Functional Analysis Of Resistance To Acid Aluminum Of GsRZFP1 In Wild Soybean (Glycine Soja)

Aluminum?Al?toxicity is one of the main factors that limits crop growth on acidic soils,which inhibits the absorption of water and nutrients on roots and thus affects the crop yield.In China,acidic soils are widely distributed in multiple provinces,particularly in the region of Southern China.Wild soybean?Glycine soja Sieb.and Zucc.?,the ancestor of soybean,has strongly resistance to adversity owing to long-term growth in adverse environments.In the early stage of this research,a total of 205 soybean materials were screened for Al-tolerant,and a wild soybean?BW69?were identified as an Al-tolerant variety.Therefore,BW69 was taken as the object of this study.All of the experimental results as follows:1.GsRZFP1?NO.100783915?was detected from the expressed profile for Al-resistant genes of Glycine soja,with a full-length sequence of 1600 bp and open reading frame length of 927 bp,which encodes 308 amino acid residues.Analysis of the protein domain revealed that GsRZFP1 protein contained three domains,concluding zf-CHY domain(65^th-145^thh amino acids),RING domain(200^th-244^th amino acids)and zinc-ribbon-6 domain(248^th-308^th amino acids).Besides,GsRZFP1 also contains a RING finger domain which belongs to the RING finger protein family.2.With the help of recombinant DNA technology,the recombinant expressed vector pCAMBIA1302-GsRZFP1 was constructed by inserting the target gene into the pCAMBIA1302 vector.Then,the recombinant vector were transformed into tobacco for subcellular localization analysis by Agrobacterium injection method.The result revealed that GsRZFP1 protein was found to be transgently expressed in the nucleus.3.Likewise,the plant expression vector pCAMBIA1301-GsRZFP1 was constructed.Afterward,heterologously transformed to Arabidopsis thaliana,positive seedlings of T₁generation were obtained through hygromycin screening.The result of DNA sequence revealed that GsRZFP1 successfully integrated into the Arabidopsis genome.Furthermore,RT-qPCR analysis indicated that GsRZFP1 is overexpressed in different Arabidopsis thaliana.After treated with AlCl₃ the relative root length of GsRZFP1 transgenic Arabidopsis thaliana was significantly higher than that of the wild type,suggesting that the Aluminum resistance of the transgenic Arabidopsis thaliana is more significantly than the wild type.4.The content of proline and malondialdehyde?MDA?in root were measured both in the wild type and GsRZFP1 transgenic Arabidopsis.As a result,Proline content was highly increased under 50?M AlCl₃ treatment with two times folds,while that was almost the same in the absence of AlCl₃,respectively.Coincidently,similar trend was found for MDA concentration in wild type and transgenic linesafter treated with 50?M AlCl₃.In addition,the increase of MDA in wild type plants was more signficant than the GsRZFP1 transgenic Arabidopsis thaliana,showed a more extremely damage on cells.In short,the results of this study indicated that the overexpression of GsRZFP1 enhanced the tolerance of transgenic Arabidopsis thaliana to acid aluminum stress.