Pear cellulose synthase gene PbrCSLD5 and application thereof

A cellulose synthase, gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions

Active Publication Date: 2021-10-22
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, although many CESA genes have been reported to be involved in cellulose synthesis in somatic cells, CESA / CSL genes responsible for cellulose synthesis in pollen tubes are still rarely reported.

Method used

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  • Pear cellulose synthase gene PbrCSLD5 and application thereof
  • Pear cellulose synthase gene PbrCSLD5 and application thereof
  • Pear cellulose synthase gene PbrCSLD5 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Pear PbrCSLD5 Gene Isolation Cloning and Overexpression Vector Construction

[0026] Take 3 μg of ‘Dangshan Suli’ pollen RNA, and use one-step gDNA removal and cDNA synthesis kit (Transgen, China) for reverse transcription, the method refers to the instruction manual. According to the analysis of the multiple cloning site of the pCAMBIA-1301 vector and the restriction site on the coding region sequence of the PbrCSLD5 gene, the pollen cDNA of 'Dangshan Suli' was used as a template, and SEQ ID No.3 and SEQ ID No.4 were used as primers , the full-length gene PbrCSLD5 was cloned. Xba I and BamH I were selected as endonucleases. According to the general principle of primer design, use Snapgene software to design primers SEQ ID NO.5 and SEQ ID NO.6 with restriction sites. The 50 μL reaction system includes: 200ng PbrCSLD5Q full-length DNA, 1× buffer (TransStartFastPfu Buffer ), 10 mM dNTP, 1U Taq polymerase (TransStart FastPfu DNA Polymerase) (the aforementioned...

Embodiment 2

[0028] Example 2: Pear pollen tube ODN experiment and detection of cellulose content and gene expression

[0029] (1) Pear pollen tube ODN experiment

[0030] The ODN sequence of PbrCSLD5 was designed using the RNAfold Web server (https: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi), and Snap Gene 2.4.3 (https: / / www. snapgene.com) to assess the match of candidate as-ODN sequences to target regions. The candidate as-ODN sequence and the corresponding sense-ODN sequence were synthesized with phosphorous oligonucleotides, wherein the primer sequence corresponding to as-ODN-PbrCSLD5 is SEQ ID No.9, and the primer sequence corresponding to s-ODN-PbrCSLD5 is SEQ ID No.10. ODN primers need to be thio-modified and purified by HPLC. The experiment was started after the mixture of ODN sequence and Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) was incubated in the medium at 25°C for 15 min. The mixture was added to the pollen tube culture solution (pre-c...

Embodiment 3

[0037] Example 3: Genetic transformation of Arabidopsis thaliana and molecular identification of transformed plants

[0038] Col-0 Arabidopsis was infected with Agrobacterium containing the PbrCSLD5 overexpression vector (Clough and Bent, 1998). The specific method is as follows:

[0039] 1. Streak and activate Agrobacterium with solid LB medium containing 50mg / L K+ and 100mg / L R+, and culture in an incubator at 28°C for 36 hours;

[0040] 2. Use a sterilized toothpick or gun tip to pick up the single clone on the line, put it into a 100mL Erlenmeyer flask, add 30mL of liquid LB medium containing 50mg / L K+ and 100mg / L R+, and place it on a shaker at 28°C Incubate at 200rpm for 12 hours;

[0041] 3. Use a 50mL centrifuge tube to collect bacteria by centrifugation at 5000rpm for 20 minutes;

[0042] 4. Resuspend the bacteria in an equal volume of transformation medium [1 / 2MS; 5% sucrose (w / v, g / 100mL); 10 μg / L 6-BA; adjust the pH to 5.7 with KOH; 0.025% surfactant ( v / v)] in...

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Abstract

The invention discloses a pear cellulose synthase gene PbrCSLD5 and an application thereof. The gene has a nucleotide sequence as shown in SEQ ID No.1. The PbrCSLD5 gene is over-expressed in arabidopsis thaliana through an agrobacterium-mediated genetic transformation method to obtain a transgenic plant, and biological function verification shows that the cloned PbrCSLD5 gene has the effect of regulating and controlling the synthesis of pear pollen tube cell wall cellulose.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to pear cellulose synthase gene PbrCSLD5 and its application, in particular to a gene PbrCSLD5 capable of regulating pear pollen tube cell wall cellulose synthase synthesis obtained by separating and cloning from 'Dangshansu pear'. Background technique [0002] Pear is a perennial woody plant of the genus Pyrus L. in the subfamily Amygdaloideae of the Rosaceae family. It is the third largest fruit tree species in my country, with a long history of cultivation and a wide range of planting areas (Teng Yuanwen, 2017). In flowering plants, pollen tubes are important organs responsible for the transfer of male gametes to female gametes during sexual reproduction (Hulskamp et al., 1995; Lord and Russell, 2002). Pollen tube cell wall is one of the important factors to maintain the shape of pollen tube cells (Edlund et al., 2004). The cell wall of pollen tube is composed of cellulose...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/11C12N15/84A01H5/02A01H6/74
CPCC12N9/1059C12N15/827
Inventor 吴巨友李贤汤超王鹏齐开杰朱晓璇蔡漪铃
Owner NANJING AGRICULTURAL UNIVERSITY
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