벼와 토마토에서 유전자가위를 이용한 HY5 유전자 편집

박현서 원광대학교 일반대학원 2022 국내석사

HY5 유전자는 asn-arg/lys-leu의 서열로 구성된 bZIP 도메인을 갖고 있으며, 식물의 발달과 생합성, 다양한 신호전달체계에서 많은 역할을 하는 유전자이다. 애기장대에서 유전자의 기능에 대한 많은 연구가 진행되었지만 작물인 벼(Oryza sativa)와 토마토(Solanum lycopersicum)에서는 연구가 많이 진행되지 않았다. 애기장대의 HY5 유전자의 orthologues 분석과 phylogenetic tree 분석을 통해 벼와 토마토에서 OsHY5.1, OsHY5.2, OsHY5.3, SlHY5 유전자를 각각 확인했다. 확인된 유전자를 기반으로 OsHY5s와 SlHY5 유전자를 클로닝 하였으며, CRISPR/Cas9 system을 이용해 유전자를 편집했다. 편집한 유전자는 deep-sequencing과 sanger-sequencing을 통해 유전자 염기서열의 편집을 확인하였다. OsHY5.1 유전자는 target1에서 36bp, 39bp, 25bp의 염기가, target2에서 159bp의 염기가 결실된 것이 확인되었다. 편집된 식물체의 아미노산 서열을 바탕으로 단백질 서열을 분석하였고 target1의 25bp와 target2의 159bp가 삭제된 식물체에서 단백질의 비정상적 번역으로 early stop codon이 생성된 것을 확인했다. OsHY5.2 유전자는 target1에서 46bp, 40bp의 염기가, target2에서 14bp의 염기가 결실되었다. 세 패턴 모두 단백질의 비정상적 번역에 의해 early stop codon이 생성된 것을 확인했다. OsHY5.3 유전자는 아데닌(A)과 티민(T)이 삽입된 두 개의 패턴과 6bp가 삭제된 한 개의 패턴으로 총 3개의 패턴을 확인했다. 세 패턴 모두 종자의 크기가 왜소하고 뿌리 발육이 좋지 못했으며 발아율이 현저하게 낮은 것을 볼 수 있었다. 또한, 티민이 삽입된 패턴의 경우 야생형과 비교하여 엽색이 뚜렷하게 옅은 것을 확인할 수 있었다. 아데닌이 삽입된 패턴은 단백질의 비정상적 번역에 의해 종결 코돈이 생성되지 않는 것을 확인했으며, 티민이 삽입된 패턴은 그 위치에 바로 종결 코돈이 생성돼 early stop codon을 확인했다. 6bp가 삭제된 패턴은 도메인의 앞쪽 단백질 2개가 결실된 것을 확인하였다. SlHY5 유전자는 2bp, 49bp의 염기가 삭제된 두 패턴을 확인했다. 두 패턴 모두 단백질의 비정상적 번역에 의한 early stop codon을 확인할 수 있었다. 엽색이 녹색인 야생형과 비교하여 돌연변이 식물체에서는 옅은 연두색을 띠었고 잎의 끝에서부터 하얗게 말라가다가 결과적으로 회복이 되지 않는 표현형을 보였다. 본 연구에서는 벼와 토마토에서 CRISPR/Cas9 기술을 이용하여 벼에서 총 11개 패턴의 순계 계통 식물체와 토마토에서 2개 패턴의 순계 계통 식물체를 각각 확보하였고 토마토 야생형 식물체에서 조직별로 HY5 유전자의 발현량을 확인하였으며 염기의 삭제 또는 삽입에 의한 단백질의 이상 번역, 이에 따른 표현형을 확인했다. 또한, 토마토에서 HY5의 과발현 식물체 2개 계통을 확보하였다. 추후 연구를 통해 앞서 확보한 돌연변이 식물체와 함께 과발현 식물체를 확보하여 벼와 토마토에서 HY5 유전자의 기능을 밝히는 연구가 진행되어야 할 것으로 생각된다. The HY5 gene is a transcription factor comprising a bZIP domain family with asn-arg/lys-leu motif. It has been found to play a variety of roles in plant developmental stage, biosynthesis, and several signaling pathways in Arabidopsis thaliana. However, there are few studies of the HY5 gene in rice (Oryza sativa) and tomato (Solanum lycopersicum). Through the orthologues analysis of Arabidopsis HY5 gene, three of OsHY5.1, OsHY5.2, OsHY5.3, and single SlHY5 genes are isolated in Rice and Tomato, respectively. Cloning of OsHY5 and SlHY5 was conducted based on the separated genes, and genes were edited using the CRISPR/Cas9 system. Editing of the target gene was confirmed through deep-sequencing and sanger-sequencing. The OsHY5.1 gene had 36bp, 39bp, and 25bp bases deleted from target1 and 159bp bases deleted from target2. Protein sequences were analyzed based on the amino acid sequence of the edited plants, and 25bp of target1 and 159bp of target2 were found to have been generated early stop codons by abnormal translation of proteins in the deleted plants. The OsHY5.2 gene was deleted 46 bp and 40 bp bases from target1 and 14 bp bases from target2. In all three patterns, it was confirmed that early stop codon was produced by abnormal translation of proteins. The OsHY5.3 gene identified a total of three patterns: two patterns in which adenine (A) and thymine (T) were inserted and one pattern in which 6bp was deleted. In all three patterns, it was confirmed that the seed size was small, the root showed an impaired phenotype, and the germination rate was significantly low. In addition, in the case of a pattern in which thymine was inserted, it was confirmed that the leaf colour was clearly pale green compared to the wild type. It was confirmed that the pattern in which adenine was inserted did not produce a termination codon by abnormal translation of the protein, and the pattern in which thymine was inserted created immediately at that location, confirming the early stop codon. In the pattern in which 6bp was deleted, it was confirmed that two proteins in the front of the domain were lost. The SlHY5 gene identified two patterns in which 2bp and 49bp bases were deleted. Compared to the wild type, the leaf colour turn pale green, and it showed blight phenomenon from the end of the leaf and eventually died. In both patterns, early stop codon due to abnormal translation of proteins were confirmed. In this study, CRISPR/Cas9 technology was used in rice and tomato to obtain a total of 11 patterns in rice and 2 patterns in tomato, the expression level of the HY5 gene by WT tissue in tomato was confirmed, and abnormal translation of proteins by deletion or insertion of bases, and phenotypes accordingly were confirmed. In addition, two lines of overexpressed plants of HY5 gene were secured in tomato. Through further research, it is thought that research should be conducted to reveal the function of the HY5 gene in rice and tomato by securing overexpression plants along with mutant plants previously secured.

