Effects of fructose sodium diphosphate on expression of CHOP and JNK in endoplasmic reticulum stress and islet apoptosis in rats with type 2 diabetes

AIM: To study the effects of fructose sodium diphosphate (FDP) on the expression of CHOP and c-Jun N-terminal binase(JNK) in endoplasmic reticulum stress and islet apoptosis in the rats with type 2 diabetes mellitus (T2DM). METHODS: T2DM model was established in male Wistar rats by feeding of high lipid diet and injection of streptozotocin. The rats were divided into 4 groups (n=8):normal control group, T2DM model group, T2DM+low-dose FDP (2 mL穔g^-1穌^-1, ip) group and T2DM+high-dose FDP (5 mL穔g^-1穌^-1, ip) group. The rats in the treatment groups received FDP for 8 weeks. The levels of fasting blood glucose (FBG), fasting serum insulin (FINS) and insulin sensitivity index (ISI) were measured. TUNEL was used to detect the islet apoptosis. The protein levels of CHOP and JNK were determined by the method of immunohistochemistry. RESULTS: (1) Compared with normal control group, FBG, FINS, the expression of CHOP and JNK, and apoptosis in T2DM model group were significantly increased (P<0.01). The level of ISI was significantly decreased. (2) Compared with T2DM model group, the levels of FBG and FINS, the expression of CHOP and JNK, and apoptosis in high-dose FDP group were significantly decreased. The level of ISI was significantly increased (P<0.01). However, the level of FBG, the expression of CHOP and JNK, and apoptosis in low-dose FDP group were significantly decreased. Compared with low-dose FDP group, the levels of FBG and FINS, the expression of CHOP and JNK, and apoptosis in high-dose FDP group were significantly decreased. The level of ISI was significantly increased (P<0.01 or P<0.05). CONCLUSION: FDP may prevent islet cells from apoptosis in T2DM rats by decreasing the expression of CHOP and JNK.     

整合农艺性状和分子标记数据构建新疆野苹果核心种质

AIM: To explore the effect of traditional Chinese medicine Qiliqiangxin on cardiomyocyte apoptosis after myocardial infarction (MI) in a rat model. METHODS: MI was induced in rats by ligation of the anterior descending coronary artery. The survivors were randomly divided into 3 groups: sham operation group, MI group and Qiliqiangxin treatment group (4 g穔g^-1穌^-1). After 28 days, the infarction size was measured. Apoptotic index (AI) was determined by TUNEL. The expression of Fas was detected by the method of immunohistochemistry in non-infarcted zones (NIZ), and the expression of xanthine oxidase (XO) and caspase-3 in myocardial tissue from NIZ was detected by Western blotting. In addition, XO activity, and O₂ ^-? and OH? scavenging activity of myocardial tissues in the NIZ were measured by colorimetry. RESULTS: Compared with MI group, AI and the expression of Fas and caspase-3 in the NIZ were significantly depressed in Qiliqiangxin treatment group. Moreover, the activity of XO was significantly decreased while O₂ ^-? and OH穝cavenging activity was significantly increased in Qiliqiangxin treatment group. Ventricular remodeling was attenuated. No significant difference in infarct size and XO expression level between Qiliqiangxin treatment group and MI group was observed. CONCLUSION: Qiliqiangxin may inhibit apoptosis of cardiomyocytes in the NIZ of rats by reducing reactive oxygen species and depressing the expression of Fas and caspase-3.     

两种砧木对年橘果实品质与产量的影响

AIM: To investigate the effect of ovariectomy (OVX) and simvastatin (SVS) on the expression of osteoprotegerin (OPG) in bone callus of rats. METHODS: Simvastatin (10 mg穔g^-1穌^-1) was injected subcutaneously to tissue overlying the site of fractured tibiae of ovariectomized rats for a treatment period of 5 days. Vehicle reagent was used as control in sham and OVX rats. Bone callus were harvested at time points of 1, 2 and 4 weeks after fracture and the expression of proliferating cell nuclear antigen (PCNA) and OPG was measured by the method of immunohistochemistry. RESULTS: Compared with OVX+vehicle group, the expression of PCNA decreased by 17.3%, 32.1% and 26.1% in OVX+vehicle group than that in sham+vehicle group (P<0.01), and increased by 60.1%, 67.7% and 67.7% in OVX+SVS group (P<0.01) at the time points of 1, 2 and 4 weeks after fracture, respectively. The expression of OPG was the lowest in OVX+vehicle group. The significant differences of OPG expression between OVX+SVS group and OVX+vehicle group at the 3 time points (P<0.01), and significant differences of OPG expression between sham+vehicle group and OVX+vehicle group at 1st week and 2nd week after fracture (P<0.05) were observed. CONCLUSION: Ovariectomy decreases the expression of OPG in bone callus of rats, while simvastatin increases it, indicating that simvastatin can indirectly inhibit the function of osteoclasts.     

