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Narcissus tazetta var. chinensis chitinase, coding gene and application thereof

A chitinase and coding gene technology, applied in the field of plant genetic engineering, can solve problems such as hindering the production and development of narcissus, no research reports on narcissus chitinase, ornamental and commercial degradation, etc.

Active Publication Date: 2014-11-05
林晓红
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Narcissus will be infected with a variety of diseases during the cultivation process, resulting in the general degradation of its ornamental and commercial properties, which seriously hinders the production and development of narcissus
However, there is no research report on chitinase from Chinese narcissus

Method used

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  • Narcissus tazetta var. chinensis chitinase, coding gene and application thereof
  • Narcissus tazetta var. chinensis chitinase, coding gene and application thereof
  • Narcissus tazetta var. chinensis chitinase, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] (1) Total RNA extraction and cDNA synthesis from narcissus leaves

[0023] For the extraction of total RNA, refer to the instructions of the polysaccharide polyphenol plant total RNA extraction kit of Beijing Biotech Biotechnology Co., Ltd., and take 1ug of RNA to reverse transcribed into cDNA. Trans Script II First-Strand cDNA Synthesis SuperMix was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0024] (2) Cloning of the target fragment

[0025] According to the existing narcissus transcriptome sequence, the conserved region of the chitinase gene sequence was found by NCBI database comparison, and the chitinase gene cassette NBS gene sequence was obtained. The software Primer Premier5.0 was used to design specific primers. The primer sequence is:

[0026] Chit3-F: 5′-TCCGTGCCGTCGATAATATCGGAAG,

[0027] Chit3-R: 5'-GAAATGAAATAATGGAGGGGGAAAT.

[0028] PCR amplification system and procedure of butyrase gene:

[0029] PCR amplification total system (25μl): template cDNA ...

Embodiment 2

[0043] Example 2 Specific Expression of Narcissus Chitinase in Chinese Narcissus

[0044] In order to study the response of the narcissus chitinase gene to narcissus base rot, virus disease and brown spot disease, this application uses qRT-PCR to detect the narcissus chitinase gene in normal plants, the initial stage of the disease, the roots and leaves of the severe disease stage. Expressive characteristics in. Among them, the specific steps of qRT-PCR detection include:

[0045] Dilute n reverse transcription cDNA products 10 times as a template, and the reaction system is 10μL: SYBR Premix Ex TaqTM (2×) 5μL, 10μM upstream and downstream primers each 0.2μL, cDNA template 1μL, add ddH 2 0 to 10μL, each sample was repeated twice, and a negative control without template was set for different primers, and the Chinese narcissus housekeeping gene Actin was used as an internal reference. The instrument used for qPCR is Roche Light Cycler 480, using 96 plate, and the reaction program ad...

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Abstract

The invention provides a Narcissus tazetta var. chinensis chitinase, a coding gene and application thereof. Primers are designed according to chitinase gene sequences obtained by prediction based on Narcissus tazetta var. chinensis transcriptome data, a chitinase gene is cloned from Narcissus tazetta var. chinensis chitinase and has a sequence shown as SEQ ID NO:1, and an amino acid sequence of chitinase is translated and is shown as SEQ ID NO:2. On the basis of bioinformatic analysis of the chitinase gene, the qRT-PCR detection technology verifies that the chitinase has significantly increased expression quantity under the induction of narcissus bulb rot, brown patch and virus diseases, and can be applied to the disease-resistant process of narcissus.

Description

Technical field [0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to a Chinese narcissus chitinase and its coding gene and application. Background technique [0002] Chinese narcissus (Narcissus tazetta var.chinensis) is a perennial bulb plant of the genus Amaryllidaceae Narcissus. Its bulb head is large, the flower color is rich, and the flower is fragrant. It can be cultivated indoors with clean water. It is one of the ten traditional famous flowers in China. Chinese narcissus has high ornamental value and economic value, and is one of the traditional flowers that people love. Narcissus will be infected with a variety of diseases during the cultivation process, leading to the general degradation of its ornamental and commercial properties, and seriously hindering the production and development of narcissus. Therefore, it is particularly important to improve the disease resistance of narcissus itself. [0003] Chitinase is...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56A01P3/00
CPCC12N9/2442C12Y302/01014
Inventor 林晓红潘东明
Owner 林晓红
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