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在继代培养中贡蕉胚性悬浮细胞的分化能力和染色体数目的变化 总被引：3，自引：0，他引：3
将贡蕉[Musa acuminata cv.Mas(AA)]胚性悬浮细胞通过不同时间的培养后,对其体胚发生能力和染色体数目进行了分析。结果表明,随着培养时间的延长,贡蕉胚性悬浮细胞的体胚发生能力下降,继代培养1.5a的悬浮细胞体胚发生能力为1.76×104个/mLPCV(packed cell volume,细胞密实体积)胚性悬浮细胞,继代培养3a后下降到0.85×104个/mL PCV胚性悬浮细胞。整个胚性细胞悬浮系为混倍体,细胞的染色体数目变化从3个到70个不等,既有含染色体数目为整倍体的细胞,也含有大量染色体数目为非整倍体的细胞;继代培养1.5a时,含正常二倍体染色体数目的细胞比例为15.8%,继代培养3.0a时下降到9.7%。 相似文献
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根癌农杆菌介导荔枝遗传转化研究 总被引：3，自引：0，他引：3
利用带有内含子的GUS基因(uidA)的瞬时表达,研究影响根癌农杆菌介导荔枝胚性愈伤组织遗传转化的若干因素。结果表明:在AGL-1、LBA4404和EHA105三种菌株中,EHA105对荔枝胚性愈伤组织的侵染力最强;采用2d的共培养时间既有较高的瞬时表达率,又避免了农杆菌的过度生长;0.5×10~8个细胞/mL的菌液密度为最佳侵染浓度;继代培养15d的胚性愈伤组织处于最旺盛分裂的时期,是转化的合适受体;愈伤组织转化前干燥处理对uidA瞬时表达率的提高有一定的促进作用。使用优化的农杆菌侵染条件获得了稳定表达uidA基因的荔枝抗性愈伤组织。 相似文献
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农杆菌介导霞多丽葡萄胚性细胞系遗传转化条件的优化 总被引：3，自引：1，他引：2
为建立葡萄(Vitis vinifera)遗传转化技术体系,以霞多丽葡萄(Chardonnay)胚性细胞系为靶组织,采用GUS检测法,对影响农杆菌介导葡萄遗传转化效率的主要因素进行了研究。结果表明,超声波处理时间长短对转化效率有较大影响,在所试的0.5、1、5、10 min 4种不同时间的超声波处理中,以5 min时转化效率较好,平均达到9.11个蓝色斑点;AS浓度对转化效率有明显差异,当浓度为50μmol/L时,平均蓝色斑点数为8.89个,100μmol/L时,达到12.44个;DTT质量浓度对转化效率也有较大影响,在所试的3种质量浓度(1,2,3 mg/L)中,以3 mg/L为最佳,有16.67个蓝色斑点。通过GUS瞬时表达检测,确立了农杆菌介导葡萄胚性细胞系遗传转化的几个最适影响因素,从而为葡萄遗传转化技术体系的建立奠定了基础。 相似文献
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【目的】提高番木瓜体细胞胚发生的同步性及其植株再生率,为番木瓜大量快繁、细胞工程和分子育种技术研发提供技术基础。【方法】以紫晖110～120 d果实的未成熟合子胚为外植体,经胚性愈伤组织诱导、液体悬浮培养和体细胞胚发生过程,建立均质胚性细胞悬浮系和高效植株再生技术体系,重点比较了不同质量浓度2,4-二氯苯氧乙酸(2,4-Dichlorophenoxyacetic acid,2,4-D)对胚性愈伤组织诱导、不同质量浓度6-苄基氨基嘌呤(6-Benzylaminopurine,6-BA)+萘乙酸(1-naphthlcetic acid,NAA)组合和活性炭(activated carbon,AC)对子叶期体细胞胚萌发与生根的影响。【结果】4 mg·L-12,4-D可诱导62.86%未成熟合子胚形成胚性愈伤组织。经5个继代周期的筛选培养,可建立由单细胞和小细胞团组成的均质胚性细胞悬浮系。采用液体培养方式能诱导大量球形胚的形成,被转移至含5 g·L-1AC的半固定培养基成熟培养30 d后,可获得大量子叶期体细胞胚。在含0.4 mg·L-1<... 相似文献
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红姜花胚性细胞悬浮体系的建立和原生质体培养 总被引：1，自引：0，他引：1
以红姜花(Hedychium coccineum)未成熟花丝和花药为试材,研究了红姜花胚性愈伤组织的诱导、细胞悬浮体系的建立以及原生质体的培养。结果表明:在MS+4mg/L 2,4-D+4mg/L NAA+1mg/L 6-BA+30g/L蔗糖+7g/L琼脂上经过120d培养诱导出了愈伤组织,愈伤组织在增殖培养基上经过继代筛选获得浅黄色、松散易碎的胚性愈伤组织。胚性愈伤组织通过3个月的悬浮培养,得到均质稳定的胚性细胞悬浮系。以胚性悬浮细胞(ECS)为起始材料分离原生质体,酶解试验表明,在原生质体分离过程中,用于原生质体分离的酶液需要保持一定的渗透压以保护原生质体不被破坏。当甘露醇的浓度为0.14mol/L时,原生质体产量最高,达到2.25×105个/mL PCV ECS(packed cell volume,PCV)。在看护培养系统中,原生质体经过7d的培养,细胞第一次分裂,经过14d培养,细胞分裂频率达到12.3%,28d时,细胞团形成频率达到4.2%。所形成的细胞团具有典型的胚性细胞特征。 相似文献
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《果树学报》2010,(5)
采用火箭琼脂糖凝胶电泳法测定了培养15 d后的香蕉悬浮细胞液体培养基中的阿拉伯半乳糖蛋白(ara-binogalactan-proteins,AGPs)的含量,发现具有体胚发生能力的贡蕉、大蕉和龙牙蕉过山香品种的胚性悬浮细胞(em-bryogenic suspension cells,ECS)培养基中的AGPs含量分别达到36、48、56 mg.L-1,而在不能进行体胚诱导的香芽蕉威廉斯品种的非胚性悬浮细胞(non-embryogenic suspension cells,NECS)的培养基中检测不到AGPs,说明悬浮细胞分泌到培养基中的AGPs含量与香蕉悬浮细胞的胚性状态有一定的关系。在香蕉ECS的悬浮培养过程中,添加不同浓度的βGlcY试剂(β-glucosyl Yariv)到悬浮培养基中可引起胚性细胞与非胚性细胞(ECS/NECS)的比值下降;在香蕉的ECS体胚诱导过程中,添加不同浓度βGlcY试剂到体胚诱导培养基中,引起体胚发生频率下降。这些结果表明,AGPs在香蕉悬浮细胞的胚性保持过程中起到重要的作用,并且具有剂量效应。 相似文献
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《园艺学报》2016,(11)
AcSERK1是菠萝(Ananas comosus)体细胞胚发生初期特异表达的基因,为探讨其转录调控规律,对其5′上游调控序列的转录起始位点及启动特性进行了鉴定。采用hiTAIL-PCR技术从菠萝基因组DNA中克隆了AcSERK1完整的5′上游调控序列,利用5′RACE技术鉴定出其转录起始位点位于起始密码子(ATG)上游258 nt(G)处。以AcSERK1 5′上游完整调控序列取代pBI121中的CaMV 35S启动子,构建植物表达载体AcSERK1(–2090/+258)︰︰GUS,并在菠萝不同组织器官中进行瞬时转化分析,发现AcSERK15′上游完整的调控序列(–2090/+258)能够启动GUS在胚性细胞中特异性表达,与AcSERK1表达规律相同,说明该启动子为胚性细胞特异性启动子,对调控外源基因在体细胞胚早期特异表达有重要意义。 相似文献