Protective effect of pyrrolidine dithiocarbamate on kidneys in type 2 diabetic rats

AIM: To investigate the protective effect of pyrrolidine dithiocarbamate (PDTC) on the kidneys in type 2 diabetic rats. METHODS: High-fat diet and a small dose (27 mg/kg) of streptozotocin-induced diabetic rats were treated with or without PDTC (50 mg穔g^-1穌^-1, ip) for 1 week, and age-matched nondiabetic animals were also used for comparison. The concentration of malondialdehyde (MDA)and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined by commercial kit. The ratio of urine microalbumin/creatinine was measured by an automatic biochemical analyzer. The morphological changes of renal glomerulus were observed by HE/Masson staining and transmission electron microscopy. The expression of inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT) in the renal tissues was examined by the method of immunohistochemistry. RESULTS: PDTC-treated rats had lower blood glucose level and urine microalbumin/creatinine ratio than those in untreated diabetic rats. The levels of tissue MDA in diabetic rats were significantly higher, and the activity of SOD and GSH-Px was lower than those in normal control rats (P<0.05). The renal damage in diabetic rats was significantly improved after PDTC treatment. PDTC administration markedly attenuated the expression of iNOS and the production of NT in renal glomerulus and tubule in diabetic rats. CONCLUSION: PDTC not only reduces blood glucose level, but also protects the diabetic rats from diabetic nephropathy by diminishing the expression of iNOS and the production of NT.     

Artesunate negatively controls expression of cyclin D1 and activity of AP-1 by inhibiting the activation of ERK1/2 protein in HSC-T6 cells

AIM: To investigate the effects of artesunate(Art) on the expression of ERK1/2, AP-1 and cyclin D1 in rat hepatic stellate cells (HSCs), and to elucidate the molecular mechanism of Art against hepatic fibrosis. METHODS: HSC-T6 cells were treated with platelet-derived growth factor BB(PDGF-BB) to induce cell proliferation. The cells were divided into control group, PDGF-BB group, PDGF-BB+Art groups (with 6.25 mg稬^-1, 25 mg稬^-1or 50 mg稬^-1 of Art) and PDGF-BB+PD98059 group. The level of collagen type I in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of ERK1/2 and cyclin D1 were measured by RT-PCR. The protein levels of p-ERK1/2 and cyclin D1 in HSC-T6 cells were detected by Western blotting. The activity of AP-1 was analyzed by electrophoretic mobility shift assay. RESULTS: The concentration of collagen type I was significantly higher in PDGF-BB group than that in control group (P<0.05), and decreased in PDGF-BB+Art group and PDGF-BB+PD98059 group in comparison with that in PDGF-BB group (P<0.05, P<0.01). The protein level of ERK1/2 in PDGF-BB+Art group (50 mg稬^-1) was lower than that in PDGF-BB group (P<0.05), and was even lower in PDGF-BB+PD98059 group (P<0.01). The mRNA expression of cyclin D1 in PDGF-BB+Art groups (25 mg稬^-1and 50 mg稬^-1) and PDGF-BB+PD98059 group were significantly lower than that in PDGF-BB group (P<0.05). The protein levels of p-ERK1/2 and cyclinD1 were the highest in PDGF-BB group, and significantly lower in PDGF-BB+Art groups (6.25 mg稬^-1, 25 mg稬^-1 and 50 mg稬^-1) and PDGF-BB+PD98059 group (P<0.05, P<0.01). The AP-1 binding activity in HSC-T6 cells was down-regulated by Art. CONCLUSION: Artesunate inhibits the proliferation of HSC-T6 cells in vitro by inhibiting the activation of ERK1/2, thus down-regulating the activity of AP-1 and expression of cyclin D1.     

