Creation Of New Genetically Modified Soybean Germplasm With Seed-specific Expression Of SiDGAT1 Gene

Soybean is an important oil crop in the world,and soybean oil demand ranks first among edible vegetable oils.Breeding new soybean varieties with high oil content is one of the effective ways to solve the problem of insufficient soybean oil demand.Traditional breeding methods h ave low efficiency and long time,and are limited by germplasm resources.With the development of science and technology,genetically modified technology is gradually emerging.This technology can obtain target traits by changing plant DNA seuquences.The combination of transgenic technology and traditional breeding methods can effectively promote the development of agricultural breeding technology.There are many existing methods of soybean genetic transformation,but the most commonly used method is the Agrobacterium-mediated method,which has the advantages of short time-cycle,easy operation,high transformation efficiency and low copy number.Soybean transformation efficiency is low and it is greatly affected by genotype,which makes soybean genetic transformation relatively difficult and restricts the development of soybean transgenic breeding to a certain extent.Diacylglycerol acyltransferase（DGAT）is the rate-limiting enzyme in the synthesis of triacylglycerol（TAG）,which plays a key role in controlling the terminal steps of triacylglycerol synthesis in plant seeds.The Si DGAT1 gene encodes diglyceryl acyltransferase of sesame,with a full-length c DNA of 1623 bp.The previous research of our team proved that the Ca MV35S constitutive promoter was used to drive the gene expression in Arabidopsis and soybean,and the seed oil content of overexpressed Si DGAT1plants was significantly increased.The seed-specific promoter can specifically express the target gene in plant seeds,so as to avoid the waste of energy caused by the over-expression of the target gene in all theorgans of the transgenic plant,and may adversely affect the growth and development of the plant.In this study,we constructed PBA002-P8-Si DGAT1 plant seed-specific expression vector.The Si DGAT1 gene was transferred into Dongnong 50 and Kennong 18respectively by using Agrobacterium-mediated genetic transformation of soybean cotyledonary node.Theresearch results are as follows:（1）We constructed theplant seed-specific expression vector PBA002-P8-Si DGAT1.（2）Using Agrobacterium-mediated genetic transformation of soybean cotyledonary node,Si DGAT1 gene was transformed into soybean cultivars Dongnong 50 and Kennong 18.Atotal of 12T₀ transgenic soybean plants were obtained,and fourpositive transgene plants with DN50 as the receptor,eight positiveplants with K18 as the receptor.（3）There were 4 T₁generation transgenic soybean lines（K18-4-1,K18-5-1,K18-6-1,D50-7-1）were identified by using 130 mg/L glufosinate resistance identification,PCR amplification,Western blot,et al.Then,the q RT-PCR method was used to detect the expression level of the Si DGAT1 in different tissues and organs of T₂ generationtransgenic soybeans lines.The results showed that Si DGAT1 had the highest expression level in transgenic seeds.（4）The oil content of T₁ generation transgenic soybean lines and Kennong 18 was measured by near-infrared grain analyzer.The results showed that the overexpression of Si DGAT1 gene could increase the oil content of transgenic lines compared with control Kennong 18.And,it was found that the overexpression of Si DGAT1 gene could affect the seed size of transgenic lines.