The Functional Identification Of Oleosin And Ara Hi Promoters And In Silico Analysis Of Transcription Factor Gene Wri1in A. Hypogaea

Peanut (Arachis hypogaea L.), one of the world’s four largest oilseed crops, is the oil andeconomic crop grown worldwide. It has the highest income of single yield total yield andexport volume in our country. Recently, as the development of the research in isolation andcloning the genes of oil lipids metabolic and tissue-specific promoters, Using geneengineering to elevate peanut’s quality has become a hot topic in the study of peanutmolecular breeding. In our research, we transfect PBI121(containing CaMV35S),pB-OL1584(containing Oleosin promoter) and pB-AH1716(containing Ara h1promoter) toArabidopsis using Agrobacterium inflorescence dip method. And then we do dokanamycinscreening and polymerase chain reaction(PCR) to identify the transgenic plants. We also useGUS histochemical staining for examining the tissue-specific expression of promoter,fluorescence photometric method for determinating GUS enzymatic activity in transgenicplants, real time fluorescence quantitative PCR(RT-qPCR) for detecting GUS relativeexpression in transcription level, and electronic cloning technology for obtaining the WRI1gene in peanut. Our major results are as follows:1. Obtaining of the transgenic ArabidopsisUsing Agrobacterium inflorescence dip Arabidopsis, we got two lines of T5generationtransgenic plants containing Oleosin promoter with a transfection efficiency of0.28%, twolines of T2generation transgenic plants containing Ara h1promoter which transfectionefficiency is0.3%, and five lines of T4generation transgenic plants containing CaMV35Spromoter which transfection efficiency is0.7%.2. The expression analysis of peanut Oleosin promoterBy Analyzing GUS enzymatic activation of the transgenic Arabidopsis containingOleosin promoter, we found that different tissues of the same plant have significantdifferences in GUS enzymatic activation, and the GUS enzymatic activation is highest in root,followed by pod, stem and leaf. What’s more, different plants also have a significantdifference in GUS enzymatic activation among the same organ. GUS enzymatic activity is4.01nmol/mg(protein)/min in the root of O2-18which has the highest enzymatic activityamong all the plants,2.60nmol/mg(protein)/min in the pod and0.73nmol/mg(protein)/min inthe leaf. Detecting the relative expression level of GUS gene in the transcriptional level, wefound the same tissue specificity in different plants as well as the enzymatic activation. This indicate peanut Oleosin promoter can drive the tissue–specific expression of GUS inArabidopsis: highest in root, followed by the pod，and little expression in stem and leaf.3. The expression analysis of peanut Ara h1promoterIn the transgenic Arabidopsis containing peanut Ara h1promoter, different tissues showsignificant differences in the GUS enzymatic activation: highest in root,1.748nmol/mg(protein)/min, followed by pod,0.946nmol/mg(protein)/min and lowest in stem,0.592nmol/mg(protein)/min. Then we detect the relative expression of GUS gene in thetranscriptional level, and found the same results as the enzymatic activation. Thus, weconclude that peanut Ara h1promoter can drive the tissue–specific expression of GUS inArabidopsis in the same way as Oleosin promoter does: highest in root, followed by the pod，and little expression in stem and leaf.4. comparison of the expression characteristics of three promotersComparing the GUS enzymatic activation of different promoters in Arabidopsis:I1-89(CaMV35S promoter),O2-18(Oleosin promoter)and A2(Ara h1promoter), we foundthat the enzymatic activation of O2-18and A2is0.66and0.29times of I1-89in the root,1.14and0.46times in the stem,1.04and1.00times in the leaf, and2.03and0.74times in the pod.This indicates that the expression of peanut Oleosin promoter in Arabidopsis is higher thanthat of CaMV35S promoter in the pod but lower in root, and there is no significant differencein stem and leaf. However, the driving of GUS by Ara h1promoter in Arabidopsis is lowerthan CaMV35S promoter in all tissues.5. cloning and sequence analysis of transcription factor WRI1gene in peanutThe cDNA sequence of transcription factor WRI1(AhWRI1) was obtained throughelectronic cloning method from peanut. We predicted and analysed the characteristics ofsequence、protein encoded as well as amino acid sequence similarity with other plants ofAhWRI1.Results showed that: AhWRI1gene contains a full length open reading frame of780bp which encodes259amino acids, and the relevant protein is hydrophilic which containstwo typical AP2domains, it shows high similarity with Arabidopsis and Brassica in proteintertiary structure. The amino acids conserved sequence of AhWRI1showed81.87%-100%homologies with Arabidopsis and Brassica. AhWRI1disordering degree is71.8%,3.8%lower than Arabidopsis thaliana, while2.5%higher than Brassica. Subcellular localization ofAhWRI11reveal that the protein lies in the nucleus and the possibility of proteintranscription、replication、regulation and combination is respectively0.244,0.226,0.152.