신물질 HY1553의 HeLa 세포에서의 세포사멸 유도

문상익 건국대학교 2004 국내석사

한글초록:최근 개발되고 있는 항암제 연구는 세포 주기의 조절에 근거한 자연스런 세포 사멸을 유도하는 방향으로 나아가고 있다. 기존의 대다수 항암제들은 비 선택적으로 정상세포와 암세포에 작용하여 심각한 부작용 및 내성을 유발 할 뿐만 아니라, 다시 재발될 수 있는 가능성을 많이 내포하고 있다. 그러나 세포 생물학의 발전과 더불어 세포 내에서의 성장 기전에 대한 획기적인 발견과 이해에 따라 성장 신호전달과 관련된 인자들의 항암제 적용이 활발하게 이루어 지고 있다. 그 결과, 최근 출시된 여러 항암제들은 암세포의 성장 혹은 생존 체계에 관련된 인자들의 활성을 억제함으로써 세포분화의 비정상적인 촉진, 세포주기 억제 및 세포사멸을 유도하여 암세포의 효과적인 제거 가능성을 보여주었다. 이러한 세포 주기 조절 인자의 조절을 가능케 하는 천연물의 탐색 또한 활발하게 진행되고 있어 암세포에 대한 성장 조절 억제 후보 물질이 꾸준히 발굴되고 있다. 본 연구는 이러한 천연물을 이용하여 암세포에 대한 항암 활성과 항암 기전의 규명, 항암물질로서의 가능성을 밝히고자 하였다. 방선균 배양액으로부터 분리한 HY1553는 몇몇 종류의 암세포(HeLa, HL-60, A549, HepG2 cells)를 농도 의존적으로 억제하며 정상세포에서는 억제현상을 보이지 않았다. 이와 같은 결과를 바탕으로 활성을 보이는 각 암세포의 DNA 분포도를 flow cytometry를 이용하여 관찰한 결과 세포 사멸을 확인할 수 있었으며 가장 뛰어난 활성을 보인 HeLa의 경우 32.2 mM에서 시간 의존적인 세포주기 억제 및 세포사멸 현상을 관찰 할 수 있었다. 위의 결과에 따른 단백질 변화량 관찰결과 세포 주기에서 G1 phase 진행에 중요한 조절 인자인 Cdk4와 Cdk6의 단백질 발현양이 시간 의존적으로 줄어듦을 관찰할 수 있었으며 Cyclin D의 감소 또한 가시적으로 확인 할 수 있었다. 또한 CKI인 p21의 단백질 증가를 관찰할 수 있었다. p21의 증가와 더불어 Cyclin D―Cdks complex의 감소는 pRb의 탈 인산화를 초래하며 이에 따른 전사인자인 E2F1의 감소를 확인 할 수 있어서 HY1553이 G1 arrest에 관여한다는 사실을 알 수 있었다. HY1553이 HeLa 세포에서 세포주기 조절 억제와 동시에 세포사멸에 영향을 미친다는 사실을 확인하기 위해 세포사멸 경로의 주요인자인 ICE family와 Bcl-2 family 및 tumor suppressor gene인 p53의 단백질 발현 양상을 관찰 한 결과, caspase-3, caspase-8 및 caspase-9의 전구체가 활성 caspase로 cleavage되는 것을 확인 할 수 있었다. Bcl-2 family의 경우 anti-apoptotic member인 Bcl-2 및 Bcl-XL의 단백질 발현 양에는 변화가 없었으나 pro-apoptotic member인 Bax의 단백질양이 증가함을 확인할 수 있었으며 Bid에서는 변화를 관찰 할 수 없었다. 즉, p53에 의해 Bax의 활성이 유도되고, 그로 인하여 mitochondria의 cytochrome C의 유출이 유도되어, 이에 따른 caspase-9, caspase-3의 활성화와 최종적인 PARP fragmentaion을 확인할 수 있었다. 본 연구에서 수행된 실험을 종합하면 방선균에서 분리한 HY1553은 HeLa 세포에서 p53이 증가해 p21의 발현을 유도, Cdks의 활성을 저해하여 세포주기의 억제와 세포사멸을 유도함을 확인 하였다. 이로써 HY1553에 의한 세포주기 조절 경로와 세포사멸 경로를 확인 할 수 있었으며 따라서 천연물을 이용한 항암제로서의 가능성을 제시하였다 영문초록:During the last few decades, several novel drugs including Gleevec and Iressa have been made to selectively induce apoptosis targeted into cancer cells. Targeted cancer cells undergo programmed cell death by death signals. Some natural products are known to regulate cell growth in response to oncogene activation and induces cell cycle arrest and apoptosis in tumor cell lines. I in order to discover a novel inducer of apoptosis in the fermented broth of actinomycetes were screened. One of them showed an apoptotic activity. The structure of the isolated compound was determined based on the interpretations of NMR spectroscopy and mass spectrometry, and confirmed a novel compound, named HY1553. Its chemical structure was (E)-3-benzyl-6,8-dihydroxyoct-2-en-4-one. For the purpose of confirmation of apoptotic activity with novel compound HY1553 in cancer cell, various anti-cancer activity analyses were excuted including MTT assay, TUNEL assay, Western blotting and Flow cytometry analysis. The result of an MTT assay, with the exception of normal cells and several cancer cells, proliferative activity of most of cancer cells was low level. The kinase activities of G1 and S Cdks (Cdk4, Cdk2) were inhibited by increments of p21 in HeLa cells treated HY1553 32.2 mM. In addition, HY1553 induced dephosphorylation of pRb, so that E2F1 was inhibited . These data suggested that HY1553 mediates Cdks at G1 and S phase which was resulting in an arrest of cells in the G1 phase. HY1553 induced an p53 mediation and mitochondrial dependent apoptosis in the case treated its 80 mM. Bax, pro-apoptotic Bcl-2 family member, activated by p53, triggered the release of cytochrome C from mitochondria in HeLa cells. In consequence of cytochrome C release, active caspase-9 cleaved procaspase-3 and which activated caspase-8 or led to DNA fragmentation. HY1553 prevented from proliferation of HeLa cells through the p53-Bax-mitochondria pathway. As a result, HY1553 induces cell cycle arrest through the Cdk-mediation and ultimately leads to programmed cell death, and it might be a potential candidate for an effective cancer theraputic agent in human cervical adenocarcinoma

