In Vitro Molecular Evolution And Function Analysis Of Water Stress Protein In Nostoc Flagelliforme

Nostoc flagelliforme commonly known as Facai,it is a terrestrial nitrogen-fixing cyanobacterium.The Nostoc flagelliforme has a strong ability to tolerate drought.It contains abundant drought tolerant gene resources and it is an ideal material for studying drought tolerant genes.Cloning their drought tolerant genes will provide candidate genes or theoretical references for plant drought resistance genetic engineering.WspA was first isolated from Nostoc commune,it has a certain drought tolerance,and it was accompanied by ?-galactosidase activity.Studies have shown that there are many WspA homologous proteins in the Nostoc flagelliforme colloid sheath,which may be a unique class of protein in the schizophum Nostoc.Using the genomic DNA of Nostoc flagelliforme as a template,the water stress protein gene sequences of Nostoc flagelliforme were cloned by PCR and named WspA3 and WspA4.Through bio informatics analysis,two proteins were found belong to hydrophilic acidic protein and distributed in the cytoplasm.The WspA3 and WspA4 genes were ligated with the expression vector pET-32a to construct a recombinant expression vector and transformed into E.coli BL21 for prokaryotic expression.The simulation of drought stress with polyethylene glycol 6000(PEG6000)found that it was able to tolerate 60%PEG6000 stress and showed strong drought tolerance.To improve the growth enhancement activity of WspA3 and WspA4 in Nostoc flagelliforme drought resistance to promote the host cell growth,a base mutation was introduced into the WspA gene sequence by error-prone PCR and transformed into E.coli BL21 to construct a mutant gene expression library.Mutated active WspA proteins were screened in test tubes according to the growth of the strains under drought stress simulated by 60%PEG6000,a total of 6 proteins with increased drought tolerance were obtained,including 2 WspA3 proteins and 4 WspA4 proteins.the mutant strain P-galactose glucoside relative vitality were 1.4-3.5 times that of the wild type protease relative vitality.The six proteins were modified by DNA family shuffling,no mutant protein sequences with enhanced drought tolerance were obtained,but their phenotypes changed significantly,indicating that the DNA family shuffling played a role in promoting the evolution of evolved proteins.The WspA3,WspA4,WspA3-sf1,and WspA4-sf1 genes were transferred into higher model tobacco plants to over express,to study the potential drought resistance of WspA3-sf1 and WspA4-sf1 genes.It Successfully obtained tobaccos transformed with WspA3,WspA4 and WspA3-sf1 genes,and the transgenic tobacco was treated with 10%PEG6000 simulated drought stress for 5 days.The activity of antioxidant enzymes in transgenic tobacco was higher than that in control,and the content of malondialdehyde decreased compared with the control.The results showed that the gene was involved in the regulation of tobacco antioxidant enzymesand accumulation of membrane stability regulators under drought stress.The change of tobacco physiological index of WspA3-sf1 gene was more prominent,indicating that the gene has stronger drought tolerance.