棉花GhRGPL2基因分离鉴定及其在组织发育和逆境胁迫中的表达调节

在棉花cDNA文库中分离了1个RGP蛋白基因同源序列,命名为GhRGPL2,编码含有359个氨基酸的RGP蛋白.随后,分离获得GhRGPL2基因全序列,基因结构分析表明:它含有3个内含子,分别位于第106和107密码子之间,第190个密码子内部,第246和247密码子之间.Northern杂交分析表明.GhRGPL2在幼根和胚珠中表达量较高.而且,该基因的表达受胚珠发育调节.GhRGPL2基因在胚珠发育早期开始表达,在开花后10 d达到表达峰值,随后基因表达活性逐渐下降.在高盐和干旱胁迫下,GhRGPL2在棉花幼根中的表达量升高.     

棉花纤维细胞分化相关基因的cDNA阵列分析

利用陆地棉(Gossypiumhirsutum)品种徐州142(野生型)开花前1-3d的胚珠构建了一个高质量的cDNA文库,从该cDNA文库中提取13056个质粒,用Biomek2000高密度点阵系统,将这些质粒点于强化硝酸纤维素膜上.分别用徐州142野生型和无絮突变体开花前1-3d的胚珠mRNA反转录标记探针与cDNA阵列杂交,筛选到8个在野生型胚珠中高表达的基因,其中4个在GenBank中有同源序列,4个为未知新基因.Northern杂交表明有两个基因在野生型中比突变体中表达量显著提高.     

棉纤维醛酮还原酶基因促进拟南芥主根伸长

为了研究棉纤维醛酮还原酶基因在参与细胞伸长发育中所起的作用,本研究利用RT-PCR技术从处于快速伸长发育时期的棉花纤维组织中克隆得到棉花醛酮还原酶(aldo/keto reductase,AKR)基因c DNA,该基因全长读码框为1134 bp,编码377个氨基酸。氨基酸序列生物信息学分析表明Gh AKR蛋白具有较高的保守性,包含氧化还原酶、醛酮还原酶结合位点和跨膜信号序列等功能序列和位点;进化树分析显示Gh AKR与可可(Tc AKR)的亲缘关系较近。q RT-PCR和酶活性分析表明Gh AKR基因与纤维细胞的快速伸长发育密切相关,尤其在开花后5-15 d纤维快速伸长发育时期具有较高的表达。构建35S::Gh AKR过量表达载体并转化野生型拟南芥(哥伦比亚生态型),转基因拟南芥的主根伸长发育获得了显著的促进,与野生型相比,转基因拟南芥主根伸长增加了约1.4倍。拟南芥根中甲醇和乙醇含量分析显示,转基因拟南芥根中具有较高的甲醇和乙醇积累。这些结果表明棉纤维Gh AKR基因与纤维细胞伸长发育联系紧密,可能通过增加细胞内的醇含量进一步促进细胞的伸长。本研究可为棉花Gh GAKR基因参与纤维伸长发育分子机制解析,以及利用基因工程培育优质棉花品种提供一定参考。     

棉纤维细胞色素单加氧酶基因GhCYP714A1促进活性氧的累积

本研究利用RT-PCR技术从陆地棉纤维组织中克隆得到了1个棉花细胞色素P450基因(Cytochrome P450714A1)的全长c DNA序列,将该基因命名为GhCYP714A1。序列分析发现,该cDNA包含1563 bp的完整开放读码框,编码521个氨基酸的蛋白质,理论分子质量为57.31 kDa;氨基酸序列生物信息学分析显示,Gh CYP714A1蛋白具有跨膜结构域和多个蛋白结合位点;进化树分析结果表明,棉花Gh CYP714A1与禾草SiCYP714B1在进化上亲缘关系上较近;半定量RT-PCR的组织表达特异性分析表明GhCYP714A1基因与纤维突起形成发育有关。本研究构建了pET28a-GhCYP714A1原核表达载体并进行体外诱导表达,获得分子质量约为57.31 kDa的重组蛋白。将GhCYP714A1基因转化烟草验证其参与活性氧产生的功能,与非转基因的野生型烟草相比,转基因烟草叶片中具有较高的H_2O_2累积。本实验结果为深入研究该基因在棉纤维发育中的作用奠定了基础。     

