Expression And Function Of ?-ketoacyl-CoA Synthase PnKCS1 From Pohlia Nutans

Antarctica is located at the southern end and occupies one-tenth of the total land area of the earth.However,it only distributes more than 850 kinds of plants.Its extreme weather such as cold,dry and strong ultraviolet radiation is the main factor restricting plant growth.Bryophytes,belonging to the lower group of higher plants,are the most widely distributed and abundant higher green plants in the Antarctic continent,which is the main producers in this ecosystem.The adaptation mechanism of Antarctic bryophytes to extreme environment is closely related to its unique functional genes and physiological functions.Cuticle wax,a component of the cuticle covering the surface of plants,plays an important role in resisting abiotic stresses such as drought,high salt,and strong ultraviolet.The wax of the stratum corneum is mainly composed of very long chain fatty acids(VLCFAs)and their derivatives.Very long chain fatty acids refer to fatty acids with acyl chains of 18 carbon atoms and above,which are formed by condensation,reduction,dehydration,and re-reduction catalyzed by the fatty acid elongases(FAE)located in the endoplasmic reticulum membrane.Among them,?-ketoacyl-CoA synthase(KCS)catalyzes the condensation reaction,which is the rate-limiting step in the synthesis of VLCFAs.KCS can change the resistance of plants to abiotic stresses by affecting the formation of cuticular wax.In this study,a highly responsive gene was screened from the transcriptome of Pohlia nutans under UV-B stresse,and it was preliminarily identified as ?-ketoacyl-CoA synthase gene by bioinformatics analysis,named PnKCS1,and analysis the biological function of PnKCS1.The specific results are as follows:1.Bioinformatics analysis of the PnKCS1The open reading frame(ORF)of PnKCS1 is 1542 bp,encoding 513 amino acids.The molecular weight of the protein is 57.91 kDa and the isoelectric point is 8.77.Multiple sequence alignment showed that PnKCSl had a typical KCS conserved domain,FAE1_CUT1_RppA and ACP_syn_?C.PnKCS1 has the highest similarity with Physcomitrella patens(XP₀24395138.1),which is 91.4%,and the similarity with KCS of Arabidopsis thaliana,Selaginella vulgaris and other species is more than 75.0%,indicating that this gene is higher conservative in the evolution of plants.Phylogenetic tree analysis showed that PnKCSl has a common evolutionary ancestor with KCS of other species;and clustered with KCS of Physcomitrella patens,indicating that they are closely related.According to the results of subcellular localization and transmembrane domain prediction,it is speculated that PnKCS1 is located in the cytoplasm and has two transmembrane domains,which may be a transmembrane protein located in the inner membrane system of cells.2.Study on the function of PnKCSl in Saccharomyces cerevisiaeThe yeast expression vector pYES2-PnKCS1 was constructed and overexpressed in Saccharomyces cerevisiae(INVSc1).The total fatty acid content of yeast was analyzed by GC-MS.It was found that compared with the negative control(yeast containing pYES2),overexpression of PnKCS1 had no effect on the types of fatty acids in yeast cells,and the main components were C16:0,C16:1,C18:1 and C22:0;but it significantly affects the ratio of its components.Among them,the content of C16:0 was reduced by 6.70%,the content of C18:1 was increased by 6.90%,and the content of very long chain fatty acid C22:0 was increased by 37.4%.This indicates that PnKCS1 can promote the accumulation of very long chain fatty acid in yeast.3.The effect of PnKCS1 on cuticle wax of Arabidopsis thalianaThe PnKCS1-pRI101 expression vector was constructed and introduced into Arabidopsis to obtain an overexpressed PnKCS1 pure line Arabidopsis(hereinafter referred to as overexpressed Arabidopsis,AtOE).The cuticle wax compositions of wild-type(Col-0)and overexpressed Arabidopsis(AtOE)silique,stems and leaves were compared by GC-MS.The results showed that in contrast with wild-type Arabidopsis,the total wax content in silique,stem and leaf of overexpressed Arabidopsis increased by 12.3%,19.8%and 15.2%,respectively.The contents of alkanes,fatty alcohols,fatty aldehydes and ketones in waxy components of overexpressed Arabidopsis silique were 1.10,1.15,1.23 and 1.12 times higher than those of wild type,respectively.Except for the content of fatty aldehyde,the content of other waxy components(alkanes,fatty alcohols and ketones)in stems was enhanced,which was higher 20.7%,20.4%and 