Ecological Safety Evaluation And Unintended Effect Study Of The Multiple Gene Poplar(P.× Euramericana ‘Guariento’)

Transgenic technology can improve the traits of tree in genetic level and solve the problems that conventional techniques can‘t. The application of transgenic technology can speed up the breeding process of poplars. By using transgenic technology, transgentic poplars with salt tolerance, disease and insect resistance, herbicide resistance, reduced lignin content and increased growth etc.have been produced successfully. With the promotion of genetically modified plants, the concern of their safety inceased. Therefore, the safety assessment must be carried out before actual promotion. At present, researchs on ecological safety of genetically modified plant are focused on transgenic crops, while the safety assessment of transgenic trees are rare. Most of the researches focused on soil bacteria in the rhizosphere of transgenic trees by using spread plate method. The transgenic plants probably affect the surrounding environment and other plants. Besides, due to the import of exogenous genes, the original sequence has been changed, which might influence primary and secondary metabolic processes, so there might be some unintended change in metabolite. In this research, we use multiple-gene transgenic poplar(P.×euramericana‘Guariento’)(five exogenous genes include the following: Vgb, encoding aerobic Vitreoscilla hemoglobin(VHb); SacB,encoding levansucrase, which is involved in Bacillus subtilis fructan biosynthesis; BtCry3 A, encoding the Bt endotoxin from Coleoptera; OC-I, an anti-insect gene, encoding rice cystatin; and JERF36, a tomato gene, encoding the jasmonate(JA)/ethylene(ET) response factor protein.) and its receptor polar as plant materials, applied Biolog-ECO Ecological plate and bacterial 16 S rDNA V4 region sequencing methods to study the carbon metabolism of soil microorganisms, soil bacterial community composition and structural diversity. T the effects caused by exogenous gene in metablism and wood properties of poplar were also studied using Liquid chromatography-mass spectrometry and gas chromatography- mass spectrometry, the main conclusions are as follows:1. No horizontal transfer of foreign genes occurred. Using genomic DNA from understory plant leaves and soil microorganisms, series of PCR were conducted by using specific primers of the 5 transgenes(Vgb, SacB, BtCry3 A, OC-I and JERF36). No any target PCR product was observed in all samples analyzed. This result suggested that no horizontal transfer of transgenes occurred between transgenic poplar and other plant species or microorganisms during 8 years.2. Transgenic poplar had no obvious effects on soil microbial quantity and carbon met-abolism capacity. Land planted with five clones of transgenic poplars, land planted with non-transgenic poplar and land without any plants as blank control were used as experimental materials. Three microorganisms in non-rhizosphere soil from the land of transgenic poplar were studied by using dilution plate culture method. Results showed that the number of bacteria, fungi and actinomycetes in non-rhizosphere soil between transgenic poplar and non-transgenic poplars did have some changes in different growing period, but the changes have no obvious regular pattern. Biolog method was applied to investigate the influence of transgenic poplars on functional diversities of the rhizosphere soil microbial community. Single carbon utilization level in rhizosphere soil microbial had been comparative analyzed. Results showed that transgenitic polar has no obvious effects on rhizosphere soil microoganisms in capacity of carbon source utilization, richness indexes, dominance indexes and evenness indexes compared with non-transgenitic polar. The principal component analysis(PCA) of carbon utilization indicated that an obvious difference was existed between no plants soil and the other treatments, but the distribution of microbial system between transgenic poplar and non-transgenic poplar was not obvious.3. The effect on composition and structure of community diversity of soil bacteria in the rhizosphere from transgenic poplar trees were not reveled. We used the amplitude sequencing method to analyze the V4 regions of the 16 S rRNA gene of rhizosphere soil microbial to identify changes in bacterial diversity and community structure in two transgenic poplar lines(D5-20 and D5-21), one wild-type line(D-50) and uncultivated soil(CK). After chimera filtering, 468 133 sequences in thedomain Bacteria remained. There were ten dominant taxonomic groups(with >1% of all sequences) across the samples. 241 out of 551 genera(representing a ratio of 97.33%) were common in all samples. A Venn diagram showed that 1,926 operational taxonomic units were shared by all samples. We found a significant reduction in Chloroflexi, in the microorganisms in the rhizosphere soil planted with poplars(transgenetic or non-transgenetic poplar). Taken together, the results showed few statistical differences in the bacterial diversity and community structure between the transgenic poplars and control poplars, suggesting that this genetic modification would hardly impact on the bacterial communities in the rhizosphere.4. Widespread diversities in wood physical property and chemical property of transgenic poplar were displayed. Comparative analysis in height, diameter at breast height, wood physical property and chemical property between 5 transgenic clones and 1 non-trangenic clone were carried out. The results show that the differences were exisited in not only transgenic lines, but also in control. Compared with the non-transgenic lines, height, contents of holocellulose and cellulose in the transgenic lines increased, the highest increased were 4.57%, 6.21% and 5.01% respectively. However, the total lignin and acid soluble lignin decreased, including total lignin highest decreased 17.06%, but acid soluble lignin was 13.99%. While the diameter at breast height, the fiber length, fiber width, and acid insoluble lignin had variations of uncertain. It is concluded that the introduced exogenous gene can generate widespread diversities in non-target target wood traits.5. The contents of transgenic poplar wood metabolites have changed, but the elements haven‘t. Non-target unbiased testes of the transgenic poplar wood metabolomics were conducted by using liquid chromatography- mass spectrometry and gas chromatography-mass spectrometry. In the 2 transgenic lines(D5-20, D5-21) and 1 non-transgenic line(D5-0), we detected 197 metabolites, finding no increase and deletions phenomenon in the variety of metabolites after genetic modified. However, 52 metabolites contents significantly changed in one transgenic line(D5-20) and 83 significantly changed in the other transgenic line(D5-21) compared to control(D5-0). Only 13 metabolites appeared significant difference in the content betweent the two transgenic lines and PCA analysis showed there has no significant difference between the two transgenic lines. Furthermore, the transgenic and non-transgenic poplar clones can be distinguished by metabolic groups.