Analysis of Protein Interaction between HY5 and bZIP17 in Stress Response

이혜비 경상국립대학교 대학원 2023 국내석사

Plants are sessile organisms whose growth and development are influenced by the ambient environmental conditions, therefore the study of their environmental stress responses is highly significant. Various transcription factors are involved in plant stress responses, and the basic leucine zipper (bZIP) family, to which ELONGATED HYPOCOTYL5 (HY5) and bZIP17 belong, are known to play key role in various stress responses. HY5 is known to have multiple physiological and transcriptional functions in plant development, including photomorphogenesis. bZIP17, along with bZIP28, is an endoplasmic reticulum (ER)-anchored transcription factor that is involved in the unfolded protein response (UPR) under ER stress conditions. In this regard, bZIP28 regulates the expression of ER stress response genes in competition with HY5 and its function as a transcription factor is relatively well studied, but the relationship with HY5 and the transcriptional role of bZIP17 are largely unknown. In this study, we characterize the role of bZIP17 as a transcription factor removing its transmembrane domain. Further, identified its subcellular localization to the nucleus, and analyzed its protein-protein binding interaction with HY5 and its function under stress. Using yeast two-hybrid assay and biomolecular fluorescence complementation (BiFC) analysis, examined their binding and found that HY5 and bZIP17 interaction in vivo. To investigate the function of HY5 and bZIP17 in ER stress, we compared the phenotypes of hy5 mutant and 35S::bZIP17-SRDX transformant with an EAR repression domain (SRDX) in tunicamycin (Tm) and azetidine-2-carboxylic acid (AZC) medium, which induce ER stress, and found that the hy5 mutant was tolerant and the 35S::bZIP17-SRDX transformant was sensitive, suggesting that they have opposite functions in ER stress. To gain further insights into their function at the transcriptional level, we found that when treated with dithiothreitol (DTT) or mannitol, which induce ER stress, the expression of bZIP17 in hy5 mutant plants increased and the expression of HY5 remained unchanged or decreased in 35S::bZIP17-SRDX transgenic plants, indicating that HY5 and bZIP17 are involved in each other's expression under ER stress conditions. Overall, the results suggest that bZIP17 may play an important role in stress response through interaction with HY5. 식물은 대표적인 고착생물로서 그 주변 환경에 따라 생장과 발달에 영향을 받기 때문에 환경 스트레스 반응에 대한 연구는 매우 중요하다. 식물의 스트레스 반응에는 다양한 전사인자들이 관여하는데 ELONGATED HYPOCOTYL5 (HY5)와 bZIP17이 속한 basic leucine zipper (bZIP) family는 다양한 스트레스 반응에 중요한 역할을 한다고 알려져 있다. HY5는 대표적으로 photomorphogenesis를 포함한 식물의 발달 등 여러 생리 및 전사 기능을 한다고 알려져 있다. bZIP17은 bZIP28과 함께 endoplasmic reticulum (ER)-anchored transcription factor로 ER 스트레스 조건에서 unfolded protein response (UPR)에 관여한다고 알려져 있다. 이때, bZIP28은 HY5와 서로 경쟁적으로 ER stress response genes의 발현을 조절하며 그 전사인자로서의 기능이 비교적 매우 잘 연구되어 있지만 bZIP17의 HY5와의 관계 및 전사적 역할은 거의 알려지지 않다. 본 연구에서는 bZIP17의 전사인자로서의 특징과 기능을 알아보기 위해 transmembrane domain을 제거하고 핵 내로 위치하도록 클로닝한 다음 HY5와의 결합과 스트레스에서의 기능 분석을 수행하였다. 둘의 결합을 보기 위해 Yeast two-hybrid와 biomolecular fluorescence complementation (BiFC) assay를 진행한 결과, HY5와 bZIP17은 in vivo에서 결합을 하는 것을 보였다. ER 스트레스에서 HY5와 bZIP17의 기능을 조사하기 위해 ER 스트레스를 유발하는 tunicamycin (Tm)과 azetidine-2-carboxylic acid (AZC)배지에서 hy5 돌연변이체와 35S::bZIP17-SRDX 형질전환체의 표현형을 비교한 결과, hy5 돌연변이체는 내성을 가진 표현형을, 35S::bZIP17-SRDX 형질전환체는 민감성을 가진 표현형을 보임으로써 ER 스트레스에서 상반된 기능을 하는 것으로 나타났다. 전사 수준에서의 기능에 대한 추가 통찰력을 얻기 위해 ER 스트레스를 유도하는 dithiothreitol 또는 mannitol로 처리했을 때 hy5 돌연변이 식물에서 bZIP17의 발현이 증가하고 35S::bZIP17-SRDX 형질 전환 식물에서는 HY5의 발현이 변하지 않거나 감소하는 것을 발견하여 HY5와 bZIP17이 ER 스트레스 조건에서 서로의 발현에 관여한다고 예상되어진다. 이러한 결과는 bZIP17이 HY5와의 상호작용을 통해 스트레스 반응에 중요한 역할을 할 수 있음을 시사한다.

Translation of HY5 with Upstream Open Reading Frame (uORF) under Endoplasmic Reticulum (ER) Stress Conditions

박지예 경상대학교 대학원 2020 국내석사

Effects of Dietary Vitamin Levels on Physiological Responses and Reproductive Performance in Sows and Growth of Their Progeny

