Abu 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: sefian_ib@yahoo.com; lixu@webmail.hzau.edu.cn; chunshenggao@webmail.hzau.edu.cn 2Agricultural 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: hepingli@mail.hzau.edu.cn 4College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, P.R. China; emails: yucailiao@mail.hzau.edu.cn *Corresponding author. Emails address: yucailiao@mail.hzau.edu.cn; ycliao06@yahoo.com.cn (Y.-C. Liao)

Expression of Fusarium trehalose synthase

genes, TPS1 and TPS2 enhances salinity

stress tolerance into wheat crops

outlines

Introduction.

Materials and Methods

I. Plasmids

II. Plant transformation and tissue culture

III. Molecular characterizations of transgenic TPS1 and

TPS2 wheat plants

IV. 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).

Introduction

The overexpression of trehalose biosynthetic

genes in some plants such like tobacco, potato,

rice, Arabidopsis, tomato, alfalfa, and maize has

led to an improvement in abiotic stress tolerance

(Holmstrom, et al., 1996; Yeo, et al., 2000; Garg, et

al., 2002; Avonce, et al., 2004; Cortina and

Culianez-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 these

transgenic plants was successfully improved.

The aims of this study are to improve drought and salinity

tolerance in wheat

Introduction

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.

2. MATERIALS AND METHODS

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 Methods

Plasmids

A B

pAHC25-Ubi-TPS1 or TPS2

1.159 KB

TPS

1

TPS

2

PA

HC

25

Agrobacterium Gene gun

Wheat transgenic approaches

Others

Electroporation

PEG

Pollen tube

…..etc

Materials and Methods

Plant transformation and tissue culture

BIOLISTIC

9

Bombardment parameters

Pressure: 1100 psi

Distance: 9 cm

Vacuum: 28 inches

Materials and Methods

1cm D

5 mm C B 5 mm 1 mm A

5 mm F 1 cm G 1 cm E 1cm f

10

Materials and Methods

The basic process of biolistic transformation of wheat

Materials and Methods PCR and Southern blot analysis

14Kb

5 kb

D A

400bp

M H2o CK M1 M2

300bp

B M H2o CK P1 P4 P6

H2O CK P1 X1 M

405bp

330bp

C

Fig. 2 Molecular characterizations of transgenic TPS1 and TPS2 wheat plants

(A), PCR analyses from TPS1 transgenic line and non transgenic line. TPS1

gene specific fragment 405 bp by primers TPS1P1/ TPS1P2. (B), PCR analyses

from TPS2 transgenic line and non transgenic line, TPS2 gene specific

fragment 330 bp by primers TPS2P1/ TPS2P2. (C), PCR analyses of the cross

between two transgenic, TPS1 (M1) X TPS2 (P1) lines, resulted in X1 two

genes 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 Methods Salinity stress tolerance assay:

In all experiments, the T2 generation seeds of

TPS1and TPS2 transgenic lines were used for

assays. One independent transgenic line M1

and three independent transgenic lines P1, P4,

and P6 along with non transgenic line Y158 or

Y12 as a control, were used to represented

TPS1 and TPS2 transgenic wheat plants,

respectively.

RESULTS AND DISCUSSIONS

+7day

B

**

*

*

A C

D

E

Fig. 3 Salinity stress tolerance for TPS1 transgenic line and non-transgenic

(Y158). (A), the appearance of T2 lines of TPS1 M1 and Y158 non transgenic

seedlings under salt stress 200 mM NaCl . (B), the appearance of T2 lines of

TPS1 M1 and Y158 non transgenic seedlings, 7 days recovery from 350 mM

NaCl concentration. (C), roots length after 10 days recovery. (D), root dry

weight after 10 days recovery. (E), Plant fresh weight (biomass) after 10 days

recovery.

*, ** indicate significant differences at P <0.05, <0.01 respectively. Three

replications

B

+7 day

***

* *** ***

C

** *** ***

D

*** *** ***

E

A

Fig. 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 transgenic

seedlings during 200 mM NaCl concentration.

B)The appearance of T2 lines of TPS2 three transgenic lines and Y12 non

transgenic 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.

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±0.01

ns 1.0±0.1 1.0±0.22

SR% 45.7±6.4 45.2±15.4 0 0

Table 2 Comparison of root length (R.L ,cm), root dry weight (R.D ,mg) and plant fresh

weight (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 in

350 mM NaCl.

Conclusion:

Over-expression of either TPS1 or TPS2

gene resulted in an enhanced salinity

tolerance in wheat without growth effects.

Trehalose genes played a role in root growth

under salt stress.

This also presents trehalose potential as a

candidate for engineering salt tolerance in

wheat crops.

Acknowledgement

This work was supported by the Ministry of

Agriculture of China and for a Doctoral

fellowship from the Chinese Exchange

program