利用花粉管通道法获得转雪花莲凝集素基因(sgna)小麦

利用适用于禾谷类作物的表达载体pGU4AGBar和pGBIU4AGBar,采用花粉管通道法,将人工合成的雪花莲凝集素基因sgna导入了优良冬小麦品系西农2208和西农132,经PCR和Southern blot鉴定,证明获得了20株导入了sgna基因的转基因植株,转化率约为0．28％—0．84％,并通过Western blot鉴定检测到了目的蛋白的表达。     

利用花粉管通道法获得转雪花莲凝集素基因（sgna）小麦

利用适用于禾谷类作物的表达载体pGU4AGBar和pGBIU4AGBar,采用花粉管通道法, 将人工合成的雪花莲凝集素基因sgna导入了优良冬小麦品系西农2208和西农132,经PCR 和Southern blot鉴定,证明获得了20株导入了sgna基因的转基因植株,转化率约为0.2 8%～0.84%,并通过Western blot鉴定检测到了目的蛋白的表达。     

花粉管通道法介导的铁皮石斛转基因技术

该研究以含有GFP和GUS基因的质粒和农杆菌为载体,采用花粉管通道法对铁皮石斛进行转基因技术研究。结果表明:(1)铁皮石斛种子萌发和原球茎生长对卡那霉素的最低致死浓度分别为90和150 mg·L~(-1)。进一步研究证实,在筛选转化种子和原球茎时,可分别向培养基中添加100和150 mg·L~(-1)的卡那霉素进行选择培养。(2)以携带GFP和GUS基因的质粒(pSuper1300和pBI121)和农杆菌为载体,用无菌去离子水重悬质粒pSuper1300和pBI121至浓度为100 ng·μL~(-1),用2%蔗糖+1/2MS+0.1%silwet-77+0.1%AS或5%蔗糖+0.1%silwet-77+0.1 mmol·L~(-1)AS重悬携带质粒pSuper1300和pBI121的农杆菌至菌液浓度为OD_(600)=0.7~0.8;在授粉后0.5~2.5 h内使用柱头滴加法导入携带外源基因的质粒或农杆菌溶液,收集成熟的转化种子,经选择培养及PCR检测发现,几乎所有处理的转化材料均能检测出外源GFP和GUS基因片段。另外,与农杆菌相比,以质粒为载体进行转化,可获得更高的结实率。该研究结果为铁皮石斛的基因工程育种提供了参考。     

小麦花粉管通道及子房注射法转化Anti-TrxS基因

为了获得抗穗发芽转基因小麦材料,以13个小麦品种(品系)为受体材料,用花粉管通道及子房注射法进行了硫氧还蛋白反义基因(Anti-TrxS)的遗传转化。花粉管通道法共转化小花2036朵,收获T0代种子1616粒,结实率为79．4％,将T0代种子大田点播,获T1代苗1424株,出苗率为88．1％。对T1代株系叶片基因组DNA进行PCR检测,31个株系中有16个株系呈阳性反应。子房注射法共转化小花1206朵,收获种子89粒,结实率为7．4％,T1代共出苗41株,出苗率为46．1％,对郑新991T1代株系进行PCR检测,电泳结果呈阳性反应。     

花粉管通道法介导PRSV-CP基因dsRNA转化番木瓜

以番木瓜‘蔬罗Ⅰ号’植株为受体材料,采用花粉管通道技术将番木瓜环斑病毒外壳蛋白(PRSV-CP)基因3′-端同源序列dsRNA转入番木瓜子房中。用PCR方法对T0代种子进行了分子检测,获得53株转基因番木瓜,转化率达到8.9%;对获得的T0代转基因番木瓜进行了田间抗病性鉴定,结果表明,转基因番木瓜植株对番木瓜环斑病毒(PRSV)表现为不同程度的抗性,可以推迟发病。     

河套蜜瓜花粉管通道法转化hALR基因

为利用河套蜜瓜为生物反应器生产可用于治疗肝病的蛋白因子,以人胎肝mRNA为模板,经RT-PCR扩增获得了471 bp cDNA片段.克隆到pMD18-T载体,命名为pMD-ALR,测序分析结果与GenBank中报道的hALR编码序列完全相同.酶切回收hALR片段,插入到含有甜瓜果实黄瓜素(Cucumisin)基因启动子的果实特异性表达载体pPZP-CGN中,构建了hALR甜瓜果实特异性表达载体pPZP-CAN.花粉管通道法转化试验大田自花授粉花63朵,收获T0代果实27颗,结实率为42.9%.提取T0代种子无菌苗DNA,经PCR、PCR-southern和斑点杂交检测有13株呈阳性.     

花粉管通道技术转化番木瓜的初步研究

以番木瓜"solo Ⅱ"号植株为受体材料,用花粉管通道法进行了番木瓜环斑病毒外壳蛋白基因(PRSV-CP)278 bp片段的遗传转化.采用质粒DNA和农杆菌菌液两种导入液,分别处理花187和232朵,收获成熟番木瓜105和30个.座果率分别为56.15%和12.93%,随机选择每种载体的种子100粒播种.成株率分别为61%和60%.对T1幼苗(除含空载体外)全部进行PCR检测.检测结果为质粒DNA和农杆菌菌液两种导入液转化所得的幼苗阳性率分别为50.54%和51.22%.     

