Populus tomentosa gene PtomiR390a and its application

A technology of poplar tomentosa and gene, which is applied in poplar tomentosa gene PtomiR390a and its application field, can solve the problems of inability to achieve forest yield and salt tolerance, and achieve the effect of improving the ability of salinity and alkali stress and improving biomass

Inactive Publication Date: 2017-12-01
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these salt-tolerant transgenic studies are all about transferring salt-tolerant genes identified in othe...

Method used

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  • Populus tomentosa gene PtomiR390a and its application
  • Populus tomentosa gene PtomiR390a and its application
  • Populus tomentosa gene PtomiR390a and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Poplar Total RNA Extraction and Reverse Transcription Synthesis of cDNA

[0030] 1. Poplar total RNA extraction

[0031] Using young poplar leaves as materials, the medicine spoon, pestle, mortar, etc. used in RNA extraction were burned with 95% alcohol until cooled before use. Take a spoonful of quartz sand and heat it in the heater for 10 minutes to cool down before use. 1.5mL centrifuge tubes, plastic products, etc. are all RNase-treated (RNase Free) AXYGEN products.

[0032] The total RNA was extracted according to the operation steps of Huashun Server-SPIN DNA-free Plant RNA Isolation Kit:

[0033] 1) Add lysate (500 μL RL+100 μL 10% PVP+10 μL 2-ME) into a 1.5 mL centrifuge tube;

[0034] 2) Mix young poplar leaves (100mg) and quartz sand under liquid nitrogen freezing conditions and quickly grind them into powder. Before the liquid nitrogen is completely volatilized, transfer all the powder to the pre-prepared lysate, and quickly blow with a pipette ...

Embodiment 2

[0048] The design of embodiment 2 PCR primers and PCR amplification obtain target fragment

[0049] 1. PCR primer design

[0050] Log in to miRBase (http: / / www.miRBase.com), and query the precursor sequence of the poplar PtomiR390a gene. Specific primers for the PtomiR390a precursor sequence were designed. The upstream primer PtomiR39a0-F sequence of the PtomiR390a fragment is: 5'-AGAATCTGTTAAGCTCAGGAG-3' (SEQ ID No.3), and the downstream primer PtomiR390a-R sequence is: 5'-AACCCATAGAACTCAGGATAG-3 '(SEQ ID No.4); the target fragment is 119bp.

[0051] Primers were synthesized by BGI (Beijing). The reaction reagents used in PCR are all TaKaRa products: dNTPMixture (2.5mM each), Taq DNA polymerase (5U / μL), with 10×PCR Buffer (0.1M Tris-HCl[pH8.3]), 0.5M KCl) and MgCl 2 (25mM).

[0052] 2. PCR amplification of the target fragment

[0053] Using poplar gDNA as a template to amplify the PtomiR390a gene fragment, the system for amplifying the PtomiR390a gene fragment:

[0054...

Embodiment 3

[0066] Example 3 PtomiR390a-pcxSN vector construction and transformation of Agrobacterium GV3101

[0067] After gel recovery of the amplified fragments, the vector pcxSN was digested with XcmI and ligated with the recovered fragments. The ligation product was transformed into Escherichia coli DH5α competent cells, and the target gene fragment was amplified by PCR and verified by enzyme digestion to obtain transformants, and the strains were preserved.

[0068] Extract the PtomiR390a-pcxSN plasmid from the positive transformant Escherichia coli respectively, and the steps follow the extraction steps of the Plasmid Mini Kit kit, and the specific operations are as follows:

[0069] 1) Take the bacterial solution cultured to the logarithmic phase in an EP tube, centrifuge at 13400rpm for 1min to collect the bacterial cells, and discard the supernatant.

[0070] 2) Add 250 μL of pre-cooled Solution I to which RNase A has been added, and vortex to resuspend the bacteria and mix wel...

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Abstract

The invention belongs to the technical field of plant gene engineering and particularly relates to a populus tomentosa gene PtomiR390a and its application. The invention discloses a populus tomentosa gene PtomiR390a, and its nucleotide sequence is shown as SEQ ID No.1; the invention further provides an expression carrier of the populus tomentosa gene PtomiR390a, a host of the expression carrier and an application in improving the aspen biomass and promoting the salt resistance of the aspen. After being transcribed, the populus tomentosa gene PtomiR390a can levelly modulate the aspen biomass; the biomass of the populus tomentosa is improved through the surplus expression of the populus tomentosa gene PtomiR390a, and the populus tomentosa's ability of resisting threat of salt and alkali in a nature environment is improved. A cultivating method provided by the invention cultivates high-quality aspen variety with high biomass and high salt resistance.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the Populus tomentosa gene PtomiR390a and its application. Background technique [0002] There are about 1.5 billion mu of various saline soils in my country, accounting for 26.3% of the world's saline soil area, and the area of ​​secondary salinization is still increasing (Yang Jinsong, 2008). Soil salinization has become an important limiting factor for the sustainable development of agricultural and forestry production in my country. Therefore, it is urgent to carry out the breeding of new varieties of salt-tolerant forest trees. Although the salt tolerance of forest trees has been improved to a certain extent by using traditional breeding techniques, the breeding cycle is long and it is difficult to aggregate multiple excellent salt-alkali tolerance genes in a short period of time (Luo Zijing et al., 2017). There is a broad prospect of using ge...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/84A01H5/00
CPCC12N15/113C12N15/8218C12N15/8261C12N15/8273C12N2310/14
Inventor 罗克明许长征何夫
Owner SOUTHWEST UNIVERSITY
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