Phyllostachys edulis PeAPX5 gene and application

A kind of gene, the technology of moso bamboo, applied in Moso bamboo PeAPX5 gene and application field, can solve the problems such as restricting the production of bamboo forest

Active Publication Date: 2021-05-11
INT CENT FOR BAMBOO & RATTAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high temperature and drought worldwide are the main

Method used

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  • Phyllostachys edulis PeAPX5 gene and application
  • Phyllostachys edulis PeAPX5 gene and application
  • Phyllostachys edulis PeAPX5 gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Cloning of Example 1 Phyllostachys pubescens PeAPX5 gene

[0048] Using bamboo leaves as materials, total RNA was extracted according to the instructions of the Trizol RNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), and 1 ng of RNA was reverse-transcribed into cDNA according to the reverse transcription kit (Promega, USA), and digested with RNase cDNA product. According to the bamboo genome database http: / / www.forestrylab.org / db / PhePacBio / ExtractSeq / phe / index.php, Prime Primer 5.0 software was used to design primers to amplify the PeAPX5 gene.

[0049] Upstream primer: 5′-ATGGCGAAGAACTACCCGGC-3′

[0050] Downstream primer: 5′-TATGCATCAGCAAACCCCAGTTC-3′

[0051] Polymerase Chain Reaction:

[0052] 20μL reaction system: 10×PCR Buffer 2.0μL, 2.5mM dNTP Mix 2.0μL, 10μM upstream and downstream primers 1.0μL each, cDNA template 2.0μL, 5U / μL LA Taq DNA polymerase 0.2μL, ddH 2 O 11.8 μL.

[0053] PCR reaction program: pre-denaturation at 94°C for 5min; denat...

Embodiment 2

[0055] The construction of embodiment 2 plant expression vector pCAMBIA2300-PeAPX5

[0056] Primers were designed for polymerase chain reaction, and EcoRI and HindIII double restriction sites were introduced into the upstream and downstream of the target gene PeAPX5 respectively, and the product was connected to the pGEM-T Easy vector (Promega Company), transformed into DH5α competent cells, and sequenced. Extract the plasmid, connect the PeAPX5 gene fragment digested with EcoRI and HindIII to the pCAMBIA2300-CaMV35S vector digested with the same enzyme, transform, extract the plasmid, and perform sequence determination.

[0057] Upstream primer PeAPX5-F: 5′-AG GAATTC ATGGCGAAGAACTACCCGGCCG-3′

[0058] Downstream primer PeAPX5-R: 5′-CCG AAGCTT TTATGCATCAGCAAAC-3′

[0059] ⑴Using the cDNA of moso bamboo leaves as a template for PCR reaction

[0060] 20μL reaction system: 10×PCR Buffer 2.0μL, 2.5mM dNTP Mix 2μL, 10μM upstream and downstream primers 1.0μL each, cDNA templat...

Embodiment 3

[0070] Example 3 Transformation of Arabidopsis thaliana with the plant expression vector pCAMBIA2300-PeAPX5

[0071] (1) Transformation of Agrobacterium strain GV3101 by freeze-thaw method

[0072] Add 1 ng of the recombinant expression vector plasmid to 100 μL of competent cells GV3101, ice-bath for 10 minutes, freeze the competent cells in liquid nitrogen for 5 minutes, quickly transfer them to a 37°C constant temperature water bath for 5 minutes, and place them on ice for 5 minutes. Add 600 μL of LB liquid medium to the centrifuge tube, shake and culture in a shaker at 28°C for 2-3 hours, and recover the bacteria. Draw 60 μL of the bacterial liquid and spread it on the YEP solid medium containing Kan resistance (50 mg / mL) and Rif (50 mg / mL) resistance, and invert the plate in a constant temperature shaker at 28 ° C for about 2-3 days until the growth White colonies. After picking a single clone colony for PCR detection, select positive clones for shake culture.

[0073] ...

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Abstract

The invention discloses a phyllostachys edulis PeAPX5 gene and application. Sequences of the phyllostachys edulis PeAPX5 gene and encoded protein thereof are as shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively. The PeAPX5 gene is cloned from phyllostachys edulis for the first time, and the biological function of the PeAPX5 gene is verified by over-expressing the PeAPX5 gene in arabidopsis thaliana. The PeAPX5 gene has the function of regulating and controlling plant stress resistance, a powerful tool can be provided for phyllostachys edulis transgenic research, and valuable candidate genes can be provided for phyllostachys edulis resistance molecular breeding.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the PeAPX5 gene of moso bamboo and its application. Background technique [0002] Moso bamboo (Phyllostachys edulis) is a scattered bamboo species of Gramminales (Gramminales) Bambooideae (Bambusoideae) Phyllostachys (Phyllostachys), it has great potential in water conservation, soil and sand fixation, and biomass energy development. It is an important ecological , economic and cultural resources. However, high temperature and drought worldwide are the main adversity factors restricting the production of bamboo forests. Bamboo plants need sufficient water during the growth process, especially the formation and growth of bamboo shoots, which are very closely related to water supply. Drought has seriously affected the yield and quality of bamboo. Therefore, it is very necessary to improve bamboo species and cultivate new bamboo varieties with strong drought res...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12N15/53C12N15/82C12N15/84A01H5/00A01H6/20
CPCC12N9/0065C12N15/8273C12Y111/01011
Inventor 李雪平宋笑龙李夷骞
Owner INT CENT FOR BAMBOO & RATTAN
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