Screening And Functional Characterization Of Cotton Genes That Are Involved In Plant Response And Defense To Aphid Attack

Cotton aphid (Aphis gossypii Glover) is one of the most important pests of the commercial crop cotton in the world. The adults and nymphs of cotton aphids collectively cause direct damage to leaves or young stems of cotton with their piercing-sucking mouths to acquire plant phloem liquids. At the same time, they also lead to indirect damage to cotton by transmitting plant viruses and triggering soot disease from honeydew secreted by aphids. The damage from cotton aphids has effects on cotton output and cotton fiber quality as result of the increased sugar content. The insecticides are used frequently because of cotton aphids characterized by small size, strong fertility, short lifespan and serious overlapping generation and so on. On account of the unreasonable use of insecticides, cotton aphids have produced resistance to many insecticides, which further make the control of cotton aphids more difficult. In addition, cotton aphid damage has a tendency to rise further with the extension and application of Bt cotton. It is regarded as an effective and environmentally friendly strategy to control aphids using plant resistance to them, and one of the most important methods of integrated management system of aphid damage, so it is focused on by worker on plant protection. With the development of research on plant resistance to insects, many of the aphid-resistant crop cultivation have brought huge ecological and economic benefits to agricultural production. Many research results also showed that aphids-resistant crops can reduce to the transmission of plant aphids-mediated virus. However, new problems also began to appear that the virulence of plant resistant genes to aphid has been produced in17kinds of aphids, which accounts for more than half of all arthropod biological type with virulence. To delay and prevent the occurrence of aphid biological type, it is of great significance to actively seek new aphid-resistant genes, reveal the molecular mechanism of interaction between plants and aphids, and realize long-lasting use of aphids-resistant gene in a production system of crop. From the perspective of cotton resistance to cotton aphids, as the main test materials with red-chicken-feet-leaf cotton (aphid-resistant varieties), we constructed the subtracted cDNA library of cotton leaves induced by cotton aphids with SSH (Suppression subtractive hybridization) method. The bioinformatics analysis showed that a total of253ESTs (Expression Sequence Tag) on cotton aphid induction were acquired from the cDNA subtracted library. As the source of their information, GhTCTP1and GhPSAK1, which were related to cotton aphid attacks, were cloned, and the functions and mechanisms of their resistance to aphids were studied. The main results were as follows:1. Construction and analysis on the subtracted cDNA library of cotton leaves induced by cotton aphidsA subtracted cDNA library of cotton leaves induced by cotton aphids was constructed with SSH method.253independent clones from the subtracted cDNA library were randomly chosen and analyzed. The size of the inserts varied from250to2000bp. The clones were sequenced and the expressed sequence tags (ESTs) were analyzed through homology search. From opinion of stress, there were38cDNA clones on biotic stress,95cDNA clones on abiotic stress,93cDNA clones on biotic and abiotic stress, and27cDNA clones on others. This suggested that plant induced defense to cotton aphids was relatively complex and involved several signal transduction pathway, and there were cross-talk among different signal pathways. In addition, we identified some interesting genes, Such as Glycogen Synthase Kinase3(GSK3), Translationally Controlled Tumor Protein (TCTP), MYB proteins, Plant cytochrome P450(P450s), NAC, Rac protein, and some proteins on photosynthesis.2. GhTCTP1sequence, phylogenetic analysis and its expression level under the cotton aphid attack or after wounding inductionA translationally controlled tumor protein gene, GhTCTP1(Accession number in GenBank: KC787689), was identified according to information from the subtracted cDNA library. GhTCTP1belonged to TCTP subfamily and edcoded a putative protein of168amino acids. GhTCTPl shared high similarity to plant TCTPs from rice, wheat and Arabidopsis and so on, with95same amino acids, and GhTCTPlwas close relative to TCTP of Vernicia fordii during their evolution. GhTCTP1expression level was suppressed in cotton roots, hypocotyls, cotyledons and leaves under cotton aphid attack, but its expression level was up-regulated in the wounded cotton leaves, which suggested that GhTCTP1expression level was relative to the induction from cotton aphids or wounding.3. GhTCTP1overexpressing in Arabidopsis thaliana can improve the resistance of transgenic plants to green peach aphidsGhTCTP1overexpression transgenic Arabidopsis plants were acquired by Agrobacterium-mediated transformation. We chose three Lines (Line1, Line2and Line10) from them to study their resistance to green peach aphids (Myzus persicae). The choice test demonstrated that the green peach aphids had lower choice for transgenic Arabidopsis, but higher for wild type Arabidopsis. No-choice