Cloning And Transcriptional Properties Of AcSKP1 Gene And Its Application In Breeding In Onion(Allium Cepa L.)

Onion(Allium cepa L.)is leading commercial vegetable crop grown in at least 139 countries in the world.The onion genome is huge,and the research in the basic field of molecular biology is relatively weak,especially the research on onion functional genes is rare.SKP1 is a kind of small molecular weight protein commonly found in eukaryotes.Its main biological function is to participate in the formation of SCF complex and regulate it through the 26 S protease pathway.Ubiquitin-mediated protein degradation in organisms is involved in multiple biological processes including plant pollen development.In this experiment,the onion restorer line(DH17)and the sterile line(230)were used as materials to analyze the onion by molecular biology methods such as chromosome walking,RACE test,sequence alignment analysis and Agrobacterium-mediated genetic transformation.The AcSKP1 gene has a variable splicing phenomenon in the onion fertile line and the sterile line,and the functional verification of the AcSKP1 gene promoter was initially carried out.On this basis,a stable and efficient SSR molecular marker(WH-SSR-1)of onion Ms locus was developed,which has been successfully applied in breeding practice.The results of this study are as follows:(1)The full-length sequence of the onion AcSKP1 gene was cloned.The restorer material(DH17)contained 5659 bp,and the sterile material(230)contained 5636 bp.The alignment analysis revealed that there was a large amount between the restorer line and the sterile line material.The mutation sites are mainly InDel and SNP sites.These variant sites provide an information resource for development of molecular markers.(2)Preliminary studies confirmed that there were alternative splicing in the restorer material(DH17)and the sterile material(230).Subsequently,we obtained a full-length transcript of each of them,named AcSKP1F1 and Acskp1S1.The AcSKP1F1 gene is 4761 bp in length and contains 10 exons with a length of 1396 bp and 9 introns with a coding sequence of 1056 bp.The Acskp1S1 gene is 4746 bp in length and contains 11 exons with a length of1434 bp.An intron with a coding sequence of 1410 bp.The gDNA alignment of AcSKP1F1 and Acskp1S1 showed 95.41% similarity,and the amino acid sequence similarity was 92.02%.This provides a sequence basis for subsequent functional studies.(3)The promoter sequences of the restorer line(DH17)and the sterile line(230)AcSKP1gene were cloned,named AcSKP1 Fp and Acskp1 Sp,respectively,and the AcSKP1 Fp and Acskp1 Sp sequences were found to contain the core promoter elements TATA-box and CAAT-box.Etc.,both also found a large number of light-inducing components ATCA-motif,CAAT-box,AE-box,Box 4,GT 1,I-box,etc.;the main difference is in the hormone-inducing component,AcSKP1 Fp contains 2 MeJA components One Auxin inducing element and one GA inducing element,and these elements were not found in Acskp1 Sp.This further suggests a difference in the function of the AcSKP1 gene between the restorer line and the sterile line.(4)The AcSKP1Fp/Acskp1Sp::GUS and AcSKP1Fp/Acskp1Sp::GFP chimeric gene promoter functional verification vectors based on pBI121,pGF and V1001 binary vectors were successfully constructed,and the Agrobacterium tumefaciens GV3101 engineering strain was obtained.Mustard,preliminary results showed that Acskp1 Sp was stained blue by GUS dye solution and AcSKP1 Fp was not stained blue.(5)Using the original site of AcSKP1,a highly efficient and stable SSR molecular marker capable of distinguishing the onion Ms site was developed and named as WH-SSR-1.The WH-SSR-1 label has only 1 protocol 102 bp band amplification in homozygous dominant materials,while the homozygous recessive material has only one 99 bp band amplification,and the hybrid material exists in 2 bands.Amplification.This result was not only verified in two BC1 segregating populations,but was further validated in 32 breeding materials.Subsequently,we used this marker to directly select the maintainer strain and the sterile strain from the OP population,and then directly construct the sterile line and the maintainer line.