Genetic Transformation And The Relevant Function Identification Of PnCCH10 Gene In Populus Simonii×Populus Nigra

At present,soil heavy metal pollution is increasingly serious due to vegetation utilization and human activities.Phytoremediation is an emerging technology to improve the soil environment contaminated by heavy metals.Copper is one of the main pollutants in soil heavy metal pollution,and it is also a trace element needed for plants to maintain normal life activities.In order to adapt to the level of copper in the environment,plants have formed a complex regulatory network to maintain cellular copper homeostasis,and copper chaperones are important players in achieving this mechanism.Based on the preliminary work of the research group,this study constructed a plant expression vector for the copper chaperone protein genes PnCCH5,PnCCH10 and PnCCH19 of Populus simonii × Populus nigra.The Arabidopsis thaliana wild-type plants and cch mutant plants were genetically transformed by the method of floral dip with PnCCH10 gene expression vector.And the PnCCH10 gene was transformed to P.simonii × P.nigra by Agrobacterium-mediated leaf disc method.Arabidopsis thaliana and P.simonii × P.nigra plants transformed with PnCCH10 gene were obtained,and related molecular biology and physiological indicators were identified.The main findings are as follows:(1)Use of cloned genes PnCCH5(MF977407.1),PnCCH10(MF977410.1)and PnCCH19(MF977413.1),the Arabidopsis overexpression vector pCAMBIA1302-PnCCHs was constructed by restriction enzyme ligation.After transformation of the Agrobacterium strain GV3101,an Agrobacterium strain containing the recombinant plasmid was obtained.At the same time,the P.simonii ×P.nigra overexpression vector pROKII-PnCCHs of PnCCH5,PnCCH10 and PnCCH19 genes was constructed,and the recombinant plasmid was obtained by electroporation into Agrobacterium strain EHA105.(2)The Arabidopsis thaliana overexpression vector constructed using the PnCCH10 gene was used to genetically transform Arabidopsis thaliana wild-type and cch mutant plants.Screening with hygromycin and obtaining T2 plants.Genomic DNA of two types of T2 transgenic plants was extracted,and 9 PnCCH10-WT transgenic lines and 10 PnCCH10-cch transgenic lines were obtained by PCR.The identified transgenic plants were collected and stored.(3)Using the PnCCH10 gene of P.simonii × P.nigra overexpression vector,the wild type plant was genetically transformed by Agrobacterium-mediated leaf disc method.Positive plants were obtained after screening using a medium containing kanamycin.Genomic DNA of positive transgenic plants was extracted and identified by PCR using specific detection primers,and 16 PnCCH10 transgenic Populus simonii lines were obtained.The expression of PnCCH10 gene in transgenic lines was identified by qRT-PCR method,and five transgenic lines with high expression of PnCCH10 gene were selected for physiological index determination.(4)The wild type and transgenic lines of P.simonii × P.nigra were treated with modified 1/2 Hoagland nutrient solution containing normal copper ions(0.5 pmol/L)and high concentration of copper ions(10 ?pmol/L)for 7 d.The malondialdehyde(MDA)content of the treated plants was measured,and physiological indexes such as superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)enzyme activity were measured.The results showed that under the normal concentration of copper ion treatment,the MDA content in the leaves of transgenic lines 2 and 13 was significantly lower than that in the wild type control plants,and the MDA content in the roots of transgenic lines 5 was significantly increased.Leaf and root SOD activity of each transgenic line was significantly increased;The POD enzyme activities of the leaves in the 3,5,7 and 13 transgenic lines and roots in the transgenic lines 2,5 and 13 were significantly reduced.The CAT activity of the leaves of the transgenic lines 13 and the roots of transgenic lines 3,5,7 and 13 were significantly increased.After excessive copper stress treatment,there was no significant change in MDA content and SOD activity of each PnCCH10 overexpressing transgenic line compared with wild type plants.Except that there was no significant difference in root POD activity of the transgenic lines 7,the POD activities of the leaves and roots of the other transgenic lines were significantly decreased.Except that there was no significant difference in the CAT activity of the leaves in transgenic lines 5,the CAT activity of the roots of each transgenic line was significantly decreased,and the CAT activity of the leaves was significantly increased.These studies laid the experimental foundation for obtaining the transformant material of P.simonii × P.nigra,further elucidating the function of poplar CCH copper chaperone protein and playing a role in maintaining cellular copper homeostasis.