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Application of dendrobium officinale embryonic development later-period rich protein DoLEA43 to promoting formation of plant callus

A technology of embryonic development and Dendrobium candidum, applied in the field of plant genetic engineering, to achieve the effect of increasing the induction rate

Active Publication Date: 2019-11-15
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, other functions of LEA protein, such as promoting callus formation, have not been reported.

Method used

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  • Application of dendrobium officinale embryonic development later-period rich protein DoLEA43 to promoting formation of plant callus
  • Application of dendrobium officinale embryonic development later-period rich protein DoLEA43 to promoting formation of plant callus
  • Application of dendrobium officinale embryonic development later-period rich protein DoLEA43 to promoting formation of plant callus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Full-length cloning of DoLEA43 gene

[0018] The specific experimental methods are not indicated in the following examples, and can be carried out according to conventional methods. The conditions are as described in J. Sambrook et al. "Molecular Cloning Experiment Guide", F. Osper et al.

[0019] Dendrobium officinale Kimura et Migo used in the examples was collected from Guanzhai Mountain, Fujian Province, and the protocorms obtained after aseptic sowing of its seeds were further multiplied to obtain protocorm-like protocorms, which were preserved in the Chinese Academy of Sciences as germplasm resources In the tissue culture room of South China Botanical Garden, cultured on 1 / 2MS + 0.1% activated carbon + 2% sucrose + 0.2% agar powder pH 5.4 medium, at 26±1℃, 40μmol m -2 the s -1 , 12-h light / 12-h dark, 60% relative humidity culture room. Reverse transcriptase M-MLV was purchased from Promega Company, its article number is: M1701; DNase I (RNaseFree) wa...

Embodiment 2

[0021] Example 2: Acquisition and Identification of DoLEA43 Overexpression Transgenic Arabidopsis

[0022] 1. Construction of the overexpression vector of the abundant protein gene DoLEA43 in late embryonic development of Dendrobium officinale

[0023]The pCAMBIA1302 vector is a 35S promoter (35Spromoter) containing a cauliflower mosaic virus (CaMV), and the late embryonic development of Dendrobium officinale abundant protein gene DoLEA43 (as shown in the 1st to 432nd base of SEQ ID NO.1) Behind the promoter, an overexpression vector of the abundant protein gene DoLEA43 in late embryonic development of Dendrobium candidum was constructed, and the recombinant expression vector was named pCAMBIA1302-DoLEA43. The specific construction steps are as follows:

[0024] According to the sequence on the pCAMBIA1302 vector, design two linker primers, the upstream primer linker: 5'-CGAACGATAG CCATGG -3', downstream primer adapter: 5'-GTCAGATCTA CCATGG T-3'. The underline indicates ...

Embodiment 3

[0044] Example 3: Effect of Overexpression of DoLEA43 on Callus Formation of Transgenic Plants

[0045]Seeds of DoLEA43 overexpressed transgenic Arabidopsis strains and wild-type Arabidopsis seeds were sterilized with 1% NaClO for 10 minutes, rinsed with sterile water for 6 times, and sowed on 1 / 2MS+15g / L sucrose+8g / L agar Powdered solid medium (pH 5.7). Synchronized in the dark at 4°C for 2 days, moved to the culture room, and cultured at 22±2°C, 16-h light / 8-h dark conditions. Take 6-day-old seedlings for callus induction experiment, cut with a sharp scalpel at the junction of cotyledons and hypocotyls at about 0.7mm, carefully pick up the hypocotyl containing roots with a toothpick and move to 1 / 2MS+15g / In the solid medium (pH value 5.7) of L sucrose+8g / L agar powder, cultivate in the dark for 12 days, and count the callus induction rate of wild-type Arabidopsis and DoLEA43 overexpression transgenic Arabidopsis lines. Such as Figure 5 , Callus induction of DoLEA43 over...

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Abstract

The invention discloses application of dendrobium officinale embryonic development later-period rich protein DoLEA43 to promoting the formation of plant callus. An encoding gene DoLEA43 of the dendrobium officinale embryonic development later-period rich protein DoLEA43 is obtained from dendrobium officinale protocorm through cloning, the dendrobium officinale embryonic development later-period rich protein DoLEA43 as the position regulation factor participates in the formation of the plant callus, the over-expression of the dendrobium officinale embryonic development later-period rich proteinDoLEA43 is proved through transgenic and functional identification, and the callus inductivity of a transgenic plant is increased. Thus, the dendrobium officinale embryonic development later-period rich protein DoLEA43 or the encoding gene thereof have important theoretical significance and application value in the plant tissue regeneration process and especially play an important role in preserving plant germplasm and generating active substances through the callus.

Description

Technical field: [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the application of DoLEA43, an abundant protein in late embryonic development of Dendrobium officinale, in promoting the formation of plant callus. Background technique: [0002] During the maturation process of plant seeds, the content of storage substances in the body increases, the water content gradually decreases, and some soluble sugars and proteins accumulate in the cotyledon or endosperm of the seeds, which are used as energy storage or to enhance the dehydration tolerance of the seeds (Angelovici et al. 2010; Leprince et al. 2017). Dehydration tolerance is the tolerance of seeds to low water content or dehydration. The seeds are dehydrated during the maturation process. During this process, a large amount of hydrophilic substances are synthesized to maintain the cell structure, prevent plasma membrane damage and important protein denaturati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8261
Inventor 段俊何春梅俞振明
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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