Cloning and Functional Analysis of Low Temperature Response Gene,GhZAT10, in Upland Cotton at Seedling Stage

[Objective] The aim of this study is to identify chilling tolerant genes which can provide a basis for breeding upland cotton cultivars with tolerance to chilling. [Method] The GhZAT10 (Zinc finger of Arabidopsis thaliana 10) gene was cloned, and its expression in roots, stems, and leaves was analyzed with cold treatment by quantitative real-time polymerase chain reaction (qRT-PCR). Then we analyzed the structural characteristics of GhZAT10, protein properties and constructed the phylogenetic tree by bioinformatics methods. Next, we constructed subcellular localization vector 35S::GhZAT10-GFP by Gateway technology. Finally, we studied the effect of GhZAT10 in chilling response by virus induced gene silencing of cotton. [Result] The Open reading frame(ORF) length of GhZAT10 gene is 813 bp (base pair), encoding 270 amino acid residues. The expression of GhZAT10 in roots was higher than that in stems and leaves, and up-regulated after chilling stress in these tissues. GhZAT10 does not possess signal peptides or transmembrane helices; its activity is closely related to the phosphorylation regulation. GhZAT10 has homology with the ZAT10 of Theobroma cacao, Arabidopsis thaliana and Citrus sinensis. GhZAT10 protein is located in the nucleus. The GhZAT10-silenced cotton plants were more sensitive to low temperature than wild type. [Conclusion] GhZAT10 belongs to C₂H₂ zinc finger protein, and plays a positive role to response the chilling stress in upland cotton.     

陆地棉小GTP结合蛋白基因GhRop4的克隆及其表达分析

【目的】对陆地棉小GTP结合蛋白基因(Small GTP-binding protein)GhRop4(AY965614.1)在多种逆境胁迫下的应答表达模式进行研究分析,为棉花抗逆相关基因克隆及棉花抗逆分子机制研究奠定基础。【方法】利用生物信息学方法分析了GhRop4基因的结构特征和进化树关系,并通过荧光定量PCR(Real-time quantitative polymerase chain reaction,qRT-PCR)的方法分析Gh Rop4基因的组织表达特异性和不同逆境诱导条件下的表达模式。【结果】以陆地棉c DNA为模板克隆得到GhRop4基因,其开放阅读框为588 bp,编码包含195个氨基酸残基的Ⅰ型Rop蛋白。氨基酸多重序列比对表明,GhRop4与其它植物Rop蛋白高度同源,符合Rop蛋白结构特点。qRT-PCR分析表明,GhRop4基因在棉花幼苗根、茎、叶、子叶和下胚轴中均有表达,且在子叶和茎中表达水平较高。GhRop4基因对高盐、干旱、低温和棉花黄萎病菌处理都有一定程度的响应。【结论】Gh Rop4基因在陆地棉的逆境胁迫适应过程中可能具有重要的作用。     

陆地棉干旱胁迫基因GhRBCO的克隆与表达分析

Rubisco蛋白(ritmlose-1,5-bisphosphosphate carboxylase/oxygenase,Rubisco)的上调表达是植物应对非生物胁迫的特征之一。本研究在前期蛋白质组学分析的结果上,利用同源克隆获得陆地棉‘KK1543’在干旱胁迫下差异表达的基因GhRBCO。GhRBCO基因编码区长747 bp,编码249个氨基酸;比对氨基酸序列及同源性分析发现,GhRBCO与其它植物中的RBCO蛋白的一致性较高,表明该基因在进化过程中是相当保守的。系统进化树结果显示,GhRBCO与雷蒙德氏棉、亚洲棉和黄花嵩的同源性最高。利用荧光定量PCR技术在15%PEG胁迫下研究该基因表达变化,表达量在棉花的根、茎和叶中都表现为先缓慢升高后降低的变化趋势,在根中表达量在24 h达到第一次最高;在茎中于12 h表达量最低。该基因的表达量在棉花的根、茎、叶中都呈上调表达水平。结果表明,GhRBCO基因可能参与棉花调节非生物胁迫机制。本研究结果为进一步研究GhRBCO基因的功能奠定了一定的基础。     

