Cloning Of SgLRPK1 And SgCRK1 Gene And Transformation In Stylosanthes Guianensis

Stylosanthes guianensis,a kind of excellent tropical legume forages that is mainly used for grazing, soil and water conservation, intercropping orchard and hay meal processing with high yield and excellent quality, which tolerance to heat, drought and resistance to acid etc, is native to central and South America. However, it is vulnerable to chilling injury. A variety of hormone signaling pathways related in the body of plant, which was identified and transmitted by the receptor- like protein kinases(RLK)that responses to plant biotic stress and abiotic stress. Therefore, it is important to study the molecular mechanism of RLK and the cold resistance of Stylosanthes guianensis for increasing the cold resistance and promoting the production.Two kinds of receptor-like protein kinase were cloned and analyzed from transcriptome database in this paper. The PCR method was used to clone the partial fragments of Sg LRPK1 and Sg CRK1, and the RACE or hi TAIL-PCR methods were used to amplify the unknown sequences base of the known fragments. The results show that the ORF of Sg LRPK1 gene is 1812 bp, encoding 604 amino acids, with a conserved LRR repeat extracellular, a transmembrane region and intracellular kinase domain. The c DNA of Sg CRK1 gene is 861 bp, encoding 287 amino acids with two unknown functional areas.Overexpressing vector was constructed with the complete encoding region of Sg LRPK1 and Sg CRK1 gene and the p Cambia3301 expression vector with 35 S promoter.Besides, two target sites of Sg LRPK1 gene connect of CRISPR/Cas9 to construct the targeting vector. Several vector was carried into Stylosanthes guianensis 184 by Agrobacterium mediated method, the overexpressing of Sg LRPK1 won six strains of transgenic plants of Stylosanthes guianensis and four of them were positive plants;Overexpression of Sg CRK1 obtain 4 strains of transgenic plants with 3 strains positive plants among them; Sg LRPK1 gene-targeting expression vector acquire 18 strains oftransgenic plants that 10 strains as positive plants, respectively.The relative expression of Sg LRPK1 gene and Sg CRK1 gene showed the trend of first increasing and then decreasing used by real-time fluorescence quantitative PCR detection follow the low temperature stress. Under low temperature treatment, the Sg LRPK1 gene expression showed an upward trend in the former 12 h, above 6 times than before treatment when it reached the highest in the 12 h, and still relatively high after the downward trend in the 24 h. The relative expression of Sg CRK1 gene was achieved the highest in treatment 24 h,which reached above 8 times than before treatment, and then decreased. Cold treatment could increase the relative expression of two RLK, which indicated that the Sg LRPK1 gene and Sg CRK1 gene could respond to the cold resistance of the Stylosanthes guianensis.