北京大学朱玉贤教授课题组在棉纤维伸长机制方面取得进展

<正>北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室朱玉贤教授课题组通过比较野生型和无长绒、无短绒突变体棉花胚珠的蛋白质组学数据发现,核苷糖合成途径在棉纤维快速伸长期最显著高调,而植物激素乙烯可能通过促进     

利用低温真空干燥技术对棉纤维起始伸长的扫描电镜观察

棉纤维由棉花受精胚珠的表皮细胞伸长加厚而成,其起始伸长过程是棉纤维发育的重要阶段.为了更好地观察棉纤维在发育过程中的形态变化,利用低温真空干燥技术,对棉花胚珠表皮细胞突起和伸长过程进行扫描电镜观察,获得了理想的观察结果.通过与传统的叔丁醇-乙腈干燥法处理样品的扫描电镜图片进行比较,利用低温真空干燥技术处理的样品形态饱满,轮廓清晰,能真实地观察样品形态.所采用的方法为扫描电镜的使用提供了一个典型范例,对其它植物或含水量较高的生物样品的制样和观察实验具有重要的参考价值.     

白光下拟南芥隐花素双突变体及其野生型的蛋白质组学比较分析

隐花素是植物的蓝光受体,在植物中调节多种光形态建成,包括抑制下胚轴的伸长、子叶的伸展和调节植物的开花时间等,但隐花素依赖蓝光调节光形态建成的分子机制仍然不清楚．采用双向凝胶电泳对生长在白光下的拟南芥隐花素双突变体及野生型的幼苗全蛋白进行分离,通过考马斯亮蓝染色,获得了分辨率和重复性较好的双向电泳图谱．选取了55个差异蛋白质点采用MALDI—TOF—TOF—MS进行肽质谱指纹图分析,最终有33个蛋白质点得到了可靠鉴定．其中在crylcry2中相对于野生型下调的有9个蛋白,其他蛋白均表现为上调．这些在白光处理下差异表达的蛋白质主要是参与碳水化合物代谢、能量代谢、细胞组织结构、抵抗压力和解毒、蛋白质的折叠和细胞壁的形成等功能,而且亚细胞定位主要是在叶绿体和线粒体．对其中5个蛋白质点的基因进行了半定量RT—PCR分析,发现这几个蛋白质点的表达与基因的表达基本一致．这些分析结果表明光控制拟南芥的发育是通过共调节许多相关基因的表达而实现的．     

金鱼蛋白磷酸酶2A—B＂家族Alpha和Gamma调节基因的cDNA克隆及mRNA表达

以金鱼卵巢组织作为材料,运用RTPCR和cDNA末端快速扩增（RACE）技术克隆得到蛋白磷酸酶2A（PP2A）调节亚基B＂家族中γ基因（GSPR）cDNA全序列和α基因（PR130）cDNA部分序列．结果显示γ基因cDNA全长1885bp,编码一个含457个氨基酸的蛋白质．而α基因cDNA长1781bp,编码的多肽共含426个氨基酸,系v亚基的部分蛋白序列．它们与已知其他物种对应的B＂家族蛋白质均有着很高的同源性．结构预测分析发现,α和γ两条多肽均存在PP2A调节亚基B＂家族成员共有的两个EFHAND结构域．用RT—PCR的方法检测了这两个基因在金鱼不同组织和胚胎发育不同时期的mRNA表达水平．结果表明,γ亚基在金鱼多种组织和各个胚胎发育时期中均有较高表达且表达量比较平均,仅在卵巢和精巢组织中最为丰富．与γ亚基表达模式相比,α亚基表达呈现明显的组织和胚胎发育阶段差异性,在卵巢和肌肉组织中最高,在肾脏和鳃中最低,在原肠胚、神经胚、脑泡分化和体色素等胚胎发育时期的表达较弱,而在其他时期中的表达较强．据此,推测α、γ两调节亚基可能在金鱼不同组织和胚胎发育过程中起着特定的作用．此外,进一步的分析发现,α基因除参与PP2A全酶功能外还可能具有独立的功能．     