陆地棉蔗糖磷酸合成酶基因家族的鉴定及表达分析

【目的】蔗糖磷酸合成酶(Sucrose phosphate synthase,SPS)是调控植物蔗糖代谢合成途径的关键酶,在植物光合产物的积累与分配方面发挥着重要作用,然而棉花中SPS基因的系统研究尚很少开展。本研究旨在对陆地棉SPS基因进行全基因组鉴定,并对它们的表达特性进行系统分析。【方法】基于已公布的陆地棉基因组序列,利用生物信息学和荧光定量聚合酶链式反应(Quantitative real-time polymerase chain reaction,qRT-PCR)等方法对陆地棉SPS家族基因的蛋白结构、进化关系、基因结构特征、染色体定位、基因复制和表达特性进行分析。【结果】(1)在陆地棉基因组中,共鉴定到10个Gh SPS基因(Gh SPS1-Gh SPS10);(2)Gh SPS蛋白具有植物SPS家族特有的两个保守的蛋白结构域和3个相对保守的蛋白磷酸化位点;(3)进化分析表明,Gh SPS蛋白可聚为A、B和C共3个亚族,其中A亚族成员最多,包含6个GhSPS蛋白;(4)位于同一亚族的GhSPS基因具有相似的外显子-内含子分布模式,但是外显子/内含子数目在不同亚族间差异很大;(5)GhSPS基因均匀地分布在陆地棉A亚组和D亚组的5条染色体上,片段复制可能导致了GhSPS基因在陆地棉基因组中的扩增;(6)转录组分析表明,不同亚族GhSPS基因具有不同的组织表达模式,A亚族Gh SPS基因在被检测的各个组织均有较高的表达,B亚族GhSPS基因主要在叶片中高表达,C亚族Gh SPS基因主要在纤维、叶片和花瓣中高表达;(7)进一步荧光定量PCR分析表明,GhSPS4在叶片中表达量很高,GhSPS1在叶片和花瓣中表达量较高,Gh SPS7和Gh SPS10在被检测的各个组织均有较高的表达,该结果与转录组分析结果相对一致。【结论】陆地棉SPS基因家族包含10个成员,分布在5条染色体上,可分为3个亚族,不同亚族成员呈现出不同的表达模式,为后续深入解析陆地棉SPS家族基因的功能奠定理论基础。     

通过GbF3’H基因单独沉默及其与GbCHI和GbDFR基因共沉默研究其在海岛棉中抗枯萎病功能

【目的】通过沉默海岛棉GbF3’H基因及共沉默GbF3’H、GbCHI和Gb DFR基因,研究其在海岛棉抗枯萎病中的作用。【方法】以海岛棉抗病材料06-146为研究对象,GhCLA1基因为阳性对照,空载体为阴性对照,构建海岛棉TRV2-Gb F3’H沉默载体,协同课题组前期构建的TRV2-CHI和TRV2-DFR载体,利用病毒诱导的基因沉默技术(Virus-induced gene silencing,VIGS)分别进行Gb F3’H基因单独沉默以及GbF3’H、GbCHI和Gb DFR这3种基因共沉默试验。通过实时荧光定量聚合酶链式反应(Quantitative real time-polymerase chain reaction,q RT-PCR)分析各处理样品中基因沉默情况;设置室内接种枯萎病菌试验测定病情指数,分析各沉默材料对枯萎病的抗性差异。【结果】q RT-PCR结果显示,海岛棉GbF3’H基因沉默后其在海岛棉根、茎和叶中的表达量比空载体对照低,Gb F3’H、GbCHI和GbDFR这3种基因共沉默后其在海岛棉根、茎和叶中的表达量均比空载体对照低。病情指数调查结果显示,野生型<空载体对照相似文献   

