Cloning,bioinformatics Analysis And Transgenic Study Of Self Incompatibility PsdSBP1 Gene In Almond

Almond is popular all over the world due to its unique flavor and value,and is also one of the important economic fruit trees in the world.At present,the world Almond planting area reached 1.9 million hectares,annual output more than 2.1 million tons.However,Almond belongs to gametophyte self-incompatibility tree species,and this genetic mechanism can avoid self-fertilization and improve its genetic diversity.However,due to a large number of self-pollination phenomena in Almond production,the fruit setting rate is low,thus seriously reducing the annual yield of Almond.Therefore,the exploration of the self-incompatibility mechanism of Almond can not only help us to understand and understand the pollination and fertilization mechanism of almond,but also further select the corresponding means to improve the fruit setting rate in the production process.Moreover,molecular biology technology can also be used to breed excellent self-incompatibility varieties.In this study,we first identified the members of the RING Fing gene family based on the tonsil genome,and identified the existence of SBP1 gene in tonsil by RING Fing members,which can be used for subsequent cloning and transgenic research of SBP1 gene in tonsil.The SBP1 gene was cloned from the main cultivars of Almond‘Zhipi’collected from the Almond fruit tree resource nursery of Forest Farm in Shache County,Kashgar,Xinjiang for the first time by homologous cloning technology,and was also named as PsdSBP1.In addition,bioinformatics technology was used to analyze the gene characteristics,and then fluorescence quantitative and subcellular localization techniques were used to detect the expression and distribution of PsdSBP1 in different tissues of‘Zhipi’.Finally,PsdSBP1 binary expression vector and Agrobacterium tumefaciens were constructed to study the transformation of Petunia.Results are as follows:（1）A total of 375 RING Finger genes were identified from the tonsil genome,and the protein length and molecular weight were different,which were hydrophilic proteins.Subcellular localization predicted that most member proteins were distributed in the nucleus,and the protein domains were divided into three types:RING-H2,RING-HC and RING-v.The phylogenetic tree was divided into 10 groups,and the members of the same type of protein domain were not highly clustered together,indicating that the sequences of 375 RING Finge protein sequences in almonds were different from those outside the RING domain.And protein conserved Motif and domain types in IV,VI and X three groups of protein members have similar Motif and domain types,and the number of exons and introns is more similar.375 RING Finger genes were distributed on 8 chromosomes and 6 fragments,and there were 53 pairs of fragment duplicates and 73 pairs of tandem duplicates.There were 97,32,295 and 225 pairs of RING Finger members in Prunus dulcis,Arabidopsis thaliana,Oryza sativa,Malus domestica and Prunus persica,respectively.The selective evolutionary pressure indicated that the amygdala fragment and tandem repeat genes and interspecies homologous genes were subject to purification selection.The results of cis-acting elements showed that 375 RING Finger genes were widely involved in many processes such as hormone regulation,stress,growth and development.And has obvious tissue-specific expression characteristics.GO enriched multiple RING Finger proteins with ubiquitination function,KEGG annotated a ubiquitin-mediated pathway,and identified specific gene functions of multiple RING Finger members.Finally,Prudul26A005303P1 was identified as SBP1 protein type among RING Finger members,and SBP1gene was identified in Almond.（2）A SBP1 gene,named PsdSBP1（NCBI accession number:MZ078142）,was cloned from cultivated Almond cultivar‘Zhipi’by homologous cloning.The length of PsdSBP1 is 1085 bp and the open reading frame is 1017 bp,which has 338 amino acids.Multiple alignment of homologous SBP1 protein showed that PsdSBP1 protein sequence was completely consistent with Prudul26A005303P1 protein sequence.Compared with Pt SBP1 protein sequence,PsdSBP1 protein sequence had only one amino acid,and had a highly conserved RING-HC domain at the end of the protein sequence.The amino acid types of SBP1 protein sequences in different families and genera were different from 10 to 80.Protein interaction network showed that PsdSBP1 had interaction with Cullin1 protein,MYB86 and Myb-related protein Zm38 proteins with floral organ development function.The fluorescence quantitative results showed that PsdSBP1 was expressed in the styles,pollens,styles,petals and leaves of almond.Subcellular localization showed that PsdSBP1 was localized in the nucleus.（3）The mercuric chloride disinfection time,sucrose content and hormone ratio of regeneration system of petunia were screened.The results showed that the seeds of petunia disinfected by mercuric chloride for8 min had no pollution.The germination rate of 20 g sucrose content in 1/2 MS medium was the highest.The hormone proportion of 2.5 mg/L IBA and 1 mg/L ZT was most suitable for Petunia explants differentiation.In addition,the binary expression vector p CAMBIA1300-35S-3flag-PsdSBP1 of PsdSBP1gene was successfully constructed,and the recombinant plasmid was obtained and transformed into Agrobacterium tumefaciens by freeze-thaw method.Subsequently,2.5 mg/L hygromycin,450 mg/L cephalosporin,bacterial concentration OD₆₀₀=0.5 and culture time 48 h were selected as explants for genetic transformation system of petunia.The transformed Petunia seedlings were subjected to PCR detection,and the target fragment was detected to be consistent with the positive photo fragment,which proved that PsdSBP1 gene was transferred into Petunia and positive Petunia plants were obtained.