Medicago sativa WL525 polygalacturonase MsPG1 and encoding gene and application thereof

A technology of polygalactose and alfalfa, which is applied in the field of plant genetic engineering, can solve problems that have not yet been solved, and achieve the effect of improving aluminum toxicity and great application value

Active Publication Date: 2020-04-14
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 related to the gene sequence of alfalfa aluminum tolerance PG coding

Method used

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  • Medicago sativa WL525 polygalacturonase MsPG1 and encoding gene and application thereof
  • Medicago sativa WL525 polygalacturonase MsPG1 and encoding gene and application thereof
  • Medicago sativa WL525 polygalacturonase MsPG1 and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, the cloning of alfalfa MsPG1 gene

[0031] 1. Acquisition of plant material

[0032] Take the leaf tissue of alfalfa WL525 after Al treatment for RNA extraction;

[0033] 2. Extraction of RNA

[0034] Total RNA was extracted with "TransZol Up Plant Total RNA Extraction Kit", 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);

[0035] 3. Full-length cloning of genes

[0036] According to the nucleic acid sequence and protein function annotation results of Medicago truncatula 'A17'A_27_P130781, the full-length gene of Medicago truncatula WL525MsPG1 was obtained.

[0037]Combining the nucleotide sequence results of A_27_P130781 with NCBI's ORF Finding (http: / / www.ncbi.nlm.nih.gov / gorf) prediction, the ORF reading frame of the Medicago WL525MsPG1 gene was found. According to the obtained sequence, the specific primers ORF-F (5'-ATGTTTC...

Embodiment 2

[0038] Example 2, Sequence Information and Homology Analysis of Medicago WL525MsPG1 Gene

[0039] The full-length open reading frame sequence of the alfalfa WL525MsPG1 of the present invention is 1215bp, and the detailed sequence is shown in SEQ ID NO.1. According to the sequence of the open reading frame, the amino acid sequence of the alfalfa WL525MsPG1 protein is deduced, with a total of 404 amino acid residues, a molecular weight of 43.83 kDa, and an isoelectric point (pI) of 9.52. The detailed sequence is shown in SEQ ID NO.2.

[0040] Using the complete amino acid sequence of the PG gene, the 51 PG amino acid sequences from different plant species were compared using the MUSCLE program in MEGA6.0. The amino acid sequences of 51 PGs were obtained from the NCBI website (http: / / www.ncbi.nlm.nih.gov / genbank / ). A phylogenetic tree was constructed using the neighbor joinhg (NJ) method. The main parameters are set as follows: distance model, Poisson model; gene tree robustnes...

Embodiment 3

[0042] Example 3, Expression differences of alfalfa WL525MsPG1 gene under abiotic stress

[0043] 1. Material acquisition: Alfalfa WL525 was treated with Al, and samples were taken at 0h, 1h, 3h, 6h, 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;

[0044] 2. The extraction of RNA, the determination of the integrity, purity and concentration of RNA and the acquisition of cDNA refer to Example 1;

[0045] 3. Design specific primers for real-time fluorescent quantitative PCR analysis of gene expression in various tissues, according to the obtained alfalfa WL525MsPG1 gene sequence, design specific primers for quantitative analysis of MsPG1 gene in Real-time PCR, primers qPG1-F(5'-TCCAGGGCATGGTATCAGCA-3'), primer qPG1-R(5'-CAAATCCACCTCCACCCTGC-3'), internal reference gene is elongation factor EF-α gene, primer is EF-F(5'-GCACCAGTGCTCG...

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Abstract

The invention discloses medicago sativa WL525 polygalacturonase MsPG1 and an encoding gene and application thereof, and relates to the technical field of plant genetic engineering. The polygalacturonase MsPG1 comprises polypeptide represented by the amino acid sequence as shown in SEQ ID NO: 2. The polypeptide is a variant form polypeptide. The invention also discloses an active fragment and an active derivative of the protein MsLEA-D34. The invention further provides an encoding gene of the polygalacturonase MsPG1. The gene comprises a nucleotide sequence as shown in a nucleotide sequence SEQID NO: 1, a degenerate sequence and a variation sequence of the nucleotide sequence SEQ ID NO: 1, and a sequence capable of hybridizing with the nucleotide sequence SEQ ID NO: 1. According to the method, the MsPG1 transgenic plant is cultivated by utilizing a genetic engineering technology, the aluminum toxicity resistance of the plant is remarkably improved, and an important theoretical basis isprovided for the cultivation and breeding work of a new aluminum toxicity resistant medicago sativa variety. The method has great application value.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to an alfalfa WL525 polygalacturonase MsPG1 and its coding gene and application. Background technique [0002] Alfalfa (Medicago sativa L.) is a perennial herbaceous plant of the genus Alfalfa, which is widely planted all over the world. Alfalfa grows rapidly, has a long green period, strong reproductive ability and high nutritional value. It is an important feed crop and plays an important role in animal husbandry production. With the decrease of rainfall, the frequent human activities, the increase of soil desertification, and the sharp deterioration of the ecological environment, the grassland vegetation has been seriously degraded, and the yield of alfalfa has been greatly affected. The continuous development of animal husbandry puts forward higher requirements on the yield and quality of alfalfa. Therefore, cultivating new varieties of alfalfa with high yiel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/82A01H5/00A01H6/20
CPCC12N9/2402C12Y302/01015C12Y302/01067C12N15/8271
Inventor 安渊李姣姣周鹏苏连泰吕爱敏张钰靖
Owner SHANGHAI JIAO TONG UNIV
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