Screening Of Arabidopsis Thaliana Mutants With Abnormal Pollen Tube Growth Mediated By Latrunculin B

Pharmacological and genetic experiments have shown that actin cytoskeleton plays an important role in regulating the polar growth of pollen tube in angiosperms.Predecessors have done a lot of research on actin cytoskeleton in the process of polar growth of pollen tube and achieved a lot of original results.However,the molecular mechanism of actin cytoskeleton accurately regulating the polar growth of pollen tube remains to be studied in many aspects.For example,the signaling network that regulates actin cytoskeleton is not perfect.How various signal molecules cooperate to regulate the organization and dynamics of actin cytoskeleton in different time and space.Actin cytoskeleton precisely regulates which cell activities in the process of polar growth of pollen tube.In order to explore the function and mechanism of actin cytoskeleton in regulating pollen tube growth,forward genetic screening was carried out based on actin polymerization inhibitor Latrunculin B.The mutants with abnormal pollen germination and pollen tube growth were screened by exogenous application of Latrunculin B,during pollen germination of Arabidopsis thaliana EMS mutagenic plants.The main results are as follows:1.An experimental system for screening abnormal mutants of Arabidopsis thaliana pollen tube growth mediated by exogenous Latrunculin B was successfully established.2.Six thousand EMS mutants were screened in the M1 generation,and 407 utants with abnormal pollen germination and pollen tube growth were obtained when treated with 3nM Latrunculin B.3.In M2 and M3 generations,38 homozygous lines with stable phenotypes ere screened,including the mutants R-1—R-20,which were resistant to exogenous atrunculin B treatment for pollen germination and pollen tube growth,and the utants S-1—S-18,which were sensitive to Latrunculin B treatment.4.The homozygous verification of 21 mutants was carried out and hybridized with wild type.These lines still had a stable phenotype in F1 pollen germination and pollen tube growth to exogenous Latrunculin B treatment.5.Homozygous lines of four mutants(R-1,S-1,S-2 and S-3)were selected from F2 genetic population,and it was confirmed that R-1,S-1,S-2 and S-3 were single gene recessive mutations.6.Preliminary observation of actin organization and FM4-64-labeled endocytic vesicles and internalization rate in R-1,S-1,S-2 and S-3 mutants.To sum up,through forward genetic experiments based on exogenous application of LatB,we preliminarily screened some mutants with defects in polarity growth of pollen tubes.These mutants can be used as genetic materials for phenotypic analysis and identification of new gene functions.It may provide new experimental materials and research ideas for revealing the mechanism of actin cytoskeleton involved in polar growth regulation.