Heterologuos Expression Of Cold Shock Protein Gene HhCsp From Halobacillus Halophilus Significantly Improves Salt Tolerance Of Arabidopsis Thaliana

Cold shock protein?Csp?,as a responsive protein,is found in almost all gram-positive and gram-negative bacteria.When cells are exposed to cold and other abiotic stresss,Csp functions as a molecular chaperone of RNA and non-specifically binds to single-stranded nucleotides,preventing the formation of mRNA secondary structure,avoiding the termination of transcription and translation,and ensuring that cells normal metabolism.In addition,Csp acts as a transcription activating factor and binds to promoters to increase the expression level of genes.The Csp genes of Escherichia coli and Bacillus subtilis have been cloned,evaluated for their function,and used to transform plants for improvement of their tolerance to drought and other abiotic stresses.The extreme microorganisms that survive in extreme abiotic stresses have unique cell structures and environmental adaptation mechanisms.Halobacillus halophiles produces various resistance solutes such as Csp to tolerate the high salt environment.In this study,the sequence of the HhCsp gene of H.halophiles were analysied for its bioinformatics characteristics,optimized according to the codon preference of the maize genome,constructed into dicotyledonous expression vector,and used to transform Arabidopsis thaliana by Agrobacterium mediation,to explore the possibility of heterologous expression for improvement of tolerance to high salt and drought stresses,and to lay foundation for the next step in the transformation of maize and other crops..The main results are as follows:?1?The full-length cDNA sequence of the HhCsp gene was 195 bp long and encodes 65amino acids.Homology modeling analysis showed that the HhCsp protein was a barrel-like structure of five inverted folded?chains,and the surface of this structure contained aromatic amino acids.The sequence of the HhCsp protein shared 83.58%similarity with the BsCspB protein of B.subtilis,contained the typical conserved domains of the Csp proteins,such as RNA binding motifs RNP1?KGFGFI?and RNP2?VFVHF?.Therefore,it was speculated that the encoding protein of the HhCsp gene have the same function of the moclecular chaperone.?2?According to the codon preference of the maize genome,the sequence of the HhCsp gene was optimized,resuting in the increase of the G/C content from 39.71%to 50.26%,the replacement of 10.25%bases and 30.76%codons,85.35%similarity with the original sequence,reduction of free energy from 70.6 to 40.39.The rare codons were eliminated to make the frequency of the codon usage consistent with the maize genome.In order to facilitate its highly efficient transcription in maize.?3?In order to ensure the safety of the transgenic plants,we submitted the optimized sequences to the NCBI database and performed safety screenings with known homologues of toxic protein sequences,comparison of allergens,and comparison of anti-nutritional factors.The HhCsp gene is safe and can be used for subsequent transformation.?4?In order to verify the optimized gene stress resistance function,we first constructed the dicotyledon expression vector pCAMBIA2300-35S-HhCsp.After digestion and sequencing,it was shown that the vector was constructed correctly.Agrobacterium-mediated transformation of Arabidopsis?Colombia ecotype?,after PCR positive test and RT-PCR validation,indicating that the five independent transformation events have been successfully expressed.The transformation events were self-bred to the T₃ generation and homozygous lines were obtained for subsequent resistance identification.?5?On 1/2 MS hypertonic medium supplemented with high concentrations of mannitol and NaCl,the root development and seed germination rate of the transgenic lines and wild type Arabidopsis were inhibited with increasing mannitol and NaCl concentrations.However,the root development and germination rate of the transgenic lines were significantly better than the wild-type controls,indicating that the root development and seed survival rate of the HhCsp transgenic lines were significantly better than the untransformed controls under high salt.?6?In the nutrient soil culture,after 20 days of natural drought,the growth of the transgenic lines and the wild-type Arabidopsis thaliana were inhibited,but only some of the transgenic lines were wilted,while the wild-type almost all withered.One week after rehydration,part of the transgenic lines returned to normal growth,whereas the wild type all died of dryness.However,when the seedlings were irrigated with 350 mmol/L NaCl to simulate hyperosmotic stress,the transgenic lines were only partially wilted and all of the wild type plants died.These results indicate that our optimized HhCsp gene can significantly improve salt tolerance and drought tolerance of Arabidopsis seedlings in recipient plants.?7?The above experiments have demonstrated that heterologous expression of HhCsp can significantly improve the drought resistance and salt resistance of Arabidopsis.Therefore,we constructed the monocot expression vector pTF101.1-Ubi-HhCsp,transformed by Agrobacterium-mediated method.The embryogenic callus of 18-599 red inbred line of maize was obtained with 5 transformation events.Self-cross breeding and molecular identification were being carried out in order to obtain homozygous lines and carry out resistance verification experiments.The above results indicate that the HhCsp gene has been integrated into the Arabidopsis thaliana genome,and its heterologous expression significantly enhaces salt tolerance of the transgenic lines.The optimized HhCsp gene is hopeful for transgenic research of abiotic tolerance in maize and other crops.