Cloning And Functions Of Stay-green Gene(LeSGR1) In Tomato (Lycopersicon Esculentum)

Stay-green mutations in a variety of plants have similar phenotype: delayed senescence, a block to chlorophyll degradation or delayed degradation. However, the molecular mechanism of stay-green underlying different genetic background is quite different. After reviewing the advances and achievements in research on physiological-biochemical (chlorophyllase metabolic pathway), hereditary feature and molecular biology of stay-green mutants, this research addressed the SGR gene that regulates stay-green phenotype. SGR is a nuclear gene. Its specific expression is induced by senescence; hence, it is an SSG gene. Its functional deletion results in stay-green phenotype of higher plants. Research on it is still in its infancy. We investigated the SGR homologous gene, LeSGR1, from AC++and gf mutant of tomato, and obtained the main results as follows:(1) The SGR homologous gene cloned from tomato is named as LeSGR1. Its cDNA is 1 206 bp in length, with a molecular mass of 30 kDa and isoelectric point of 8.74. It consists of an 816 bp ORF which encodes a 272-amino-acid sequence, a partial 5'UTR of about 145 bp, and a 3'UTR of 259 bp. According to the blast results in GenBank database ,the cDNA nucleotide sequence shares 78 % and 75 % identity with arabidopsis thaliana and soybean, respectively, and 66% identity with monocotyledons plants which have a relatively distant genetic relationship.(2) Bioinformatics prediction on LeSGR1 gene nucleotide and encoded amino acid residue sequence indicates the encoded protein possibly has 4 phosphorylation sites at serines 202, 205 and 217, and threonine 82, and 3 O-glycosylation sites at amino acid residues 6, 216 and 219, respectively. This protein has no trans-membrane structure. There are 9 obviousβ-folds and 7α-helices in its secondary structure. The active subcellular motif may locate in a chloroplast.(3) The DNA sequence of LeSGR1 gene is approximately 2.1 kbp, in which 3 introns respectively of 100 bp, 1118 bp and 167 bp separate 4 exons apart. The exon-intron boundary of the gene follows Chambon's rule for the splice donor and acceptor sites. The 0.9 kbp upstream flanking region of 5′UTR in LeSGR1 gene contains several TATA-box, GC-box, CAAT-box ,and other cis-acting elements such as ethylene reaction element, heat shock element(HSE), MBS element, defense and pressure response element, Salicylic acid response (TCA-element).(4) LeSGR1 gene expression analysis reveals the mRNA level is very low in young leaves and mature leaves, barely detected .However, the mRNA of LeSGR1 is expressed considerably in aged leaves, which implies the specific expression of LeSGR1 is induced by senescence and this gene is an SSG gene. The LeSGR1 expression is upregulated obviously in tomato fruit, especially in mature period B and the periods afterward. The LeSGR1 expression is affected in tomato ethylene mutant compared with that in AC++ tomato. This indicates ethylene upregulates SGR gene expression. Hypohydration and dark both induce SGR gene expression.(5) According to siRNA sequence design principles, a RNAi expression vector of targeting gene LeSGR1 was constructed and transformed into AC++ tomato. Eight transgenic plants were obtained, which are the necessities for further probe into the functions of LeSGR1 gene.(6) There is no significant difference between the chlorophyll content in mature leaves and MG fruits of Ac++ and that of gf mutant tomatoes. Nevertheless in aged leaves and ripe fruits, chlorophyll content is obviously higher in gf than in AC++. The aged leaves of the gf mutant appears stay-green phenotype, and its ripe fruits become rusty due to chlorophyll non-degradation.(7) The SGR gene cloned from gf mutant has one mutated nucleotide (A→T) in the highly conserved region of SGR gene, and this mutation results in an amino acid change (Arg→Ser). After developing an overexpression vector 35S∷L eSGR1, a recombinant plasmid was transformed successfully into gf mutant. This research provides the foundation for further study on the relationship between this nucleotide mutation and the stay-green phenotype, on the molecular mechanism of gf stay-green.