花粉管通道法转基因技术的细胞胚胎学机理探讨

本文从细胞胚胎学出发,从理论上对花粉管通道、花粉管通道法的转化机理进行了研究。认为,外源DNA进入胚囊的途径,即外源DNA沿着连接柱头与胚囊的花粉管外界面渗入胚囊;外源DNA转化的受体应为合子;外源DNA转化的时期应限定在精卵融合至合子分裂前这一段时期,此时合子细胞壁尚未封闭;外源DNA转化受体的机制可能是外源DNA与处于原生质体状态的合子的随机融合。强调在应用此方法时,应对植物雌蕊结构以及受精经历的时间有全面了解,并列出了一些重要植物授粉后受精过程各阶段的时间,对外源DNA导入部位和方法提出了建议,并分析了花粉管通道法转基因技术转化率较低的原因。     

利用花粉管通道技术导入大豆抗病虫目的基因

采用花粉管通道技术,将几丁质酶基因导入14个大豆品种,共导入1125朵花,花朵的成活率为47.8%,将Bt基因导入11个大豆品种,共导入260朵花,花朵的成活率为40.8%。8个转Bt基因品种中的D1代共收获192粒种子,获得幼苗132株,出苗率为68.75%,把转Bt基因的132株植株用X-Gluc溶液检测,没有发现阳性反应,将转Bt基因的132株植株进行PCR检测,得到5株阳性转化植株,对D1代获得的5株阳性转化植株的D2代,进行PCR检测,得到D2代稳定遗传的阳性转化植株2株。     

我国小麦品质改良的发展方向

小麦是我国的主要粮食作物之一,也是我国最重要的商品粮食和贮藏品种。据农业部统计,１９９７年小麦种植面积４．５亿亩,占粮食播种面积的２６．６％,产量１．２３亿吨,占粮食总产量的２４．９％。从总体上讲,我国的小麦品种的蛋白质含量与国外品种差异不大,但是蛋白质质量差,表现为面筋强度弱,面团流变学特性差,加工品质不好。随着市场经济的发展,人民生活水平的提高,“标准粉”、“富强粉”已逐渐失去市场竞争力,各种专用粉,如面包粉、糕点粉、饺子粉、面条粉、方便面用粉和富硒、铁、钙等保健粉日趋进入市场。为适应“专用…     

Actin organization and regulation during pollen tube growth

Pollen is the male gametophyte of seed plants and its tube growth is essential for successful fertilization. Mounting evidence has demonstrated that actin organization and regulation plays a central role in the process of its germination and polarized growth. The native structures and dynamics of actin are subtly modulated by many factors among which numerous actin binding proteins (ABPs) are the most direct and significant regulators. Upstream signals such as Ca²⁺, PIP₂ (phosphatidylinositol-4,5-bis-phosphate) and GTPases can also indirectly act on actin organization through several ABPs. Under such elaborate regulation, actin structures show dynamically continuous modulation to adapt to the in vivo biologic functions to mediate secretory vesicle transportation and fusion, which lead to normal growth of the pollen tube. Many encouraging progress has been made in the connection between actin regulation and pollen tube growth in recent years. In this review, we summarize different factors that affect actin organization in pollen tube growth and highlight relative research progress.     

蛋白质可逆磷酸化对花粉管生长的调控作用

花粉管极性生长受多种信号与代谢过程的调控,主要包括Rop GTPase信号途径、磷脂酰肌醇信号通路、Ca²⁺信号途径、肌动蛋白动态变化、囊泡运输、细胞壁重塑等,这些过程都受到蛋白质可逆磷酸化作用的调节。如：(1) Rop调节蛋白(GEF、GDI和GAP)的可逆磷酸化可以改变其活性,从而调节Rop GTPase;同时,蛋白激酶还可能作为Rop下游的效应器分子参与Rop下游信号途径的调节;(2) 蛋白质可逆磷酸化作用既能够激活/失活质膜上的Ca²⁺通道或Ca²⁺泵,又参与调节胞内贮存Ca²⁺的释放,从而调控花粉管尖端Ca²⁺梯度的形成;此外,蛋白激酶还作为Ca²⁺信号的感受器,磷酸化相应的靶蛋白,参与Ca²⁺信号下游途径的调节;(3) 肌动蛋白结合蛋白(ADF和Profilin)的活性也受到蛋白质可逆磷酸化的调节,进而调控肌动蛋白聚合与解聚之间的动态平衡;(4) 蛋白质磷酸化作用调节胞吞/胞吐相关蛋白的活性,并调控质膜的磷脂代谢,从而参与调控囊泡运输过程;(5) 胞质丝氨酸/苏氨酸蛋白激酶和蔗糖合酶的可逆磷酸化可以调节其在花粉管中的功能与分布模式,参与花粉管细胞壁重塑;(6) 转录调节蛋白与真核生物翻译起始因子的可逆磷酸化可以改变其活性,从而调控RNA转录与蛋白质合成。文章主要综述了花粉管生长过程中重要蛋白质的可逆磷酸化作用对上述关键事件的调节。     