정재학 서울대학교 대학원 2019 국내박사

Effects of Dietary Vitamin Levels on Physiological Responses and Reproductive Performance in Sows and Growth of Their Progeny Reproductive performance is one of the most important economic values in mordern pig production. Dietary supplementation of vitamins is essential during gestation and lactation sows to prevent reproductive failure. Most of pigs are consumed vitamins as premixes to meet requirements of vitamins in commercial diets (Chae, 2000; NRC, 2012). The amounts of vitamins in the premix may be substantially higher than the requirement because premixes lose vitamin potency depending on feed processing practices and the length and manner of storage (Lauridsen and Jensen, 2005; NRC, 2012). It is well known that most of feed companies in Korea supplemented vitamin in pig diets at levels almost 3 to 10 times to requirement of NRC (2012) because they considered safety margin such as stress or any environmental conditions. Consequently, this is one of the reasons for rising feed cost in Korea. The current study was conducted to investigate the effect of dietary vitamin levels on physiological responses, blood profiles and reproductive performance in gestating sows (Experiment 1), and in lactating sows (Experiment 2), and to evaluate the effects of dietary Hy-D® in diet of gestating sows on physiological responses, blood profiles, reproductive performance and their progeny growth (Experiment 3). Experiment I. Effects of Dietary Vitamin Levels on Physiological Responses, Blood Profiles and Reproductive Performance in Gestating Sows This study was conducted to evaluate the effects of dietary vitamin levels on physiological responses, blood profiles and reproductive performance in gestating sows. A total of 52 F1 multiparous sows (Yorkshire × Landrace) with average body weight (BW) of 223.49 ± 31.65 kg, average backfat thickness of 18.5 ± 4.9 mm, and a average parity of 6.38 ± 2.69 were allotted to one of 4 treatments considering BW, backfat thickness, and pairty in complete randomized design with 13 replicates. Treatments were 100% (V1), 300% (V3), 600% (V6) and 900% (V9) of NRC (2012) requirements. The gestating diet was formulated based on corn-SBM and contained 3,265 kcal of ME/kg, 12.00% crude protein. In lactation period, all sows were fed the same commercial lactating diet. There was no significant difference in body weight of gestating sows, however backfat tended to increase as higher levels of vitamin was provided to gestating sows (P< 0.10). The body weight change of lactating sows was increased when sows were fed higher level of vitamin (P<0.01). The feed intake of lactating sows tended to decrease when sows were fed increasing level of vitamin (P=0.06). Different levels of dietary vitamin did not show any difference in the number of total born, born alive, stillbirth piglets as well as body weight of piglets. The blood concentration of serum 25(OH)D3 of sows at 90 day of gestation showed a linear increment as dietary vitamin increased (P< 0.01). Furthermore, the serum vitamin E level of sows during gestation was higher linearly as dietary vitamin increased (P< 0.05). Consequently, current vitamin requirement of NRC (2012) is enough for gestating sow and additional supplementation of vitamin in gestating diet did not show any beneficial response during gestation. Experiment II. Effects of Dietary Vitamin Levels on Physiological Responses, Blood Profiles and Reproductive Performance in Lactating Sows This experiment was conducted to evaluate the effects of dietary vitamin levels on physiological responses, blood profiles and reproductive performance in lactating sows. A total of 48 F1 multiparous sows (Yorkshire × Landrace) with average body weight (BW) of 236.7 ± 24.27 kg, average backfat thickness of 20.83 ± 5.68 mm, and a average parity of 4.08 ± 0.69 were allotted to one of four treatments considering BW, backfat thickness, and pairty in complete randomized design (CRD) with 12 replicates. Treatments were 100% (V1), 300% (V3), 600% (V6) and 900% (V9) of NRC (2012) requirements. The lactating diet was formulated based on corn-SBM contained 3,300 kcal of ME/kg, 13.43% crude protein. There were no significant differences in body weight of lactating sows at 24 hours postpartum and 21 day of lactation and backfat thickness with dietary vitamin levels. No differences in average daily feed intake of lactating sows and weaning to estrus interval (WEI) were observed when sows were fed various level of vitamin in lactation period. There had no detrimental effect on total litter weight and piglet weight at 21 day of lactation as various levels of dietary vitamin supplemented. The concentrations of serum 25(OH)D3, vitamin E, calcium and phosphorus of sows in V6 and V9 treatments are significantly high compared to those of sows in V1 and V3 treatments. The concentrations of 25(OH)D3 and serum phosphorus of piglet in V6 and V9 treatments were higher compared to those in V1 and V3. Also, the concentrations of 25(OH)D3 and serum phosphorus were significantly elevated as increasing vitamin was supplied to sows (linear, P=0.001 and P=0.002, respectively). Although, serum concentration of vitamin in sows and piglets was increased as increasing level of vitamin in lactating sow diet, increasing level of vitamin failed to improve litter performance. This result demonstrates that supplementing vitamin levels over 3 times of vitamin requirement has no beneficial effect in lactating sows. Experiment III. Effect of Dietary