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Stress-resistant gene SiRLK35 of setaria italica L.Beauv. as well as encoding protein and application

A kind of millet and gene technology, applied in the field of genetic engineering, can solve the problems of millet being undamaged and achieve the effect of enhancing plant stress resistance

Active Publication Date: 2017-06-13
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lag in genome sequencing, millet has not received the attention of the international scientific community for a long time

Method used

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  • Stress-resistant gene SiRLK35 of setaria italica L.Beauv. as well as encoding protein and application
  • Stress-resistant gene SiRLK35 of setaria italica L.Beauv. as well as encoding protein and application
  • Stress-resistant gene SiRLK35 of setaria italica L.Beauv. as well as encoding protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Cloning of SiRLK35 gene in millet

[0045] Search the NCBI database, design specific primers based on the sequences provided in the database, and use the extracted millet RNA as a template. PCR reaction system (20 μl): cDNA: 1 μl, upstream and downstream primers 0.5 μl, dNTP: 2 μl, HiFi : 1μl, 10×BufferII: 2μl, ddH2O: 13μl; PCR reaction steps: 94°C 5min, 94°C 30s, 53°C 30s, 72°C 1min20s, 34 cycles, 72°C 10min; PCR product electrophoresis detection results are as follows figure 1 shown. A full-length sequence of the SiRLK35 gene with a full length of 1188bp was obtained ( figure 1 ). The sequence obtained by PCR was subjected to T 4 The ligase was inserted into pMD18T to transform Escherichia coli DH5α, and the sequence was sequenced as shown in SEQ ID No.1. Bioinformatics analysis shows that the gene encodes 392 amino acids, and the sequence is shown in SEQ ID No.2. It belongs to the class of serine / threonine protein kinases.

[0046] The specific pr...

Embodiment 2

[0049] Example 2: Analysis of the expression pattern of the gene SiRLK35 under drought, salt stress and different hormone treatments

[0050] 1. Analysis of the expression pattern of the gene SiRLK35 under drought and salt stress

[0051] 1. Extraction of RNA from millet

[0052] The millet seedlings with consistent growth were treated as follows: (1) 20% PEG simulated drought for 0h, 6h, 12h and 24h; (2) 200mmol / L NaCl for 0h, 6h, 12h and 24h respectively. 1 g of each treatment was taken, quick-frozen in liquid nitrogen, and stored in a -80°C refrigerator.

[0053] All test equipment is autoclaved for 1 hour, mortar, pestle, and medicine spoon are dried, wrapped in tin foil and baked in an oven at 180°C for 6-8 hours, and set aside;

[0054] According to the method provided by the Trizol kit, take 0.1g of plant material in a liquid nitrogen pre-cooled mortar, grind it to powder, transfer it to a 1.5ml centrifuge tube, add 1ml Trizol and mix well; place at room temperature f...

Embodiment 3

[0072] Embodiment 3: Construction and identification of SiRLK35 prokaryotic expression vector

[0073] 1. Construction of SiRLK35 gene prokaryotic expression vector

[0074] 1. With the PCR product of the SiRLK35 full-length gene of embodiment 1 as template, with SiRLK35S12:5'- GGATCC ATGGAAGCCTTCATGGGCATC-3' (restriction site is underlined) (SEQ ID NO.9), SiRLK35A12: 5'- GTC GAC TAAATTT GACTTCATTTTCAGCCAGCTG-3' (underline shows restriction site) (SEQ ID NO.10) is a primer;

[0075] 2. PCR reaction system (20μl): cDNA: 1μl, upstream and downstream primers 0.5μl each, dNTP: 2μl, HiFi: 1μl, 10×BufferII: 2μl, ddH2O: 13μl; PCR reaction steps: 94℃ for 5min, 94℃ for 30s , 53°C for 30s, 72°C for 1min20s, 34 cycles, 72°C for 10min; obtain the target band;

[0076] 3. Digest prokaryotic expression vector pET28a and SiRLK35 gene fragments with BamH I / Sal I, after purification and recovery, use T 4 The ligase was ligated in a circulating pump at 22°C for 5 minutes to obtain the rec...

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Abstract

The invention discloses a stress-resistant gene SiRLK35 of setaria italica L.Beauv. as well as encoding protein and an application. Receptor-like protein kinase SiRLK35 is separated from setaria italica L.Beauv., the nucleotide sequence of the receptor-like protein kinase SiRLK35is shown in SEQ ID NO.1, and the gene encoding protein product belongs to serine / threonine protein kinase. The expression pattern of the gene in a stress condition is researched, the prokaryotic expression vector of the gene is established, the gene can be expressed in the protein level as is verified, the functions of the gene are primarily verified with a dot assay through a gene prokaryotic expression system, meanwhile, the growth curve of the gene under a salt treatment condition is detected, the gene is heterologously transformed into rice, a corresponding transgenetic plant is obtained, the functions of the transgenetic plant in salt resistance and stress resistance are identified primarily, targeted quality and character improvement on setaria italica L.Beauv. is further performed purposefully, the new stress-resistant germplasm of setaria italica L.Beauv. is innovated, and a guidance function is performed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to millet stress-resistant gene SiRLK35, its encoded protein and its application. Background technique [0002] Drought is one of the important limiting factors affecting the normal growth and development of plants. Due to the uneven distribution of water resources in time and space, as well as the worldwide water shortage caused by environmental changes, it affects 64% of the world's cultivated land. As far as my country's current situation is concerned, the annual water shortage in agricultural production is 30 billion cubic meters, and the grain loss caused by drought is about 40 billion catties per year. The shortage of water resources has seriously threatened food security. [0003] When plants are subjected to drought stress, the most direct effect is dehydration of plant cells. After the cell is dehydrated, the cell membrane and cell wall are separated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/10C12N15/82A01H5/00
CPCC12N9/1205C12N15/8273
Inventor 刘炜王一帆潘教文王庆国李臻
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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