Produção e emissão de β-cariofileno em laranjeiras e Arabidopsis thaliana pela superexpressão de terpeno sintases e avaliação da expressão gênica nas vias biossintéticas para produção de terpenos
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Data
2019-09-24
Autores
Santos, Mateus de Almeida
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Universidade Estadual Paulista (Unesp)
Resumo
As citriculturas brasileira e mundial têm sido assolada pelo huanglongbing (HLB), doença associada às bactérias Candidatus Liberibacter spp., transmitidas pelo psilídeo Diaphorina citri. Não há cura para a doença e o manejo do HLB é feito com plantio de mudas sadias, erradicação de plantas sintomáticas e controle do inseto vetor. Pomares de citros intercalados com goiabeiras no Vietnã, tiveram menor ocorrência de psilídeos e de plantas com HLB. O composto volátil β-cariofileno, emitido por goiabeiras foi caracterizado como repelente ao psilídeo. Laranjeiras das variedades Pera e Valência foram geneticamente modificadas (GM) com genes da terpeno sintase (TPS) e da farnesil difosfato sintase (FPPS) de Arabidopsis thaliana, com a expectativa de aumentar a produção e emissão de β-cariofileno, tornando estas plantas repelentes. Em testes biológicos e químicos foram selecionadas plantas transgênicas que apresentaram incremento no acúmulo e emissão de β-cariofileno, fazendo com que o efeito repelente deste sesquiterpeno, superasse a atração que laranjeiras exercem sobre D. citri. De maneira similar, foram geradas Arabidopsis thaliana GM para superexpressar TPS de laranjeira doce. As plantas GM foram caracterizadas quanto à expressão do transgene e dos genes da via biossintética dos terpenos com 1-deoxixilulose 5-fosfato sintase (DXS), geranil difosfato sintase (GDPS) e farnesil difosfato sintase (FPPS) e quanto ao acúmulo e emissão de β-cariofileno. Foi demonstrada a viabilidade da transformação genética de laranja doce com TPS de A. thaliana, aumentando a produção de β-cariofileno a níveis superiores do que a produção endógena deste composto. Em A. thaliana, a superexpressão da TPS também aumentou a produção do composto repelente. Houve alteração na expressão em genes chave da via biossintética dos terpenos, mas por outro lado, não houve alteração fenotípica observável, exceto o incremento na produção do β-cariofileno. Nos dois casos, onde foram utilizadas terpeno sintases de Arabidopsis e de laranjeira, a manutenção dos íntrons se mostrou preponderante para o aumento na produção e emissão de β-cariofileno. Os resultados apresentados demonstram a viabilidade desta estratégia biotecnológica para produzir compostos terpênicos repelentes e gerar plantas que possam combater a epidemia de HLB.
Citrus in Brazil and around the world has been devastated by Huanglongbing (HLB), a disease which is associated to Candidatus Liberibacter spp. bacteria and transmitted by the psyllid Diaphorina citri. There is no cure to such disease and the HLB management is done by planting healthy seedlings, eradicating symptomatic plants and controlling the insect vector. Citrus orchards interspersed with guava trees in Vietnam had less occurrence of psyllids and fewer plants with HLB. The volatile compound β-caryophyllene, emitted by guava trees, has been considered a repellent to the psyllid. Pera and Valencia sweet oranges varieties were genetically modified (GM) with Arabidopsis thaliana’s terpene synthase and with farnesyl dyphosphate synthase (FPPS) genes, expecting to raise the production and emission of β- caryophyllene, turning such plants into repellents. In biological and chemical trials, transgenic plants with incremental β- caryophyllene accumulation and emission were selected, leading to a sesquiterpene repellent effect stronger than the attraction that citrus trees exert over D. citri. Similarly, genetically modified Arabidopsis thaliana were generated to overexpress sweet orange TPS. Such genetically modified plants were characterized regarding the transgene expression and to the genes of terpene biosynthesis pathway with 1-deoxyxyllulose 5-phosphate synthase (DXS), geranyl diphosphate synthase (GDPS) and farnesyl diphosphate synthase (FPPS) and to the accumulation and emission of the β-caryophyllene. The GM sweet orange plants expressing A. thaliana TPS had increased increased β- caryophyllene production to levels which are higher than endogenous production of such compound. In A. thaliana, the TPS overexpression also increased the repellent compound production. There were alterations in the expression of key genes of the terpene biosynthetic pathway, however, there was no observable phenotypic alteration, except the β-caryophyllene production increment. Arabidopsis and sweet orange terpene synthases harboring íntrons, showed a preponderant increase in β-caryophyllene production and emission. The results showed the viability of such biotechnological strategy in order to produce repellent terpene-compounds and generate plants that could fight the HLB epidemy.
Citrus in Brazil and around the world has been devastated by Huanglongbing (HLB), a disease which is associated to Candidatus Liberibacter spp. bacteria and transmitted by the psyllid Diaphorina citri. There is no cure to such disease and the HLB management is done by planting healthy seedlings, eradicating symptomatic plants and controlling the insect vector. Citrus orchards interspersed with guava trees in Vietnam had less occurrence of psyllids and fewer plants with HLB. The volatile compound β-caryophyllene, emitted by guava trees, has been considered a repellent to the psyllid. Pera and Valencia sweet oranges varieties were genetically modified (GM) with Arabidopsis thaliana’s terpene synthase and with farnesyl dyphosphate synthase (FPPS) genes, expecting to raise the production and emission of β- caryophyllene, turning such plants into repellents. In biological and chemical trials, transgenic plants with incremental β- caryophyllene accumulation and emission were selected, leading to a sesquiterpene repellent effect stronger than the attraction that citrus trees exert over D. citri. Similarly, genetically modified Arabidopsis thaliana were generated to overexpress sweet orange TPS. Such genetically modified plants were characterized regarding the transgene expression and to the genes of terpene biosynthesis pathway with 1-deoxyxyllulose 5-phosphate synthase (DXS), geranyl diphosphate synthase (GDPS) and farnesyl diphosphate synthase (FPPS) and to the accumulation and emission of the β-caryophyllene. The GM sweet orange plants expressing A. thaliana TPS had increased increased β- caryophyllene production to levels which are higher than endogenous production of such compound. In A. thaliana, the TPS overexpression also increased the repellent compound production. There were alterations in the expression of key genes of the terpene biosynthetic pathway, however, there was no observable phenotypic alteration, except the β-caryophyllene production increment. Arabidopsis and sweet orange terpene synthases harboring íntrons, showed a preponderant increase in β-caryophyllene production and emission. The results showed the viability of such biotechnological strategy in order to produce repellent terpene-compounds and generate plants that could fight the HLB epidemy.
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Palavras-chave
Huanglongbing, Repelência, Transformação genética, Repellency, Genetic transformation, Diaphorina citri