Hy-D® in Diet of Gestating Sows on Physiological Responses, Blood Profiles, Reproductive Performance and Their Progeny Growth This study was carried out to investigate the effect of dietary Hy-D® in diet of gestating sows on physiological responses, blood profiles, reproductive performance and their progeny growth. A total of 30 F1 gilts (Yorkshire × Landrace) with average body weight (BW) of 149.91 ± 10.83 kg, average backfat thickness of 23.1 ± 4.3 mm were allotted to one of 3 treatments considering BW, backfat thickness, and pairty in complete randomized design with 10 replicates. All experimental diets for gestating gilts were formulated based on corn-soybean meal Treatments are as followed: 1) CON : corn-SBM based diet without supplementation of Hy-D® , 2) HD5 : corn-SBM based diet with Hy-D® 0.05%, 3) HD10 : corn-SBM based diet with Hy-D® 0.10%. In lactation period, all sows were fed the same commercial lactating diet. There were no significant differences in body weight, backfat thickness of gestating sows by treatments and differences were not observed in body weight, daily feed intake and WEI of lactating sows. Backfat thickness of sows fed 0.10% Hy-D® was significantly increased (P<0.01). There was no significant difference in litter size among treatments, however total litter of sows fed higher level of Hy-D® was significantly improved (linear, P=0.041). The blood concentration of 25(OH)D3 when sows were fed Hy-D® was significantly increased in 35 d, 70 d of gestation period and 0 day of lactation period. On the other hand, no significant difference was observed in the serum concentration of 1,25(OH)2D3 in sows. When gestating sows were fed Hy-D®, the concentration of 25(OH)D3 in blood of their piglets was significantly higher (P=0.026). Also, the elevated levels of 25(OH)D3 at 24 hours postpartum and 1,25(OH)2D3 at weaning were observed as maternal Hy-D® regimen was increased (linear, P=0.013 and P=0.047, respectively). Body weight of weaning pig at 5 weeks was linearly increased when Hy-D® was provided to sows (linear, P=0.041). Average daily gain in late weaning period was significantly higher in the Hy-D® group. These results demonstrated that 0.1% of Hy-D® in gestating diet increased reproductive performance of sows and total litter weight. Also, supplementation of Hy-D® in diet of gestation sows improved blood concentration of 25(OH)D3 in both sows and piglets subsequently greater growth performance was observed in their progeny. 본 실험은 임신돈 및 포유돈 사료 내 비타민 수준이 모돈의 생리학적 변화와 번식성적 및 자돈의 성장에 미치는 영향을 알아보기 위하여 수행되었다. 총 3 개의 실험으로 구성되어 있으며, 1) 임신돈 사료 내 비타민 수준이 모돈의 생리학적 변화, 번식성적 및 혈액 성상에 미치는 영향, 2) 포유돈 사료 내 비타민 수준이 포유돈의 생리학적 변화, 번식성적 및 혈액성상에 미치는 영향, 3) 임신돈 사료 내 Hy-D®의 수준별 첨가가 모돈의 번식성적과 혈액성상 및 자돈의 성장에 미치는 영향을 평가하였다. 실험 1. 임신돈 사료 내 비타민 수준이 모돈의 생리학적 변화, 번식성적 및 혈액 성상에 미치는 영향 본 연구는 임신돈 사료 내 비타민 수준이 모돈의 생리학적 변화, 번식성적 및 혈액 성상에 미치는 영향을 평가하기 위하여 수행되었다. 총 52두의 F1 경산모돈(Yorkshire × Landrace)을 공시하였으며, 4처리, 13반복, 반복당 1두씩 체중과 산차에 따라 완전임의배치법으로 구배치 하였다. 실험처리구는 비타민의 첨가수준(NRC(2012) 요구량의 1, 3, 6, 9배)에 따라 구분하였다. 포유기에는 동일한 포유돈 사료를 급여하였다. 임신기동안 체중에는 처리구에 따른 유의적인 차이가 나타나지 않았지만, 비타민 수준이 높아질수록 임신기 등지방 두께가 증가하는 경향이 나타났다 (P<0.10). 포유기에는 비타민을 높은 수준으로 급여한 모돈의 체중이 급격하게 감소하는 결과가 나타났으며 (P<0.01), 포유기 사료섭취량 또한 비타민 수준에 따라 유의적으로 감소하는 경향이 나타났다 (P=0.06). 임신돈 사료 내 비타민 수준은 총산자수, 사산수, 생시자돈수에 영향을 미치지 않았으며, 또한 복당 체중 및 자돈의 체중에 있어서도 처리구간 유의적인 차이가 나타나지 않았다. 임신기 혈액 성상에 있어서 임신 90일령에 모돈의 혈중 25(OH)D3 농도가 비타민 수준이 증가함에 따라 통계적으로 유의하게 증가하는 결과가 나타났다. 또한 임신기동안 모돈의 혈중 vitamin E 농도 또한 증가하였다. 포유기 모돈의 혈액 성상 및 돈유의 유성분에 있어서는 처리구간 유의적인 차이가 나타나지 않았다. 임신기 사료 내 비타민 수준이 높아질수록 포유자돈의 혈액 내 25(OH)D3 및 비타민 E의 농도가 유의적으로 증가하는 결과가 나타났다. 결론적으로 임신기 사료 내 비타민을 높은 수준으로 급여하였을 때 임신돈 및 포유자돈의 혈중 비타민 농도를 증가시켜주는 결과가 나타났지만, 모돈의 번식성적에 어떠한 긍정적인 효과도 주지 못하였으며, 포유기 사료섭취량 및 체중이 감소하는 결과를 나타내었다. 따라서 임신돈 사료 내 비타민 수준은 현재 NRC (2012)의 비타민 요구만큼 첨가하여도 충분한 것으로 판단된다. 실험 2. 포유돈 사료 내 비타민 수준이 포유돈의 생리학적 변화, 번식성적 및 혈액성상에 미치는 영향 본 실험은 포유돈 사료 내 비타민 수준이 모돈의 생리학적 변화, 번식성적 및 혈액 성상에 미치는 영향을 평가하기 위하여 수행되었다. 총 48두의 F1 경산모돈(Yorkshire × Landrace)을 공시하였으며, 4처리, 12반복, 반복당 1두씩 체중, 등지방두께 및 산차에 따라 완전임의배치법으로 구배치 하였다. 실험처리구는 비타민의 첨가수준(NRC(2012) 요구량의 100, 300, 600, 900%)에 따라 구분하였다. 실험결과 포유돈 사료 내 비타민의 수준이 증가하여도 분만 21일차 모돈의 체중 및 등지방 두께에 있어서 유의적인 차이가 나타나지 않았다. 또한, 수준별 비타민 공급은 포유기 모돈의 사료섭취량이나 재귀발정일에도 영향을 미치지 않았으며, 포유기 자돈의 성장에도 어떠한 긍정적인 효과도 나타나지 않았다. 하지만 포유돈 사료 내 비타민의 수준이 높아질수록 모돈의 혈중 25(OH)D3 (linear, P<0.01), 비타민 E (linear, P<0.01), 칼슘 (linear, P<0.01)과 인 (linear, P<0.01) 농도가 유의적으로 높아졌으며, 자돈 또한 혈중 25(OH)D3 (linear, P<0.01) 및 인 (linear, P<0.01)의 농도가 높아졌다. 결론적으로 포유돈 사료 내 비타민 증가는 모돈과 자돈의 혈중 비타민 농도를 높이지만 포유능력을 개선시키지 못하였다. 따라서 포유돈 사료 내 비타민은 NRC 요구량만큼만 공급하면 되는 것으로 판단된다. 실험 3. 임신돈 사료 내 Hy-D®의 수준별 첨가가 모돈의 번식성적과 혈액성상 및 자돈의 성장에 미치는 영향 본 연구는 임신돈 사료 내 Hy-D®의 수준별 첨가가 모돈의 번식성적과 혈액성상 및 자돈의 성장에 미치는 영향을 평가하기 위하여 수행되었다. 총 30두의 F1 초산돈(Yorkshire × Landrace)을 공시하였으며, 3처리, 10반복, 반복당 1두씩 체중과 산차에 따라 완전임의배치법으로 구배치 하였다. 실험처리구는 Hy-D®의 첨가수준(0, 0.05% 및 0.10%)에 따라 구분하였으며, 포유기에는 동일한 포유돈 사료를 급여하였다. 임신돈 사료 내 Hy-D®의 첨가는 임신돈의 체중 및 등지방에 영향을 미치지 않았으며, 포유기 체중, 사료섭취량 및 WEI 에 있어서도 처리구간 유의적인 차이가 나타나지 않았다. 하지만 Hy-D®를 0.10% 급여받은 모돈의 등지방 두께가 대조구에 비해 유의적으로 증가하였다 (P<0.01). 임신돈 사료 내 Hy-D®의 첨가는 모돈의 포유성적에 영향을 주지 않았지만, 복당자돈생시체중이 증가하였다 (linear, P=0.041). Hy-D®를 급여받은 모돈의 혈중 25(OH)D3 농도가 임신 35일령, 70일령 및 분만시에 유의적으로 높아졌다. 또한 모돈이 Hy-D®를 공급받았을 때 그 자돈의 생후 24시간 이내 혈중 25(OH)D3 농도가 유의적으로 높아지는 결과가 나타났다 (P=0.026). 이유 후 자돈의 성장성적에 있어서, 이유 5주령에 자돈의 체중이 Hy-D®를 공급한 처리구에서 유의적으로 높은 결과가 나타났다 (linear, P=0.041). 이러한 결과로 보았을 때 임신기 사료 내 0.1%의 Hy-D®첨가는 모돈의 복당자돈생시체중을 증가시켰으며, 모돈과 자돈의 혈중 25(OH)D3의 농도를 높여주어 이유 후 자돈의 성장에 긍정적인 영향을 미치는 것으로 판단된다.

