miR408 and application of biological material related to miR408

A biological material and plant technology, applied in the field of plant genetic engineering and biology, can solve problems affecting normal growth, achieve the effects of increasing utilization efficiency, delaying lignification of stems, and reducing lignin content of stems

Pending Publication Date: 2021-10-01
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, underexpression, knockout, or overexpression of these functional genes can significantly affect normal growth

Method used

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  • miR408 and application of biological material related to miR408
  • miR408 and application of biological material related to miR408
  • miR408 and application of biological material related to miR408

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The construction of embodiment 1 plant expression vector pCAMBIA-2300-35S-pagmiR408 vector

[0070] 1. Obtaining the target gene: using the cDNA of wild-type 84K poplar as a template, using pagmiR408-F and pagmiR408-R as primers (introducing KpnⅠ and BamHI recognition sites and Protected bases), amplified by RT-PCR, the size of the obtained PCR products was identified by agarose gel electrophoresis, and then gel recovery was performed, and the recovered products were sequenced to obtain the recognition sites and protected bases containing KpnⅠ and BamHI The recovered product of the gene encoding the precursor of miR408 (or the gene encoding miR408) shown in SEQ ID NO.2 (abbreviated as pagmiR408). The nucleotide sequence of miR408 is shown in SEQ ID NO.1. The nucleotide sequence of the precursor of miR408 is shown in SEQ ID NO.3.

[0071] The primer sequences are as follows:

[0072] pagmiR408-F: 5'-GG GGTACC AGAGACAGATGAAGACGGGG-3' (the underline is the recognition...

Embodiment 2

[0076] Obtaining and qRT-PCR analysis of embodiment 2 transgenic plants

[0077] 1. Obtaining of transgenic plants

[0078] Agrobacterium-mediated leaf disc method (this method is described in the non-patent literature "Gang Z, Kang ZS, Sun YF, Han QM, Xiao-Fei LU (2005) Establishment of gene transformation receptor system for poplar 84K. Journal of Northwest Sci -Tech University of Agriculture and Forestry 33:87-90), the pCAMBIA-2300-35S-pagmiR408 constructed in Example 1 was transformed into 84K poplar to obtain transgenic plants.

[0079] The genomic DNA of leaves of transgenic plants was extracted respectively, and the genomic DNA of leaves of transgenic plants was used as a template, the genomic DNA of wild-type 84K poplar leaves was used as a negative control, and pCAMBIA-2300-35S-pagmiR408 was used as a positive control. The 5' end of the 35S promoter of -35S-pagmiR408 designs upstream primer (35S-F: 5'-CCACGTCTTCAAAGCAAGT-3') and uses the recognition site of the enzym...

Embodiment 3

[0085] Example 3 Analysis of growth phenotypes of overexpressed transgenic plants and wild-type plants

[0086] 1. Plant growth analysis: the plant height, base diameter, number of internodes, internode 5 (IN5) and internode 15 of wild-type 84K poplar and overexpression miR408 transgenic lines 1, 5 and 6 grown in the greenhouse for 6 months The internode length and internode width of the internode (IN15) were measured, and 10 strains were measured for each strain, and the results were averaged.

[0087] The measurement results of plant height and base diameter were analyzed, and the results of plant height were as follows figure 2 As shown in middle a and c: the growth status and plant height of the overexpressed miR408 transgenic lines 1, 5 and 6 (indicated by "#1", "#5" and "#6" respectively in the figure) were significantly higher than those of the wild Type 84K poplar (indicated by "WT" in the figure); the results of the base diameter are as follows figure 2 Shown in m...

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Abstract

The invention discloses miR408 and the application of a biological material related to the miR408. The invention firstly discloses the application of miR408 in improving saccharification efficiency of plant stems or delaying lignification of the plant stems or reducing lignin content of the plant stems or reducing lignin content of secondary cell walls of the plant stems or promoting plant growth. The invention further discloses a preparation method of the transgenic plant. Tests prove that overexpression miR408 can promote height and thickness increasing growth of poplars, postpone lignification of stems and reduce the lignin content of the stems; under the condition of no acid pretreatment, the saccharification efficiency of the poplar stems by 84.76%-92.44% is remarkably improved, so that the utilization efficiency of biomass energy is improved, and the method has important application value and environmental protection benefits for cultivating efficient biomass energy tree species in forestry production.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and biotechnology, and specifically relates to the application of miR408 and related biological materials. Background technique [0002] Populus 84K (Populus alba × Populus glandularis, Populus alba × Populus glandulosa) was introduced from South Korea in 1984 by Zhang Qiwen, a researcher at the Institute of Forestry, Chinese Academy of Forestry. 84K poplar is male and does not fly catkins; it has strong disease resistance, cold resistance and drought resistance. Its growth rate is fast, and it is an excellent industrial timber tree species and urban and rural greening tree species (Zhou Yongxue, Fu Yuqin, Fan Junfeng, Liu Yuyuan, Gao Jianshe, Wang Jun, Fu Jun, Li Junan. (2007). Growth characteristics and hybrid compatibility of 84k poplar. Journal of Northeast Forestry University, 35(10), 11-12.). 84K poplar grows rapidly and is easy to transform. An efficient genetic transformation sys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/82C12N5/10A01H5/04A01H5/06A01H5/12A01H6/00
CPCC12N15/113C12N15/8261C12N15/8255C12N2310/141
Inventor 林金星郭亚玉许会敏
Owner BEIJING FORESTRY UNIVERSITY
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