| Soybean is an important food crop in China,with a long history of planting.Soybean protein is often used as an important source of plant protein,which is of great significance in food processing.The amino acids in soybean protein components are relatively average as a whole,but they are slightly deficient in sulfur-containing amino acids,which makes sulfur-containing amino acids one of the limiting factors for the nutritional quality of soybean protein.Recent studies have shown that there are intersections between methionine and cysteine synthesis pathway in plant aspartic acid family,in which CGS and SAT play an important role.In this experiment,soybean cystathionine gamma synthase GmCGS1 and soybean serine acetyltransferase GmSAT5 were cloned based on soybean Jilin 26.The following results were obtained:1.GmCGS1 and GmSAT5 genes were cloned from soybean Jilin 26.The CDS region of GmCGS1 gene is 1610 bp in length and encodes 536 amino acids with a molecular weight of 58.14 kDa.The CDS region of GmSAT5 gene is 861 BP in length and encodes 286 amino acids with a molecular weight of 30.36 kDa.2.The GmCGS1 and GmSAT5 genes were constructed on the overexpression vectors PCHF-3300 and PCHF-1301,and the above plasmids were transformed into Agrobacterium tumefaciens through the competence of EHA105 Agrobacterium tumefaciens.3.The positive and reverse interference fragments of GmCGS1 were constructed on the PFG-5941 interference vector,and the above plasmids were transformed into Agrobacterium tumefaciens through the competence of K599.4.PCHF-1301-GmCGS1 and PCHF-1301-GmSAT5 were successfully transformed into soybean hairy roots by soybean genetic transformation system and identified by GUS staining.Meanwhile,PFG-5941-GmCGS1 interference vector was transferred into soybean hairy roots and identified by fluorescence microscopy.5.Among the results of amino acid determination by applying methionine and threonine,applying 50 mg/L threonine and 100 mg/L methionine slightly increased the content of methionine in seeds,and the effect of applying methionine was slightly better.When methionine was applied at a higher concentration,such as 250 mg/L,there was no significant change in methionine concentration in seeds.The threonine content in seeds decreased slightly with the application of 50mg/L threonine,and further decreased when the concentration increased to 250mg/L.6.Among the results of determination of crude protein and oil content in the seeds of Williams 82 soybean with external application of amino acids,the protein content increased slightly when methionine and threonine were applied at 100 mg/L and 50 mg/L,respectively,while the effect on oil content was concentrated at low concentration.Under 50 mg/L,the oil content in the seeds was slightly reduced by external application of methionine.The application of threonine decreased oil content at 150 mg/L concentration.Overexpression of GmCGS1 gene had the highest lysine content in hairy root samples.Overexpression of GmSAT5 and interference of GmCGS1 detected alanine in hairy root samples,and the content of GmSAT5 was slightly higher.In all over-expressed and interfered hairroot samples,lysine content increased significantly,but phenylalanine content did not change significantly.7.The plant height,stem diameter,pod number per plant,grain number per plant,100-grain weight,grain weight per plant,node number of main stem and branch number of the external application of amino acid harvesting materials were analyzed.The results showed that the external application of 50mg/L threonine had a significant promoting effect on grain number per plant;the external application of 100mg/L methionine had a significant promoting effect on 100-grain weight and grain weight per plant.8.The interaction of the two genes in the aspartic acid metabolic pathway is not significant.External application of 100mg/L methionine could promote the expression of GmCGS1 to some extent.External application of 50mg/L threonine slightly promoted the expression of GmSAT5. |
PDF Full Text Request