The Duplication And Functional Divergence Analysis Of Starch Synthase Gene Family In Plants

Starch is the major reserve polysaccharide of plants. Starch in crop grains is presently widely used not only in foodstuff and feedstuff, but also as raw material of industry and medicine. At present, the biosynthetic pathway and molecular mechanisms of plant starch has been basically clear, the key enzyme and starch synthase genes has carried out extensive research in genetics and biochemistry. In order to analyze the molecular evolution mechanism of starch biosynthesis in land plants and to explore the possible evolutionary basis which the plants with different evolution level owned different ability to synthesize starch, in this study, the molecular evolution of SS gene family were studied. Based on the starch synthase gene family as the research object, we combined the molecular biology technology and bioinformatics method identified and cloned the SSV gene which is the new isoform of SS gene family. Then, we selected the representative plants with the sequenced whole genome from lower plants to higher plants as our analysis of objects to analyze the duplication mode, the sequence feature, evolutionary selection pressure and the functional divergence of SS gene family. The main results of this study as follow:1.We cloned the 2190bp full length cDNA of ZmSSV in maize elite inbred 18-599 (GenBank accession number:KP192927). By searching and aligning sorghum genome as query of maize homologous sequence, and then we cloned SbSSV which the full length of ORF was 2.1kb (GenBank accession number:KP192926). Besides, we identified SSV in. other plants, included algae, moss, dicots and monocots by searching genome database. The putative protein sequence had the conserved regions including starch catalytic domain and glycosyltranferase domain, which were specific characteristics of starch synthase in plants. The phylogenetic analysis of SS showed that SSV was closed with the known SSFV, which they were produced by an early gene duplication, so we inferred that the SSV identified by us was the new isoform of SSs.2.The expression levels of ZmSSV and SbSSV were detected by real-time PCR. The result showed that ZmSSV mainly expressed in leaf and kernal at the grain filling stage. The transcript level increased after 10 DAP kernal with prolonging the grain filling and it’s level was obviously lower than those of 15 DAP kernel. The expression level of SbSSV in leaf was significantly higher than root and stem. Its expression was induced by light and had the circadian rhythm. The similar expression profiles were found in other homologues of SS, we inferred that SSV may be very important to starch synthesis in grain.3.We performed a comprehensive phylogenetic evolution analysis of SS gene family in ten plants genome (included the green algae, the moss, the lycophyte, the dicots, the monocots). Phylogenetic relationships of SS in all plant lineages showed that SS subfamily had been divided into two groups, each group included three separate clades. One group included GBSS, SSI and SSII; The other one included SSIII, SSIV and SSV. SS gene family was widely distributed in eukaryotes organism from alga to monocot, indicating that SS may originate from the early gene duplication and then they maybe occur functional divergence.4.Each SS subfamily have different copies in different plants because they experience different gene duplication and loss. In total, SS genes expanded among angiosperm genome and the number of duplication of SS gene in angiosperm was obviously more than lower plants. After functional divergence, crop could efficiently synthesize storage starch. The result also showed that the genome duplication or large fragment of chromosome duplication contributed to the expansion of SS genes.5.The gene structure of SS were highly conserved in each subfamily. The number of exon and intron in higher plants was more than lower plants. The structure was more complex than lower plants, inferring that they may have more fined expression and regulation based in intron.6.By multiple alignment and conserved sequence analysis, we found all SS were highly conserved in C-termini with GT-5 and GT-1, but little sequence similarity with N-termini. Besides, each subfamily had special conserved motif which may be related with substrate specificity of the enzyme. We also analyzed protein sequence among internal subfamily and found they had various motif among plants at different evolutionary level, inferring that the motif may be related with the ability and quantity of starch synthesis.7.Molecular evolutionary investigation indicated that some amino acid sites were affected by positive selection and induced to shift their evolutionary rate, it was likely to have contributed to functional divergence after diversification of the orthologous. But 95% sites were under purifying selection or neutral selection, so SS were relatively conservative during evolution.The starch which plant with different evolution level synthesized is variant on type, quantity, structure and purpose. The possible molecular evolutionary mechanisms in higher plants, especially cereal crops, is that SS genes expanded among angiosperm genome and retained in the subsequent evolution. Then, they had formed endosperm starch metabolic pathway with a fine expression and regulation, at the same time the formation of a specific conserved domains and its partial amino acid sites under positive selection, which promoted the ability of starch synthesis in cereal crops. Therefore higher plants have more stronger and efficient in starch biosynthesis.