Retinoic acid inhibits renal interstitial myofibroblast proliferation in a rat unilateral ureteral obstruction model

AIM: To investigate the effect of retinoic acid on proliferation of renal interstitial myofibroblasts in a rat model of the left unilateral ureteal obstruction (UUO). METHODS: UUO model was established by unilateral ligation of ureter in 36 SD rats. Rats were divided into 2 groups: Retinoic acid-treated group and control group, each with 18 rats. UUO rats were treated with either daily subcutaneous injection of 10 mg/kg body weight of all trans-retinoic acid or vehicle alone two days before the operation until being sacrificed. Groups of 6 rats were killed on day 3, 7 and 12 after ligation of the left ureter. The percentage of renal tubular lesion, interstitial fibrosis score, the number of interstitial myofibroblasts, the number of proliferating myofibroblasts and the expression of TGFβ-1 mRNA were determined. RESULTS: There were significant accumulation and local proliferation of myofibroblasts in the interstitium of the UUO rats. On day 7 of the UUO model, the percentage of tubular lesions and interstitial fibrosis score were significantly lower in the retinoic acid-treated group than those in control group[(15.9±2.0)%vs(27.3±2.2)%和(0.47±0.12)vs(1.65±0.18),P<0.01]. The numbers of interstitial myofibroblasts and proliferating myofibroblasts in the retinoic acid-treated group were significantly lower than those in the control group[(12.2±2.2)cells/HPF vs(29.5±1.8)cells/HPF和(1.4±0.6)cells/HPF vs(4.3±0.8)cells/HPF,P<0.01]. Furthermore, the expression of TGFβ-1 mRNA was significantly lower in the retinoic acid-treated group than that of the control group (P<0.01). CONCLUSION: Retinoic acid suppresses interstitial fibrosis in a rat UUO model by inhibiting the accumulation and local proliferation of myofibroblasts in the renal interstitium.     

Changes of hydroxyl radical,malondialdehyde and total antioxidant capa-city in intestines after orthotopic liver autotransplantation in rats

AIM: To explore the pathological changes of small intestines after orthotopic liver autotransplantation in rats and to analyze the correlation between these changes and the levels of hydroxy radical (稯H),malondialdehyde(MDA)and total antioxidant capacity(T-AOC). METHODS: Thirty-six Sprague-Dawley rats were randomly divided into sham operation group (group S, n=6) and model group (group M). According to the period after liver reperfusion, the rats in group M were divided into 5 sub-groups: 2 h after reperfusion (group M1, n=6), 4 h after reperfusion (group M2, n=6), 8 h after reperfusion (group M3, n=6), 16 h after reperfusion (group M4, n=6), and 24 h after reperfusion (group M5, n=6). After anesthesia, the rats in group S involved laparotomy and vascular dissection without hepatic vascular exclusion and perfusion. The rats in other groups received orthotopic liver autotransplantation. The intestinal tissues starting from 5 cm to terminal ileum were removed 2 h, 4 h, 8 h, 16 h and 24 h after reperfusion. The morphological changes of intestinal epithelial basement membrane were observed under optical microscope. The levels of 稯H, MDA and T-AOC were detected. RESULTS: (1) In model groups, the morphological damages in the intestines were significant compared to group S, especially 8 h after reperfusion. The intestines showed massive epithelial lifting down the sides of villi and a few tips being denuded. The repair of pathological damage in the intestines 24 h after reperfusion was observed. (2) Compared to group S, the levels of 稯H in the intestines significantly increased in group M2, M3 and M4 (P<0.05). The levels of MDA in the intestines significantly increased in group M1, M2 and M3 (P<0.05). The levels of T-AOC significantly decreased in group M1, M2, M3 and M4 (P<0.05). CONCLUSION: Orthotopic liver autotransplantation increases the levels of 稯H and MDA, diminishes T-AOC and induces reversible pathological changes in intestines.     

Binding characterization of cholecystokinin receptors in rat pulmonary interstitial macrophages

