This study investigated the influence of sodium bicarbonate (NaHCO3) and progesterone on acrosome reaction and proportion of polyunsaturated fatty acid (PUFA) composition boar sperm. The sperm were diluted with semen extender and incubated with NaHCO3 and progesterone at 38℃, 5% CO2 for 6 h. Plasma membrane integrity and acrosome reaction were analyzed using SYBR14/propidium iodide (PI) and FITC-PNA/PI doubling staining method, and proportion of PUFA was analyzed using gas chromatography. In results, Plasma membrane integrity was significantly decreased in 50 mM NaHCO3 group and acrosome reaction was significantly increased by over the 100 mM NaHCO3 group compared to control group (p < 0.05). In addition, progesterone significantly increased decreased plasma membrane integrity at 100 mM progesterone and acrosome reaction at over the 5.0 µM progesterone (p < 0.05), but there was no difference among the 5.0 to 100 µM groups. PUFAs were significantly decreased in 100 mM NaHCO3 and 50 µM progesterone treatments compared to control group. In summary NaHCO3 and progesterone induce acrosome reaction and reduce PUFA composition in boar sperm, therefore, the results maybe help to understand basically knowledge for the acrosome reaction and PUFA composition in boar sperm.
높은 안전성과 견고한 기계적 특성을 가진 고체상 슈퍼커패시터는 차세대 에너지 저장 장치로서 세계적 관심을 끌고 있다. 슈퍼커패시터의 전극으로서 경제적인 탄소 기반 전극이 많이 사용되는데 수계 전해질을 도입하는 경우 소수성 표 면을 가진 탄소 기반 전극과의 계면 상호성이 좋지 않아 저항이 증가한다. 이와 관련하여 본 연구에서는 전극 표면에 산소 플라즈마 처리를 하여 친수화된 전극과 수계 전해질 사이의 향상된 계면 성질을 기반으로 더 높은 전기화학적 성능을 얻는 방법을 제시한다. 풍부해진 산소 작용기들로 인한 표면 친수화 효과는 접촉각 측정을 통해 확인하였으며, 전력과 지속시간을 조절함으로써 친수화 정도를 손쉽게 조절할 수 있음을 확인하였다. 수계 전해질로 PVA/H3PO4 고체상 고분자 전해질막을 사 용하였으며 프레싱하여 전극에 도입하였다. 15 W의 낮은 전력으로 5초간 산소 플라즈마 처리를 시행하는 것이 최적 조건이 었으며 슈퍼커패시터의 에너지 밀도가 약 8% 증가하였다.
In the present study, we examined the effect of straw size on spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. Hanwoo semen was collected from three bulls and diluted with an animal protein-free extender, divided into two groups, namely, 10 million spermatozoa in 0.25 mL and 20 million spermatozoa in 0.5 mL straw, and cryopreserved. In Experiment 1, the motility and motility parameters of the frozenthawed spermatozoa were evaluated. After freezing-thawing, the spermatozoa motility parameters fast progressive, straight line velocity, and average path velocity were compared between the 0.25 mL straw and 0.5 mL straw groups. They were 35.2 ± 1.0 and 32.3 ± 0.7%, 34.6 ± 0.7 and 31.8 ± 0.5 μm/s, 51.4 ± 1.3 and 47.1 ± 1.1 μm/s, 0.25 mL straw and 0.5 mL straw groups, respectively. In Experiment 2, the viability, acrosome membrane integrity, and mitochondrial membrane potential of the frozen-thawed spermatozoa were assessed. After freezing-thawing, the percentages of spermatozoa with live, intact acrosomes and high mitochondrial membrane potential were compared between the in 0.25 mL straw and 0.5 mL straw groups. They were 48.0 ± 2.6% and 35.6 ± 2.8% between the 0.25 mL straw and 0.5 mL straw groups. In Experiment 3, the plasma membrane integrity of frozen-thawed spermatozoa was compared. After freezingthawing, the plasma membrane integrity was higher for the in 0.25 mL straw group than the 0.5 mL straw group. They were 62.0 ± 2.2 and 54.1 ± 1.3% between the 0.25 mL straw and 0.5 mL straw groups. In conclusion, our results suggest that freezing semen in 0.25 mL straw improves the relative motility, viability, and acrosomal, mitochondrial membrane potential, and plasma membrane integrity of Hanwoo bull spermatozoa.
