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- Title
番木瓜 CpTIFY10A-like 基因克隆及表达分析.
- Authors
吕金慧; 王府润; 陈萍
- Abstract
【Objective】This study cloned the papaya CpTIFY10A-like gene sequence and analyzed its expression characteristics, in order to provide a theoretical reference for exploring the function of this gene and the molecular mechanism of resistance to papaya ring spot virus(PRSV).【Method】Combined with the anti-PRSV transcriptome data of the previous research of the research group,the up-regulated gene CpTIFY10A-like was cloned using RACE technology. The fulllength cDNA was obtained and the coding sequence(CDS)was analyzed,then bioinformatic analysis of this gene was conducted. Three-month-old papaya plants were treated differently with the identified PRSV. With plants infected with PRSV and showing symptoms(CK+),plants infected with 1.0 mL/L CTS-N infected with PRSV and showing symptoms (B)were the treatment groups. The plants irrigated with clear water(without the addition of CTS-N)and not infected with PRSV(CK-)were as the control group. qRT-PCR was used to detect the expression of PRSV gene and CpTIFY10A-like gene in papaya leaves under different treatments. At last,connected the CDS sequence of CpTIFY10A-like gene to pET28asumo vector to construct a prokaryotic expression vector. The reconstruction vector was transformed into competent cell Rosetta(DE3)and induced protein expression by different concentrations of IPTG. The prokaryotic expression of this gene was detected by SDS-PAGE electrophoresis.【Result】The results demonstrated that the gene was 1352 bp in fulllength, of which the CDS sequence was 822 bp,and it encoded 273 amino acid residues. The theoretical isoelectric point (pI)of the encoded protein was 9.23,the instability coefficient was 62.97,and the average hydrophilicity coefficient was 0.458. It belonged to unstable hydrophilic basic protein. Subcellular localization predicted that the gene was located on the nucleus. The secondary structure of the protein expressed by this gene was mainly random curl and α-helix,and also contained a small amount of β-turn fold and extended chain. Amino acid sequence of CpTIFY10A-like protein had high similarity with that of TIFY protein in Z. jujuba and Pyrus x bretschneideri,they both had specific TIFY domain and belonged to TIFY family. Phylogenetic analysis revealed that the CpTIFY10A-like gene was clustered into the same branch as of the homologous genes of Ziziphus jujuba. This showed that their kinship was close. Using qRT-PCR to detect papaya leaves in different treatments,it was found that the relative expression of PRSV gene was ranked as CK+(1.02)>B(0.39)>CK- (0),and the relative expression of CpTIFY10A-like gene was ranked as B(53.12)>CK+(1.15)>CK-(1.02),and the expression of CpTIFY10A-like gene in the leaves of PRSV infected papaya treated with CTS-N was significantly increased(P< 0.05,the same below),while the expression of PRSV gene was significantly decreased. The molecular weight of the protein expressed by CpTIFY10A-like gene in prokaryotic cells was consistent with the theoretical molecular weight(29.36 kD). In the induction of different concentrations of IPTG,the higher the IPTG concentration,the greater the protein expression,it indicated that the gene was successfully expressed in prokaryotic cells.【Conclusion】CTS-N can induce the efficient expression of CpTIFY10A-like gene,which plays a regulatory role in the jasmonic acid pathway to prevent and control PRSV. It indicates that the CpTIFY10A-like gene plays an important regulatory role in the anti-PRSV process of papaya.
- Subjects
AMINO acid sequence; AMINO acid residues; BASIC proteins; JUJUBE (Plant); PROTEIN structure; CYTOKININS
- Publication
Journal of Southern Agriculture, 2020, Vol 51, Issue 6, p1308
- ISSN
2095-1191
- Publication type
Article
- DOI
10.3969/j.issn.2095-1191.2020.06.009