Study On Mechanism Of The Ethylene Signaling In Lotus Response To Cadmium Stress

Lotus(Nelumbo nucifera)is one of the ten famous flowers in China.As an aquatic flower with high ornamental,edible and medicinal value,lotus is widely cultivated all over the world.In recent years,many studies have shown that lotus has a strong adsorption capacity and a certain tolerance to heavy metal Cd,which can be used as a potential plant material to remove Cd in polluted water.Ethylene,as a "stress hormones",can be increased by Cd stress in plant.However,the molecular mechanism of ethylene in lotus in response to Cd stress has not been clear.Therefore,in this study,’Weishanhuhonglian’ seedlings were used as experimental materials and transcriptome sequencing technology was used to analyze lotus response to Cd.The gene expression related to Cd stress in lotus was analyzed,and the key gene of ethylene signal transduction NnEIN2(LOC104601274),was cloned and analyzed.Meanwhile,DEGs responded to Cd stress in AP2/ERF transcription factors were preliminarily analyzed.This study explored the lotus response mechanism of heavy metal stress and ethylene signal pathway at the molecular level,providing a theoretical basis for phytoremediation and breeding of lotus.1 Transcriptome analysis of lotus under cadmium stressIn this study,transcriptome sequencing was used to analyze lotus treated with Cd.By filtering and assembling the clean data,a total of 95484 high quality unigene were obtained.A total of 15877 genes were identified as DEGs,including 8948 down-regulated genes and 6929 up-regulated genes.The GO and KEGG enrichment analysis of DEGs showed that they were mainly involved in the metabolic process,cell,membrane,catalytic activity,binding and other processes.Based on transcriptome data and previous studies,the related genes involved in ethylene signal transduction pathway and photosynthesis,Cd absorption,transport,antioxidant and detoxification in lotus were further analyzed.2 Cloning and functional certification of NnEIN2 in response to CdIt was found that in lotus under Cd stress,the key gene NnEIN2(LOC104601274)in ethylene signal transduction was significantly down-regulated,which was identified as target gene.Combined lotus genome in NCBI and transcriptome data with cloned NnEIN2 sequence,it showed that NnEIN2 has a 3912 bp open reading frame(ORF),encoding 1303 amino acids;N terminal(1-650aa)of NnEIN2 protein contained 11 transmembrane domains(11-459 aa),of which the Nramp-like protein domain was located at 38-394 aa.The C terminal(651-1303 aa)contained an NLS sequence(L1271-L1278),which highly conserved.S650 and F651 was cutting site of N-terminal and C-terminal.The homology of NnEIN2 with other species ranged from 45 to 61%.qRT-PCR analysis of NnEIN2 expression under different treatments showed that NnEIN2 expression could be reduced by Cd stress,and be increased by exogenous ACC,and be down-regulated by STS.In addition,an overexpression vector of 35S::NnEIN2 was constructed.Through transient transform in tobacco leave,it was found under Cd stress,NnEIN2 might play the same role as exogenous ACC in lotus to increase damage of leave and lead to chlorophyll degradation.3 Preliminary analysis of NnERFs in lotus response to CdIt was found that many AP2/ERF transcription factors were related with transcriptional regulation of lotus under Cd stress,in which were filtered as DEGs,further evolutionary tree was constructed.It was found that 35 of them belonged to ERF subfamily,which was an important family in lotus response to Cd.Through amino acid sequence alignment and conserved Motif analysis,it was found that 35 ERF subfamily members had typical highly conserved AP2/ERF domain,which contained Motif 1,namely,WLG elements.The expression levels of 23 members of the NnERF subfamily were detected under different treatments.It was found that,except NnRAP2-2-like,the expression levels of other 22 members were significantly affected by Cd and ethylene,and responsive degree was different.Four genes NnERF091,NnERF098,NnERF4-like and NnRAP2-12-like were cloned which were significantly responsive to Cd stress and had incomplete sequences in transcriptome.Sequencing results showed that 4 genes were present and complete in lotus.