Salty Response And Biosafety Of AhDREB1 Transgenic Hybrid Poplar

In order to enhance the utilization of salinity land and satisfy the demand of timber,it is important to cultivate some new populus species that could normally afforested on salinity lands.Because traditional breeding methods usually time-consuming and low-efficiency,genetic engineering has become a potential breeding method for its high efficiency and relative low-time consuming compared with traditional breeding.In previous study,we heterologous expressed AhDREB1 transcription factor into hybrid Populus tomentosa 401 and increased the salty resistance of host plants.To further enhance the salty resistance of AhDREB1 transgenic Populus and clarify whether AhDREB1 would cause any unintended effects or if AhDREB1 transgenic Populus has a long-term stability and biosafety in field condition,we investigated the physiology and biochemistry as well as transcriptomic changes of transgenic and non-transgenic controls under different levels of salty and found the functional stress toleranced genes that regulated by AhDREBl;and in order to further enhance the salty tolerance ability of host poplar,we have cloned three salinity resistance related genes:TaMnSOD?TaLea from Tamarisk and A tNHX from Arabidopsis with different salty toleranced functions.Then,we constructed a plant multi-genes transformation vector containing the three cloned genes as well as the AhDREB1 transcription factor and genetic transferred to poplar(P.alba×P.glandulosa cv.84K).We also made a multi-years assessment of the environmental impacts and foreign gene stability of AhDREBl transgenic populus in the field condition.Our study made a reference for salty tolerance genetic engineering breeding in poplar and have great theory and applied value.The main results were showed below:(1)The transformation of AhDREB1 significantly increased the salt resistance of poplars POD activity?SOD activity?proline content and relieve the photosynthesis and growth suppression by salt.Through comparing the transcriptome between transgenic and non-transgenic seedlings,we totally found 5 1 differential expressed genes that associated to stress responsive,including 10 transcription factors and 41 functional genes.The functional genes including resistance proteins?oxidase or oxygenase?stress proteins and so on.(2)Under non-stress condition,only 52 differential expressed genes were found,and a few genes were associated to photosynthesis or lignin synthesis,no significant unintended effects were found,while under stress condition,several differential expressed genes were associated to lignin synthesis and flower development,which indicated that foreign may influence the wood property and reproduction of host plants.(3)Through specific amplification,we successfully cloned TaMnSOD?TaLea from Tamarisk and AtNHX from Arabidopsis,then we constructed a plant multi-genes transformation vector containing the three cloned genes and an AhDREBl transcription factor cloned from Atriplex hortensis.All four genes were drived by the promoter of Arabidopsis RD29a gene(4)The foreign gene was still in the genome of recipient plants and can still expressed in all three organs.However,the transcriptional level were significantly lower than previous study,and the foreign gene transcriptional level of seedlings that re-tissue cultured form the field were also significantly lower than tissue cultured seedlings conserved in our lab for the same time.In addition,part of the stress resistance genes were significantly down-regulated in re-tissue cultured seedlings.(5)No significant differences in the number of soil microbes present were detected between the transgenic lines and the non-transgenic controls.The allelopathic activity of leaves from both the transgenic and non-transgenic lines varied with sampling time,but did not differ significantly between the transgenic and non-transgenic lines.These results indicated that the impacts on the environment of AhDREBl transgenic P.tomentosa did not differ significantly from that of the non-transformed controls for the variables observed in field trial.We also investigated the persistence of AhDREB1 gene in decomposing transgenic poplar leaves on the soil under natural conditions for successive five months,and our results showed that no fragments of the genetically modified DNA were detected after the leaves degrading for more than two months.We used a TTC test(or pollen germination method)and hybridisation to test the pollen viability and fertility,respectively,of the transgenic and non-transgenic trees and the results showed that the pollen viability of both the transgenic and non-transgenic trees was extremely low in 2016,the receptor plant may have been sterile.In a word,we made a preliminary study on the mechanism that AhDREB1 improved the salty resistance of poplar and tried to make a further improvement;we also studied the unintended effects and the long-term field safety of AhDREBl poplar,which made a reference for the transgenic breeding of poplar in the future.