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Blueberry salt-resistant drought-resistant gene VcLON2 and protein encoded by blueberry salt-resistant drought-resistant gene VcLON2 as well as application

A technology of salt tolerance and blueberry, applied in the field of genetic engineering, can solve the problem of less research and achieve the effect of improving the salt tolerance and drought resistance of plants

Active Publication Date: 2015-09-16
ZHEJIANG NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most current studies mainly focus on non-plant bodies such as archaea, prokaryotes, and animals, and relatively few studies in plants
In particular, there is no relevant report on the function of this gene in plant stress resistance (biotic stress or abiotic stress), therefore, it has important research value and prospects for the study of LON protease in plants
Especially little about blueberries VcLON2 Research on anti-stress and other functions

Method used

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  • Blueberry salt-resistant drought-resistant gene VcLON2 and protein encoded by blueberry salt-resistant drought-resistant gene VcLON2 as well as application
  • Blueberry salt-resistant drought-resistant gene VcLON2 and protein encoded by blueberry salt-resistant drought-resistant gene VcLON2 as well as application
  • Blueberry salt-resistant drought-resistant gene VcLON2 and protein encoded by blueberry salt-resistant drought-resistant gene VcLON2 as well as application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Implementation example 1, VcLON2 Gene acquisition

[0024] 1.1 Extraction of RNA

[0025] (1) Take 0.1-0.2 g of blueberry tissue in a mortar, grind it into powder with liquid nitrogen, and quickly transfer it to a 2 mL centrifuge tube preheated at 65 °C (the centrifuge tube contains 0.9 mL of CTAB extract (20 g / L CTAB, 20 g / L PVP, 100 mmol / L Tris-HCl (pH 8.0), 25 mmol / L EDTA, 2 mol / L NaCl) + 30 μL β-mercaptoethanol), shake with a vortex shaker Uniform; preheat in a 65°C water bath for 10 minutes, and mix by inverting 3 times during the period;

[0026] (2) After water bath, centrifuge at 7000 r / min for 5 min, take the supernatant, add 1 / 3 volume of 5 mol / L potassium acetate (pH 4.8) and mix well, then ice-bath for 30 min, then add 700 μL chloroform / isoamyl alcohol (24:1) Vortex to mix;

[0027] (3) Centrifuge at 12000 r / min for 5 min at 4°C, take the supernatant, add 500 μL water-saturated phenol (pH 5.2) and mix well, then add 500 μL chloroform / isoamyl alcohol (24...

Embodiment 2

[0080] Embodiment 2, the construction of plant overexpression vector

[0081] 2.1 pSH737-CaMV35S- VcLON2 Construction of Plant Overexpression Vector

[0082] To construct the overexpression vector pSH737-CaMV35S- VcLON2 ,exist VcLON2 Restriction sites BamHI and KpnⅠ were added to both ends of the start codon and stop codon, and primers VcLON2-BamHI-F were designed: 5'-CGCGGATCCATGGCGGAATCGGTCGAGCT-3'(SEQ ID NO.7) and VcLON2-KpnⅠ- R: 5'-CGGGGTACCTCATAACTTTGAGTGTTGTCTCCAAGGG-3' (SEQ ID NO.8), use blueberry cDNA (synthesized in step 1.2) for RT-PCR, recover the fragment, clone it into pMD19-T vector for identification and sequencing (see 1.3). The sequencing result obtained from the cloning in 1.3 of Example 1 VcLON2 The sequences were compared to prove that the amplified sequences were correct. PCR identification results see Figure 3-a , and then extract the plasmid by alkaline denaturation.

[0083] For the plant overexpression vector pSH737-CaMV35S, BamHI and KpnⅠ wer...

Embodiment 3

[0106] Embodiment 3, preparation and transformation of Agrobacterium competent

[0107] 3.1 Preparation of Competent Agrobacterium GV3101

[0108] (1) Pick a single colony of GV3101, inoculate it in YEP liquid medium containing Rif (50 mg / L), and culture overnight at 28°C with shaking at 180 rpm to OD 600 Value is 0.5;

[0109] (2) Transfer the bacterial solution into a 50 mL sterile centrifuge tube and place in an ice bath for 30 min;

[0110] (3) Centrifuge at 5000 rpm at 4°C for 5 min, remove the supernatant, and add 10 mL of 0.15 M pre-cooled NaCl solution to resuspend;

[0111] (4) 4°C, 5000 rpm, centrifuge for 5 min, remove the supernatant, add 2 mL of 20 mM pre-cooled CaCl 2 Solution resuspension;

[0112] (5) Divide into 100μL-200μL tubes, freeze in liquid nitrogen, and store at -80°C for later use.

[0113] 3.2 Transformation of Agrobacterium GV3101

[0114] (1) Take out the competent state from the -80℃ refrigerator, and slowly melt on ice;

[0115] (2) Take 3...

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Abstract

The invention discloses a blueberry salt-resistant drought-resistant gene VcLON2 and protein encoded by blueberry salt-resistant drought-resistant gene VcLON2 as well as application. VcLON2 has a sequence shown by SEQ ID NO.1 in a sequence list; by converting nicotiana benthamiana, the salt resistance and drought resistance of a transgenic plant can be obviously improved. The gene plays an important role in cultivating a salt-resistant and drought-resistant plant (especially a crop).

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to blueberry salt-tolerant and drought-resistant LON2 protease genes VcLON2 And its coded protein and application. Background technique [0002] Abiotic stresses such as drought and soil salinization are the main environmental factors limiting crop production today, and plant production is closely related to people's lives. Drought occurs frequently and in a wide range. Most countries in the world are affected by drought. Drought has become one of the most serious natural disasters in the world, causing economic losses of hundreds of billions of dollars every year. In terms of soil salinization, about 1 billion hectares of land in the world are salinized to varying degrees, accounting for about 10% of the world's total land area, and my country has nearly 100 million hectares of salinized land. Therefore, improving the salt tolerance and drought resistance of crops has be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/50C12N15/10C12N15/82A01H5/00
Inventor 陈文荣叶美娟余柯达邵俊怡吴洁慧郑晓梅
Owner ZHEJIANG NORMAL UNIVERSITY
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