Study On Oleosin PsOLE1 Gene And Its Promoter Related To Oil Accumulation In The Seeds Of Prunus Sibirica

Prunus sibirica L.,also known as Siberian apricot,has an oil content of up to 50%in the seeds,which has emerged as a novel potential source of biodiesel in China,and thus deserved for further research and exploitation.In this study,the cloning and bioinformatics analysis of PsOLE1 gene and its promoter from the seeds of P.sibirica were carried out,and the expression vector was constructed for Pro_PsOLE1::GUS,and the T3 generation of transgenic A.thaliana plants were obtained.Both GUS histochemical staining and expression analysis were performed to determine tissue expression specificity of the PsOLE1 promoter(Pro _PsOLE1).In addition,in order to effectively explore the effect of PsOLE1 overexpression on oil content and its fatty acid（FA）compositions of P.sibirica seeds,the expression vectors of Pro_PsOLE1::PsOLE1 and35S::PsOLE1 were constructed,respectively,and then the analyses of plant phenotype,and seed oil content and FA compositions as well as gene expression involved specifically in TAG biosynthesis were conducted on the T3 generation of transgenic A.thaliana lines with Pro _PsOLE1::PsOLE1 and 35S::PsOLE1.All these should help to elucidate the possible role of the PsOLE1 gene and its specific promoter in oil accumulation of P.sibirica seeds.The main findings obtained in this study are summarized as follows:1.The full length of PsOLE1 gene with 683 bp was successfully cloned from the P.sibirica seeds,which contained 5′untranslated region（5’UTR）with 10 bp,open reading frame（ORF）with 447 bp and 3’UTR with226 bp,and encoded 148 amino acids.The secondary and tertiary structures of PsOLE1 protein were mainlyα-helix.Analysis of subcellular localization indicated that PsOLE1 protein was located in cell membrane.2.The PsOLE1 promoter with 2251 bp in length was successfully cloned from the P.sibirica seeds,designated as Pro _PsOLE1.This promoter contained a variety of stress responsive elements（such as WUN-motif,ARE,LTR,etc.）and seed specific regulatory elements（GCN4-motif,RY-elements,and AACA-motif）.To explore tissue expression characteristics of PsOLE1 promoter,the expression vector of Pro _PsOLE1::GUS was constructed and transformed into A.thaliana.The results from an integrated analysis of GUS histochemical staining,RT-PCR and qRT-PCR detection showed that GUS gene exhibited a relatively high expression level in the seeds of transgenic A.thaliana,and thus concluded that the PsOLE1 promoter from P.sibirica seeds is a seed-specific promoter.3.The phenotypes of T3 generations of transgenic A.thaliana lines with Pro _PsOLE1::PsOLE1 and35S::PsOLE1 were analyzed.It was found that compared with wild type（WT）plants,the root length,plant height and seed weight increased in the transgenic lines of 35S::PsOLE1 and Pro _PsOLE1::PsOLE1,but a significant increase of seed weight was detected for transgenic lines of Pro _PsOLE1::PsOLE1.In addition,compared with the WT seeds,the oil content of seeds for transgenic 35S::PsOLE1 and Pro _PsOLE1::PsOLE1was increased by 4%and 8%,respectively,and notably,the contents of most FA compositions（except C20:0）increased in different degrees,of which the contents of unsaturated FAs（C18:1,C18:2 and C18:3）in the seeds of transgenic Pro _PsOLE1::PsOLE1 lines were increased by 16%,26%and 20%respectively,while only11%,19%,and 13%were increased in the seeds of transgenic 35S::PsOLE1 lines.These results indicated that PsOLE1 gene expression driven by seed-specific promoter of Pro _PsOLE1 could effectively promote seed development and unsaturated FA accumulation in the seeds of transgenic A.thaliana,which may contribute to increase seed oil content.4.The expression changes of functional genes relevant to TAG biosynthesis in the seeds of transgenic A.thaliana with 35S::PsOLE1 and Pro _PsOLE1::PsOLE1 were analyzed by qRT-PCR.The results showed that compared with the WT seeds,the expression levels of LPAT2,PDAT1,DGAT1 and OBAP1A were all up-regulated in the seeds of transgenic A.thaliana with 35S::PsOLE1 and Pro _PsOLE1::PsOLE1,indicating that expression of PsOLE1 gene driven by its specific promoter(Pro _PsOLE1)could result in abundant expression of key functional genes involved specifically in TAG biosynthesis.This study focused on the cloning,genetic transformation,and functional identification of the oil body protein gene PsOLE1 and its specific promoter,all of which could help to explore functional mechanism of PsOLE1 and its promoter for seed development and oil accumulation of P.sibirica,and may also provide important foundation for molecular improvement in the oil crops and tree species.