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。     

陆地棉‘徐州142’纤维不同发育时期蛋白质组比较分析

该研究以陆地棉‘徐州142’为材料,于开花后5、10、15、20、25、30d分别取样(棉铃)代表6个不同时期的棉纤维,利用双向电泳技术比较了棉纤维发育不同时期蛋白质组的变化,以明确不同发育阶段差异表达蛋白质及其功能与纤维发育之间的关系,为棉花产量和品质的改良提供理论依据。结果显示:(1)随着棉花的发育,棉纤维中的总蛋白含量逐渐降低,在开花后5d(5DPA)时棉花纤维中总蛋白的含量最高,达到11.7%,同时2-DE图谱中发现了15个明显的差异蛋白点;(2)运用MALDI-TOF-MS对差异蛋白质点鉴定结果发现,有5个明显的差异蛋白与棉花纤维发育相关,分别是GhSAM、GhPGK、GhCSD、GhFB和GhMDH蛋白;(3)开花后不同时期棉纤维蛋白质组的2-DE图谱表明,GhSAM、GhPGK1和GhMDH 3个蛋白在整个发育期都表达,但GhCSD从15DPA开始表达,GhFB从20DPA开始表达,而且GhSAM在15DPA时蛋白表达量最高,GhPGK1在10DPA时表达量最高,GhMDH、GhCSD和GhFB都是在30DPA时表达量最高。研究表明,棉纤维发育不同时期存在着差异蛋白,且在纤维发育不同时期蛋白的表达量也存在差异,同时这些差异蛋白参与能量代谢、碳代谢、细胞周期调控和发育等途径。     

过表达或沉默棉花GhPCBER基因株系的获得及其茎叶木质素和木脂素含量研究

编码苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)的基因PCBER属于PIP亚家族,是苯丙烷代谢途径中参与木脂素合成的关键基因。该研究构建了棉花GhPCBER基因的植物过表达载体并转化拟南芥,同时构建了VIGS(virus induced gene silencing,病毒诱导的基因沉默)载体转化棉花,采用实时荧光定量PCR技术对GhPCBER基因在不同组织中的表达进行分析;对野生型和转基因植株茎叶组织中的木质素和木脂素含量进行测定分析。结果表明:(1)成功构建了GhPCBER植物过表达载体pGWB17-GhPCBRE以及基因沉默重组载体pTRV2-GhPCBER;经遗传转化获得6株转棉花GhPCBER基因抗性拟南芥植株,同时获得15株GhPCBER基因沉默棉花植株(5株为一组)。(2)PCR检测表明,6株转基因拟南芥均为过表达株系,其中株系1、2、3相对表达量更高,且在茎、叶组织中的表达量分别较野生型提高了7～14倍和6～16倍,表明GhPCBER基因成功在拟南芥中过表达;GhPCBER基因沉默棉花植株的茎、叶组织中的表达量分别比野生型棉株约下降12%和26%,表明烟草脆裂病毒(TRV)体系(pTRV2-GhPCBER)成功抑制了GhPCBER基因的表达。(3)转GhPCBER基因拟南芥茎、叶中木质素和木脂素含量较野生型均显著降低;GhPCBER基因沉默棉花植株茎、叶中木质素和木脂素含量较野生型均极显著降低;组织化学染色观察发现GhPCBER基因沉默棉花植株茎秆颜色明显比野生型染色浅,也证明沉默基因棉花植株茎秆中的木质素含量减少。(4)苯丙烷代谢通路中8个相关基因的实时荧光定量PCR分析发现,过表达或抑制GhPCBRE基因均会导致苯丙烷代谢途径发生重新定向。     

棉花LIM结构域基因(GhLIM1)的克隆和表达分析

LIM结构域蛋白是一个重要的发育调控因子,参与基因转录,细胞骨架建成和信号传导等许多发育调控过程,胞质骨架是形成和稳定细胞形态以及传递物质,能量和信息的重要成分。为研究棉花纤维细胞发育过程中胞质骨架的形成和作用机理,通过棉花纤维EST序列整合,从陆地棉徐州142胚珠（含纤维）中扩增并克隆出棉花LIM结构域基因的编码区段。该棉花LIM结构域基因（GhL1M1)长848bp,包含一个570bp的开放阅读框,推导的氨基酸序列（189个氨基酸）与拟南芥,烟草和向日葵的LIM结构域蛋白有极高的同源性,而且两个LIM结构域完整,RT-PCR和Northerm杂交分析表明,该基因（GhL1M1）在陆地棉的根,茎尖,上胚轴,叶片,花蕾,花药,胚珠和不同发育时期的陆地棉纤维（4DPA、12DPA、18DPA）以及海岛棉纤维（18DPA）和中棉纤维（12DPA）中均有表达,但GhL1M1基因在茎尖,纤维和有纤维的胚珠中表达量更高,因此GhL1M1基因应与棉花纤维发育有密切关系。     

