雷蒙德氏棉HSP70基因家族的进化分析及其同源基因在陆地棉中的表达分析

热激蛋白70家族(HSP70)是一类在植物中高度保守的分子伴侣蛋白,在细胞中协助蛋白质正确折叠。文章利用隐马可链夫模型(HMM)在雷蒙德氏棉(Gossypium raimondii L.)全基因组范围内进行HSP70基因家族成员进化分析,共得到30个HSP70家族成员。利用生物信息学对雷蒙德氏棉HSP70基因的结构、染色体分布、基因倍增模式以及系统进化进行分析,结果表明,HSP70基因家族根据亚细胞定位结果可分为不同的基因亚家族,各亚家族中HSP70基因具有相对保守的基因结构;染色体片段重复和串联重复是雷蒙德氏棉HSP70基因家族扩增的主要方式。通过对不同物种的HSP70基因家族进行系统进化分析可知,HSP70亚组的分化发生在单细胞植物形成前,且细胞质型HSP70成员大量扩增。比较陆地棉棉纤维发育不同时期的深度测序表达谱,发现HSP70基因可能参与棉纤维的生长发育。本研究结果有助于了解棉属植物HSP70基因家族的功能,以期为深入研究棉纤维发育过程中的分子调控机理提供基础。     

Potato Expressed Sequence Tag Generation and Analysis using Standard and Unique cDNA Libraries

To help develop an understanding of the genes that govern the developmental characteristics of the potato (Solanum tuberosum), as well as the genes associated with responses to specified pathogens and storage conditions, The Canadian Potato Genome Project (CPGP) carried out 5′ end sequencing of regular, normalized and full-length cDNA libraries of the Shepody potato cultivar, generating over 66,600 expressed sequence tags (ESTs). Libraries sequenced represented tuber developmental stages, pathogen-challenged tubers, as well as leaf, floral developmental stages, suspension cultured cells and roots. All libraries analysed to date have contributed unique sequences, with the normalized libraries high on the list. In addition, a low molecular weight library has enhanced the 3′ ends of our sequence assemblies. Using the combined assembly dataset, unique tuber developmental, cold storage and pathogen-challenged sequences have been identified. A comparison of the ESTs specific to the pathogen-challenged tuber and foliar libraries revealed minimal overlap between these libraries. Mixed assemblies using over 189,000 potato EST sequences from CPGP and The Institute for Genomics Research (TIGR) has revealed common sequences, as well as CPGP- and TIGR-unique sequences. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

陆地棉纤维优势表达基因GhRACK1的克隆与序列分析

纤维品质改良是我国棉花育种的主要目标之一,纤维特异或优势表达基因的挖掘是利用基因工程手段改良纤维品质的关键。根据苏棉12纤维中优势表达的GhRACK1 EST序列设计引物,通过RACE技术克隆了GhRACK1基因的全长cDNA。推导的氨基酸序列含有4个串联的WD基序,属于WD40重复家族,与已知的RACK1蛋白同源性达70％以上,PDB模拟的蛋白三维结构也与已知的RACK1蛋白结构相似。荧光定量PCR分析表明GhRACK1在纤维中的表达量比叶片中高20倍以上。研究结果为棉花纤维品质改良基因工程提供了新的基因资源。     

陆地棉脂肪酶基因克隆及氨基酸序列分析

根据陆地棉(Gossypium hirsutum L.)EST序列设计一对引物,采用RT-PCR方法从萌发的棉籽中获得了脂肪酶(triacylglycerol acylhydrolases,EC 3.1.1.3)基因,该基因编码483个氨基酸;比对结果显示,棉籽脂肪酶与拟南芥、水稻、蓖麻等脂肪酶相似性较低,具有由Ser-Asp-His组成的三联体催化活性中心,在亲核Ser残基周围有GXSXG保守序列.软件预测显示该脂肪酶为可溶性蛋白,分子量约为55.4 kD,等电点为9.07,不含N端信号肽,亚细胞定位可能是过氧化物酶体或胞质溶胶.     