In this study, we examined number, motility and plasma membrane integrity of spermatozoa from six regions of epididymis in bull. Six testicles with epididymides were castrated from six bulls (mean±standard error, age of days = 441.3±9.6, body weight (kg) = 367±8.4, scrotal circumference (cm) = 30.7±0.4) at Hanwoo Research Institute, NIAS and transported to laboratory within 1 hour. Testicular weight, length, width and circumference were recorded. Epididymis in each bull was randomly used for recovery of spermatozoa. Epididymis was divided into six regions: efferent duct (ED), caput, corpus, proximal cauda (Pcauda), distal cauda (Dcauda) and vas deferens (VD). In experiment 1, we examined sperm number of each region of epididymis. Each region of epididymis contained different number of spermatozoa: ED (37.8±15.7 × 106cells/ml, 8.2%), caput (93.6±18.8 × 106cells/ml, 20.2%), corpus (33.0±8.5 × 106cells/ml, 7.1%), Pcauda (104.2±23.5 × 106cells/ml, 22.5%), Dcauda (180.5±32.5 × 106cells/ml, 39.0%) and VD (14.0±5.0 × 106cells/ml, 3.0%). In experiment 2, sperm motility of each epididymal region was examined by computer assisted sperm analysis (SCA, MicroOptic) system. Sperm motility was divided into 4 groups (fast progressive, slow progressive, non-progressive and immotile) based on WHO guideline. Percentages of fast progressive of Pcauda and Dcauda (11.0±2.3 and 15.4±3.6%) were significantly higher than that of ED, Caput, Corpus and VD which is 0.1±0.1, 1.5±0.6, 1.9±0.7 and 0.3±0.2%, respectively (p<0.05). In experiment 3, percentage of intact plasma membrane spermatozoa of each regions were examined by hypoosmotic swelling test. Percentages of intact plasma spermatozoa were not significantly different among six regions of epididymis: ED, caput, corpus, Pcauda, Dcauda and VD which is 68.0±8.6, 74.0±5.3, 68.5±6.2, 70.8±5.5, 71.0±5.8 and 64.6±10.8%, respectively. In conclusion, in the present study, we found out distribution, motility and plasma membrane integrity of spermatozoa from six regions of epididymis in Hanwoo bull. These results will be contributed to basic research about spermatozoa transportation and characters in epididymis of bull.
In this study, we examined total number, motility and plasma membrane integrity of epididymal spermatozoa from cauda epididymis of bull after preservation at 4ºC. Totally, 23 testicles were castrated from 23 bulls (mean±standard error, age of days = 426.0±7.3, body weight (kg) = 379.7±8.4, scrotal circumference (cm) = 31.0±0.4) at Hanwoo Research Institute, NIAS, and transported to laboratory and preserved on 1, 4 and 6 days at 4 ºC. As control, epididymal spermatozoa recovery from 7 testicles was conducted after transportation to laboratory immediately. In experiment 1, we compared total number of spermatozoa among groups. Total number of spermatozoa from epididymis was not significantly on different preservation day of 0, 1, 4 and 6 which is 1778.0±304.7, 1824.8±343.9, 1228.4±91.7, 1201.8±178.6×106 cells/ml, respectively). In experiment 2, we examined spermatozoa motility and motility parameters (VCL (μm/s), VSL (μm/s), VAP (μm/s), LIN (%)) by computer assisted sperm analysis (SCA, MicroOptic) system. Percentage of motile on 0 and 1 day (88.9±5.2 and 85.8±6.1) was significantly higher than that on 4 and 6 days (32.6±6.5 and 34.3±8.25). Percentage of VCL (μm/s) on 0 and 1 day (93.5±7.6 and 83.0±14.9) was significantly higher than that on 4 and 