Expression And Regulatory Mechanism Of β-Conglycinin Gene By RNAi In Soybean

RNA interference (RNAi) is a post-transcriptional gene-silencing phenomenon induced by double-strand RNA. In plants, hpRNA(hairpin RNA) has been widely used to analyze gene function because of its high specificity and efficacy. Soybean storage protein gene expression is regulated by both transcriptional and post-transcriptional processes. Thus it has become possible to get more information about the 11S and 7S regulatory mechanisms by RNAi. Aαsubunit gene(CG-3) ofβ-conglycinin was isolated by PCR from genomic DNA in Nannong 99-10 (GenBank No.AB051865).Then the CG-3 gene inserted into the vector pBI121,which the site was between CaMV35S and green fluorescent protein (GFP) ,to generate the plasmid vector pYXP7SαG.Aα-subunit gene fragment (containing 5'UTR,the 1st exon and the 1st intron of CG-3) and the otherα-subunit gene fragment (a part of 1st exon of CG-3) were amplified by PCR from genomic DNA in Nannong 99-10. These twoα-subunit gene fragments were ligated in an antisense orientation and inserted into the vector pBI121, which the site was between CaMV35S and nos terminator, to produce hpRNAi vector pYXP7SαRi.Transformation of four soybean cultivars ( Nannong88-1, Nannongl8-6, Yu23 and Nannong 87C-38) by infecting cotyledonary-node with A. tumefaciens strain EHA105 (containing plasmid pYXP7SαG). The results indicated that the addition of thiol compounds ( L-cysteine, dithiothreitol and sodium thiosulfate) in co-cultivation period increased the transformation efficiency of three soybean cultivars (Nannong88-1, Nannong18-6 and Yu23). There has no affect on Nannong 87C-38. The transformation efficiency of Nannong88-1 was 1.39% in system only with L-cysteine, while 2.20% in system with thiol compounds.Using the same system, transformation of seven soybean cultivars ( Nannong88-1, Nannong18-6, Yu23, Nannong87C-38, Changshusanzihuangdou, Qidongyangyandou and Bamajiuyuehuang) by infecting cot-node with A. tumefaciens strain EHA105 (containing plasmid pYXP7SαRi). The result showed that the transformation frequency was different among cultivars of soybean. Three genotypes with high transformation frequency , its Nannong88-1, Nannong18-6 and Yu23.In addition, Bamajiuyuehuang showed with high regeneration frequency.We have obtained 21 positive transformated plants by Southern blot. 13 soybean transgenic lines were cultivated to set seeds. There were 5 soybean transgenic lines from the vector pYXP7SαG, and 8 soybean transgenic lines from the vector pYXP7SαRi.The results showed that the presence of transcripts from CG-3 gene in developing seeds were supressed by RT-PCR and Northern analysis in transgenic lines with hpRNA vector.A proteomic approach was used to analyze differential expression of proteins among the seeds of the control(Nannong 88-1) ,the over-expression of CG-3 and the RNAi-CG-3 trans- genic soybean. 25 protein spots was detected on the 2-D gels by PDQuest image software, which peoteins expression was greater than 2 fold. These 25 protein spots were treated by tryptic in-gel digestion and characterized by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and peptide mass fingerprintings of all were obtained, Total 20 proteins were identified from Soybean UniGene database. These proteins are classified into five groups .①Storage proteins(a-subunit ofβ-conglycinin, proglycinin AlaBlb subunit, Glycinin A3B4 subunit).②Cell growth/division (maturation associated protein, seed maturation protein PM22 and PM32, 68 kDa LEA protein)③Transporter (sucrose binding protein homolog S-64)④Disease/defense(heat Shock 70kD protein).⑤Transcription (glycine-rich RNA-binding protein). In our study, the result seeds with reducedα-subunit ofβ-conglycinin by RNAi was apparently compensated by an increased accumulation of GY gene products GY2 and GY5.The result further indicated that a decrease in the level ofβ-conglycinin(7S) protein leads to an increased level of glycinin protein(11S). We next analyzed the total amino acid composition among the seeds of the control(Nannong 88-1) ,the over-expression of CG-3 and the RNAi-CG-3 transgenic soybean. The total amino acid content of seeds differed little among the three soybean seeds.The seed-specific promoter only express its down stream genes from mid to late stage of seed maturation,and there is no expression or much lower expression in other tissues.So the seed-specific promoter are distinguished for the improvement having brought to plant quality engineering. A promoter fragment (7SαP ) ofαsubunit gene was isolated by PCR from genomic DNA in various soybean accessions, including cultivars Nannong99-10, N2899, Nannong 88-1, and wild soybeans Jiangpu -1 and ZYD4174.The sequences of 7SαP fragment from 5 soybean accessions shared 99% homology. It indicated that the promoter of a subunit gene were conserved. Meanwhile,sequencing analysis showed that the 7SαP fragment contained several seed-specific motifs, such as RY motif, AGCCCA motif, ACGT motif and A/T rich motif. So the expression vector pBI121-7SαP was constructed with the 7SαP fragment (Nannong99-10) promoter and the GFP reporter gene for functional analysis. Arabidopsis thaliana plants were transformed by A.grobacterium mediated method. Southern blot results showed that the 7SαP had been integrated into genomic DNA of Arabidopsis thaliana. Assay of GFP expression in the seeds of transgenic Arabidopsis thaliana was determined to identify the function of 7SαP promoter. The results showed that 7SαP was a seed-specific promoter.It will be a favourite tools for directing seed-specific expression of foreign genes in the genetic engineering of crops.