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Gene chip composed of tea plant expressed sequence tags

A technology for expressing sequence tags and gene chips, which is applied in the field of tea tree gene detection and can solve problems such as large target fragments.

Inactive Publication Date: 2011-01-12
TEA RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional hybridization detection methods mainly include target fragment amplification, direct spotting or transfer to nylon membrane after electrophoresis, and UV hinge. has certain limitations

Method used

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  • Gene chip composed of tea plant expressed sequence tags
  • Gene chip composed of tea plant expressed sequence tags
  • Gene chip composed of tea plant expressed sequence tags

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The construction of embodiment 1 tea tree leaf cDNA library

[0035] 1. Extraction of total RNA from tea tree leaves

[0036] In spring, take 100 mg of vigorously growing young leaves of tea tree, quickly add liquid nitrogen to freeze and grind into a fine powder, add 1000 μL Trizol reagent (purchased from Gibco BRL) to continue grinding, and then add 1000 μL Trizol reagent, mix evenly and divide into two vials. Into a 1.5 mL centrifuge tube treated with diethyl pyrocarbonate, place on ice for 5 minutes, add 200 μL of chloroform each, mix by inverting, centrifuge at 12,000 rpm at 4°C for 10 minutes, take 500 μL of the supernatant, add an equal volume of iso Propanol, gently upside down 10 times, place at room temperature for 10 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, remove the supernatant, add 1 mL of 75% ethanol to the precipitate to wash gently, pour off the ethanol, and add 20 μL of coke after drying. Diethyl carbonate-treated double distilled wate...

Embodiment 2

[0093] The construction of embodiment 2 tea tree radicle cDNA library

[0094] 1. Extraction of total RNA from young roots

[0095] Sow the stored seeds of the tea tree Longjing 43 in quartz sand with a certain humidity, and cultivate them in a culture room at room temperature of 25°C. After the seedlings grow, take 100 mg of their young roots, quickly add liquid nitrogen to freeze and grind them into fine powder, and add 1000 μL Trizol After the reagent continues to be ground, add 1000 μL of Trizol reagent, mix evenly and divide into two 1.5mL centrifuge tubes treated with DEPC (diethyl pyrocarbonate), place on ice for 5 minutes, add 200 μL of chloroform each, mix upside down, Centrifuge at 12,000 rpm at 4°C for 10 minutes, take 500 μL of supernatant, add an equal volume of isopropanol, invert up and down gently 10 times, place at room temperature for 10 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, remove the supernatant, add 1 mL of 75% ethanol to the precipitate...

Embodiment 3

[0163] Example 3, Sequencing of Tea Tree Leaf and Young Root Expressed Sequence Tags

[0164] Using the previously constructed tea tree leaf cDNA library and tea tree radicle cDNA library as the source of sequencing EST libraries, the automatic sequencer Megabace TM 1000 Random sequencing analysis was performed on 4320 clones of the Longjing 43 shoot cDNA library, and 2963 effective sequences were obtained. After deduplication and splicing of all effective sequences, 1692 shoot expression sequence tags were obtained; 5115 of the radicle cDNA library The clone was subjected to random sequencing analysis, and 4833 effective sequences were obtained, most of which were between 300bp and 700bp in length, with an average length of 474bp. After deduplication and splicing of all effective sequences, 3482 radicle expression sequence tags were obtained. All the obtained non-redundant sequences were compared with the protein database of GenBank by BlastX analysis, and the results were m...

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Abstract

The invention discloses a gene chip composed of tea plant expressed sequence tags, which uses the tea plant expressed sequence tags as probes and comprises probes in the sequence of SEQ ID No.1-SEQ ID No.4866 in the sequence list. The gene chip not only can be used for detecting the gene expression information of different tea plant varieties under biotic or abiotic stress and screening different genes, but also can be used for constructing gene expression profiles of the tea plant varieties in different growth stages, different tissues and organs and specific cells. The chip can realize the high-throughput detection on the gene expression information, has the advantages of convenient and safe operation and reliable and effective detection result, can save a large amount of time, labor, material resources and financial resources, and has wide application prospects.

Description

technical field [0001] The invention relates to a tea tree gene detection technology, in particular to a gene chip composed of tea tree expression sequence tags. Background technique [0002] Using known gene information to specifically detect the expression information of the target gene in the sample to be tested is a commonly used detection method in the field of molecular biology, such as the commonly used Southern hybridization, Northern hybridization and PCR techniques. However, with the development of molecular biology, these techniques cannot simultaneously analyze thousands of genes under the same conditions, so they have certain limitations. It has become a trend to immobilize thousands of genes on the carrier by in situ synthesis (in sitil synthesis) probes or synthetic target gene fragments and then use cross-linking, and then use hybridization to detect gene expression information , with the continuous development of this method, biochip technology has emerged....

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 陈亮马春雷姚明哲王新超金基强
Owner TEA RES INST CHINESE ACAD OF AGRI SCI