甘菊3个ClSCL6基因的克隆及表达分析

GRAS家族HAM亚家族基因是维持植物茎端分生组织(shoot apical meristem,SAM)未分化状态的重要因子,并影响着植物的成花转化进程。该研究基于转录组数据中甘菊(Chrysanthemum lavandulifolium)HAM亚家族基因同源序列设计引物,利用RT PCR技术从甘菊中克隆得到3个HAM类基因。序列分析结果表明,所克隆的3个基因开放阅读框长度分别为1 845、1 479和1 881 bp,分别编码614、492和626个氨基酸。Blastp分析显示,3个基因的编码蛋白均含有典型HAM亚家族蛋白特征,并与菊科植物黄花蒿(Artemisia annua)SCL6蛋白具有较高的一致性,分别达到了94.39%、91.90%和94.27%。进一步分析表明,3个基因的编码蛋白与所有拟南芥GRAS家族中的SCL6蛋白进化关系最近,故将其分别命名为ClSCL6a、ClSCL6b和ClSCL6c。荧光定量分析显示,3个ClSCL6基因均在甘菊茎中表达量最高,而在根和花中表达量普遍较低。在不同发育时期的花器官中,3个ClSCL6基因均有表达,其中ClSCL6a在管状花花粉散开前达到表达高峰,ClSCL6b和ClSCL6c则在小花蕾时期表达量最高,在其他时期表达水平差异不大。该研究结果为进一步研究ClSCL6在菊花成花转化过程中的功能奠定了基础。     

黄秋葵八氢番茄红素脱氢酶基因的克隆与分析

该研究根据黄秋葵(Hibiscus esculentus L.)转录组测序获得的八氢番茄红素脱氢酶基因(HePDS)序列(GenBank登录号为MG372370)设计引物,克隆验证得到1条HePDS基因全长为2 020 bp cDNA,开放阅读框(ORF)包含1 686个碱基;预测其编码561个氨基酸,理论分子量为62.62 kD,等电点为8.155;编码的蛋白与海岛棉(Gossypium barbadense)、雷蒙德氏棉(Gossypium raimondii)、陆地棉(Gossypium hirsutum)同源蛋白的相似性均在93%以上,均含有1个保守的二核苷酸结合域和1个类胡萝卜素结合域,显示其高度的保守性。荧光定量PCR 分析表明,HePDS基因在黄秋葵根、茎、叶、花和果荚中均有表达;叶发育过程以嫩叶中表达最高,果实发育中以花后2 d表达量最高。类胡萝卜素含量随着叶、果实发育逐渐升高,成熟叶的含量最高,果实以花后4 d含量最高,且HePDS基因的表达与类胡萝卜素含量存在密切的相关性。该研究结果为进一步探讨HePDS基因的功能和调控机制,以及采用VIGS和CRISPR/Cas9技术开展黄秋葵基因功能的反向遗传学研究奠定了基础。     

海岛棉GAUT基因家族的鉴定及其在棉纤维发育中的表达分析

本研究鉴定了海岛棉全基因组中的GAUT基因家族,对其进行了生物信息学及纤维发育过程中的表达分析。结果表明,海岛棉GAUT家族包含37个成员,分为3个亚组,分布在海岛棉18条染色体上。GAUT氨基酸序列保守基序有4个,保守性较强,所有GAUT蛋白均定位在高尔基体膜。根据GAUTs在海岛棉纤维发育不同时期的表达变化,将其分为起始期高表达、纤维伸长期高表达、次生壁增厚期高表达、全时期低表达等4类模式。其中,GAUT05、GAUT06、GAUT07、GAUT23、GAUT24、GAUT26等在纤维发育某个时期具有优势表达的基因可能在纤维发育过程中发挥着重要作用。上述结果为研究海岛棉GAUT基因家族在棉纤维发育中的功能提供了参考依据。     

棉花MADS框蛋白基因(GhMADS1)的克隆

作为转录因子,MADS框蛋白基因在植物花器官发育中有着重要的功能。为研究棉花花器官发育的分子机理,以棉花花器官突变体CHV1(cotton homeotic variant)和徐州142正常植株为材料,利用棉花EST数据库资料,通过EST序列整合,从陆地棉徐州142花蕾中克隆出一个MADS框蛋白的编码区段,GenBank登录号为AF538965。该片段(GhMADS1)长713bp,包含一个711bp的开放阅读框,推导的氨基酸序列(236个氨基酸)与葡萄、烟草、矮牵牛、拟南芥和金鱼草等的AGL2组MADS框蛋白有很高的序列相似性。系统进化分析同样将GhMADS1基因归人AGt2组MADS框蛋白。RT-PCR分析显示,该基因在陆地棉的花瓣、雄蕊、胚珠和纤维中表达,特别是在花瓣中表达量最高,而在根、茎、叶等营养器官和棉花同源异型突变体CHV1(所有花器官均变为苞叶状叶性器官)的变异花蕾中不表达。这些结果说明GhMADS1基因可能在棉花花器官发育中有着重要的功能。     

