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Molecular analysis of transgenic rice overexpressing UDP-glycosyltransferase from Brassica rapa
Me-Sun Kim,Hye-Jung Lee,Joonki Kim,Dal-A Yu,Sanguk Byeon,Kwon-Kyoo Kang,Illsup Nou,Yong-Gu Cho 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07
Secondary plant metabolites undergo several modification reactions, including glycosylation and physiological functions. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, a UDP-glucosyltransferase cDNA was isolated from Brassica rapa hereinafter referred to as BrUGT. It has a full-length cDNA of 1,236 bp that contains a single open reading frame of 834 bp which encodes a polypeptide of 277 amino acid residues with a calculated mass of 31.19 kDa. BLASTX analysis hits a catalytic domain of glycos_transf_1 super family (c112012) that belongs to the glycosyltransferases group 1 with tetratricopeptide (TPR) regions. UGT gene expression analysis showed high mRNA transcripts in pistil, followed by petal, seed and calyx of flower in Brassica rapa. Furthermore, we constructed a recombinant pFLCIII vector carrying the BrUGT gene under the control of ubiquitin promoter and NOS terminator and transformed into rice using Agrobacterium tumefaciens. The UGT overexpressing rice lines were then characterized at the physiological and molecular levels. To further understand the biological function of BrUGT, transcriptional profiling of the gene in transgenic rice lines under cold, salt, PEG, H2O2, ABA and drought stress condition is underway.
Dal-A Yu,Hye-Jung Lee,Joonki Kim,Me-Sun Kim,Marjohn Nino,Sanguk Byeon,Jeongmi Kim,Illsup Nou,Kwon-Kyoo Kang,Yong-Gu Cho 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07
Since global climate changes drastically, pre-harvest sprouting (PHS) is expected to pose serious problems in rice production. CBL-interacting serine/threonine protein kinases (CIPKs) have been implicated to play important role in regulating various abiotic stresses such as cold, salinity and drought. In this study, to understand the function of this gene under pre-harvest sprouting in rice, a cDNA clone encoding CBL-interacting protein kinase 15 (CIPK15) was isolated from rice flowers. This gene is 2,818 bp long with 1,332 bp coding region that encodes a polypeptide of 443 amino acids. We constructed a recombinant vector carrying the OsCIPK15 under the control of the CaMV 35S promoter and Tnos terminator and transformed into rice using Agrobacterium tumefaciens. Insertion of the gene was verified in transformants using HPT resistance test and genomic PCR. Transcriptional profiling using tissues of wild type, Gopum, revealed expression of the gene in whole plant tissues with level of expression highest in the seeds suggesting possible role in dormancy. Comparative expression analysis of the gene in transgenic and wild type through semi-quantitative RT-PCR and real-time PCR showed higher expression in transgenic rice lines. Moreover, screening in the mist chamber showed overexpression lines that were resistant to the PHS. This result suggests the involvement of OsCIPK15 in the regulation of pre-harvest sprouting.
Modification of Starch Composition Using Downregulation of GBSS1 gene in Japonica Rice
Hye-Jung Lee,Moo-Geun Jee,Dal-A Yu,Me-Sun Kim,Franz Nogoy,Mingmao Sun,Sailila E. Abdula,Kwon-Kyoo Kang,Illsup Nou,Yong-Gu Cho 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07
There is a great consideration on rice eating quality aside from improving its tolerance to various stresses. High yielding and pest and disease tolerant rice is highly desirable but it is more commercially important if it also has a high eating quality. There are various factors contributing to the good eating quality of rice. This study focuses on modifying the expression of GBSS1 genes which are responsible for amylopectin and amylose synthesis in rice by using RNAi and antisense techniques. We have developed 40 transgenic plants with RNAi-GBSS1 gene and 60 transgenic lines with antisense-GBSS1 gene. The transgenic plants show diverse amylose contents in rice seed. We selected candidate lines according to PCR, RNA expression and amylose contents. A semi-quantitative RT-PCR was carried out to measure the expression level of GBSS1 gene at several time points after the flowering of transgenic plants. The expression level of GBSS1 gene in rice grains decreases over time and the mRNA expression among the transgenic plants were lower compare to its wild type. In the SEM analysis, the starch granule of wild type Gopumbyeo has very large structures accompanied with small ones around the area. However, the starch structures in transgenic plants were smaller and more uniform in size and shape throughout the viewing area
Modification of starch composition using RNAi targeting of SSS1 gene in rice
Hye-Jung Lee,Moo-Geun Jee,Dal-A Yu,Me-Sun Kim,Joonki Kim,Seon-Kyeong Song,Kwon-Kyoo Kang,Yong-Gu Cho 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07
An increasing preference for good eating quality of rice among consumers has become one of the important considerations in rice breeding. Amylose content is a leading factor affecting eating quality of rice. Amylose composition is determined by the relative activity of soluble starch synthase (SSS) and granule-bound starch synthase (GBSS). This study focused on modifying the expression of SSSI gene which is responsible for amylopectin and amylose synthesis in rice by using RNA interference (RNAi) technology. The transgenic rice plants showed various amylose content in rice grains. Favorable rice lines were selected according to genomic PCR, transgene expression and amylose contents analysis. A semi-quantitative RT-PCR was carried out to determine the expression level of SSSI gene after flowering of transgenic rice and wild type. Down-regulation of SSSI gene in transgenic plants was evident in the decreasing expression in rice grains. Accordingly, scanning electron microscopy (SEM) analysis revealed uniform size with smooth curves starch granules in down-regulation rice lines, in contrast with the non-uniform granules in wild type. Results indicated that RNAi-SSSI transgenic lines produced low amylose contents that fell between glutinous and non-glutinous rice. This study showed that down-regulation of endogenous SSSI may improve the eating quality in rice.
Kim, Joonki,Lee, Hye-Jung,Nogoy, Franz Marielle,Yu, Dal-A,Kim, Me-Sun,Kang, Kwon-Kyoo,Nou, Illsup,Cho, Yong-Gu The Korean Society of Plant Biotechnology 2016 식물생명공학회지 Vol.43 No.3
Seed size traits are controlled by multiple genes in crops and determine grain yield, quality and appearance. However, the molecular mechanisms controlling the size of plant seeds remain unclear. We performed functional analysis of BrPATL4 encoding Sec14-like protein to determine the genetic architecture of seed size, shape and their association analyses. We used 60 $T_3$ transgenic rice lines to evaluate seed length, seed width and seed height as seed size traits, and the ratios of these values as seed shape traits. Pleiotropic effects on general architecture included small seed size, erect panicles, decreased grain weight, reduced plant height and increased sterility, which are common to other mutants deficient in gibberellic acid (GA) biosynthesis. To test whether BrPATL4 overexpression is deleterious for GA signal transduction, we compared the relative expression of GA related gene and the growth rate of second leaf sheath supplied with exogenous $GA_3$. Overexpression of BrPATL4 did not affect GA biosynthesis or signaling pathway, with the same response shown under GA treatment compared to the wild type. However, the causal genes for the small seed phenotype (D1, SRS1, and SRS5) and the erection of panicles showed significantly decreased levels in mRNA accumulation compared to the wild type. These results suggest that the overexpression of BrPATL4 can control seed size through the suppression of those genes related to seed size regulation. Although the molecular function of BrPATL4 is not clear for small seed and erect panicles of BrPALT4 overexpression line, this study provides some clues about the genetic engineering of rice seed architecture.