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Method for extracting total RNA from Stevia rebaudiana Bertoni pollen

An extraction method and technology of stevia flowers, applied in the field of plant molecular biology, to achieve high quality total RNA, reduce operation difficulty, and maintain the effect of pollen vitality

Pending Publication Date: 2020-09-25
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the technical problem of isolating pollen and extracting total RNA from tiny stevia flowers, and to provide a method suitable for extracting total RNA from stevia pollen

Method used

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  • Method for extracting total RNA from Stevia rebaudiana Bertoni pollen
  • Method for extracting total RNA from Stevia rebaudiana Bertoni pollen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The invention is a method for extracting total RNA from stevia pollen, comprising the following steps:

[0030] (1) Preparation of reagent materials: sterile 10% sucrose solution, Trizol reagent, water-saturated phenol-chloroform-isoamyl alcohol (25:24:1) mixture, isopropanol, 70% ethanol (RNase-free H2O Preparation), RNase-free H2O, 400-mesh stainless steel filter, RNase-free centrifuge tube (1.5mL) and pipette tips of various specifications.

[0031] (2) Collection of anthers: From 8 to 10 in the morning, collect the flower buds that are about to open on the same day from the stevia variety "Zhongshan No. 6" at the full flowering stage, peel off the tubular corolla with tweezers, take out the anthers together with the stigma, and put them into a 10% sucrose solution. Gently invert and mix to make a pollen suspension.

[0032] (3) Separation of pollen: After enough anthers are collected, use a pipette to absorb the above pollen suspension, pass through a 400-mesh stai...

Embodiment 2

[0035] Add 50 μL of RNase-free H2O to the stevia pollen total RNA precipitate obtained in Example 1, place at 4°C for 30 minutes to fully dissolve the RNA, and store in a -80°C refrigerator for later use;

[0036] Determine RNA content with ethidium bromide fluorescence method: prepare 1% agarose gel, take 2 μL RNA sample for electrophoresis, stain with ethidium bromide for 15 minutes after electrophoresis, irradiate under short-wavelength ultraviolet light, and visually inspect the quality and content of RNA the height;

[0037] Such as figure 2It is the result of agarose electrophoresis detection of total RNA extracted from Stevia "Zhongshan No. 6". The bands are 25S, 18S, and 5S from top to bottom. It can be seen from the figure that the 28S and 18S bands of the RNA are bright and clear, and there is no DNA contamination, no degradation, good RNA integrity, and good quality and yield.

Embodiment 3

[0039] Add 50 μL of RNase-free H2O to the stevia pollen total RNA precipitate obtained in Example 1, place at 4°C for 30 minutes to fully dissolve the RNA, and store in a -80°C refrigerator for later use;

[0040] Using the nucleic acid protein detection method, the purified RNA solution was measured for its absorbance value under 230nm, 260nm and 280nm wavelength light with a nucleic acid protein detector, and the RNA OD value detection and purity determination were performed. The results are shown in Table 1.

[0041] 230nm 260nm 280nm 260 / 230 260 / 280 0.018 0.046 0.023 2.541 1.918

[0042] The values ​​of A230, A260, A280, A260 / A230 and A260 / A280 were 0.018, 0.046, 0.023, 2.541, 1.918 respectively, and the value of A260 / A280 was greater than 2.0, indicating that the "Zhongshan No. 6" stevia total RNA extracted by this method It is pure RNA without protein, polysaccharide and polyphenol pollution.

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Abstract

The invention discloses a method suitable for extracting total RNA from Stevia rebaudiana Bertoni pollen, and belongs to the field of plant molecular biology. The method mainly comprises the followingsteps of performing collecting on anthers of flowers about to bloom on the same day of Stevia rebaudiana Bertoni, performing immersing on the anthers in a 10% sucrose solution to dissociate pollen, performing filtering to remove impurities such as anther walls and the like, and carrying out centrifugal collecting on the pollen to obtain the total RNA; and carrying out extracting of the total RNAby adopting a Trizol method. The operation method for separating pollen from tiny Stevia rebaudiana Bertoni and extracting total RNA is disclosed for the first time, the method is simple, convenient and efficient, the operation difficulty of pollen collection and RNA extraction is greatly reduced, the total RNA quality of the obtained Stevia rebaudiana Bertoni pollen is relatively high, and subsequent research is facilitated.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology, and in particular relates to a method for extracting stevia plant pollen total RNA. Background technique [0002] Stevia ( Stevia rebaudiana Bertoni) is an emerging economic crop. The steviol glycosides contained in its leaves not only have the characteristics of high sweetness and low calories, but also have medicinal value such as lowering blood sugar and blood pressure. With the development of the Stevia industry, the cultivation of fine new varieties is important and urgent, and the research on pollen-specific expression genes is very important for Stevia genetics and breeding. However, stevia flowers and pollen are so tiny that collecting pollen is extremely difficult. [0003] Liquid nitrogen grinding is widely used in molecular biology experiments, especially the separation of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The temperature of liquid nitrogen is ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 杨永恒原海燕徐晓洋佟海英张婷张永侠孙玉明
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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