Cloning The Promoter Of CsFBXL And Selection Of Suitable Internal Reference Gene By SYBR Green QRT-PCR In Develpoment Of Cucumber

FBXL subfamily is large member of F-box proteins family, FBXL subfamily was used to be participation in the plant ubiquitin-proteasome pathway, and FBXL subfamily played an important role in development of the plant. Cs FBXL gene is a member of the FBXL subfamily, through the previous study found that the expression of Cs FBXL genes was effected by hormones and high salinity, which has different extant responses. However the molecular mechanism of the induction of Cs FBXL remains unclear. In this study, we isolated the promoter of Cs FBXL basis on the cucumber DNA. Deletion analysis of the Cs FBXL promoter against biotic and abiotic stress was carried out via an Agrobacterium-mediated transient transformation assay in tobacco.With the molecular biology development in deeply, the research of gene expressions in cucumber development process have been from qualitative analysis to quantitative analysis. In many study, the researchers usually detect gene expression by using of q RT-PCR. q RT-PCR should choose appropriate reference gene in order to get reliable results. However, the reference gene could express least steadily under different treatments, plant and so on.. Therefore, the reference gene need to evaluation verification is necessary before it’s used to be q RT-PCR.The results of analysis: 1. Based on the DNA sequence of Cs FBXL and the completion of cucumber genome sequencing, there is prediction the sequence of promoter with the DNMAN.A 2.2kb promoter sequence was isolated by PCR technique. 2. With the plant CARE database, the promoter includes the massive elements. The results showed that the promoter included the core promoter region such as TATA-box and CAAT-box. The promoter accord with the characteristics of promoter in eukaryotic. Furthermore, the promoter also has some elements for example AE-box、ARE、CGTCA-motif、P-box、SARE. 3. There were construct different length of expression vecter by different promoter region fragments replaced with Ca MV35 S. 4. The Agrobacterium-mediated transient transformation in tobacco results show that the length of full promoter was enough for GUS expression against SA and Me JA induction, and the result simlar as the region fragment have the responsive elements of SA(TCA-element) and Me JA(CGTCA-motif); when the MBS elements in the length of full promoter, the GUS gene expression against PEG-6000 and Na CL, when the promoter fragment lost the element of MBS, the GUS expression could not agaist the PEG-6000 and Na CL; the result similar to low temperature and high temperature, when the promoter lost the element of LTR, the GUS expression could not agaist the low temperature; the promoter only the element of Me JA(CGTCA-motif), the GUS expression agaist the high temperature. 5. the length of full promoter has different extent for GUS expression against PEG-6000, Na Cl and cold, and its abolished the response of GUS to high temperature. However, deletion of the full fragment promoter has different different extent for GUS expression response of SA and Me JA induction. 6. The ge Norm-M analysis results:EF1a-1,ACT and UB1-ep were the best reference genes in root tissue samples;UBQ,UB1-ep and ACT2 were the most stable reference genes in fruit tissue samples; TUA-1,UBQ and ACT3 were the most stable reference genes in seed tissue samples. 7. The Norm Finder analysis results: UB1-ep, ACT3 and TUA-1 were most stable reference genes in root, stem and leaf tissue samples, respectively. TUA-1, ACT3 and EF1a-1 were most stable reference genes in floral, fruit and seed tissue samples. 8. The Stabillty Index analysis results: ACT1 was the best reference gene during cucumber development processes.