Cloning And Functional Analysis Of SlCAT2Gene In Tomato

Amino acid transporters, which mediate the transport of amino acids, play animportant role in plant growth and development. Based on sequence similarities, theplant transporters can be classified into two major superfamilies: the ATFs (the aminoacid transporter family) and the APCs (the amino acid polyamine choline transporter).The CATs family (cationic amino acid transporters) is subfamily of the APCs. TheAPCs are poorly studied in plant and only a few members of the CATs family havebeen studied.In this paper, we cloned SlCAT2a and SlCAT2b gene from tomoto, studied theirgene sequence and the protein characteristics. We analysed the phylogenetic relations ofCATs famiy proteins and the subcellular localizations of SlCAT2a. We also analysedtheir temporal and spatial expression patterns. More, we constructed the overexpressionvectors SlCAT2a and SlCAT2b and the antisense vector AsCAT2. The plant expressivevectors were transformed into tomato by Agrobacterium-mediated method. In addition,we studied the substrate specificity of SlCAT2a and SlCAT2b proteins to investigate thepotential physiological function of them in plant growth and development. The mainresults are as follows:1. Based on gene sequence homology, two possible EST sequences of tomatoSlCAT2protein were found from tomato genome database (SGN), named SlCAT2a andSlCAT2b. We found their1476bp were the same and only the5’ nucleotide sequenceexsited difference. In addition, SlCAT2a and SlCAT2b were located on chromosome10,the position of10exons on the genome were the same.2. We cloned SlCAT2a and SlCAT2b using RT-PCR. The result showed that thecloned full-length cDNA of SlCAT2a was1950bp in length, which encodes650aminoacids, and SlCAT2b was1635bp in length, which encodes545amino acids. SlCAT2aprotein contained14putative transmembrane domains and SlCAT2b protein contained12putative transmembrane domains in silico analysis. SlCAT2a and SlCAT2b proteinswere an ortholog of AtCAT2and PtCAT2in phylogenetic analysis. SlCAT2a localizdto the tonoplast through the subcellular localizations of the SlCAT2a protein fusioned tothe green fluorescent protein (GFP).3. Real-time PCR was used to investigate the temporal and spatial expressionpatterns of SlCAT2a and SlCAT2b. These results indicated that the SlCAT2a was mainly expressed in flower organ. The expression level was higher obviously than other organs.SlCAT2b gene was detected in various tissues, but there were not obvious difference.SlCAT2a was found to be expressed in all the parts of flowers at anthesis stages, but itsexpression level was the highest in stamen. SlCAT2b gene was detected in various partsof flowers, but there were also not obvious difference.4. To study the physiological function of SlCAT2, the overexpression vectors ofSlCAT2a and SlCAT2b and the antisense vector AsCAT2were constructed. The plantexpressive vectors were transformed into tomato by Agrobacterium-mediated method.We used multiple methods to identify transgenic plants, including GUS staining, PCRand medium selection including antibiotics. We more tested the expression levels ofSlCAT2a and SlCAT2b in transgenic plants by RT-PCR.5. Observing the phenotypes of transgenic plants, we found the inflorescencemorphology abnormal, flower numbers increased by statistics, flower stalks appeared inthe cambium of transgenic plants AsCAT2. These phenotypes indicate that SlCAT2maybe involved in the process of flower development.6. The yeast expression vectors of SlCAT2a and SlCAT2b were constructed. Toanalyze the substrate specificity of SlCAT2a and SlCAT2b, the yeast heterologousexpression systems were used. Because the yeast growth of the experimental group andcontrol group on a variety of nutrient medium are synchronous, so it’s not clear aboutthe substrate specificity of SlCAT2a and SlCAT2b, and whether they have differences.