Function Of G Protein In Nitrogen Response Of Brassica Napus

Rapeseed is one of the important oil crops.Nitrogen fertilizer input can significantly improve the yield of rape.However,excessive nitrogen fertilizer application will cause environmental pollution and resource waste.At present,exploring the genetic potential of rapeseed by transgenic technology is one of the effective ways to improve nitrogen efficiency.Recent studies have shown that heterotrimeric guanine nucleotide binding protein(G protein)mediates the response of rice,wheat and Arabidopsis to nitrogen.However,little is known about G protein in Brassica napus,and the physiological and molecular mechanism of G protein involved in nitrogen response remains to be further studied.In this study,we investigated the effects of overexpression of Arabidopsis G protein on growth and nitrogen efficiency in Brassica napus.At the same time,the G protein family members in Brassica napus were identified by bioinformatics methods at the whole genome level,and their physicochemical properties,subcellular localization and expression patterns were analyzed.The main research results are as follows :1.Effect of G protein on growth and development of rape seedlingThe overexpression vectors of Arabidopsis G protein driven by Ca MV35 S promoter(35S-GPA1,35S-AGB1-AGG1,35S-AGB1-AGG2 and 35S-AGB1-AGG3)were transformed into rapeseed “K407” by Agrobacterium-mediated transformation.The results of nitrogen treatment experiments showed that under either low or high nitrogen conditions,overexpression of GPA1,AGB1-AGG1,AGB1-AGG2 and AGB1-AGG3 genes make the whole plant biomass,aboveground biomass,root biomass,root length and leaf area of B.napus seedlings higher than those of wild-type plants.2.Effects of G protein on nitrogen uptake,nitrogen accumulation,enzyme activities related to nitrogen metabolism and expression of genes related to nitrogen uptake and transport metabolism in rapeseed seedlingsUnder low nitrogen conditions,overexpression of GPA1,AGB1-AGG1,AGB1-AGG2 and AGB1-AGG3 could significantly increase the nitrogen content and nitrogen accumulation in the top and root of rapeseed compared with the wild type,and thus had higher nitrogen absorption efficiency,while there was no significant difference between the wild type and the high nitrogen condition.In addition,the activities of key enzymes in nitrogen metabolism were analyzed,and it was found that the activities of glutamine synthetase(GS)in leaves and roots of plants overexpression GPA1,AGB1-AGG1,AGB1-AGG2 and AGB1-AGG3 were higher than those of wild type.The expression levels of nitrate transporter(NRT),NR and GS family genes in leaves and roots of transgenic rape plants were analyzed by q RT-PCR.The results showed that compared with the wild type,the overexpression of GPA1,AGB1-AGG1,AGB1-AGG2 and AGB1-AGG3 in B.napus significantly up-regulated or down-regulated most NRT,NR and GS genes under high or low nitrogen conditions.The results showed that overexpression of G protein affected the expression of genes involved in nitrogen uptake and transport metabolism,especially increased the expression of GS family genes,thereby increasing nitrogen uptake in rapeseed.3.Identification and expression analysis of G protein family genes in B.napusTwenty-two G protein family members were identified in Brassica napus,encoding two Gα subunits,seven Gβ subunits and thirteen Gγ subunits,respectively.Subcellular localization results showed that G protein was membrane protein.The transcriptome sequencing results of different tissues showed that Gα and C-type Gγ genes(except Bn GG10 and Bn GG12 were highly expressed in flowers and ovules)were almost not expressed in most tissues,while Gβ and A-type Gγ genes were highly expressed in most tissues.The results of q RT-PCR analysis showed that G protein showed different expression patterns under low nitrogen treatment.In roots,Gα and five C-type Gγ genes were up-regulated under short-term low nitrogen treatment.In leaves,five C-type Gγ genes were highly expressed,while the expression of Gα was down-regulated.In summary,the results showed that Arabidopsis G protein was involved in the low nitrogen response of rape,and could improve nitrogen uptake efficiency by increasing nitrogen uptake and assimilation of rape under low nitrogen conditions.At the same time,the identification and analysis of G protein genes in Brassica napus showed that Gα and five C-type Gγ coding genes responded to low nitrogen stress.These results have important theoretical basis and reference value for further study on the molecular mechanism of G protein regulating nitrogen uptake and metabolism in rapeseed.