Arabidopis thaliana AAP1 gene cloning and plant expression vector construction method

Abstract

The invention discloses an arabidopis thaliana AAP1 gene cloning and plant expression vector construction method, and the method is as follows: total RNA of an arabidopis thaliana seedling is extracted by a TRIzol method, the OD260 / OD280 ratio is determined to be 2.014; gel recovery of PCR (polymerase chain reaction) products is performed, recovered products are connected with a PMD-18T carrier, after conversion is completed, plasmid is extracted, PCR and enzyme digestion verification of the extracted plasmid is performed, a 1600bp DNA band is obtained by PCR verification of the plasmid; sequencing reveals that the gene ORF1458bp encodes a polypeptide of 485 amino acids, after respective enzyme digestion of a target gene and pCAMBIA3300 vector by use of restriction enzyme EcoR I and Xba I, the target gene is directionally connected to a plant expression vector, after conversion is completed, plasmid is extracted, and PCR and enzyme digestion verification of the extracted plasmid is performed; a 1600bp DNA band is obtained by PCR verification of the plasmid; by enzyme digestion verification, a 8300bp pCAMBIA3300 vector and a 1600bp target fragment are respectively obtained. The vector is successfully constructed, and is renamed P3300-AAP1.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and relates to a method for cloning Arabidopsis AAP1 gene and constructing a plant expression vector. Background technique [0002] The synthesis of plant seed storage proteins is regulated by multiple factors, and protein accumulation largely depends on the supply of amino acids. If the supply of amino acids is sufficient, the protein content in plants can be greatly increased. Amino acid permease (AAP) gene is an important amino acid transporter in Arabidopsis, which plays an important role in tap root, lateral root, root hair and root epidermal cells. Studies by Yong et al. have shown that overexpression of AAP1 gene can significantly improve Arabidopsis The absorption capacity of neutral amino acids, glutamate and histidine makes the content of amino acids in the root group significantly increase, and then the protein content increases. AAP is an integral membrane protein that couples ...

AI Technical Summary

Problems solved by technology

Since the AAP1 gene has the function of transporting amino acids, which can increase the amino acid content in plants, we imagine that the AAP1 gene can be transferred into common corn species to breed high-protein corn. High-protein corn has the characteristics of high essential amino acid content and nutrient enrichment. It is a high-quality feed for animal husbandry; the feeding value of common corn is higher than that of other grains, and the feed value of high-protein corn is better than that of common corn, and the application and key technology innovation of AAP1 in corn have not been reported abroad

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