雄性不育相关基因msLTP的反义RNA验证

根据普通白菜雄性不育相关的脂质转移蛋白基因(msLTP)的cDNA序列设计引物,从普通白菜花蕾的cDNA中扩增出312bp的片段,然后将该片段连接至双元载体pBI12l中,得到反义RNA植物表达载体并导入农杆菌LBA4404菌株中,通过农杆菌介导法转化菜心;利用PCR和Southern blot分析检测得到了25株转基因植株,转基因植株的花粉部分畸形或空瘪,花粉离体萌发率为38.56%,较未转化植株的萌发率(76.32%)降低了37.76个百分点.研究表明,反义RNA技术使msLTP基因沉默而导致了菜心转基因植株的部分花粉发育不良,说明msLTP基因在普通白菜和菜心等花粉发育中具有重要作用.     

白菜小孢子发育相关基因BcMF3的分离及序列分析

利用cDNA-AFLP技术对‘矮脚黄’白菜(Brassica campestris L．ssp．chinensis Makinovar．communis Tsen et Lee cv,Aijiaohuang)雄性不育两系不育株系与可育株系的表达差异进行分析,结果以A18和T16引物为引物对在可育的中蕾和大蕾中筛选出一条474bp特异表达的差异条带BA18-T16,配合RACE方法扩增了该片段的3’和5’末端,获得了该基因的cDNA全长序列BcMF3。BcMF3全长2082bp,开放阅读框长度为1755bp,编码584个氨基酸。序列分析结果表明,BcMF3基因与油菜、大白菜和拟南芥的花药特异表达果胶甲酯酶(PME)基因具有较高的相似性,由此推定,BcMF3基因为白菜花药特异表达的果胶甲酯酶基因。     

一个白菜花粉发育相关的新基因BcMF7的鉴定

利用cDNA-AFLP技术分析了白菜(Brassica campestris L. ssp. chinensis Makino, syn. B. rapa L. ssp. chinensis)雄配子减数分裂的胞质分裂突变体(male meiotic cytokinesis, mmc)和野生型植株花蕾基因表达的差异, 克隆了其中一个在野生型花蕾中特异表达的基因BcMF7, 序列分析发现该基因的最大开放阅读框为390 bp, 编码129个氨基酸, 推测的蛋白质分子量为13.75 kD, 含有8个蛋白激酶C磷酸化位点, 2个酪蛋白激酶II磷酸化位点, 2个酪氨酸激酶磷酸化位点, 2个N-糖基化位点以及2个N-豆蔻酰化位点等, 这些位点均与细胞内信号传导、蛋白定位以及黏附等过程有关. 时空表达特点分析发现BcMF7在花粉中特异表达, 其转录本最早在花粉发育的四分体时期开始出现, 在单核小孢子时期表达丰度达到最高, 随后在成熟花粉粒形成时期表达量又有所下降. 推测BcMF7可能在花粉发育的四分体时期到成熟花粉粒形成过程的信号传导中发挥重要作用.     

传粉导致的转基因白菜与其近缘种属材料间的基因流动

从形态、染色体及分子水平上证实,转基因不结球白菜(Brassica campestris ssp. chinensis var. utilis Tsen et Lee)中编码除草剂Basta抗性的bar基因能在田间条件下,经自然传粉,以较高频率侵入芜菁(B. campestris ssp. rapifera)、结球白菜(B. campestris ssp. pekinensis)和不结球白菜(B. campestris ssp. chinensis)的基因组中,也能少量侵入同属异种的甘蓝型油菜(B. napus)基因组中; 在温室人工辅助授粉条件下,除在上述种中的基因漂移率提高外,bar基因尚能以一定频率侵入同属的黑芥(B. nigra)、埃塞俄比亚芥(B. carinata)、芥菜(B. juncea)基因组中,但始终未能得到转基因白菜与结球甘蓝(B. oleracea)、萝卜(R. sativus)的杂种.转基因白菜与十字花科的7种常见杂草经温室人工辅助授粉,也均未得到抗性杂种.     

