| Heavy metal contamination in soil is a global problem,seriously affecting human health,crop yield and ecological security.Phytoremediation is an environmentally friendly soil remediation technology that can improve heavy metal contamination in soil.Phytoextraction in phytoremediation,in which plants absorb heavy metals and transport them to the aerial parts of the plant tissues,is the most environmentally friendly method to reduce the total amount of heavy metals in soil,and has broad application prospects.Hyperaccumulators or highaccumulating transgenic plants with biomass are important biological materials for phytoextraction.The scientific efforts and practices of phytoremediation in recent years have shown that the creation of hyperaccumulators by transgenic technology targeting the HMA and Nramp transporter families is a powerful approach for phytoremediation of heavy metalcontaminated soils.In previous studies,three different metal transporters SpHMA2,SpHMA3 and SpNramp6 from the hyperaccumulator Sedum pumbizincicola were confirmed to have Cd transport capacity.SpHMA2 and SpNramp6 are located on the plasma membrane,and SpHMA3 is located on the tonoplast.Treatment of heavy metals Cd,Pb,Cu,Zn and Mn can all strongly stimulate their expression.In order to create potential plant extract biomaterials,our laboratory overexpressed three single genes SpHMA2,SpHMA3 and SpNramp6,and two combined genes SpHMA2&SpHMA3 and SpHMA2&SpNramp6 in K407 rape with high biomass and environmental adaptability,and found that the ability of these transgenic engineered rapes to transport and accumulate Cd in the aerial part was superior to that of the wild-type at the seedling stage.Furthermore,the accumulation of each heavy metal was enhanced in the aerial parts of each line of transgenic rape in mixed Cd,Pb,Cu,Zn,and Mn heavy metal-contaminated soils.These results provide an important basis for the successful use of SpHMA2,SpHMA3 and SpNramp6 transgentic engineered rapeseed as phytoextraction biomaterial for Cd and various heavy metal contaminated soil.Based on the above research results,the genetic stability and environmental safety of transgenic rapeseed with SpHMA2,SpHMA3 and SpNramp6 were preliminarily analyzed by combining molecular and physiological tests.The specific research results obtained are as follows:1.The target genes of SpHMA2,SpHMA3 and SpNramp6 transgentic engineered rape has integrated genetic stability.Through specific PCR technology,the genetic material of T1-T3 generation of each line of transgenic engineering rapeseed was detected for the main functional elements SpHMA2,SpHMA3 and SpNramp6 genes.The results of agarose gel electrophoresis showed that there were obvious bands in the corresponding bp length of SpHMA2,SpHMA3 and SpNramp6 genes.In addition,the flanking sequences on the left and right sides of the T-DNA insertion position of the transgenic rapeseed genome were amplified by TAIL-PCR technology,and the amplified fragments were sequenced and analyzed by highthroughput sequencing technology and BLAST technology.The base sequence alignment of the rapeseed genome has successfully found the insertion position,so it can be seen that the exogenous genes SpHMA2,SpHMA3 and SpNramp6 have been successfully integrated into the rapeseed genome and stably inherited of multiple generation.2.The target genes of SpHMA2,SpHMA3 and SpNramp6 transgenic engineered rape has genetic stability of expression.The relative expression levels of exogenous genes SpHMA2,SpHMA3 and SpNramp6 in T1-T3 transgenic rapeseed were determined by realtime quantitative PCR technology.The results showed that SpHMA2,SpHMA3 and SpNramp6 genes were expressed tens to thousands of times in each line of transgenic rapes in three generations,indicating that these genes were expressed stably.3.The target genes of SpHMA2,SpHMA3 and SpNramp6 transgenic engineered rape has the stability of Cd and other heavy metals adsorption.The Cd content per unit weight of each line of the wild-type and T2 generation transgenic rapeseed after high-concentration Cd treatment was measured by atomic spectrophotometer.The SpHMA2-OE1/OE3、SpHMA3-OE33/OE34、SpNramp6-OE1、SpHMA2&SpHMA3-OE2 和 SpHMA2&SpNramp6-OE4/OE5 strains had better ability to adsorb or transport Cd in the aerial part.The same method was used to measure the Cd content per unit weight of the aerial parts of the wild-type and T2 generation transgenic rapeseed lines treated with low concentrations of Cd at the flowering stage.The fluorescence intensity of the roots,stems and leaves of wild-type and T3 generation transgenic rapeseed after Cd treatment was observed by confocal laser,and the fluorescence intensity of the aerial part of transgenic rapeseed was higher,which intuitively indicated that transgenic rapes would transport Cd upwards.FE-SEM and EDAX instrument were used to analyze the content of heavy metals in the cross-sections of roots,stems and leaves of wildtype and T3 generation transgenic rapeseed treated with mixed heavy metals.It was found that the content of transgenic rapeseed was higher in stems and leaves demonstrating a synergistic transport of Cd and multiple heavy metals.The results indicated that these transgenic rapeseeds had stable adsorption properties of heavy metals such as Cd.4.The survival competitiveness of SpHMA2,SpHMA3 and SpNramp6 transgenic rapes is the same as that of the wild type.By measuring and evaluating the seed germination rate and emergence rate,pollen vigor and other comprehensive agronomic traits of wild-type and transgenic rapeseed,the dissemination mode and ability of SpHMA2,SpHMA3 and SpNramp6 transgenic engineered rapeseed in polluted soil,as well as comprehensive agronomic traits were found.Traits are indistinguishable from wild type.It indicated that the transgenic process did not affect the survival competitiveness of the engineered rapeseed.5.The target genes of SpHMA2,SpHMA3 and SpNramp6 transgenic rapes have no gene flow and are environmentally safe.Through specific PCR detection technology,the genetic material of weeds around 3km of transgenic rape field in full bloom stage was detected for the target genes SpHMA2,SpHMA3 and SpNramp6.The results of agarose gel electrophoresis showed that there was no target band in the corresponding bp length of SpHMA2,SpHMA3 and SpNramp6 genes,indicating that the target genes of transgenic rapeseed were less likely to be transferred to other plants.6.SpHMA2,SpHMA3 and SpNramp6 transgenic rapes with did not affect the diversity of field organisms.SpHMA2,SpHMA3 and SpNramp6 transgenic rapeseed did not affect the organism diversity of the field.Records of the number and species of insects and the quality and quantity of weeds in the farmland around the transgenic rapeseed is no different from that of the wild type.It is preliminarily judged that the genetically engineered rapeseed will not change the population and quantity of field organisms,further explain their environmental safety.In summary,it was researched that the exogenous genes SpHMA2,SpHMA3 and SpNramp6 have been stably integrated into the K407 rapeseed genome,which could stably and effectively transport and adsorb Cd and various mixed heavy metals.In terms of environmental safety,the transmission mode and transmission ability of SpHMA2,SpHMA3 and SpNramp6 transgenic engineered rapeseed did not change,and their growth status,life cycle and adaptability did not change,and their genetic material was more likely to be transferred to other plants.Small,and will not change the population and number of field organisms.Therefore,the SpHMA2,SpHMA3 and SpNramp6 transgenic engineered rapeseeds are genetically stable and environmentally friendly,and can be promoted as phytoremediation materials under heavy metal pollution in soil. |
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