최근 전세계적으로 기능성 성분이 강화된 유전자교정 작 물의 생산 및 소비가 증가하고 있다. 하지만 유전자교정 작물의 유전독성에 관한 소비자의 우려도 증가하고 있어 과학적인 자료 확보 및 정보 공유에 대한 인프라가 필요한 실정이다. 본 연구에서는 CRISPR/Cas9 시스템을 활용한 유 전자교정 토마토 동결건조물(LTT)이 DNA나 염색체에 직접 적인 손상을 일으키고 형태적 또는 기능적 이상을 유발하 는지 여부를 확인하기 위해 유전독성 평가를 수행하였다. 이를 위하여 미생물 복귀돌연변이시험, 염색체이상시험, 골수세포를 이용한 체내 소핵시험을 국제적으로 표준화된 OECD Guidelines에 따라 시험을 진행하였다. 복귀돌연변이 시험에서 LTT는 S9의 존재 여부와 관계없이 Salmonella typhimurium 균주 TA98, TA100, TA1535 및 TA1537, 그리 고 Escherichia coli WP2 uvrA에서 복귀돌연변이를 유발하 지 않았다. LTT는 CHL 세포의 수적이상 중기상과 구조적 이상 중기상 등의 염색체 이상을 유발하지 않았다. 또한, LTT는 다염성 적혈구에서 소핵화된 다염성 적혈구의 빈도 를 증가시키지 않았다. 이러한 연구를 통해 CRISPR/Cas9 시스템을 활용한 유전자교정 토마토의 안전성을 검증하고, 향후 CRISPR/Cas9 시스템을 활용한 유전자교정 작물의 유 전독성을 평가하는 기초 자료로 사용될 수 있을 것이다.

Zinc finger nucleases (ZFNs) have been used for targeted mutagenesis in eukaryotic cells. Custom-designed ZFNs can induce double-strand breaks (DSBs) at a specific locus. Our custom ZFN dimer was designed 3-finger of left and 4-finger of right with 2 kb size using 2A. A Ti-plasmid vector, pTA7002 containing the target site of SSS4A gene for a ZFN pair, that was shown to be active in yeast, was integrated in the rice genome. This promising technique for genome engineering was induced into 4 exon region of SSS4A gene in rice genome using Agrobacterium-mediated transformation. The SSS4A full-length cDNA was 5,070 bp consisting of a 318 bp 5′-untranslated region (UTR), a complete ORF of 2,928 bp encoding a polypeptide of 975 amino acids and a 3′-UTR of 1,824 bp. The vector is based on glucocorticoid receptor inducible gene expression system. Thus, SSS4A::ZFN expression was tightly controlled and the phenotype in low concentrations 10uM of the glucocorticoid hormone dexamethasone (DEX). In plant cells, transient ZFN expression is achieved by direct gene transfer into the target cells. For an alternative, ZFN delivery and production of mutant plants using a tobacco transient expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of plants. ZFN activity was determined by PCR and sequence analysis of the target site. ZFN induced plants were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1and 100 bp and insertions ranging between 1 and 10 bp. Our results describe an alternative to direct gene transfer for ZFN delivery and for the production of mutated rice.

UDP-glucose 4-epimerase (UGE; EC 5.1.3.2) is an enzyme that plays an essential role in the interconverts UDP-D-glucose (UDP-Glc) and UDP-Dgalactose (UDP-Gal). Five members of the Chinese cabbage (Brassica rapa) UDP-glucose 4-epimerase gene family, designated BrUGE1 to BrUGE5, have been cloned and characterized. Quantitative PCR shows that the BrUGE1and BrUGE4 mRNA are most abundant among other BrUGE genes, accounting for more than 55% of total BrUGE transcripts in most of the tissues examined. All genes showed organ specific expression pattern, two of which (BrUGE1 and 4) actively responded after Pectobacterium carotovorum subsp. carotovorum infection, while four genes (BrUGE-1, -3, -4 and -5)were shown to respond considerably against salt, drought and abscisic acid (ABA) treatments. To better understand the function of the UGE gene, we constructed a recombinant pART vector carrying the BrUGE1 gene under the control of the CaMV 35S promoter and nos terminator and transformed using Agrobacterium tumefaciens. We then investigated BrUGE1 overexpressing rice lines at the physiological and molecular levels under biotic and abiotic stress conditions. Bioassay of T3 progeny lines of the transgenic plants in Yoshida solution containing 120 mM Nacl for 2 weeks, confirmed that the BrUGE1 enhances salt tolerance to transgenic rice plants. Also T3 progeny lines of the transgenic plants, when exposed to infection caused by Xanthomonas oryzae pv oryzae, showed tolerance to bacterial blight. These results showed that BrUGE1 can be used as potential genetic resource for engineering Brassica with multiple stress resistance.

