|Other Abstract||The functional divergency among duplicated genes is considered as an important genetic driving force in the evolution of plantgenome. Itis well known that the genesinvolved in signal transduction and response to stress are often origin from gene duplication. Here, we analyzedadaptive evolution and functional divergence analysis on phosphatidylserine decarboxylase gene family from Populus euphratica. Furthermore, functional verification of phosphatidylserine decarboxylase (PSD) have also been carried out using transgenic poplars and Arabidopsis plants, which all together aimed to explore mechanism of P. euphratica adapt to extreme environments. The results are shown as following:
(1)The evolutionary analysis of PSDsfrom P. euphratica. Based on sequences alignment of DNA and protein of PSDs, by Blast and Pfam screening, three homologous to PSDwere identified to encode phosphatidylserine decarboxylase in P. euphratica genome. Phylogenetic evolutionary analysis showed that three putative PSDsclustered into two groups, subfamily I and subfamily II. One copy was found in subfamily I across most plants, while more copies could be found in subfamily II, which duplication events occurred in family-specific manner in plants, for example, in subfamily II, PSDsfrom Populus species have undergone a family-specific gene duplication event, and result in PeuPSD2 and PeuPSD3 in P. euphratica. Adaptive evolution analysis was conducted using branch-site model of PAML software, the results showed that PeuPSD1 underwent strong positive selection (&omega= 999.0P <0.001), and an adaptive evolutionary amino acids site (220N, asparagine) was also detected.
(2) The function divergence of PeuPSDsfrom P. euphratica. According to the domain specificto PSDs subfamily, we predicted that PeuPSD1 located at mitochondria, while PeuPSD2 and PeuPSD3 located at the inner membrane system. The expression pattern analysis showed that PeuPSD1, PeuPSD2 and PeuPSD3 expressed with similar patternsbut at differential levels, for example, PeuPSD1 was expressed in leaves, roots, xylem and phloem at low levels, while PeuPSD2 and PeuPSD3 were expressed at high levels. Moreover, the expression level of PeuPSD2 was highest in phloem, while that of PeuPSD3 was in xylem, suggesting that the expression pattern of PeuPSDshave diversified. To verified the adaptive evolutionary site in PeuPSD1, PeuPSD1and its mutant (N220S) were transformed in Yeast cell (Pichia pastorisGS115 strain), respectively. In SDS-YPDA yeast growth media, yeast expressing PeuPSD1 grew more quickly than in that of mutant strain, suggesting that the adaptive site 220N in PeuPSD1 could contribute to improve the tolerance to osmotic stress in transgenic yeast cells.
(3) Function verification of PeuPSD1using transgenic transformation. To verify the role of PeuPSD1in plants, PeuPSD1have been promoted by 35Spromotorand over-expressed in P. alba and Arabidopsis thaliana plants, respectively through transgenic technology. Under normal conditions, the root length of 35S::PeuPSD1 seedlings is significantly longer than that of wild type (Col-0) and mutant (atpsd1) (p<0.05). After 7-day treatment with high salt, the root length of 35S::PeuPSD1seedlingswas the longest while that of mutant was the shortest (p<0.05). Similar results were also found in these Arabidopsis lines when cultured in soil. Furthermore, after salt treatment, the MDA content in transgenic P. albaleaves and roots was lower than that of in WT poplars, suggesting that plasma membrane intransgenic lines was less damaged. In termsof anti-oxidase activity, such as SOD, CAT, and POD, which were all increased significantly, while H2O2contentwas decreased in transgenic plants, indicating that increasing anti-oxidase activities could catalysethe decomposition of H2O2to water and oxygen and reduceddamageto plants. Similar results were also found in these transgenic plants, including P. albaand Arabidopsis plants treated with drought. Altogether, overexpression of PeuPSD1 could significantly improve the tolerance of transgenic A. thaliana and P. alba to drought and salt stresses.
In conclusion, the three genes PeuPSD1, PeuPSD2, and PeuPSD3 fromP. euphratica have diversified notonly in expression patterns, but also in function since occurrence of gene duplication specific-in-family.Among of them, PeuPSD1 has undergone positive selection and an adaptive evolutionary site (220N) were identified, which all together could contribute to improve the tolerance of P. euphratica to drought and salt stresses.|