Pear transcription factor PbrMYB169 and application thereof

A technology of transcription factors and genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of reducing fruit quality, achieve the effect of reducing agricultural costs and achieving environmental friendliness

Active Publication Date: 2019-04-12
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Specific accumulation of stone cells in pear pulp reduces fruit quality

Method used

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  • Pear transcription factor PbrMYB169 and application thereof
  • Pear transcription factor PbrMYB169 and application thereof
  • Pear transcription factor PbrMYB169 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Analysis of spatio-temporal expression pattern of PbrMYB169 gene

[0029] Different tissue samples of ‘Dangshan Suli’ were collected from Gaoyou Orchard, Jiangsu Province (2015). Pear varieties with high and low stone cell content were collected from the National Sand Pear Germplasm Resource Garden. Total RNA was extracted by CTAB method (Porebski et al., 1997), and the quality of the extracted samples was detected by spectrophotometer and agarose gel. Take 3 μg of extracted total RNA, and use one-step gDNA removal and cDNA synthesis kit (Transgen, China) for reverse transcription, and the method refers to the instruction manual. The primers used in the fluorescent quantitative PCR were gene-specific primer pairs: SEQ ID No.5 and SEQ ID No.6; GAPDH was used as an internal reference gene, and the fluorescent quantitative kit was purchased from Roche Company. The instrument used for Real-timePCR was Roche 480 quantitative PCR instrument, and the reaction syste...

Embodiment 2

[0031] Example 2 Isolation and Cloning of PbrMYB169 Gene

[0032]Take 3 μg of early pulp RNA of ‘Dangshan Suli’, and use one-step gDNA removal and cDNA synthesis kit (Transgen, China) for reverse transcription, and the method refers to the instruction manual. The amplification gene primer pair is SEQ ID No.3 and SEQ ID No.4. The 50 μL reaction system includes 200ng cDNA, 1× buffer (TransStart FastPfu Buffer), 10mM dNTP, 1U Taq polymerase (TransStart FastPfu DNA Polymerase) ( The aforementioned buffer solution and Taq polymerase were purchased from TRANS Company), 500 nM of the aforementioned primers. The PCR reaction was completed on the eppendorf amplification instrument according to the following program: 95°C, pre-denaturation for 2 minutes, denaturation at 95°C for 20 seconds, annealing at 60°C for 20 seconds, extension at 72°C for 1 minute, 35 thermal cycles, extension at 72°C for 10 minutes, Store at 4°C. Produces a single PCR band product.

[0033] After the PCR prod...

Embodiment 3

[0034] Embodiment 3 Construction of Plant Transformation Overexpression Vector

[0035] According to the multiple cloning site of the pCAMBIA-1301 vector and the analysis of the enzyme cutting sites on the coding region sequence of the PbrMYB169 gene, Xba I and BamH I were selected as endonucleases. According to the general principles of primer design, primers SEQ ID NO.3 and SEQ ID NO.4 with restriction sites were designed with Snapgene software. The annealing temperature of PCR amplification is 55° C., and the PCR reaction system and amplification procedure are the same as in Example 2. Recover the target band and connect it to the pEASY vector.

[0036] The total volume of the double enzyme digestion system was 40 μL, which contained 12 μL of pEASY-PbrMYB169 plasmid, 4 μL of 10×Buffer (purchased from NEB Company), 0.8 μL of Xba I, 0.8 μL of BamH I and 22.2 μL of water. The total volume of the pCAMBIA1301 vector double enzyme digestion system was 40 μL, which contained 8 μ...

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Abstract

The invention discloses a pear transcription factor PbrMYB169 and an application thereof. The pear transcription factor PbrMYB169 is separated from Dangshan crisp pears, and has MYB family member PbrMYB169 genes capable of adjusting and controlling development of sclereid of pear fruits, the nucleotide sequence of the pear transcription factor PbrMYB169 is as shown in SEQID No.1, and the coding amino acid sequence of the pear transcription factor PbrMYB169 is as shown in a sequence table SEQID No.2. Through an agrobacterium tumefaciens mediated heredity conversion method, PbrMYB169 transcription factors are converted into arabidopsis thaliana, and transgenic plants are obtained; biological function verification indicates that the PbrMYB169 gene cloned through the pear transcription factorPbrMYB169 has the function of promoting synthesis of arabidopsis thaliana inflorescence stem lignin, besides, the PbrMYB169 transcription factor has the advantage of being capable of adjusting and controlling a plurality of genes, and an efficient way is provided for molecular breeding. New genetic resources are provided for reduction of molecular breeding of synthesis of organism lignin, new heredity resources are provided for implementing green agriculture, and reduction of the agriculture cost and realization of an environmental-friendly purpose can be facilitated through development and utilization of the heredity resources.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and relates to pear transcription factor PbrMYB169 and its application, in particular to the isolation and cloning of a MYB family member PbrMYB169 gene that regulates the development of pear fruit stone cells from 'Dangshansu pear'. Background technique [0002] Pear is a perennial woody plant belonging to the genus Pyrus L. of the Rosaceae subfamily Amygdaloideae. It is the third largest fruit tree species in my country and has a cultivation history of more than 3,000 years (Teng Yuanwen, 2017). In addition to Hainan Province, pears are cultivated in other regions, especially in Hebei, Anhui, Jiangsu and other provinces in my country, where the cultivation area is very wide. In 2016, my country's pear production area reached 1.12 million hectares, with an annual output of 19.5 million tons, accounting for 71.3% of the world's total. However, the competitiveness of Chinese pear fruit in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N1/21C12N15/11A01H5/00A01H6/20
CPCC12N15/8255C07K14/415
Inventor 吴俊薛程薛雍松张绍铃张明月李甲明齐开杰
Owner NANJING AGRICULTURAL UNIVERSITY
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