Response of the enzymes to nitrogen applications in cotton fiber (Gossypium hirsutum L.) and their relationships with fiber strength

To investigate the response of key enzymes to nitrogen (N) rates in cotton fiber and its relationship with fiber strength, experiments were conducted in 2005 and 2006 with cotton cultivars in Nanjing. Three N rates 0, 240 and 480 kgN/hm2, signifying optimum and excessive nitrogen application levels were applied.The activities and the gene expressions of the key enzymes were affected by N, and the characteristics of cellulose accumulation and fiber strength changed as the N rate varied. Beta-1,3-glucanase activity in cotton fiber declined from 9 DPA till boll opening, and the beta-1, 3-glucanase coding gene expression also followed a unimodal curve in 12—24 DPA. In 240 kgN/hm² condition, the characteristics of enzyme activity and gene expression manner for sucrose synthase and beta-1,3-glucanase in developing cotton fiber were more favorable for forming a longer and more steady cellulose accumulation process, and for high strength fiber development.     

棉花叶绿体基因RNA编辑位点的测定及分析

陆生植物叶绿体RNA编辑是转录后基因表达调控的一种重要方式。该文在预测棉花（Gossypium hirsutum）叶绿体基因RNA编辑位点的基础上,选取中棉10（CRRI 10）为实验材料,采用PCR、RT-PCR及测序等方法,确定CRRI 10的27个叶绿体蛋白编码基因共有55个编辑位点,均是C→U的转换。与棉种柯字310（C310）的编辑位点比对后发现,CRRI 10多出accD-468和rpoC1-163两个编辑位点,同时缺失psbN-10。利用生物信息学分析这3个位点,rpoC1-163和psbN-10的编辑可能会改变各自蛋白的二级结构。对CRRI 10中55个编辑位点上游的顺式作用元件（？30–？1）分析显示,共有8组顺式作用元件的相似性达到60%或以上,推测各组中的编辑位点可能由相同的反式作用因子来识别。     

Comparative Development of Lint and Fuzz Using Different Cotton Fiber-specific Developmental Mutants in Gossypium hirsutum

A series of fiber-specific mutants, or germplasms, have been recently used in the study of fiber development. In the current study, scanning electron microscopy （SEM） was used to investigate developmental differences in lint and fuzz initiation in different genotypes （Gossypium hirsutum） of upland cotton. These fiber mutants included dominant naked seed N1, recessive naked seed n2, Xuzhou-142 lintless-fuzzless （XZ142WX）, Xinxiangxiaojilintless-fuzzless （XinWX）, Xinxiangxiaojilinted-fuzzless （XinFLM）, with TM-1, the cytogenetic and genetic experimental standard stock, as the control. Characteristics of fiber initiation were analyzed from -1 to ＋1 days post anthesis （dpa） and at 4 and 5 dpa for fuzz initiation. Our data suggested that lint initiation centered on day of anthesis （0dpa）, and elongated significantly at 1dpa, while fuzz initiation began at 4dpa, although the shape of fuzz protrusions differed from that of lint fibers. Fiber initiation occurred first on the ovule funicular crest. Compared to TM-1, there was a noted retardation in development and fiber protrusion in N1 and XinFLM. Microscopy data also demonstrated that lintless-fuzzless mutants （XZ142WX and XinWX） developed irregular protrusions during early developmental stages, which were unable to grow into fiber.     

Comparative Development of Lint and Fuzz Using Different Cotton Fiber-specific Developmental Mutants in Gossypium hirsutum

