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Pear lignin synthesis gene PbMC1a/1b and application of pear lignin synthesis gene PbMC1a/1b in genetic improvement of fruit quality

A gene and transgenic cell line technology, applied in the field of plant genetic engineering, can solve problems such as no related reports

Active Publication Date: 2019-12-17
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, previous studies mainly focused on the participation of model or herbaceous MCs in the process of stress-induced cellular PCD, but whether the PCD process initiated and executed by MCs is involved in the synthesis of stone cells and lignin in pear fruit has not been reported.

Method used

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  • Pear lignin synthesis gene PbMC1a/1b and application of pear lignin synthesis gene PbMC1a/1b in genetic improvement of fruit quality
  • Pear lignin synthesis gene PbMC1a/1b and application of pear lignin synthesis gene PbMC1a/1b in genetic improvement of fruit quality
  • Pear lignin synthesis gene PbMC1a/1b and application of pear lignin synthesis gene PbMC1a/1b in genetic improvement of fruit quality

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1, the cloning of pear PbMC1a / 1b gene full-length cDNA

[0042] The pear gene database was searched with Arabidopsis AtMC1 as the keyword, and 11 nucleotide sequences with the most similar sequences were obtained, and the nucleotide sequence with the highest score was selected and named as PbMC1a / 1b. According to the PbMC1a / 1b gene sequence, a specific primer pair for amplifying the sequence was designed using Primer Premier 5.0.

[0043] Specific steps are as follows:

[0044] Using the cDNA of Dangshansu pear as a template, high-fidelity enzymes were used to amplify. The amplification system is shown in Table 1, and the amplification procedure is shown in Table 2. The sequences of the amplification primers are:

[0045] PbMC1a / 1b-F: 5'-gagaacacgggggactctagaATGGCATTATATTGGCTTGTACAAG-3'

[0046] PbMC1a / 1b-R: 5'-gcccttgctcaccatggatccTAGGGAGAAGGGTTTTGCATACA-3'

[0047] use DNA gel recovery kit (Axygene, USA) was used to purify and recover the amplified produc...

Embodiment 2

[0069] Example 2, analysis of changes in stone cells and lignin content in pulp during fruit development

[0070] The content of stone cells in pulp was determined by freeze separation method (Syros et al., 2004). Take three fruits of the same size (more young fruits are taken), remove the pericarp, take the edible part of the fruit according to the quartering method, weigh 100g, place it in a -20°C refrigerator for 24 hours, take it out to thaw at room temperature, add 200ml of distilled water, and mash the tissue Machine (1000-1500r·min -1 ) mash for 5min. Then transfer the homogenate to a 1000ml beaker, stir with a glass rod for 1min, and let it stand for 5min, so that the stone cells are fully precipitated at the bottom of the beaker, pour out the upper suspension, and suspend the precipitate in 0.5M hydrochloric acid solution for 30min, during which time Stir once every 5 minutes, remove the floating matter, rinse 5-6 times with distilled water, collect the suspension f...

Embodiment 3

[0073] Example 3, Analysis of the expression pattern of PbMC1a / 1b during fruit development

[0074] The expression pattern of PbMC1a / 1b gene was analyzed by real-time fluorescent quantitative PCR (qRT-PCR), and the quantitative reagent was QuantiNova TM SYBRGreen PC (QIGEN, Germany), the method refers to the instruction manual, and the reaction system is shown in Table 7.

[0075] Table 7 Quantitative PCR reaction system

[0076]

[0077] Tublin from pear was used as an internal reference gene, and the cDNA obtained by reverse transcription was used as a template. Each sample was repeated three times, and the reaction program is shown in Table 8. After the reaction, use 2 -ΔΔCt Algorithms perform calculations on gene expression. The internal reference gene qRT-PCR primers are:

[0078] Tublin-F: 5'-TGGGCTTTGCTCCTCTTAC-3'

[0079] Tublin-R: 5'-CCTTCGTGCTCATCTTACC-3'

[0080] Table 8 Quantitative PCR reaction program

[0081]

[0082]

[0083] In this experimen...

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Abstract

The invention discloses a pear lignin synthesis gene PbMC1a / 1b and application of the pear lignin synthesis gene PbMC1a / 1b in genetic improvement of fruit quality. The PbMC1a / 1b gene is a cysteine-like protease isolated and cloned from Dangshansu pear with high lignin content, an applicant names the gene as PbMC1a / 1b, the sequence is shown as SEQ ID NO.1, and the corresponding amino acid sequenceis shown as SEQ ID NO.2. A gene construction over-expression vector is introduced into arabidopsis thaliana, obtained transgenic plants are verified by a biological function, the cloned PbMC1a / 1b genecan promote the cell walls of fibers between ducts, lignocellulosic fibers and vascular bundles to be obviously thickened, lignin accumulation in stems is promoted and plant growth is inhibited, theexpression of genes related to lignin biosynthesis is increased, new genetic resources is provided for molecular design and breeding of fruit quality, and the PbMC1a / 1b gene is an important candidategene for future genetic engineering to improve fruit quality breeding.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular to pear lignin synthesis gene PbMC1a / 1b and its application in genetic improvement of fruit quality. Background technique [0002] Pear is the third most widely planted fruit tree in my country. 'Dangshan Suli' originated from Dangshan County in my country and has been the main planted variety in my country for a long time. In recent years, due to factors such as reduced quality of varieties and poor field management, the content of stone cells in pear fruit has increased, and the texture of the pulp has become rougher. These changes adversely affected the flavor and quality of pears (Rogers et al., 2004). Stone cells in pear fruit are the key determinants of pear quality. They exist in the fruit in the form of separation or aggregation. Stone cells are rich in lignin and cellulose (Donaldson, 2001; Chang et al., 2006). Pear fruit stone cell is a sclerenchyma cell formed b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/50C12N15/82C12N15/11A01H5/00A01H6/20A01H6/74
CPCC12N9/63C12N15/8255C12N15/8243
Inventor 陶书田贡鑫张绍铃谢智华徐佳慧赵梁怡
Owner NANJING AGRICULTURAL UNIVERSITY
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