Preliminary Study On The Function Of Callose Synthase 6 In Physcomitrella Patens

Physcomitrella patens belongs to the genus of the genus Funariaceae,belonging to the non-vascular group,and is a representative of early landing plants.After being infested with Botrytis cinerea,the P.patens forms a mastoid structure that resists pathogens in the cell wall.Callose is the main component of the formation of mastoids and is synthesized by the callose synthase.In the genome of the P.patens,a total of 12 genes encode the callose synthase.The expression level of the callose synthase PpCalS6 gene in the plant on the second day after inoculation with B.cinerea was significantly increased by transcriptome sequencing and real-time fluorescent quantitative PCR.It indicated that callose may be involved in the defense response of P.patens to B.cinerea.Other studies have reported that callose may also be involved in plant growth and development.This study intends to study the function of PpCalS6 by reverse genetics method to confirm whether callose participates in resisting pathogenic bacteria and affecting plant growth and development in P.patens.The results of this study are as follows:1.This study successfully constructed PpCalS6 knockout plants.The PpCalS6-PTN182 knockout vector was constructed first,and then PpCalS6-PTN182 was transferred into the Protoplast of P.patens by PEG-mediated genetic transformation using homologous recombination technology.PpCalS6 knockout plants(PpCalS6-PTN182 plants)were successfully obtained by G418 twice different concentration screening(25 ?g/mL,50 ?g/mL)and molecular level verification.2.The content of callose in the wild-type(WT)and PpCalS6-PTN182 plants in the protoplasts and gametes was determined by ELISA.The results showed that the callose content of the P.patens WT in the protoplast(5.742 ?g/g)and gametophore phase(12.146 ?g/g)was significantly higher than that of PpCalS6-PTN182 plants in the protoplast(3.799 ?g/g)and gametophore stage(8.556 ?g/g).It is indicated that PpCalS6 is involved in the synthesis of callose in P.patens.3.After the B.cinerea was inoculated with WT and PpCalS6-PTN182 plants,the hyphae formed on the third day of infection,indicating that both genotype plants were infected with B.cinerea.Observed and compared,the degree of infection of PpCalS6-PTN182 plants was significantly higher than that of WT.At the same time,the content of callose in both bodies increased with the infestation time.At the same infestation time,the content of callose in WT plants was higher than that in PpCalS6-PTN182.It is speculated that callose is a conservative defense response mechanism in P.patens.4.By comparing the gametophyte structure,there was a significant difference between the PpCalS6 knockout plants and the wild type phenotype.The protoplast division of WT and PpCalS6-PTN182 was observed by microscopy.It was found that the protoplast division time of PpCalS6-PTN182 was later than that of WT.It has been reported that callose is related to the formation of cell plates,indicating that the dysplasia of PpCalS6 knockout plants may be related to the failure to form normal cell plates at the time of cell division.And at the same time point,the number of dividing cells of PpCalS6-PTN182 and the presence of the lateral branches of the protoplasts were less than those of WT.It is speculated that the inability of PpCalS6 knockout variants to form normal gametes may be associated with the regulation of transport dysregulation of intercellular auxin.