A series of fiber-specific mutants, or germplasms, have been recently used in the study of fiber development. In thecurrent study, scanning electron microscopy (SEM) was used to investigate developmental differences in lint and fuzzinitiation in different genotypes (Gossypium hirsutum) of upland cotton.These fiber mutants included dominant nakedseed N1, recessive naked seed n2, Xuzhou-142 lintless-fuzzless (XZ142WX), Xinxiangxiaojilintless-fuzzless (XinWX),Xinxiangxiaojilinted-fuzzless (XinFLM), with TM-1, the cytogenetic and genetic experimental standard stock, as the control.Characteristics of fiber initiation were analyzed from -1 to 1 days post anthesis (dpa) and at 4 and 5 dpa for fuzz initiation.Our data suggested that lint initiation centered on day of anthesis (Odpa), and elongated significantly at 1dpa, while fuzzinitiation began at 4dpa, although the shape of fuzz protrusions differed from that of lint fibers. Fiber initiation occurred firston the ovule funicular crest. Compared to TM-1, there was a noted retardation in development and fiber protrusion in N1and XinFLM. Microscopy data also demonstrated that lintless-fuzzless mutants (XZ142WX and XinWX) developed irregularprotrusions during early developmental stages, which were unable to grow into fiber.     

两个棉花Rac蛋白基因的克隆与表达分析

为研究棉花纤维起始和伸长的分子机理,在棉花纤维EST序列分析的基础上,从棉花纤维中扩增并克隆了2个棉花Rac蛋白的cDNA基因,分别命名为GhRacA和GhRacB。GhRacA cDNA长959bp,推测的编码蛋白包含211个氨基酸。GhRacB cDNA长920bp,编码195个氨基酸的蛋白。GhRacA和GhRacB蛋白均含有GTP／GDP结合和激活区域、Effector区和碱性氨基酸区。GhRacB的C末端有保守的异戊烯基化位点CSIL,而GhRacA没有明显的异戊烯基化位点。序列比较分析表明,GhRacA和GhRacB是2个新的棉花Rac蛋白。RT-PCR分析表明,GhRacA和GhRacB在根、下胚轴、茎、叶和纤维中都有表达,但均在棉花纤维起始和伸长时期有优势表达,推测2个基因在棉花纤维的早期发育中可能有重要的功能。     

Changes in the level of tubulin subunits during development of cotton (Gossypium hirsutum) fiber

The levels of tubulin protein in developing cotton ( Gossypium hirsutum L. cv. Stoneville 825) fibers were measured from 8 to 28 days post-anthesis using commercially available monoclonal antibodies against alpha- and beta-tubulin. As the monoclonal antibodies against alpha- and beta-tubulin were prepared from yeast tubulin and chick brain tubulin, respectively, indirect immunofluorescence microscopy was used to establish that the two monoclonal antibodies recognized microtubule structures in cotton fibers. Western blots of electrophoretically separated proteins in crude extracts of cotton roots and fibers showed that single polypeptides with the expected apparent molecular weight for tubulin subunits were recognized by the antisera. An enzyme-linked immunosorbent assay was used to quantify tubulin levels. From 10 to 20 days post-anthesis the level of tubulin protein increases approximately three-fold. After 20 days post-anthesis, the amount of tubulin relative to total fiber protein reaches a plateau or decreases slightly. The rapid rise in tubulin is correlated with the elongation of the fiber and an increase in cellulose synthesis.     

棉花GhCCR1基因结构及表达分析

根据棉花GhCCR1基因的cDNA序列设计引物,采用PCR技术从棉花中克隆了GhCCR1基因的DNA序列,并采用半定量RT-PCR方法分析了GhCCR1基因在不同发育阶段棉纤维中的表达情况.结果表明:GhCCR1编码区DNA序列长度为1 161 bp,包含4个外显子和3个内含子,内含子富含AT,所有外显子/内含子交接点都遵从gt/ag剪接规则.半定量RT-PCR检测表明,GhCCR1基因在不同发育阶段的棉纤维中均有表达,在开花后20 d的棉纤维中表达量最高,说明该基因可能参与调控棉纤维细胞的伸长和次生壁的增厚过程.     

