| As for the problems such as plant diseases and insect pests easily occurring in the process of poplar planting,a large number of studies were carried out on genetic breeding by the researchers.A lot of transgenic plants have been tested in the field,and a large number of studies on the safety evaluation of transgenic plants were also carried out.However,there were few sustained and systematic studies on the effects of transgenic poplar on the biodiversity of the surrounding environment.The paper mainly focuses on ecological security of transgenic polar.On the one hand,the experimental forest of transgenic 741 poplars were used as the material,the quantity of the composition and variation of bacterial community in different seasons and different parts of transgenic 741 poplars were systematically analyzed using the technology of soil microorganism culture method and high-throughput sequencing method.On the other hand,the construction and verification of poplar chloroplast transformation vector were studied,in order to obtain transgenic plants with more safety through chloroplast transformation technology.It is possible to solve the problem of ecological risk caused by pollen transmission and provide a new tool for the breeding of transgenic poplar.The main results are as follows:(1)The quantitative dynamic changes of culturable bacteria,actinomycetes and fungi in bulk soil,rhizosphere soil and rhizoplane samples of 7 lines of Bt transgenic 741 poplar(Cry1Ac and Cry3A)and non-transgenic 741 poplar(CK)were statistically analyzed by cultivation method.The results showed that there were significant differences in the quantity of three microorganisms in different months for 2 years(P<0.01).It shows that the quantities of microorganisms in the root are greatly affected by season.In general,the quantities of microorganisms in the same type of samples remained at the same order of magnitude.Based on the analysis of the differences between CK and the transgenic lines under the same sampling time,it indicated that the quantities of microorganisms of CK were different with some transgenic lines,and the differences between the lines were random.(2)Two transgenic lines,Bt Cry1 Ac transgenic line Pb29 and Bt Cry3 A transgenic line CC84 were selected,and CK was used as control.The changes of the number of culturable microorganisms in 3 lines during the growth of 5 years were analyzed.The results showed that the number of microorganisms was low in June,but increased gradually from July to September,and the number started to decline at the beginning in October.Moreover,the number of microorganisms in rhizosphere samples was higher than that of bulk soil and rhizoplane samples.There was a significant difference in the number of bacteria in the bulk soil and rhizosphere soil from June to October(P<0.01),and there were significant differences of actinomycetes and fungi in the bulk soil(P<0.05).However,there were no significant differences in the number of bacteria,actinomycetes and fungi between the 3lines(P>0.05).Based on the correlation analysis of the number of microorganisms in 3lines for 25 months,it was found that there was a significant correlation between the number of samples in the same kind of samples,which showed that the number of microorganisms in the 3 kinds of samples showed a consistent change in the overall dynamic change in the 5 years.These results showed that the growth of transgenic 741 poplars had no influence on the number of soil microorganisms in root system.(3)The composition of bacterial communities in different parts of the root system(bulk soil,rhizosphere and rhizoplane)of three lines,non transgenic line,Bt Cry1 Ac transgenic line Pb29 and Bt Cry3 A transgenic line CC84,were analyzed by the Illumina sequencing technology.The results showed that Proteobacteria was dominant in all samples(56.2%),followed by Acidobacteria(11.9%),Bacteroidetes(10.7%),Actinobacteria(9.1%),Candidate division TM7(3.6%),Chloroflexi(2.8%),Planctomycetes(1.5%),Verrucomicrobia(1.5%)and Gemmatimonadetes(1%).The above9 bacterial phyla can account for about 98% of the total proportion,but the proportion of each phylum is different in different kinds of samples.Based on the analysis of the variance of the main bacteria phyla in different samples,we found that most of the samples were different between different months(P<0.05).But there were no significant differences between different lines.(4)Alpha diversity analysis results showed that there were no significant differences in bacterial community richness and diversity index among different months and different lines.However,there were significant differences among different sampling sites.The results showed that the bacterial community composition was not affected by the transgene.The results of Beta diversity analysis showed that the majority of samples in different months or lines were overlapped,indicating that the bacterial community structure in different months and different lines was similar.Further LDA Effect Size analysis also found that in June,there were some differences in the abundance of some species,and in August,the differences were gradually reduced,and there was no difference between the lines in October.It also shows that changes in abundance of individual species occur only temporarily,and the physical and chemical environmental factors of soil contribute greatly to the emergence of different species.Pearson correlation analysis was used to analyze the correlation between the alpha diversity index of bacteria and the physical and chemical properties of root soil.It was found that p H was positively correlated with Simpson index,but negatively correlated with Shannon index.There was a significant negative correlation between AK and Simpson index,and there was no significant correlation between the physicochemical factors and diversity index.AP has a greater contribution to community structure than other factors.(5)The chloroplast transformation vector of Bt Cry1 Ac gene was successfully constructed.The aad A gene was used as the screening marker,and rbc L gene and acc D gene,rps12/7 gene and ndh B gene were the homologous fragments of chloroplast genome of tobacco and poplar.Chloroplast transformation of tobacco and Populus × euramericana‘Neva’ was carried out by gene gun bombardment.After 3-4 rounds screening,the Bt gene was detected by PCR,and exogenous Bt gene was detected in 6 lines of tobacco and Populus × euramericana ‘Neva’,respectively.Bt Cry1 Ac toxicity test showed that the toxic protein content of T-4 was the highest in the 6 tobacco transgenic lines,which was 19.32ng/m L,and P1-1 was the highest in the 6 poplar transgenic lines,which was 6.48 ng/m L.However,the expression of foreign proteins was generally low,indicating that there are a large number of copies of the chloroplast genome of tobacco and Populus × euramericana‘Neva’ without homogenization.Based on the above research results,we analyzed from multi-perspectives and a long period.It showed that the planting of transgenic 741 poplars had no significant influence on the quantity and community composition of root microbial.This study could provide a more systematic understanding of the risk assessment of natural ecosystem after the release of transgenic 741 poplars.The research on chloroplast transgenic technology of poplar can also provide new research ideas for genetic modification of insect-resistant poplar with higher ecological security. |
PDF Full Text Request