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Key gene PeRR12 for regulating adventitious root formation and stem development of poplars as well as expression protein and application of key gene PeRR12

A key gene, poplar technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of few targeted, systematic and in-depth research reports, etc., to reduce the number of adventitious roots, increase plant height, and rooting rate rising effect

Pending Publication Date: 2018-12-28
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although in-depth studies have been conducted on ARRs genes in herbaceous model plants such as Arabidopsis, there are few targeted and systematic in-depth research reports in woody plants to explain their mechanism of action.

Method used

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  • Key gene PeRR12 for regulating adventitious root formation and stem development of poplars as well as expression protein and application of key gene PeRR12
  • Key gene PeRR12 for regulating adventitious root formation and stem development of poplars as well as expression protein and application of key gene PeRR12
  • Key gene PeRR12 for regulating adventitious root formation and stem development of poplars as well as expression protein and application of key gene PeRR12

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Cloning of the PeRR12 gene by RACE technology

[0028] Based on the results of the applicant's previous research on the expression profile chip of poplar adventitious roots, Oligo 6 was used to design the RACE primers at the 3' end and perform 3' RACE to obtain the 3' cDNA end fragment, which was cloned into a T-vector, and the insert fragment was screened by PCR. Sequencing, Blast confirmed that the above fragments were homologous to genes related to other plants. In the same way, design the RACE primers at the 5' end according to the obtained correct 3' cDNA end fragment sequence, perform 5' RACE, obtain the 5' cDNA end fragment, clone it into T-vector, and perform PCR screening on the insert fragment. sequencing. The specific primers and main process are as follows:

[0029] PeRR12 3'Outer primer: 5'-CCGATCCTTGAACAATCCTCTC-3';

[0030] PeRR12 3'Inner inner: 5'-CAACCCTCAGATGCTTCATGTGAACCCA-3';

[0031] PeRR12 5'Outer primer: 5'-CCCATCTTTTGACCTCACTAATG-3'...

Embodiment 2

[0052] Embodiment 2PeRR12 gene plant expression vector construction

[0053] The overexpression vector of PeRR12 gene was constructed by pathway cloning technology. Using specific PCR primers (PeRR12 ORF primers in Example 1), PCR amplification was performed using cDNA as a template, and the PeRR12 gene ORF was constructed into an entry vector. The entry vector is pCR TM 8 / GW / TOPO TM vector (Invitrogen). The reaction system is: Fresh PCR product (purified) 10-20ng; Salt solution 1μL; pCR TM 8 / GW / TOPO TM vector 1μL; add sterile ddH 2 O to make up 6 μL. The reaction procedure is: stand at room temperature for 30 min.

[0054] Pick positive clones from the screening culture plate for PCR detection and sequencing verification, the entry vector with PeRR12 gene and the plant expression vector pH35GS for LR reaction. Vector plasmid such as figure 1shown. The reaction system is: linearized dentry clone 100ng; purified destination vector (100ng / μL) 1.5μL; LR Clonase IIenzy...

Embodiment 3

[0055] The genetic transformation of embodiment 3PeRR12 gene

[0056] The constructed pH35GS-PeRR12 overexpression vector was transformed into Agrobacterium strain EHA105 (Invitrogen) by liquid nitrogen freeze-thaw method, and the PeRR12 gene was transformed into poplar through Agrobacterium-mediated. Experimental results such as Figure 2-6 As shown, among them, figure 2 For real-time quantitative molecular detection of overexpressed PeRR12 gene; image 3 Comparison of the overall morphology of the transgenic poplar with overexpression of PeRR12 gene and non-transgenic poplar (CK); Figure 4 is the rooting time and rooting rate of transgenic poplar and non-transgenic poplar (CK) overexpressing PeRR12 gene; Figure 5 It is the comparison of the number of adventitious roots and plant height of transgenic poplar overexpressing PeRR12 gene and non-transgenic poplar (CK); Figure 6 It is the phenotype analysis of PeRR12 transgenic lines under NAA treatment. From the results,...

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Abstract

The invention discloses a key gene PeRR12 for regulating adventitious root formation and stem development of poplars as well as an expression protein and an application of the key gene PeRR12. The nucleotide sequence of the key gene PeRR12 is shown as SEQ ID NO.1. The gene PeRR12 is transferred into the poplars, transgenic poplars overexpressing the gene PeRR12 show delayed rooting time, reduced rooting rate and reduced adventitious root quantity; rooting defects of the transgenic poplars can be partially overcome after addition of exogenous auxin NAA, rooting time is advanced, rooting rate isincreased obviously, and the adventitious root quantity is increased obviously; meanwhile, compared with the nontransgenic poplars, height of the transgenic poplars is increased remarkably, which proves that overexpression of the gene PeRR12 promotes growth of poplar stems.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a key gene PeRR12 for regulating the formation of adventitious roots and stem development of poplar, its expressed protein and its application. Background technique [0002] Poplar has the characteristics of fast growth, strong adaptability, and wide application. It is widely cultivated in mid-latitude regions as the main tree species for timber forests, shelter forests, and surrounding greening, and has important economic and ecological values. Although most poplars are relatively easy to take root from cuttings, there are still many good clones that are difficult to take root from cuttings. Therefore, research on the molecular mechanism of adventitious root development in poplar not only has important theoretical significance, but also has potential application value. However, the regulation of plant adventitious root development is very complex an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/00
CPCC07K14/415C12N15/8261
Inventor 胥猛刘思安刘鑫
Owner NANJING FORESTRY UNIV
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