草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box 基因家族编码高度保守的转录因子, 参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆(Eustoma grandiflorum)花器官发育的分子调控机制, 根据MADS-box基因保守序列设计简并引物, 用3'-RACE方法从 草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明, 这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性, 分别属DEF/GLO、AG-like和SEP-l ike亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示, 这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域, 每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示: EgDEF1 在萼片、花瓣、雄蕊及胚珠中高丰度表达, 在心皮中微量表达; 而EgGLO1在花瓣和雄蕊中高丰度表达, 在萼片中微量表达; 在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达, 但表达的丰度存在差异, 在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

利用mRNA荧光差异显示技术规模化筛选棉纤维特异表达基因

以陆地棉（Gossypium hirsutum L.）品种TM-1开花后9d、21d、27d三个不同发育时期的棉花纤维为材料,利用mRNA荧光差异显示 (FDD) 技术,筛选到109个差异显示的cDNA片段。在此基础上,结合两轮反Northern杂交筛选和Northern杂交分析,获得了多个仅在棉花纤维细胞中特异表达或在纤维中优先表达基因的cDNA片段,序列测定和数据库搜索分析表明,这些cDNA片段中的多数还未有报道。本工作为克隆上述基因的全长cDNA,并进一步研究它们在棉纤维发育中的功能奠定了基础。     

Characterization and expression of plasma and tonoplast membrane aquaporins in elongating cotton fibers

Cotton fiber (Gossypium hirsutum L. and G. barbadense L.) is a good model for studies of plant cell elongation and cell wall biogenesis. Aquaporins are ancient membrane channel proteins that facilitate the permeation of water across biological membranes. We studied GhPIP1-2, encoding plasma membrane intrinsic protein, and GhgammaTIP1, encoding tonoplast intrinsic protein, during cotton fiber development. The full-length cDNAs of GhPIP1-2 and GhgammaTIP1 were obtained through 5' RACE. The deduced amino acid sequences of GhPIP1-2 and GhgammaTIP1 share high sequence identity with aquaporins from diverse plant species. Phylogenetic analysis of GhPIP1-2 and GhgammaTIP1 with other plant aquaporins showed that GhPIP1-2 belongs to the PIP1 group of the PIP subfamily and GhgammaTIP1 belongs to the gammaTIP group of the TIP subfamily. GhPIP1-2 and GhgammaTIP1 contain three and two introns, respectively. Genomic Southern blot analysis indicated that GhPIP1-2 and GhgammaTIP1 have several copies and multiple homologous genes in allotetraploid cotton. Northern blot analysis with gene-specific probes and real-time PCR demonstrated that GhPIP1-2 and GhgammaTIP1 are predominantly expressed during cotton fiber elongation, with the highest expression levels at 5 days post-anthesis. Moreover, expression patterns of the two genes in G. hirsutum and G. barbadense are similar, whereas the expression levels in G. barbadense are much lower than that in G. hirsutum. The high and preferential expression of GhPIP1-2 and GhgammaTIP1 during fiber cell elongation suggests that they may play important roles in supporting the rapid influx of water into vacuoles during cotton fiber cell expansion.     

Molecular cloning and characterization of five genes encoding pentatricopeptide repeat proteins from Upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

The pentatricopeptide repeat (PPR) protein family is one of the largest and most complex families in plants. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding RNA. PPR proteins have been implicated in many crucial functions broadly involving organelle biogenesis and plant development. In this study, we identified many genes encoding PPR protein in Upland cotton through an extensive survey of the database of Gossypium hirsutum. Furthermore, we isolated five full-length cDNA of PPR genes from G. hirsutum 0-613-2R which were named GhPPR1–GhPPR5. Domain analysis revealed that the deduced amino acid sequences of GhPPR1–5 contained from 5 to 10 PPR motifs and those PPR proteins were divided into two different PPR subfamilies. GhPPR1–2 belonged to the PLS subfamily and GhPPR3–5 belonged to the P subfamily. Phylogenetic analysis of the five GhPPR proteins and 18 other plant PPR proteins also revealed that the same subfamily clustered together. All five GhPPR genes were differentially but constitutively expressed in roots, stems, leaves, pollens, and fibers based on the gene expression analysis by real-time quantitative RT-PCR. This study is the first report and analysis of genes encoding PPR proteins in cotton.     

