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Pear PbrNSC gene and application thereof

A gene, pear fruit technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of complex anabolic pathways, unable to meet the needs of high-quality pear fruit for industrial development, and achieve the effect of achieving environmental friendliness and reducing agricultural costs.

Active Publication Date: 2022-05-06
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The anabolic pathway of fruit stone cells is very complex, not only involving the anabolism and mutual transformation of various components, but also involving many genes, and each gene contains multiple copies (Zhong et al., 2018). It can be seen that fruit stone cells Cell formation is the final result of multi-gene coordinated regulation, which undoubtedly has a huge impact on our efforts to improve stone cell traits through traditional hybrid breeding. In the short term, it cannot meet the industrial development and market demand for high-quality pome fruit.

Method used

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  • Pear PbrNSC gene and application thereof
  • Pear PbrNSC gene and application thereof
  • Pear PbrNSC gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 PbrNSC Gene Isolation and Cloning and Construction of Overexpression Vector

[0026] 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. 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 PbrNSC gene, Xba I and BamH I were selected as endonucleases. According to the principle of general primer design, the primers SEQ ID NO.3 and SEQ ID NO.4 with restriction sites were designed with Snapgene software. The 50 μL reaction system included 200ng cDNA, 1× buffer ( GXL Buffer), 0.2μM dNTP, 1.25U GXL polymerase ( GXL DNA Polymerase) (the aforementioned buffer and GXL DNA polymerase were purchased from TaKaRa Company), 0.2 μM of the above primers. The PCR reaction was completed on the eppendorf amplification instrument according to t...

Embodiment 2

[0028] Example 2 Analysis of spatiotemporal expression pattern of PbrNSC gene

[0029] Different tissue samples of ‘Dangshan Suli’ were collected from Gaoyou Orchard, Jiangsu Province (2015). 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. 3 μg of extracted total RNA was used for reverse transcription using the one-step gDNA removal and cDNA synthesis kit (Transgen, China), and the method was referred 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 system was: 10 μL of 2×SYBR GreenI Master Mix, 0.4 μL of upstream and downstream primers (10 μM), 2 μL of cDNA, and 7.2 μ...

Embodiment 3

[0031] Embodiment 3 pear fruit transient transformation

[0032] Use Agrobacterium containing PbrNSC overexpression or gene silencing vector to inject into 'Dangshan Suli' about 35 days after flowering through Agrobacterium-mediated method, the method is as follows:

[0033] 1. Activate the Agrobacterium containing the correct plasmid on solid medium, and grow in an incubator at 28°C for 48 hours;

[0034] 2. Add 30mL containing R to a 100mL Erlenmeyer flask + with K + The liquid LB culture medium, pick the activated Agrobacterium into the activated Agrobacterium with a pipette tip, and grow in a shaker at 28°C and 200rpm for 12 hours;

[0035] 3. Pour the bacterial liquid into a 50mL centrifuge tube and centrifuge at 6000rpm for 15 minutes to collect all the bacterial cells;

[0036] 4. Use an appropriate amount of induction medium (10mM MgCl 2 , 10mM MES, 200mM acetosyringone, pH 5.6) to resuspend the precipitate, adjust the OD value to 0.8-1.2, and induce it with a deco...

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Abstract

The invention discloses a pear PbrNSC gene and application thereof. The invention discloses a PbrNSC gene which is separated from Dangshan pears and has the function of regulating and controlling the development of secondary cell walls of stone cells of pear fruits. The nucleotide sequence of the PbrNSC gene is as shown in SEQ ID No.1. Through instantaneous overexpression in pear fruits and a gene silencing technology, it is proved that PbrNSC can change the accumulation process of cellulose and lignin in secondary cell walls of fruit stone cells; in the PbrNSC-overexpressed transgenic arabidopsis thaliana inflorescence stem, the monomer contents of cellulose, lignin and G-type lignin are obviously increased, and the secondary cell walls of conduits and interbundle cells are obviously thickened. Therefore, the PbrNSC gene participates in regulating the formation of the secondary cell wall of the stone cell of the pear fruit.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, relates to pear PbrNSC gene and application thereof, and specifically relates to the separation and cloning of a PbrNSC gene for regulating the development of secondary cell walls of pear fruit stone cells from 'Dangshansu pear'. Background technique [0002] Pyrus L. belongs to Rosaceae, Amygdaloideae, Maleae, and Malinae (Teng Yuanwen, 2017), and is an important fruit tree crop in China. There are more than 3000 years of cultivation history (Lombard, 1987). There are at least 22 species of Pyrus recognized worldwide, of which white pear (Pyrus bretschneideri), sand pear (Pyrus pyrifolia), autumn pear (Pyrus ussuriensis), Xinjiang pear (Pyrus sinkiangensis) and Western pear (Pyrus communis) are the main ones. cultivar (Vavilov, 1951). White pears, sand pears, Qiuzi pears, and Xinjiang pears are also collectively referred to as "Oriental pears" or "Asian pears" because their main produc...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/08A01H6/20
CPCC07K14/415C12N15/8255C12N15/8205
Inventor 吴俊薛程薛雍松汪润泽张明月李甲明李晓龙赵渴姣秦梦帆
Owner NANJING AGRICULTURAL UNIVERSITY
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