陆地棉NAC转录因子基因GhNAC6的克隆、表达和耐盐性分析

【目的】NAC(NAM-ATAF-CUC)家族是植物细胞内特有的1类转录因子家族,参与植物的抗逆过程。为了研究棉花抗逆的分子机制,本研究从陆地棉TM-1中克隆到1个NAC转录因子基因并研究了其功能。【方法】根据其序列同源性和进化分析结果,将该基因命名为GhNAC6,本文对GhNAC6进行了生物信息学和表达分析,并利用病毒诱导基因沉默技术在棉花中沉默GhNAC6的表达。【结果】GhNAC6全长为1629 bp,包含2个内含子,其中编码区为900 bp,编码1个相对分子质量为33.9×10~3、等电点为6.18的蛋白。GhNAC6启动子区段包含多个与诱导表达和组织特异表达相关的顺式作用元件。转录组数据分析结果表明,GhNAC6在棉花发育的不同时期具有时空表达差异性;实时荧光定量核酸扩增检测结果显示GhNAC6还受到植物激素水杨酸、乙烯利、茉莉酸甲酯和逆境胁迫低温、高温、高盐、伤口的诱导。干涉GhNAC6基因发现,降低GhNAC6基因的表达能增强棉花对盐胁迫的耐受性。【结论】研究结果表明GhNAC6可能参与了棉花发育、逆境响应和激素信号传导的过程。     

GhCPS基因沉默对棉花幼苗生长和内源激素含量的影响

利用RT-PCR从棉花中获得493 bp的GhCPS(赤霉素(GA)合成酶基因)片段,经EcoRⅠ/KpnI双酶切后连入pTRV-RNA2质粒,获得了重组载体pTRV-GhCPS;用该载体转化农杆菌后,室内盆栽条件下侵染棉花品种欣抗4的子叶苗。结果表明,病毒诱导沉默GhCPS基因的棉花幼苗中GhCPS的表达量仅为空载体对照的52%。与空载体对照相比,沉默GhCPS的棉花幼苗第1叶和第2叶的内源GA4含量分别降低33%和60%;第2叶ABA含量降低48%,IAA、ZR和iPA含量均无显著变化;株高降低64%。第1叶和第2叶的叶面积分别减小26%和47%,总叶绿素含量分别增加29%和63%,类胡萝卜素含量分别增加27%和46%。第1叶的净光合速率、气孔导度、胞间CO2和蒸腾速率分别增加23%、40%、9%和31%。可见,利用病毒诱导基因沉默技术技术沉默GhCPS后可调控棉花幼苗叶片内源激素平衡,控制棉花幼苗的生长,并提高单位叶面积的光合能力。     

Isolation and Characterization of GhLEA3 Gene from Upland Cotton and Its Expression in Response to Low Temperature Stress

[Objective] Based on the analysis of cotton GhLEA3 gene structure and its expression model in chilling stress, we investigated the resistance function of GhLEA3 in response to cold stress. [Method] We cloned GhLEA3 from upland cotton by homologous sequence cloning way. We analyzed the properties of the GhLEA3, and constructed phylogenetic tree by bioinformatics analysis. We constructed the transient expression vector 35S∷GhLEA3-GFP by In-Fusion connection technology and studied the subcellular localization of GhLEA3. The expression of GhLEA3 gene in leaves of TM-1 at three-leaf stage under low temperature stress treatments (4 ℃, 24 h) was performed. Agrobacterium tumefaciens mediated floral dipping method was used to transform the gene with expression vector 35S∷GhLEA3-GFP into Arabidopsis thaliana wild type. Low temperature germination experiment at 4 ℃ was conducted to verify germination ability of the transgenic Arabidopsis thaliana T₃ seed. The transgenic Arabidopsis seedlings were treated at 4 ℃(low temperature), and all the leaves were taken to measure their electrical conductivity. [Result] The coding sequence of GhLEA3 gene is 1 218 bp, which encodes 405 amino acids. GhLEA3 protein includes 4 functional domains of PF02987. Phylogenetic analysis showed that the similarity of amino acid sequences between upland cotton GhLEA3 and Arabidopsis thaliana AtLEA3 was 52.6%. GhLEA3 might be mainly localized in vacuoles and small vesicles. GhLEA3 was up-regulated in leaves after low temperature treatment. The germination rate of transgenic Arabidopsis thaliana of T₃ generation at low temperature was significantly higher than that of wild type, and its electrical conductivity of leaves after low temperature treatment was significantly lower than that of wild type. [Conclusion] GhLEA3 belonged to the member of LEA3 family. Phylogenetic analysis showed that GhLEA3 had the closest relationship with AtLEA3. GhLEA3 was induced by low temperature stress and may play an important role in improving cold resistance.     