Isolation of VOZ1 as a Novel Target of HY5 and COP1 in Arabidopsis

김유정 경상국립대학교 대학원 2023 국내석사

ELONGATED HYPOCOTYL5 (HY5) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) regulates diverse plant growth and development responses as regulators. In a previous study, we observed that about 86 proteins that interact with HY5 or COP1 using yeast two-hybrid screening. VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) transcription factor is one of the proteins that has been shown to interact with HY5 and COP1 in the previous study and is known to act as a critical regulator of plant growth and development. Recently, it is revealed that VOZ1 is involved in stress response as well as plant growth and development in Arabidopsis. However, it is unclear that the interaction between VOZ1 and other proteins and the function by interacting with other proteins. Here, we demonstrate that VOZ1 interacts with HY5 and COP1, the master regulators of plant growth, development, and stress responses. We show that VOZ1 and HY5, and COP1 physically interact by yeast two-hybrid in yeast and Bimolecular fluorescence complementation (BiFC) assays in planta. Moreover, we also show that 35S::GFP-VOZ1 is located in the nucleus and cytosol. Furthermore, 35S::GFP-HY5 and 35S::GFP-COP1 are located in the nucleus. VOZ1, HY5, and COP1 are commonly located in the nucleus and expected to interact in the nucleus. In Arabidopsis, VOZ1 is known to have a similar function to VASCULAR PLANT ONE-ZINC FINGER2 (VOZ2). To investigate the function of the VOZ1 transcription factor, we made 35S::VOZ1-SRDX lines. In this paper, we observed that 35S::VOZ1-SRDXs are sensitive to salt stress as compared to the wild type, and VOZ1 regulates the expression of salt-related genes to gain tolerance in salinity. Furthermore, we observed that interactions between MAP kinases (Mitogen-activated protein kinases) as key regulators of salt stress with VOZ1, suggesting that VOZ1 regulates stress responses by binding with these regulatory proteins. Thus, our work reveals that VOZ1 is a key target for HY5 and COP1 and MAP kinases and functions as a positive regulator in salt stress. ELONGATED HYPOCOTYL5 (HY5)와 CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)은 regulator로서 다양한 식물의 생장과 발달 과정을 조절한다. 따라서 이들의 기능을 파악하기 위해 yeast two-hybrid 실험을 이용해 다른 식물 전사인자들과 상호작용하는 지 확인하였으며 그 결과, 86개의 HY5 또는 COP1과 결합하는 전사인자 library를 구축하였다. 그 중 VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) 전사인자는 HY5와 COP1 모두와 결합을 보였으며 식물의 생장과 발달 과정에서 매우 중요한 regulator의 기능을 하는 것으로 아주 잘 알려져 있으며, 최근 연구에서는 VOZ1이 식물 생장과 발달과정 뿐만 아니라 스트레스 반응에도 관여하고 있는 것으로 밝혀졌다. 하지만 VOZ1이 다른 조절 단백질들과 상호작용 해 기능하는지에 대해서는 아직까지 많은 연구가 이루어지지 않았다. 본 연구는 VOZ1과 식물 내 주요 조절 단백질인 HY5와 COP1의 결합단백질로서 VOZ1의 단백질 결합과 환경 스트레스 저항성을 분자 수준에서 연구하고자 하였다. Yeast two-hybrid를 통해 이들 사이 결합을 확인한 결과 VOZ1이 HY5와 COP1과 yeast 상에서 물리적으로 결합한다는 것을 확인할 수 있었다. GFP 형광 단백질과 fusion하여 이들의 위치를 확인한 결과 VOZ1은 핵과 세포질에 존재하였고 HY5는 핵에 위치하였으며 COP1은 주로 핵에 위치하나 세포질에도 일부 존재함을 확인하였다. 또한 이들이 같은 식물 세포 내에서는 어떻게 위치하였는지 확인하기 위해 이들을 같은 세포에 동시에 넣어서 위치를 확인한 결과 이들이 같은 위치에 존재한다는 것을 확인하였으며 공통적으로 위치하는 핵에서 결합이 있을 것이라는 것을 예측할 수 있었다. Bimolecular fluorescence complementation (BiFC) assay를 이용해 plant cell 내에서 이들의 결합을 확인한 결과 HY5와 VOZ1은 핵에서 결합을 보였으며 VOZ1과 COP1은 주로 핵에서 결합을 보였으나 일부 세포질에서도 결합을 보이는 것을 확인할 수 있었다. VOZ1은 식물 내에서 같은 전사인자 과에 속하는 VASCULAR PLANT ONE-ZINC FINGER2 (VOZ2)와 유사한 기능을 한다고 잘 알려져 있다. 따라서 VOZ1의 기능을 자세히 파악하기 위해 Chimeric Repressor gene Silencing Technology (CRES-T)를 이용해 35S::VOZ1-SRDX plant line을 형성하여 VOZ1의 기능을 파악하고자 하였다. HY5, COP1과 VOZ1 모두 식물 내에서 salt stress에 관여하고 있다고 연구결과가 있기 때문에 이들 사이 연관성을 찾고자 NaCl이 첨가된 배지에 hy5 k/o, cop1-4, voz1 k/o, 35S::VOZ1-SRDX를 배양한 결과 이들은 모두 높은 농도의 NaCl 배지에서 잘 자라지 못하였으며 따라서 이들이 상호작용하여 salt stress 내에서 positive regulator로 작용할 것이라 예측할 수 있었다. VOZ1이 salt stress에 어떻게 관여하는 지 알아보기 위해 yeast two-hybrid 실험을 통해 stress response에 관여한다고 알려진 MAP kinases(MPKs)와의 결합을 확인한 결과 VOZ1이 MPK3, MPK6와 결합한다는 것을 확인하였으며 이들의 constitutive active form과 결합을 확인한 결과 MPK3, MPK4, MPK6의 constitutive active form과 결합을 하는 것을 확인할 수 있었다. 이러한 결과를 통해 VOZ1이 다양한 조절단백질과 식물 내에서 결합하여 기능하며 salt stress를 조절할 것이라는 것을 확인할 수 있었다.

Biochemical Characterization of HY5-interacting Proteins Using Tandem Affinity Purification of HY5 bZIP Protein

레티트랑 경상대학교 대학원 2019 국내석사

Identification and functional analysis of Arabidopsis transcription factor interactome involving HY5 and COP1 in plant development