생강연작재배지 토양에 녹비작물의 재배가 토양 양분의 변 화와 뿌리썩음병원균 경감효과를 알기위해 헤어리베치, 크림 손클로버, 오차드글라스를 80일간 재배 후, 토양에 환원하여 토 양분석 및 real-time PCR 분석한 결과는 다음과 같다. 녹비작 물의 생육은 크림손클로버가 가장 좋았으며, 질수흡수량은 크 림손클로버가 가장 높게 나타났으며, 토양의 인산함량은 오차 드글라스가 가장 낮게 나타났다. T-N 함량은 헤어리벳치 > 크 림손클로버 > 오차드글라스 순으로 나타났다. 또한 토양 내 뿌 리썩음 병원균 밀도분석을 위해 Pythium zingiberum 균 특이 5.8S rDNA를 이용하여 real-time PCR 분석한 결과 헤어리베 치, 오차드그라스 및 크림손클로버 처리의 Ct값은 대조구보다 낮게 나타났다. 이상의 결과를 요약해볼 때 생강연작재배지에 서 녹비처리 후 토양은 대조구에 비해 EC가 감소되고, 몇 몇 무 기성분은 증가하였으며, 뿌리썩음 병원균 밀도는 감소하였다.

본 연구는 시설하우스 내 토마토 연작장해 경감을 위해 단기 녹비작물 재배가 토마토의 생육특성 및 수량에 미치는 영향을 알아보기 위해 토마토 시설재배지에 헤어리베치와 호밀을 단파 및 혼파로 구분하여 파종하였으며, 생육된 녹비작물의 양분공 급량과 녹비작물의 토양환원 후 토양의 이화학적 특성 그리고 녹비작물 환원 후 후작물인 토마토의 생육특성 및 토마토 시들 음 병원균의 밀도를 조사한 결과, 녹비작물의 질소(N), 인(P2O5), 칼륨(K2O), 칼슘(CaO) 및 마그네슘(MgO) 공급량은 헤어리베 치의 경우 각각 26.2, 5.8, 10.2, 6.6, 및 1.5 ㎏/10a였으며, 호밀 은 각각 9.1, 4.2, 11.8, 3.8, 및 3.1 ㎏/10a였고, 혼파의 경우는 단파와 유사한 경향이었다. 토마토 시설재배지에서 녹비작물 의 토양환원 전･후 토양의 pH는 대조구와 별다른 차이 없이 6.37-6.52 범위였으며, EC는 헤어리베치(2.64 dS/m) 및 호밀 (2.62 dS/m) 처리가 대조구(헤어리베치-2.80 dS/m, 호밀 -2.91 dS/m)에 비해 낮았다. 토양 중 유기물, T-N 및 avail. P2O5 함량은 녹비작물 처리가 대조구에 비해 증가하는 경향이 었다. 녹비작물 토양환원에 따른 후작물인 토마토 생육은 녹비 작물 처리는 대조구에 비해 우수한 생육을 보였으나, 관행처리 와는 비슷한 생육을 보였다. 또한 녹비처리에서 토마토 시들음 병원균의 밀도가 현저하게 감소하였다. 이상의 결과를 종합할 때, 토마토 시설재배지에서 녹비작물의 시용은 비료로서 충분 한 가치가 있으며, 녹비작물 토양 환원 후 토양화학성을 개선함 으로써 시설하우스 토마토 연작재배지의 연작장해를 경감할 수 있을 것으로 사료된다.

