Preliminary Analysis Of SNARE Disassembly Factors SNAP In Arabidopsis Thaliana

Proper sorting and secretion of proteins and other substances in cells is critical for the growth and development of organisms,the resistance to biotic and abiotic stresses,and signal transduction.There are four main steps of vesicle transport,namely,budding,movement,tethering and fusion.In the last step,SNRAEs play an important role in mediating vesicles contacting with target membrane and membrane fusion.Arabidopsis genome has 64 genes encoding SNAREs.Studies in yeast and mammals have shown that after membrane fusion,the protein complex consisting of four SNAREs is disassembled by a NSF/SNAP complex,which is essential for cell viability.However,function of the NSF/SNAP complex in plants has not been reported.Here,we provide preliminary functional studies of Arabidopsis GS,AS1 and AS2,genes encoding the putative components of the NSF/SNAP complex,?-SNAP,?-SNAP1 and?-SNAP2,respectively.Main results and conclusions of this thesis are as follows:?1?GS is constitutively expressedBy GUS reporter analysis,we showed that GS was constitutively expressed.Besides,functional loss of GS did not show obvious growth defects.?2?AS1 is probable a pseudogeneWe could not clone AS1 referenced coding sequence and found that the GUS signal only showed in Pro_AS1:GUS lines rather then AS1 genomic:GUS lines.Besides,we found that,in1135 ecological types of Arabidopsis,there were 655 strains had insertions,deletions,or SNPs of AS1 that casued AS1 premature termination or frame shift.Thus,we guess that AS1is likely a pseudogene.?3?AS2 is constitutively expressedHistochamical GUS staining of AS2 genomic:GUS showed that AS2 was constitutively expressed,and its expression was high in root meristem,shoot meristem,and gametophytes.?4?Functional loss of AS2 resulted in defective female gametogenesisBecause there were no mutant materials of AS2 available in Arabidopsis stock centers,we generated AS2 loss-of-function mutant,as2,by CRISPR/Cas9.By silique analysis,we determined that as2/+had around 50%seed set.Pollination with Pro_LAT52:GUS pollen on wild type or and histochamical GUS staining at 12 hours after pollination?HAP?showed that half of ovules in as2/+pistils did not show GUS signals.These results suggested that as2 embryo sacs failed to attract pollen tubes.Optical sections of developing as2/+ovules showed that functional megaspore of as2 did not go through mitosis at stage 3-III during ovule development.The result indicated that AS2 is essential for female gametogenesis.?5?Functional loss of AS2 resulted in defective male gametogenesisBy Alexander staining,DAPI staining,and SEM,we determined that functional loss of AS2 resulted in pollen abortion.By optical sections and plastic sections of developing as2/+anthers,we determined that around half of the microspores in as2/+started to be abnormal between stage 10 and stage 11.At this stage,large vacuoles within the unicellular microspores begin to fragment into smaller ones in the bicellular microspores in wild type.By contrast,large vacuoles remain at stage 11 in a portion of as2/+microspores,finally leading to pollen abortion.The result indicated that AS2 is essential for male gametogenesis.?6?AS2 localizes in the cytoplasmTo confirm the defective male and female gametogenesis was indeed due to functional loss of AS2,we generated a CRISPR/Cas9-resistant AS2,crAS2.A GFP translational fusion driven by Pro_UBQ10,Pro_UBQ10:GFP-crAS2,was introduced into as2/+.Among transgenic progenies,we obtained several Pro_UBQ10:GFP-crAS2;as2 lines in T1,indicating that crAS2rescued lethality of as2.By confocal laser scanning fluorescence imaging of the stable transgenic plants of Pro_UBQ10:GFP-crAS2,we determined that AS2 is localized in the cytoplasm.Our results suggested that functional loss of GS did not show obvious growth defects,AS1 is probable a pseudogene and AS2 is critical for gametogenesis.Future reseach should be focused on characterizing the mechanism of AS2 in plant development.