Application of populus trichocarpa PtrbHLH186 gene in regulation and control of secondary xylem development of 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: 2022-03-18
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 populus trichocarpa PtrbHLH186 gene in regulation and control of secondary xylem development of trees
  • Application of populus trichocarpa PtrbHLH186 gene in regulation and control of secondary xylem development of trees
  • Application of populus trichocarpa PtrbHLH186 gene in regulation and control of secondary xylem development of trees

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0030] (1) Cloning of PtrbHLH186 gene sequence

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

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

[0033] Table 1 is used for the upstream and downstream primer sequences of PtrbHLH186 gene cloning

[0034]

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

Embodiment 2

[0053] Example 2: Obtaining of Populus trichocarpa PtrbHLH186 overexpression plant

[0054] (1) Transformation of Agrobacterium by freeze-thaw method

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

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

[0057] ③Centrifuge for 15 seconds, remove the supernatant, retain 300-400 μl of the supernatant, resuspend, plate on LB (kanamycin + gentamicin) solid medium, and incubate at 28°C for about 48 hours.

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

[0059] ⑤ Carry out PCR with the bacterial solution as a template to confirm positive clones, refer to Example 1...

Embodiment 3

[0083] Example 3: Application of Populus trichocarpa PtrbHLH186 gene in regulating the development of secondary xylem of trees

[0084] (1) Analysis of the growth phenotype of the independent resistant strains obtained in Example 2 and the wild-type plants of Populus trichocarpa

[0085] Growth phenotype analysis includes the following indicators:

[0086] Tree height measurement: Measure the height from the soil surface to the terminal buds of 1, 2, 3, and 4-month-old wild-type and transgenic greenhouse plants overexpressing PtrbHLH186, with at least 3 biological replicates of wild-type plants and transgenic plants.

[0087] Number of stem nodes: The number of stem nodes was counted for 1, 2, 3, and 4-month-old wild-type and transgenic greenhouse plants overexpressing PtrbHLH186, and wild-type plants and transgenic plants had at least 3 biological replicates.

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

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Abstract

The invention relates to application of a populus trichocarpa PtrbHLH186 gene in regulation and control of secondary xylem development of trees, and belongs to the technical field of gene engineering. In order to solve the problems that the need for wood quantity and quality is increased day by day and the biological function of the PtrbHLH186 transcription factor is unknown, a PtrbHLH186 overexpressed populus trichocarpa transgenic plant is obtained through an agrobacterium-mediated populus trichocarpa genetic transformation system, and then a series of technical means are utilized for analysis and discovery that the PtrbHLH186 overexpressed populus trichocarpa transgenic plant is found to have the advantages that the PtrbHLH186 overexpressed populus trichocarpa transgenic plant; the overexpression of the PtrbHLH186 transcription factor affects the growth and development of plants and the formation of wood, which is mainly embodied in that the lignin synthase gene is regulated by the gene to affect the deposition of lignin in the secondary cell wall of a tree, so that the morphological structure and distribution of xylem cells are changed; the results show that the PtrbHLH186 transcription factor has a certain regulation effect on the development of the secondary xylem of the tree. The research content provided by the invention has important theoretical guiding significance for cultivating new varieties of high-quality woods and forests.

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, house construction, etc., and is also an important renewable resource in the world. my country is a big country in the processing, 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 day by day. The formation of tree wood is a highly orderly development process. The vascular cambium of the stem continuously divides and differentiates inward to form secondary xylem composed of vessel cells, fiber cells, and ray cells. The secondary xylem is conti...

Claims

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Application Information

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Patent Type & Authority Applications(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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