AIM: To investigate the binding characteristics of cholecystokinin receptors in rat pulmonary interstitial macrophages (PIMs). METHODS: The PIMs were isolated from rat lung tissues, purified using the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. The PIM membrane was obtained by supercentrifuge. Receptors for CCK in PIMs were examined using labeled CCK-8S as ligand. The specificity of -CCK-8S binding to PIMs membrane and the subtypes of CCK receptors were determined by competitive inhibition experiments with CCK-8S, CCK-A and CCK-B receptors selective antagonists (CR1409 and CR2945). The effects of time and incubation temperature on the specific binding were also observed. RESULTS: The specific binding of -CCK-8S was not detected in normal rat PIMs, but was detected in the rat administrated with LPS for 48 h. The capacity of ligand-receptor binding was dependent on the incubation temperature and time. Scatchard analysis of the saturation curves suggested that the presence of CCK receptors with high affinity[Kd=(0.68士0.28) nmol/L] and low binding capacity [Bmax=( 32.50±12.70) pmol·g^-1in PIMs. By means of competitive inhibition studies, the specific binding of [³H]-CCK-8S to rat PIMs was inhibited by unlabelled CCK-8S[IC₅₀=(3.20士1.13)nmol/L],CCK-AR specific antagonist CR 1 409 and CCK-BR specific antagonist CR 2945[IC₅₀=(2.30士0.08)nmol/L].CONCLUSION:These results suggest the presence of two subtypes of CCK一AR and CCK-BR and provide a structural basis for CCK to play a pivotal role in PIMs.     

龙眼TPI基因的克隆及其在体胚发生过程中的表达分析

 采用RT-PCR结合RACE法，通过T/A克隆测序，获得龙眼胚性愈伤组织磷酸丙糖异构酶基因（TPI）两个类型的全长序列TPIⅠ（GenBank登录号：FJ595244）和TPIⅡ（GenBank登录号：GU073294），序列全长分别为1 084 bp和1 113 bp，其ORF都由765 bp核苷酸组成，编码254个氨基酸，与其它植物的TPI在核苷酸序列和推导的氨基酸序列的相似性较高。qRT-PCR结果分析表明，在龙眼体胚发生过程中，TPI在松散型胚性愈伤组织阶段的转录水平比较低，在胚性愈伤组织Ⅱ阶段转录水平急剧升高，并一直持续到胚性紧实球形结构阶段，球形胚阶段大幅下调，之后至子叶形胚阶段都维持在较低水平，初步证明TPI及其编码蛋白在龙眼启动体胚发生和早期体胚发育中行使重要的功能。     

低温胁迫下龙眼碳酸酐酶基因（CA）的克隆与表达分析

 应用蛋白质组学研究低温胁迫下龙眼叶片蛋白质组变化时发现碳酸酐酶（CA）蛋白下调表达。利用RT-PCR方法克隆CA基因的全长cDNA,GenBank登录号JN033201,长度为1 119 bp,包括1个966 bp的开放阅读框,编码321 bp的氨基酸序列,同源性分析表明,12个不同植物同源性为81% ~ 88%。龙眼CA基因具有典型的CA结构域,并且非常保守。实时荧光定量分析结果表明,CA在龙眼根、茎、叶中都有表达,为组成型表达,在叶中的表达量最高,茎和根中的表达量最少。CA基因在低温胁迫下随着低温胁迫时间的延长而发生变化。将CA在大肠杆菌中表达,获得1个约40.5 kD的外源蛋白。推测CA表达与低温胁迫有关。     

马铃薯C-8,7甾醇异构酶基因(StSI1) cDNA克隆及表达

 以拟南芥C-8, 7甾醇异构酶的氨基酸序列为信息探针搜索GenBank数据库, 对高度同源的马铃薯EST序列进行拼接、引物设计和RT2PCR扩增, 扩增产物测序结果证实获得一个马铃薯C-8, 7甾醇异构酶基因(StSI1) 的全长cDNA序列。序列分析结果显示, StSI1全长886 bp, 包含59 bp的5′非编码序列、161 bp的3′非编码序列和一个长度为666 bp编码221个氨基酸的开放阅读框, 分子量约为25 kD。氨基酸结构分析显示该蛋白的N端含有一个长度由35个氨基酸残基组成的信号肽, C端成熟肽区域含有典型的类EBP结合域。氨基酸比对分析表明, StSI1与已知C-8,7甾醇异构酶同源性介于32.9% ～61.3%之间, 与拟南芥AtSI1相似性最(61.3% ) 。RT-PCR 表达谱分析显示, StSI1在马铃薯的块茎芽眼和表皮组织中均能表达, 并且该基因的表达水平受贮藏温度升高和光照增强的正向调节。     

