The Growth And Ecological Safety Evaluation Of Multiresistant Transgenic Populus × Euramericana Cv. ’74/76’

Populus is an important forest tree species,and plays a huge ecological and economical role in the world.It is the first choice among woody model plants due to its advantages of rapid growth and simple genome.Insect pests and salinity are important factors restricting poplar yield in China.With the development of transgenic technology,a number of poplar varieties with resistance genes have been successfully transformed and entered the stage of environmental release,productive testing and commercial application.Since poplar is a perennial woody plant,its generation cycle is much higher than that of crops,the unintended effects and ecological safety of transgenic poplar have become the focus of attention.The poplars carrying CrylAc-Cry3A-BADH genes and CrylAc-Cry3A-NTHK1 genes have good insect resistance and certain salinity and alkali tolerance through indoor feeding insects and pot experiments.In order to further evaluate the field resistance,growth performance and ecological safety of transgenic poplars,comparative experimental forests were planted in Baoding,Cangzhou and Tangshan,Hebei Province.Using ecological methods,the field insect resistance and possible ecological safety of multiresistant transgenic Populus x euramericana cv.’74/76’(transgenic 107 poplar)were analyzed,including the impact on arthropod community,rhizosphere and endophytic microorganisms,in order to provide scientific basis for the safe and rational application of multiresistant transgenic 107 poplar.The main findings are as follows:(1)The leaves of six transgenic lines grown in the field were subjected to PCR amplification,and detection of Bt toxin proteins in long branches,short branches,phloem and roots.It was confirmed that the foreign gene was stably present in the transgenic lines,and the Bt toxin proteins could be expression throughout the growing seasons,the leaf toxin protein content was the highest,and the root was the lowest.The average expression of Cry3A toxin protein(8931.75 μg/g)was much higher than that of Cry1Ac toxin protein(71.55 μg/g),and the content of toxin protein in long and short branche leaves in August was higher than that in June and October,but the root and phloem did not significant seasonal changes.Based on NGS sequencing technology,seven integration sites of T-DNA were detected in six transgenic lines,of which the B3 line was double-site integration,and the T-DNA integrated in genome of other lines were single copies.A total of 21 genes were found within 20 kb upstream and downstream of the insertion site.The T-DNAs of B1,B2 and B3 lines(chromosome 6)were integrated into the gene region,and the rest of the lines were integrated into the intergenic region.No significant changes in the expression of the insertion site and its adjacent genes were found by transcriptome sequencing.Analysis of the base sequence near the integration site showed that the content of AT bases within 1000bp above and below the 7 insertion sites was higher than the average content of chromosomes.(2)The growth of transgenic 107 poplars was evaluated in the fields of Mancheng,Yanshan and Luannan experimental forest.The results showed that the height and DBH yield of transgenic 107 poplar were significantly lower than those of transgenic acceptor 107 poplar,and the adaptability of each line in different forest was significantly different,as follows:Mancheng>Luannan>Yanshan,there is an interaction effect between the growth yield of each line and the test site.Transcriptome analysis of the two transgenic lines and the control leaves showed that a total of 944 DEGs were identified in the A1 and B1,including 575 in the A1 line and 678 in the B1 line,including 43 and 59 transcription factors,respectively.Most transcription factors belong to bHLH,WRKY,ERF families,and the expression trend is mainly up-regulated.The GO functional enrichment results of DEGs in A1 and B1 lines are similar.DEGs are mainly enriched in metabolic processes,cellular processes and single-organism processes in biological processes,catalytic activity and binding in molecular function,and cells,cellular part,and membrane in celluclar compoment.The results of DEGs enrichment in KEGG pathway showed that A1 line had 131 DGEs enriched in 90 metabolic pathways,B1 line had 141 DGEs enriched in 84 metabolic pathways,of which 68 pathways were both enriched in A1 and B1 lines,including plant hormone signal transduction,phenylpropane biosynthesis,zeatin synthesis and other important pathways.In the above three pathways,13 and 19 genes were screened in the A1 and B1 lines respectivly,and the expression levels were significantly changed,mainly related to hormone and lignin synthesis-related genes.Among which the primary auxin-responsive gene(CH3),Cytokinin dehydrogenase(CKX),shikimic acid/quinic acid hydroxyl cinnamoyl transferase(HCT)and peroxidases synthesized from four lignin monomers were co-enriched and expressed in A1 and B1 lines.These genes have the same expression trend in A1 and B1 lines,indicating that the differential expression of some phytohormone and lignin synthesis-related genes caused the slow growth of height and DBH of the transgenic lines.(3)The arthropod communities of transgenic poplar 107 in three experimental forests were investigate,and 10,485,5,538 and 13,098 arthropods were counted in Mancheng,Yanshan and Luannan forests.The three experimental forests have similar arthropod species.All were dominated by lepidopteran insects.The target insects Lepidoptera occurred the most in Luannan,accounting for 45.83%of the total community,and Coleoptera occurred the most in Yanshan,accounting for 7.67%of the total community.The transgenic lines of all the three experimental forests had a certain inhibitory effect on Lepidoptera and Coleoptera insects,and had insect resistance selectivity,but had no obvious inhibitory or proliferation effect on natural enemies and neutral insect populations.The niche breadth and overlap analysis of functional groups showed that,compared with non-transgenic 107 poplar,the natural enemies in the transgenic 107 poplar arthropod community had a stronger following effect on pests,and the predation relationship was intense.The introduction of insect-resistant genes increased the time and double dimension niche breadth of lepidopteran pests;The trends of the number of arthropod species,diversity index,uniformity index and dominance concentration index in the field of transgenic 107 poplar and non-transgenic 107 poplar were basically consistent with seasons changed.When the outbreak of lepidopteran insects in the community is severe,the community diversity and evenness of the transgenic lines are higher,the dominance index is lower,and the arthropod community is more stable.Compared to no-transgenic 107 poplar,the functional group structure and community stability index of transgenic 107 poplar were changed,the relative abundance of phytophagous insects in the transgenic community decreased,and the proportion of neutral insects and predatory natural enemies increased.In terms of the similarity of community structure,the arthropod communities of the forests in different regions were significantly different,and the arthropod communities of the transgenic lines and the control were highly similar.