test showed that the aphid number from transgenic Arabidopsis was more than that from wild type. These results suggested that transgenic Arabidopsis was higher resistant to green peach aphids than wild type, and GhTCTP1overexpression may improve the Arabidopsis resistance to green peach aphids.4. Overexpressing GhTCTP1transgenic Arabidopsis resistance mechanisms research on green peach aphidsSince GhTCTP1overexpression may improve Arabidopsis resistance to green peach aphids, the study of its mechanisms of aphid-resistance was necessary. To attain this aim, we measured of two kinds of important nutrient content (Soluble sugar and total free amino acid), activities of three enzymes (Phenylalanine ammonia enzyme, PAL; peroxidase, POD; superoxide dismutase, SOD), and observed the leaf callus deposition with aniline blue staining. The content of two nutrient substance was different in leaves between transgenic Arabidopsis and wild type Arabidopsis. The activities of PAL, SOD and POD in leaves from transgenic Arabidopsis were higher than wild type Arabidopsis. The callose amount in transgenic Arabidopsis leaf was more than wild type Arabidopsis leaf. These results suggested that GhTCTPl overexpression in Arabidopsis improved some aphid-resistant indexes and increased Arabidopsis resistance to green peach aphids. In addition, the expression levels of key genes in SA-related pathway were upregulated in transgenic Arabidopsis under aphid attack in comparison with wild type, suggesting GhTCTPl might regulated plant resistance to aphid by SA-related pathway.5. Analysis on GhTCTPl overexpressing transgenic cotton resistance to cotton aphidsChoice tests were carried out between GhTCTPl overexpression transgenic cotton Line2, Line4, Line5, Line6and wild type cotton Coker312. Choice tests showed that the number of cotton aphids from leaves of Line4or Line6was lower than that from leaves Coker312at6h,12h,24h under cotton aphid attack, and there was significant or very significant difference between them. The number of cotton aphids from leaves of Line2or Line5was no significant difference than from leaves of Coker312at6h,12h,24h under cotton aphid attack. However, the number of the former were little lower than the latter. No-choice tests were performed on leaves from Coker312, red-chick-feet-leaf cotton (JJ, aphid-resistant cultiyar), and four transgenic lines (Line2, Line4, Line5, Line6). The results confirmed that the cotton aphid number on leaves from red-chick-feet-leaf cotton, Line2, Line4, Line5, Line6was lower than that from Coker312at24h,48h under cotton aphid infestation. All these results suggested that T1generation of GhTCTP1overexpression transgenic cotton were higher resistant to cotton aphids than Coker312.6. GhPSAK1sequence, phylogenetic analysis and its transcripts under the cotton aphid infestation or after wounding inductionA photosynthesis-related cotton gene, GhPSAK1(Accession number in GenBank:KF246683), was identified. It belonged to PSI-PsaK family and encoded a putative protein of162amino acids. GhPSAKl was highly homologous with GhPSAKl of alfalfa, soybeans, wheat and Arabidopsis thaliana. Phylogenetic tree showed that GhPSAK1was closely relative to soybean and alfalfa during their evolutions. Cotton GhPSAKl expression levels rised at0h,12h,24h under cotton aphid attack or after wounding induction and reached highest level at24h. However, GhPSAKl transcripts declined gradually at48h and72h. In this process, GhPSAK1expression levels of cotton infested by cotton aphids raised more quickly than that induced by wounding, but the former decreased more slowly than the latter.7. Effects on resistance to green peach aphids in overexpressing GhPSAK1Arabidopsis.The analysis on resistance to green peach aphids (Myzus persicae) was performed between GhPSAK1overexpression Arabidopsis thaliana lines (Line1, Line2and Line10) and wild type (WT). Choice assays showed that aphid population on transgenic Arabidopsis leaf was smaller than that on WT at6h,12h,24h under attack from green peach aphids, which confirmed that green peach aphids had lower choice for transgenic Arabidopsis than WT. No-choice assay demonstrated that the number of green peach aphids on transgenic Arabidopsis was less than that on WT at24h,48h under attack from green peach aphids. All these results suggested that overexpression of GhPSAK1in Arabidopsis improved plant resistance to green peach aphids.8. Aphid-resistance mechanism of GhPSAK1overexpressing transgenic ArabidopsisThe callose amount in transgenic Arabidopsis leaves was more than that of wild type. In addition, The contents of the soluble sugars and total amino acids were also altered in leaves of transgenic Arabidopsis plants in comparison with wild type. Activities of SOD (superoxide dismutase) and POD (peroxidase) in transgenic leaves were higher than those of wild type. These results suggested that GhPSAK1overexpression could improve aphid-resistant condition of Arabidopsis, and the transgenic Arabidopsis showed higher resistance to green peach aphids than wild type. In addition, the transcriptions of key genes related on SA pathway were upregulated in transgenic Arabidopsis under aphid infection, suggesting GhPSAK1might regulate plant resistance to aphid by SA-related pathway.