龙眼体胚发生早期miR166初级体的克隆与表达分析

为了解龙眼miR166初级体基因Pri-miR166 S53结构特点及其前体和成熟体在龙眼体胚发生早期的表达模式,采用SMART~(TM) RACE试剂盒和PCR扩增技术克隆龙眼体胚早期miR166基因的初级体(Pri-miR166 S53),确认其转录起始位点,并预测其含有的潜在ORF;利用龙眼基因组数据库提取其启动子序列,预测其含有的顺式作用元件;用实时荧光定量PCR对龙眼体胚发生早期及不同激素处理下的胚性愈伤组织中miR166基因的前体(Pre-miR166 S53)和成熟体(miR166a.2)表达模式进行分析。结果表明:获得长317 bp的Pri-miR166 S53基因初级体序列,对其进行翻译,得到一条长13个氨基酸序列的miPEP(MLCFVDALFLIST)。利用生物信息学软件分析Pri-miR166 S53基因的启动子序列发现,除了具有TATA/CAAT-box外,还含有生长素、脱落酸、乙烯、水杨酸、茉莉酸甲酯及spl、HSE等特异作用元件。实时荧光定量PCR分析表明,在2,4-D调控的龙眼体胚发生早期过程中,从胚性松散型愈伤组织发育到球形胚的过程中,Pri-miR166 S53基因的前体pre-miR166 S53和成熟体miR166a.2都表现为下调趋势;而在无2,4-D调控的龙眼体胚发生早期中,pre-miR166 S53和miR166a.2表达模式不同。此外,pre-miR166S53随ABA和乙烯处理浓度升高呈下调表达,而对不同浓度2,4-D处理无应答;miR166a.2随2,4-D、ABA处理浓度升高呈下调表达,而在乙烯处理下呈上调表达。上述研究结果提示miR166前体和成熟体在对外源激素应答的模式上并不呈简单线性相关,可能存在多层次、多方位的调控。     

梨矮化砧木‘中矮1号’生长素氢转运体基因PcAHS及其启动子的克隆与表达分析

以梨（Pyrus communis L.）紧凑型矮化砧木‘中矮1号’及其母本‘锦香’新梢韧皮部为试材,根据转录组测序结果设计特异性引物,克隆到1个长1 239 bp的基因序列。该序列在两个品种间不存在差异,均编码412个氨基酸,其氨基酸序列与苹果（np_001280772.1）、梅（xp_008242099.1）、毛果杨（xp_002306421.2）、草莓（xp_00428771.1）的生长素氢转运体基因（AHS）编码的氨基酸序列的相似性在67% ~ 99%之间,命名为PcAHS。qRT-PCR分析发现,‘中矮1号’新梢韧皮部中PcAHS的表达量均低于母本‘锦香’,推测其启动子序列存在差异。‘中矮1号’及其母本‘锦香’PcAHS 基因上游启动子长度分别为828 bp和888 bp,二者相似性89.1%。序列分析发现‘中矮1号’PcAHS基因启动子有一段58 bp（–496 bp ~–553 bp）的缺失;利用植物顺式作用元件数据库PLACE和PLANTCARE分析表明,‘中矮1号’启动子含有一个‘锦香’没有的BPBF转录因子结合元件P-box。推测‘中矮1号’PcAHS基因启动子特有的片段缺失和P-box转录因子结合元件可能是导致其表达量低并通过影响生长素的运输最终引起矮化的原因。     

龙眼胚胎F3H基因的cDNA克隆及序列分析

采用IEF-SDS-PAGE双向电泳技术,对‘立冬本'龙眼合子胚发育不同时期的蛋白质进行分离,通过MALDI-TOF-TOF鉴定到其中一个上调差异表达的蛋白为黄烷酮-3-羟化酶 (flavanone 3-hydroxylase,F3H)。以‘立冬本’龙眼胚胎cDNA为模板,采用RT-PCR和RACE技术,获得F3H基因cDNA 1 404 bp全长序列。序列分析表明,F3H基因共编码365个氨基酸,其cDNA序列与柑橘、苹果、棉花等F3H基因的同源性均高达80%以上,该基因在GenBank中登录号为EF468104。     

