Fuctional Analyses Of CsABVC19 And CsPDR From Cucumis Sativus L.response To Propamocarb

Cucumber(Cucumis sativus L.) is one of the most valuable vegetable crops, and widely cultivated in the world. Due to its good flavor, cucumber has become one of the most popular vegetable species,occupies an important position in the market. Nevertheless, cucumber growth is often affected by various diseases and insect pests, especially downy mildew, which severely declines the quality and production.Propamocarb(propyl-3-(dimethylamino) propylcarbamate, PM) is abroad-spectrum carbamate fungicide pesticide, and is widely used in agricultural production for its high efficient control of pathogens, especially cucumber downy mildew. However, appl ication of propamocarb always caused environment contamination and pesticide residue.In recent years, with the improvement of living standards, people pay more and more attention to food safety problems, especially pesticide residue.Hence, study about low pesticide residues of cucumber has an important significance to reduce the use of pesticides, cucumber production costs, environmental pollution, prevent the harm to human health preferably.ABC transporter genes play an important role in detoxificationmet abolism in plants, involving ina variety of adverse environmental stress. In this study, low residual line D0351 and high residual line D9320 were used as materials, the two cucumber ABC transporter gene s were cloned, and the research content involves bioinformatics analysis, expression analysis, the genetic transformation, the main results are as follows,(1)Based on the RNA-seq results from the transcriptome changes of cucumber fruit(low residual line D0351) in response to PM treatment by using Solexa Illumina, 29 ABC transporter genes showed differential expression, phylogenetic analysis shows that the AB C transporters genes belong to 5 ABC subfamily respectively, including 6 genes ABCB,7 genes ABCC, 10 genes ABCG, 2 genes ABCF, and 4 genes ABCI.(2)Using the q RT-PCR technology, the expression of CsABC19,CsPDR,and CsMRP was determined under PM stress. The results found that the expression of Cs ABC19 in cucumber D0351 and D9320 was up-regulated and down-regulated, respectively. The expression of Cs PDR in cucumber D0351 and D9320 was both up-regulated.The expression of Cs MRP in cucumber D0351 and D9320 was both down-regulated. Treated with PM, the expression of gene at different time in different tissues showed that the fold change of the expression of Cs ABC19 and Cs PDR was higher in the low residual line D0351 than in the high residual line D9320, and responsed the signal to PM quickly,which could play an important role in the PM treatment early.(3)Expression levels in different tissues showed that Cs ABC19 and CsPDR expression level in the reproductive organs(fruit peels, fruit pulps, female and male flowers) is higher in D0351 than that in D9320, on the contrary, expression level in the vegetative organs(leaves, stems, roots) is lower in D0351 than that in D9320, was constitutive expression,which had tissue and variety difference.(4)Cs ABC19 and CsPDRwere cloned, belonged to ABCF subfamily and ABCG subfamily, respectively. Multiple alignment of Cs ABC19 and other ABC protein amino acid showed that the amino acid sequences were highly conserved in the motifs of Walker A, Walker B and ABC signature.Cs PDR, which encodes a full ABC protein,contains two transmembrane domain s and two nucleotide binding domains.The results of analysis of promoter indicated that they had not only basic regulating element of eukaryotic gene transcription, but included cis-elements involved in responses to hormones and stresses. At the same time, expression vectors of Cs ABC19 and Cs PDR were constructed.(5)Using Agrobacterium co-cultivation method, Cs ABC19 was introduced into Arabidopsis, transgenic homozygous Arabidopsis lines were obtained, WT and transgenic homozygous Arabidopsis were treated with PM and sucrose.The results showed that CsABC19 improved transgenic plants the adaptability and tolerance of PM and sugar stress, and obvious decrease in PM residues found in CsABC19 transgenic lines than WT.(6)The Cs ABC19 with GFP-fusion protein expression vector was successfully constructed, CsABC19 localized in the nucleu, membrane,cytoplasm by transient expressing of GFP-fusion protein.(7)The sensitivity of Kan in cucumber 649 and D9320 was studied. The Kan concentration of adventitious bud inducting in cucumber 649 and D9320 was 75 mg/L and 50 mg/L,respectively. The Kan concentration of root inducting in cucumber 649 and D9320 was both 50 mg/L. The best time for immersed or co-cultured was 20 min or 3 d.(8)Five transgenic cucumber plants were obtained and positive plants by identification analysis.