棉花苯丙氨酸解氨酶基因家族的生物信息学分析

【目的】苯丙氨酸解氨酶是苯丙烷类代谢的关键酶,对调节植物生长和抗逆有重要作用。从全基因组层次了解棉花苯丙氨酸解氨酶基因家族的进化和功能,可为相关研究提供数据支撑。【方法】利用HMMER和BLASTP从棉花基因组筛选苯丙氨酸解氨酶基因家族,利用MEGA-X进行进化分析,利用MCScanX进行共线性分析,并利用NCBI SRA的数据进行表达分析。【结果】本研究分别从陆地棉、海岛棉、亚洲棉和雷蒙德氏棉基因组中筛选到12、13、7、8个苯丙氨酸解氨酶基因,依据进化分析和共线性关系数据,我们推测四种棉花的二倍体祖先起初应有6个苯丙氨酸解氨酶基因。顺式作用元件分析结果表明,苯丙氨酸解氨酶基因家族含有较多的光响应、激素响应、胁迫响应和生长发育相关的元件。苯丙氨酸解氨酶的蛋白序列具有保守的基序。陆地棉的12个苯丙氨酸解氨酶在低温、高温、盐和PEG胁迫下有不同的表达方式。【结论】本研究明确了苯丙氨酸解氨酶在棉花基因组中的数量、理化性质、进化和表达特征,为苯丙氨酸解氨酶的深入研究提供了参考。     

棉花酰基辅酶A结合蛋白(ACBP)家族基因的发掘及在非生物胁迫抗性中的功能鉴定

酰基辅酶A结合蛋白(ACBP)家族基因在植物正常生长发育、响应逆境胁迫及生物膜修复等方面具有重要作用。本研究基于陆地棉(Gossypium hirsutum)遗传标准系TM-1基因组序列信息,鉴定并获得21个棉花ACBP家族基因成员的全序列和染色体定位等信息。聚类分析表明这21个基因分属于I~IV类。依据与拟南芥的同源性,将棉花ACBP家族基因命名为GhACBP1~GhACBP6等6大亚类。转录组分析表明,该家族基因在不同组织、不同发育时期表达差异较大。不同逆境诱导分析表明,GhACBP1、GhACBP3和GhACBP6显著受盐、旱、低温、高温逆境胁迫诱导,而GhACBP4和GhACBP5对逆境胁迫响应不强烈。进一步分析表明,GhACBP3和GhACBP6的表达受过氧化氢(H2O2)、水杨酸(SA)、茉莉酸(JA)、脱落酸(ABA)和乙烯(ET)的诱导。病毒诱导的基因沉默(VIGS)试验表明,沉默GhACBP3和GhACBP6亚类基因会降低棉花植株对干旱和盐的耐性。目标基因沉默后,植株干物质积累下降,株高变矮,超氧化物歧化酶(SOD)和过氧化物酶(POD)活性降低,丙二醛(MDA)含量升高,表明GhACBP3和GhACBP6在棉花抗旱、耐盐中发挥作用。研究为利用ACBP家族基因进行棉花抗逆性研究及应用提供参考。     

Genome-Wide Identification and Analysis ofGRGene Family in Cotton

[Objective] Glutathione reductase (GR) gene family is involved in biological processes such as plant growth and abiotic stress response, but its characteristics and functions in cotton have not been known yet. This study aims to explore the role of GR genes in cotton genome evolution and abiotic stress response through the whole genome identification and characterization of GR genes, thus providing a theoretical basis for future studies on the roles of the GR genes in enhancing abiotic stress tolerance in cotton. [Method] The GR genes in Gossypium hirsutum, G. barbadense, G. raimondii and G. arboreum were all identified using bioinformatics software. The physicochemical properties, sequence characteristics, chromosomal location, phylogeny and expression patterns were analyzed. [Result] A total of 18 GR genes were identified. The number of GR genes in G. hirsutum, G. barbadense, G. raimondii and G. arboreum was 6, 6, 3 and 3, respectively. Phylogenetic analysis revealed that GR genes were divided into two sub-groups. The genes in the same subgroup exhibited similar gene structure in relation to exon-intron ratios. The ratios of the non-synonymous mutations (K_a) and homologous mutations (K_s) were all less than 1, indicating that the GR genes underwent strong purification selection during their evolution process. The analysis of the expression patterns of GR genes in upland cotton indicated that all the GR genes responded actively to the stress environment; but under different abiotic stresses, the gene expression patterns were significantly different. [Conclusion] The study explored the evolution and function of the GR gene family in the four cotton genomes, providing a theoretical basis for future studies of cotton GR genes.     

