The Study Of Ubiquitin Pathway Genes SorUBC And SorRma1 On The Role Of Abiotic Stress Resistance In Black Nightshade (Solanum Nigrum L.)

Plants'growth and normal physiological metabolic levels will occur disorder when suffered with the salt,drought,pests,bacteria and other stress.Solanum nigrum L is not only a typical cadmium super-accumulation of plants,but also has good adaptability in drought,pest and other stress,therefore it is an ideal gene bank for studying plant resistance mechanism.The ubiquitin/26S proteasome pathway is the most efficient and highly selective pathway for protein degradation at present and is involved in the life activities of eukaryotes and plays a very important role in the plant's abiotic stress.In the ubiquitination,E2 ubiquitin-binding enzyme and E3 ubiquitin ligase have a critical role.In order to study the structure and function of E2 ubiquitin-binding enzyme SorUBC gene and E3 ubiquitin ligase enzyme SorRmaI gene in Solanum nigrum L;Gene expression in adult plants and its expression characteristics in the response to non-biological stress of Solanum nigrum L.In this paper,the full-length cDNA of SorUBC gene was first homologously cloned and analyzed by biological informatics.Amino acid sequence to predict the structure and function of its protein;The expression characteristics of the two genes in the tissues of Radix Solanaceae?roots,stems,leaves,flowers and fruits?were analyzed by qRT-PCR;Meanwhile some physiological responses?POD,SOD and MDA?to study under different abiotic?drought,salt,alkali and high temperature and the low temperature?,speculated that nightshade SorUBC and SorRmaI genes in the early response to abiotic regulation;Finally,the PBI121-SorUBC and PBI121-SorRmaI plant overexpression vectors were constructed and transferred into the model plant Arabidopsis to obtain the transgenic T₀ generation seed.Through the above experimental contents,we provided some theoretical basis for the study of ubiquitin gene in plant abiotic stress.This study had obtained the following results:1?The test material is wild type Solanum nigrum L,predict the characteristics of E2 ubiquitin binding enzyme and provide theoretical basis for the application of E2ubiquitin binding enzyme in plant stress.The research cloned E2 ubiquitin binding enzyme UBC gene full-length coding region sequence by electronic cloning and RT-PCR method from Solanum nigrum L,named SorUBC,GenBank accession number:KU233684.Sequence analysis showed that the coding region of SorUBC gene was 888bp,encoded 295 amino acids.Protein molecular weight was 25 kD,the theoretical isoelectric point pI=5.23.In the 266²84 amino acid position of the protein,there is a transmembrane helix region,and the ubiquitination site predicted that it contained 8ubiquitination sites.The SorUBC protein has a UBCc domain while the Solanum nigrum L SorUBC also contains a C-terminal extension structure and an N-terminal extension structure.The homology analysis of SorUBC protein showed that the amino acid sequence of the gene was the highest in potato and tomato,followed by tobacco,95%and 91%.2?The expression profiles of the E2 ubiquitin-binding enzyme SorUBC and E3ubiquitin ligase enzyme SorRmaI were analyzed by qRT-PCR,the results showed that the two genes expressed in roots,stems,leaves,flowers and fruits of Solanum nigrum L,SorUBC gene expression level:leaf>fruit>flower>stem>root,SorRmaI gene expression level:leaf>flower>stem>fruit>root,the expression of these two genes in leaves was higher than that in other organs,and the difference was significant.3?Solanum nigrum L under abiotic stress treatment,such as drought,salt,alkali,high temperature and low temperature were carried out,and the expression characteristics of SorUBC and SorRmaI genes in the roots and leaves were analyzed by qRT-PCR at treated with 0,1,3,5,9,12 h.The results showed that this two genes expressed both in the roots and the leaves of Solanum nigrum L In the abiotic stress,there existed the early response of the upward trend of SorUBC gene in 3h or 5h and SorRmaI gene in 1h or 3h in roots.The expression of genes in the leaves decreased,SorUBC gene expression with salt stress and alkaline stress was first decreased and then increased,the maximum value of 9h,the early response of SorRmaI gene were up-regulated and reached the peak at the 9h in the alkali treatment,other stress gene expression in leaves changed little.On the whole,the expression of genes in Solanum nigrum L roots compared with the control group showed an increasing trend,and the expression levels and change trend at 5h and 9h in roots were significantly higher than that in leaves.4?Gene expression analysis were carried out as above,and meanwhile selected the leaves under drought,salt and high temperature stress at different time?0,1,3,5,9h?tested part of the enzyme activity?POD,SOD,MDA?of Solanum nigrum L.The results showed that the difference of malondialdehyde?MDA?content was not significant,indicating that the plant was less harmful in the above stress.At the same time,the activities of POD and SOD in the plant were decreased first and then increased,and the activity of SOD was decreased after 1h of stress,and the activity of POD and SOD increased with the control group compared with the difference was significant.The results showed that POD and SOD were involved in the repair process of Solanum nigrum L,and the late stage of stress?9h?relieved the damage caused by reactive oxygen species.5?In order to further verify the function of the E2 ubiquitin-binding enzyme SorUBC and E3 ubiquitin ligase SorRmaI,we constructed the overexpression vector of SorUBC gene and SorRmaI gene,named PBI121-SorUBC and PBI121-SorRmaI respectively.Through Agrobacterium tumefaciens carried the recombinant plasmid into Colombian wild-type Arabidopsis thaliana,and harvested the transgenic T₀ generation of seeds.