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Chinese fir plant sulfopeptidin clpsk2 gene and its application

A technology of plant sulfopeptide and Chinese fir, which is applied in the field of genes, can solve the problems of poor stability and low degree of somatic embryo synchronization, and achieve the effect of promoting growth and promoting callus proliferation

Active Publication Date: 2019-05-07
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have shown that PSK-α can promote the formation of somatic embryos, and the somatic embryogenesis system of Chinese fir has been established, but the degree of synchronization of somatic embryogenesis is not high and the stability is poor

Method used

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  • Chinese fir plant sulfopeptidin clpsk2 gene and its application
  • Chinese fir plant sulfopeptidin clpsk2 gene and its application
  • Chinese fir plant sulfopeptidin clpsk2 gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] fir plant sulfopeptine gene CLPSK2 cloning, the specific steps are as follows:

[0027] 1) Using the CTAB method to extract RNA from Chinese fir callus to obtain RNA with high purity, OD 260 / OD 280 The value is 1.8-2.0, OD 260 / OD 230 The value is 2.0.

[0028] 2) Use POCHE company's inversion kit, the template is the above-mentioned extracted RNA, and synthesize the first strand of cDNA according to the instructions, and store the obtained cDNA at -20°C.

[0029] 3) PCR amplification of the target fragment: using cDNA as a template, use TOYOBO's high-fidelity enzyme KOD-Fx for PCR amplification, 25 μL PCR amplification system: 12.5 μL 2×PCR Buffer, 5 μL dNTP (2mM), 0.75 μL ForwardPrimer, 0.75 μL Reverse Primer, 1.0 μL cDNA, 0.5 μL KOD-Fx polymerase, 4.5 μL ddH 2 O. The specific primer sequences are shown in the table below:

[0030] ClPSK2-F: 5'-ATGAAAAAAATTTCATTTCAACAGGGAAG-3';

[0031] ClPSK2-R: 5'-TGGGTTCTTGTGGTGTTGTGTATAAATG-3'.

[0032] PCR reaction con...

Embodiment 2

[0040] phytosulfopeptine gene CLPSK2 Construction and transformation of the overexpression vector, the steps are as follows:

[0041]1) Add restriction sites: Since there are no restriction sites on both sides of the target gene cloned in Example 1, it is necessary to add restriction sites to connect with the PBI121 vector. Using the cloning vector PMD19-T positive recombinant plasmid with the target fragment as a template, by rebuilding primers, add Xba I and Bam HI two enzyme cutting sites.

[0042] 2) Plasmid extraction: SDS alkaline lysis method was used to extract plasmids.

[0043] 3) 20μL PCR reaction system: 1.0μL 10×PCR Buffer, 0.4μL 10mMdNTP, 1.2μL 25mMMgCl 2 , 1.0 μL Forward Primer, 1.0 μL Reverse Primer, 1.0 μL plasmid, 0.1 μL rTaqpolymerase, 13.3 μL ddH 2 O. Primers used:

[0044] CLPSK2Xba I-F: 5'-TCTAGAATGAAAAAAATTTCATTTCAACAGGG-3';

[0045] CLPSK2Bam HI-R: 5'-GGATCCTGGGTTCTTGTGGTGTTGT-3';

[0046] Reaction conditions: 94°C for 5min; 95°C for 30s, 5...

Embodiment 3

[0057] CLPSK2 The application of genes, the main process is as follows:

[0058] 1. Tissue culture of transgenic plants

[0059] 1) Medium formula: basic medium: MS+20g / l sucrose, agar 6.8g / l, pH 5.8. Callus induction medium: MS+2.0g / l 2,4D+0.2g / l 6-BA+0.5g / l hydrolyzed casein+30g / l sucrose, agar 6.8g / l, pH5.8. Regenerated somatic embryo induction medium: MS+30g / l sucrose, pH5.8.

[0060] 2) Callus induction: Sterilize the seeds of wild-type Col seeds and transgenic Arabidopsis T2 strains P1-1, P1-5, P1-21, P2-2, P2-4, and P2-12, and point On the basic medium and the A medium with kanamycin added, culture at 23°C under light. When Arabidopsis thaliana grew four true leaves, the explants were harvested and callus induction culture was carried out.

[0061] The wild-type Col and the transgenic positive plants (contained the target fragment after PCR verification) were inoculated into the callus induction medium after scratching the leaves and rhizomes with a scalpel. The wi...

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Abstract

The invention discloses Chinese fir phytosulfopeptine CLPSK2 Gene and its application, Chinese fir phytosulfopeptide CLPSK2 The DNA sequence of the gene is shown in SEQ ID NO.1. Chinese fir phytosulfopeptine of the present invention CLPSK2 The gene, confirmed by experiments, can not only promote the growth of roots, but also promote the proliferation of callus in function, and will have wide application in plant breeding and growth.

Description

technical field [0001] The invention belongs to the field of gene technology, in particular to Chinese fir phytosulfopeptin (PSK) CLPSK2 Genes and their applications. Background technique [0002] Chinese fir is one of the unique evergreen coniferous tree species in southern my country. It grows fast, has a straight and round stem shape, is corrosion-resistant, and has excellent wood quality. It has important ecological and economic values. Chinese fir has a history of more than 3,000 years of cultivation, with a cultivation area of ​​853.86Mha, accounting for 25-30% of my country's forest reserve area, and commercial timber supply accounting for about 25% of my country's plantation forest. Since the 1950s, my country has begun to carry out genetic improvement of Chinese fir in a planned way, and through a series of breeding methods, a large number of excellent provenances, families and excellent clones of Chinese fir have been bred. At present, the development of breeding...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/00
CPCC07K14/415C12N15/8205C12N15/8261
Inventor 陈金慧吴华施季森成铁龙陆叶王丹丹鲁路
Owner NANJING FORESTRY UNIV
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