Cloning And Functional Analysis Of CpSAMDC And CpSPDS Genes From Cherry (Cerasus Pseudocerasus Lindl.)

Polyamines(PAs),as a kind of physiological active substance,play an important role in flowering,fruitsetting,fruit growth and the development of plants.Chinese cherry is an excellent variety independently selected by Guizhou Province.It is widely planted in Southwest China,such as Guizhou Province,because of its excellent fruit quality,good storability,high industrial value and high economic benefit.However,its fruit yield is easily affected by severe weather,such as cold,hail and rainstorm.Previously,it was justified that the shelter cultivation was an effective measure to improve the quality and yield of cherry,the research group further carried out a transcriptome analysis on the leaves and fruits of cherry as exposure to the shelter covering.The transcriptome data showed that there was a significantly elevation in the expressions of polyamine metabolism-related genes under the shelter covering compared without the shelter.In the present work,we cloned the full-length sequences of CpSPDS and CpSAMDC,and carried out the bioinformatics analysis of CpSPDS and CpSAMDC.Subsequently,to further understand the expression of the genes in different tissues of cherry,the gene expressions and polyamine contents in four different tissues were quantified as exposure to shelter covering.Further,the two genes were transformed into Arabidopsis so as to identify their function.The main results were as follows: 1)Spermidine synthetase gene was cloned from cherry by RT-PCR,named CpSPDS(Accession number: MK045261);S-adenosylmethionine gene,named CpSAMDC,(Accession number: MK045262).The total length of the CpSPDS c DNA sequence was 1,243 bp,including 10 open reading frames(ORFs).The longest ORF was 1,101 bp in length,encoding a SPDS zymogen containing 366 amino acid residues.Cluster analysis indicated that CpSPDS has the highest homology similar with sweet cherry(XP?021826297 Prunus avium)and peach(XP?020413638 Prunus persica),with 100% and 99% respectively;CpSAMDC c DNA is 1,698 bp in length,including 12 ORFs,and the longest ORF is 1,074 bp,encoding SAMDC zymogen containing 357 amino acid residues.2)CpSAMDC and CpSPDS proteins are unstable proteins and are both hydrophilic proteins.They have no transmembrane movement and are non-secretory proteins;CpSPDS has a typical spermidine synthase structure.CpSAMDC has a typical S-adenosylmethioninase structure.Through the transient transformation of Arabidopsis protoplasts to express the target gene and the GFP fusion protein,it was found that CpSPDS is localized in both nucleus and cytoplasm,consistent with the subcellular localization of other polyamine synthesis-related enzymes,indicating that the gene mainly functions in the cytoplasm and functions.3)qPCR analysis showed that different gene expression levels in different tissues inside and outside the shelter were analyzed,and the results showed that each gene was expressed in different tissues.Among these four tissues,the expression levels of CpSPDS and CpSAMDC genes under shelter were higher than those outside the shelter,which was consistent with the results of the transcriptome sequencing.The results showed that the fruitsetting rate under the shelter covering was significantly higher than that the shelter-free,which suggested that the expression levels of CpSPDS and CpSAMDC genes may play an important role in fruitsetting of cherry.4)The determination of polyamine accumulation showed that the content of three polyamines under the shelter covering was higher than that the shelter-free.Among them,Spd content in alabastrums and fruitlets of the sheltered tree was 3.29-times,and 2.46-times higher than that of the shelter-free,respectively.The Spm content in alabastrums of the sheltered was 2.75-folds as high as that of the shelter-free,and in mature fruits and leaves,were around 1.26-times and 1.91-times of that the shelter,respectively.In the case of Put,its content in alabastrums was about 1.62 times higher than that of shelter-free,and in fruitlets,1.35 times higher than that in the unsheltered.In leaves,the differences were not statistically significant between the shelter and shelter-free.In the mature fruits,the Put content in the shelter covering was about 1.30 times higher than that of the shelter-free.5)Plant expression vectors pCAMBIA1301-CpSPDS and p CAMBIA1301-CpSAMDC were successfully constructed and genetically transformed into the wild type of Arabidopsis thaliana by means of a floc infection method,and Arabidopsis strains transfected with p CAMBIA1301-CpSPDS and p CAMBIA1301-CpSAMDC genes were successfully obtained by PCR.Eight CpSPDS transgenic Arabidopsis lines and eight CpSAMDC transgenic Arabidopsis plants were obtained.The q PCR results displayed that CpSPDS in transgenic Arabidopsis 4 had the highest expression and CpSAMDC had the highest expression in transgenic Arabidopsis 3;the determination of polyamine content showed that the three major polyamines in transgenic CpSPDS Arabidopsis were higher than the wild type.The contents of Put and Spm in transgenic CpSAMDC was higher than that of wild type,and the content of Spm was slightly lower than that of wild type.The Spd contents of CpSPDS transgenic Arabidopsis was obviously higher than that of wild type and CpSAMDC transgenic Arabidopsis.