Cloning And Bioinformatic Analysis Of Self-incompatible PsdCUL1 Gene From Almond And Transgenic Petunia

Amygdalus subgenus of Rosaceae,also known as Padanmu,is one of the four dried fruits with high medicinal value and nutritional value.Almond belongs to gametophyte self-incompatible tree species,self-pollination is not normal fruiting,if the pollination tree configuration is improper,flowering can not be normal pollination will cause a substantial reduction in yield.Therefore,exploring the self-incompatibility characteristics of almond can not only deeply understand the physiological characteristics of almond pollination and fruiting,and find suitable pollination varieties in production,but also break the original self-incompatibility characteristics by molecular biotechnology,and obtain self-compatible tree species with good quality is also the focus of the current development of almond industry.In this study,the self-incompatible PsdCUL1 gene of Prunus persica ‘Zhipi’ was obtained by homologous sequence cloning method.The evolution characteristics of PsdCUL1 gene were analyzed by codon preference,and the expression of PsdCUL1 gene was detected by fluorescence quantitative analysis.The bioinformatics analysis of PsdCULs gene family members was carried out to understand the characteristics of PsdCULs gene,and the regeneration system and genetic transformation system of Petunia hybrida were further optimized.The results were as follows:(1)A new full-length sequence of CUL1 gene was cloned from cultivated tonsil by homologous cloning technology,named PsdCUL1(NCBI accession number is MZ073343).The ORF open reading frame is 2217 bp,encoding 738 amino acids with a molecular weight of 85.86 kDa,which belongs to hydrophilic protein.The codon preference analysis of CUL gene shows that its evolution is greatly affected by natural factors.The real-time fluorescence quantitative results showed that the expression level of PsdCUL1 gene in pollen was the highest,and it had obvious indigenous tissue-specific expression characteristics.(2)Bioinformatics analysis of PsdCULs gene family members showed that 15 PsdCULs gene family members were screened from almond.There were significant differences in protein sequence length,molecular weight and isoelectric point between the members,which were mainly distributed in the cytoplasm and mainly existed in Pd01,Pd03,Pd04,Pd05 and Pd08 chromosomes to regulate plant growth.Most members of PsdCULs were expressed in ovary and pollen.The expression activity of anther was inhibited after cold stress,and the expression of ovary was less affected by cold stress.(3)In order to construct the tissue culture system of petunia,the medium and hormone concentration were screened,and the most suitable medium formula at different stages was determined: 1/2 MS medium was the sowing medium;the optimal hormone combination for inducing adventitious bud differentiation was 1.0 mg/L 6-BA and 0.2 mg/L NAA;the optimal IBA concentration for inducing adventitious roots was0.1 mg/L.(4)The binary expression vector pCAMBIA1300-35S-PsdCUL1-MYC was constructed.The Agrobacterium was transformed by freeze-thaw method,and the petunia was infected by leaf disc method.The optimal concentration of kanamycin was 50 mg/L,the optimal concentration of cephalosporin was 500mg/L,the pre-culture time was 48 h,and the infection time was 5 min.The genetic system of petunia was optimized.Finally,PCR detection showed that PsdCUL1 positive plants were successfully obtained.