A rubber tree lignin synthesis regulation related protein hbmyb85 and its coding gene and application

A gene and encoding technology, applied in the biological field, can solve the problems of poor physical and mechanical properties, low density, and restrictions on the development of rubber trees, and achieve the effect of promoting the synthesis and accumulation of lignin

Inactive Publication Date: 2017-12-19
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Although the economic and social benefits of rubber tree wood have been widely recognized by the international community, rubber tree is a fast-growing tree species. Its wood material is brittle, its density is low, and its physical and mechanical properties are poor. Development as a plantation timber resource

Method used

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  • A rubber tree lignin synthesis regulation related protein hbmyb85 and its coding gene and application
  • A rubber tree lignin synthesis regulation related protein hbmyb85 and its coding gene and application
  • A rubber tree lignin synthesis regulation related protein hbmyb85 and its coding gene and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the discovery of HbMYB85 protein and its coding gene

[0043] Obtain the EST sequence database of leaves, stems, roots, flowers, and latex of rubber trees, screen out high-abundance expression DNA fragments in stems, and conduct homologous comparison analysis with existing DNA fragments to determine an EST assembled sequence of about 1000bp (contig ). A pair of specific primers were designed based on contig: 5'-GCATTAGTGGAACTTGTTCAT-3' and 5'-TTTTCTCACTACGTACGTACG-3'. Total RNA was extracted from the bark of the rubber variety "Reyan 7-33-97" and reverse transcribed into cDNA. Using cDNA as a template, specific primers were used for PCR amplification. The amplified product was subjected to 1% agarose electrophoresis, and the target band was recovered and cloned into the pMD18-T vector for sequencing.

[0044] Sequencing results showed that the amplified product contained a gene encoding a new protein.

[0045] The new protein discovered was named HbMYB8...

Embodiment 2

[0046] Embodiment 2, the expression level comparison of HbMYB85 gene in Hevea different parts

[0047] Total RNA was extracted from the leaves, stem top, stem middle, stem lower part, cortex and xylem of the rubber variety "Reyan 7-33-97", and the total RNA was reverse transcribed into cDNA. The CFX96 (Bio-Rad company) real-time fluorescent quantitative PCR detection system was used to analyze the expression level of HbMYB85 gene in different parts of rubber tree.

[0048] The primers used for real-time fluorescent quantitative PCR detection of HbMYB85 gene are as follows:

[0049] HbMYB85-QTF: 5'-AGAGGCCTTCTAACTGAAGCA-3';

[0050] HbMYB85-QTR: 5'-TTTATGGAAGGATTCATGCG-3'.

[0051] The amplification reaction program of real-time fluorescence quantitative PCR: pre-denaturation at 95°C for 3min; denaturation at 95°C for 10s, annealing at 60°C for 10s, extension at 72°C for 30s, 39 cycles.

[0052] The relative expression level of HbMYB85 gene was normalized with 18S gene as in...

Embodiment 3

[0054] Example 3, Subcellular localization analysis of HbMYB85 protein

[0055] Sequence 2 of the sequence listing is inserted between the XbaI and EcoRI restriction sites of the pCAMBIA1205-GFP vector from the 69th-899th nucleotide at the 5' end to obtain the recombinant plasmid pCAMBIA1205-GFP-HbMYB85.

[0056] The pCAMBIA1205-GFP vector or the recombinant plasmid pCAMBIA1205-GFP-HbMYB85 were respectively introduced into the EHA105 Agrobacterium strain to obtain the recombinant Agrobacterium.

[0057] The recombinant Agrobacterium was treated with 150 μM acetosyringone and 10 mM MgCl 2 MES buffer (pH5.6, 10mM) resuspended, adjust OD 600nm =0.8, and then injected into tobacco leaves, cultured for 2 days and then observed under a laser confocal microscope with 488nm excitation.

[0058] see results figure 2 . Unlike GFP, the fluorescence of HbMYB85-GFP is mainly concentrated in the nucleus. The results showed that HbMYB85 protein had nuclear localization properties.

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Abstract

The invention discloses a regulatory protein HbMYB85 synthesized by virtue of rubber tree lignin as well as an encoding gene and application of the regulatory protein HbMYB85. The protein is named HbMYB85 protein and is shown as follows (a) or (b): (a) a protein formed by amino acid sequences shown by a sequence 1; and (b) a regulatory protein which is derivated by the sequence 1 and formed by replacement and / or deletion and / or addition of one or more amino acid residues on the amino acid sequences shown by a sequence 1 and synthesis of thickness of a plant secondary cell wall and / or lignin. An HbMYB85 gen also belongs to the protection range of the invention. The invention also protects a method of cultivating a transgenic plant. The method comprises the following steps: introducing the HbMYB85 gene into a target plant to obtain the transgenic plant with the thickness of the plant secondary cell wall and / or the content of the lignin higher than that or those of the target plant. The regulatory protein HbMYB85 has important significance of promoting thickness increase of the plant secondary cell wall and accumulation of the lignin and particularly quality genetic improvement of rubber woods and cultivation of a new wind-resistant variety.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a protein HbMYB85 related to the regulation of rubber tree lignin synthesis and its coding gene and application. Background technique [0002] Forest wood is an indispensable and important industrial raw material in the construction and development of the national economy, and it also plays a huge ecological benefit in the construction of the ecological environment. As the public continues to pay attention to the overexploitation of natural forests and the loss of biodiversity, it has gradually become a global consensus in the development of modern forestry to develop sustainable plantations and protect existing natural forest resources. As a country with serious shortage of forest resources, the contradiction between timber supply and demand is particularly acute. Therefore, accelerating the genetic improvement of high-quality fast-growing plantation tree species and imp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8255
Inventor 仇键校现周罗世巧刘彤魏芳杨文凤高宏华
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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