The Evolution Of CesA/Csl Superfamily And Gene Function Of CesA In Saccharum

Sugarcane is the world’s largest crop by production biomass that mainly contains cellulose, and is considered to be the most successful domesticated C4 crop. The biological characteristics of high biomass made the crop as the most efficient source for producing fuel ethanol. However, due to the complex of genetic background in sugarcane, the gene function and genetic basis are still uncovered so far. In this study, aim to investigate the molecular basis for the gene involving in cellulose accumulation, we identified the cellulose synthase genes by comparative genomics and explored the gene function by using model plant Arabidopsis the main results as follows.（1） Based on the comparative genomics analysis between sorghum and Saccharum species, hybridization probes for forty-six cellulose synthase superfamily genes were designed for screening a high genome coverage Saccharum spontaneum BAC library. The results showed that, besides CesA8, forty-five genes of orthologous cellulose synthase gene superfamily were identified and sequenced from the BAC libraries. The absent orthologous gene in BAC libraries, CesA8 was cloned by using cDNA template PCR. Phylogenetic analysis suggested that there are genes in cellulose synthase family while 35 are belong to cellulose synthase-like with seven families. The seven cellulose synthase-like were designated as subfamilies CslA, CslC, CslD, CslE, CslF, CslH and CslJ, with number of gene member 8,5,5,3,10,3, and 1 members, respectively.（2） The cellulose synthase gene superfamily in momocotyledon （sugarcane, rice, maize and sorghum） and dicotyledon（Arabidopsis and grape） were divided into three clusters based on the phylogeny and conserved domains. Cluster â…  was comprised of three phylogenetically related families, CesA, CslD and CslF. Cluster â…¡ was consisted of CslB, CslE, CslG, CslH and CslJ. CslB is a dicot-specific family while CslH is monocot-specific. Genes of the dicot-specific CslG family form mostly genome-specific monophyletic clusters. CslJ is possibly a new family according to recent studies and our current analyses. Naturally, cluster III was composed of CslA and CslC. Among these, the CslA and CslC families are closely related to each other and are the most evolutionarily divergent from the cellulose synthase superfamily.（3） The phylogenetic genetic analysis were performed for momocotyledon （sugarcane, rice, maize and sorghum） and dicotyledon （Arabidopsis and grape） of cellulose synthase gene family. The results suggested that there were at least six ancestor genes for the two classes and ScCesA2 was rapidly evolving compared to the other genes. Based on previous studies for the CesA family in the model plants（Arabidopsis and rice） and phylogenetic genetic classification, ScCesAl, ScCesA6 and ScCesA9 were predicted to play important roles in primary cell wall formation, and ScCesA10, ScCesAll and ScCesA12 were indispensable for secondary cell wall formation.（4） Based on the hierarchical cluster analysis, the ScCESA/CSL family exhibited a co-expression pattern of mature stage of five tissue types in three Saccharum species. Group IA, ScCesA1,-6 & 9 had high co-expression in the young tissues typical of the primary cell wall in addition to the internode 3 of Mo16081, indicating that ScCesAl,-6 & 9 may form a cellulose complex for primary cell wall biosynthesis. However, ScCesA10-11 & -12 in Group IC showed a co-expression pattern that overlapped with Group IA in young and stem tissues except in the internode 3 & 8 of MOL6081, which represent the transition stage from primary to secondary cell wall synthesis. Therefore, ScCesA10,-11 & -12 could be played as a cellulose synthase complex involved in secondary cell wall synthesis.（5） The SALK₀84627, CS478543 and CS478543 are the T-DNA insertion mutants for AtCesA4, AtCesA7 and AtCesA8 in Arabidopsis, respectively. Phenotype observation suggested that the mutant plants were dwarf, partial abortive pollen and the declined of seed setting rate for the T-DNA insertion mutants of Arabidopsis. Moreover, sections from top, middle, and base of stems in the mutant plants formed irregular xylem cells, lead to the collapse of the xylem. These results suggested that these three genes were involved in secondary cell wall synthesis, and there was no mutual redundancy phenomenon.AtCesA4, AtCesA7 and AtCesA8 are to ScCesA10, ScCesA12 and ScCesA11, respectively. By GatewayTM system, the three genes were transformed into expression vector （pMDC206）, and subsequencently transferred into Agrobacterium, for further sugarcane gene function research.