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【目的】建立梨悬浮细胞体系，并利用悬浮细胞体系获得高质量悬浮细胞，进行遗传转化和原生质体的分离。【方法】以新梨7号花药为试材，诱导获得梨花药愈伤组织，研究了细胞悬浮系建立和影响悬浮细胞增殖的主要因子。【结果】花药在1/2 MS+1.0 mg·L-12,4-D+0.4 mg·L-1NAA+30 g·L-1蔗糖+7 g·L-1琼脂(pH值为5.8～6.0)培养基上诱导的愈伤组织，经4～5次继代得到了黄白色、颗粒状，状态相对较好的愈伤组织；将愈伤组织接种于液体培养基中，于28℃，200 r·min-1的黑暗条件下悬浮振荡培养，经4～5次悬浮继代培养建立了悬浮细胞系，适宜的启动和增殖培养基为：MS+1.5 mg·L-12,4-D+1.0 mg·L-16-BA+30 g·L-1蔗糖，细胞生长曲线呈“S”型，活细胞率达81.98%，近圆细胞率达83.66%，可作为遗传转化的受体，转化率为20.00%。也可直接用于原生质体的分... 相似文献
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超声波辅助农杆菌介导八棱海棠转rolC基因 总被引：7，自引：1，他引：7
应用超声波辅助农杆菌介导法,对八棱海棠进行rolC基因转化,以期提高转化效率并获得转基因植株。利用gus基因瞬间表达的方法研究了超声波处理时间、处理时期和农杆菌悬浮液中乙酰丁香酮(As)浓度对rolC基因转化率的影响。结果表明,叶盘在D600nm为0．6且含有75 mg／L As的农杆菌悬浮液中侵染2 min后,用功率为100 W的超声波处理30 s,再浸泡2．5 min,然后放到再生培养基上共培养3 d,能获得最佳的gus基因瞬间表达率。最佳处理条件下转化683枚八棱海棠叶片,共得到138个抗性愈伤组织和15株抗性苗,转化率为2．2％。GUS染色、PCR及Southern blotting检测结果显示,有12个八棱海棠株系的基因组中整合了完整的外源rolC基因。 相似文献
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WANG Li-jun WANG Jian-hui WANG Yu-juan FAN Su-jing ZHU Li-yan YANG Xiu-hong 《园艺学报》2016,32(3):405-410
AIM: To investigate the role of renin-angiotensin system(RAS) disequilibrium in hyporeactivity and injury of aorta after tourniquet shock(TS) by observing the changes of aortic contractile reactivity and RAS components after TS. METHODS: Male C57BL/6 mice(8 months old) were divided into 7 groups including control group and 6 model groups. The mice in model groups were sacrificed at reperfusion of 10 min, 1 h, 2 h, 4 h, 6 h and 12 h. The mice in control group were not subjected to tourniquet ligation. The Doppler flowmetry was used to determine the limb blood flow. The carotid artery catheter was applied to detect the blood pressure. The isolated vascular tension tester was available to measure the reactivity of the aorta. HE staining combined with transmission electron microscopy was used to evaluate the morphology of injured aortas. The protein expression of AT1 receptor, Mas receptor, ACE and ACE2 was measured by Western blot. The serum contents of Ang Ⅱ and Ang(1-7) were detected by ELISA. RESULTS: Compared with control group, the blood flow in model groups decreased gradually with the prolongation of reperfusion time. The blood pressure increased at 10 min after reperfusion, and then decreased gradually. Accordingly, vascular reaction to norepinephrine(NE) increased at 10 min and then descended. The vascular reactivity reached the lowest level at 4 h. Morphological injury score increased gradually. Vascular AT1 receptor and ACE2 proteins were reduced, while Mas receptor and ACE proteins were up-regulated compared with control group. The content of Ang Ⅱ in the serum elevated, while the content of Ang(1-7) was reduced. CONCLUSION: The mechanism of aortic reaction to NE increased temporarily in the early stage of shock and then decreased. It may be related to the morphological injury of aorta and the imbalance of RAS. 相似文献
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LIU Fan WANG Xiao-ying SHAHIN Ahmed MUHAMMED Rafi-N LI Shu-min YANG Xiu-hong 《园艺学报》2017,33(7):1264-1270
