Studies On Isoenzymes And Related Genes For Sex Identification Of Schisandra Chinensis

Schisandra sphenanthera Rehd.et Wils.(Schisandra,Schisandraceae),a traditional authentic Chinese medicinal material,has the effects of nourishing the kidney,astringent and astringent,and improving qi and vitality.However,due to climate change and over-picking,resources and suitable habitats of wild S.sphenanthera have gradually decreased.Therefore,protection and sustainable use of S.sphenanthera is imperative.As a dioecious plant,the main medicinal part of S.sphenanthera is fruit,but the identification of males and females is a major problem.Therefore,research on its gender is beneficial to the introduction,cultivation and breeding of S.sphenanthera,and has practical significance.In this study,S.sphenanthera flowers and leaves of male and female were used as materials to reveal the differences between male and female plants of S.sphenanthera from the isoenzyme bands,cloning of sex-related genes AP3,PI of S.sphenanthera and their expression,so as to provide method and experimental data support for the differentiation of male and female of S.sphenanthera.The main conclusions are as follows:1.The isoenzyme pattern(superoxide dismutase(SOD),peroxidase(POD),esterase(EST),polyphenol oxidase(PPO))of the female and male of S.sphenanthera were different.Among them,SOD isoenzymes have two female-specific enzyme bands(SOD6,SOD7)and three male-specific enzyme bands(SOD2,SOD3,SOD5);POD isoenzymes have two female-specific enzyme bands(POD2,POD4)and one male-specific enzyme band(POD3);EST isoenzymes have one male-specific enzyme band(EST2);PPO isoenzymes has a female-specific enzyme band(PPO2).Isoenzyme bands can be used as a reference for sex identification of S.sphenanthera.2.The full-length cDNA of S.sphenanthera AP3 gene and the conserved fragment and 3' end of S.sphenanthera PI gene were cloned by the combination of homologous cloning and RACE technology.The CDS sequence of AP3 gene was 591 bp,encoding 197 amino acids.The conserved fragment of PI gene was 299 bp,and the 3' end was 671 bp.BLAST comparison revealed that the S.sphenanthera AP3 and PI genes had the highest homology with Kadsura japonica KjAP3 and KjPI genes,99%and 95%.And the SsAP3 gene GeneBank accession number is MN908946.3.Protein structural analysis showed that SsAP3 protein is a stable nuclear-localized hydrophilic protein with protein phosphorylation as the major serine.Evolutionary analysis showed that S.sphenanthera SsAP3,together with Kadsura japonica and four species of Illiciaceae(Illicium floridanum,I.henryi,I.parvifolium,I.anisatum),gathered near the base of the development tree,indicating that Schisandraceae is a primitive group of plant origin and closely related to Illiciaceae4.The difference between SsAP3 and SsPI genes in female and male of S.sphenanthera leaves was detected by real-time fluorescence quantitative PCR.The results showed that the expression levels of SsAP3 and SsPI genes in male S.sphenanthera leaves were very significantly higher than those of female(P<0.01).SsAP3 and SsPI genes may be one of the reasons for the sex differentiation of S.sphenanthera.This study initially revealed the differences in four isoenzyme and expression of sex-related genes SsAP3 and SsPI between female and male plants in S.sphenanthera.Through this study,it provides an alternative to morphology for the sex identification of S.sphenanthera,and provides some theoretical basis and experimental data support for the sex identification in expand breeding and cultivation.It has important practical value.