6 days (36.6±5.1 and 39.5±5.5) (p<0.05). Percentage of VSL (μm/s) on 0 day (28.0±2.1) was significantly higher than that on 1, 4 and 6 days (20.2±3.0, 9.0±2.0 and 8.5±1.6, p<0.05). Percentage of VAP (μm/s) on 0 and 1 days (49.4±3.8 and 41.3±6.6) was significantly higher than that on 4 and 6 days (18.2±3.0 and 19.3±2.8, p<0.05). Percentage of LIN (%) on 0 day (30.7±2.6) was significantly higher than that on 4 and 6 days (23.4±2.7 and 21.1±1.0, p<0.05). Motility of spermatozoa was divided into 4 groups (fast progresive, slow progressive, non-progressive and immotile) based on WHO guideline. Percentage of fast progressive on day at 0 was significantly higher than that on 1, 4 and 6 days (0, 1, 4 and 6 days vs. 19.8±1.9, 10.2±1.1, 2.6±1.0 and 2.3±1.2%, respectively). In conclusion, cauda epididymal spermatozoa should be recovered within one day after preservation at 4 ºC to recover high quality of epididymal spermatozoa in Hanwoo bull
본 연구에서는 막 증류법에 사용되는 PVDF 분리막을 나노입자로 개질하여 젖음 현상에 미치는 영향을 확인하였다. 나노입자를 분리막에 적용하기 위하여 널리 사용되는 dip-coating 방법의 경우, 나노입자에 의한 기공 막힘 현상과 나노입자와 표면과의 부착력이 약하다는 문제점이 있다. 이를 해결하기 위하여, plasma 전처리를 통해 분리막 표면의 기공 크기를 키워주었으며, Fenton 반응을 통해 분리막 표면에 SiO2의 성장 작용기인 OH기를 생성시켜 주었다. 이로부터 SiO2를 성장시켜 준 뒤, fluoroalkylsilanes (FAS)를 이용하여 SiO2 표면의 소수성 처리를 통해 막을 준비하였다. 상기 방법을 통해 준비된 분리막과 처리 전분리막의 젖음 정도를 막증류 법으로 비교하였다.
The present study was conducted to investigate the effect of BHT supplementation on sperm motility, viability, acrosomal integrity and plasma membrane integrity after frozen-thawing. One ejaculate was collected from one fertile Hanwoo bull by using artificial vagina at Hanwoo Research Institute. The ejaculate was transferred to laboratory immediately and diluted with pre-warmed semen extender (Optixcell, France) (1:1). Sperm dilutions were extended to a final concentration of 40 x 106 sperm/ml, and divided into 5 groups according to BHT concentration (0, 0.5, 1.0, 2.0 and 4.0 mM) and cryopreserved in LN2 tank until evaluation. Frozen-thawed semen was transferred to 1.5 ml tube and incubated for 0, 2 and 4h. Sperm motility and motility parameters (total motility, VSL with 25μm≥, VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were evaluated by sperm class analysis (SCA, IVOS, Spain). There were not significant effects of BHT supplementation (0, 0.5, 1.0 and 2.0 mM) on total motile and VSL with 25μm≥ at 0, 2 and 4h. However, 4.0 mM of BHT supplementation showed negative effect on total motile (26.3%), VSL with 25μm≥ (1.3%) at 0 h (p<0.001). The viability and acrosomal integrity of spermatozoa were evaluated by Trypanblue/Giemsa staining method and divided into 4 groups; live and intact acrosome (LIA), live and damaged acrosome (LDA), dead intact acrosome integrity (DIA), dead damaged acrosome (DDA). There were no significant differences of LIA, LDA, DIA and DDA on various BHT concentrations at 0 and 2 h. However, 4.0 mM BHT supplementation showed decreased LIA compared with 0, 0.5, 1.0 and 2.0 mM BHT at 4 h (34.6, 37.1, 43.6, 45.4 and 14.7% vs. 0, 0.5, 1.0, 2.0 and 4.0 mM, irrespectively; p<0.01). Addition of 4.0 mM of BHT showed negative effect on plasma membrane integrity compared with that of 0, 0.5, 1.0 and 2.0 BHT at 2 h (71.9, 64.2, 64.6, 67.5 and 31.7 % vs. 0, 0.5, 1.0, 2.0 and 4.0 mM, irrespectively; p<0.05). In conclusion, various BHT concentrations on optixcell extender showed no improvement on sperm motility, viability and plasma membrane integrity.
