Biochemical Characteristic And Physiological Functions Analysis Of OsGLP1 In Rice

Proteins with 30 to 70% identity to wheat germin were designated germin-like proteins(GLPs). It has been shown that GLPs widely exist in angiosperm and participate in many processes that are important for plant development and defence. However, their exact biochemical and physiological functions remain limited. To further elucidate the physiological function of Os GLP1 and its molecular mechanisms, Os GLP1 or Os GLP1-His overexpressing and CRISPR/Cas9 sg RNA-mediated targeted gene modification of Os GLP1 transgenic rice plants were constructed first, then phenotypes of transgenic rice plants and related physiological indicators were analysed in this study. Main results were described as follow:(1)Generation of Os GLP1 or Os GLP1-His overexpressing transgenic rice plantFragments containing complete ORF sequences for Os GLP1 or Os GLP1-His were cloned from DNA of Japonica rice Dong Jing leaves using PCR, then over-expression vectors p Ox-Os GLP1 and p Ox-Os GLP1-His with purification tag were constructed respectively. The vectors were transformed into rice callus via Agobacterium-mediated transformation technology, overexpressing Os GLP1 and Os GLP1-His transgenic rice plants were generated successfully. The results of western bloting showed that the protein Os GLP1 increased remarkablely in transgenic plants.(2)CRISPR/Cas9 sg RNA-mediated targeted gene modification of Os GLP1The p YLCRISPR/Cas9-Os GLP1 vectors were transformed into Japonica rice Dong Jing via Agobacterium-mediated transformation technology, 10 independent transgenic lines were obtained with Hygromycin selection. Targeted sequence assays in T0 transgenic rice plants revealed that all lines of Os GLP1 carried mutations, and most of the targeted mutations were chimera or biallelic, few were heterozygous and homozygous. 3 transgenic lines carrying mutated Os GLP1 and lacked detectable HPT transgene fragment in T1 transgenic rice plants were obtained by sequencing the products of the corresponding site-specific genomic PCR and amplifying HPT.(3)Os GLP1 exhibited high heat stability and no SOD activitySOD activity has not been increased in leaves of Os GLP1 overexpressing transgenic plants, but SOD activity increased in leaves of transgenic plant carrying mutated Os GLP1. The protein Os GLP1 still was detectable in the supernatant by western bloting after the extract from the leaves of Os GLP1 overexpressing transgenic plants was heated at 70? for 60 min or at 85? for 10 min, it indictated that Os GLP1 was a protein with high heat stability. After the crude supernatants from Os GLP1 overexpressing transgenic plants and WT plants was heated at 70? for 45 min and centrifuged, then concentrated with 15 m L Millipore centrifugal filter devices, respectively, finaly, the concentrate was collected and analysed by Native-PAGE. One of the bands only could be observed in the sample from Os GLP1 overexpressing transgenic plants after CBB staining. The protein in the band was extracted and detected by western bloting after analyzed in SDS-PAGE, the results showed the protein in the band is Os GLP1, but no evident difference in SOD activity was observed between the samples from Os GLP1 overexpressing transgenic plants and WT plants whether SOD activity was assayed by colorimetric method or by in-gel activity staining. Therefore, Os GLP1 was a protein lacked SOD activity.(4) Mutation of Os GLP1 result in plant height and tiller number decreased and brown spot produced on leaf bladesCompared with WT plants, Cas9-Os GLP1 mutants showed semi-dwarf and tiller number decreased at the heading stage. Moreover, there were some brown spots on the leave blades of Cas9-Os GLP1 mutants. DAB staining or NBT staining showed that Cas9-Os GLP1 mutants had lower content of H2O2 and O2.- than that of WT plants and Os GLP1 overexpressing transgenic rice plants. MV treatment showed that Cas9-Os GLP1 mutants were more tolerance to oxidative stress than WT plants and Os GLP1 over expressing transgenic rice plants. The above results suggested that there is no relationship between leaf brown spot formation and oxidative stress.(5)Os GLP1 subcellular localizationUsing protoplast transient expression studies to test for Os GLP1 subcellular localization, Os GLP1 was located in endoplasmic reticulum in rice protoplasts. Hovever, in the leaves of tobacco plants, discontinous GFP signal was detected in the periphery of leaf cells, so Os GLP1 is highly likely to be localized in the extracellular. Because the cell membrane was not isolated from cell wall, it was not sure that Os GLP1 was in the extracellular or in the cell wall.