Cloning And Functional Verification Of P5CS Gene In Apricot Kernel

Kernel-apricot is a generic name of the genus Apricot,which is based on almonds and is a major product.However,due to the fact that the use of apricot apricots often suffers from low temperature and cold damage,the development of the apricot industry is seriously affected.Therefore,the cultivation of cold-resistant seed varieties through genetic engineering has become an urgent task.In this study,we used RT-PCR technology to clone the c DNA of leaves from " Weixuan1 hao " as a template,and cloned P5 CS gene(Wx P5CS)from apricot " Weixuan1 hao " for bioinformatics analysis.The Wx P5 CS was analyzed by q RT-PCR.The spatio-temporal expression pattern of the gene;construction of an eukaryotic expression vector and transformation of yeast cells,analysis of the growth of transgenic cells under temperature stress.The result is as follows:(1)Sccessfully cloned the WxP5 CS gene,bioinformatics analysis showed that the ORF was 2280 bp in length and encoded a total of 759 amino acids with a relative molecular weight of 82227.09 Da and an isoelectric point of 6.64.The homology with the P5 CS protein in Gen Bank was very high;the conserved domain analysis revealed that the protein contains the aldehyde-deletion of the amino acid kinase(AAK)superfamily,?-glutathyl phosphate reductase(GPR),and NAD(P)+ dependent enzyme.Superfamily of ALDH-SF and AA_Kinase;transmembrane domain analysis P5 CS protein found no transmembrane domain.(2)Using the Clustal 4.0 software to perform multiple sequence alignments on seven species,it was found that the amino acid sequence of the P5 CS gene encoded by the P5 CS gene has a high similarity.It can be inferred that the structure of the P5 CS protein is very conservative.The phylogenetic tree was constructed using MEGA 6.0 software.The results showed that the kernel apricot “Weixuan1hao” was closely related to aconite leaves,tomato,kiwifruit,and rice,but had a distant relationship with Arabidopsis thaliana.(3)The expression pattern of Wx P5 CS gene in different organs and the analysis of expression patterns under low temperature conditions showed that the expression level of Wx P5 CS gene was different in different organs,the highest in flowers,the difference was not significant in other parts,showing the specificity of tissues and organs.Under the low temperature stress,with the extension of time,“Weixuan1hao” and the control “Longwangmao” showed different expression patterns: the former showed a continuous upward trend,while the latter showed a first rising and then a downward trend.(4)The recombinant plasmid p YES2-Wx P5 CS was transformed into yeast cells to analyze the growth of yeast cells under low temperature and high temperature.The results showed that the expression of Wx P5 CS gene enhanced the stress resistance of yeast.