The purpose of this study is to evaluate the effects of astaxanthin added to freezing buffer on semen parameters, total sperm oxidation stress after post-thawing of boar sperm and lipid peroxidation (LPO) which is caused by reactive oxygen species (ROS) in sperm membrane. Varying concentrations of astaxanthin (0, 10, 50, 100 and 500 μM) were used in the freezing buffer during cryopreservation to protect the DNA of thawed miniature pig sperm. Semen parameter was measured using computer assisted sperm analysis (CASA) for sperm motility and determine ROS rate, oxidative stress of boar sperm using fluorescence-activated cell sorting (FACS). Sperm motility was higher (p<0.05) in the astaxanthin group than in the control group. Sperm motility and the number of progressive motile sperm was higher (p<0.05) in the astaxanthin 500 μM group (66±1.7%) than in the control group (49.8±4%). In ROS evaluation, the astaxanthin group lowered intracellular O2 and H2O2 in viable sperm. The Yo-Pro-I/HE and PI/H2DCFDA staining as revealed using flow cytometry was lower in astaxanthin groups than in the other groups. As the result, we found that astaxanthin could protect the sperm plasma membrane from free radical and LPO during boar sperm post-thawing.
The purpose of this study was to examine the effects of taurine and vitamin E on ovarian granulosa cells damaged by bromopropane (BP) in pigs. We evaluated cell viability, plasma membrane integrity (PMI) and apoptotic morphological change in porcine ovarian granulosa cells. The cells were treated with 1-BP (0, 5.0, 10, and 50 μM), 2-BP (0, 5.0, 10, and 50 mM), taurine (0, 5.0, 10, and 25 mM), and vitamin E (0, 100, 200, and 400 μM) for 24 h. 10 μM 1-BP and 50 μM 2-BP inhibited viability and PMI, and induced apoptosis in porcine ovarian granulosa cells (p < 0.05). Cell viability and PMI were increased by taurine (10 and 25 mM) and vitamin E (100 and 200 μM), and apoptosis decreased (p < 0.05). Finally, the porcine ovarian granulosa cells were co-treated with BPs (10 μM), taurine (10 mM) and/or vitamin E (200 μM). Cell viability and PMI in the co-treated cells were increased, and apoptosis was decreased. In conclusion, taurine and vitamin E can improve cell viability and inhibition of apoptosis in porcine ovarian granulosa cells damaged by bromopropane.
본 연구는 고온과 고습 조건 하에서 양파 화구의 총 단백질 발현과 원형질막 H+ATPase 영향을 조사하고자 조생종 '신선황'과 중생종 '맵시황'를 이용하여 실험을 수행하였다. '신선황'과 '맵시황' 품종의 양파 회구 개화전, 반개화, 그리고 만개 단계에서 단백질의 양생에는 차이를 보이지 않았지만 결실 단계에서는 유도 및 비유도되는 단백질이 현저하게 나타났다. 양파 화구 개화 후 실시한 고온과 고습처리 18일째의 양파두 품종의 단벡질도 현저하게 감소하였고 특히 고온처리구에서 더 감소되는 경향을 보였다. 원형절막 H+ATPase 발현을 western-blot으로 살펴본 결과, '신선황' 과 '맵시황'의 경우 대조구에서 원형질막 H+ATPase 단백질 발현이 유지 되는 반면에, 고온처리구에서는 두품종 모두 원형질막 H+ATPase가 발현 되지 않았다. 고습처리구에서 중생종 '맵시황'약 원형질막 H+ATPase는 발현 되었는데 조생종 '신선황'에서 발현되지 않았다. 이는 양파 종자 성숙단계에서 고온 조건을 조우하면 고습처리 보다는 단백질 및 원형질막의 H+ATPase피해가 더 심각함을 보여 주는 결과이다.
