Vectors Construction And Their Transformation Of Peanut Aiming To Improve Seed A-tocopherol Component

Peanut(Arachis hypogaea L.)is the world's fourth largest oilseed crop grown mainly in subtropical and tropical regions.China is the largest country of peanut production and consumption in the world.Vitamin E is an important class of lipid-soluble compounds with antioxidant activities that plays a very important role in plant,animal and human being.Plant oil is the main resource of the vitamin E in human nutritious.The content of activatedα-tocopherol is very low in plant,including peanut.To improve the rate of vitamin E component in peanut,we have tried to improve peanut vitamin E content by employing gene engineering.We reported here the cloning of homogentisic acid prenyltransferase(VE₂),2-methyl-6-phytylbenzoquinol(VE₃)andα-tocopherol methyltransferase(VE₄)genes,embryo-specific expression vectors construction and their transformation.To make vitamin E genes express specifically in peanut embryo,two promoters already cloned in T-vectors were isolated with 99.8%and 100%identity to the sequences interested on GenBank,respectively.They are 7S seed storage protein gene promoter of soybean and 2S seed storage protein gene promoter of Brassica napus.The promoters region were 795bps and 1058bps in length.Extracting total RNA from leaves of Arabidopsis thaliana and designing the primers according to the published sequence of VE₂,VE₃ and VE₄ genes on GenBank, we obtained those three genes,with 97%,100%and 100%identity to the conterpart sequences on GenBank,respectively,by RT-PCR.Those three genes were first constructed on intermediate expressing vector pSPROK with 7S promoter upstream to VE2 gene and 2S promoter upstream to VE3 and VE4 genes,respectively.Then the intact expression units with genes of interest flanked by specific promoters and nos terminals were cut and transferred to plant expressing vector pCAMBIA1300.Three plant expression vectors,named pCAMBIA1300VE2.7S,pCAMBIA1300VE3.2S and pCAMBIA1300VE4.2S,were obtained afterwards.Sequencing results showed that all the three genes were correctly inserted in plant expressing vector pCAMBIA1300.To improve the tocopherol biosynthetic pathway and to increase the total amount of vitamine E,a plant bivalent expression vector,pCAMBIA1300 VE2.VE3,was constructed.It contains VE₂ and VE₃ genes,each has their own promoter and nos terminal.To improve both tocopherol and the rate of oleic acid component in peanut, another plant bivalent expression vector pCAMBIA1300AT was constructed.It has intact ultra-expression units of VE₄ and complete anti-RNA expression unit of FAD2.Three vectors,pCAMBIA1300VE4.2S,pCAMBIA1300AT and pCAMBIA13002a, have been used to transform peanut variety Minghua 6,mediated by agrobacterium C58. Through many resistant transgenic seedling were obtained,only three,three and six seedlings with respective to genes of VE2,anti-RNA of FAD2 and VE4,and anti-FAD2 were shown to carry the genes of interest in the cells,after subjected to PCR assay,with transformation rate of 2.0%,2.0%and 3.85%.Further evaluation must be done after seed harvest,since those genes are all directed by seed specifically expressed prmoters.In a word,we obtained five plant expressing vectors involved in increasing vatamine E contant.Plant expressing vectors pCAMBIA1300VE4-2S, pCAMBIA1300AT and pCAMBIA13002a were being used to transform peanut mediated by Agrobacterium and some transfomant seedlings were got,which provides basis in improving vitamin E and fatty acid composition in peanuts.