Isolation of full-length GsLTP
The full-length sequence information of GsLTP was derived from extending the 254 bp partial EST to 689 bp by Phrap software. The extended GsLTP gene shares high similarity with genes in the LTP family, indicating its association with fatty acid transport in soybean.
Characterisation of GsLTP gene and protein sequences
After sequencing, multiple sequence alignment was performed with known LTP sequences. This comparison revealed high homology and the expected splice sites without nucleotide mutations. BLASTx analysis revealed that GsLTP is 369 bp in length, and encodes a predicted polypeptide of 122 amino acids. The molecular weight of the protein is ~13 kDa and the predicted isoelectric point (pI) is 8.74. Amino acid sequence analysis with the NCBI BLASTp program revealed similarity with LTP proteins in a various plants, further indicating membership of the LTP gene family. Therefore, the gene was named GsLTP and submitted to GenBank (accession number FJ825765) after confirming its sequence.
Identification of transgenic tobacco lines
To identify transgenic tobacco lines, total DNA was isolated and the inserted fragment was verified by PCR (Figure 1). The pBI121-GsLTP construct was successfully transferred into tobacco line #1, #3, #5, #6, #8, #10, #11, #12, #14, #15, #16, #17, #18, #19, #20, #21, #23, #24, #25, #26, #27 and #28. Total DNA was isolated from positive seedlings and digested with BamHI for Southern blotting (Figure 2) and RT-PCR (Figure 3). Southern blot hybridization results showed that three transgenic tobacco lines yielded positive electrophoretic bands (lanes 4, 5 and 7), indicating that the GsLTP gene was successfully transferred into the plant genome. Conversely, tobacco lines without a hybridization signal indicated no gene integration. The signal in lanes 4 and 7 implied single-copy insertion into the genome. However, lane 5 has two hybridization bands, indicating the insertion of two gene copies. As expected, the positive control displayed one band, validating the appropriateness of the experimental procedure (Fig. 2). RT-PCR was also used to investigate gene transcription in plants. The successfully amplified fragments in lanes 3, 6, 8, 10, 12, 14 and 16 confirmed these independent lines harbor the target gene (Fig. 3). After reliable identification, two transgenic lines (#85 and #96) were chosen for further functional analysis.
Overexpression of GsLTP enhances drought and salt stress tolerance in transgenic tobacco but not improves low temperature resistance
The results in Tables 1 and 2 reveal no significant difference between transgenic plants and non-transgenic plants regarding chlorophyll content under low temperature treatment (p = 0.2248, a = 0.05). However, significant differences in chlorophyll content were observed under drought (p = 0.0077, a = 0.05) and salinity (p = 0.0101, a = 0.05) treatments. The chlorophyll content of transgenic seedlings was also significantly higher than that of control seedlings under drought treatment. We, therefore, concluded that transgenic seedlings displayed higher tolerance to drought conditions than control seedlings. In addition, the MDA content of transgenic seedlings in each treatment was significantly higher than that of control seedlings (p<0.0001, a = 0.05). This suggests that transgenic seedlings were more resistant to osmotic stress than control seedlings. Under all treatments, there was no significant difference in the conductivity between transgenic and control seedlings. Thus, transgenic seedlings did not demonstrate higher tolerance to osmotic stress than control seedlings under any of the treatments.
There was a significant difference between transgenic and control plants in terms of Pro content (p<0.0001, a = 0.05) under low temperature and salinity treatments, but the difference was less significant (p = 0.0413, a = 0.05) under drought treatment. Because the Pro content of transgenic seedlings was much higher than that of control seedlings, transgenic seedlings exhibited better resistance to osmotic stress.
Tobacco seedlings harboring the GsLTP gene displayed greater resistance than control seedlings based on membrane permeability and enzymatic activity. Based on chlorophyll content, the GsLTP gene significantly enhanced tolerance to drought but did not enhance tolerance to low temperature stress or salinity stress. Based on the MDA and Pro content, the LTP gene significantly enhanced resistance to low temperature, drought and salinity. The accumulation of these osmotic protective substances in transgenic seedlings in response to stress is an important biochemical indicator for measuring tolerance to low temperature, drought and salinity. Our results clearly showed that resistance of transgenic tobacco seedlings to drought and salinity was improved (Fig. 4). Compared with control seedlings, all physiological parameters tested were enhanced to various degrees in transgenic tobacco seedlings. Thus, GsLTP improved the tolerance of tobacco plants to drought and salinity stresses.
Table 1. Variance analysis of chlorophyll content, MDA content, Conductance and Pro content under different treatment.
Pr>F
|
Source
|
Chlorophyll content
|
MDA content
|
Conductance
|
Pro content
|
Low temperature
|
Treat
|
0.2248
|
<0.0001
|
0.2317
|
<0.0001
|
(4℃)
|
Data
|
<0.0001
|
<0.0001
|
0.0303
|
<0.0001
|
|
Repeat
|
0.4631
|
0.9256
|
0.6446
|
0.8066
|
Drought treatment
|
Treat
|
0.0077
|
<0.0001
|
0.7990
|
0.0413
|
|
Data
|
0.0001
|
0.0005
|
0.0076
|
<0.0001
|
|
Repeat
|
0.5453
|
0.6738
|
0.8940
|
0.3755
|
Salt treatment
|
Treat
|
0.0101
|
<0.0001
|
0.0795
|
<0.0001
|
|
Data
|
<0.0001
|
<0.0001
|
0.0041
|
<0.0001
|
|
Repeat
|
0.3382
|
0.8806
|
0.7632
|
0.8378
|
Table 2. T-tests (LSD) for chlorophy content, MDA content, Conductance and Pro content in different treatment.
T-tests (LSD)
|
Treat
|
Low temperature (4°C)
|
Drought treatment
|
Salt treatment
|
Chlorophyll content
|
control
|
0.71847a
|
0.70110b
|
0.65486a
|
LTP
|
0.73573a
|
0.89190a
|
0.94614a
|
MDA content
|
control
|
0.0129333a
|
0.0110952a
|
0.0123810a
|
LTP
|
0.0078667b
|
0.0080000b
|
0.0071905b
|
Conductance
|
control
|
81.727a
|
73.631a
|
80.064a
|
LTP
|
86.061a
|
76.024a
|
86.879a
|
Pro content
|
control
|
0.045333b
|
0.036000b
|
0.049238b
|
LTP
|
0.077000a
|
0.052667a
|
0.082952a
|
Note:control:wild type plant; LTP: Seedlings transformed with LTP gene