狗蔷薇生长素输出载体蛋白基因PIN1 和PIN2的分离与表达分析

 植物体内生长素输出载体PIN 蛋白基因的表达变化间接反映了生长素的运输与积累状态。 为分析生长素在狗蔷薇（Rosa canina）类原球茎发生初期的作用，以狗蔷薇叶片愈伤形成的不定根为材 料，分离了生长素输出载体蛋白基因PIN1 和PIN2 的cDNA，分别命名为RcPIN1（GenBank 登录号 KF543362）和RcPIN2（GenBank 登录号KF543363）。RcPIN1 全长2 266 bp，编码621 个氨基酸；RcPIN2 全长2 248 bp，编码643 个氨基酸，两者推导蛋白结构差异微小，在N 端和C 端均有5 个跨膜区域，中 间1 个亲水区。Blast 比对发现两个基因与多种植物PIN 基因具有高度同源性（> 70%）。半定量RT-PCR 分析表明，RcPIN1 在根、茎中表达量高于叶和花，RcPIN2 在根中表达水平高于茎、叶和花；在狗蔷薇类 原球茎发生初期，TDZ 诱导培养下愈伤—不定根RcPIN1 和RcPIN2 表达上调，而TDZ + TIBA 抑制培养 下两基因表达不活跃，且类原球茎形成率降低。试验结果表明生长素的极性运输与积累参与了调控狗蔷 薇类原球茎的初期形态建成。     

龙眼转醛醇酶基因的克隆与原核表达

转醛醇酶作为磷酸戊糖途径非氧化阶段的关键酶,在调节植物PPP对环境胁迫的应答中起重要作用.该研究应用RACE方法克隆了龙眼(Dimocarpus longan Lour.)花芽中转醛醇酶的基因,获得一段长度为1 655 bp的cDNA,其中包括一个1 320 bp的开放阅读框,已登陆GenBank,登陆号为FJ472991(GI:217795375).将转醛醇酶全长cDNA在大肠杆菌中表达,获得一个约55 kD带有组氨酸标签的外源蛋白.RT-PCR结果显示,TAL mRNA在龙眼成花逆转花芽中有较明显的表达量上调,说明TAL在一定程度上影响了龙眼正常成花.     

菠萝生长素极性运输载体基因AcPINs和AcAUXs的分离与表达分析

乙烯已经被广泛应用于菠萝人工催花,但其分子机制不是很清楚。以菠萝(Ananas comosus L.‘Perola’)茎尖为材料,采用RT-PCR结合RACE方法得到3个生长素极性运输输出载体基因(Ac PIN1、Ac PIN2和Ac PIN3)和2个生长素极性运输输入载体基因(Ac AUX1和Ac AUX2)的c DNA及基因组DNA全长。Ac PIN1、Ac PIN2、Ac PIN3、Ac AUX1和Ac AUX2的c DNA全长分别为2 690、2 388、2 057、2 156和1 580 bp,其开放读码框长度分别为1 854、1 917、1 530、1 479和1 392 bp,分别编码617、638、509、492和463个氨基酸;其基因组DNA全长分别为3 602、3 208、4 204、5 457和2 436 bp,从起始密码子到终止密码子的长度分别为3 244、2 780、3 947、5 264和2 321 bp。氨基酸序列多重比对及系统发育树结果显示Ac PINs和Ac AUXs分别属于植物PINs和AUX/LAXs家族。荧光定量PCR结果表明,菠萝茎尖经200和1 200 mg·L-1乙烯利诱导处理后,Ac PINs的表达上调较多,其中Ac PIN2在处理后的早期(1~2 d)和后期(28~37 d)显著上调,另外两个PIN家族基因Ac PIN1和Ac PIN3在处理后的大部分时间都明显提高。Ac AUX1的上调表达量相对较少,且相对表达量显著提高也主要集中在处理初期(1 d)和后期(28~37 d),而Ac AUX2的上调表达则只在处理后的前期(1、2、9 d)。研究结果表明,乙烯利诱导菠萝成花过程中,存在着生长素极性运输,且生长素极性输出在此过程中的作用可能更重要。     

龙眼14-3-3基因的克隆与原核表达

 以龙眼(Dimocarpus longan Lour. ) 为试材, 应用同源克隆和RACE方法从花芽中获得了调控 蛋白14-3-3的全长cDNA序列, GenBank登录号为FJ479618 (GI: 218202931) 。该cDNA全长1 121 bp, 包括一个783 bp的开放阅读框, 编码261个氨基酸, 序列比较分析显示14-3-3 cDNA具有较高的保守性。半定量RT-PCR分析结果表明, 14-3-3 mRNA在龙眼叶芽、叶片、花芽和成熟的花中都有表达, 但在花芽中表达量最大。构建了pET14-3-3原核表达系统, 将14-3-3全长cDNA在大肠杆菌中表达, 获得一个分子量约为34 kD的可溶性融合蛋白, 经Western blotting验证, 该蛋白为14-3-3蛋白。