Identification Of Autophagy As Well As Cloning And Analysis Of Autophagy Gene Atg8 In Pollen Mother Cells Of Brassica Napus(L.)and Althaea Rosea(L.)

Autophagy is a conserved mechanism for the degradation of cellular contents in order to recycle nutrients or break down damaged or toxic material in eukaryotic cells under biotic/abiotic stress and normal growth conditions.It plays crucial roles in cellar homeostasis,cell reconstruction,autoimmunity and anti-stress.In plants,autophagy has been known for some time to be important for nutrient remobilization during sugar and nitrogen starvation,leaf senescence,disposal of protein aggregates and oxidative stress,but recent research has uncovered it possibly involved in plant growth and organ development during normal growth conditions as a housekeeping function.However,the study of plant autophagy is just begin in organ development under normal conditions also its generative patterns,real mechanisms and functions remain unknown,especially in plant reproductive cells.Consequently,fluorescent microscopy observation(monodansylcadaverine(MDC)staining),transmission electron microscopy(SEM)observation and autophagy related gene(Atg8)expression analysis approach were employed to study of the autophagy generative patterns and mechanism of action in pollen mother cells(PMCs)of Althaea rosea(L.),Lilium davidii Duchartre and Brassica napus(L.)(MF and MS).The main results are the follows:(1)MDC staining results show that the studied plants PMCs all contain blue fluorescent spots which are in different sizes and quantities.These blue spots are located in edge and uniform distribution in PMC of Althaea rosea(L.)and Lilium davidii Duchartre,respectively.But they are diffuse distribution in PMCs of Brassica napus(L.).While the density and quantity of blue spots in PMCs of Brassica napus(L.)MF are more than the MS line's.(2)Ultrastructural observation results show that the PMCs of Brassica napus(L.)MF line and Althaea rosea(L.)all contain pre-vacuole vesicles which include a large number of autophagic bodys.The pre-vacuole vesicles are in different sizes and quantities,but the vesicles in Althaea rosea(L.)PMCs are more abundant than Brassica napus(L.)MF's PMCs.When observe carefully we can find some vesicles are engulfing autophagosomes.(3)Autophagy related gene 8s(Atg8)were isolated from Brassica napus(L.)and Althaea rosea(L.)BnAtg8a?BnAtg8b and ArAtg8a(GenBank accession numbers are:KC896379?KC896380?KC896383,respectively).Their protein homology are above 80%when compare with other plant ATG8s.And the conserved regions of them are same with the other plant ATG8s.In addition,we also obtained the BnAtg8a/b full lenth gDNA sequences which contain five exons and four introns.(4)Expression analysis of BnAtg8a and ArAtg8b display that they have different levels of expression in PMCs of Brassica napes(L.)(MS or MF)and Althaea rosea(L.),respectively;BnAtg8a is highly expression in anthers(PMC phage,meiophase and Tetrad stage)and young roots of Brassica napus(L.),but has low or no expression in stems and leaves;However,ArAtg8a has some extent expression in PMCs,stems and leaves.(5)Two plant expression vectors(2×35S::eGFPBnAtg8a and 2×35S::BnAtg8a)areconstructed.And transformation of Brassica napus(L.)with these two vectors show that the the average transformation efficiency is more than 2.89%;Finally,obtained transgene plants:MFE(8 lines),MFA(8 lines),MSE(6 lines)and MSA(3 lines).These transgene lines' phenotypes have no obvious difference compared with wild type in the vegetative growth stage.But the differencebetween them remains unknown within the reproductive growth stage or under some specialtreatments.All of the results above suggestion:Autophagy may exist in PMC developing process of all plants in this study.The existence of autophagy in PMCs may have some universality in different Plants.Also the autophagy degree among different plants may have species specificity.The autophagy could lead to male sterile in MS line,through the way of damaging PMC developing process,while in MF line it could lead to the uniform distribution of cytoplasm between all PMCs in the sporangium and eliminate the non-identity of cytoplasm of the PMCs caused probably by the random asymmetric division observed at sporogenous cell phage.BnAtg8 over expression transgene plants can use for the study of autophagy molecular mechanism and ATG8 tissue localization in PMCs of Brassica napus(L.).In addition,BnAtg8 transgene lines can use to estimate the effective of autophagy in Brassica napus(L.)anti-stress process.And offer some suggestions for agricultural industry of autophagy related gene modificated plants.