风信子HoMADS2基因的克隆及表达分析

利用同源克隆策略,从风信子中分离出一个MADS box基因,命名为HoMADS2。序列比较分析表明,HoMADS2与B类MADS box蛋白具有较高的同源性。分子进化树分析显示,HoMADS2与PI家族类聚在一起。同时,在HoMADS2的Kbox和C末端区域均具有PI家族的特征序列。以上序列分析结果表明,HOMADS2可能是尸,的一个同源基因。RNA分子杂交结果显示,HoMADS2在四轮花器官中均表达,其表达模式不同于双子叶植物中尸,同源基因。利用风信子离体花器官再生系统研究表明,HoMADS2在再生花芽中的表达不同于HoMADS1和HAG1,该基因在再生花芽发育过程中组成型表达,不受外源细胞分裂素和生长素的影响。     

Effects of Temperature on Induction of Regenerative Stamens in vitro Culture,Microsporogenesis and Pollen Development in Hyacinthus orientalis L.

In order to culture the regenerated stamens of hyacinth in vitro to maturity, the effects of temperature on regeneration of stamens, microsporogenesis and pollen development were studied. Results showed: the proper temperature for stamen regeneration was 25℃. The temperature going down gradually was advantageous to the microsporogenesis and pollen development. The most suitable temperatures for differentiation of microsporocyte, meiotic division and pollen first mitosis were 20–25℃, 20℃ and 10℃ respectively. Under such temperature condition, it was possible to culture the regenerated stamens to maturity, and pollen grains in the stamens had the higher germination frequency. On the contrary, unsuitable temperature condition will make microsporogenesis and pollen development stop at certain development stage, it will finally result in pollen abortion.     

不同风信子品种在南京地区的物候期及生长特性分析

对从荷兰引进的17个风信子(Hyacinthus orientalis L. )品种在南京地区的物候期、观赏特性和生长状况进行了调查分析.调查结果表明,17个风信子品种的耐寒性较强,出苗期最早在1月下旬,多数品种的出苗期在2月中下旬;绝大多数风信子品种的叶片展开期集中在3月14日至20日;花现色期多为2月底至3月初,第1朵花的开花期在3月中上旬;大部分品种的单花序开花持续时间和群体开花持续时间较长,分别达20～22 d和27～31 d.17个风信子品种的花均具有非常浓烈的香味,花蕾与花脉颜色相同,花色从花脉辐射至花瓣边缘并逐渐变淡,花色艳丽, 观赏性强. 少数品种具1个花枝, 多数品种具2个花枝, 花茎长度12.7～21.9 cm, 多数品种的花茎长度在17 cm以上;主花序长度多在11 cm以上,约占整个花茎长度的2/3;单花序花朵数为20～61.各品种的花瓣数和花药数基本一致,多以6为基数;17个风信子品种的花粉均有一定的萌发能力,其中9个品种的花粉萌发率较高,均超过40%.多数品种的叶片数为6或7,叶片长18～29 cm,宽2.2～3.6 cm.除品种'阿姆斯特丹'、'简波斯'和'福特'外,其余品种均可以分生出籽球,其中,品种'蓝星'、'蓝夹克'和'爱丽斯'的籽球分生能力最强,分生率达66%以上;不同风信子品种的籽球直径也各不相同,多数籽球直径较大,为3～5 cm.结果显示,风信子可作为早春花卉在南京进行种植,但具体种植条件尚待深入研究.     

东方山羊豆Cu/ZnSOD基因的克隆及表达分析

超氧化物歧化酶是一种广泛存在于真核生物中的金属酶类,在植物的抗逆性中起到重要的作用。文章采用RACE方法,从东方山羊豆中克隆了Cu/ZnSOD基因,并对其进行了初步分析。该基因cDNA序列全长935 bp,开放阅读框600 bp,编码199个氨基酸,蛋白质分子量为20.35 kDa。通过实时荧光定量PCR结果分析,该基因在东方山羊豆叶中表达量最多,茎中次之,根中最少。在NaCl和PEG诱导下,Cu/ZnSOD基因表达量先上调后下降。NaCl诱导24 h后,该基因的表达量显著低于对照。ABA胁迫抑制了该基因的表达。亚细胞定位结果表明,Cu/ZnSOD蛋白定位于叶绿体中。实验结果证明,Cu/ZnSOD基因主要在东方山羊豆的绿色组织中表达,在抵抗渗透性胁迫方面起到一定作用。     

