| Cucumber?Cucumis sativus L.?is crisp and versatile,it's also a kind of vegetable which is mainly cultivated in greenhouse.The growth of cucumber is easily affected by the environment and soil,and downy mildew is a mainly susceptible disease of cucumber.Propamocarb is a type of systemic fungicide which posseses a low toxicity and short half-value period.It has a good effect on preventing and treating of downy mildew,too.However,the application of propamocarb can cause high pesticide residue in cucumber plants easily,it will result in new food safety problems.the related researches on low pesticide residues in cucumber which can significantly enhance the quality and safety of cucumber.Our research group have sequenced the transcriptome of a low pesticide residue type‘D0351'previously,then screened a CsDREB1 whose expression is significantly decrease under propamocarb stress.We speculate CsDREB1 is the key gene of modulating pesticide residue in cucumber by regulating other genes.This study involved cloning,expression pattern analysis,subcellular localization and other molecular bio-techniques.The results of the research are as follows:?1?The CDs sequence of CsDREB1 gene was cloned by PCR technology.The length is 603bp,encoding 200 amino acids,including a 63 amino acids AP2 domain which indicate CsDREB1 is a member of AP2 super family.?2?The molecular weight of CsDREB1 is 22.26KD,which attributes to unstable hydrophilic protein,without obvious signal peptide and transmembrane region were predicted by bioinformatic methods.The relationship between cucumber CsDREB1 and melon CmDREB1D is the closest.Besides,CsDREB1 belongs to the DREB-A1 subgroup.?3?The expression pattern of CsDREB1 in different species,different time and different tissues was analyzed by qPCR.The study found that the relative expression of CsDREB1 in'D0351'is lower than'D9320'under propamocarb stress;Compared with control,the relative expression of CsDREB1 in'D0351'is significant decrease under propamocarb stress.However,there is no obvious change in‘D9320';Analyzing expression pattern of CsDREB1 in different organs which found that the main differently expressed organ is fruit.?4?The fusion products of CsDREB1 and green fluorescent protein?GFP?were imported into Arabidopsis protoplasts.Confocal laser microscopy indicated that CsDREB1 encode protein was located in the nucleus,and it is a nucleoprotein.CsDREB1 plays whose transcription factor in the nucleus primarily.?5?The pBI121-CsDREB1 plant overexpression vector was constructed and the CsDREB1gene was introduced into wild-type Arabidopsis through inflorescence inoculation.The wild type Arabidopsis and the transgenic Arabidopsis were respectively subjected to the imperfections of propamocarb(MS medium with 2 mmol.L-1 propamocarb).It was observed that in the control group,the germination rate and root length of the transgenic Arabidopsis thaliana and wild type Arabidopsis did not show significant difference;under propamocarb stress,the germination rate and root length of the transgenic Arabidopsis thaliana were significantly lower than those of the wild type.This indicated that CsDREB1 could response to the stress of promecarb,and the overexpression CsDREB1 Arabidopsis plants'tolerance to propamocarb significantly reduced.?6?By analyzing the expression patterns of CsDREB1 under other stress conditions which found that the expression of CsDREB1 was significantly decreased under the treatments of abscisic acid,high salt,drought,and Corynespora cassiicola.However,the expression of CsDREB1 was significantly increased under jasmonic acid treatment.?7?CsDREB1 was imported into cucumber High pesticide residues type'D9320'by Agrobacterium-mediated method,and 11 CsDREB1 transgenic plants were confirmed by PCR,whose F1 cucumber seeds were harvested. |
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