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Taxodium hybrid 'zhongshanshan' ThSHR3 gene for promoting plant growth and application of taxodium hybrid 'zhongshanshan' ThSHR3 gene

A Zhongshan fir and plant technology, which is applied in the field of plant genetic engineering, can solve the problems of clone rooting ability decline, plant growth dwarfing, rooting ability difference, etc., and achieve growth speed enhancement, stem internode length elongation, and length increase Effect

Active Publication Date: 2020-11-24
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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Problems solved by technology

At present, Zhongshan fir clones are mainly propagated by twig cuttings. In recent years, forestry workers have been committed to improving the rooting rate of Zhongshan fir through the improvement of Zhongshan fir cutting technology. However, during the long-term selection process of Zhongshan fir, it was found that , the rooting ability of different clones is significantly different, even if the cutting technology is optimized, this phenomenon cannot be changed
And with the increase of the physiological age of the clones, the rooting ability of the clones selected in the early stage declines, and the plant growth also appears to be dwarfed. The growth and development of Zhongshan fir are in urgent need of further research.

Method used

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  • Taxodium hybrid 'zhongshanshan' ThSHR3 gene for promoting plant growth and application of taxodium hybrid 'zhongshanshan' ThSHR3 gene
  • Taxodium hybrid 'zhongshanshan' ThSHR3 gene for promoting plant growth and application of taxodium hybrid 'zhongshanshan' ThSHR3 gene
  • Taxodium hybrid 'zhongshanshan' ThSHR3 gene for promoting plant growth and application of taxodium hybrid 'zhongshanshan' ThSHR3 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Cloning of ThSHR3 gene by RACE technology

[0026] 1) Extraction of RNA and inversion of cDNA

[0027] Zhongshan fir 406 (Taxodium mucronatum) collected in the nursery of Nanjing Zhongshan Botanical Garden in July 2019 ×T. distichum )(T.hybrid'Zhongshanshan 406') Adventitious root as material. Use RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (TIANGEN Company) to extract the total RNA from Shanshanshan. Before extracting RNA, all pipette tips, centrifuge tubes, and mortars should be soaked in 0.1% DEPC water overnight, autoclaved for 40 minutes, and then dried. All solutions should be prepared in 0.1% DEPC water. The extracted RNA was treated with DNase (Tiangen Biochemical Technology Co., Ltd.) to make it purified. The concentration and purity of total RNA were measured with NanoDrop 2000 (Thermo Scientific). And use 2.0% agarose gel electrophoresis to separate the total RNA. Afterwards, the 3'RACE and 5'RACE kits of Invitrog...

Embodiment 2

[0050] Embodiment 2: Construction of ThSHR3 gene plant expression vector

[0051] Using Gateway directional cloning technology, the obtained ORF fragment of the target gene is transferred to the target expression vector, including two parts: BP reaction and LR reaction. The carrier map used in this embodiment is as follows figure 1 shown.

[0052] 1) The purpose of the BP reaction is to transfer the gene fragment to the entry vector, the reaction system: vector ThSHR3 ORF fragment 10-20ng, Salt solution 1μL, pCRTM8 / GW / TOPOTM vector (entry vector) 1μL, add Nuclease-free Water to total The volume was 6 μL, mixed gently, and transferred to ice after reacting at 22°C for 60 minutes; 6 μL of the reaction product from the previous step was transformed into TOP10 competent cells, spread on LB screening culture plates, and single clones were picked for bacterial liquid PCR detection. The primers were gene-specific upstream primers and T7 primers on the carrier, and the positive clon...

Embodiment 3

[0054] Example 3: Sequence alignment and evolution analysis of ThSHR3 gene

[0055] The protein sequence of ThSHR3 was compared with the SHR protein sequences of other plants using ClustalX2 software. The protein sequence of ThSHR3 was compared with the Arabidopsis protein sequence AtSHR in TAIR, the poplar protein sequence PeSHR1, PeSHR2, and the Sequoia protein sequence ThSHR1, ThSHR2 in PeSHR3. The results showed that the N-terminus of ThSHR protein was not conserved, while the C-terminus was relatively conserved. Like the SHR proteins of other species, ThSHR protein included the basic sequences of LHRI, VHIID, LHRII, PFYRE and SAW that are unique to GRAS family members.

[0056]Further use MEGA 7.0 software to construct a phylogenetic tree, the construction method is the maximum likelihood method, and the bootstrap detection is 1000 times. In this study, the phylogenetic tree analysis was performed on the amino acid sequence of ThSHR3 and the published 45 GRAS amino acid ...

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Abstract

The present invention discloses a taxodium hybrid 'zhongshanshan' ThSHR3 gene for promoting plant growth and an application of the taxodium hybrid 'zhongshanshan' ThSHR3 gene, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the ThSHR3 gene is shown in SEQ ID NO.1 and an amino acid sequence of an expressed protein is shown in SEQ ID NO.2. Sequence alignment and evolution analysis of the ThSHR3 gene show that the ThSHR3 gene belongs to a SHR gene subfamily. Populus davidiana x P.bolleana is transformed by constructing a gene pH35GS-ThSHR3 vector. Compared with the control plant, the ThSHR3 gene is highly expressed in the Populus davidiana x P.bolleana, the expression amount of the target gene is increased by 500 times or more, and the plant growthrate is significantly higher than that of the control plants, indicating that the ThSHR3 gene is an important gene that regulates plant development and affects plant morphology, has important application value in the field of forest genetic engineering and can be used to adjust plant growth and development speed or plant morphology.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a Zhongshan fir ThSHR3 gene for promoting plant growth and its application. Background technique [0002] Taxodium hybrid'zhongshanshan' is a general term for excellent clones with certain super-parental traits, bred by the Institute of Botany, Chinese Academy of Sciences in Jiangsu Province from the hybrid combination of Taxodium trees. It has rapid growth, strong stress resistance, With the characteristics of good landscape and high yield, it has been widely used in the construction of coastal shelterbelts, road and urban and rural greening, farmland forest network and tidal flat afforestation in my country. At present, Zhongshan fir clones are mainly propagated by twig cuttings. In recent years, forestry workers have been committed to improving the rooting rate of Zhongshan fir through the improvement of Zhongshan fir cutting technology. Howeve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00
CPCC07K14/415C12N15/8261
Inventor 宣磊王芝权华建峰裴笑笑殷云龙
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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