(4)From 2018 to 2020,the Mancheng experimental forest counted 10,485,15,077 and 8,930 arthropods respectively.The composition of each year was roughly similar,and Lepidoptera or Hymenoptera dominated.The number of target Lepidoptera insects was stable,and Coleoptera was increasing.Except in 2019,the transgenic lines had better control effects on Lepidoptera and Coleoptera,and had no persistent adverse effects on non-target insect populations.The niche breadth and overlap analysis showed that the natural enemies in the entire arthropod community in the experimental forest had a good following effect on the pests,and the transgenic lines had wider time and double dimension niches than the control lepidopteran pests(except in 2019).The trend of arthropod structural characteristic index changes with time is basically the same.In some seasons,the diversity index and evenness index of transgenic lines were higher,and the dominant index was lower,that is,the stability of transgenic lines was stronger.Community functional group structure analysis showed that the relative ratio of predatory natural enemies to pests in the transgenic lines was higher,and the ability of natural pest control of the community was stronger.The community structure differences between the transgenic lines and non-transgenic lines were smaller than that of different years.(5)The analysis of soil physicochemical properties of transgenic 107 poplar lines showed that most of the rhizosphere soil physicochemical factors were significantly differ between different seasons,but there was no significant difference between transgenic 107 poplar and acceptor 107 poplar.Sequencing analysis and identification of bacterial 16S rDNA V5-V7 region showed that there were no significant difference between transgenic 107 poplar rhizosphere,rhizosphere and phloem bacteria compared with acceptor recipient 107 poplar.The bacterial community of transgenic 107 poplar in the rhizosphere soil was dominated by Proteobacteria,Acidobacteria and Firmicutes,accounting for 85%of the total rhizosphere soil community,in the root was dominated by Proteobacteria,which alone accounted for 95%of the total root community,and in the was phloem dominated by Proteobacteria and Firmicutes,which accounted for 97%in the total phloem community.The difference in phyla abundance was the largest in different parts grouping,followed by different season groupings,and that was the smallest between transgenic lines and controls was.The bacterial community richness index(Chaol index),diversity index(Shannon index),lineage diversity(PD-whole tree index)and evenness index(Peilou index)of rhizosphere soil were higher than those of root and phloem,and the difference between root system and phloem was significant(the difference was relatively small).The bacterial community structure was significantly different form that of roots and phloem,but the community structure was similar between roots and phloem.There was no significant difference in each characteristic index,and the community structure was similar between different seasons and different lines.In addition,the abundance of nitrogen-fixing bacteria,nitrifying bacteria,denitrifying bacteria,and hydrogen-oxidizing bacteria in the rhizosphere and endogenous samples of Populus 107 was not affected by the transgene.The correlation analysis between soil physical and chemical properties and bacterial community showed that the rhizosphere bacterial community was significantly affected by soluble salts,total salts,pH value and alkali-hydrolyzed nitrogen factors,while the root and phloem bacterial community had a low correlation with various soil factors,and only a few factors had a significant correlation with a few low abundance bacteria phyla.(6)Sequencing analysis and identification of fungal ITS2 region showed that there was no significant difference of rhizosphere,root and phloem fungal community between transgenic and acceptor 107 poplars.In the total fungal community composition was dominated by Ascomycota and Mucoromycota,among which Mucormycota mainly existed in the endophytic samples.The greatest differences were found among different parts in abundance,and there were also great differences among different seasons and different lines.The community richness index(Chaol index),diversity index(Shannon index),lineage diversity(PD-whole tree index)and evenness index(Peilou index)of the rhizosphere soil were higher than those of root and phloem,and no significant differences were found between seasons and different line groups.Cluster analysis and principal component analysis showed that the fungal community structure of rhizosphere soil was significantly different from that of roots and phloem.The roots and phloem were close to each other,but the community structure of rhizosphere soil was also significantly different.Fungal communities could not be clearly distinguished between different seasons and different lines.Pathotrophic fungi were dominant in all samples,and the most funguilds were found in the roots.There were no significant differences in the trophic type and funguilds of fungi among different seasons and different lines.The rhizosphere soil fungal community was mainly affected by pH value and hydrolytic nitrogen factors,the root community had little correlation with soil physical and chemical properties,and the phloem community was mainly affected by total salt,soluble salt and hydrolytic nitrogen factors.