巴旦杏授粉试验及花粉管生长的荧光显微观察

对巴旦杏4个主栽品种的授粉试验结果表明：自花授粉试验中“鹰嘴”、“双果”坐果率分别为19．74％和4．29％,“纸皮”和“麻壳”坐果率为零;自然授粉状态下4个主栽品种坐果率在9．37％－20．13％之间;人工异花授粉试验中4个品种9个授粉组合坐果率绝大多数都在40％以上,其中混合花粉授粉试验结果最好;人工养蜂条件下的坐果率明显高于无蜂自然授粉的坐果率,昆虫传粉可达到人工混合花粉授粉的效果。授粉试验还表现出巴旦杏雌蕊柱头的有效授粉时间为开花后花期的前4d,授粉越早,越有利于坐果。荧光显微观察表明,巴旦杏花粉管最早约于授粉第7天通过花柱基部到达子房室,第8天前后到达胚珠。     

Pollen germination and pollen tube growth in Fraxinus pennsylvanica

With regard to adaptation of green ash (Fraxinus pennsylvanica Marshall) to ecological conditions in Croatia, pollen germination and pollen tube length after 2, 4 and 6 hours were examined in vitro at 10, 15, 20 and 25°C during two years 2001 and 2002. Narrow leaved ash (F. angustifolia Vahl) pollen served as a control in 2002. The year, time and temperature, and the interaction between time and temperature were significant for both germination percentage and pollen tube length. Interactions year × temperature and year × time were significant for pollen tube length only. The highest germination percentage (17.86% in 2001 and 19.40% in 2002) of green ash pollen was at 15°C after 6 hours. The pollen tube length was greatest at 20°C (393.46 μm) in 2001 and 25°C (899.50 μm) in 2002 after 6 hours. Narrow leaved ash pollen had the highest germination percentage (19.22%) at 20°C after 6 hours and was significantly reduced at 25°C. The pollen tube length was greatest at 25°C (518.90 μm) after 6 hours. It can be concluded that green ash pollen has satisfactory germination in ecological conditions in Croatia and that the optimum temperature for pollen germination is higher than 20°C.     

Sugar composition of pollen grain and pollen tube cell walls

The sugar composition of pollen grain and pollen tube cell walls was studied for Camellia japonica, C. sasanqua, C. sinensis, Tulipa gesneriana and Lilium longiflorum. In all species, the main components of pollen grain walls were arabinose, galactose, glucose and uronic acid. On the other hand, the pollen tube walls consisted mostly of glucose. The pollen tube wall of C. japonica was fractionated into hemicellulose, α-cellulose and pectic substance fractions in yields of 61, 19 and 3 %, respectively. The hemicellulose fraction was composed essentially of glucose. The sugar composition of the pollen tube wall was not influenced by the nature of exogenously supplied sugars. Rapid growth of the pollen tube seemed to correlate with the synthesis of hemicellulosic glucan.     

从柱头到胚囊——花粉管发育研究进展

花粉管的生长和发育是植物有性生殖过程中重要环节。本文首先介绍了花粉管的结构及其脉冲生长方式。然后介绍了花粉粒在柱头上萌发、花粉管在花柱道中生长及其进入胚囊的一些特点,同时还介绍了雌蕊在花粉管生长发育所起的重要作用。     

Effect of smoke derivatives on in vitro pollen germination and pollen tube elongation of species from different plant families

Plant‐derived smoke stimulates seed germination in numerous plant species. Smoke also has a positive stimulatory effect on pollen germination and pollen tube growth. The range of plant families affected my smoke still needs to be established since the initial study was restricted to only three species from the Amaryllidaceae. The effects of smoke‐water (SW) and the smoke‐derived compounds, karrikinolide (KAR₁) and trimethylbutenolide (TMB) on pollen growth characteristics were evaluated in seven different plant families. Smoke‐water (1:1000 and 1:2000 v:v) combined with either Brewbaker and Kwack's (BWK) medium or sucrose and boric acid (SB) medium significantly improved pollen germination and pollen tube growth in Aloe maculata All., Kniphofia uvaria Oken, Lachenalia aloides (L.f.) Engl. var. aloides and Tulbaghia simmleri P. Beauv. Karrikinolide (10^?6 and 10^?7 m ) treatment significantly improved pollen tube growth in A. maculata, K. uvaria, L. aloides and Nematanthus crassifolius (Schott) Wiehle compared to the controls. BWK or SB medium containing TMB (10^?3 m ) produced significantly longer pollen tubes in A. maculata, K. uvaria and N. crassifolius. These results indicate that plant‐derived smoke and the smoke‐isolated compounds may stimulate pollen growth in a wide range of plant species.