Cloning Of AiiA And Its Inducible Expression In Eucalyptus Urophylla×E.grandis

Eucalyptus, belonging to the Myrtaceae family, is the most widely planted hardwood crop in tropical and subtropical regions, because its growth is superior to that of other hardwood crops, it adapts well to the environment, and its wood can be used for multiple purposes. Eucalyptus is the most important forest tree in China, covering more than3million hectares of commercial plantations. Eucalyptus urophylla×E. grandis can reach a height of up to50m, and its wood is widely used as timber for heavy structures and bridges. It is highly prized in China for its superior wood properties, rooting ability and disease resistance. Plantation forestry of E. grandis×E. urophylla supplies high-quality raw material for pulp, paper,"wood, and energy while it reduces the pressure on native forests and associated biodiversity. However, diseases such as bacterial wilt, fungal infection, and gray mold disease have seriously endangered the Eucalyptus crop in China, especially that of the hybrid E. grandis×E. urophylla. Traditional methods for the prevention and treatment of these diseases involve the use of chemical pesticides, which often cause environmental pollution and ecological damage. In contrast, the process of producing adevelopment of disease-resistant trees through genetic engineering can potentially be faster, more controllable and predictable and cheaper than traditional breeding.N-acyl-homoserine lactones(AHL) are metabolites of mostly gram-negative bacteria critical and signaling molecules in bacterial quorum-sensing systems. They can activate the expression of pathogenic genes and induce diseases when AHL reach a threshold concentration. Therefore, reducing the concentration of AHLs is a key point of the diseases’ control in plants.The callus induction and shoot regeneration were studied systematically in elite clone of Eucalyptus urophylla×E. grandis by comparing the variety of combinations of different plant growth regulators, such as N-phenyl-N’-[6-(2-chlorobenzothiazol)-yl] urea (PBU). The results showed that sterile clones of seedling stem segments were used as explants and cultured in a modified MS medium supplemented with the synthetic growth regulator2mg·L-1PBU and0.05mg·L-1IAA. After cultivation for5d,96%of explants formed callus. Callus were transferred to MS medium containing1mg·L-16-BA and0.05mg·L-1NAA to induce bud formation, and90.8%of the callus induced by PBU exhibited adventitious bud formation. Shoot elongation was then stimulated on1/2MS supplemented with0.8mg-L-1PBU and0.05mg·L-1IAA for20d. For rooting, the elongated shoots were cultivated on root induction medium containing0.5mg·L-1IBA. Plantlets were then successfully transplanted to a greenhouse for growing into mature plants. Preliminary reveal callus embryonic phenomenon is affected by environmental stress also reveal the molecular mechanism of non-embryonic phenomenon.The AHL-Lactonase, expressed by aiiA gene which widespread in Bacillus sp,can hydrolyze AHLs. The study have cloned the aiiA gene from Bacillus subtilis by PCR, The sequence analysis indicated that the clone was consisted of751nucleotides (nt), coding250amino acids.The nucleotide sequence showed87%-96%identities with those of the aiiA gene that have reported. A plant expression vector of the aiiA gene was constructed and named PCAMBIAb-PPP3-aiiA which the aiiA was controlled by the PPP3promoter. This work laid the foundations for future transgenic research on aiiA gene function.pGEX-4T-1was constructed and transformed into bacteria strain of E.coli BL21for construction of genetically engineeringed bacterium strain. After9h induction with0.2mmol·L-1IPTG at25℃, the expression of AIIA protein reached to maximal level. AIIA fusion protein was obtained in E.coli system. It was observed that recombinant Escherichia coli producing AIIA proteins had AHL-degrading activity and could attenuate the plant pathogenicity of pathogen.According to the sequences of pathogen-inducible plant promoters PPP3at GenBank, PPP3promoters were cloned from tobacco genome. It was used to replace cauliflower mosaic virus35S promoter of pCAMBIA1301. The recombinant plasmid was used to transform Agrobacterium tumefaciens GV3101. The inducibility of the PPP3promoters in tobacco leaf was evaluated by Agrobacterium tumefaciens-transient genetic transformation assye. Real-time quantitative PCR was used to screen the PPP3promoters with high inducible expression. The results showed that the gus transcript level under the control of PPP3promoters increased respectively27.94fold and17.69fold after inoculation with Ralstonia solanacearum and SA. The PPP3promoter had the advantages such as low basal activity and high expression activity.The recombined plasmid was transformed into Eucalyptus urophylla x E.grandis by Agrobacterium-mediated transformation. The results of PCR and Southern blotting showed that the aiiA genes were successfully integrated into the Eucalyptus urophylla×E.grandis genome. The result of reverse transcription polymerase chain reaction (RT-PCR) showed aiiA had partly basal expression. The transcript efficiency of aiiA was calculated by real-time quantitative PCR. The transcript efficiency of aiiA under the control of PPP3promoters increased respectively43.88,30.65and18.95fold after inoculation with Ralstonia solanacearum, E.carotovoraf.sp.zeaeSabet and C.quinqueseptatun.The result of plant inoculation showed that ransgenic Eucalyptus urophylla×E. grandis expressing AIIA exhibit significantly enhanced resistance to disease comparing to non-transgenic ones. The transgenic plants could delay the wilt symptom development and make disease index reduced. The activity of PPO, PAL, PPO and SOD increased significantly than non-transgenic Eucalyptus urophylla×E.grandis. Inoculated with spores of Phytophthora capsici, transgenic E.urophylla×E.grandis showed markedly enhancement of disease resistance. All of the results indicated that the resistance of plants could be enhanced by introducing aiiA gene into the genomic of plants.