Short-tube lycoris S-adenosylmethionine synthetase and its coding gene and use

A technology for adenosylmethionine and synthase, which is applied to Lycoris S-adenosylmethionine synthase and its encoding gene and application fields, and can solve crop yield reduction, ecological environment deterioration, influence on plant growth and development, etc. question

Inactive Publication Date: 2013-11-06
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Saline-alkali soil will affect the growth and development of plants

Method used

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  • Short-tube lycoris S-adenosylmethionine synthetase and its coding gene and use
  • Short-tube lycoris S-adenosylmethionine synthetase and its coding gene and use
  • Short-tube lycoris S-adenosylmethionine synthetase and its coding gene and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1. Cloning of gene LrSAMS

[0014] The Lycoris radiata S-adenosylmethionine synthase gene was obtained by establishing a full-length cDNA library of Lycoris radiata treated with NaCl, and comparing the cDNA sequence with the NCBI database for homology.

[0015] 1.1 Construction of a full-length cDNA library of Lycoris radiata treated with NaCl

[0016] According to the operating procedure of Clontech's Creator SMART cDNA Library Construction Kit, a full-length cDNA library of Lycoris radiata treated with NaCl was constructed.

[0017] 1.2 LrSAMS full-length sequence

[0018] After a large number of sequencing of the library, a highly homologous sequence with the S-adenosylmethionine synthase gene of other organisms was found. After sequence alignment, it was confirmed that the full length of the Lycoris radiata S-adenosylmethionine synthase gene was obtained. Sequence, named LrSAMS. Sequence analysis showed that the open reading frame of the gene was 1206bp, encoding 40...

Embodiment 2

[0022] Example 2. Expression of LrSAMS under salt treatment

[0023] In order to verify whether LrSAMS was induced by salt treatment, RNA from Lycoris radiata leaves treated with 400mM NaCl for 1, 3, 6, 9, 12, 24, 48 h was extracted and semi-quantitative RT-PCR was performed. Design primers SAM-F (5'-ACAAAGGTTGACAGGAGTGG-3') and SAM-R (5'-GAAGGCATCAA ACCAGAGAG-3') to amplify a fragment of approximately 430bp; actin was used as an internal reference, and primer actin-F (5'-CTATTCTTCGTCTTGATCTCGC- 3') and actin-R (5'-CAAACTCATCGTA CTCTCCCTT-3') amplified fragment length is 576bp. by figure 2 It can be seen that when the plants are exposed to salt stress, the expression of LrSAMS gene increases, indicating that LrSAMS is induced by salt stress; with the extension of NaCl treatment time, the gene expression also gradually increases. The expression level of LrSAMS has increased significantly after 3 hours of NaCl treatment. The maximum expression level was after 24h.

Embodiment 3

[0024] Example 3. Functional verification of LrSAMS gene

[0025] 3.1 Highly efficient expression of LrSAMS gene in E. coli

[0026] In order to show the coding function of the LrSAMS gene, the present invention cloned the LrSAMS gene into the Nde I and Xho I sites of pET29a(+) of novogen company, and transformed the E. coli BL21(DE3) to obtain high-efficiency expression.

[0027] The present invention realizes the high-efficiency expression of LrSAMS by inserting the LrSAMS gene into the multiple cloning site of the pET29a(+) high-efficiency expression vector. Design and synthesize a pair of specific primers according to the LrSAMS gene, and PCR amplify the gene coding sequence with specific restriction sites at both ends. The primer sequence is:

[0028] SAM-F: 5’-GGCATATGGCCGACACCTTCCTC-3’

[0029] SAM-R: 5’-CCCTCGAGAGCT GCTGGCTTCTCCC-3’

[0030] The LrSAMS gene and vector were digested with restriction endonucleases (Nde I and Xho I) and then ligated. The ligation product was transf...

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Abstract

The invention provides a short-tube lycoris S-adenosylmethionine synthetase and its coding gene LrSAMS and use. After salt treatment, as time went on, gene LrSAMS expression is improved. The coding gene LrSAMS or a DNA sequence which is homologous with the coding gene LrSAMS and can code the same functional protein is introduced into escherichia coli and compared with the original strain, the recombinant strain has higher high-concentration NaCl and KCl resistance. The short-tube lycoris S-adenosylmethionine synthetase and its coding gene have important theoretical and practical significances for research on a plant salt-resistant mechanism, improvement of plant salt and alkali resistance, and improvement of relative characters, have important effects in improvement of plant salt and alkali-resistant gene engineering, and have a wide application prospect.

Description

Technical field [0001] The present invention relates to the fields of molecular biology and genetic engineering, and more specifically, to Lycoris radiata S-adenosylmethionine synthase (SAMS) and its coding genes and applications. Background technique [0002] With the modernization of industry, the expansion of irrigated land and greenhouse area, soil secondary salinization is becoming more and more serious. Taking saline as an example, there are nearly 1.5 billion acres of saline-alkali land in the country, and more than 20 million hectares of saline-alkali wasteland need to be cultivated. Saline soil will affect the growth and development of plants, reduce crop yields, and make the ecological environment worse. How to improve crop resistance to osmotic stress and make full use of saline-alkali land resources to increase arable land, increase food production and save water resources has become a major issue in tapping the production potential of saline-alkali land and maintaini...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N5/10C12N1/21A01H5/00
Inventor 李晓丹汪仁夏冰江玉梅贺佳彭峰
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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