Hypoosmotic swelling test (HOST) is used for evaluating the plasma membrane function and fertilizing ability in mammal spermatozoa. However, HOS solutions and experimental conditions have not been determined clearly for assessing canine spermatozoa. This study was conducted to examine the HOS solutions and assay conditions, including incubation time (30 to 120 min), storage temperature (4, 17 and 20℃), semen status (fresh and frozen). Maximum spermatozoal plasma membrane swelling was obtained in an 150 mOsm Na-citrate/Fructose solutions with an incubation time for 45 min. The storage temperature and semen status affected the percentage of HOS positive spermatozoa. The HOS test adapted to canine spermatozoa in this study was simple and highly consistent assay with good repeatability. The optimal condition of HOST in canine spermatozoa is an 150 mOsm Na-citrate/Fructose solutions with an incubation time for 45 min regardless of semen storage temperature and semen status.
폴리메틸펜텐 막(polymethylpentene membrane, PMP)을 Ar, NH3 플라즈마로 표면 처리하고, 처리 전후의 투과 도와 선택도의 변화를 관찰하였다. Ar 플라즈마로 처리하였을 때 O/C의 비율이 증가하며 친수성기 (OH, COOH, C=O)의 도입이 확인되었고 NH3 플라즈마로 처리하였을 때 아민, 아미노기가 도입되었다. 플라즈마 처리된 폴리메틸펜텐막에서 CO2의 투과도와 N2,에 대한 선택도 (Actual Separation Factor)의 최적조건은 Ar 플라즈마 처리 (30 W-6 min)의 경우 각각 182 Barrer [10-10;cm3(STP)cm/cm2.s.cmHg]와 6.17이며, NH3, 플라즈마 처리 (30 W-8 min)의 경우 각각 144 Barrer [10-10/cm2(STP)cm/cm2.s.cmHg] 와 6.13을 얻었다.
우수한 열적, 화학적, 기계적 성질을 가진 폴리이미드 막의 표면을 Ar, NH3 플라즈마로 처리한 후 혼합기체(CO2/N2=20/80 vol%) 의 투과 실험을 통하여 플라즈마 처리 조건이 기체 투과도와 분리성능에 어떠한 영향을 미치는지를 조사하였다. 투과실험은 30℃, 5atm에서 variable volume method에 의해 행하여졌다. 표면개질된 폴리이미드 막의 투과거동에 대해 처리시간, 출력세기, 가스주입 유량 및 반응기 내의 압력과 같은 플라즈마 처리 조건의 영향을 조사하였다. 플라즈마 처리된 막의 표면은 FTIR-ATR, ESCA, AFM으로 분석하여 처리전후의 변화를 관찰하였다. 또한 플라즈마 처리시간에 따른 etching 효과와 흡수성은 weight loss와 contact angle를 측정하여 조사하였다. 그리고 투과 실험에 있어서 반응 온도의 변화에 따른 영향도 함께 연구되었으며, saturator를 이용한 dry 상태와 wet 상태에서 혼합가스에 대한 폴리이미드 막의 기체투과특성에 대한 실험 역시 수행되었다.
생물 발효 공정에서 생산되는 부탄올 수용액으로부터 부탄올을 농축시키기 위한 투과증발 공정의 flux와 선택도를 향상시키기 위하여 저온 plasma 처리 공정으로 막을 제조하였다. 플라즈마 처리 조건인 공급 power, 반응 시간, 단량체 공급 속도 등에 따른 영향을 조사하여 (W/FM)t의 최적값이 4.0389times10(sup)9 J.min/kg임을 확인하였으며, 이에 따른 최적의 막 제조 공정을 확립하였다. 여러 가지 유기 화합물로서 저온 플라즈마 처리된 막에 대하여 부탄올 분리 성능을 조사하였으며, 플라즈마 처리된 막에 대하여 물과 부탄올의 상대적 sorption 비와 접촉각을 측정하여 표면 분석을 하고자 하였으며, 그 상관 관계를 조사하였다. 접촉각과 상대적 sorption 비가 증가함에 따라 부탄올의 선택도는 0.186에서 3.525로 향상되었고, 부탄올 질량 flux는 0.042에서 0.567 kg/m2.hr로 향상됨을 볼 수 있었는데, 이는 막의 소수서이 증가함에 따라 막과 부탄올과의 친화력이 증가하였기 때문인 것으로 판단된다. 또한 사용된 유기 화합물과 물 또는 부탄올과의 친화력 정도를 알아보기 위해 heat of mixing을 측정하여 분리 성능과 비교하였으나 뚜렷한 경향성을 얻지 못하였다. 이것은 플라즈마 처리 시 유기 화합물이 분해되어 새로운 화합물을 형성하게 되어 유기 화합물 본래의 특성을 잃어버리기 때문인 것으로 생각된다.
