Microstructure And Development Related Gene Analysis Of Spores And Sporangia In Huperzia Serrata (Thunb.) Trev

Huperzia serrata(Thunb.)Trev.,also known as the Qian Cengta,has received much attention due to its inclusion of Huperzine A.Hup A is derived from the wild plant of H.serrata.There are no other natural products that can replace Hup A.The shortage of resources has become a bottleneck restricting the development of Hup A.In this study,the microscopic observation of the occurrence and development of the spores and sporangia of H.serrata was carried out by optical microscopy.The viability and physiological indexes of the artificially cultured spores were determined,and the genes related to spore growth were also screened for Hs KNOX1 and Hs KNOX2.In addition,genetic diversity analysis was carried out in the closely related species of Huperzia serrata using SSR molecular marker technology,and the genetic relationship between H.serrata,Huperzia crispata(Ching)Ching,Huperzia sutchueniana(Herter)Ching and Huperzia se Lago(L.)Bernh.ex Schrank et Mart.was established.The results are as follows:(1)The microscopic structure of the spores and sporangia of the genus H.serrata can be seen that the sporangium wall of the H.serrata is divided into the inner layer and the outer layer,and the cell wall in the mature stage reaches 4-5 layers;spores of H.serrata from the beginning of growth to the maturity of spores,the number of spores in the sporangia is increasing and then released;the tapetum acts as the innermost layer of the sporangia wall,close to the inner wall of the spore,and plays an important role in the development of spores;At the end of the period,the velvet layer began to degenerate and fracture,which also indicated that the velvet layer played less role in the later stage of development.(2)The viability of the spores of H.serrata was determined in different culture periods.Firstly,the determination method was screened and finally determined to be methylene blue method.The results showed that the viability of the spores of H.serrata decreased with the increase of culture time.At the same time,the SOD activity,soluble protein and proline content of the spores of H.serrata were determined.The content of soluble protein decreased sharply with the increase of culture time.The content of proline also increased with the culture.The increase in the number of days has gradually declined.The SOD enzyme activity increases with the increase of the number of culture days,and the ability to scavenge free radicals.(3)Hs KNOX1 and Hs KNOX2 genes of H.serrata were cloned successfully.There are1524 bases in ORF region of Hs KNOX1 gene and 756 bases in Hs KNOX2 gene.The bioinformatics analysis of these two genes showed that Hs KNOX1 protein was composed of 507 amino acids and Hs KNOX2 protein was composed of 251 amino acid residues.The sequence of HSKNOX1,Hs KNOX2 and protein of H.serrata was compared with the homologous sequence in the database,and the result showed that H.serrata had the closest relationship with Taxodium cephala;Hs KNOX1 protein belonged to KNOX protein.KNOX class II of the family,while Hs KNOX2 protein belongs to KNOX class I of the KNOX protein family.The functional domains of the two proteins are analyzed,which conforms to the typical KNOX protein family.The subcellular localization of the three proteins is predicted by the online software,and both proteins are located in the nucleus.The online software analysis can lay a theoretical foundation for further study of protein hierarchy.(4)Through screening SSR primers in different populations of H.serrata,and SSR validation in 15 individuals of natural populations of three closely related species,H.crispata,H.sutchueniana and H.se Lago.It was found that all of the loci were polymorphic,that is,the ratio of polymorphic loci reached 100%.The size of major gene fragments of each locus in four species of H.serrata preliminarily showed that there were close relationships among four species of Huperzia,among which the relationship between H.serrata and H.se Lago was the closest.Through validation,polymorphic primers for genetic diversity analysis of Huperzia were further screened.