Effects Of Overexpression Of Rape Autophagy-related Gene BnATG8 On The Growth And Development Of Brassica Napus L.

Autophagy(AP)is one of the most important ways for eukaryotes to degrade and reuse cytoplasmic components during normal growth and development or under biological and abiotic stresses.It is important for maintaining cell homeostasis and developmental model reprogram,autoimmune and antioxidant stress and so on.In the early,studies on autophagy activity are focused on yeast and mammalian cells.In the past two decades,the study of plant autophagy has increased sharply but it is mainly confined to the vegetative growth process and the biological or abiotic stress conditions.So it is not clear whether there is autophagy in the normal sexual reproduction of plants and what their functions are.ATG8(Autophagy related gene 8)is one of the most important genes involved in autophagy activity.Previously our group found that there were structures similar to autophagesome in pollen mother cells of Hollyhock and Brassica napus under electron microscopy,and cloned the homologous gene(BnATG8)of AtATG8 from Brassica napus L..In addition,after fused with green fluorescent protein gene GFP,gene Bn ATG8 was intergrated into the genomes of rape male fertility(MF)and sterile line(MS),resulting in corresponding overexpression lines,named MFAG and MSAG.The results showed that branche number of MFAG(T1)increased,but plant height and thousand kernels weight(TKW)decreased compared with MF.Compared with MS,the branche number of MSAG(T1)was decreased,but the plant height and TKW increased.In order to understand the genetic stability of these two transgenic lines,the genetic stability of T2 and T3 plants were analyzed in 2015 and 2016 respectively.In addition,the dynamics of autophagy activity in pollen mother cells(PMCs)was analyzed by GFP detection and staining of specific agent MDC for autophagic body.The results are the follows:(1)Compared with MF/MS,T2-MFAG and T3-MFAG transgenic lines showed a significant decrease in bolting rate,plant height and TKW,but had a significant increase in branche numbers.However,T2-MSAG and T3-MSAG transgenic lines displayed a significant increase in bolting rate,plant height and TKW,but displayed a significant decrease in branche numbers,compared with MS line.There was no significant difference between T3 and T2 generations.(2)Morphological observation showed that whether in T2-MFAG or T3-MFAG lines,their filaments became short,anthers became small with less number of chambers and PMCs and thenumber and viability of pollen grains decreased,comparerd with MF line.In contrast,in T2-MSAG and T3-MSAG lines,their filaments became long,anthers became large with 4chambers and more PMCs,and the number and viability of pollen grains increased comparerd with MS line.(3)RT-PCR analysis showed that BnATG8 of rape itself was expressed in all lines,the Bn ATG8,GFP and HYG carried by the expression vector were expressed only in all of the transgenic lines and their expression levels were varied in different plants.Each of BnATG8,GFP and HYG was completely not expressed in MF and MS line.(4)Laser confocal microscopy observation showed that GFP gene expressed in PMCs,tetrads and microspores of MSAG and MFAG plants.In the cytoplasm of PMCs and microspores,GFP fluorescence mainly distributed in the area close to cell membrane.But in tetrads,fluorescence maily concentrated in surrouding of nucleus.Fluorescence intensity was gradually increased from PMCs to microspore stage.(5)MDC staining revealed that PMCs,tetrads and microspores all showed blue fluorescence in both of the above transgenic lines and MF/MS line,and there was no significant difference in fluorescence intensity and distribution between transgenic lines and MF/MS line or among PMCs,tetrads and microspores.(6)Physiological studies showed that the salt tolerance of MSAG lines was higher than that of MS,while the salt tolerance of MFAG lines was lower than that of MF in both of T2 and T3 generation.The above results indicate that: 1)the overexpression of ATG8 gene has a very important effect on the growth,fertility and stress resistance of rapeseed,especially fertility.2)the traits of transgenic lines obtained can be stable genetic,and possibly have a certain practical value in agricultural production,especially MSAG system.