Expression of Fusarium threhalose synthase genes, TPS1 and TPS2 enhance salinity stress tolerance into wheat crops
Expression of Fusarium trehalose synthase genes, TPS1 and TPS2 enhances salinity stress tolerance into wheat cropsAbu Sefyan I. Saad1, 2, Xu Li1, Chun-Sheng Gao1, He-Ping Li1,3, Yu-Cai Liao1,4*1Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070, Hubei, P.R.China，emails: email@example.com; firstname.lastname@example.org; email@example.comAgricultural Research Corporation (ARC), PO Box 126, Wad Medani, Sudan.3College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, P.R. China; emails:firstname.lastname@example.orgCollege of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, P.R. China; emails:email@example.com*Corresponding author. Emails address: firstname.lastname@example.org; email@example.com (Y.-C. Liao)
outlines Introduction. Materials and MethodsI. PlasmidsII. Plant transformation and tissue cultureIII.Molecular characterizations of transgenic TPS1 and TPS2 wheat plantsIV. Salinity stress tolerance assay Results and Discussions Conclusion
Introduction Trehalose is a non-reducing disaccharide sugar, widely distributed in anhydrobiotic micro-organisms (bacteria, fungi, etc.), and plants These anhydrobiotic organisms can survive in drought conditions over a long period of time and recover within hours when in contact with water (Hottiger, et al. 1994; Elbein, et al., 2003). In plant, trehalose is produced from glucose by trehalose-6- phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) pathway similar to that reported in several species of bacteria (Avonce, et al. 2006). Trehalose serves as sugar storage, metabolic regulator and protects against abiotic stress by the stabilization of proteins structures and biomembranes Elbein, et al.,( 2003).
IntroductionThe overexpression of trehalose biosyntheticgenes in some plants such like tobacco, potato,rice, Arabidopsis, tomato, alfalfa, and maize hasled to an improvement in abiotic stress tolerance(Holmstrom, et al., 1996; Yeo, et al., 2000; Garg, etal., 2002; Avonce, et al., 2004; Cortina andCulianez-Macia, 2005; Suarez, et al., 2009; Jiang,et al. 2010).In wheatTPS from E. coli improve drought (A)and Salinity stress tolerances (B) (Abebe et al.2003).Therefore, the stress tolerance of thesetransgenic plants was successfully improved.
Introduction The aims of this study are to improve drought and salinity tolerance in wheat through genetic transformation by inserting two stress response genes (trehalose-6-phosphate synthase, TPS1, and trehalose-6-phosphate phosphatase, TPS2), which were isolated from Fusarium graminearum.
pAHC25-Ubi-TPS1 or TPS2Materials and Methods PAHC25Plasmids TPS1 TPS2 A B 1.159 KB Fig.1 Structure of two a recombinant plasmids pUbiTPS1 and pUbiTPS2 each containing bar gene and ubiquitin promoter from maize used for transformation, A, the plasmid vector pAHC25, with the bar gene, ubiquitin promoter and TPS1 gene; B, the plasmid vector pAHC25, with the bar gene, ubiquitin promoter and TPS2 gene.
Materials and MethodsPlant transformation and tissue culture Wheat transgenic approaches Agrobacterium Gene gun Others Electroporation Pollen tube PEG …..etc
Materials and Methods BIOLISTIC Bombardment parameters Pressure: 1100 psi Distance: 9 cm Vacuum: 28 inches 9
Materials and Methods The basic process of biolistic transformation of wheat 1 mm 5 mm 5 mm 1cmA B C DE 1cm 1 cm F 5 mm G 1 cm f 10
Materials and MethodsPCR and Southern blot analysis M H2o CK M1 M2 M H2o CK P1 P4 P6 A B D 14Kb 400bp 300bp 5 kb H2O CK P1 X1 M C 405bp 330bpFig. 2 Molecular characterizations of transgenic TPS1 and TPS2 wheat plants(A), PCR analyses from TPS1 transgenic line and non transgenic line. TPS1gene specific fragment 405 bp by primers TPS1P1/ TPS1P2. (B), PCR analysesfrom TPS2 transgenic line and non transgenic line, TPS2 gene specificfragment 330 bp by primers TPS2P1/ TPS2P2. (C), PCR analyses of the crossbetween two transgenic, TPS1 (M1) X TPS2 (P1) lines, resulted in X1 twogenes specific fragments by four primers specific for both genes. (D),southern Blotting for genes, TPS1 transgenic lines and non transgenic line(Y158) and three TPS2 transgenic lines alone with non transgenic (Y12).
Materials and MethodsSalinity stress tolerance assay:In all experiments, the T2 generation seeds ofTPS1and TPS2 transgenic lines were used forassays. One independent transgenic line M1and three independent transgenic lines P1, P4,and P6 along with non transgenic line Y158 orY12 as a control, were used to representedTPS1 and TPS2 transgenic wheat plants,respectively.
A C ** D * E * B +7dayFig. 3 Salinity stress tolerance for TPS1 transgenic line and non-transgenic(Y158). (A), the appearance of T2 lines of TPS1 M1 and Y158 non transgenicseedlings under salt stress 200 mM NaCl . (B), the appearance of T2 lines ofTPS1 M1 and Y158 non transgenic seedlings, 7 days recovery from 350 mMNaCl concentration. (C), roots length after 10 days recovery. (D), root dryweight after 10 days recovery. (E), Plant fresh weight (biomass) after 10 daysrecovery. *, ** indicate significant differences at P <0.05, <0.01 respectively. Threereplications
A C *** *** *** * D *** *** ** E *** *** *** B +7 dayFig. 4 Salinity stress tolerance for TPS2 transgenic line and non-transgenic(Y12).A)The appearance of T2 lines of TPS2 P1, P4 and P6 and Y12 non transgenicseedlings during 200 mM NaCl concentration.B)The appearance of T2 lines of TPS2 three transgenic lines and Y12 nontransgenic seedlings, 7 days recovery from 350 mM NaCl concentration.C) Roots length after 14 days recovery.D) Root dry weight after 14 days recovery.E) Plant fresh weight (biomass) after 14 days recovery. **, *** indicates significant differences at P <0.05, <0.01 respectively.
Table 2 Comparison of root length (R.L ,cm), root dry weight (R.D ,mg) and plant freshweight (F.W ,g) after 14 days recovery of transgenic TPS2 & TPS1 plants and non-transgenic Y12 or Y158 grown in normal conditions, survival rate of transgenic lines in350 mM NaCl. TPS2 TPS1 CK1(Y158) Ck1(Y12) Ck(Y158) Ck(Y12) R.L(cm) 11.0±0.79ns 10.6±0.19 ns 11.1±0.6 13.9±0.26 R.D(mg) 26.0±1.6 ns 18.2± 0.93 ns 24.4±2.7 32.8±1.5 F.W(g) 0.7±0.04 ns 0.6±0.01ns 1.0±0.1 1.0±0.22 SR% 45.7±6.4 45.2±15.4 0 0
Conclusion: Over-expression of either TPS1 or TPS2gene resulted in an enhanced salinitytolerance in wheat without growth effects. Trehalose genes played a role in root growthunder salt stress. This also presents trehalose potential as acandidate for engineering salt tolerance inwheat crops.
AcknowledgementThis work was supported by the Ministry ofAgriculture of China and for a Doctoralfellowship from the Chinese Exchangeprogram