Preliminary Functional Study Of Lipase Gene LpGDSL In Lilium Pumilum

Saline-alkaline land as a reserve land resources is very worthy of attention,the effect of saline-alkaline stress on the growth and development of plants has also received a lot of attention,and Lilium pumilum is a plant with strong saline-alkaline tolerance.Therefore,we performed transcriptome sequencing analysis on the bulb of Lilium pumilum after treatment with 20 mM NaHCO₃.The analysis of the results showed that the expression level of lipase gene was significantly increased as compared with that in the untreated plant.In order to explore the response of lipase to saline-alkali stress,we successfully cloned the LpGDSL gene and performed homologous sequence alignment and evolutionary tree analysis.The specific research results are as follows:1、The subsequent amino acid sequence alignment confirmed that LpGDSL had high homology with the amino acid sequence of GDSL protein in multiple plant species and predicted that it had a highly conservative domain SGNH,which belonged to lipolytic enzyme.LpGDSL protein is a relatively stable hydrophobic protein,with a segment of N-terminal signal peptide.It is predicted that the protein may be localized in the whole cell to exert effects.2、After obtaining the pYES2-LpGDSL recombinant yeast strain,stress culture is carried out,and the recombinant yeast has better growth performance compared with the empty carrier yeast and can respond to stress sensitively;Through analysis of qPCR data,it was found that the LpGDSL gene was expressed in all tissue parts of Lilium davidii,and its expression reached the highest in the leaves,while it was relatively less in the seeds and roots.The results showed that the expression of LpGDSL gene showed an up-regulation trend compared with the control group under a variety of stresses,and the response of LpGDSL gene to saline-alkali stress was significantly involved in the stress response of plants.3、The LpGDSL gene was constructed on a protein expression vector,and the optimal conditions for protein induction of pGEX-LpGDSL were determined as follows:1mM IPTG,30℃,and 4h,and the purified protein was obtained.The measurement results of the protease activity of LpGDSL showed that it was a plant lipase gene with lipase activity.The analysis of bacterial liquid tolerance showed that the bacterial liquid tolerance to the expression of LpGDSL protein was inhibited under different stress conditions.Compared with pGEX-6P-3vector,the bacterial liquid tolerance to stress was obviously demonstrated.4、The stress treatment was applied to the aseptic seedlings and soil-cultured seedlings of pEH12-LpGDSL overexpressed Lilium pumilum.Compared with the wild type,the overexpressed Lilium pumilum strain showed obvious salt and alkali tolerance.The expression level of LpGDSL protein in plants after stress was higher than that of wild type.The measurement of ROS-related indicators showed that over-expressed Lilium pumilum had significantly better tolerance than the wild type and was less vulnerable to salt-alkali stress.LpGDSL gene endows plants with saline-alkali tolerance by improving the degree of bolting and lignin content of plants,so as to protect plant cells from saline-alkali stress.5、The yeast two-hybrid was used to preliminarily screen out the protein that might interact with LpGDSL from cDNA library,and the LpBCP protein related to saline-alkali tolerance was screened out.The BIFC experiment again verified that the LpGDSL protein could interact with LpBCP,and it could determine the interaction with LpGDSL protein was obtained.The promoter fragment of LpGDSL was cloned and analyzed,and the B3transcription factor related to stress tolerance was screened for verification.The B3transcription factor can act on the LpGDSL promoter to regulate the expression of downstream genes,and the action mechanism of LpGDSL gene was further explored.