| Amino acid permease(AAP) is a kind of transmembrane transport protein. It is widely involved in the uptake and transport of amino acids in plant. Amino acid permease family consists of eight members in Arabidopsis thaliana, called At AAP(1-8).Endosperm specificity promoter can promote target gene to express in the endosperm effectively and quickly. Our laboratory constructed a PTF101.1-GT1-AAP1 plant expression vector with endosperm specificity successfully in the early stage. In this study, the method of agrobacterium mediated transformation of Arabidopsis was used, through the resistance screening of two generations, and finally obtained positive plants of T2 generation as the transgenic materials.In this study, the nucleic acid and protein sequences of At AAP family were analyzed with bioinformatics method. The expression levels of At AAP(1-8) gene in the roots, stems, leaves, flowers and siliques of wild-type and transgenic plant were analyzed by q RT-PCR. The three physiological indexes of wild-type and transgenic plants were measured, which aimed to analysis the relationship between tissue expression quantity of At AAP family and the index of amino acids. The three physiological indexes included the content of free amino acids, total protein and 20 kinds of amino acids. Meanwhile, the analysis of tissue expression and physiological index in transgenic plants aims to increase the amino acid content of reproductive organs by overexpression of At AAP1 gene, and to make the AAP genes used in industrialization. The main results were as follows: 1: Bioinformatics results showed that: At AAP gene located at chromosome 1 and 5, the number of amino acids was among 466-493, and they were all basic amino acids. Protein secondary structure was mainly α-helix. At AAP family contained transmembranes, and they were all membrane transport protein. Gene structure contained introns and exons. At AAP gene family had the elements of hormonal response, stress response, organization express and physiological regulation. Phylogenetic tree showed that At AAP family had the closest relative with the Bn AAP family. 2: The relative expression quantity of At AAP family was different in roots, stems, leaves, flowers and siliques. Relative expression level of At AAP1 and At AAP7 gene was highly in siliques; Relative expression level of At AAP2 in siliques, flowers and leaves was higher than other tissues; Relative expression level of At AAP3 gene was highly in leaves and roots; Relative expression level of At AAP4 gene was highly in stems; Relative expression level of At AAP5 and At AAP6 gene was highly in leaves; Relative expression level of At AAP8 gene was highly in flowers. 3: The method of infection of inflorescence mediated by agrobacterium was transformed into Arabidopsis thaliana. Through the resistance screening of two generations, 24 positive plants of T2 generation were obtained as the transgenic materials. 4: The expression level of At AAP family appeared different degrees of up-regulation and down-regulation in roots, stems, leaves, flowers and siliques of transgenic plants. It was up-regulated clearly in roots, siliques and stems. At AAP1 and At AAP6 were up regulated in roots. At AAP3, At AAP5 and At AAP7 were up regulated in stems. At AAP1 and At AAP2 were up regulated in siliques. 5: The changes of physiological indexes in each tissue of wild-type and transgenic plants were analyzed and founded that the contents of free amino acids, total protein and 20 kinds of amino acids in reproductive organs of transgenic plants were higher than wild-type by overexpression of At AAP1 gene. |
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