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Protein MsLEA-D34 of medicago sativa 'WL525' at late embryonic development period and coding gene of protein MsLEA-D34

A technology of WL525 and alfalfa, applied in genetic engineering, plant genetic improvement, fermentation, etc., can solve the problems of lack of LEA protein and plant flowering time regulation, and achieve improved gene expression, early flowering time, and excellent salt tolerance Effect

Active Publication Date: 2020-04-03
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the relationship between LEA protein and the regulation of plant flowering time

Method used

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  • Protein MsLEA-D34 of medicago sativa 'WL525' at late embryonic development period and coding gene of protein MsLEA-D34
  • Protein MsLEA-D34 of medicago sativa 'WL525' at late embryonic development period and coding gene of protein MsLEA-D34
  • Protein MsLEA-D34 of medicago sativa 'WL525' at late embryonic development period and coding gene of protein MsLEA-D34

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, the cloning of alfalfa MsLEA-D34 gene

[0032] 1. Acquisition of plant material

[0033] Take the leaf tissue of normal growing alfalfa 'WL525' and extract RNA;

[0034] 2. Extraction of RNA

[0035] Total RNA was extracted with "TransZol Up Plant Total RNA Extraction Kit" (Beijing Quanshijin), the integrity of RNA was identified by gel electrophoresis, and the purity and concentration of RNA were determined by a spectrophotometer (Thermo Scientific Nanodrop 1000);

[0036] 3. Full-length cloning of genes

[0037] According to the nucleic acid sequence and protein function annotation results of Medicago truncatula 'A17' MTR_1g072090, the full-length MsLEA-D34 gene of Medicago truncatula 'WL525' was obtained.

[0038] The extracted RNA was reverse-transcribed (TransGen 1st Strand cDNA Synthesis Kit), using the first-strand cDNA as a template, using primers MsLEAD34-F (5'-ATGAGTCAAGAACAGCCCAGA-3') and MsLEA D34-R (5'-TCACCCGCTCTTCGTGTTC- 3') Perform PCR,...

Embodiment 2

[0040] Example 2, Sequence Information and Homology Analysis of Medicago 'WL525' MsLEA-D34 Gene

[0041] The full-length open reading frame sequence of the alfalfa 'WL525' MsLEA-D34 of the present invention is 795 bp, and the detailed sequence is shown in the sequence shown in SEQ ID NO.1. According to the sequence of the open reading frame, the amino acid sequence of the alfalfa 'WL525' MsLEA-D34 protein is deduced, with a total of 264 amino acid residues, a molecular weight of 27.30 kDa, and an isoelectric point (pI) of 4.65. See SEQ ID NO.2 for the detailed sequence display sequence.

[0042] The open reading frame sequence of Medicago truncatula 'WL525' MsLEA-D34 and the amino acid sequence of its encoded protein were searched for nucleotide and protein homology in NCBI using the BLAST program, and it was found that it was similar to the LEA gene of Medicago truncatula at the amino acid level The similarity is extremely high, and phylogenetic tree analysis shows that ther...

Embodiment 3

[0043] Example 3. Expression difference of alfalfa 'WL525' MsLEA-D34 gene under abiotic stress

[0044] 1. Material acquisition: Alfalfa 'WL525' Al, drought, and salt treatments were sampled at 0h, 0.5h, 1h, 3h, 6h, 9h, 12h, and 24h. The samples were respectively wrapped with aluminum platinum paper, put into liquid nitrogen, and then transferred to -80°C ultra-low temperature refrigerator for storage until use;

[0045] 2. The extraction of RNA, the determination of the integrity, purity and concentration of RNA and the acquisition of cDNA refer to Example 1;

[0046] 3. Design specific primers for real-time fluorescent quantitative PCR analysis of gene expression in various tissues. According to the obtained gene sequence of alfalfa 'WL525' MsLEA-D34, design a primer for quantitative analysis of MsLEA1 gene in Real-time PCR Specific primers, primer QMsLEA-D34-F (5'-AGAGGGAGTGATTGGAGCAG-3'), primer QMsLEA-D34-R (5'-TCGTGTTCTGGTTGAGCGT-3'), internal reference gene EF-α primer...

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Abstract

The invention discloses protein MsLEA-D34 of medicago sativa 'WL525' at late embryonic development period and a coding gene of the protein MsLEA-D34, and relates to the field of plant genetic engineering. The protein MsLEA-D34 comprises polypeptide as shown in an amino acid sequence SEQID NO:2, polypeptide in variation form of the polypeptide, active fragments of the protein MsLEA-D34 and active derivatives; and besides, the invention provides the coding gene of the protein MsLEA-D34, and the coding gene comprises a nucleotide sequence as shown in nucleotide sequence SEQID NO:1, degenerate sequences and variation sequences of the nucleotide sequence SEQID NO:1, and a sequence hybridized with the nucleotide sequence SEQID NO:1. According to the protein MsLEA-D34 and the coding gene disclosed by the invention, through a genetic engineering technique, cultivation of MsLEA-D34 transgenic plants is realized, adjustment of flowering time and increase of stress resistance of the MsLEA-D34 transgenic plants can be realized, an important theory basis is provided for cultivation of medicago sativa new species, adjustment and control of the flowering time and breeding work, and the protein MsLEA-D34 has great application value.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a late embryo developmental protein MsLEA-D34 of alfalfa 'WL525' and its coding gene. Background technique [0002] Alfalfa (Medicago sativa L.) is a perennial herb of the legume family. Because of its long green period, rapid growth, and high nutritional value, it is known as the "king of grass" and plays an important role in animal husbandry production. Forage legumes are economically important plants in the world, and flowering time has an important impact on yield. Therefore, understanding its flowering mechanism and rationally regulating the flowering time are very important for the yield and quality of forage grass. [0003] LEA protein (late embryogenesis abundant proteins, LEA) is highly expressed in the late embryonic development of plants, and later studies have found that it will also accumulate in large quantities when plants are under adversity stress. LEA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/54
CPCC07K14/415C12N15/827C12N15/8273
Inventor 安渊吕爱敏李姣姣苏连泰于晨
Owner SHANGHAI JIAO TONG UNIV
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