Cloning Of SPL Gene Family And Functional Analysis Of FmSPL2 In Fraxinus Mandshurica

Fraxinus mandshutrica is one of the precious broad-leaved tree species in Northeast China.It has been listed as a gradual endangered species.Fraxinus mandshurica were used as materials,the SBP domain of poplar was used to blast the local transcriptome database and selected 28 FmSPL family genes,named FmSPL1?FmSPL28.Phylogenetic analysis of FmSPL family genes of Fraxinus mandshurica were analyzed.Compared the expression patterns of SPL family genes in Fraxinus mandshurica with Arabidopsis and Poplar trichocarpa.Besides,the response patterns of FmSPL family genes to hormone and stress treatment were analyzed.The plant expression vectors were constructed.Then we transformed the leaves of tobacco,and FmSPL2 transgenic tobacco plants were used to analyse the function of FmSPL2,the main results are as follows:1.Clone and analysis of FmSPL gene familyPreliminary analysis of amino acid physicochemical properties showed that 28 FmSPLs encode 133?1085 amino acids,protein isoelectric point(pI)is between 5.44?9.77,molecular weight(Mw)is between 11.05?113.6 kDa.All of FmSPL proteins selected are unstable and hydrophobic.Phylogenetic analysis of Arabidopsis thaliana,Poplar trichocarpa and Fraxinus mandshurica revealed that these SPLs were divided into 7 groups.There are 10 pairs of paralogous proteins in Fraxinus mandshurica.The evolutionary relationship between Poplar trichocarpa and Fraxinus mandshurica was closer in the same group.2.Expression pattern of SPL gene familyThe expression pattern of FmSPLs in different tissues of Fraxinus mandshurica was compared with the tissue-specific expression patterns of Arabidopsis and Poplars obtained from public databases.It was found that the expression pattern of SPL family gene has tissue specific,and expression patterns of most structurally similar SPLs gene vary widely.Analysis of the expression patterns of the FmSPL gene in different months revealed that the expression level of FmSPL2 was the highest expression in May,3.74 times than that in August;FmSPL9 was the highest expression in June,3.43 times than that in May;the expression patterns of FmSPL7 and FmSPL10 were similar,both of them was highest expressed in August,was 91 times,125 times than that in July,respectively.FmSPL14 had the highest expression in May,which was 2.25 times than that of August;FmSPL16 had the highest expression in June,which was 19 times than that of July.Further analysis of the expression patterns of these genes in the treatment materials revealed that the expression patterns of the FmSPLs genes in the treatment materials were different,but all responded to stress and hormone signals.3.Functional analysis of FmSPL2Using FmSPL2 transgenic tobacco as experimental material,we confirmed that FmSPL2 located in the nucleus.After 28 days of subculture,the average height of FmSPL2 transgenic tobacco seedlings and wild-type tobacco is 10.33±0.33cm and 8.33±0.53cm,the difference is not obvious.The average root length of the transgenic lines and wild type is 1.80±0.41cm and 3.26±0.19cm(0.01<p<0.05),the difference is significant;the average number of roots of the transgenic lines and wild type is 17±2 and 31±3(p<0.01);the difference is extremely significant.Growth of transgenic tobacco leaves is significantly inhibited.FmSPL2 overexpressed transgenic tobacco plants flowered earlier than wild type about 7-10 days.The result shows that FmSPL2 has the function of promoting flower development.The results of NaCL treatment showed that FmSPL2 improved the tolerance of transgenic tobacco to salt stress.These results indicate that FmSPL2 is a multifunctional regulatory gene that participates in the development of plants at different growth stages and involves in the regulatory response of plants to salt stress.