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A method for enhancing the salt tolerance of fruit mulberry by using long-chain non-coding rna gene rootstock

A genetically modified, salt-tolerant technology that is applied in the fields of biology and agriculture to achieve the effects of reducing risks, improving salt tolerance, and broadening the planting range

Active Publication Date: 2021-11-23
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the application of lncRNA in the salt-resistant breeding and cultivation of mulberry

Method used

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  • A method for enhancing the salt tolerance of fruit mulberry by using long-chain non-coding rna gene rootstock
  • A method for enhancing the salt tolerance of fruit mulberry by using long-chain non-coding rna gene rootstock
  • A method for enhancing the salt tolerance of fruit mulberry by using long-chain non-coding rna gene rootstock

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the cloning method of mulberry LncR-SR gene

[0040] 1. Use the nucleic acid extraction kit to extract the total RNA of mulberry leaves, and use the reverse transcription kit to reverse the RNA to cDNA. The reaction system is as follows:

[0041]

[0042] After adding the above system, mix well, react in a water bath at 42°C for 90 minutes, immediately ice-bath for 3 minutes after the reaction, and continue to react in a water bath at 75°C for 15 minutes. The reaction product is mulberry cDNA, which is stored at -20°C.

[0043] 2. Design a pair of specific primers LncR-SR-5 and LncR-SR-3, the specific sequences are as follows:

[0044] LncR-SR-5: 5'-TCATCCCCAGGAGACCTCCATTCT-3'; (SEQ ID NO.3)

[0045] LncR-SR-3: 5'-TTGGCGCAAAACCTTGAGCTT-3'; (SEQ ID NO.4)

[0046] Using mulberry leaf cDNA as a template for PCR amplification, the system is as follows:

[0047]

[0048] The reaction procedure is as follows:

[0049]

[0050] After the reaction, the ...

Embodiment 2

[0051] Embodiment 2: the construction of mulberry LncR-SR plant expression vector

[0052] 1. according to the nucleotide sequence of the isolated mulberry LncR-SR gene, design primers LncR-SR-F and LncR-SR-R with restriction sites, and its sequence is specifically as follows:

[0053] LncR-SR-F: 5'-GGATCCTCATCCCCAGGAGACCTC-3'; (SEQ ID NO.5)

[0054] LncR-SR-R: 5'-GAGCTCTTGGCGCAAAACCTTG-3'; (SEQ ID NO.6)

[0055] Using mulberry leaf cDNA as template, carry out PCR reaction, PCR amplification system and amplification conditions are the same as embodiment 1.

[0056] 2. Take the PCR product and connect it to the pMD18-T Simple cloning vector, transform the ligated product into Escherichia coli DH5a competent cells, screen positive clones for resistance to ampicillin (50mg / L), extract the recombinant plasmid DNA after PCR identification of the bacteria solution, and perform enzyme digestion identification followed by sequence determination.

[0057] 3. Digest the recombinant p...

Embodiment 3

[0058] Embodiment 3: the acquisition of transgenic mulberry hairy root

[0059] 1. The constructed LncR-SR plant expression vector was transformed into Agrobacterium rhizogenes K599 competent cells by freeze-thaw method, and positive clones were screened for kanamycin (50 mg / L) resistance.

[0060] Pick Agrobacterium (a single colony of Agrobacterium carrying a recombinant plasmid) and inoculate it in LB liquid medium (trypsin 10g / L, yeast powder 5g / L, sodium chloride 10g / L, kanamycin 50mg / L) , 28°C, 250rpm, shaking culture for about 48h to late logarithmic growth; dilute the bacterial solution 10 times with MS culture solution, and set aside.

[0061] 2. Sow the plump Guisangyou No. 12 seeds on the seedbed and cover them with a small shed for protection and cultivation. When the seedlings grow two true leaves, set the seedlings at a distance of 5 cm. Inject the above-mentioned Agrobacterium rhizogenes suspension into the epicotyls of the seedlings, bury the epicotyls with mo...

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Abstract

The invention discloses a method for enhancing the salt-tolerant ability of fruit mulberry by using long-chain non-coding RNA gene-transformed stock. The present invention utilizes a long-chain non-coding RNA (lncRNA) derived from mulberry trees, named LncR-SR, transforms mulberry seedlings through the transgenic technology mediated by Agrobacterium rhizogenes, and obtains the hairy roots of mulberry trees with transgenic LncR-SR genes. The obtained transgenic hairy roots are used as rootstocks to obtain fruit mulberry seedlings through grafting. The salt tolerance of the fruit mulberry seedlings obtained by the method is significantly improved, and the sugar content of the mulberry produced by the fruit mulberry cultivated in the high-salt soil is obviously higher than that of the fruit mulberry cultivated in the normal soil. The invention provides a method for the cultivation of salt-tolerant fruit mulberry seedlings, improves the salt tolerance of fruit mulberry, broadens the planting range of fruit mulberry, improves the quality of mulberry, and can reduce the concern about the safety of genetically modified organisms. Improving the salt tolerance of other fruit trees provides a reference and has broad application value.

Description

technical field [0001] The invention relates to the technical fields of biology and agriculture, in particular to a method for enhancing the salt tolerance of fruit mulberry by using long-chain non-coding RNA gene rootstocks. Background technique [0002] As the world population continues to increase, land salinization is increasing. The area of ​​salinized soil in my country has exceeded 37 million hectares, of which the cultivated land salinized area has reached 9.209 million hectares, accounting for 6.62% of my country's total cultivated land area. Soil salinity Staining has become an important factor affecting the healthy development of my country's agriculture and forestry. Mulberry is the material basis of sericulture production, and it is also an important ecological tree species for water and soil conservation, windbreak and sand fixation, and improvement of saline-alkali land. In addition, mulberry is rich in nutrition, sweet and sour, and has multiple health care f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/06A01H6/00
CPCC12N15/113C12N15/8273
Inventor 冀宪领盖英萍刘壮
Owner SHANDONG AGRICULTURAL UNIVERSITY