Cloning And Expression Analysis Of The Key Genes Of Ubiquitin-26S Proteasome Pathway In Sweet Almond (Prunus Dulcis Mill.)

In flowering plants, a widely used mechanism to prevent inbreeding and promote outcrossing is known as self-incompatibility (SI). Most almonds display highly SI. The poor efficiency of outcrossing makes the amount of progeny very low. So the investigation of the molecular mechanisms of SI will benefit the genetic breeding of almond. It has recently been determined that SLF (S-locus F-box) genes contral SI on the pollen side. SLF encodes an F-box protein that is a component of ubiquitin ligase (SCF complex), raising the possibility that the ubquitin/26S proteolytic system plays a central role in the discrimination of self/nonself pollen in the RNase-based GSI.We use one cultivar of almond 'Pioneer' as plant materials. Firstly, there are several ESTs of key enzymes in ubquitin/26S proteolytic system in our lab achieved previously. Using these ESTs and resources from NCBI, we have finished cloning seven genes by RACE and RT-PCR technology, including ubiquitin activating enzyme (El), ubiquitin conjugating enzyme (E2), two components of Ubiquitin ligase (cullin and Skpl), SLF and two alleles of S-RNases. Up to now, we have achieved partial CDS sequence of El and full-length CDS sequences of the other genes. These results will facilitate the investigation of their functions.Secondly, we have studied the expression mode of the genes listed above in different organs of almond, including anthers, pistils, petals, sepals and leaves. Results of RT-PCR revealed that SLF expressed exclusively in anthers, and S-RNase exclusively in pistils, suggesting that they may participate in SI responses. Other genes expressed in all of the five organs. We presumed that as a main pathway of protein degradation, the ubquitin/26S proteolytic system invole in lots of metabolizable processes in different organs.Last, in order to investigate whether SLF and S-Rnase will interact with each ather, we performed yeast two-hybrid assay. First, the full-lenth CDS of the two genes was cloned, named PdSLFl and PdSm, Afterwards, we constructed two expression vectors for yeast, pSos and pMyr, respectivly. Then, we validated that PdSLFl and PdSm were inserted accurately into expression vectors, and will expresse in the same reading frames with Sos bait and Myr signal peptide. The results of yeast two-hybrid assays showed that there are no physical interactions between PdSLF and S-RNase, consistent with the results achieved from P.inflata.