Apple MdSHN1 gene and application of apple MdSHN1 gene in improving waterlogging tolerance of plants

A technology of waterlogging tolerance and gene, applied in apple MdSHN1 gene and its application in improving plant waterlogging tolerance, can solve the problems of reducing petunia's tolerance to flooding, and achieve the effect of enhancing waterlogging tolerance

Active Publication Date: 2022-05-31
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, in the waterlogging tolerance study of petunias, it was found that compared with wild-type plants, PhERF2-silenced plants showe

Method used

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  • Apple MdSHN1 gene and application of apple MdSHN1 gene in improving waterlogging tolerance of plants
  • Apple MdSHN1 gene and application of apple MdSHN1 gene in improving waterlogging tolerance of plants
  • Apple MdSHN1 gene and application of apple MdSHN1 gene in improving waterlogging tolerance of plants

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Embodiment 1

[0050] Cloning of embodiment 1 apple MdSHN1 gene

[0051] (1) RNA extraction and reverse transcription of Pingyi sweet tea leaves

[0052] 1. Extraction of apple total RNA

[0053] The extraction method of total RNA from apple leaves was slightly optimized according to the instructions of the RNA extraction kit (Shenggong). The specific process is as follows:

[0054] (1) Add 500μl Buffer Rlysis-PG into 1.5ml RNase-free centrifuge tube for later use.

[0055] (2) Add 50 mg of plant tissue samples ground by liquid nitrogen to the above-mentioned 1.5 ml centrifuge tube, shake and mix immediately, and place at room temperature for 5 min.

[0056] (3) Centrifuge at 13,400g, 4°C for 3min, and transfer the supernatant to a new 1.5mL RNase-free centrifuge tube.

[0057] (4) Add 1 / 2 volume of absolute ethanol and mix well.

[0058] (5) Put the adsorption column into the collection tube, add all the solution to the adsorption column with a pipette, let it stand for 1 min, centrifu...

Embodiment 2

[0076] Embodiment 2 plant overexpression vector construction

[0077] In order to study the function of the MdSHN1 gene, the pCAMBIA2300-GFP vector was digested with Xbal and Kpn enzymes, the recovered product was ligated with the target gene, and transformed into DH5α competent cells, and sequenced after bacterial screening to form the MdSHN1 overexpression vector. The MdSHN1 overexpression vector was transformed into Agrobacterium GV3101 for use.

Embodiment 3

[0078] The acquisition of embodiment 3 transgenic Arabidopsis

[0079] Arabidopsis thaliana was infected by the inflorescence infection method, referring to the method of Clough et al. (1998). Select 4-week-old robust Arabidopsis plants, cut off the fruit pods that have bloomed before the first infection, and water enough to keep the soil moist. To increase transformation efficiency, approximately one week later, perform a second infection. After the seeds are mature, the T0 generation seeds are collected in a dry centrifuge tube, stored after the seeds are dried, and placed at 4°C for vernalization for later use. The plant resistance of the plant overexpression vector pCAMBIA2300-GFP is kanamycin, which can be integrated into the plant genome along with the target gene during transformation, so kanamycin is used to screen the Arabidopsis seeds obtained after infection.

[0080] First, disinfect the obtained Arabidopsis seeds: soak in 5% sodium hypochlorite for 5 minutes, an...

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Abstract

The invention discloses an apple MdSHN1 gene, the nucleotide sequence of the apple MdSHN1 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the protein is as shown in SEQ ID NO.2. According to the invention, a cDNA full-length sequence of the MdSHN1 gene is cloned from a waterlogging-resistant apple rootstock Malus hupehensis leaf, a primer for amplifying the apple MdSHN1 gene is also disclosed, and an amino acid sequence of the gene is determined. Through arabidopsis thaliana transgenosis function verification and analysis, it is found that the MdSHN1 gene has a remarkable effect in the aspect of improving the resistance of a plant, the adversity resistance of the transgenic plant is improved, particularly, compared with a wild type, the tolerance of the transgenic plant is higher than that of the wild type under flooding stress, and it can be seen that the MdSHN1 gene is related to waterlogging tolerance, the waterlogging tolerance of the plant can be improved, and the stress resistance of the transgenic plant is improved. The method is of great significance to breeding of new apple varieties.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an apple MdSHN1 gene and its application in improving plant waterlogging tolerance. Background technique [0002] Apple (Malus×domestica Borkh) is one of the important fruit tree species in my country. Its area and output both rank first in the world. In recent years, due to the impact of global warming, extreme climates have occurred frequently, rainfall distribution has been uneven, and large-scale, varying degrees of waterlogging have often occurred, showing an increasing trend year by year. In actual production, orchards are often not drained in time after rainfall, or improperly irrigated, etc., resulting in excessive soil moisture content in the orchard, obstructed respiration of apple roots, shedding of apple leaves, and decline in fruit quality and yield. The whole plant dies, which has become an urgent problem in apple production. Systematic research on the re...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/84C12N15/11C07K14/415A01H5/00A01H6/20A01H6/74
CPCC07K14/415C12N15/8205C12N15/8271Y02A40/146
Inventor 白团辉郑先波宋春晖焦健陈晓菲王苗苗宋尚伟
Owner HENAN AGRICULTURAL UNIVERSITY
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