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WPGS5 or WPGS6 gene of eucalyptus and function of controlling and increasing plant biomass through overexpression

A gene and eucalyptus technology, applied in the biological field, can solve problems such as separation and identification work to be carried out

Inactive Publication Date: 2012-09-19
SINO FOREST CHINA INVESTMENTS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The functions of the above genes have been thoroughly studied in plants such as Arabidopsis thaliana and tobacco, but the isolation and identification work in forest trees has yet to be carried out

Method used

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  • WPGS5 or WPGS6 gene of eucalyptus and function of controlling and increasing plant biomass through overexpression
  • WPGS5 or WPGS6 gene of eucalyptus and function of controlling and increasing plant biomass through overexpression
  • WPGS5 or WPGS6 gene of eucalyptus and function of controlling and increasing plant biomass through overexpression

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Cloning of Eucalyptus WPGS5 and WPGS6 Genes

[0032] 1. Preparation of Eucalyptus cDNA

[0033] Fresh eucalyptus (Eucalyptus DH32-29) leaves were selected, frozen in liquid nitrogen, and then stored in a -80°C refrigerator for later use.

[0034]Referring to the method of Murray and Thompson (1980), the total tissue RNA was extracted using the Cetyltrimethyl Ammonium Bromide (CTAB) method. Specific implementation: 1) Preheat 15 ml CTAB extraction solution in 65°C water ; 2) Grind 2-3 grams of eucalyptus frozen tissue in liquid nitrogen; 3) Transfer the sample to a centrifuge tube containing CTAB extract, vortex vigorously for 30 seconds immediately, and then bathe in 65°C water for 4-5 minutes; 4) Add an equal volume of Chloroform / isoamyl alcohol, vortexed, centrifuged at 10,000 rpm for 15 minutes at room temperature; 5) Transfer the supernatant to a new centrifuge tube, and repeat the extraction once; 6) Transfer the supernatant to a new centrifuge tube A...

Embodiment 2

[0055] Example 2 Obtainment of HOG1 and ABAP1 Gene Knockout Arabidopsis Mutants and Observation of Their Characters

[0056] Referring to the method of Hiei (1994), the pSV expression vector containing the hairpin structure composed of the conservative fragment of the target gene was transformed into Agrobacterium tumefaciens strain GV3101 by freeze-thaw method, and the colony PCR identification was carried out with primers for amplifying the gene. image 3 Shown is the identification of the genomic DNA of the WPGS5 gene silencing mutant strain, Figure 4 The results of genomic DNA identification of WPGS6 gene silencing mutant strains are shown. Mass culture positive clones. Arabidopsis thaliana was transformed by flower dipping method (refer to the method published in Plant Journal by S J Clough et al. in 1998), and the current harvest was T0 transgenic seeds. The transgenic seeds of the T0 generation were screened on MS medium containing 50mg / L hygromycin, and only the p...

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Abstract

The invention relates to sequences and functions of two eucalyptus genes (namely a WPGS5 gene and a WPGS6 gene) used for increasing plant biomass, belongs to the technical field of biology. RNA (Ribonucleic Acid) in eucalyptus is extracted and is reversely transcribed to cDNA (complementary DNA), and then is subject to deep sequencing to obtain full-length cDNA sequence library of eucalyptus, the similarity comparison is further carried out between the sequence of the cDNA sequence library of eucalyptus and amino acid sequences of arabidopsis AtABAP1 protein and arabidopsis AtHOG1 protein published by an NCBI (National Center of Biotechnology Information) Genbank to obtain the cDNA sequence of the WPGS5 gene and the cDNA sequence of the WPGS6 gene, and finally, sense fragments and antisense fragments in conservated regions of the WPGS5 gene and the WPGS6 gene are selected to build gene silencing recombinant vectors, and the gene silencing recombinant vectors are transferred to arabidopsis to carry out gene silencing, so that afunctional mutant strains are obtained. As a result, the biomass of transgenic plants obtained through the silencing of the WPGS5 gene and the WPGS6 gene is remarkably improved. Therefore, the controlling functions of the WPGS5 gene and the WPGS6 gene of eucalyptus in growth of dicotyledon are proved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to eucalyptus ( Eucalyptus ) of the two genes WPGS5 and WPGS6 associated with increased biomass. Background technique [0002] Eucalyptus is the general name of Eucalyptus in the family Myrtaceae. It is an excellent tree species that integrates ornamental, timber, pulp raw materials, and medicinal uses. Due to the long growth cycle of forest trees and high genetic heterogeneity, many traits are quantitative traits controlled by polygenes. Conventional breeding methods are difficult to meet the requirements of directional breeding of new eucalyptus varieties. Therefore, people place their research focus and success on genetic engineering technology. Solve the difficult problems of conventional eucalyptus breeding, and accelerate the process of breeding new varieties of high-quality and high-yielding eucalyptus. Forest trees and other woody plants are rich in genetic resources, but many...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/63A01H5/00
Inventor ZHANG JUNPAN SHENG
Owner SINO FOREST CHINA INVESTMENTS LTD
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