Rice is one of the most important major food crops which provide the major food for more than half of global population. To improve the grain quality as well as grain yield has been the essential breeding goal in rice. The composition of amylopectin is the determinant of rice eating quality under certain threshold of protein content and the ratio of amylose and amylopectin. In this study, RBE 1 driven by CaMV-35S promoter was constructed and transformed using Agrobacterium tumefaciens. We selected single copy with low amylose content among transgenic lines. The mRNA expression was investigated using RT-PCR, and enzyme activity was determined using activity staining method in mid-milky stage endosperm. Also, the overexpression vectors for RBE 1 and SSS 1 driven by seed specific globulin promoter were constructed, respectively. Moreover, the RNA interference vectors for soluble starch synthase 1 and granule bound starch synthase 1 derived by CaMV35S promoter were constructed, respectively and transformed using Agrobacterium tumefaciens. The transgene has been confirmed by amplification of HPT and target gene. The transgenic plants obtained will be used to investigate the gene function of related starch pathway in plant cells using Gopumbyeo as a wild type rice, based on the gain-of-function and the loss-of-function. The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency. Efficient in planta GT was achieved in rice by expression of a site specific endonuclease (SSS1::ZFN) that not only cuts within the target but also the chromosomal transgenic donor, leading to an excised targeting vector.

A cDNA clone encoding CBL-interacting protein kinase 1 (CIPK1) was isolated from Chinese cabbage seedlings. The gene, BrCIPK1 consisted of 1,982 bp long with 216 bp of the 5’-untranslated region (UTR), 1,509 bp of the coding region and 257 bp of the 3’-UTR. It is highly conserved CBL-interacting module with absolutely conserved domain among the 15 amino acid NAF domain of the 15 related genes. Southern blot analysis showed a single copy number. BrCIPK1 gene was localized in the cytoplasm and peripheral region in the plant cell which is highly expressed in seedling of rice and in the shoot and pistil of Arabidopsis. Analyses of gene expression on Ubi-1::BrCIPK1 rice lines was differentially accumulated by cold, salinity and drought, indicating its biological roles in the multiple stress response pathways in plants. Further, the expression of BrCIPK1 is hijacked by rice calcineurin-B-like protein (OsCBL5). Moreover, mRNA expression of P5CS1, a gene responsible for proline biosynthesis is regulated by the BrCIPK1 during abiotic stresses resulting to improved accumulation of proline. The interaction of BrCIPK1 with OsCBL5 along with the regulation of P5CS1 explained the enhanced tolerance of transgenic rice. This gene could be used in the development of rice varieties with enhanced tolerance to abiotic stresses.

UDP-glucose 4-epimerase (UGE) catalyzes the reversible conversion of UDP-glucose to UDP-galactose. The gene, named BrUGE1, isolated from a Chinese cabbage had a total length of 1,328 bp that contains a single open reading frame (ORF) of 1,056 bp which encodes a polypeptide of 351 amino acid residues with a calculated mass of 39.0 kDa. Sequence analysis of BrUGE1 protein has the characteristic of an active site tetrad and NAD-binding motif (typically TGXXGXXG) of the extended short chain dehydrogenase/ reductase (SRD) superfamily. Expression analysis showed that BrUGE1 is tissue specific and highly expressed in stem of rice plant. Interestingly, BrUGE1 mRNA was highly accumulated by drought stress with significantly higher amount of soluble sugar. Morphological evaluation showed an increase in yield by 27%. Panicle length, number of productive tillers/hill, and filled spikelets were significantly increased by 17~20% compared to the wild type Gopum. Moreover, the growth of the wild type Gopum seedlings on galactose was increasingly inhibited with a decrease in UDP-glc epimerase 1 expression compared to the transgenic rice lines. In the Ubi-1::BrUGE1 lines, the increase of UDP-glc epimerase 1 expression was apparently sufficient to overcome the toxic effects of galactose. Taken together, the Ubi-1::BrGUE1 rice lines increased yield probably by increasing the rate of filled grains. The enhanced drought tolerance may be due to the induction of soluble sugar which may act as osmolyte to compensate dehydration during drought stress.

