Molecular Plant Breeding2016, Vol.7, No.18, 1
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room temperature.
The effects of different temperature treatments on removing the stagnancy of low-temperature germination were
compared, the effect of 25°C was the best (Zhao, 2010). The treatment of seeds were divided into three groups:
The first group is the seeds before low-temperature stratification, that is, the seed is directly wetted with sterile
water; The second group is the seeds in the low-temperature germination stagnancy, that is, the seed placed at 4°C
for 10 days after be wetted; The third group is the seeds that removed the low-temperature germination stagnancy,
that is, the seed placed at 25°C for 50 min after it was stratificated. The samples of seeds were ground into powder
in liquid nitrogen and placed in the refrigerator at -80°C for subsequent experiment.
3.2 Method
3.2.1 Preparation of protein sample and determination of the protein content
The total protein of three groups of seeds were extracted by TCA/ acetone precipitation method respectively
(Agnieszka and Juan, 2013; Vasconcelos et al., 2005; Wu et al., 2014). The concentration of total protein was
measured by Bradford method (1976), and the protein content of three samples was calculated respectively.
3.2.2 Screening for temperature-responding proteins during germination of
L. apetalum
seeds
The proteins of three groups are isolated with two-dimensional electrophoresis method. 17 cm precast linear IPG
strips (pH3-10) were chosen for the Isoelectric Focusing, sample volume was unified according to the quality of
seed. Methods refer to 2-DE process of spring soybean (
Glycine max
L.) (Tian et al., 2015) and wild wheat
(
Triticum urartu
L.) (Gharechahi et al., 2014), the methods were changed slightly in this study. Compared with the
first and second group, screening differentially expressed proteins in the third group of seeds which removed the
low-temperature germination stagnancy. PDQuest8.0 was used to analyze the 2-DE map, protein spots which
expression changed more than 3 times were screened, and identified using LC / MS-MS.
3.2.3 Analysis of the relationship between the protein expression of
L. apetalum
seeds response to low
temperature and germination at low temperature
The expression of several proteins, response to low temperature during germination, in the three groups of seeds
were compared and screened. The seeds in the low-temperature germination stagnancy were divided into six parts,
and were respectively treated for 0 min, 30 min, 45 min, 50 min, 55 min, 60 min at 25°C, for analyzing and
verifying the gene expression of different protein by RT - PCR. Part of seeds continue to be placed at 4 °C for
germination, germination rate of them was counted after 15 days. Comprehensive experimental result is to analyze
the relationship between the protein expression of
L. apetalum
seeds response to low temperature and germination
at low temperature.
The protein expression was detected by fluorescence quantitative RT-PCR, specific operations are as follows:
Total RNA of
L. apetalum
seeds was extracted using the method of Trizol (TIANGEN). Synthesizing the first
cDNA of the RNA samples refers to the operating instructions of Revert Aid First Strand cDNA Synthesis Kit
(Thermo). Based on the results of MASCOT, the primers were designed by Primer5.0, according to the
corresponding nucleotide sequence of the target protein. The primer sequences, annealing temperature and lengths
of PCR products (Table 1). With
ACTIN
as the reference gene, use UltraSYBR Mixture (With ROX II) (CWBIO)
for the purposes of gene expression analysis. Each sample was done with three biological repeats.
Table 1 Sequences of the primers used in qRT-PCR analysis for key protein and the product size
Primer
Sequences 5
，
___
3
，
Annealing Temperature
Product Size (bp)
CDCF
CTCGTGCTGTGGCTAATGAA
60.1
120
CDCR
TCTCTGCCTCCTCAAATGCT
60.01
120
P12F
GAATAGCCCTTGTCGGTTCA
60.07
109
P12R
CACTTGTCCTACCGCGAACT
60.31
109
Hsp17.6F
TCTTCAGTCCAGGCAAATCC
57.8
172
Hsp17.6R
AACAGGCGAAACAGCATCTT
58.21
172
actinF
CGACGATTCCACCACTAAGC
60.66
140
actinR
TGAGGTTCTTTACCAGCCATCT
60.13
140
