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A kind of breeding method of plant low temperature resistant gene and transgenic low temperature resistant plant

A breeding method, low temperature resistance technology, applied in the field of plant genetic engineering

Active Publication Date: 2020-03-10
NANJING XIAOZHUANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has been no literature report on the isolation of genes related to low temperature stress from Mongolian leek

Method used

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  • A kind of breeding method of plant low temperature resistant gene and transgenic low temperature resistant plant
  • A kind of breeding method of plant low temperature resistant gene and transgenic low temperature resistant plant
  • A kind of breeding method of plant low temperature resistant gene and transgenic low temperature resistant plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Screening of genes related to low temperature stress in Chinese chives

[0034] This embodiment provides a method for screening genes related to low temperature stress in Chinese chives using cDNA-AFLP technology, which specifically includes the following steps:

[0035] 1. Low temperature stress treatment

[0036] (1) Take Mongolian leek seeds and rinse them with tap water, then soak them in sterile water for 10 minutes. Then they were sterilized with 70% alcohol for 30 seconds, washed three times with sterile water, and inoculated on hormone-free MS basic medium. Take the leaves of sterile seedlings with a height of about 5 cm, cut them into 3 mm long sections from the tip to the base, and inoculate them on the callus induction medium in turn. After the callus is formed, transfer the callus to the differentiation medium In about 2 weeks, the callus differentiated into seedlings; when the seedlings of Mongolian Chinese chives grew to about 3 cm, they were t...

Embodiment 2

[0060] Example 2 Verification of TDF124 related to low temperature stress of Mongolian chives

[0061] This embodiment provides a method for verifying low temperature stress-related TDF124 using RT-PCR technology, which specifically includes the following steps:

[0062] 1. Low temperature stress treatment of Mongolian leek seedlings

[0063] According to the low temperature stress treatment method shown in Example 1, the Mongolian leek seedlings were placed in a temperature environment of 0-5°C and cultivated for 0h, 2h, 4h, 8h, 24h, 72h and 7d.

[0064] 2. Semi-quantitative amplification of TDF124

[0065] Take the leaves of Allium mongolica treated in step 1, extract the total RNA of the leaves according to the method shown in Example 1, and reverse transcribe to obtain cDNA. Using the above cDNA as a template, primers TDF124-F and TDF124-R were designed based on the sequence information of TDF124 in Example 1. The nucleotide sequence of TDF124-F is 5'-TTGGGCTTGGACTGCCCCG...

Embodiment 3

[0067] Example 3 Amplification of the full-length cDNA sequence of TDF124 related to low temperature stress of Chinese chives

[0068] This example provides a method for amplifying the full-length cDNA sequence of TDF124 associated with low temperature stress in Chinese chives, using the SMARTer RACE cDNA amplification kit (clontech, USA). Specifically include the following steps:

[0069] 1. Prepare RACE-Ready cDNA

[0070] (1) Add the reagents shown in Table 4 to the reaction tube, mix well and centrifuge briefly to obtain the Buffer Mix required for RACE-Ready cDNA preparation;

[0071] Table 4 Buffer Mix

[0072] 5XFirst-Strand Buffer 2.0 μL DTT (20mM) 1.0 μL dNTP Mix(10mM) 1.0 μL

[0073](2) Take another two reaction tubes, add 2.0 μL of RNA prepared in Example 1 and 1.0 μL of 5’-CDS Primer A to one of the reaction tubes, as the preparation system of 5’-RACE-Ready cDNA; Add 3.0 μL of RNA prepared in Example 1 and 1.0 μL of 3'-CDS Primer A...

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Abstract

The invention discloses a plant low temperature resistance gene. The coding amino acid sequence of the plant low temperature resistance gene is represented by SEQ ID NO.1, and the cDNA sequence is represented by SEQ ID NO.2. The plant low temperature resistance gene possesses important utilization potentiality in improvement of plant low temperature stress resistance or cultivation of plant with low temperature resistance. The invention also provides a recombinant vector, a transgenic cell line, and a transgenic recombinant bacterium which contain the plant low temperature resistance gene. The invention also discloses a cultivation method of transgenic low temperature resistant plant. According to the cultivation method, transgenosis of the plant low temperature resistance gene into a target plant is carried out so as to obtain the transgenic low temperature resistant plant. The cultivation method can be used for culturing transgenic plant with excellent low temperature resistance; the cultivation process is economical and rapid; and application prospect in the field of agriculture is promising.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a plant low-temperature-resistant gene, a recombinant vector containing the plant low-temperature-resistant gene, a transgenic cell line or a transgenic recombinant bacterium, and a method for cultivating a transgenic low-temperature-resistant plant. Background technique [0002] In nature, plants are inevitably under the stress of various environmental factors. Abiotic stresses such as low temperature, high temperature, drought and high salinity can easily cause damage to plants through physiological and biochemical processes such as affecting plant cell membrane components and stomatal opening. In severe cases, it will affect the yield of crops and the planting area. Abiotic adversities such as chilling injury, drought, high temperature, and salt damage are serious problems faced by global agricultural production and are also important factors rest...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/82A01H5/00A01H6/56
CPCC12N9/0071C12N15/8273
Inventor 王立科毛善国杨平陈全战张边江
Owner NANJING XIAOZHUANG UNIV
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