海岛棉DELLA蛋白基因GbGAI2的克隆及其在胚珠中表达分析

赤霉素(Gibberellins,GA)在棉花纤维的发育过程中起重要作用,DELLA蛋白是GA信号通路中的起关键作用的负调控因子。本研究采用同源克隆方法,获得了海岛棉的DELLA蛋白同源基因GbGAI2。序列比对分析结果表明,GbGAI2编码的氨基酸序列具有DELLA蛋白的典型结构域,并与陆地棉DELLA蛋白GhGAI2氨基酸有90.13%相同。半定量RT-PCR分析结果表明,GbGAI2在1-5DPA和23DPA棉胚中的表达量较高,说明该基因可能在海岛棉棉纤维发育的起始阶段和棉纤维次生壁加厚阶段起作用。     

天然棕色棉纤维发育过程中生化物质变化规律的研究

目的:比较棕色棉和白色棉纤维发育过程中生化物质含量的差异,分析棕色棉纤维色素合成与纤维发育的关系,为棕色棉育种提供理论依据。方法:以白色棉泗棉3号为对照,测定4种棕色棉各发育阶段纤维中主要生化物质含量及其动态变化规律,分析了棕色棉纤维各发育时期生化物质含量与棕色棉纤维色素含量相关关系。结果:棕色棉纤维40DPA含水率、10DPA还原糖含量低于白色棉,30-40DPA还原糖含量高于白色棉。白色棉20DPA可溶性蛋白质出现高峰期,棕色棉相应的蛋白质高峰期出现在25DPA,棕色棉纤维生长的各阶段纤维素的含量均低于白色棉;棕色棉和白色棉,含水率、还原性糖、可溶性蛋白质的含量都随着棉纤维发育不断降低,而纤维素含量不断增加。成熟棉纤维色素含量与10DPA还原性糖含量、30-40DPA纤维素含量负相关达到显著水平(p<0.05),与35DPA还原性糖含量正相关达到极显著水平(p<0.01),与15DPA纤维素含量负相关达到极显著水平(p<0.01)。结论:棕色棉纤维的各发育阶段的生化物质含量与白色棉表现明显的差异,但是二者在动态变化规律上表现一致;棕色棉色素合成与棉纤维发育过程中生化物质组成关系密切。     

SCFP,a novel fiber-specific promoter in cotton

To investigate the expression pattern of GhSCFP which was isolated from cotton fiber cDNA library, a 1006 bp upstream fragment of the gene was cloned by chromosome walking and fused to GUSand GFP respectively. Histochemical GUS and GFP fluorescence analysis revealed that the expression of the report genes driven by the promoter sequence was detectable only in outer layer cells during the seed development in the transgentic tobaccos. In transgenic cotton, strong GUS activity was observed in spherical protrusions on 0 dpa （days post anthesis） ovule surface, and in the 2-36 dpa fiber cells, while no GUS signals were detected in the root, leaves, stem, corolla, anther and stigma. Our data demonstrated that GhSCFP upstream sequence is a cotton fiber-specific promoter and this promoter will be useful in the molecular research on fiber cell development and in cotton fiber improvements by genetic modification.     

Suitable internal control genes for qRT-PCR normalization in cotton fiber development and somatic embryogenesis

The mechanisms of cotton fiber development and somatic embryogenesis have been explored sys-tematically with microarray and suppression subtractive hybridization. Real-time RT-PCR provides the simultaneous measurement of gene expression in many different samples,with which the data from microarray or others can be confirmed in detail. To achieve accurate and reliable gene expression re-sults,normalization of real-time PCR data against one or several internal control genes is required,which should not fluctuate in different tissues during various stages of development. We assessed the gene expression of 7 frequently used housekeeping genes,including 18S rRNA,Histone3,UBQ7,Actin,Cyclophilin,Gbpolyubiquitin-1 and Gbpolyubiquitin-2,in a diverse set of 21 cotton samples. For fiber developmental series the expression of all housekeeping genes had the same down tendency after 17 DPA. But the expression of the AGP gene(arabinogalactan protein) that has high expression level at the later fiber development stage was up-regulated from 15 to 27 DPA. So the relative absolute quanti-fication should be an efficient and convenient method for the fiber developmental series. The expres-sion of nonfiber tissues series varied not so much against the fiber developmental series. And three best control genes Histone3,UBQ7 and Gbpolyubiquitin-1 have to be used in a combinated way to get better normalization.     

