Pear PbrSTONE gene and application thereof

A gene and gene coding technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as increasing production costs and environmental pollution, and achieve the effects of reducing agricultural costs, achieving environmental friendliness, and improving stone cell content.

Active Publication Date: 2021-06-01
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for wood used in papermaking, lignin is an important factor affecting paper quality and papermaking process. A large amount of chemical substances such as strong acid and alkali are needed to remove lignin, which not only increases production costs, but also causes serious environmental damage. Pollution

Method used

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  • Pear PbrSTONE gene and application thereof
  • Pear PbrSTONE gene and application thereof
  • Pear PbrSTONE gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of PbrSTONE Gene Isolation Cloning and 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. The method refers to the instruction manual. According to the multiple cloning site of the pCAMBIA-1301 vector and the restriction site analysis on the coding region sequence of the PbrSTONE gene, Xba I and BamH I were selected as endonucleases. According to the general principle of primer design, 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 (TransStart FastPfu Buffer), 10mM dNTP, 1U Taq polymerase (TransStart FastPfu DNA Polymerase) (the aforementioned buffer and Taq polymerase were purchased from TRANS), and 500nM of the above primers. The PCR reaction was completed on the eppendorf amplification instrument according to the f...

Embodiment 2

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

[0029]Different tissue samples of ‘Dangshan Suli’ were collected from Gaoyou Orchard, Jiangsu Province (2015). Total RNA was extracted using the CTAB method (Porebski et al., 1997), and the quality of the extracted samples was detected by a 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, 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 SEQID 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 μL of P...

Embodiment 3

[0031] Example 3 Instant transformation of pear fruit and detection of lignin content and gene expression

[0032] (1) Instant transformation of pear fruit

[0033] Use the Agrobacterium containing the PbrSTONE overexpression vector to inject it into the 'Dangshan Suli' about 35 days after flowering through the Agrobacterium-mediated method. The method is as follows:

[0034] 1. Activate the Agrobacterium containing the correct plasmid on solid medium, and grow in an incubator at 28°C for 48 hours;

[0035] 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;

[0036] 3. Pour the bacterial liquid into a 50mL centrifuge tube and centrifuge at 6000rpm for 15 minutes to collect all the bacterial cells;

[0037] 4. Resuspend the pellet with an appropriate amount of induction medium (10mM MgCl2, 10mM...

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Abstract

The invention discloses a pear PbrSTONE gene and an application of the pear PbrSTONE gene. The PbrSTONE gene which is separated from Dangshan pears and has the function of adjusting the development of the stone cells of the pear fruits has a nucleotide sequence as shown in SEQ ID No.1, and an amino acid sequence coded by the PbrSTONE gene is as shown in SEQ ID No.2 in a sequence table. Through instantaneous overexpression in pear fruits and a gene silencing technology, it is proved that PbrSTONE can change the accumulation process of fruit stone cells and lignin; in the transgenic arabidopsis thaliana inflorescence stem of overexpression PbrSTONE, the lignin content and G-type monomer thereof are significantly increased. Meanwhile, the number of lignified inter-bundle fiber cell layers is increased, and the secondary cell wall of conduit cells is significantly thickened. Therefore, the PbrSTONE gene participates in adjusting and controlling the formation of the lignin of the stone cells of the pear fruits.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, relates to a pear PbrSTONE gene and its application, in particular to a PbrSTONE gene that regulates the development of pear fruit stone cells obtained by separating and cloning from 'Dangshan crispy pear'. Background technique [0002] Pear is a perennial woody plant of the genus Pyrus L. in the subfamily Amygdaloideae of the Rosaceae family. It is the third largest fruit tree species in my country, with a long history of cultivation and a wide range of planting areas (Teng Yuanwen, 2017). As a big exporter of pears, my country has ranked first in the world in terms of cultivation area and output of pears in recent years. However, due to the high content of stone cells in some traditional main varieties of our country, such as Dangshansu pear, the fruit quality is not good and the market competitiveness is weak. Stone cells are a unique trait in pear pulp and an important factor affecti...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/84A01H5/08A01H6/20A01H6/74
CPCC07K14/415C12N15/8255
Inventor 吴俊薛程薛雍松张明月李甲明张绍铃秦梦帆赵渴姣汪润泽
Owner NANJING AGRICULTURAL UNIVERSITY
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