김혜진 경상대학교 대학원 2015 국내박사

파이토크롬-PIF인자 상호작용 연구 및 애기장대에서의 Brachypodium HY5 유전자들의 기능 분석

권현우 전남대학교 2021 국내석사

식물의 파이토크롬은 식물의 생장 및 발달을 조절하는 적색 및 원적색 광수용체이다. 이들은 적색광을 흡수하는 Pr 및 원적색광을 흡수하는 Pfr 형태로 존재하며, 적색광에 노출되면 Pr 불활성화 형태는 생리적으로 Pfr 활성화 형태로 전환된다. 파이토크롬은 하위 신호 인자들과 상호작용하는데, 그 중에서 PIF (phytochrome-interacting factor) 인자들이 중추적인 역할을 하는 것으로 알려졌다. 이에 본 연구에서는 귀리 파이토크롬A (AsphyA), 애기장대 파이토크롬B (AtphyB) 및 숲개밀 파이토크롬A (BdphyA)의 식물 파이토크롬과 애기장대에서 알려진 8 종의 PIF (AtPIF1~AtPIF8) 인자 간의 상호작용을 분석하였다. 그 결과, AtphyB는 모든 AtPIF 인자들과 Pfr 특이적 상호작용하는 반면, AsphyA는 AtPIF2, AtPIF5 및 AtPIF6를 제외한 5 종의 AtPIF 인자들과 상호작용하는 것을 확인하였다. 흥미롭게도, BdphyA는 AtphyB와 유사하게 모든 AtPIF 인자들과 상호작용하는 것을 확인하였다. 추가적으로, AsphyA/AtphyB와 4종의 AtPIF (AtPIF1, AtPIF3, AtPIF6 그리고 AtPIF8) 사이의 상호작용에 대한 정량적 분석을 수행했다. 정량적 상호작용 분석 결과, AtPIF1과 AtPIF8은 AtphyB보다 AsphyA에 높은 친화도를 보인 반면, AtPIF3와 AtPIF6는 AtphyB에 더 높은 친화도를 보여주었다. 본 연구의 결과는 파이토크롬이 서로 다른 친화도를 가진 AtPIF 인자에 대한 특이성을 가지고 있음을 제시하였다. 본 연구의 두 번째 주제에서는 단자엽 모델 식물인 숲개밀(Brachypodium distachyon)에서 분리된 3종의 HY5 (elongated hypocotyl 5) 유전자의 기능을 애기장대에서 분석하였다. 애기장대에서 HY5는 광형태형성의 양성적 조절을 위한 핵심 전사인자로 알려져 있다. 본 연구에서는 3종의 숲개밀 HY5 유전자(BdHY5-1, BdHY5-2, BdHY5-3로 명명)를 HY5 또는 HY5 및 HYH 결핍 애기장대 (hy5 또는 hy5hyh)로 도입시킨 형질전환체를 하여 기능적 상보성을 분석하였다. 형질전환체의 광 반응성 분석 결과, hy5 및 hy5hyh 식물들은 긴 하배축을 보이는 반면, BdHY5 도입 식물체들은 야생형 애기장대 (Ws-2 또는 Col-0)와 유사하게 짧아진 하배축 길이를 보여주었다. 그리고 중력 방향으로의 뿌리 생장이 억제되는 hy5 및 hy5hyh 식물과는 달리, BdHY5 도입 식물체들의 뿌리 각도는 대조구 식물체와 유사하게 나타났다. 그러므로, 본 연구의 결과는 단자엽 식물로부터 분리된 3 종의 BdHY5 유전자가 모두 쌍자엽 식물인 애기장대에서 기능을 한다는 것을 제시할 수 있었다. Plant phytochromes are red and far-red photoreceptors that regulate plant growth and development. They exist either red light-absorbing Pr form or far-red light-absorbing -Pfr form. Upon red light exposure, the inactive Pr form converted to the physiologically active Pfr form. Phytochromes interact with many downstream signaling components, among which phytochrome-interacting factors (PIFs) are known to play a pivotal role for the negative regulation of photomorphogenesis. In this study, the interactions between plant phytochromes and eight PIFs in Arabidopsis thaliana (AtPIF1 to AtPIF8) were analyzed using Avena sativa phytochrome A (AsphyA), A. thaliana phytochrome B (AtphyB), and Brachypodium distachyon phytochrome A (BdphyA).Results showed that AtphyB interacted with all eight AtPIFs in a Pfr-specific manner, while AsphyA interacted with five AtPIFs, excluding AtPIF2, AtPIF5, and AtPIF6. Interestingly, BdphyA interacted with all the PIFs, similar to AtphyB. In addition, quantitative analysis was performed for the interactions between AsphyA/AtphyB and four AtPIFs (AtPIF1, AtPIF3, AtPIF6 and AtPIF8). Results showed AtPIF1 and AtPIF8 had a higher affinity to AsphyA than that to AtphyB, while AtPIF3 and AtPIF6 had a higher affinity to AtphyB. The results of the present study suggest that phytochromes have distinct specificity to AtPIFs with different binding affinities. In the second topic of this study, three HY5 (elongated hypocotyl 5) genes were isolated from a monocotyledonous model plant, Brachypodium distachyon. HY5 is known as a master transcription factor for the positive regulation of photomorphogenesis in A. thaliana. In the study, three B. distachyon HY5 genes, named BdHY5-1 (Bradi3g07540), BdHY5-2 (Bradi1g36750), and BdHY5-3 (Bradi2g04590), were introduced into HY5 or HY5- and HYH-deficient plant (hy5 or hy5hyh; Ws or Col-0 ecotype, respectively), and the transgenic plants was analyzed to examine functional complementation. Under light conditions such as red, far-red, blue, and white light, hy5 and hy5hyh plants exhibited long hypocotyls, whereas white light, the transgenic plants expressing three BdHY5 genes showed shortened hypocotyls similar to wild-type Arabidopsis plants (Ws-2 and Col-0). Moreover, while hy5 and hy5hyh plants exhibited defective root gravitropism, the transgenic plants showed similar root angles to wild-type plants. Therefore, the present results suggest that all three BdHY5 genes isolated from a monocotyledonous plant are functional in Arabidopsis, a dicotyledonous plant.

Protective Effect of Fermented Gelidium amansii and Cirsium japonicum Extract Mixture and Lactiplantibacillus plantarum HY7714 on UVB-induced Skin Photo-aging and Their Applications

