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Application of ptrbhlh186 gene in Populus trichocarpa in regulating secondary xylem development in trees

A technology of Populus trichocarpa and xylem, applied in the field of genetic engineering, can solve the problems that the biological functions of genes have not been studied

Active Publication Date: 2022-07-22
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the PtrbHLH186 transcription factor of Populus trichocarpa is highly expressed in stem xylem, but the biological function of this gene has never been studied so far

Method used

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  • Application of ptrbhlh186 gene in Populus trichocarpa in regulating secondary xylem development in trees
  • Application of ptrbhlh186 gene in Populus trichocarpa in regulating secondary xylem development in trees
  • Application of ptrbhlh186 gene in Populus trichocarpa in regulating secondary xylem development in trees

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Acquisition of PtrbHLH186 transcription factor of P. trichocarpa and construction of plant expression vector

[0030] (1) Cloning of PtrbHLH186 gene sequence

[0031] The total RNA of the wild-type plants of Populus trichocarpa Nisqually-1 genotype was extracted by Qiagen plant RNA extraction kit, and cDNA was obtained by reverse transcription using Takara reverse transcription kit (RR047A).

[0032] Referring to the genome sequence information of P. trichocarpa provided by the phytozome website, gene-specific primers were designed at both ends of the target gene sequence and a bio-company was entrusted to synthesize the primers. The upstream and downstream primer sequences used for PtrbHLH186 gene cloning are shown in Table 1.

[0033] Table 1 Sequences of upstream and downstream primers used for PtrbHLH186 gene cloning

[0034]

[0035] Note: The underlined part represents the restriction site, and the protection base is in front of the restriction site...

Embodiment 2

[0053] Example 2: Obtainment of PtrbHLH186 Overexpressing Plants of P. trichocarpa

[0054] (1) Freeze-thaw method to transform Agrobacterium

[0055] ①Take out the Agrobacterium competent cells and put them on ice, add about 30 ng of plasmid to the Agrobacterium competent cells, mix well, ice bath for 30 minutes, immediately put them in liquid nitrogen, freeze for 1 minute, and then immediately put them into 37℃, water bath 3min.

[0056] ②Add 1ml of liquid LB and incubate at 180-200rpm at 28℃ for 2-3h.

[0057] ③Break off for 15s, remove the supernatant, retain 300-400 μl of the supernatant, resuspend, plate on LB (kanamycin + gentamicin) solid medium, and culture at 28°C for about 48h.

[0058] ④ Pick the large clone, inoculate it in 5ml LB (kanamycin + gentamicin) liquid medium, and cultivate overnight at 220rpm and 28°C.

[0059] ⑤ Carry out PCR with bacterial liquid as a template to confirm positive clones. Refer to Example 1 for the specific method.

[0060] ⑥ Save ...

Embodiment 3

[0083] Example 3: Application of PtrbHLH186 gene in PtrbHLH186 in regulating tree secondary xylem development

[0084] (1) The independent resistant lines obtained in Example 2 and the wild-type plants of P. trichocarpa were subjected to growth phenotypic analysis

[0085] Growth phenotype analysis includes the following indicators:

[0086] Tree height measurement: The height from the soil surface to the terminal bud was measured for the wild type and transgenic greenhouse plants overexpressing PtrbHLH186 at 1, 2, 3 and 4 months old, respectively. The wild type and transgenic plants were at least 3 biological replicates.

[0087] Number of stem nodes: 1, 2, 3, and 4-month-old wild-type and transgenic greenhouse plants overexpressing PtrbHLH186 were counted for the number of stem nodes. Wild-type and transgenic plants were replicated at least 3 times.

[0088] Basal stem measurement: 1, 2, 3, and 4-month-old wild-type and transgenic greenhouse plants overexpressing PtrbHLH186...

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Abstract

The application of PtrbHLH186 gene of Populus trichocarpa in regulating the development of secondary xylem of trees belongs to the technical field of genetic engineering. In order to solve the problem that the demand for wood quantity and quality is increasing day by day and the biological function of the PtrbHLH186 transcription factor of P. trichocarpa is unknown, the present invention obtains PtrbHLH186 over-expressing PtrbHLH186 transgenic plants through the Agrobacterium-mediated PtrbHLH186 genetic transformation system. Using a series of technical means, it was found that the overexpression of the PtrbHLH186 transcription factor affects the growth and development of plants and the formation of wood, which is mainly reflected in that the gene affects the deposition of lignin in the secondary cell wall of trees through the regulation of lignin synthase genes. Furthermore, the morphological structure and distribution of xylem cells were changed. The above results indicated that the PtrbHLH186 transcription factor had a certain regulatory effect on the development of secondary xylem in trees. The research content of the present invention has important theoretical guiding significance for cultivating new varieties of high-quality timber trees.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of PtrbHLH186 gene of Populus trichocarpa in regulating the development of secondary xylem of trees. Background technique [0002] Wood is an essential and important raw material for pulp and paper making and house construction, and is also an important renewable resource in the world. my country is a big country in the production and consumption of wood products in the world. With the development of economy and society, the increase of population and the improvement of people's living standards, my country's demand for the quantity and quality of wood is also increasing. The formation of tree wood is a highly ordered developmental process. The vascular cambium of the stem divides and differentiates inwards to form secondary xylem composed of vessel cells, fiber cells, and ray cells. The secondary xylem is continuously formed by lignin depo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/00
CPCC07K14/415C12N15/8255
Inventor 李伟刘慧子李爽高境烩孙佳彤姜立泉
Owner NORTHEAST FORESTRY UNIVERSITY
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