水孔蛋白在细胞延长、盐胁迫和光合作用中的作用

水孔蛋白属于一个高度保守的、能够进行跨生物膜水分运输的通道蛋白MIP家族。水孔蛋白作为膜水通道,在控制细胞和组织的水含量中扮演重要角色。本研究的重点是属于PIP亚家族的GhPIP1;2和属于TIP亚家族的γTIP1在植物细胞延长中的作用。使用特异基因探针的Northern杂交和实时荧光PCR技术证明GhPIP1;2和GhγTIP1主要在棉花纤维延长过程中显著表达,且最高表达量在开花后5d。在细胞延长过程中,GhPIP1;2和GhγTIP1表达显著,表明它们在促使水流迅速进入液泡这一过程中扮演重要角色。而且也研究了盐胁迫植物中钙离子对水孔蛋白的影响。分别或一起用NaCl或CaCl2处理原生质体或细胞质膜。结果发现在盐胁迫条件下,水渗透率值在原生质体和质膜颗粒中都下降了,同时PIP1水孔蛋白的含量也下降了,表明NaCl对水孔蛋白的功能和含量有抑制作用。同时也观察了Ca2+的两种不同的作用。感知胁迫的胞质中游离钙离子浓度的增加可能导致水孔蛋白的关闭。而过剩的钙离子将导致水孔蛋白的上游调控。同时实验已经证明大麦的一类水孔蛋白-HvPIP2;1有更高的水和CO2转移率。本研究的目标是确定负责转运水和CO2的关键水孔蛋白...     

Genetic mapping of EST-derived microsatellites from the diploid<Emphasis Type="Italic"> Gossypium arboreum</Emphasis> in allotetraploid cotton

To increase the numbers of microsatellites available for use in constructing a genetic map, and facilitate the use of functional genomics to elucidate fiber development and breeding in cotton, we sampled microsatellite sequences from expressed sequence tags (ESTs) transcribed during fiber elongation in the A-genome species Gossypium arboreum to evaluate their frequency of occurrence, level of polymorphism and distribution in the At and Dt subgenomes of tetraploid cotton. From among ESTs derived from G. arboreum fibers at 7–10 days post anthesis (dpa), 931 ESTs were found to contain simple sequence repeats (SSRs); 544 (58.4%) EST-SSR primer pairs were developed, and 468 (86%) amplified PCR products from allotetraploid cotton ( G. hirsutum cv. TM-1 and G. barbadense cv. Hai7124). However, only 99 (18.2%) of these were found to be polymorphic and segregating in our interspecific BC₁ mapping population [(TM-1×Hai7124)×TM-1]. In these amplified and informative EST-SSRs, hexa- and tri-nucleotide repeat motifs were the most frequent, representing 40.1 and 30%, respectively, of the total. A total of 111 loci detected with these 99 EST-SSRs were integrated into our backbone map including 511 SSR loci. The distribution of the EST-SSRs appeared to be non-random, since 72 loci were anchored to the At and 37 to the Dt subgenome of allotetraploid cotton based on linkage tests. Interestingly, out of the 10 pairs of duplicate loci amplified, seven were mapped to the corresponding homeologous linkage groups and/or chromosomes. BLASTX analysis revealed that 69 of the 99 ESTs showed significant similarities to known genes. Some genes important for fiber development, such as sucrose synthase, were mapped to corresponding chromosomes. These EST-SSRs provide structural and functional genomic information that will be useful for understanding cotton fiber development.Communicated by R. Hagemann     

Expression of a phenylcoumaran benzylic ether reductase-like protein in the ovules of <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to identify differentially expressed proteins in wild-type (DP 5690) and fiberless (SL 1-7-1) cotton ovules. One protein, designated V2 was unique to ovules of the fiber producing DP 5690 line. The protein was purified from 2D-PAGE of 4 d post anthesis DP 5690 ovules and partially sequenced. The short amino acid sequence was nearly identical to the deduced amino acid sequence for cotton phenylcoumaran benzylic ether reductase (PCBER) protein. A consensus sequence was assembled from ESTs encoding cotton PCBER genes, primers were designed, and a full length gene was amplified from plasmid DNA from a 72 h etiolated cotton cotyledon library. The polymerase chain reaction generated a 950 bp product with unique EcoRI (5′) and (3′) KpnI restriction sites for directional insertion into the expression vector pPICZA. Nucleotide sequencing was performed, and the full length coding region was 924 bp encoding a protein of 308 amino acids. The molecular mass and pI measured (2D PAGE) were similar to the theoretical protein.