Applications of LsWD taken as reference gene in lagenaria siceraria var.hispida analysis under infection of cucumber green mottle mosaic virus

A technology of green mottled flowers and internal reference genes, applied in the field of molecular biology, can solve the problems of lack of internal reference genes, etc., and achieve the effect of high expression stability and wide applicability

Active Publication Date: 2019-11-12
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In order to solve the problem of the lack of systematically verified and stable expression reference genes f

Method used

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  • Applications of LsWD taken as reference gene in lagenaria siceraria var.hispida analysis under infection of cucumber green mottle mosaic virus
  • Applications of LsWD taken as reference gene in lagenaria siceraria var.hispida analysis under infection of cucumber green mottle mosaic virus
  • Applications of LsWD taken as reference gene in lagenaria siceraria var.hispida analysis under infection of cucumber green mottle mosaic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Selection of internal reference genes

[0042] 1. Gourd planting and sample collection

[0043] The gourd variety "Hangzhou Changgua" is planted in the soil rich in organic matter in the greenhouse of the Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, keeping the temperature at 20-25°C, and watering every 3 days to maintain soil moisture. CGMMV disease sap was rubbed and inoculated on the unfolded system leaves at the stage of two leaves and one heart. The specific preparation method of the disease sap was: 100 mg of CGMMV susceptible tissue homogenate in 20 times the volume of PBS buffer (0.1M PBS, pH7.5, 0.2% sodiumsulfite and 0.01M 2-mercaptoethanol), while the control healthy plants were rubbed inoculated with PBS buffer only. Other management proceeded as usual. The leaves and fruits of the gourd system infected by CGMMV and the leaves and fruits of the healthy gourd system were collected for RNA extraction, and t...

Embodiment 2

[0056] Example 2: Design of Real-time Fluorescent Quantitative PCR and Conventional PCR Detection of Internal Reference Genes of Gourd Gourd Infected by CGMMV

[0057] 1. Design of quantitative primers

[0058] Use the online primer design program Primer3.0 to design internal reference quantity primers. The main design parameters include: the length of the amplified fragment ranges from 80-250bp, the Tm value of the quantitative primer is set at 58°C-64°C, and the GC content range of the primer is 45%-55%. . The common candidate internal reference genes of gourd leaves and fruits related to CGMMV infection were LsH3, LsWD, LsACT, LsUBA52, LsPRL23, LsRAN, LsPP2A, LsGAPDH, LsEF1α, LsADP, LsTUA, LsTBP, LsRPS15, LsCYP20, LsCYP and LsL23A, and the designed primers See sequence Figure 14 Shown; the candidate internal reference genes of gourd leaves screened in the transcriptome data are LsARL, LsTPT, LsSTK, LsCNX, LsP4HB, LsSHAGGY, LsUBC, LsGAD and LsRBP, and the candidate inte...

Embodiment 3

[0062] Embodiment 3 real-time fluorescent quantitative PCR primer verification

[0063] Using gourd leaf and fruit cDNA as templates, RT-qPCR amplification was carried out with the designed quantitative primers in ABI Prism 7900HT FAST fluorescent quantitative PCR instrument. The reaction system and reaction procedure of the PCR reaction were as follows:

[0064]The reaction system is: 2×SYBR Green Master Mix, 5 μL, 0.15 μL each for upstream and downstream detection primers ( Figure 14 and Figure 15 Listed reference genes), cDNA 0.5μL, RNase Free dH 2 O 4.2 μL. The reaction program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, a total of 40 cycles.

[0065] According to the qRT-PCR analysis to obtain the melting curve as figure 2 shown, from figure 2 It can be seen that the melting curves of all candidate internal reference genes are in a single-peak state, which proves that the specificity of the quantitativ...

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Abstract

The invention belongs to the technical field of molecular biology, and specifically relates to applications of LsWD taken as a reference gene in lagenaria siceraria var.hispida leaf or fruit qRT-PCR analysis under the infection of a cucumber green mottle mosaic virus. Through the utilization of lagenaria siceraria var.hispida transcriptome data, the expression level and stability of a gene can becompared at the whole genome level; and that the disclosed LsWD gene can be stably expressed in lagenaria siceraria var.hispida leaves and fruits under the infection of a cucumber green mottle mosaicvirus can be determined by using a LsIPT or LsDdRP gene to perform verification, so that the LsWD is a reliable reference gene in the analysis of gene expression of lagenaria siceraria var.hispida under the infection of the cucumber green mottle mosaic virus detected by utilizing a qRT-PCR technology. The LsWD gene is firstly taken as the reference gene to be used for the analysis of gene expression of the lagenaria siceraria var.hispida, so that present situations that existing lagenaria siceraria var.hispida quantitative PCR detection has no reference genes can be solved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to the use of a gene as an internal reference gene in qRT-PCR analysis of gourd leaves or fruits infected by cucumber green mottled mosaic virus. Background technique [0002] Gourd (Lagenaria siceraria var. hispida), an annual vine herb of Cucurbitaceae, is a medicinal vegetable that can reduce swelling and moisturize the skin. It has been cultivated in China since ancient times. The tender fruit is a popular summer delicacy among the people. It has a delicate and soft taste and a slightly sweet taste. It can be eaten after being peeled. The ripe old fruit is not edible. In ancient China, the ripe and dried fruit shell was used as a container and also for medicinal purposes. Gourds are cultivated in all parts of the north and south of my country, and the cultivation is common in the south. In recent years, gourds have also been introduced and cultivated in the...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/94
CPCC12Q1/701C12Q1/686C12Q2521/107C12Q2545/101
Inventor 燕飞郑红英张趁华吴昕扬彭杰军鲁宇文林林陈剑平
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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