风信子花器官中HAP2基因的分离与表达研究(英文)

在离体条件下,以风信子花被片为外植体,通过控制激素的浓度可诱导花被片、雄蕊或胚珠的再生。近年来,在拟南芥和金鱼草等模式植物中已经分离出了许多控制花器官发育的同源异形基因,如AG,AP1,AP2,AP3等,其中AP2在控制花萼和花瓣形成过程中起重要作用,因此本文从风信子中分离AP2的同源基因,并对它在风信子再生系统中的表达进行了分析。根据AP2同源基因功能域的保守序列设计一对简并引物：5'-TGGGA（A/G）TC（G/T/C）CA（C/T）AT（C/T）TGGA-3'和5'-TCCCA(AGC)(CT)(GT)(AG)CC(AG) CA(CT)TT(AG)TG-3', 以再生的花被片为材料进行RT-PCR,扩增出大小约300bp的片段,序列分析表明该片段的氨基酸序列与AP2同源性高达89%。进而,利用5’和3’Race PCR,得到全长的cDNA。该基因命名为HAP2,GenBank登记号为AF134116,该基因全长1597bp,编码368个氨基酸(Fig.1）。与AP2相比,HAP2也含有10个氨基酸长的碱性功能域,其中KKSR为核定位信号。此外,HAP2也含有两个序列重复的68个氨基酸长的功能域（HAP2-R1,HAP2-R2）,HAP2-R1也含有能形成(-螺旋结构的核心区域,且与AP2-R1中的核心序列100%同源,而HAP2-R2中的核心区域与AP2-R2相比, 缺少9个氨基酸(Fig.2）。RT-PCR结合Southern 杂交结果表明(Fig.3）,HAP     

风信子花被外植体年龄对花器官分化的影响

离体培养风信子（ＨｙａｃｉｎｔｈｕｓｏｒｉｅｎｔａｌｉｓＬ．）不同年龄的花被外植体诱导花器官直接再生的实验表明：１．在ＭＳ附加６ＢＡ２ｍｇ／Ｌ、２,４Ｄ０．１ｍｇ／Ｌ的培养基上,年龄Ｖ的外植体大量衰老,基本丧失器官再生能力,处于年龄段ＩＩ～ＩＶ的外植体可发生玻璃化反应。２．玻璃化反应的外植体转移至ＭＳ附加６ＢＡ０．２ｍｇ／Ｌ、ＮＡＡ０．００５ｍｇ／Ｌ的培养基上继续培养３０ｄ后可再生正常的花被片,表明降低培养基中外源激素浓度能够阻止玻璃化反应继续发生。３．在ＭＳ附加６ＢＡ２ｍｇ／Ｌ、２,４Ｄ０．１ｍｇ／Ｌ的培养基上,外植体形态学下部可再生雌蕊状早期结构。平均每块外植体分化雌蕊状早期结构数以年龄Ⅲ的外植体最多     

Production of essential oils by flowers of Hyacinthus orientalis L. regenerated in vitro

Essential oils were produced by flowers of Hyacinthus orientalis L. that had been regenerated in vitro. The production of these oils was affected by the concentration of gibberellic acid and sucrose in the medium and by temperature. The highest concentration of essential oils was obtained when regenerated flowers were cultured in vitro for 3 weeks at 20 °C on Murashige and Skoog's medium that contained 30 g/l sucrose plus 1 mg/l gibberellic acid, whereas the highest amount of essential oils was obtained after a culture period of 3 weeks at 25 °C. The composition of essential oils from flowers that had been regenerated in vitro was compared with that from flowers grown in the field. Essential oils detected by gas-liquid chromatography included nine components in the case of the regenerated flowers and six and ten components in the case of stage 3 and stage 4 flowers grown in the field, respectively. There were four common components, namely, 1-hepten-3-ol, benzyl alcohol, phenethyl alcohol and cinnamyl alcohol. In the regenerated flowers, a single component, phenethyl alcohol, was a major constituent (75%), whereas two compounds, phenethyl alcohol (stage 3, 55%; stage 4, 48%) and cinnamyl alcohol (stage 3, 23%; stage 4, 29%) were the major constituents in the case of flowers grown in the field. Four and five other components were specific to flowers regenerated in vitro and field-grown flowers, respectively.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - ABA abscisic acid - GA₃ gibberellic acid - ACC 1-aminocyclopropane-1-carboxylic acid - ethephon (2-chloroethyl)phosphonic acid - MS medium Murashige and Skoog's medium - GLC gas-liquid chromatography - GC-MS gas chromatography-mass spectrometry - FW fresh weight     