陆地棉REM基因家族全基因组鉴定及表达分析

[目的]REM(Reproductive meristem)基因家族编码一类转录因子在植物生长发育过程中发挥重要作用,但在棉花中未见报道.[方法]基于陆地棉基因组数据和公共数据库中的转录组数据,运用生物信息学方法对陆地棉REM基因家族成员进行系统鉴定,并对该基因家族的理化性质、基因结构、组织表达特异性以及胁迫条件下的表...     

VIGS技术及其在棉花功能基因组研究中的应用进展

病毒诱导的基因沉默(Virus induced gene silencing,VIGS)是一种转录后基因沉默现象,是植物体内普遍存在的遗传免疫机制,现已被开发为快速、高效、高通量的反向遗传学技术,在植物基因功能研究中得到广泛应用。近年,利用VIGS技术进行棉花基因功能研究也取得了一定进展。本文对VIGS技术的发展、操作技术的优化进行了综述,尤其总结了VIGS技术在棉花抗病、品质改良、生长发育等基因鉴定和功能研究中的应用进展,并对其应用前景进行了展望。     

雷蒙德氏棉DnaJ蛋白的生物信息学预测及盐胁迫下的表达分析

Dna J蛋白是一类很大的蛋白家族,是近年来研究较多的与胁迫反应等相关的蛋白。本文利用生物信息学手段,系统研究了雷蒙德氏棉(Gossypium raimondii)Dna J蛋白的数目、分类、染色体定位、亚细胞定位、进化以及在旱地棉(Gossypium aridum)和克劳茨基棉(G.klotzschianum)盐胁迫下的表达模式。结果显示:雷蒙德氏棉基因组有119个Dna J蛋白,长度从73个氨基酸到2574个氨基酸不等;在13条染色体上不均匀分布,且具有不同的亚细胞定位;Dna J结构域大致被分为9个小组,每个小组都有与拟南芥同源的基因;盐胁迫下,鉴定到旱地棉和克劳茨基棉共同差异表达的Dna J基因3个,在旱地棉盐胁迫整个过程中差异表达的基因2个。雷蒙德氏棉Dna J基因家族的鉴定和分析,将为进一步研究Dna J基因家族的功能奠定一定的基础。     

陆地棉Pht1家族成员的全基因组鉴定及表达分析

【目的】磷是植物三大必需矿质元素之一,对植物的生长发育至关重要。Pht1家族的磷酸盐转运蛋白在植物磷吸收和转运方面具有重要作用,然而关于该基因的系统研究工作尚很少开展。本研究旨在进行Pht1家族成员全基因组鉴定和表达模式分析。【方法】通过生物信息学的方法对陆地棉Pht1家族成员的基因结构、编码蛋白质的结构、染色体定位、基因复制和表达情况等进行全面分析。【结果】(1)在陆地棉的基因组中共鉴定到17个磷酸盐转运蛋白基因(GhPT),其中A亚组包含8个GhPT,D亚组包含9个GhPT;(2)棉花GhPT蛋白之间序列相似性很高,并均具有12个疏水的跨膜区域;(3)进化分析表明这些GhPT蛋白主要聚为2大组(GroupⅠ和GroupⅡ),位于同一组的GhPT的编码基因大部分具有相似的内含子/外显子分布模式;(4)17个GhPT基因不均匀分布在A亚组和D亚组中的5条染色体上,串联复制和片段复制可能导致GhPT基因在陆地棉中的扩增;(5)表达模式分析表明,GhPT6和GhPT14在根中表达量最高,且同时响应低磷和低钾胁迫诱导并上调表达。【结论】这些结果将有助于深入了解Pht1家族基因的功能及离子信号途径相互作用的分子机制。     

