Gene Function Analysis Of PA Specifically Expressed In Diploid Potato Pollen

Potato(Solanum tuberosum)is an important crop in the world and plays an important role in ensuring global food security.In agricultural production,potato is often propagated with tubers,and long-term asexual propagation will cause seed potato degeneration and yield reduction.Conventional breeding of potato is to give full play to parents' elite traits through hybrid seeds.Like other sexually propagated crops,heterosis is a common phenomenon in the biological field.Rice and corn and other crops use heterosis to greatly increase the yield.More and more attention has been paid to the seed of potato,and the creation of male sterile line is a necessary process for potato to realize the breeding of seed with heterosis.Diploid potato cross breeding is to improve the potato into a seed breeding crop based on inbred lines at the diploid level,which can greatly accelerate the breeding process of new potato varieties.Nowadays,more and more attention has been paid to diploid potato cross breeding,so it is very important to explore male sterility genes and develop male sterility lines.In the Phospholipase A(PLA)superfamily of Arabidopsis thaliana,the PLA2-??PLA2-? and PLA2-? of secretory PLA2(sPLA2)family are correlated with male fertility,and the At1g61850 of patatin-like PLA(PAT-PLA)is highly expressed in early stamen development and dry pollen.In order to study the mechanism of potato male sterility,two homologous genes were obtained by homologous search in Spud DB Potato Genomic Resource using At1g61850 sequence.Through Real-time fluorescence quantitative experiment,it was found that PGSC0003DMT400005303 was specifically highly expressed in the pollen of diploid potato C065,and was named as PA(Pollen Abortion).In order to verify whether the gene can affect male reproductive ability,we used the CRISPR/Cas9 system to knock out the gene in a diploid potato C065 and obtained two frame-shifting mutants,named pa-1 and pa-2 respectively.While defecting the pollen viability,the ability of pollen tube germination in vitro and in vivo and the fruit setting rate of the mutants,we found that the pollen viability and pollen tube germination rate of the mutant were significantly decreased compared with the wild type.In addition,we analyzed RNA profiles of wild-type and mutant pollens and found that 3,539 genes in the mutant pa-1 and 2,203 genes in the mutant pa-2 showed changes at expression levels.GO analysis of Differentially expressed genes showed that the expression of sulfate transport,carboxylic acid metabolism related enzymes and ribonuclease ? genes in PA gene mutant was evidently different from those in the wild type.The results showed that PA gene was related to potato pollen fertility,but its main biological functions in affecting pollen development needed to be further studied.Potato mutant of PA gene created in this project provided materials for the study of PA gene function.The differentially expressed genes in mutants could provide reference for the search of genes concerning male sterility in potato.