Peach fruit ethylene response factor PpRAP2.12 gene as well as cloning method and application thereof

A pprap2.12, response factor technology, applied in application, genetic engineering, plant genetic improvement, etc., to achieve the effect of reducing sucrose content, increasing decomposition, and increasing VIN activity

Active Publication Date: 2022-03-01
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no successful application of transient overexpression of PpRAP2.12 in peach fruit. Therefore, it is particularly important to transiently overexpress the target gene PpRAP2.12 in peach fruit by Agrobacterium transformation.

Method used

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  • Peach fruit ethylene response factor PpRAP2.12 gene as well as cloning method and application thereof
  • Peach fruit ethylene response factor PpRAP2.12 gene as well as cloning method and application thereof
  • Peach fruit ethylene response factor PpRAP2.12 gene as well as cloning method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0023] Cloning and sequence analysis of the ethylene response factor PpRAP2.12 gene in peach fruit

[0024] 1. Total RNA was extracted from "Yulu" peach fruit, and reversed into cDNA, which was used as a PCR template; the details were as follows: using Promega's EastepTM total RNA extraction kit (Promega, United States) to extract total RNA from peach fruit, and then using HiScript® II Q Select RT Super Mix for qPCR (Vazyme, Nanjing, China) kit was reverse transcribed into cDNA as a template for PCR reaction;

[0025] 2. Use the online website NCBI-PRIMER (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design the specific amplification of the CDS region of peach fruit PpRAP2.12 gene (Gene ID: LOC18783672) Primer, upstream primer sequence: 5´-ATGTGTGGAGGTGCTATAATATCCG-3´, downstream primer sequence: 5´-TCAGAAACCTCCCCCAATCAG-3´;

[0026] 3. PCR amplification: The PpRAP2.12 gene amplification product was obtained by PCR amplification. The PCR amplification reaction system ...

specific Embodiment 2

[0032] Confirmation that PpRAP2.12 can bind to the promoter of PpVIN2 gene by yeast one-hybrid system (Y1H)

[0033] 1. Construction and identification of the bait recombinant vector pAbAi-PpVIN2pro-A containing the PpVIN2 promoter truncation (PpVIN2pro-A) and the prey recombinant vector pGADT7-PpRAP2.12

[0034] The PpVIN2 promoter contains abundant transcription factor binding sites. Since the cloned PpVIN2 promoter sequence is relatively long, and the front of the promoter sequence contains a potential binding site GCC-box (ACCGAC) for PpRAP2.12, it is planned to activate PpVIN2 The promoter was truncated, and the front part of the promoter was taken and named PpVIN2pro-A to verify whether it could bind to the PpRAP2.12 transcription factor.

[0035] Use the online website NCBI-PRIMER (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design peach fruit PpRAP2.12 gene and PpVIN2 gene promoter truncations (Gene IDs are LOC18783672, LOC18776102) specific amplification pri...

specific Embodiment 3

[0046] Validation of the transcriptional activity of PpVIN2 promoted by PpRAP2.12 using dual luciferase assay

[0047] 1. The full-length PpVIN2 promoter was constructed on pGreen Ⅱ 0800-LUC as a reporter vector, and the CDS sequence of PpRAP2.12 was constructed on pGreen Ⅱ 62-sk vector as a control and effector vector.

[0048] Use the online website NCBI-PRIMER (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design peach fruit PpRAP2.12 gene and PpVIN2 gene promoters (Gene IDs are LOC18783672 and LOC18776102, respectively). Specific amplification primers, with appropriate restriction sites and protective bases or homologous sequences of the vector at both ends of the primers (Table 2).

[0049]Table 2 Primer sequences for constructing recombinant vectors pGreen Ⅱ 62-sk-PpRAP2.12 and pGreen Ⅱ 0800-PpVIN2pro

[0050]

[0051] Note: The underline indicates the protected base; the bold indicates the restriction site, XhoI, SmaI, BamHI, SmaI

[0052] The reaction syste...

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Abstract

The invention discloses a peach fruit ethylene response factor PpRAP2.12 gene and a cloning method and application thereof, and the peach fruit ethylene response factor PpRAP2.12 gene is characterized in that the nucleotide sequence of the peach fruit ethylene response factor PpRAP2.12 gene is shown as SEQ ID NO: 1; the amino acid sequence of the protein encoded by the peach fruit ethylene response factor PpRAP2.12 is as shown in SEQIDNO: 2, and the cloning method comprises the following steps: (1) extracting total RNA (Ribonucleic Acid) of peach fruits and reversely transcribing into cDNA (Complementary Deoxyribonucleic Acid) as a template; (2) designing a primer according to the gene sequence of the PpRAP2.12; (3) PCR amplification: a PpRAP2.12 gene amplification product is obtained through PCR amplification, and the method has the advantages that the PpRAP2.12 and a PpVIN2 promoter have DNA-protein interaction, and the PpRAP2.12 positively regulates the transcriptional activity of the PpVIN2, so that the expression quantity of the PpRAP2.12 in peach fruits is effectively increased, the gene expression and activity of the acid invertase PpVIN2 can be remarkably increased, and sucrose decomposition is increased.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and in particular relates to a peach fruit ethylene response factor PpRAP2.12 gene regulating peach fruit acid invertase PpVIN2, a cloning method and application thereof. Background technique [0002] Peach( Prunus persica ) is a plant of the family Rosaceae and the genus Peach, which is prone to chilling damage during cold storage, showing symptoms such as internal browning, juice reduction, and flocculent corruption, and then loses its commercial value. Peach fruit is often accompanied by changes in soluble sugar content and metabolism-related enzyme activities under low temperature stress. Sucrose is the soluble sugar with the highest content in peach fruit, and its content not only affects the taste and flavor of peaches, but also affects the cold resistance of peaches. The content of sucrose in peach fruit decreased rapidly under low temperature storage, accompanied by the occurrence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/10C12N15/82C07K14/415A01H5/00A01H6/74
CPCC07K14/415C12N15/8218C12N15/8273C12N15/8245
Inventor 邵兴锋曹可枫韦莹莹陈义姜舒
Owner NINGBO UNIV
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