Genome-Wide Selection of MAPKKK Gene Family in Gossypium raimondii and the Expression of Orthologs in Gossypium hirsutum

[Objective] The MAPKKK gene family plays an important regulating role in response to multiple abiotic stresses and the development of plant. This study aims to identify MAPKKK genes of Gossypium raimondii and analyze their functions. [Method] In this study, based on G. raimondii genome database and bioinformatics method, G. raimondii MAPKKK family genes were identified and analyzed. Using the MEGA5, GSDS and Mapchart program, the phylogenetic tree, gene structure and chromosomes location analyses were accomplished. Based on the existing microarray data in cotton and comparative profiles of these MAPKKK genes, different expression of them in multiple abiotic stresses and the expression at different cotton fiber developmental stages were analyzed. [Result] A sum of 114 MAPKKK genes were identified systematically in G. raimondii and classified into 3 subfamilies (Raf, ZIK and MEKK) according to the gene stucture and phylogenetic tree analyses. They were distributed on all the 13 chromosomes of G. raimondii, and segmental duplication and tandem duplication events may have occurred. Compared with the recently released 78 genes of G. raimondii MAPKKK family genes, 47 sequences are exactly the same ones. [Conclusion] The results are helpful to understand the evolution and function of MAPKKK gene family. Our results provide a foundation for future functional characterizations of MAPKKK genes in cotton and probably other Gossypium plants.     

棉花AGO蛋白家族基因鉴定及表达分析

AGO蛋白在所有由小RNA介导的RNA沉默通路中发挥重要作用。在植物中,它广泛参与维持基因组的稳定、调控组织的发育、对逆境的应答以及对入侵的转基因和植物病毒的免疫。对AGO基因家族成员的研究有利于研究其功能。为揭示棉花中AGO家族的功能,本研究利用全基因组信息在陆地棉基因组中鉴定了27个AGO蛋白,并对这些家族成员的特性进行了进化分析,这些AGO蛋白被分为3类,蛋白长度介于731~1462 aa之间,第一、三亚家族蛋白编码区较多,第二亚家族蛋白编码区较少。27个基因分布在16条染色体上。8个基因在所有组织中的表达量较高,棉花AGO基因与拟南芥AGO基因在组织中的表达模式极为相似。本研究为进一步研究棉花中AGO蛋白的功能提供了指导意义。     

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。     

陆地棉KFB基因家族全基因组鉴定与表达分析

【目的】KFB(Kelch containing F-box protein)是普遍存在于各种生物体中含有F-box结构域的一类家族蛋白,参与众多生物过程。本研究旨在开展陆地棉KFB家族基因鉴定和表达分析。【方法】利用生物信息学方法从陆地棉(TM-1)基因组中鉴定KFB家族成员,与已知的拟南芥、水稻、大豆等KFB蛋白序列构建系统进化树;进一步针对第Ⅱ亚族进行基因结构、染色体定位和表达分析。【结果】从陆地棉基因组共鉴定出150个KFB家族成员,进化分析表明这些基因可分为7个亚族。陆地棉KFB第Ⅱ亚族24个基因分布在14条染色体上,有9对直系同源基因;该亚族基因结构简单,半数基因只有1个外显子;qRT-PCR结果表明,该亚族基因具有较为广泛的组织表达模式,但在不同组织的表达存在差异。【结论】这些结果显示陆地棉KFB第Ⅱ亚族基因在进化过程中出现了功能分化。     

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.