The Yeast Two-hybrid Library Was Used To Screen The Interacting Proteins Of Different Transcripts Of Upland Cotton DAD Gene

Programmed Cell Death(PCD)is a special cell death mechanism produced by organisms to maintain the stability of the internal environment,and is a basic biological phenomenon.Defender against Apoptotic Death(DAD)is an important gene that involved in the PCD pathway.This gene encodes a subunit in the oligosaccharyltransferase(OST)complex.OST is a core component of the endoplasmic reticulum that catalyzes N-glycosylation.DAD can negatively regulate PCD process,and then plays important roles in plant normal development and resistance to various stresses.Our previous studies found that three transcripts of DAD gene,named Dt1,Dt2 and At,were maintained in Gossypium hirsutum L.(an allotetraploid species with AADD genome)due to polyploidization and alternative splicing event.In addition,the three transcripts showed divergent temporal and spatial expression.In order to further reveal the coexistence mode and anti-apoptotic mechanism of these three transcripts in G.hirsutum,In order to further reveal the coexistence mode and anti-apoptotic mechanism of these three transcripts in G.hirsutum,this study will preliminarily find interaction profiles of three transcript-encoded proteins from the perspective of protein interaction,and the main results are as follows:1.The bait vectors of three transcripts were successfully constructed.The full-length sequences of the coding regions of Dt1,Dt2 and At transcripts were obtained by using specific primers and molecular cloning techniques using the cDNA of the leaf tissue of allotetraploid upland cotton as a template;The bait vectors Dt1-pGBKT7,Dt2-pGBKT7 and At-pGBKT7 for yeast two-hybrid were constructed respectively;The results of yeast growth after the bait vector transformed Y2H Gold strain showed that all three transcript-encoded proteins were non-toxic to yeast cells and had no self-activating activity.2.A yeast two-hybrid cDNA expression library was successfully constructed.A high-quality cDNA expression library for protein interaction network screening was constructed using leaf,flower and cotton fiber tissues of upland cotton as samples.The titer of library was great than 2.6×10~6 cfu,the average length of the insert segment was more than 1.0 kb,and a plasmid concentration of 1.0 mg/mL in a million-scale amplified library.3.Preliminary screening interacting proteins of coding protein of three transcripts.The bait vectors Dt1-pGBKT7,Dt2-pGBKT7 and At-pGBKT7 were co-transformed into Y2H Gold strain with the cDNA expression library,respectively.The co-transformation efficiency was between 7.15×10~4-1.25×10~5 cfu/ug.Five proteins interacting with Dt1,four with Dt2 and three with At were preliminarily screened by yeast colony PCR and sequencing identification.Five of the screened proteins were involved in the mechanism of PCD regulation in different tissues in plants.Additional findings,DUF538 family protein can interact with Dt1 and Dt2.However,no shared interacting proteins have been screened between Dt1,Dt2 and At transcript-encoded proteins.This suggests that the three transcripts of the DAD gene in Upland Cotton may be involved in different metabolic pathways and have functional differentiation.