棉花中异性纤维的多光谱检测

为有效检测棉花中与棉纤维形态、色泽极其相似的异性纤维杂质,提出一种利用多波段光谱信息融合成像检测的新方法。针对6种肉眼极难识别的异性纤维:无色塑料、黄麻、编织袋、白头发丝、白羊毛、猪鬃在7个离散波段光谱中的反射特性,获得不同波段光谱图像中异性纤维与棉纤维的图像特征差别,由此确定检测各异性纤维的最佳光谱波段。采用基于区域信息相关度权值小波分析算法将多个波段的图像进行融合,得到具有完整异性纤维特征信息的单幅杂质图像。实验结果表明,在多波段光谱融合图像中,异性纤维灰度、形态特征明显,可有效识别棉花中异性纤维杂质。     

木棉纤维的微细结构研究——胞壁层次结构与原纤尺度

通过对木棉纤维横截面超薄切片的透射电镜观察，获得了木棉纤维的胞壁层次结构、原纤尺度及排列。木棉纤维胞壁具有清晰可见的多层状结构，基本上可以分为外表皮层S、胞壁1层W1、胞壁2层W2、胞壁3层W3和内皮层IS共5个基本的层次，各层具有不同的厚度和原纤堆砌密度与排列方向。可见木棉纤维横截面最小结构单元宽度为3．2～5．0nm，与棉纤维基原纤尺寸相当。实验证明，碱液对木棉纤维的可及性存在显著的个体差异；对于同一纤维胞壁各层的溶胀也存在差异。其中W2是最易被溶胀的；内皮层的原纤容易被分离出来。而未经溶胀处理的木棉纤维电镜照片反差弱，层次结构不够细致。     

Observation of fiber ultrastructure of Ligon lintless mutant in upland cotton during fiber elongation

Lintless mutant is a super-short fiber mutanl in upland cotton only 4-8 mm in fiber length and also named Ligon cotton controlled by one dominant gene Li1. Fiber ultrastructure of the mutant (Li1) and its its wild (li1) in siti and in vitro was observed under an electron microscope to understand its cytological characteristics during the fiber cell elongation. The resulls showed that the mutant fiber in situ had thinner cytoplasm, more small vacuoles, less mitochondria, Golgi apparatus and endoplasmic reticula, and there were more starch granules which were free or packed in the amyloplast beside the cell wall than thai of wild type. It was indicated that scarcity of functional organelles and disability of transformation from starch to sugar might be associated with the tact that the mutant fiber cell was aborted too early to elongate into normal length. Mutant ovule in some media containing GA3 could produce a kind of huge callus that grew faster than normal ovules. The callus was covered with many white, loose, and semitransparent fiber-like cells that apt lo get off from ovule. These fiber-like cells were multicellular fibers generated by cell division and had black dots just like pigment glands in the stem and leaf of cotton. There were lots of micro-tubes beside cytoplasm membrane uf the multiecllular fiber, which were thought to be primary preparation for second wall deposition of multicellular fiber. It was indicated that GA3 might induce the expression of gene(s) that kept inactive in the field condition and then stimulate the original fiber cell in vitro to undergo divisionagain.     

Cloning and characterization of two putative seven-transmembrane receptor genes from cotton

Using rapid amplification of cDNA ends （RACE）-PCR, two full-length cDNAs encoding putative seven-transmembrane receptors （designated Gh7TMpR1 and Gh7TMpR2） were cloned from cotton plants. Southern blot and an ApaL1 restriction site polymorphism analyses revealed that Gh7TMpR1 was derived from the ancestral A diploid genome, while Gh7TMpR2 was from the D subgenome. Northern blot hybridization indicated that both Gh7TMpR1 and Gh7TMpR2 were expressed preferentially in the elongation phase of fiber development. Majority of the Gh7TMpR1 proteins were located within the membrane structure and displayed a punctuate pattern of distribution. Overexpression of Gh7TMpR1 in fission yeast disrupted the polar growth and caused the formation of rounded cells. These results suggest that GhT7MpR1 may play a critical role in cotton fiber development, perhaps as a signaling receptor that is involved in controlling fiber elongation.