传粉导致的转基因白菜与其近缘种属材料间的基因流动

从形态、染色体及分子水平上证实,转基因不结球白菜(Brassica campestris ssp．chinensis var．utilis Tsen et Lee)中编码除草剂Basta抗性的bar基因能在田间条件下,经自然传粉,以较高频率侵入芜菁(B．campestris ssp．rapifera)、结球白菜(B．campestris／sssp．pek／nens／s)和不结球白菜(B．campestrs ssp．ch／nens／s)的基因组中,也能少量侵入同属异种的甘蓝型油菜(B.napus)基因组中;在温室人工辅助授粉条件下,除在上述种中的基因漂移率提高外,bar基因尚能以一定频率侵入同属的黑芥(B．nigra)、埃塞俄比亚芥(B．car／nata)、芥菜(B．juncea)基因组中,但始终未能得到转基因白菜与结球甘蓝(B．oleracea)、萝卜(尺．sativus)的杂种。转基因白菜与十字花科的7种常见杂草经温室人工辅助授粉,也均未得到抗性杂种。     

白菜高效再生系统的建立及其不定芽发生的组织学观察(简报)

白菜(Brassica campestris L ssp.chinensis Maki-no)起源于欧洲的野生芸薹,有许多变种和类型,是我国尤其是长江流域及南方各省普遍栽培的重要蔬菜种类之一,在农业生产中占有重要的地位。由于白菜的植株再生频率同其他芸薹属作物相比较低,因此,影响了基因工程技术在白菜品种改良上的应用。虽经国内外多年的研究,白菜的植株再生频率有所提高,但还达不到转基因植物研究80%-90%的植株再生频率的要求。本试验在筛选获得“油冬儿”白菜适宜培养基配方的基础上,比较了“上海青”等8个白菜类蔬菜不同品种在该培养基上的离体培养再生效率,进一步完善了白菜类蔬菜的离体培养再生体系,     

红菜薹新型细胞质雄性不育系的转育研究

以新型甘蓝型油菜（Brassica napus L.）细胞质雄性不育系（Eru CMS）为不育源,通过远缘杂交结合回交的方法,并运用组织培养、单株选择、异地加代种植的手段成功地将该不育基因转育到了红菜薹上。经检测所得的红菜薹（Brassica campestris L.ssp.chinensis L.var.utilis Tsen et Lee.）不育系苗期遇低温叶片不黄化,不育性稳定,蜜腺正常,不育度和不育株率均为100%。     

甘蓝DELLA基因家族鉴定及其mRNA在嫁接体中的运输分析

DELLA基因家族参与植物激素信号转导通路的调控，其中GAI(GA insensitive)mRNA还是植物体内长距离运输的信号分子。在全基因组范围内鉴定甘蓝(Brassica oleracea var. capitata) DELLA基因家族成员并分析mRNA运输特性，可为甘蓝DELLA基因家族的开发应用提供基础数据。本研究利用甘蓝基因组数据和转录组数据，在甘蓝中鉴定了5个DELLA基因家族成员(BoRGA1、BoRGA2、BoRGL1、BoRGL2和BoRGL3)，但甘蓝基因组缺失了GAI基因。采用劈接法将甘蓝(自交系G27)接穗和菜心(Brassica campestris L. ssp. chinensis var.utilis Tsen et Lee)(四九菜心)砧木嫁接在一起，构建异源嫁接体。对砧木菜心花序轴和对应位置的实生苗菜心花序轴(对照)取样进行转录组测序。在甘蓝/菜心异源嫁接体的砧木菜心花序轴的转录组测序文库中分别鉴定到8、9、3、5、1个来自甘蓝BoRGA1、BoRGA2、BoRGL1、BoRGL2和BoRGL3的外源read。甘蓝DELLA家族基因mRNA运输并...     

改良菜心离体培养植株再生体系的研究(简报)

菜心(Brassica campestris L.subsp.chinensis Makino var.parachinensis Tsen et Lee)为十字花科芸薹属芸薹种中国白菜亚种中的一个变种,又名“菜薹”。它是我国南方特产蔬菜之一,在蔬菜的周年供应上有重要地位。目前迫切需要培育抗病虫、抗逆和具有其他优良农艺性状的新品种,以提高菜心的     

