Effect Of Sulfate,methionine And Light On The Expression Of GmCGS And Creating Of Transgenic Soybean Overexpressing β-zein

Soybean（Glycine max（L.）Merr.）is acrucial economic crop with high protein and lipid feed for human and livestock all over the world.Although the soybean possessed high protein content,the quality of soybean storage protein suffers from a low content of sulfur-containing amino acids,methionine（Met）and cysteine（Cys）.Despite several soybean materials with high content of Met were created,those materials still hard to meet the Met requirement for feeding.Thus,to improve soybean nutritional quality and to supply superior protein sources for human and livestock,a high level increasing of Met content is needed.In this study,we investigated the transcription pattern of GmCGSin soybean and created transgenic lines overexpressingβ-Zein and AtDCGSin soybean c.v.Jack,respectively,both of which were also used as crossing parentsto obtain progenies co-expressingβ-Zein and AtDCGS.The transcriptional level of GmCGS was detected in transgenic lines overexpressing the Met-insensitive form of Arabidopsis cystathionineγ-synthase（AtDCGS）and wild type（WT）soybean c.v.Jack after sulfate and methionine treatment to identify their effect on regulating the expression of GmCGS.At the stage V₄ and R₆,above materials were treated with sulfate（0 mM,1.5mM,4.5mM）,methionine（2.5 mM,5.0 mM）and purified water（CK）.Compared to WT,the transcriptional level of GmCGS was significantly decreased in transgenic lines,which indicated that the expression of GmCGS was likely inhibited after introducing exogenous gene AtDCGS.The relative expression level of GmCGS in WT was significantly increased aftertreated with 4.5 mM sulfate,whereasthe transcription level of GmCGSin transgenic lines was significantly decreased.No significantly expression difference was observed among transgenic lines,wild types and CKaftermethioninetreatment.To analyze the effect of light on the expression of GmCGS,the soybean materials were grown in the 12 h light/12 h darkness or 0 hlight/24 h darkness condition at 25°C.The expression level of GmCGS was consistently lower under consistent darkness.However,the expression of GmCGS was sharply increased once after exposuring under the light and reached to the peak at the 2 h light point,after that it was consistently decreased.The results indicated that the expression of GmCGS was triggered by light while the effect could not last for long.We acquired transgenic soybean overexpressingβ-Zein and AtDCGSboth in a constitutive manner and in a seed-specific manner.As for the transgenic lines overexpressingβ-Zein,phosphinothricin-resistance assay,PCR,RT-PCR were performed to select the positive transgenic lines.So far,there were 65 lines overexpressingβ-Zein in a constitutive manner derived from 10 T₀ plants and84 lines overexpressingβ-Zein in a seed-specific manner derived from 11 T₀ plants.One transgenic lineoverexpressingβ-Zein in aseed-specific manner was screened out forits highest Met content,which the Met content increased 14.8%compared to WT.To the transgenic lines overexpressing AtDCGS,glyphosate-resistance assay,PCR,RT-PCR were performed.There were 26 lines overexpressing AtDCGS in a constitutive manner derived from 4 T₀ plants and 33 lines overexpressing AtDCGS in a seed-specific manner derived from 13 T₀ plants.All of these materials make it possible to obtain plants overexpressing two key genes simultaneously by crossing.