AIM:To explore the role of imbalance of local renin-angiotensin system (RAS) in lung injury by observing the changes of angiotensin Ⅱ type 1 receptor (AT1R) and Mas receptor protein expression in the lung and the degree of lung injury subject to limb ischemia-reperfusion (LIR) in the mice.METHODS:Male ICR mice (n=42,8 weeks old) were randomly assigned into 7 groups (6 in each group),including control group and 6 model groups with LIR of 0.5 h,1 h,2 h,4 h,6 h and 12 h reperfusion.Tourniquets were used to block the blood flow of the hind limbs of the ICR mice and were released after 2 h ischemia to initiate reperfusion.The mice were sacrificed by eyeball blood withdrawal at different time points after reperfusion.The organ coefficient and wet/dry weight ratio (W/D) of the lung tissue were calculated.Bronchoalveolar lavage fluid (BALF) was taken for cell counting and protein concentration measurement.The histopathological changes of the lung tissues was observed,and the pathological score was calculated.The protein expression of AT1R and Mas receptor in the lung tissues was determined by Western blot.RESULTS:The organ coefficient,W/D of lung tissue,and cell number and protein concentration in BALF of model groups were significantly higher than those in control group after LIR.The pathological changes were found in the lung tissue of model mice,including alveolar capillary dilation and congestion,edema,inflammatory cell infiltration in peripheral vascular,alveolar and bronchial walls,alveolar septal thickening and inflammatory cell infiltration.The lung injury score was elevated gradually along with the extension of reperfusion time.The protein expression of AT1R began to increase at reperfusion time points of 0.5 h and 1 h.With the extension of reperfusion time,the protein expression of AT1R decreased gradually.Conversely,the protein expression of Mas receptor increased gradually with prolonged reperfusion.CONCLUSION:LIR induces acute lung injury gradually.The imbalance of AT1R and Mas receptor expression may be involved in the damage process. 相似文献
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LIANG Wei-jie CHEN Jing-fu SONG Ming-cai MO Li-qiu PAN Wan-ying Li Jian-hao FENG Jian-qiang ZHANG Wen-zhu 《园艺学报》2015,31(2):267-273
AIM: To study whether the angiotensin-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardiomyocytes against high glucose(HG)-induced injury by inhibiting nuclear factor-κB(NF-κB) pathway. METHODS: The cell viability was measured by CCK-8 assay. The intracellular levels of reactive oxygen species(ROS) were detected by DCFH-DA staining. The number of apoptotic cells was tested by Hoechst 33258 nuclear staining. Mitochondrial membrane potential(MMP) was examined by JC-1 staining. The levels of NF-κB p65 subunit and cleaved caspase-3 protein were determined by Western blotting. RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose(HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated(p) NF-κB p65, peaking at 60 min. Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30 μmol/L and HG for 24 h inhibited HG-induced cytotoxicity, evidenced by an increase in cell viability. On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage, indicated by decreases in the number of apoptotic cells, cleaved caspase-3 level, ROS generation and MMP loss. However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor(Mas receptor). Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG. CONCLUSION: Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway. 相似文献
