Preliminary Research On The Expression Pattern And Function Of Nodulin-like Gene At1g01070 In Arabidopsis Thaliana

Expression of plant genes induced by symbiotic nitrogen fixation microbes upon nodulation were initially refered as nodulins genes in legumes, which play an important role in various processes besides nitrogen fixation and the signal transduction, material transport, osmotic stress. Further study shows, some homologous genes of nodulin gene were found in non-leguminous plants, and named nodulin-like gene. Reports suggest these nodulin-like genes play important roles in non leguminous plant. Earlier experimental acquired At1g01070 over-expression transgenic arabidopsis thaliana plants through the method of agrobacterium-mediated transformation. Based on the previous study, we acquired homozygous plants of At1g01070 over-expression arabidopsis thaliana and explored the expression patterns and functions of At1g01070 by comparing with Colombia wild-type arabidopsis.The results of this study are as follows:1. We analyze the the structure of At1g01070 gene and the protein encoded by it though bioinformatic software.The result shows At1g01070 has two different transcripts through alternatively splicing pattern. ORF finder showes that the coding region of At1g01070.1 gene has 1089 nucleotides and encodes 365 amino acids, and At1g01070.2 gene has 957 nucleotides and encodes 318 amino acids. Results of phylogenetic analysis show At1g01070 is most similar to wat1-relate gene in Camelina sativa and a kind of lectin gene in Capsella rubella. Conserved domains prediction shows that protein encoded by At1g01070 contains EamA domains. The EamA domain(DUF6 domain) is considered as symbol of transport protein. Therefore,we presumed that the protein encoded by At1g01070 may have function as carrier. Proteins encoded by At1g01070 gene contain several transmembrane domains, suggests the proteins belong to membrance proteins. The spatial structure of protein encoded by At1g01070 is similar to a kind of auxin carrier WAT1, composed of several α-helix and random coil domains, which suggests protein encoded by At1g01070 gene may act as a kind of carrier.2. The expression site of At1g01070 was tested by means of GUS staining and subcellular localization. GUS staining results indicat that At1g01070 expressed in all organs such as root, stem, rosette leave, cauline leave, flower and silique. But the gene showed fibrous-specific expression in each organ and tissue,which also indicate At1g01070 has transport function. The results of subcellular localization show that the protein encoded by At1g01070 mainly located in the periphery parts of the cell, then plasmolysis experiment demonstrated the protein located in the membranous structure. We confirmed At1g01070 gene encodes the membrane-located transport protein.3.The homozygous lines of super-At1g01070.1 and super-At1g01070.2 arabidopsis were screened and identificationed by means of Kan resistance selection and PCR. To detect the relative expression level of target gene in transgenic arabidopsis, mRNA of transgenic and wild arabidopsis were tested by real-time PCR. The results show that the expression level of At1g01070.1 in super-At1g01070.1 arabidopsis has been raised 6-26 times and the expression level of At1g01070.2 in super-At1g01070.2 arabidopsis has been raised 4-20 times compared with that in wild arabidopsis.4.The results of phenotype analysis between transgenic and wild arabidopsis revealed that radicles of At1g01070 overexpressing arabidopsis were longer than that the wild. The leaves and stems of the transgenic arabidopsis growed stonger and higher than the wild under the same normal conditions. The above phenomenons suggest that over expression of At1g01070 could promote the growth of arabidopsis plants. Except that, line 91 and 111 of super-At1g01070.2 arabidopsis show a series of characteristics such as flowing later, growing faster and higher compared with the wild.This article shows Arabidopsis nodulin-like gene At1g01070 encodes transmembrane protein. At1g01070 has the function of promoting root and stem growing by over-expressing in transgenic arabidopsis. Bioinformatic analysis speculated that the gene encodes a protein with a function of auxin transport. Our study would lay a foundation of in-depth study on nodulin-like gene function in non-legume plants.