Plant Materials and gene clone
Seedings of Medicago sativa L.cv. ‘Baoding’, after 20 weeks growth in artificial climate growth chamber, were used for the full-length clone of MsCIPK. Total RNA was extracted following the protocol of Takara (Takara, Dalian, China). The total RNA integrity and concentration was qualified using the EasyPure® Quick Gel Extraction Kit (Beijing TransGen Biotech Co., Ltd, Beijing, China) and Thermo Scientific NanoDrop 2000 (Thermo Fisher Scientific Co., Ltd, Waltham, MA, USA). The forward and reverse PCR primer was 5'-ATGGCAGTTGTAGCTGCTCCCAAGC-3' and 5'-TCAGGTATCTAAGTTCAGAGATTC-3', respectively, which were designed by Primmer v5. After transforming recombinant plasmids, positive clones were selected for sequencing verification, which was performed by Yingjun Technology Co., Ltd., China. Transgenic plants were obtained via the tobacco leaf disc transformation approach. Positive transgenic plants were identified via by PCR detection, using the extracted transgenic tobacco DNA.
The basic physical and chemical properties of MsCIPK protein were predicted in ProtParam 27. The cDNA sequence was then blasted with NCBI blastx in order to identify CIPK-like genes. Protein domains were predicted using SMART 28. The secondary and three-dimensional structure of MsCIPK protein was predicted using the online GOR IV method (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html) 29. The neighbor-joining (NJ) tree was constructed with 1000 replicates using MEGA v7.0 30. The NCBI ID for the 26 Arabidopsis CIPK-like genes were AtCIPK1 (AAG28776.1), AtCIPK2 (AAF86506.1), AtCIPK3 (AAF86507.1), AtCIPK4 (AAG01367.1), AtCIPK5 (AAF86504.2), AtCIPK6 (AAF86505.1), AtCIPK7 (AAK16682.1), AtCIPK8 (AAK16683.2), AtCIPK9 (AAK16684.1), AtCIPK10 (AAK16686.1), AtCIPK11 (AAK16686.1), AtCIPK12 (AAK166877.1), AtCIPK13 (AAK16688.1), AtCIPK14 (AAK16689.1), AtCIPK15 (AAK16692.1), AtCIPK16 (AAK50348.1), AtCIPK17 (AAK64513.1), AtCIPK18 (AAK59695.1), AtCIPK19 (AAK50347.1), AtCIPK20 (AAK61493.1), AtCIPK21 (AAK59696.1), AtCIPK22 (AAL47845.1), AtCIPK23 (AAK61494.1), AtCIPK24 (AAK72257.1), AtCIPK25 (AAL41008.1) and AtCIPK26 (NP_850861.2).
Gene expression analysis
Seedings of Medicago sativa L.cv. Baoding, after 4 weeks growth in artificial climate growth chamber, were then treated with different treatments, including 200 mm/L NaCl, drought, 4 ℃ cold and 42 ℃ heat and 10 μM ABA, in order to investigate MsCIPK’s role in response to various stresses. After 2h, 4h, 8h, 12h and 24h treatment, above-ground and root tissues were immediately frozen in liquid nitrogen (-80 ℃) before RNA extraction. The PCR amplification verification was carried out with hygromycin Hyg specific primers. Gene expression analysis was performed using quantitative real-time polymerase chain reaction (qRT-PCR) by SYBR Premix Ex Taq (Takara, Dalian, China). The main PCR reaction parameters were: 95 ℃ pre-denaturation for 1 min; 40 cycles of 95 ℃ for 30 s, 60 ℃ for 30 s, and 72 ℃ for 30 s; 72 ℃ for 10 min and terminated and saved at 4 ℃. β-actin was used an the internal gene and the forward and reverse primer was 5′-TTTGAGACTTTCAATGTGCCCGCC-3′ and 5′-TAGCATGTGGGAGTGCATAACCCT-3, respectively. The forward and reverse primer for MsCIPK was 5′- TCAGCCCTACAGTCCTACCACGG-3′ and 5′- GTTCCACCTCTCTGCAGCTCATC-3′, respectively. The signal of the qRT-PCR was monitored by the CFX96 real-time system (Bio-Rad, CA, USA). The relative expression level of MsCIPK was determined by the 2-∆∆Ct method 31. Three biological and technical replicates were used to calculate the standard error (SE). The ANOVA significant test was performed in SAS software.
Functional analysis of transgenic tobacco with MsCIPK
In order to validate the functional role of MsCIPK, we treated T1 transgenic tobaccos (Line 3 and Line 7) and non-transgenic tobacco (WT) under salt, drought and cold stresses, and then evaluated the impacts on the contents of four compounds including malondialdehyde (MDA), superoxide dismutase (SOD), soluble protein (SOP), and proline (Pro). ild type tobaccos (WT) were treated with normal Hoagland nutrient solution. For salt treatment, transgenic plants were treated with 1/2 Hoagland nutrient solution and 200 mM NaCl. The salt treatment was conducted for a period of 21 days and samples were collected at every 7 days (0day, 7day, 14day and 21day) and immediately frozen in liquid nitrogen (-80 ℃) before use. For low temperature treatment, plants were treated with 4 ℃ and collected at 0h, 6h, 12h and 24h and frozen in liquid nitrogen for a total of 24 h treatment. The content of MDA was measured by the thiobarbituric acid method 32. The content of SOD was measured by the NBT method 33. The content of SOP was measured by the Coomassie Blue Staining method: SOP content (Mg/g) = C × VTV1 × FW × 1000, where C was the standard curve value (μg); VT was the total extract volume (mL); FW was the fresh weight and V1 was the volume added during measurement (mL). The content of Pro was measured by the methods proposed by Bates 34. All the measurements were performed with nine replicates. Significant tests were performed in SAS software.