Molecular probe for detecting vitality of musa spp. pollen and preparation method of molecular probe

A technology of pollen vitality and bananas, applied in the field of molecular biology, can solve the problems of difficult and precise quantitative use, and achieve the effect of high precision and practical value

Pending Publication Date: 2021-09-03
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of this type of staining reaction to display the activity of cells, tissues, organs and individuals including pollen is that it is fast and simple. The disadvantage is that its quantitative display is restricted by the shape of the cells, tissues, and organs to be detected, and it is difficult to use it accurately and quantitatively.

Method used

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  • Molecular probe for detecting vitality of musa spp. pollen and preparation method of molecular probe
  • Molecular probe for detecting vitality of musa spp. pollen and preparation method of molecular probe
  • Molecular probe for detecting vitality of musa spp. pollen and preparation method of molecular probe

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1. Preparation of a molecular probe for banana pollen vitality detection:

[0030] The molecular probe sequence used in the present invention is derived from the gene on the first chromosome of the banana A genome, and the gene is preliminaryly identified by the genes of the GDSL lipase family, which can promote plant pollen hydration, thus improving the pollination rate. . MA01_T03770.1 is screened by the analysis of the total genome identification and the analysis of the GDSL family, and the CDS sequence is shown in SEQ ID NO.1. The molecular probe design of the present invention refers to the MA01_T03770.1 gene sequence downloaded by banana a genomic database (https: / / banana-genome- hub.southgreen.fr / pahang_v2), and entrusts platinum bioca. The molecular probe nucleotide sequence is as follows: SEQ ID NO. 2 F: 5'-cgttgtgattctgtgctGCT-3 '; SEQ ID No. 3 R: 5'-gtttctcagcgaggaagtcg-3'.

Embodiment 2

[0031] Example 2. How to use banana pollen active molecular probe:

[0032] The collected fresh pollen is divided into two parts, and a portion is immediately used for the first step in dyeing, and a portion of liquid nitrogen is prepared for cDNA for the second step.

[0033] The first step: Configure TTC dyeing liquid: Dissolve the TTC powder in PBS buffer according to the mass fraction of 0.5 wt% -1 wt%, and the contaminated dyed liquid can be stored in short-term preservation under the preservation conditions. First, TTC solution is added dropwise on the slide, take a small amount of pollen to the surface of the droplet, cover the slide, and placed in a 25 ° C-35 ° C to cultivate 15 min (eg figure 1 Indicated). Observing some pollen becomes a light red, partially dark red, the better the color, the better the activity.

[0034] Step 2: The pollen preserved in liquid nitrogen is ground to extract RNA and transcribed into the CDNA stock solution required by the relevant step, an...

Embodiment 3

[0038] Example 3. Production and use of banana pollen active molecular probe kits

[0039] The banana pollen active molecular probe kit includes a component shown in Table 1. The kit is stored: -20 ° C for two years.

[0040] Table 1 Banana Pollen Active Molecular Probe Kit Product Component

[0041]

[0042] The sequence of the inner paragraph gene primer in the above kit is as follows:

[0043] SEQ ID NO.4 UBQ (f): 5'-ggcaccacaaacaacaigagg-3 ';

[0044] SEQ ID NO.5 UBQ (R): 5'-agaggagcaaggcttcccatt-3 '.

[0045] The banana pollen active molecular probe kit can be accurate and efficient for QPCR amplification, and the reaction temperature is 60 ° C. The TTC powder and microscope are included for rapid detection of pollen vitality. The TTC powder can be dissolved directly with the PBS buffer, and the ratio is 0.5% -1%, which is now dispelled.

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Abstract

The invention provides a molecular probe for detecting vitality of musa spp. pollen and a preparation method of the molecular probe. According to the invention, the optimal pollen collection, storage and use conditions are determined according to quantitative data by identifying phenotype data of specific expression of genes and pollen vitality. The high-precision pollen vitality molecular probe makes up the problem of insufficient vitality monitoring of a traditional method such as TTC enzyme staining as a basic method, and has application value on cross breeding and pollen research.

Description

Technical field [0001] The present invention belongs to the field of molecular biology, and in particular to a molecular probe for banana pollen viability detection and a preparation method thereof. Background technique [0002] banana( Musa SPP.) Basicae Forta Banania ( Musa ), It is a perennial evergreen grass character tree for a single child, and is one of the most important crops in tropics and subtropical regions. It is also the second largest fruit in the world after citrus, accounting for 16% of the world's fruit production. Cultivated bananas ( Musa Paradisiaca The variety is a ploid, and the genomic types mainly include AAA, AAB and ABB. Most cultivated bananas varieties have a high degree of hypertrophy, which leads to a large breeding of bananas, but since it is not 100% male infertility, some pollen is active. At the same time, some triploid varieties can produce non-reduced triplines in the decancing process, and a small amount of tetraploid seed can also be obtaine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/158
Inventor 张城瑜王姗姗赖钟雄班景洁林玉玲徐涵
Owner FUJIAN AGRI & FORESTRY UNIV
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