VIGS沉默CaCYC1基因对喜树叶片中喜树碱合成的影响

喜树是一种具有药用价值的木本植物,其主要次级代谢产物喜树碱具有良好的抗癌功效。本研究主要通过病毒诱导的基因沉默(virus induced gene silencing,VIGS)技术对喜树碱生物合成途径中的关键酶基因CaCYC1环烯醚萜合酶(Camptotheca acuminata iridoid synthase,CaCYC1)进行瞬时沉默,进而研究该基因对喜树碱生物合成的影响。以喜树叶片总RNA为模板,通过RT-PCR技术克隆CaCYC1基因片段并构建TRV-VIGS重组质粒,转化农杆菌GV3101后侵染喜树叶片,培养20 d后利用qRT-PCR与HPLC技术分析沉默对CaCYC1基因转录水平及喜树碱合成的影响。结果表明:与对照组相比,实验组中CaCYC1基因的相对表达量显著下降49%,叶片中喜树碱的积累量显著下降35%。上述结果表明,本研究所构建的由TRV介导的VIGS体系可有效沉默内源基因的表达,CaCYC1基因可正向调控喜树叶片中喜树碱的生物合成。     

棉花精氨酸脱羧酶基因GhADC1克隆与表达分析

以抑制性消减杂交(SSH)文库序列为基础,采用生物信息学和RT-PCR方法从陆地棉中克隆出精氨酸脱羧酶基因,命名为GhADC1,GenBank登录号为KC851856.该基因全长2954 bp,其中5’非翻译区477 bp,具有多个参与胁迫响应的顺式作用元件和一个编码8个氨基酸的微型编码序列(uORF);主编码区(mORF)2181bp,共编码726个氨基酸,其中N端具有叶绿体定位信号肽.推测的GhADC1蛋白具有PLP结合位点及Orn/Dap/Arg脱羧酶的信号位点,属于典型的Ⅲ型PLP依赖型精氨酸脱羧酶家族成员.荧光定量RT-PCR结果表明,正常条件下,GhADC1在叶片中表达量高于茎和根部组织,且在抗黄萎病品种冀棉20中的表达水平高于耐病品种中521;黄萎病菌胁迫能够快速诱导抗病品种根部GhADC1表达,而在耐病品种中未见明显差异,推测在抗病品种中GhADC1可能参与根部对黄萎病菌胁迫的早期应答.     

棉花幼苗对低温胁迫的响应及抗冷机制初步研究

【目的】探讨棉花不同组织对低温胁迫的响应,初步探讨研究抗冷的生理生化和分子机制。【方法】测定了4个抗冷性差异显著的材料在低温处理后的生物量、抗氧化酶活力和可溶性蛋白含量,同时分析了抗冷相关基因在豫2067根、茎、叶的表达情况。【结果】4℃低温处理24 h对冷敏感材料生长抑制最大,对根系生长的抑制大于地上部分;细胞膜透性测定结果表明,受低温胁迫后4个材料根系细胞膜能保持相对稳定的结构,而叶片细胞膜对低温胁迫敏感,抗冷材料叶片细胞膜稳定性大于冷敏感材料,低温胁迫后叶片细胞膜透性与棉花的抗冷性呈负相关,可以作为棉花抗冷性的鉴定指标;棉花在受到低温胁迫24 h后,抗冷材料根中SOD的活力基本不变,冷敏感材料根中SOD的活力却极显著下降,2个材料的根系中POD、CAT的活力均下降,但抗冷材料豫2067下降的幅度小于冷敏感材料,可溶性蛋白含量在抗冷材料叶茎中基本不变,在冷敏材料的叶片和茎中均下降。低温胁迫后5个与抗冷相关的基因在豫2067叶片中上调表达的多于下调表达的,上调表达的基因分别是脂肪酸脱氢酶基因、胁迫诱导蛋白基因、假定R2R3-MYB转录因子基因、b HLH1转录因子基因;在根中下调表达的多于上调表达的,下调的基因分别为b HLH1转录因子基因、胁迫诱导蛋白基因、伸展蛋白基因2,这与根系的生长受抑制的结果是一致的。【结论】推测这些抗冷相关基因在叶片中的上调表达与叶片生长受低温抑制程度低有一定的关系。     