Canola(Brassica napus) 엽에서 추출한 미세막으로부터 PEG-dextran 2상분획법을 이용하여 세포막과 세포내막을 분리하였다. U2 상에 있는 원형질막의 K+-ATPase의 특이활성도가 미세막에 비하여 25℃에서 자란 canola는 6.6배, 10℃에서 4.6배 각각 증가되었다. 원형질막(U2)은 미세막이나 세포내막(L2) 보다 cytochrome-c-oxidase 활성이 적게 나타난 반면, 세포내막에서는 K+-ATPase의 특이활성도가 가장 적게 나타났다. 10℃에서 생장한 canola의 18:3/18:2 률은 25℃보다 29.2% 더 높게 나타났다. 원형질막의 2중결합지수는 10℃에서 생장한 canola가 25℃에서 생장한 것보다 8.9% 더 증가되었으며 세포내막에서도 같은 경향으로써 10℃에서 19.7% 더 증가되는 현상을 보였다. 또한 엽록소 함량은 10℃에서 생장한 것이 25℃에 비하여 17.3% 낮았다. Canola가 저온에서 생장시 주로 C18 지방산들이 변화되어, 세포막 내에 불포화 지방산이 많았으며, 그 중에서도 리롤렌산(18:3)이 크게 변화되는 현상을 보였다. 이러한 변화는 생리적으로 canola의 세포막이 저온에 살아가기 위한 하나의 수단으로 추정된다.
Background : Water uptake and flow across cellular membranes is a fundamental requirement for plant growth and development, and plant water status is important not only for plant growth under favorable conditions but also for ability of a plant to tolerate adverse environmental conditions. Thus identification of plasma membrane water channel genes (aquaporins) in ginseng provides extensive information for functional studies and the development of markers for salinity stress tolerance. Methods and Results : For salinity treatment, the plants were grown for 4 weeks in culture medium gelled with 0.8% Phytoagar, and the old media were replaced with the fresh medium containing NaCl at 0, 50, 100, 200 and 400 mM, respectively. The samples for stress treated and non-stressed plants were collected from 6h to 72h, and frozen immediately into liquid nitrogen. According to the sequence information from the assembled transcripts, four primer pairs were designed from the aquaporin gene regions. In order to determine the pattern of aquaporins expression in ginseng seedlings to salinity stress, we conducted semi-quantitative RT-PCR. Conclusion : A tonoplast intrinsic protein 1 (TIP1)-type aquaporin is not only believed to be essential for plant life, but also to be beneficial for growth under salinity stress. Therefore, a deeper understanding of aquaporin genes in ginseng will be essential for crop improvement, which could help us to understand the molecular genetic basis for the ginseng genetic improvement and also provide the functional genetic resources for selective breeding and transgenic research.
