Cloning, Expression And Function Analysis Of Stay-green Genes In Soybean And Arabidopsis

It has a potential value of stay green gene in generation of transgenic crop varieties that improve the plant photosynthetic capacities and further overcome the pre-senescence which frequently occurred in crop production, by which for improvement of the crop yield and quality. In this study, stay green genes referred to GmSGR1 and AtNYE1, derived from soybean and Arabidopsis, respectively, have been cloned and functional analyzed. The main results were as follows:1. The open reading frame of GmSGR1 was 786 bp, encoding 261 amino acids with a molecular weight of 29.41 kDa and an isoelectric point of 8.45. GmSGR1 is located at the mitochondria and chloroplast at the subcellular level based on the molecular predication analysis. Five potential phosphorylation sites and three O-glycosylation sites have been identified in GmSGR1.2. Phylogenetic analysis suggested that GmSGR1 had high similarities with pea SGR (AB303331 and AB303332), pepper sgr (AM746208) and tobacco (EU294209). These genes possibly have same evolution ancestor.3. At the DNA level, GmSGR1 is composed of four exons and three introns. The length of these introns is 100 bp, 682 bp, and 105 bp, respectively. The bases at positions that connected the exon and intron was followed the Chambon rule.4. During the leaf natural senescence, the expression level of GmSGR1 was increased. Exogenous abscisic acid (ABA) could induce the expression of GmSGR1, whereas 6- benzylpurine (6-BA) could down-regulated the expression of GmSGR1. Thus, GmSGR1 is a typical senescence-associated gene (SAG) with up-regulated expression pattern, and responded dramatically to the ABA and 6-BA signaling.5. During the late growth stage, the contents of chlorophyll a, b and carotenoid in the leaves that located at the lower or middle part decreased in the transgenic plants that overexpressed GmSGR1, in contrast to the control (CK), showing a pattern to be higher expression of the target gene, to be lower of the above photosynthetic pigment contents. Therefore, overexpression of GmSGR1 in plants could fasten the degradation of photosynthetic pigments and promote the leaf senescence progression.6. It is found that the conserved regulatory elements such as TATA box and CAAT box were located at the GmSGR1 promoter, with the positions to be close to the translation start codon ATG. Based on the expression pattern analysis of Gus, a reporter gene that is driven by various lengths of the GmSGR1 promoter segment, it is suggested that the Gus expression level was elevated with the prolonged promoter segments. Segment lengths of the promoter such as 658 bp and 811 bp chiefly drive the reporter gene to be predominantly expressed in the vascular bundles.7. The open reading frame of AtNYE1 was 807 bp, encoding 268 amino acids with a molecular weight of 30.05 kDa and an isoelectric point of 8.57. AtNYE1 is located at the chloroplast at the subcellular level based on the molecular predication analysis. Fifteen potential phosphorylation sites and one O-glycosylation sites have been identified in AtNYE1.8. Compared to the control (CK), the contents of chlorophyll a, b and carotenoid in the transgenic plants that overexpressed AtNYE1 were all decreased during the late growth stage, showing a pattern to be higher expression of the target gene, to be lower of the above photosynthetic pigment contents. Which were similar to the results of GmSGR1 mentioned above. Therefore, Down-regulation of AtNYE1 in plants could probably delay the degradation of photosynthetic pigments and the leaf senescence progression.9. Except the TATA box and CAAT box, AtNYE1 promoter also contains other important regulatory elements that respond to the signals of development, biotic and abiotic stresses. The histochemical staining of Gus that was driven by various lengths of AtNYE1 promoter segments suggested less Gus stained when the promoter region of AtNYE1 was shorter than 1482 bp. The much stronger Gus staining when driven by 1882 bp promoter region indicated that the enhancer(s) located at -1483 and -1882 upstream of the translation start codon of AtNYE1. A constitutive pattern with strong capability endowed by 1882 bp AtNYE1 promoter suggested that this promoter has a potential value in the transgenic research and applications in the future.