The latest report on draft genome of Brassica rapa sequence has been published. To elucidate the functions of a large population of these genes and to search efficiently for agriculturally useful genes, the Full-length cDNA Over-eXpressor (FOX) gene hunting system was used. The FOX library was transformed into rice by Agrobacteriummediated transformation. Approximately 1,150 FOX-rice lines were generated. Genomic PCR analysis indicated that the average length of FL-cDNAs was 900∼1,200 bp with functional annotation of many unknown function (35.5%). Most of the randomly selected transgenic rice lines showed overexpression (92%) and barely mRNA expression in the wild type Gopum. Moreover, 94% of the 850 transgenic rice lines were moderately tolerant (slightly yellow) to cold and 9 lines were tolerant (seedling light green). For the salinity evaluation, most of the transgenic lines (85%) were highly susceptible whereas seven lines were tolerant. In addition, morphological evaluation of rice lines showed minimal phenotypic alteration (12%). About 25.1 and 22% were earlier in terms of days to heading and chlorophyll contents, respectively. Further, 18% of FOX rice lines showed lower chlorophyll contents. Filled grains, number of tillers, panicle length, culm and plant height were relatively less variable among the lines. These results provided useful genes for functional analyses in the mechanisms of identified transgenic tolerant lines.

The antimicrobial peptide possesses defence system to virus, fungi and bacteria. To study antibiotic in plant, antimicrobial peptides were obtained by PCR analysis by primers designed from antimicrobial peptides (Gene bank accession no. NM-004345), cloned in pET28 expression vector and the vector transformed into E. coli. And this gene was inserted into Ti-plasmid VB2 vector, which contained the pGD1 promoter. The expression construction was transformed into Agrobacterium EHA105 and then plant tissues of rice (Oryza sativa). Seeds from transgenic plants (T0) were germinated on selective media containing spectinomycin 50 mg/L. Selected plants and wild type were analyzed by PCR and RT-PCR with pGD1 promoter region and transgene specific primer set. All transgenic plants showed expression pattern of similar levels. We showed that the chromobody is effective in binding GFPand antimicrobial peptide gene in tobacco leaf. Most interestingly, this can be applied to interfere with the function of GFP fusion protein and to mislocalize (trap) GFP fusions to the plant cytoplasm in order to alter the phenotype mediated by the targeted proteins. Bacterial blight disease was enhanced resistance in transgenic lines. These results showed that antibiotic peptides might show a broad-spectrum antimicrobial activity.

A cDNA clone encoding CBL-interacting protein kinase 1 (CIPK1) was isolated from Chinese cabbage seedlings. The gene, BrCIPK1 consisted of 1,982 bp long with 216 bp of the 5’-untranslated region (UTR), 1,509 bp of the coding region and 257 bp of the 3’-UTR. It is highly conserved CBL-interacting module with absolutely conserved domain among the 15 amino acid NAF domain of the 15 related genes. Southern blot analysis showed a single copy number. BrCIPK1 gene was localized in the cytoplasm and peripheral region in the plant cell which is highly expressed in seedling of rice and in the shoot and pistil of Arabidopsis. Analyses of gene expression on Ubi-1::BrCIPK1 rice lines was differentially accumulated by cold, salinity and drought, indicating its biological roles in the multiple stress response pathways in plants. Further, the expression of BrCIPK1 is hijacked by rice calcineurin-B-like protein (OsCBL5). Moreover, mRNA expression of P5CS1, a gene responsible for proline biosynthesis is regulated by the BrCIPK1 during abiotic stresses resulting to improved accumulation of proline. The interaction of BrCIPK1 with OsCBL5 along with the regulation of P5CS1 explained the enhanced tolerance of transgenic rice. This gene could be used in the development of rice varieties with enhanced tolerance to abiotic stresses.