Establishment And Analysis Of BnAtg8-rape Transgenic Lines

Autophagy (AP) refers to one of the pathways that degradate and reuse the cytoplasmic constituents in eukaryotes under normal growth process, as well as biotic or abiotic stress. AP plays important roles in regulation of cell homeostasis, development mode reconstruction, autoimmune and antioxidant stress. In addition, many studies have shown that autophagy is associated with the stress resistance of plants. ATGs (autophagy related genes) are the genes involved in AP process. ATG8 is one of the most important members of ATG family, being diretly relateed to autophagosome formation. Previously, we observed the presence of autophagosome aand autophagic vacuole in the pollen mother cells (PMCs) of both male fertility (MF) and male sterile (MS) lines of rapeseed(Brassica napus L.). Furthermore, the ATG8 gene of rapeseed was cloned, named BnAtg8. In the present study, the BnAtg8 and BnAtg8-GFP were overexpressed in MF and MS plant of rapeseed. The purpase of this study is to further confirm the existence and formation mechanism of autophagosome in PMCs of plants through morphological, genetic, cellular and molecular analysis of the BnAtg8 and BnAtg8-GFP transgenic line plants. The main results are the follows:(1) Throuth PCR identification,8 MF-BnAtg8 (MFA1-8),10 MF-BnAtg8-GFP (MFAG1-10), 11 MS-BnAtg8 (MSA1-11) and 11 MS-BnAtg8-GFP (MSAG1-11) transgenic plants were obtained, using the hypocotyls of rape as receptors through agrobacterium-mediated genetic transformation and plant regeneration in vitro.(2) Based on selection on the plates with hyg, we obtained four transgene lines with sigle copy of BnAtg8 or BnAtg8-GFP transgens,MFAG-2、MFAG-3、MSA-7 and MSAG-7. Primary morphological anaylsis found that the stem of MFAG-2 became thinner, the pods number of MFAG-3 decreased and the branch numbers of both MFAG-2 and MFAG-3 and both MSA-7 and MSAG-7 increased or decresed, compared to corresponding wide type (MF or MS).(3) RT-PCR analysis showed that BnAtg8 transgene were expressed in the four transgenic lines above, but its expression level was less than than that of endo enous BnATG8.(4) Confocal microscope observation revealed that GFP signal was located mainly in root tips of MFAG-2 and MSAG-7 seedlings, but distribution sites were different from each othert:it distributrd in meristem and elongation zone in MFAG-2 line, but in MSAG-7 line, it distributed in meristem and root cap. In cotyledon protoplasts, GFP signal distributed mainly around the chloroplast.(5) MDC staining showed that MDC flourosence (or signal) was distributed mainly in root meristem and elongation zone in MFAG-2 seedlings, or meristem and root cap in MSAG-7 seedlings. MDC and GFP signal appearred in similar positions and there was no obvious difference in the location and intensity of MDC signal between transgenic and wild-type root tips.(6) Analysis of their tolerance to salt stess showed that the rates of root and hypocotyl elongatoin of MFAG-2 and MSAG-7 transgenic lines was higher than that of MF and MS line under the same salt stress (150mM NaCl,12h light/12h darkness,7 days).(7) Starvation stess analysis found that there were no significant differences in the root and hypocotyl growth between WT (MF, MS) and transgenic lines of MFAG-2 and MSAG-7 under the same condition.The result above primarily indicates that the over-expression of BnAtg8 can promote the resistance of rape to salt stress, and that the promoted salt resistance may be related to the enhanced autophay activity due to the expression of BnAtg8 transgene. Due to the limited time, we only analyzed the characteristics of morphology, physiology and molecular biology of the transgenic line above. Study is underway about the expression profiling of BnAtg8 transgene and the mechanism of auotophagosome formation in the PMCs of the transgenic lines.