Cloning And Expression Analysis Of CpRBOHD And CpTIFY10A-like Genes Of Papaya Resistant To Ring Mosaic Virus Disease

PRSV（Papaya ringspot mosaic virus）disease seriously affects the papaya planting industry.Screening anti-PRSV genes with functionally identifying them is an effective way to breed anti-PRSV papaya varieties.The anti-PRSV papaya genetic resources can enhance the papaya’s ability to resist PRSV and avoid the reduction in yield caused by papaya cultivation.This is of great significance for the cultivation of disease-resistant,yield-increasing,high-quality related papaya varieties.Using the data of "CTS-N（Chitosan-N）induced papaya anti-PRSV transcriptome" obtained in the preliminary test of this research group,two up-regulated genes CpRBOHD gene and CpTIFY10A-like gene were found.In this study,CpRBOHD gene and CpTIFY10A-Like gene was cloned by RACE（Rapid amplification of cDNA end）,the full length of the gene was obtained and bioinformatics related analysis was performed.Real-time quantitative polymerase chain reaction（qPCR）was used to identify the changes of CpRBOHD gene and CpTIFY10A-like gene expression in samples.Prokaryotic expression of the target gene was performed and SDS-PAGE electrophoresis was used to verify the prokaryotic expression effect.Fluorescence microscopy was used to observe tobacco leaf cells to determine the subcellular localization of CpRBOHD gene and CpTIFY10A-like gene.The pCXSN plasmid was used to reconstruct plant overexpression vectors,and they were used to transiently infect PRSV papaya leaves.In the transient expression process,the changes of CpRBOHD gene and CpTIFY10A-like gene expression were analyzed by qPCR,and the changes of related physiological indicators（SOD,PPO,CAT,PAL,MDA）leaves were also measured in papaya.It has laid a good foundation for further regulation of papaya’s anti-PRSV and enhancement of its anti-PRSV ability through genes.The conclusions of this study are as follows:1、The CpRBOHD gene（XM₀22044550.1）was 3095bp in full-length,of which the coding sequence（CDS）was 2760bp,and it encoded 919 amino acids.The relative molecular mass of the protein was 103.74kDa,the theoretical isoelectric point pI=9.25.This gene had no signal peptide and had 4 transmembrane domains.Bioinformatics analysis showed that its expressed protein structure predicted that the proportion of α-helix（43.63%）,extended chain（14.47%）,β-turn angle（4.57%）,random curl（37.32%）.Phylogenetic analysis revealed that the CpRBOHD gene was clustered into the same branch as of the homologous genes of Durio zibethinus、Gossypium arboreum and Gossypium raimondii.qPCR analysis showed that the expression level of CpRBOHD gene was significantly up-regulated in the infected leaves with PRSV after treatment with CTS-N.This gene contains 4 transmembrane domains,so the amino acids at 757-919aa were selected for prokaryotic expression.The prokaryotic expression vector pET28aCpRBOHD-sumo was successfully constructed.The protein detected was 18.73kDa by SDS-PAGE electrophoresis.This gene constructed the pNC-Green-CpRBOHD-SubN vector and found that the subcellular localization in tobacco cells was on the plasma membrane.2、The CpTIFY10A-like gene（XM-022035541.1）was 1352 bp in full-length,of which the CDS was 822 bp,and it encoded 273 amino acids.The relative molecular mass of the protein is 29.36kDa,the theoretical isoelectric point pI=9.23,no signal peptide and transmembrane domain.The encoded protein belonged to the basic protein of TIFY-family.Bioinformatics analysis showed that its expressed protein structure predicted that the proportion of α-helix（15.02%）,extended chain（8.42%）,β-turn angle（2.20%）,random curl（74.36%）.Phylogenetic analysis revealed that the CpTIFY10A-like gene was clustered into the same branch as of the homologous genes of Ziziphus jujuba and Pyrus x bretschneideri.qPCR analysis showed that the expression level of CpTIFY10A-like gene was significantly up-regulated in the infected leaves with PRSV after treatment with CTS-N.The prokaryotic expression vector pET28a-CpTIFY10A-like-sumo was successfully constructed.The protein detected was 29.36kDa by SDS-PAGE electrophoresis,which was consistent with the theoretical prediction.This gene constructed the pNC-Green-CpTIFY10A-like-SubN vector and found that the subcellular localization in tobacco cells was on the nucleus.3、After papaya inoculation with PRSV virus for 16 days,obvious symptoms of PRSV appeared on the leaves,and the PRSV virus was simultaneously identified at the molecular level using RT-PCR（Reverse Transcription-Polymerase Chain Reaction）technology.The plant overexpression vectors pCXSN-CpRBOHD vector and pCXSN-CpTIFY10A-like vector were re-constructed,and were transiently expressed on the papaya leaves infected with PRSV The expression of CpRBOHD gene and CpTIFY10A-like gene were significantly increased by qPCR technology.At the same time,the enzyme activities of SOD,PPO,CAT,and PAL in papaya leaves were increased,and the content of MDA was relatively increased.This indicateed that the up-regulation of CpRBOHD gene and CpTIFY10A-like gene had a certain relationship with papaya anti-PRSV.