Ionic Homeostasis and Expression of Na+ Related Genes of Cotton under Different Salt and Alkali Stresses

[Objective] Maintaining intracellular ion homeostasis is one of the important salt-tolerant mechanisms of crops. This study aims to analyze differences in response characteristics of cotton ionome and salt-tolerant gene expression under different saline-alkali stresses, which provides a basis for understanding the mechanism of salt tolerance and improving salt tolerance of cotton. [Method] Using Lumianyan 24 as the experimental material, three kinds of salt and alkali stress types (salt stress, alkali stress, and mixed salt-alkali stress) and two concentration gradients (low and high concentrations) were set under pot cultivation conditions. Meanwhile, non-saline-alkali stress treatment was set as control. The dry matter weight of cotton plants and root morphological parameters including root length, root surface area, and root volume were measured in this study. The concentrations of 13 elements such as P, Na, K, Ca and Mg in different organs of cotton plants were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). The relative expressions of salt tolerance related genes GhDFR1, GhSOS1, GhNHX1 and GhAKT1 were determined by the quantitative real-time polymerase chain reaction method. [Result] 1) Salt and alkali stresses significantly inhibited cotton growth. The growth inhibition rate of cotton under mixed salt-alkali stress treatments (48.7%–57.9%) was significantly higher than that under salt stress (27.6%–49.9%) and alkali stress (21.2%–35.5%) treatments. Under salt stress and mixed salt-alkali stress treatments, both shoot and root growth of cotton were significantly inhibited, dry matter weight, root length, root surface area and root volume were significantly reduced, while root growth was less inhibited under alkali stress treatments. 2) Under three types of saline-alkali stresses, Na content and Mo content in different organs of cotton plant increased significantly, but N content in leaves and roots decreased. 3) Under salt stress treatments, the uptake of Ca, Mg, Fe, Mn and Zn in cotton was inhibited, and the ion balance was maintained by promoting the transport of these ions and P, K. 4) In addition to Ca, Mg, Fe, Mn and Zn, P uptake was also inhibited under alkali stress treatments, but K uptake and P, K, Ca, Mg, Fe, Mn and Zn transport were promoted. 5) Under mixed salt-alkali stress treatments, especially under high salinity and pH conditions, most of nutrients uptake was inhibited, and the transport capacity of Ca, Mg, Zn, Mn and Fe was reduced. 6) The relative expression of GhSOS1 and GhAKT1 genes increased significantly under salt stress treatments, but increased first and then decreased under alkali stress and mixed salt-alkali stress treatments. The relative expression levels of GhSOS1 and GhAKT1 genes under three types of saline-alkali stresses were alkali＞salt＞mixed saline-alkali stress. With the increase of soil salinity and pH value, the relative expression of GhNHX1 gene increased first and then decreased. The expression levels of GhNHX1 gene were salt＞alkali＞mixed salt-alkali stress. [Conclusion] Due to high salinity and pH value, mixed salt-alkali stress significantly inhibits cotton growth and ions uptake, which restricts the transport of P, K, Ca, Mg, Zn, Mn and Fe. The decrease of K and Na regulation ability leads to ions imbalance.     

Effect of Coronatine on AsA-GSH Cycle of Cotton Seedling under Low Temperature Stress

