Cloning And Function Analysis Of Gene GmPLMT In Soybean

Phospholipids are is the basic substance of life,is an important component of human and animal and plant tissue cell membrane,is and an indispensable substance to normal metabolism and healthy survival of the human body.And phosphatidylcholine is important metabolites of plant growth and development and is also the main lipid component of plant cell membrane.Soybean phosphatidyl transferase(GmPLMT)is a key enzyme in lecithin synthesis which exists in the synthetic route of soybean phosphatidylcholine.In order to find out the function of the GmPLMT in the soybean,this study takes Jiyu No.72 soybean as test materials.GmPLMT gene was cloned and the plant expression vector was constructed.Then the genetic transformation of arabidopsis thaliana and soybean was carried out to obtain the transgenic plants respectively.Meanwhile,the amount of gene expression and biochemical index related to lipid metabolism were determined.The results were as follows:1: By using sequence in NCBI to design primers,the GmPLMT gene was cloned.The length of the gene obtained is 504 bp,which includes a 504 bp ORF.2: The plant expression vector pCAMBIA3301-GmPLMT was constructed and transformed into arabidopsis thaliana and soybean by the method of agrobacterium-mediated transformation.By screening Using grass butyl phosphonic herbicides,and blotting PCR and southern blotting to commonly verify,it was proved that a lot of arabidopsis thaliana of T3 generation and 1 transgenic soybean of T1 generation were achieved and GmPLMT gene in the form of a single copy was integrated into the soybean and arabidopsis thaliana genome.3:By using the method of Fluorescence quantitative PCR to determine the amount of gene expression of GmPLMT during different growth period of soybean,the amount of gene expression of GmPLMT in the root of the mature stage of soybean was highest,reaching 0.66,which is 7.4 times as much as that of the same period in blade,indicating that GmPLMT gene is mainly expressed in the root in mature soybean.The variation of the amount of gene expression of GmPLMT in soybean with different maturity was increased first and then decreased.The amount of the gene expression of the first sample was 0.061,and then it gradually decreased to 0.004 of the fourth sample,which is also the lowest value of the whole soybean ripening process.After that,it gradually increased,and the content decreased rapidly to 0.061 after the eighth maximum of 0.25.It can be seen that the amount of gene expression of GmPLMT is dynamic during the gradual maturation of soybean seeds.In order to study the changing relationship between GmPLMT gene and lipid metabolism in maturation process,,samples were taken every 5 days and sampled 9 times totally in the process of soybean seed maturation.The results were as follows: phospholipids in soybean seeds,fatty acid content were determined.Through correlation analysis,it was found that GmPLMT gene was positively correlated with phosphatidyl choline in soybean seeds of different maturity and the correlation coefficient is 0.5.The content of phosphatidyl choline was positively correlated with linoleic acid and linolenic acid,and the correlation coefficient was 0.74 and 0.82 respectively.It can be seen that the amount of gene expression of GmPLMT can directly increase the content of phosphatidyl choline and fat in soybean seeds,and indirectly increase the content of polyunsaturated fatty acids in soybean seeds.4: By using the method of Fluorescence quantitative PCR to determine the amount of gene expression of GmPLMT during different growth period of arabidopsis thaliana of T3 generation,the amount of gene expression of GmPLMT was highest in the flowering period,reaching 0.139.And in the mature period the amount of gene expression of GmPLMT was lowest,only 0.0054,which is 38.8% as much as that in the flowering period.It is shown that the amount of gene expression of GmPLMT of flowering period in the transgenic T3 generation of arabidopsis thaliana is the highest.By using variety of biochemical methods to determine the phospholipids(phosphatidylcholine,phosphatidyl ethanolamine,phosphatidyl inositol),fatty acid content and crude fat content in the seeds of transgenic arabidopsis thaliana and taking wild type arabidopsis thaliana as a control,the contents of total phosphorus,phosphatidyl choline and phosphatidyl ethanolamine in transgenic arabidopsis were increased by 2.02 times,2.09 times and 1.36 times respectively.The content of linoleic acid and linolenic acid were increased by 4.51% and 3.39% respectively.The results were consistent with the correlation analysis in soybean.It can be seen that the transfer of GmPLMT gene can not only increase the content of phosphatidyl choline substantially and total phosphorus but also increase the content of polyunsaturated fatty acid in plant seeds.