ThHAM1 gene related to development of roots of taxodium zhongshansha and application for ThHAM1 gene

A technology for root development and Chinese fir, applied to the ThHAM1 gene and its application field, can solve the problems of the decline of the rooting ability of the clonal line, the difference in rooting ability, and the insignificant effect, and achieve the effect of reducing the number of adventitious roots and slowing down the growth rate.

Active Publication Date: 2019-12-10
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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Problems solved by technology

However, during the long-term breeding process of Zhongshan fir clones, it was found that the rooting ability of different clones was significantly different, and with the growth of the physiological age of the clones, the rooting ability of the clones bred in the early stage declined, even if the cutting technology was continuously optimized. , the effect is not obvious, so it is necessary to conduct in-depth research on its rooting mechanism through molecular means to better solve this problem
[0004] In summary, the HAM gene plays an important role in plant root development, but there is no related report in Zhongshan fir. In-depth research on the regulation of HAM gene on the development of Zhongshan fir adventitious roots will be helpful for further improvement of Zhongshan fir and even bald cypress. Rooting traits are significant

Method used

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  • ThHAM1 gene related to development of roots of taxodium zhongshansha and application for ThHAM1 gene
  • ThHAM1 gene related to development of roots of taxodium zhongshansha and application for ThHAM1 gene

Examples

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Effect test

Embodiment 1

[0019] Example 1: Cloning of ThHAM1 gene by RACE technology

[0020] 1) Extraction of RNA and inversion of cDNA

[0021] Using Zhongshanshanshan 406 (Taxodium mucronatum♀×T.distichum♂) (T.hybrid ‘Zhongshanshan 406’) plants as materials, total RNA was extracted using RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (TIANGEN Company). 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 NanoDrop2000 (ThermoScientific). And use 2.0% agarose gel electrophoresis to separate the total RNA. Afterwards, the 3'RACE and 5'RACE kits of Invitrogen were used to obtain the nucleic acid sequence between the 3' end and the 5' end of the gene. ...

Embodiment 2

[0043] Embodiment 2: ThHAM1 gene plant expression vector construction

[0044] 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.

[0045] 1) The purpose of the BP reaction is to transfer the gene fragment to the entry vector, the reaction system: vectorThHAM1 ORF fragment 10-20ng, Salt solution 1μL, pCRTM8 / GW / TOPOTMvector (entry vector) 1μL, add Nuclease-free Water to a total volume of 6μL, Mix gently, react at 22°C for 60 minutes, and then transfer to ice; transform 6 μL of the reaction product from the previous step into TOP10 competent cells, spread on LB screening culture plates, and pick single clones for bacterial liquid PCR detection, the primers used are gene Specific upstream primers and T7 primers on the carrier, and the positive clones after detection were fu...

Embodiment 3

[0047] Example 3: Genetic transformation of ThHAM1 gene

[0048] The ThHAM1 gene was transformed into Agrobacterium by liquid nitrogen freeze-thaw method, and then the hybrid Populus montana was transformed by leaf disc method. Operation steps: add at least 100ng of recovered and purified expression vector to EHA105 competent cells, mix gently, ice bath for 30min, quick freeze in liquid nitrogen for 1min, heat shock at 37°C for 3min, quickly place on ice for 1-2min, add 800μL LB Culture medium, 28°C, 100rmp recovery for 3h, 4000rmp centrifugation for 3min, suck off 800μL LB medium, mix the remaining bacterial solution, smear on the LB plate containing antibiotics, 28°C inverted culture for 30-48h, positive clones detected by PCR, positive Propagate the cloned colonies, inoculate in 120mL LB liquid medium containing corresponding antibiotics, and culture at 28°C for about 24 hours until OD 600 The value is about 0.5, divide the bacterial solution into 50mL centrifuge tubes, ce...

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Abstract

The invention discloses a ThHAM1 gene related to development of roots of taxodium zhongshansha and application for the ThHAM1 gene and belongs to the technical field of plant gene engineering. A nucleotide sequence of the ThHAM1 gene is shown as SEQ ID NO. 1, and an amino acid sequence of an expression protein of the ThHAM1 gene is shown as SEQ ID NO. 3. Populus davidiana x P.bolleana is convertedby constructing a gene GbF3'H1 vector, compared with a control plant, the ThHAM1 gene performs efficient expression in the Populus davidiana x P.bolleana, following the situation that variation of aphenotype occurs, a growth speed of a plant is slowed with roots being most obvious, the number of adventitious roots is reduced, a length is shortened, and the expression quantity of a target gene isincreased by 200-500 times, so that illustrated that the ThHAM1 gene is an important gene for regulating and controlling development of the plant roots and influencing a plant morphology, and the ThHAM1 gene has an important application value in the field of forest tree gene engineering and can be used for regulating a plant growth and development speed or the plant morphology.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a ThHAM1 gene related to the root development of Zhongshan fir and its application Background technique [0002] Root is an important organ formed by plants for long-term adaptation to terrestrial habitats. It is a bridge for material exchange between soil and aboveground parts of plants, and is the basis for plant prosperity and resistance to adversity. Rooting traits play an important role in the genetic improvement of forest trees, and the study of the molecular mechanism of adventitious root development in forest trees is not only of theoretical significance, but also of potential application value. In recent years, researchers at home and abroad have isolated a number of functional genes from the adventitious roots of different plants. They are involved in a series of physiological and biochemical activities such as material transportation, signal transduct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/06
CPCC07K14/415C12N15/8261
Inventor 宣磊王芝权杨颖华建峰於朝广裴笑笑殷云龙
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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