[Preliminary analysis of the role of GmSnRK1.1 and GmSnRK1.2 in the ABA and alkaline stress response of the soybean using the CRISPR/Cas9-based gene double-knockout system].

Sucrose non-fermenting related protein kinases (SnRKs) are a ubiquitous Ser/Thr protein kinase in the plant kingdom. These kinases play important roles in plant growth, development, metabolism and resistance to environmental stresses. The soybean (Glycine max L.) genome has four SnRK1 genes, of which GmSnRK1.1 and GmSnRK1.2 are predominant and participate in multiple stress response pathways. To dissect the mechanism of the role of GmSnRK1.1 and GmSnRK1.2 proteins in response to ABA and alkaline stresses, we constructed a dual-gRNA CRISPR vector to specifically knock out GmSnRK1.1 and GmSnRK1.2. The resultant constructs were transformed into soybean cotyledon nodes to induce hairy roots by agrobacteria (Agrobacterium rhizogenes). The soybean hairy roots obtained were genotyped, and the results showed that GmSnRK1.1 and GmSnRK1.2 were efficiently doubly knocked out in 48.6% hairy roots. We also generated control hairy roots that over-expressed GmSnRK1. The materials were treated with 25 µmol/L ABA for 15 days and the results showed that the growths of wild-type and GmSnRK1 over-expressed roots were significantly inhibited than GmSnRK1.1 GmSnRK1.2 double-knockout roots, as the controls displayed less root lengths and fresh weights. However, after treating with 50 mmol/L NaHCO3 for 15 days, we found that the growths of GmSnRK1.1 GmSnRK1.2 double-knockout roots were significantly inhibited than the wild-type and GmSnRK1 over-expressed control roots, as the knockout groups contained less root lengths and fresh weights. These results implied that the GmSnRK1.1 GmSnRK1.2 double knockout mitigated hairy root sensitivity to ABA and resistance to alkaline stress. Taken together, we established the CRISPR/Cas9 system to perform gene double knockout in the soybean and by using this technique, we determined the roles of GmSnRK1.1 and GmSnRK1.2 in response of abiotic stresses.

[Purification and composition analysis of polysaccharide RCPS from Rhodiola crenulata].

Hot water extracting and ethanol precipitating method was employed to isolate polysaccharides. RCP (Rhodiola crenulata polysaccharide) was fractionally precipitated with EtOH. RCP3 (Rhodiola crenulata polysaccharide 3) was one of the three fractions. RCPS was obtained after RCP3 was purified by deproteination; decolourization and gel chromatography on Sephadex G-100. The homogeneity and molecular masses of RCPS were proved by HLGPC. The amount of total carbohydrates of RCPS was measured with phenol-sulfuric acid method. IR spectrometry and UV-spectrophotometer were used to determine the characteristic absorption of RCPS. The monosaccharides contained in the RCPS were analyzed by GC. The amount of total carbohydrates in RCPS is 99.11%. The molecular weight was 27 876. IR spectrometry analysis indicated that RCPS showed typical signals of acid polysaccharide, including signals at 3 424.83, 2 934.10, 1 742.11, 1 438.96, 1 261.40, 1 103.54 and 832.86 cm(-1); UV-spectrophotometer analysis indicated that RCPS showed a signal of polysaccharide at 195 nm and no signals of protein, nucleic acid at 260 and 280 nm. The monosaccharide constituents of RCPS were Rha, Ara, Xyl, Man, Glu, Gal and GalA, and their molar proportions were 1 : 2.96 : 0.21 : 0.26 : 0.08 : 0.58 and 0.15, respectively.