transgenic Progeny

[0110] a. Transgenic cotton

[0111] After culturing the Agrobacterium containing the vector plasmid on YEB bacterial medium supplemented with kanamycin 50 mg / L, rifampicin 100 mg / L and streptomycin 300 mg / L for 2 to 3 days, pick a single colony and inoculate it in the culture medium containing the same antibiotic. Suspension culture was carried out overnight at 28°C on a shaker at 200 rpm / min in YEB liquid medium. Centrifuge the bacterial solution at 4000rpm / min for 10 minutes, resuspend the pellet with 1 / 2MS liquid medium containing 30g / L glucose and 100μmol / L acetosyringone, and adjust the OD 600 The value is about 0.4 to 0.6, which is used as an infection solution for later use.

[0112] Cotton R15 (a tetraploid wild-type upland cotton, as the transgenic female parent) seeds were placed in 1 / 2MS0 medium [1 / 2MS salt (purchased from DUCHEFA M0221) + 5g / L glucose + 7g / L agar powder, pH 6.0], ger...

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Abstract

The invention provides application of a plant RDL1 gene or RDL1 protein in improving properties of crops seeds, and a method for improving properties of crops seeds. Particularly, the invention provides a gene engineering method for promoting the crops seeds to enlarge and cotton fiber to lengthen by utilizing the plant RDL1 gene. The invention also provides a vector containing the RDL1 gene, a host cell, a method for preparing transgenic plants and a method for obtaining seeds with improved properties through the transgenic plants. The method can increase the volume of the seeds and the weight of the seeds, and lengthen seed fiber and / or increase the strength of the seed fiber, plays a positive role in improving the yield and properties of crops, and has extensive application prospects.

Description

technical field [0001] The invention belongs to the fields of plant bioengineering and plant improvement genetic engineering. Specifically, the present invention relates to the isolation of the cotton fiber-specific expression gene RDL1 cDNA and the construction of an overexpression vector, as well as a method for promoting excellent traits such as seed enlargement and fiber growth of transgenic crops by transferring the RDL1 gene. Background technique [0002] Crop seeds are important raw materials in agriculture and industry such as grain, cotton, and oil, and their properties are directly related to the quality of seeds and the quality of processed products. These traits mainly include the size of the seed volume, the weight of the seed, the length of the seed fiber (for utilizing the seed fiber) and / or the strength of the seed fiber. [0003] Cotton is an important economic crop, and cotton fiber is an important raw material for the textile industry. In 2006, the world...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C07K14/415A01H1/00A01H4/00
CPCC07K14/415C12N15/8261Y02A40/146
Inventor 陈晓亚徐冰缑金营上官小霞毛颖波林芝萍王凌健
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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