风信子中抗青霉素内生菌的分离、筛选和活性检测

通过用初筛培养基培养,从风信子叶子中分离出一种抗青霉素的内生菌,进行离体培养和抑菌活性检测。结果表明所筛选的菌株的发酵产物对细菌和真菌均有一定的抑菌活性。其中发酵液提取物对枯草芽孢杆菌有一定的抑菌活性,对苹果干腐病原菌和烟草赤星病原菌的抑菌率分别是94.8%和93.8%,而菌体内的活性物对蜡状芽孢杆菌和枯草芽孢杆菌都有较高的活性,对苹果干腐病原菌、烟草赤星病原菌和苹果轮纹病原菌的抑菌率都在80%以上。     

风信子品种间杂交与幼胚离体培养研究

选用5个风信子品种进行花粉生命力测定,对完全双列杂交获得的杂种种子剥胚进行离体培养.试验表明:(1)风信子不同品种花粉萌发率差别较大,其中Fondante的花粉萌发率最高为74.45%,Amsterdam的最低为12.68%,其他3个品种的花粉萌发率介于二者之间.(2)品种间自交杂交亲和力存在一定差异,结实率大于30%的组合从高到低依次为Fondante×Atlantic(61.2%)、Fondante×Fondante(54.0%)、Carnegie×Carnegie(42.6%)、JanBos×JanBos(36.0%)、Carnegie×Atlantic(32.7%)、Atlantic×Fondante(30.0%),组合Atlantic×Amster-dam、Carnegie×JanBos、Amsterdam×Fondante、Amsterdam×JanBos、JanBos×Amsterdam未得到果实,其余组合结实率介于0～30%之间.(3)适宜风信子杂交幼胚离体培养的初代培养基为MS+100 mg/L谷氨酰胺,其次为MS+0.2 mg/LIBA+0.02 mg/L6-BA+100 mg/L谷氨酰胺...     

The Exogenous Hormornal Control of the Development of Regenerated Flower Buds in Hyacinthus orientalis

The critical role of exogenous hormone on inducing the initiation of different floral organs in the regenerated flower bud and controlling their numbers was further evidenced. The initiation of the flower buds was first induced from the perianth explants of Hyacinthus orientalis L. cv. White pearl by a combination of 2 mg/L 6-BA and 0.1 mg/L 2,4-D, and then a continuous initiation of over 100 tepals (a flower bud of H. orientalis in situ has only 6 tepals) was successfully controlled by maintenance of such a hormone concentration. However, a change of hormonal concentration (2 mg/L 6-BA and 0-0.000 1 mg/L 2,4-D) caused cessation of continuous initiation of the tepals but gave rise to induction of stamen initiation. Keeping the changed hormone concentrations could successfully control the continuous initiation of over 20 stamens (a flower bud of H. orientalis in situ has only 6 stamens). The experiment showed that the number of identical floral organs in the regenerated flower buds can be controlled by certain defined concentrations of the exogenous hormones, and the amount of the induced identical floral organs has no effect on the differentiation sequence of the different floral organs in the regenerated flower bud. Based on a systematic research on controlling the differentiation of the floral organs from both the perianth explants and the regenerated flower buds by the exogenous hormones in H. orientalis over the past decade, the authors put forward here a new idea on the role of phytohormone in controlling the automatic and sequential differentiation of the different floral organs in flower development. The main points are as follows: 1. the development of flower bud in plant is a process in which all of the floral organs are automatically and sequentially differentiated from the flower meristem. 2. Experiments in vitro showed that the effect of exogenous hormones in controlling the initiation of different floral organs is strictly concentration dependent, i.e., one kind of the floral organ can continuously and repeatedly initiate from the flower meristem as long as it is maintained in that specific concentration of the exogenous hormone which is suitable for the initiation of that particular kind of floral organ. 3. It shows that the flower buds in situ must be automatically able to adjust the endogenous hormonal concentrations just after the completion of the differentiation of one whorl of floral organ to suit the differentiation of the next whorl. Thus, the phytohormone in different concentrations takes after many change-over switches of the organ differentiation and plays a connective and regulatory role between the differentiation of every two whorls of the floral organ. In other words, these change-over switches play the roles of inhibiting the expression of the genes which control the initiation of the floral organs in the first whorl, meanwhile, activating the expression of the genes which control the initiation of the floral organs in the second whorl during the successive initiation of the different floral organs from the flower bud. It results in the automatic and sequential initiation of the various floral organs from the floral meristem.      

