Functional Identification And Differential Expression Analysis Of Trehalose-6-phosphate Synthase Gene Family In Maize

Trehalose, a non-reducing disaccharide composed of two glucose moieties linked with a-d-gluc-opyranosyl-1,1-a-d-glucopyranoside is widely distributed in bacteria, fungi, invertebrate animals and plants. Microorganisms accumulate trehalose to protect their proteins, nucleic acid and biomembrane. Except for relative high accumulation in some 'resurrection plants'such as Selaginella lepidophylla, trehalose accumulates in a trace level in higher plants. However, trehalose and/or trehalose-6-phosphate played important roles in embryogenesis, inflorescence architecture, cell shape and plant architecture, sugar sensing and signal transduction under abiotic stress. The abiotic stress tolerance of plants which overexpressing exogenous trehalose synthesis genes was improved dramatically. Even those which overexpressing their endogenous trehalose synthesis genes, the abiotic stress tolerance of Arabidopsis and rice were also improved dramatically. Therefore, exploring maize endogenous trehalose-6-phosphate synthase (TPS) genes relative to trehalose metabolism will contribute to elucidate the mechanism of low trehalose content in maize, also provide theory support for overexpressing exogenous and endogenous trehalose synthesis genes in maize.According to searching the sequenced genomic BAC bank of maize inbred line 'B73',totally eighteen TPS gene copys were found. Senquecne aligment and genomic loacation analysis revealed these genes could be further divided into twelve TPS genes. They were nominated as TPSI-TPS12 according to their protein homologous to yeast TPS1. TPS3 has three copys in genome, TPS1, TPS4, TPS5 and TPS1 1 have two copys in genome, TPS1, TPS6, TPS1,7P58, TPS9, TPS10 and TPS12 only have one copy in genome. Followed the grouping principle used in Arabidopsis thaliana, maize TPS gene family could be grouped in two subfamilies depending on whether they display most similarity to yeast TPS1 or TPS2. The first subfamily only consists of TPS1; the second subfamily consists of the rest TPS genes. Primers used to clone the open reading frame (ORF) were designed based on the predicted sequences of TPS genes. Homologous amplifications were carried out in our local inbred line'18-599'. TPSI-TPS9 were cloned from our local inbred. The ORF of TPSl is 2820 bp encoding for a 939 amino acid protein; the ORF of TPS2 is 2607 bp encoding for a 868 amino acid protein; the ORF of TSP3 is 2592 bp encoding for a 863 amino acid protein; the ORF of TPS4 is 2673 bp encoding for a 890 amino acid protein; the ORF of TPS5 is 2739 bp encoding for a 912 amino acid protein; the ORF of TPS6 is 2595 bp encoding for a 864 amino acid protein; the ORF of TPS7 is 2736 bp encoding for a 911 amino acid protein; the ORF of TPS8 is 2568 bp encoding for a 855 amino acid protein; the ORF of TPS9 is 2556 bp encoding for a 851 amino acid protein. Similarity of amino acid of TPS1 to TPS2-TPS9 which belong to classⅡsubfamily was very low, from 33%-36%. However, classⅡmembers have relative high similarity to each other, from 51%-94%, the similarity of TPS5 and TPS7 was as high as 91%, and the similarity of TPS8 and TPS9 was as high as 94%. In order to identify the functional proteins which having TPS and/or Trehalo-6-phosphate phosphatase (TPP) activities, the cloned ORFs were insert into yeast shutter vector pRS6, constructed plasmids were used to transform the yest TPS1(tps1△) mutant YSH290 and the TPS2 (tps2△) mutant YSH450. Based on the yeast complementation tests, TPS1 and TPS3 were proved to have TPS activity; TPS3 and TPS4 were proved to have TPP activity.Seedlings of'18-599'at the two-leaf stage were transplanted into a plastic mesh grid for aquaculture and grown hydroponically. At three-leaf stage, identical seedlings were subjected to 150 mmol/L NaCl stress and 12℃cold stress. At time zero (control) and at 0.5,1,2,4,6,8,12,24 and 48 h after the stress treatment, leaves and roots were sampled separately from three seedlings, real-time quantitative PCR(qRT-PCR) and reverse transcripttion PCR(RT-PCR) were used to assay the expression profile of the nine TPS genes under stresses.TPS genes were response to both salt and cold stress in both leaf and root. Except for downregulating of TPS4 in leaf, other eight TPS genes were upregulated at different stage in response to salt stress both in leaf and root.TPS1,TPS2,TPS3 and TPS5 were upregulated at different stage in response to cold stress in leaf. TPS4 was constantly downregulated under cold sress in leaf.TPS6,TPS7,TPS8 and TPS9 were all downregulated in forepart and metaphase, and upregulated in anaphase in leaf under cold stress; TPS1,TPS3 and TPS4 were all upregulated under cold stress in leaf. These suggested that the members of maize TPS gene family may participate in the signal transduction under abiotic stress.Seuqunce diversity were found among three sequenced clones of TPS1, it seemed that TPS1 underwent alternative splicing. Reamplification the ORF of TPS1 was conducted by using leaf cDNA samples of 0 h control and 24 h under salt and cold stress. Two spliced isoforms:TPS1 and TPS1 A were identified in 0 h control, and five spliced isoforms:TPS1, TPS1 A, ZTPS1B,TPS1 C and TPS1 D were identified in stress conditions. Compared to splicing of TPS1,TPS1 A belonged to intron retention, the fourth, the seventh and the eighth were retentd in mature mRNA.TPS1B belonged to 5' splice sites selection, compared to splicing of TPS1, it used the distal 5'splice site when splicing the seventh intron.TPS1C belonged to intron retention, compared to splicing of TPS1, the fourth intron was retentd in mature mRNA.TPS1D was much more complicated, compared to splicing of TPS1, it used proximal 5'splice site and distal 3'splice site when splicing the first intron, and the seventh exon was excluded from the mature mature mRNA.TPS1 encoded for a protein for a 939 amino acid protein containing the TPS domain and putative TPP domain like other plant functional TPS1 proteins; TPSIA encoded for 369 amino acid protein; TPS1 B encoded for 462 amino acid protein; TPS1 C encoded for 369 amino acid protein; TPS1 D encoded for 133 amino acid protein. Multiple sequence aligment among the proteins encoded by TPS1,TPS1A, ZTPSIB, TPSIC and TPS1D revealed the detachment of intact TPS protein. This detached plant TPS protein which only containing the TPS domain may has higher TPS activity.In conclusion, most of maize TPS genes were active transcribed. These active transcribed TPS genes showed different response to salt and cold stresses, some of them show encoding for proteins which having TPS and/or TPP activity. Maize TPS genes may participate in the signal transduction under abiotic stress.