| Banana(Musa spp.) serve as staple food for over 400 million people in Sub-Saharan Africa, South America, Central America, and Asia, and they are also the second of the most popular fruits in the world. Cold temperature and Fusarium wilt are two of the most destructive plant diseases of banana production. Transgenic modifying of banana to improve its agronomic performance and stress-tolerance is of great importance for promoting banana production in the world. In view of the banana cold-tolerance breeding, Compared to banana(Musa spp. Cavendish, AAA Group), Dajiao(Musa spp.ABB Group) has superior cold tolerance. It is usually regarded as a germplasm resource with superior cold-tolerance, Our previous comparative transcriptome analysis for banana and Dajiao identified several cold-tolerance candidate gens in Dajiao. In this study, two Dajiao candidate genes: ICE1 and MYBS3(Mp ICE1 and Mp MYBS3) coding for transcription factors were cloned and their genetic characterizations were conducted. On the basis of efficient banana transformation system, the function of Mp ICE1 and Mp MYBS3 on the improvement of banana cold tolerance was studied. In view of banana diseaseresistance breeding, primary quantitative proteomics analysis of Foc TR4 during early developmental and 24 types of VCGs genome provided new strategies for finding key genes and signal transduction pathways. In this study, we characterized two key genes of ergosterol biosynthesis metabolism(Fo ERG6 and Fo ERG11), and constructed host-induce gene silencing vectors. Later, the vectors were used for transformation of Cavendish banana embryogenic cell suspensions(ECSs) using our previous protocol. From theory to practice to verify the HIGS in resistance to fusarium wilt is feasible and effective.In order to establishment a specific “Gene-Deletor” method in fruits, the promoter of the floral meristem determination gene LEAFY of Arabidopsis, were used to drive recombinase gene FLP, and constructed “Gene-deletor” vector p BIN19-LFY-FLP. This “Gene-deletor”vector was solely introduced into Arabidopsis, The elimination situation of exogenousgenes in transgenic clones was analyzed and the specificity of fruit special promoter in Arabidopsis was detected, Based on efficient banana transformation system, the‘Gene-deletor’ vector was successfully transferred into banana, which provided a foundation for banaan biosafety research. The main results were as follows:1. The characteristics and Bioinformatic Analysis of the cold-tolerant Mp ICE1 and Mp MYBS3In this study, two cold-tolerance related transcription factors were cloned from cold-tolerant Dajiao, designated as Mp ICE1(Genbank accession number: KM379133) and Mp MYBS3(Genbank accession number: KM379134). Biological functions of Mp ICE1 and Mp MYBS3 were analyzed. According to the comparison with Musa acuminata and Musa balbisiana genome, it was found that Mp ICE1 was located on Chromosome 10, The ORF length of Mp ICE1 is 1,680 bp, which encodes a protein composed of 559 amino acids, The predicted molecular mass of Mp ICE1 is 59 k Da with p I = 5.09. Amino acid sequence alignment and phylogenetic analysis showed that Mp ICE1 containing the conserved b HLH and ACT-like domains, homolog of ICE1 in Arabidopsis thaliana. The subcellular localization showed that Mp ICE1 located in rice nucleus. Mp MYBS3 located on Chromosome 2, The ORF of Mp MYBS3 is 981 bp, which encodes a protein composed of326 amino acids. The theoretical molecular mass of Mp MYBS3 is 35.59 k Da, p I = 9.17.There are a conserved 1R domain in Mp MYBS3. Homolog of MYBS3 in rice. The subcellular localization showed that Mp MYBS3 also located in rice nucleus.2. Mp ICE1/Mp MYBS3 overexpression enhances cold tolerance in bananaThe effect of overexpressing Mp ICE1/Mp MYBS3 on cold-tolerance were evaluated in the transgenic and control plants exposed to a low temperature of 10 oC, Molecular detection showed that overexpression of Mp ICE1/Mp MYBS3 resulted in the greater accumulation of the cold responsive genes compared to WT(control). To investigate the resulting physiological and biochemical changes that might contribute to the improved cold tolerance of transgenic plants, electrolyte leakage, MDA and proline contents were measured against a time course of cold treatment. These research results show that the Mp ICE1/Mp MYBS as crucial transcription factors of cold signaling confers the cold tolerance of banana.3. Expression of FoERG6/FoERG11 in Cavendish banana confers resistance to Foc TR4To investigate weather feasibility and practicality of the host-induced gene silencing(HIGS)in the prevention and control of banana fusarium wilt. We considered Fo ERG6 and Fo ERG11 of Foc TR4 as target genes, constructd RNAi vectors in order to interfered theFo ERG6 and Fo ERG11 genes. Embryogenic cell suspensions(ECSs) of Cavendish banana were genetically transformed using Agrobacterium tumefaciens harboring HIGS vectors.Finally we successfully got the expression of Fo ERG6 and Fo ERG11 ds RNAs transgnic plants. Encouraged by the in vitro data, we tested whether expressing Fo ERG6 and Fo ERG11 ds RNAs in transgenic plants could inhibit growth of Foc TR4. To this end,greenhouse inoculation and field trials were used to test disease-resistance of transgenic plant. Resistance to Foc TR4 was assessed upon inoculating roots of 2-months-old banana plants with 2 × 106 Fg macroconidia per m L-1 in greenhouse. At 14 d postinoculation(dpi),the transgenics exhibited reduced incidence on roots. Quantitative RT-PCR con?rmed almost complete silencing of the two ERG6 genes and three ERG11 genes, respectively.Field trials test results also show that expression of Fo ERG6 and Fo ERG11 ds RNAs significantly reduce the incidence of transgnic plants.4. Construction and the elimination situation of “Gene-deletor” vectorIn this study, we constructed “Gene-deletor” vector p BIN19-LFY-FLP. This“Gene-deletor” vector was solely introduced into Arabidopsis, we have got 3 transgenic plants. GUS staining was used to detect the deletion effect of transgenes in roots, stems,leaves, anthers, and fruits.The results showed that among 53 tobacco plants transformed.The results showed that among 200 seeds transformed with p BIN19-LFY-FLP, there were only 23 seeds show visible GUS activities, 177 seeds did not show any visible GUS acticities, indicating that the transgenes were completely deleted in the fruits of the transgenic Arabidopsis plants, and 88.5 % of the elimination efficiency could be obtained in this study. In this present research, 5 transgenic banana plants have obtained, and its deletion effect is under evaluation. We need to screen fruit specific promoter in the next step of work, and clear all exogenous genes from banana fruits, aiming to thoroughly solve safety problems in the transgenic banana. |
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