Identification Of Protrins Interacting With Salvia Miltiorrhiza Bunge PPO Using Yeast Two-hybrid System

Salvia miltiorrhiza Bunge is a perennial herb of the genus Lepidoptera,whose rhizome is a traditional Chinese medicine,with the effect of promoting blood circulation and removing blood stasis.The water-soluble active ingredient of Salvia miltiorrhiza is mainly composed of salvianolic acid,which is represented by salvianolic acid B.Previous studies have shown that polyphenol oxidase(PPO)can respond to drought stress and positively regulate the accumulation of salvianolic acid B,but its mechanism is not clear.The aim of this study is to explore the physiological function and expression characteristics of PPO by means of molecular biology.By searching for the proteins interacting with PPO,we can study the possible anti-stress ability of PPO in Salvia miltiorrhiza.Through the experiment,we get the following conclusions:1.RNA was extracted from the hairy roots of Salvia miltiorrhiza,and the first strand of cDNA was obtained by reverse transcription,then ligated with T vector.The T-PPO plasmid and yeast vector pGBKT7 were digested by EcoR I and Nde I,and the recombinant plasmid pGBKT7 was digested with EcoR I and Nde I,and linked up by T4 ligase,then the pGBKT7-PPO plasmid was successfully constructed.The recombinant plasmid pGBKT7-PPO and pGBKT7 were transformed into Y2 HGold yeast competent and cultured on SD/-Trp agar plate.3 days later,the size and number of these colonies were similar.The results showed that the recombinant plasmid was not toxic to yeast.Transform the recombinant plasmid pGBKT7-PPO into Y2 HGold yeast competent and cultured on SD/-Trp?SD/-Trp/X-?-Gal?SD/-Trp/X-?-Gal/AbA agar plate,then the results showed that the bait does not activate the repoter genes.2.A total of 475 single colonies were observed on the SD/-Leu medium that was spread 1/10000 dilutions.The titer was 4.75×107 cfu/mL > 2×107 cfu/mL,which met the requirements of the libraries.48 clones were detected by colony PCR,randomly.The rate of recombinant was 85.4%,and the length of inserted fragment was between 250 bp and 2500 bp,the average length is 668 bp.3.Screening the protein interacting with PPO from the cDNA library of Salvia miltiorrhiza by yeast two-hybrid system.8 colonies were observed on the SD/-Leu/-Trp agar plate that was spread 1/1000 dilutions.The number of colonies being screened was 1.08 × 106 cfu/m L > 1 × 106 cfu/mL.The hybridization efficiency was 8%,which was much higher than 2%.243 single clones which might interact with the PPO were obtained on the SD/-Leu/-Trp/-His agar plate,and then transferred to the QDO/X/A agar plate for further screening which turned out to be 191 colonies.To increase the chance of rescuing the positive prey plasmid,streak 2~3 times on QDO/X,each time picking a single blue colony for restreaking.To identify the gene responsible for positive interaction,rescue the plasmid from yeast cells and transform them into DH5? and select for the prey plasmid on LB plus Amp.80 positive colonies were selected and sent to sequence.4.The positive clones were sequenced and the BLAST nucleotide similarity was analyzed.Finally,nine proteins with related functional studies were obtained,including ?-L-arabinofuranosidase,phospho-2-dehydro-3-deoxyheptonate aldolase,ferredoxin-nitrite reductase,NADH dehydrogenase,long-chain acyl-CoA synthase,4CL,trafficking protein,hypersensitive-induced response protein,transforming growth factor-? receptor-associated protein.5.The recombinant plasmid pCAMBIA1304-PPO was constructed by enzyme-cutting the T-PPO plasmid and pCAMBIA1304 via Spe I,then linked up with T4 ligase,thus overexpression vector and antisense vector were constructed.Agrobacterium was used as the medium to infect the leaves of Salvia miltiorrhiza,which laid the foundation for further study of PPO response to different stress.