陆地棉XTH基因家族全基因组鉴定及在纤维发育过程的表达分析

本研究从陆地棉TM-1基因组中鉴定出72个XTH家族基因,编码木葡聚糖内转糖苷酶/水解酶(XTH,xyloglucan endotransglycosylase/hydrolase),分别命名为Gh XTH01~Gh XTH72,分析了其基因结构、保守基序、系统进化、理化性质、亚细胞定位,并探究其在棉纤维发育不同时期的表达规律。结果表明,XTH家族基因分布在除At 07、Dt 07以外的24条棉花染色体上,根据系统发育树,将XTH家族基因分为3个亚组;XTH氨基酸序列有3个保守基序,保守性较强;多数XTH蛋白定位在细胞外。根据XTHs在纤维发育不同时期的表达量变化,将其分为4类。通过构建陆地棉与拟南芥XTH氨基酸序列进化树,推测Gh XTH15、Gh XTH28、Gh XTH36、Gh XTH49、Gh XTH59、Gh XTH62、Gh XTH63等基因在棉纤维发育过程中发挥重要作用。通过比较XTH家族基因在不同纤维品质陆地棉品种中的表达差异,推测在优质棉花品种中优势表达基因Gh XTH03、Gh XTH12、Gh XTH17、Gh XTH22、Gh XTH23、Gh XTH28、Gh XTH33、Gh XTH44、Gh XTH46、Gh XTH59等在纤维发育伸长过程中可能发挥着重要作用。上述结果为研究陆地棉XTH基因家族在棉纤维发育中的功能提供了参考依据。     

棉花细胞核雄性不育两用系差异表达基因分析

应用cDNA-AFLP对棉花ms5ms6双隐性核雄性不育两用系的不育株和可育株花粉发育的3个时期—造孢细胞时期、花粉母细胞时期和花粉粒时期进行对比分析,共得到17个差异表达片段,它们分别属于11种表达模式,其中14个片段可以在NCBI数据库中找到同源序列,功能分析表明这些片段所编码的基因可能参与了信号转导、转录、能量代谢、细胞壁发育等相关过程。Northern杂交结果证明检测片段的表达模式与cDNA-AFLP结果吻合。同时还在可育花药中发现了与玉米T型细胞质雄性不育恢复因子RF2基因高度同源的育性恢复因子类基因。     

一个陆地棉羧基末端蛋白酶cDNA的克隆及其表达分析

利用PCR筛选方法从陆地棉纤维cDNA文库中分离出1个基因序列,命名为ChCtp。该cDNA全长1917bp,编码1个含473个氨基酸残基的多肽。GhCtp蛋白与拟南芥和水稻中的一类羧基末端蛋白酶具有较高的同源性,在GhCtp的N-末端有1个精氨酸富集区,而C-末端有1个Pfam数据库中编号为DUF239的高度保守区域;该蛋白的N-末端还存在1个在拟南芥和水稻羧基蛋白酶中所缺乏的ATP/GTP结合区A序列。亲水性分析表明,GhCtp为1个可能的跨膜蛋白。从表达特征来看,GhCtp不属于纤维细胞特异表达或优势表达基因,并且它在棉花不同组织中或不同纤维发育时期的表达强度均很低。     

陆地棉GhSDP1及其启动子的克隆与表达分析

三酰基甘油脂肪酶(SDP1)是催化三酰甘油降解的关键酶,在植物油脂代谢调控中起着重要作用。克隆棉花SDP1并研究其在3种胁迫下的表达分析,为解析棉花SDP1的生物学功能提供依据。以陆地棉品种冀丰1271为试材,克隆GhSDP1编码序列和上游启动子序列;利用PlantCARE分析GhSDP1启动子区顺式作用元件;qRT-PCR检测逆境胁迫下GhSDP1的表达谱;通过烟草瞬时表达pGhSDP1启动子+GUS载体检测启动子活性。结果表明,GhSDP1的编码序列为2 541 bp,其在盐、低温和干旱胁迫下呈差异表达模式。pGhSDP1除具有启动子所必需的TATA-box和CAAT-box等基本顺式作用元件外,还含有多个与光响应、激素响应及逆境应答等相关的顺式作用元件。棉花pGhSDP1启动子能驱动GUS蛋白高效表达,具有较强的启动子活性。研究揭示了棉花GhSDP1参与胁迫应答的新功能。