Functional Analysis On Apsbt Alternative Splicing From Agapanthus Praecox During Cropreservation-Induced Programmed Cell Death

Subtilisin like proteases(SBT)are multifunctional serine proteases related to plant development and defense.In this paper,the full-length SBT gene was screened and cloned by comparing the transcriptome data of Agapanthus praecox ssp.orientalis’Big Blue’in cryopreservation.ApSBT.1 and ApSBT.2 are 2316 bp and 2468 bp in length,which of ApSBT.1 encodes 771 amino acids,and the predicted protein size is 82.69kDa.Due to the alternative splicing of intron retention type in ApSBT.2,the retained intron contains a termination codon PTC,so the translated protein sequence contains only 115 amino acids,and the predicted protein size is 13.18 kDa.ApSBT.1 is a hydrophilic protein of the peptidases S8family.It has typical structural characteristics of subtilisin like protease,including I9 and S8 domains,while ApSBT.2 contains only one I9 domain of subtilisin like protease.The comparative analysis of homologous sequences showed that the SBT protein of Agapanthus praecox is closely related to the SBT genetic evolution of monocotyledons such as Palmaceae and Gramineae.Subcellular localization analysis showed that ApSBT.1mainly located in cytoplasm and ApSBT.2 in nucleus.Study found that the expression of ApSBT.1 and ApSBT.2 genes in Agapanthus praecox EC was response to the complex stress of cropreservation.Through the detect of DNA ladder,TUNEL,ROS,membrane lipid peroxidation and PCD-related caspase like enzyme activity in transgenic Arabidopsis,found that OH~-was the main component of ROS in transgenic Arabidopsis,and the membrane lipid peroxidation of transgenic Arabidopsis with ApSBT.1 gene was significantly higher than that of wild type,the transgenic Arabidopsis thaliana with ApSBT.2 gene was slightly lower than that in wild type.In addition,ApSBT.1 increased the activity of Caspase-1,-3 and-6,while ApSBT.2 decreased the activity of caspase-6.ApSBT.1 advanced and accelerated the PCD process in cryopreservation,while ApSBT.2 weakened the PCD process and showed partial inhibition of ApSBT.1,which improved the stress tolerance of cryopreservation.In the meantime the experiment of survival rate of transgenic Arabidopsis under single stress,found ApSBT.1 under oxidation was the lowest,only 0.68 times over wild type,and the survival rate of ApSBT.2under salt stress was the highest,1.4 times over WT.Therefore,the differences of expression and survival rate indicate that the response modes of ApSBT.1 and ApSBT.2 to abiotic stress are different.But the relevant mechanism needs further study.In conclusion,the present study reveals the role of subtilisin-like protease in abiotic stress-induced PCD,and provides a theoretical basis for the subsequent development of protease targeted cryoprotectants.