김민교 경희대학교 대학원 2022 국내박사

The interest in health, which was considered the exclusive property of the middle-aged and older people, is emerging as a global concern, transcending age and gender, as new infectious diseases such as Corona 19 threaten the health of people around the world due to climate change caused by environmental pollution. Accordingly, the functional material and food market that can promote health is steadily growing. In addition, the inner beauty market, which pursues healthy beauty from the inside, is also growing rapidly due to a change in perception to maintain a more youthful and beautiful health along with an increase in the number of self-care people who want to take care of their health. Collagen, which decreases with age and causes a decrease in skin elasticity, hyaluronic acid related to skin moisture, and ceramide, which is a major lipid component of the stratum corneum of the skin, strengthens the skin barrier and prevents moisture evaporation, have been known as skin health materials. These functional materials are being applied to cosmetics and functional foods for skin health. Recently, as natural materials-derived materials are in the spotlight, fermented natural extracts and probiotics are being researched and developed as major skin-related functional materials. Fermented natural extracts are fermented extracts of herbs, medicinal plants, and seaweeds with beneficial microorganisms such as lactic acid bacteria. In Korea, the industrialization of skin health functional materials has been made based on the research results of natural extracts that help maintain skin health from UV damage or skin moisturize. Photo-aging refers to damage to the skin caused by ultraviolet rays and oxidative stress, which increases the production of free radicals by decreasing the expression of antioxidant enzymes. It induces changes in the intracellular signal transduction system and increases the activity of matrix metalloproteinases (MMPs), thereby promoting skin photo-aging such as reduction of skin elasticity and generation of wrinkles. Ultraviolet rays are divided into UVA, UVB, and UVC depending on the wavelength of the ultraviolet rays. Among them, UVB has more than 1,000 times the intensity compared to other wavelengths, so it causes skin erythema, sunburn, etc. when exposed to acute exposure, and causes dryness when exposed to chronic conditions. It is the main cause of photo-aging by causing deep, rough wrinkles and skin cancer. In this study, the photo-aging inhibitory effects of natural fermented extracts and probiotics derived from Jeju natural resources and breast milk were investigated in human fibroblasts and SKH-1 hairless mice. In addition, a mass production process was developed for the application of inner beauty functional materials. In Chapter 1, Gelidium amansii and Cirsium japonicum, which have excellent procollagen synthesis ability, were selected and the mixture was fermented with lactic acid bacteria to confirm the effect of preventing photo-aging of the skin. The effect of fermented Gelidium amansii and Cirsium japonicum extract mixture (FGCM) on UVB-induced photo-aging was evaluated in human dermal fibroblasts (Hs68 cell) and SKH-1 hairless mice. In vitro, FGCM increased type I procollagen levels and suppressed UVB-induced matrix metalloproteinase-1 (MMP-1) expression more effectively than G. amansii and C. japonicum extract mixture (GCM). In vivo, oral administration of FGCM significantly inhibited UVB-induced the number and total depth of wrinkles in the dorsal skin of mice. FGCM suppressed UVB-induced epidermal thickening, and attenuated UVB-induced MMP-13 expression and MMP-2 and MMP-9 activities in dermal tissue. Furthermore, FGCM increased skin hydration and blocked transepidermal water loss in the dorsal skin of mice compared with the UVB-irradiated group. These data indicate that FGCM exerts potent anti-photo-aging activities by improving wrinkle formation and dryness. Probiotics, defined as living microorganisms that enter the body and have beneficial health effects when consumed in moderation, recent clinical studies have reported that probiotics may exert other health-promoting effects beyond gut health. In addition to lowering blood cholesterol, preventing tooth decay, alleviating menopausal symptoms, and immune modulation, it has been studied to exert skin potential such as atopic dermatitis, healing burns and scars, skin rejuvenation properties and improving the skin's innate immunity. In Chapter 2, we evaluated the effect of Lactiplantibacillus plantarum HY7714 against UVB-induced photo-aging in human dermal fibroblasts and hairless mice. The results showed that L. plantarum HY7714 treatment effectively rescued UVB-reduced procollagen expression through the inhibition of UVB-induced matrix metalloproteinase (MMP)-1 expression in human dermal fibroblasts. Data from a western blot showed that L. plantarum HY7714 inhibited phosphorylation of Jun N-terminal kinase (JNK), thereby suppressing UVB-induced phosphorylation and expression of c-Jun. Oral administration of L. plantarum HY7714 clearly inhibited the number, depth, and area of wrinkles in hairless mouse skin. Histological data showed that L. plantarum HY7714 significantly inhibited UVB-induced epidermal thickness in mice. Western blot and zymography data also revealed that L. plantarum HY7714 effectively inhibited MMP-13 expression as well as MMP-2 and -9 activities in dermal tissue. For the industrialization of L. plantarum HY7714 as a probiotic with skin photo-aging inhibition, high-concentration powdering technology and maintaining shelf life stability are essential. Since functional probiotic raw materials has to maintain 1.0 ×1011CFU/g for shelf life stability, this study aimed to produce high-concentration powder of L. plantarum HY7714 and maintain stability. The number of viable cells for each process after optimization was 5.90×109cfu/ml in culture medium and 4.2×1011CFU/g in freeze-dried powder to produce high-concentration probiotic powder. As a result of the stability test, 1×1011CFU/g was maintained until 16 weeks to develop a commercial probiotic with a skin photo-aging inhibitory function. These results provide further insight regarding the skin biological actions of L. plantarum HY7714, a potential skin health ingredients. Overall, it was confirmed that fermented Gelidium amansii and Cirsium japonicum extract mixture (FGCM) and Lactiplantibacillus plantarum HY7714 on UVB-induced skin photo-aging inhibitory effects and could expand industrial applicability as skin health ingredients that can be safely consumed as food. Due to this, its applicability is expected to be expanded not only in the recently growing inner beauty food market, but also in the cosmetics and personal care markets. Chronic ultraviolet (UV) light causes skin photo-aging, characterized by fine and coarse wrinkle formation and dryness. Among the native plants of Jeju-island, Gelidium amansii and Cirsium japonicum, which have excellent procollagen synthesis ability, were selected and the mixture was fermented with lactic acid bacteria to confirm the effect of preventing photo-aging of the skin. The effect of fermented Gelidium amansii and Cirsium japonicum extract mixture (FGCM) on UVB-induced photo-aging was evaluated in human dermal fibroblasts (Hs68 cell) and SKH-1 hairless mice. In vitro, FGCM increased type I procollagen levels and suppressed UVB-induced matrix metalloproteinase-1 (MMP-1) expression more effectively than G. amansii and C. japonicum extract mixture (GCM). In vivo, oral administration of FGCM significantly inhibited UVB-induced the number and total depth of wrinkles in the dorsal skin of mice. FGCM suppressed UVB-induced epidermal thickening, and attenuated UVB-induced MMP-13 expression and MMP-2 and MMP-9 activities in dermal tissue. Furthermore, FGCM increased skin hydration and blocked transepidermal water loss in the dorsal skin of mice compared with the UVB-irradiated group. These data indicate that FGCM exerts potent anti-photo-aging activities by improving wrinkle formation and dryness. Keywords: Anti-wrinkle, Cirsium japonicum, Gelidium amansii, Skin hydration, Dryness, Fermented Gelidium amansii and Cirsium japonicum extract mixture (FGCM), Photo-aging Ultraviolet (UV) irradiation alters multiple molecular pathways in the skin, thereby inducing skin damage, including photo-aging. In recent years, probiotics have gained interest due to their beneficial effects on skin health, such as inhibiting atopic dermatitis and improving skin immunity or inflammation. However, little is known about the effects of probiotics on UVB-induced photo-aging. In this study, we evaluated the effect of Lactiplantibacillus plantarum HY7714 against UVB-induced photo-aging in human dermal fibroblasts and hairless mice. The results showed that L. plantarum HY7714 treatment effectively rescued UVB-reduced procollagen expression through the inhibition of UVB-induced matrix metalloproteinase (MMP)-1 expression in human dermal fibroblasts. Data from a western blot showed that L. plantarum HY7714 inhibited phosphorylation of Jun N-terminal kinase (JNK), thereby suppressing UVB-induced phosphorylation and expression of c-Jun. Oral administration of L. plantarum HY7714 clearly inhibited the number, depth, and area of wrinkles in hairless mouse skin. Histological data showed that L. plantarum HY7714 significantly inhibited UVB-induced epidermal thickness in mice. Western blot and zymography data also revealed that L. plantarum HY7714 effectively inhibited MMP-13 expression as well as MMP-2 and -9 activities in dermal tissue. For the industrialization of L. plantarum HY7714 as a probiotic with skin photo-aging inhibition, high-concentration powdering technology and maintaining shelf life stability are essential. Since functional probiotic raw materials has to maintain 1.0 ×1011 CFU/g for shelf life stability, this study aimed to produce high-concentration powder of L. plantarum HY7714 and maintain stability. The number of viable cells for each process after optimization was 5.90×109 CFU/mL in culture medium and 4.2×1011 CFU/g in freeze-dried powder to produce high-concentration probiotic powder. As a result of the stability test, 1×1011 CFU/g was maintained until 16 weeks to develop a commercial probiotic with a skin photo-aging inhibitory function. Collectively, these results provide further insight regarding the skin biological actions of L. plantarum HY7714, a potential skin health ingredients. Key words Lactiplantibacillus plantarum, photo-aging, probiotics, ultraviolet B (UVB), Skin hydration, Anti-, Industrialization, shelf life stability, Optimization, Skin health ingredients