[Objective] In order to explore the stress-resistant mechanism of coronatine, an indoor experiment was conducted to study the effect of coronatine on ascorbate-glutathione circulatory system of the vegetative organs of cotton seedling under low temperature stress. [Method] Xinluzao 57 was used as the experimental material, when the seedlings reached the two-leaf stage, they were treated at 25 ℃ and 4 ℃ after spraying water, marked as CK and LT; the same treatment was conducted after spraying 0.01 μmol·L^－1 coronatine, marked as COR and (LT+COR). After 1 d of treatment, the roots, stems and leaves of cotton seedling were collected to determine the antioxidant contents and antioxidant enzyme activities. [Result] Compared with the control treatment (CK), the APX, MDHAR activities and DHA, GSH, glutathione contents decreased, the GPX activity, AsA and ascorbate contents increased, and no significant effect on DHAR and GR activities were observed in roots, stems and leaves of cotton seedling after spraying water at low temperature (LT), while in leaves, the GSSG content decreased, AsA-DHA ratio and GSH-GSSG ratio showed the maximum increase. Compared with the LT treatment, spraying COR at low temperature (LT+COR) showed increased APX, MDHAR, DHAR, GPX, GR activities and AsA, DHA, GSH, ascorbate contents; glutathione content in roots, stems and leaves of cotton seedling, and GSSG content, AsA-DHA ratio and GSH-GSSG ratio obviously changed in leaves. [Conclusion] Spraying COR at low temperature could regulate the AsA-GSH metabolism and alleviate the damage caused by low temperature to cotton seedling. COR had the strongest relieving effect on seedling leaves.     

棉花转录因子GhWRKY4基因的克隆及特征分析

 本研究从陆地棉中克隆得到GhWRKY4基因,全长cDNA是1281 bp,包含1083 bp的开放阅读框,编码的360个氨基酸属于IIcWRKY转录因子家族。分析GhWRKY4基因结构表明其有三个外显子和两个内含子。GhWRKY4蛋白的亚细胞定位实验表明其定位在洋葱表皮细胞核。实时荧光定量分析结果显示GhWRKY4在棉花的根、茎、叶和花中均有表达。染色体步移得到了GhWRKY4的5’端侧翼序列,生物信息学软件预测表明GhWRKY4包含一系列和胁迫基因调控相关的反式作用元件,表明GhWRKY4参与植物对盐害和冷害胁迫的反应。     

陆地棉水通道蛋白GhNIP6.1基因的克隆及表达分析

根据EST拼接的序列设计引物,利用RT-PCR技术,从陆地棉品种苏棉18中克隆获得了一个水通道蛋白基因GhNIP6.1。该基因开放阅读框包含903个核苷酸,编码300个氨基酸,分子量为30.97kD,理论等电点为8.99。GhNIP6.1蛋白的生物信息学分析表明:GhNIP6.1含有6个跨膜区,由5个环相连,其中环A、C和E在细胞膜外,环B、D和蛋白的N、C末端都位于细胞膜内,N末端79个氨基酸,C末端18个氨基酸,具有MIP家族典型的保守氨基酸序列;该蛋白的三级结构同拟南芥的AtNIP6.1非常相似,亚细胞定位也同AtNIP6.1一致,可能位于质膜中。进化分析发现,GhNIP6.1蛋白同拟南芥的AtNIP6.1蛋白相似性最高。进一步扩增棉花核基因组获得了2021bp的DNA序列,它包含5个外显子和4个内含子,所有外显子/内含子交接点都遵从gt/ag剪接规则。半定量分析表明,该基因在根、茎和叶中都有表达,其中真叶含量较高,子叶中没有表达。     

棉花冠菌素不敏感因子GhCOI1基因分离和对大丽轮枝菌的抗性分析

为了解决黄萎病对棉花的危害,利用基因工程技术培育抗病棉花品种是当前育种的主要目标。从棉花中克隆了一个黄萎病抗性基因GhCOI1,其编码冠菌素不敏感因子。GhCOI1基因序列全长2 213 bp,编码一个600氨基酸的多肽,分子量为68 k Da,等电点p I是7.06。GhCOI1含有典型的F-box和LRR结构域。组织表达分析表明,该基因在根器官优势表达,并且其表达受到大丽轮枝菌浸染诱导。利用病毒诱导基因沉默(Virus-induced gene silencing,VIGS)技术,抑制了GhCOI1基因在棉花植株中的表达;通过大丽轮枝菌接菌分析表明,GhCOI1基因沉默能够引起植株的抗性下降,发病加重。研究结果表明GhCOI1参与棉花对大丽轮枝菌抗性调控,因此,其可以作为棉花抗病育种的候选基因。