27.5%than wild type;the content of waxy components in leaves was low,and only some waxy components were detected,among which the content of alkanes and fatty acids in overexpressed Arabidopsis was 112.5%and 352.6%of that in wild type,respectively;the content of alcohols had no significant change.These results indicate-that overexpression of PnKCSl can promote the wax content of plants by increasing the accumulation of very long chain fatty acids.4.The effect of PnKCS1 on the response of Arabidopsis to abiotic stressesIn order to study the effect of overexpression of PnKCSl on abiotic stresses of plants,Arabidopsis was treated with UV-B,NaCl,drought,oxidative stress and abscisic acid(ABA).Under normal conditions,overexpression of PnKCS1 had no significant effect on the growth status of Arabidopsis,but significantly reduced the water loss rate and chlorophyll extravasation rate of detached leaves.After UV-B stress,compared with the wild type,the overexpression of Arabidopsis leaves showed less yellowing and shrinkage,lower H2O2 level and decreasing Malondialdehyde(MDA)content;the activity of superoxide dismutase(SOD)and the relative water content of leaves in overexpressed Arabidopsis were 2.71 and 1.31 times higher than those in wild type,respectively;furthermore,the expression levels of AtHY5,AtCu-Zn-SOD1,AtNHX1,AtP5CS1 in overexpressed plants were significantly up-regulated in comparison to wild type.This indicated that overexpression of PnKCS1 could improve the resistance of Arabidopsis to UV stresses.When cultured in 1/2 MS containing 100 mM and 125 mM NaCl,the seed germination rate and early root length of the overexpressed Arabidopsis were significantly higher than those of the wild type.Among them,the root length was 1.46 and 1.96 times that of the wild type,respectively.Under salt stress,compared with the wild type,the water loss rate,chlorophyll extravasation rate,H2O2 and MDA levels in the leaves of overexpressed Arabidopsis decreased significantly,while the free proline(Pro)content,SOD and POD activity increased significantly;the expression levels of salt stress-related genes AtRD29A,AtAVP1,AtNHX1,AtP5CS1,AtCAT1 were also significantly up-regulated.These results indicate that overexpression of PnKCS1 can improve the resistance of plants to salt stress.Mannitol and PEG 6000 were used to simulate drought stress to analyze the effect of overexpression of PnKCS1 on drought stress resistance of plants.Under the treatment of 0.1-0.3 M D-Mannitol,the growth status of overexpression Arabidopsis was better than that of wild type,and the root length of overexpression Arabidopsis was about 1.87 times of that of wild type under the treatment of 0.3 M D-Mannitol.After 16.0%PEG treatment,the water loss rate,chlorophyll permeability rate,H2O2 and MDA level of leaves were significantly lower than that of wild type.After PEG treatment,the expression levels of drought stress related genes AtNHXl,AtP5CS1,AtCu-Zn-SOD1 and AtCAT2 were significantly increased.Abiotic stress usually leads to oxidative stress.Under the treatment of different concentrations of H2O2,the root length of overexpressed Arabidopsis was higher than that of the wild type;the water loss rate and chlorophyll extravasation rate of detached leaves were lower than that of the wild type.This indicates that overexpression of PnKCS1 Arabidopsis can improve the resistance of plants to drought and oxidative stress.Abscisic acid(ABA)plays an important role in regulating the growth and development of plants and coping with abiotic stresses.When cultured on 1/2 MS with 0.5 ?M ABA,the germination rate of overexpressed Arabidopsis was above 50.0%,while the germination rate of wild type was only 6.30%.After being treated with 100 ?M ABA for 24 h,the water loss rate of wild-type detached leaves was significantly higher than that of overexpressed After treatment with 100?M ABA for 2.5 h,the expression levels of AtRD29B,AtAREB2 and AtRAB18 were higher than those of wild type.This suggests that overexpression of PnKCS1 can reduce the sensitivity of Arabidopsis to ABAIn summary,PnKCS1 can promote the synthesis of very long chain fatty acids,thereby increasing the wax content of plants,reducing the permeability of the epidermis,preventing water loss,and improving the resistance of plants to abiotic stresses.This paper provides a theoretical basis for revealing the role of PnKCS1 in the adaptation of Antarctic Pohlia nutans to extreme environments,and promotes the development and utilization of polar biological resources.