不结球白菜雄性不育系及其保持系花药发育的细胞学观察

以不结球白菜（Brassica campestris ssp．chinensis Makino）雄性不育系及其保持系为试验材料,选择不同发育阶段的花蕾,取其花药,制成石蜡切片和超薄切片,经染色后在电子显微镜下观察。结果表明,不结球白菜雄性不育系与保持系的花药发育有明显的不同：不育系花药发育受阻于花粉母细胞分化期,形成1～3个药室,并形成正常的四分体小孢子,此时细胞组织逐步解体,形成空腔花药;最后向内皱缩;保持系花粉母细胞能形成正常的四分体,进而形成小孢子,最终形成充满正常花粉粒的花药。     

Isolation and Molecular Characterization of RALF Like Gene BcRALF from Brassica campestris ssp.chinensis var.parachinensis

The rapid alkalinization factor (RALF) gene is a new found plant polypeptide signal molecule, wide spreading in higher plants. In this study BcRALF was cloned from Brassica campestris sspchinensis Makino varparachinensis based on BcMF14 (GenBank accession number EF523516) from Bcampestris ssp. chinensis var. communis cv. Aijiaohuang. The sequence of this gene was 273 bp (GU086228) and was identified as a rapid alkalization factor gene according to its high identity with Boleracea var botrytis BoRALF1 and Arabidopsis thaliana RALFL9. Protein characteristics and sequence structure were predicted, and moreover, many bioactive sites were found. The results showed that the characteristics of the BcRALF protein consistent with its category as a peptide signal molecule.     

快速碱化因子基因BcRALF在菜心中的克隆及序列分析

快速碱化因子是近年来新发现的一种植物多肽类信号分子,广泛存在于高等植物中。通过已得到的普通白菜的快速碱化因子基因BcMF14（GenBank序列登录号EF523516）的核苷酸序列,在其编码框两侧设计引物,从菜心中克隆出该类信号分子基因,命名为BcRALF（登陆号：GU086228）。序列同源比对表明该基因与花椰菜、拟南芥等的快速碱化因子基因有很高的相似性,证明BcRALF属于快速碱化因子家族。蛋白质特征预测以及蛋白序列结构分析发现BcRALF蛋白包含有多个生物活性位点,符合其作为多肽类信号分子类蛋白的特征。     

Characterization and functional analysis of a novel PCP gene BcMF5 from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino)

Brassica campestris Male Fertile 5 (BcMF5), a novel member of the pollen coat protein class A (PCP-A) gene family, was identified from Brassica campestris L. ssp. chinensis Makino (Chinese cabbage-pak-choi). Temporal and spatial expression analysis showed that BcMF5 is a late-expressed PCP gene related to the process of determining pollen fertility. Functional analysis by hairpin RNA (hpRNA)-mediated RNA interference also showed that the expression of BcMF5 is inhibited, which resulted in the low germination ability of the pollen and also in an abnormality of the pollen exemplified by a collapsed germination furrow. This demonstrates that the expression of BcMF5 is closely related to the tapetum. Further, the expression profile of the BcMF5 promoter in Arabidopsis was also analyzed. This analysis indicated that the BcMF5 promoter began expression in the early stage of anther development and drove high levels of glucuronidase (GUS) expression in anthers, pollen, and the pollen tube in the late stage of pollen development, but did not drive any expression in petals, sepals, or pistils. Together with the functional analysis, the hypothesis that BcMF5 may have a sporophytic or gametophytic expression pattern is presented.     

Morphological and functional characterization of BcMF13 in the antisense-silenced plants of Brassica campestris ssp. chinensis var. parachinensis

The gene Brassica campestris male fertility 13 (BcMF13, GenBank accession number EF158459) was isolated as a reproductive organ-specific gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis). It is exclusively expressed in stage four and five flower buds of fertile lines and is most strongly expressed in stamens. Here, we report a functional characterization of this BcMF13 gene in the antisense-silenced plants. The inflorescence of the BcMF13 mutant was compacted with anthers curved outside. The fertility of this mutant was greatly reduced with less than 5 seeds per silique. Under scanning electron microscopy, the mutant demonstrated numerous shriveled pollen grains with deep invaginations. The frequency of normal pollen grains was just 45.34%. The pollen mother cell, the tetrad, and the mature pollen of the BcMF13 mutant were abnormal resulting in the poor pollen vitality. Germination test in vivo suggested BcMF13 delayed the pollen tubes’ extension in the style. All these indicated BcMF13 had a vital role in pollen development of Chinese cabbage.