外源激素对风信子再生花芽发育的控制

外源激素在诱导再生花芽花器官发生和控制它们的数目中的关键作用被进一步证实。首先通过２ｍｇ／Ｌ ６－ＢＡ和０．１ｍｇ／Ｌ ２,４－Ｄ的激素组合诱导风信子Ｈｙａｃｉｎｔｈｕｓ ｏｒｉｅｎｔａｌｉｓ Ｌ．ｃｖ．Ｗｈｉｔｅ ｐｅａｒｌ）花芽从花被外植体发生,然后保持这种激素浓度,成功地控制了１００多片花被片的连续发生（自然情况下一个风信子花芽仅有６片花被片）。改变激素浓度（２ｍｇ／Ｌ ６－ＢＡ和０～０．０     

绵羊CCNG1基因克隆及表达分析

细胞周期蛋白cyclin G1(CCNG1)是一个重要的细胞周期调控因子,参与哺乳动物颗粒细胞增殖、卵母细胞成熟等繁殖生物学过程,但其在绵羊中鲜有报道。为了研究CCNG1基因对绵羊发情调控以及季节性繁殖的影响,文章对绵羊CCNG1基因进行了克隆和组织表达谱分析,利用Real-time PCR对该基因在多浪羊(常年发情)与中国美利奴羊(季节性繁殖)发情周期不同阶段性腺轴组织的表达变化进行了实时检测。获得了绵羊CCNG1基因部分cDNA序列,其中编码区全长885 bp,编码294个氨基酸。CCNG1蛋白结构经预测存在多个磷酸化位点和蛋白激酶C磷酸化位点。CCNG1基因在所检测绵羊各组织中均有表达,但在卵巢与肾脏中为高丰度表达;CCNG1在不同绵羊品种发情周期不同阶段性腺轴组织的表达变化规律基本一致,卵巢、子宫、松果体、垂体均是在发情期达到峰值。但是在发情期和发情后期卵巢CCNG1的表达量存在显著的品种间差异(P<0.01)。研究结果表明,CCNG1可能通过参与卵泡的生长发育继而达到对绵羊发情和季节性繁殖的调控。     

Molecular cloning and structural analysis of a novel Rac gene osRACB in rice (Oryza sativa L.)

Rac is a subfamily of small GTP-binding protein family. Its molecular weight is between 20 and 30 kilodaltons. As a signal protein, Rac directly or indirectly participates in many physiological processes, such as the regulation of cytoskeleton and the transduction of stress-induced signal. So Rac is also named ?molecular switch? The switch is based on the cycle from a GTP-bound 憃n?to a GDP-bound 憃ff?state[1]. In the superfamily of GTP-binding protein, only heterotrimeric G protein, Ra…     