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Picloram,ABA和TDZ对香蕉体细胞胚胎发生的影响 总被引：5，自引：1，他引：5
研究Picloram、ABA和TDZ对贡蕉未成熟花序来源的胚性细胞悬浮系体细胞胚胎发生的影响。结果表明, 8.28 μmol/L Picloram替代2, 4-D可获得15.56%的胚性愈伤组织诱导率; 在体细胞胚诱导初期加入ABA则抑制体细胞胚的发生, 并造成其萌发时严重愈伤化; TDZ可有效地改善体胚萌发率和植株转换率, 其中0.2 μmol/L TDZ处理的体胚萌发率和植株转换率分别从对照的17.28%、16.49%提高到72.77%和67.05%。 相似文献
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LI Xiu-juan SHEN Hui WEI Lin-yu WANG Guo-hong ZHANG Li-bing LI Chao-kun LI Xin-juan WANG Lu ZHAO Hong-gang SONG Jing-gui LI Dong-liang 《园艺学报》2017,33(9):1587-1592
AIM: To investigate the neuroprotective effect of progesterone against adenosine triphosphate (ATP)-injured human neuroblastoma SH-SY5Y cells.METHODS: The SH-SY5Y cells in the logarithmic phase were divided into different groups according to the progesterone and ATP concentrations. The cell viability was measured by CCK-8 assay. The membrane permeability was detected using fluorescent dye YO-PRO-1. Cytosolic Ca2+ concentration was measured with fluorescent dye Fluo-3/AM. The expression of purinergic P2X7 receptor was assessed by Western blot.RESULTS: The viability of the SH-SY5Y cells was significantly decreased (P<0.05) and YO-PRO-1 uptake was obviously increased (P<0.05) in a concentration-dependent manner compared with control group when SH-SY5Y cells were treated with ATP at 1, 3, 5 and 7 mmol/L for 2 h. The viability reduction of the SH-SY5Y cells induced by ATP was obviously counteracted by treatment with progesterone at 3, 10 and 30 nmol/L for 30 min (P<0.05) as compared with ATP group. YO-PRO-1 fluorescence enhancement induced by ATP in SH-SY5Y cells was significantly reduced (P<0.05) by progesterone (30 nmol/L) or P2X7 receptor antagonist KN-62 (500 nmol/L) pretreatment for 30 min, and no obvious difference between treatments with progesterone and KN-62 was observed. Cytosolic Ca2+ fluorescence intensity in normal group was a little, but that in ATP group was increased (P<0.05). Progesterone or KN-62 pretreatment significantly decreased the cytosolic fluorescence intensity of Ca2+ induced by ATP (P<0.05). However, no obvious difference between treatments with progesterone and KN-62 was found. The expression of P2X7 receptor in ATP group was significantly higher than that in control group (P<0.05), and progesterone inhibited ATP-induced P2X7 receptor expression (P<0.05).CONCLUSION: Progesterone inhibits P2X7 receptor expression, membrane pore formation, intracellular Ca2+ increase and cell death induced by ATP, so progesterone may protect SH-SY5Y cells against ATP-induced injuries. 相似文献
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根癌农杆菌介导的西瓜遗传转化研究 总被引：8，自引：1，他引：7
利用带有内含子的GUS基因的瞬时表达,研究影响根癌农杆菌介导的西瓜遗传转化的若干因素。结果表 明:西瓜子叶块外植体对潮霉素较为敏感,15 mg/L是适宜的筛选浓度,可明显抑制非转化组织的生长;脱菌过程中 采用500 mg/L的头孢霉素,对外植体生长影响较小;农杆菌菌株EHA105对西瓜子叶块的侵染能力较强;预培养有 利于转化;共培养3-4 d有利于提高转化频率并避免了农杆菌的过度生长;OD600值为0.3的菌液侵染10min效果最 佳;共培养培养基中添加乙酰丁香酮可提高转化频率。 相似文献