본 연구는 간이 수경재배법을 이용하여 보리의 알루미늄 스트레스 내성과 민감성 품종을 간편하고 빠르게 screen하는 방법을 소개하고, 선별된 품종간의 뿌리의 생장, 뿌리 조직의 염색, 알루미늄 함량, 원형질막의 H+ -ATPase의 발현 변화를 조사하여 분석하였다. l. 보리 65가지 품종을 간이 수경재배법을 이용하여 20uM 알루미늄을 24시간 처리 후 뿌리생장의 차이로 내성 세 품종(자예2, 자예6, 모치무기)과 민감성 세 품종(흰쌀, 올쌀, 품2)을 선별하였다. 2. 알루미늄에 내성 품종은 알루미늄 처리 농도(0, 5, 10, 20uM )에 따라 뿌리 생장 감소폭이 적었으나, 민감성 세 품종은 상대적으로 낮은 5uM 농도에서부터 80%의 생장이 억제되었다. 3. 내성인 자예2와 민감성인 품2의 알루미늄 처리 후, 농도별(0, 5, 10, 20uM ), 시간별(3, 6, 12, 24시간)로 0.2% hematoxylin으로 염색 시 주로 apex에 3시간 이후부터 염색되었으며, 민감성 품2가 내성인 자예2에 비해 농도와 시간에 따라 그 피해 정도가 매우 심각하였다. 4. 20uM 로 24 시간 처리된 뿌리 apex(10 mm)의 알루미늄 함량을 측정한 결과, 내성인 자예2는 주당 47.1 nmol의 함량을 보여 주었으나, 민감성인 품2는 주당 64.9 nmol의 높은 함량을 보여 주었다. 5. 24시간 동안 20uM 알루미늄을 처리한 뿌리 원형질막 H+ -ATPase 발현을 western blotting을 통해 분석한 결과, 내성인 자예2는 차이가 없었으나, 민감성 품2는 현저히 억제되었다. 이로 보아 원형질막 H+ -ATPase가 알루미늄의 내성 기작에 관여하는 것으로 보인다. 6. 본 연구를 통해 간이 수경재배와 hematoxylin을 이용한 염색으로 간단하고 빠르게 보리의 알루미늄 내성과 민감성 품종의 screening을 할 수 있었고, 보리뿐 아니라 쌀, 밀 등의 다른 종자에도 적용할 수 있을 것이다.
There are many species as bioenergy crops and have different cold sensitivity in each. Cold-tolerant camelina and rapeseed, -sensitive jatropha, were used to investigate the cold stress response. Various physiological parameters such as leaf length, width, electrolyte leakage, stomatal conductance and chlorophyll fluorescence were measured to determine the growth rate treated with cold (2℃) for 5 days. Cold treated jatropha was damaged seriousiy but camelina and rapeseed were withstand. In order to investigate the cold-response on plasma membrane H+-ATPase activity isolated from leaves and roots of camelina, rapeseed and jatropha crops were exposed to cold stress. There were an increase in the activity of leaves and roots plasma membrane in cold-tolerant crops (camelina, rapeseed) while decreased the activity in cold-sensitive crop (jatropha). By western-blot analyses, the protein expression of plasma membrane H+-ATPase isolated from leaves and roots of camelina and rapeseed was increased in the presence of cold stress, but not in jatropha. These results may suggest that increased plasma membrane H+-ATPase of crops are closely related with cold-tolerant.
Aluminum (Al) toxicity in plants is one of the major limitations to crop growth on acid soils. The Al-induced change of H+-ATPase expression has been regarded as an important mechanism for Al tolerance in soybean. To investigate whether translational regulation of plasma membrane H+-ATPase is involved in the response to Al stress, we conducted western - blot of this protein. The results show that western - blot of plasma membrane H+-ATPase in the "Sowon" (Al tolernace) significantly increased in translational expression level, while citric acid (50 μM) with Al (50 μM) treatment has not effected. In contrast, Al sensitive cultivar "Poongsannamool" inhibited expression level of plasma membrane H+-ATPase with Al treatment. Two - dimensional gel analysis were performed to determine the protein induction patterns of control and Al (50 μM, 24 h) treated soybean. There are many changes of plasma membrane proteins in both cultivars under Al stress. Especially "Sowon" was significantly enhanced the expression of the plasma membrane H+-ATPase in Al treatment. But protein expression of "Poongsannamool" was less than "Sowon". These results suggest that the regulatory role of plasma membrane H+-ATPase may involved the tolerance mechanism in soybean roots. At the present, transcriptional level of H+-ATPase is under investigation.