猪Gli1基因的克隆、表达谱分析及脂肪组织特异性表达载体的构建

Hedgehog(Hh)信号通路对动物脂肪沉积具有抑制作用,并且从果蝇到脊椎动物具有高度保守性,但在家猪研究中鲜见报道。文章选择家猪Hh通路的转录激活因子Gli1进行研究,通过RT-PCR结合RACE技术,首次获得家猪Gli1基因cDNA全长,利用Real-time PCR对家猪Gli1基因在不同组织中的表达丰度进行了分析,并构建了真核表达载体和脂肪组织特异性表达载体。结果表明:猪Gli1基因cDNA全长3 576 bp,基因组序列全长10 715 bp,共12个外显子,编码1 106个氨基酸。生物信息学分析表明,猪Gli1为不稳定亲水性蛋白,不具有跨膜结构域和信号肽序列,但具有锌指结构与核定位序列。对7个物种的Gli1蛋白序列和基因组序列相似性进行分析,发现各物种间序列相似性均在80%以上,说明Gli1在物种间高度保守。组织表达谱分析表明,Gli1仅在成体猪舌组织中表达;在家猪脂肪组织发育进程中,Gli1仅在出生1周的猪脂肪组织中检测到微弱表达,但1月龄及3月龄猪脂肪组织中均检测不到表达,由此推断猪Gli1表达与脂肪组织发育呈负相关。最后,将猪Gli1编码区克隆到真核表达载体pIRES2-EGFP,体外转染实验证明该载体能够正确表达猪Gli1,另外还构建了脂肪组织特异性表达载体,为构建脂肪组织特异性转基因动物奠定基础。     

Molecular cloning and expression analysis of a monosaccharide transporter gene OsMST4 from rice (Oryza sativa L.)

Two putative glycosyltransferases in Arabidopsis thaliana, designated reduced residual arabinose-1 and -2 (RRA1 and RRA2), are characterized at the molecular level. Both genes are classified in CAZy GT-family-77 and are phylogenetically related to putative glycosyltranferases of Chlamydomonas reinhardtii. The expression pattern of the two genes was analyzed by semi-quantitative RT-PCR using mRNA extracted from various organs of bolting Arabidopsis thaliana plants. In addition, promoter::gusA analysis of transgenic Arabidopsis thaliana containing a fusion between either the RRA-1 or -2 promoter fragment and the gusA reporter gene showed that whereas the RRA1 promoter was primarily active in the apical meristem, the expression pattern of the RRA2 promoter was more diverse but also highly active in the meristematic region. In addition, T-DNA mutant insertion lines of both RRA-1 and -2, were identified and characterized at the molecular and biochemical level. Monosaccharide compositional analyses of cell wall material isolated from the meristematic region showed a ca. 20% reduction in the arabinose content in the insoluble/undigested cell wall residue after enzymatic removal of xyloglucan and pectic polysaccharides. These data indicate that both RRA-1 and -2 play a role in the arabinosylation of cell wall component(s).     

禾谷缢管蚜阳离子通道基因RSC1的克隆、分子特性及诱导表达分析

【目的】SC1通道(sodium channel 1)是昆虫体内一种重要的离子通道,被认为是一种开发新型杀虫剂的神经靶标。本研究拟克隆禾谷缢管蚜Rhopalosiphum padi的SC1通道基因,并初步分析其生理功能及其与SC1类通道、电压门控钠离子通道、电压门控钙离子通道的进化关系。【方法】采用RT-PCR技术,克隆了禾谷缢管蚜SC1基因完整的开放阅读框;利用实时荧光定量PCR技术,分析禾谷缢管蚜成蚜在不同浓度的高效氯氟氰菊酯诱导下SC1基因表达变化。【结果】获得了禾谷缢管蚜SC1基因(命名为RSC1)完整的开放阅读框(Gen Bank登录号为KU640190),其长度6 687bp,编码2 228个氨基酸。RSC1具有SC1通道的结构特征,有一个不同于电压门控钠离子通道和电压门控钙离子通道的特殊DEEA模体(motif)。系统进化分析结果显示,RSC1与电压门控钠离子通道组成一个进化枝,电压门控钙离子通道组成另外一个进化枝,SC1与电压门控钠离子通道在进化上有更近的起源关系。实时荧光定量PCR分析结果表明,LC15,LC35和LC503种剂量的高效氯氟氰菊酯处理6 h后,禾谷缢管蚜RSC1基因表达量相对于清水对照显著下调,表达量分别为对照的0.57,0.82和0.78倍;3种剂量的高效氯氟氰菊酯处理24 h后,禾谷缢管蚜RSC1基因表达量分别为对照的2.19,1.33和1.19倍,其中LC15(0.1484 mg/L)胁迫下RSC1基因的表达量显著上调。【结论】SC1类通道与电压门控钠离子通道在进化起源上有更近的关系。RSC1通道可能是高效氯氟氰菊酯的次级靶标。由于RSC1和其同源基因只存在于节肢动物中,脊椎动物尚未发现该类基因,因此这类通道可能作为开发新型杀虫剂的神经靶标。