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。     

Genome-Wide Identification and Expression Analysis of TLP Genes in Gossypium hirsutum L.

[Objective] Thaumatin-like proteins (TLPs) are important pathogenesis-related proteins that function during disease defense-related responses, participating in the defense reactions triggered by several kinds of pathogen infections. A genome-wide analysis of TLP genes could help increase our understanding of their molecular mechanism in response to Verticillium dahliae infections in cotton (Gossypium hirsutum L.). [Method] The TLP family members in the genomes of the G. hirsutum L. were identified and expression analyses of TLP genes in response to Verticillium dahliae inoculation were conducted using bioinformatics and real-time fluorescent quantitative PCR methods, respectively. [Result] In total, 88 TLP genes were identified in G. hirsutum. These TLPs were classified into 10 groups based on their amino acid sequences and a phylogenetic analysis. A gene structure analysis revealed that the number of exons ranges from 1 to 5, and the number of introns ranges from 0 to 4. TLP genes in the same group shared similar structures. Additionally, most TLP proteins contain five motifs that are arranged in the following order: 5, 4, 2, 3 and 1. In total, 87 TLP genes are distributed on 20 of the 26 chromosomes, and 42 and 45 TLP genes are distributed in the A and D subgroups, respectively. The real-time fluorescent quantitative PCR analysis showed that the expression levels of six candidate TLP genes were induced in both tolerant cotton cultivar GZ-1 and susceptible cotton cultivar 86-1, and showed a higher expression in the former. [Conclusion] These results provide a foundation for future studies of the functions and regulatory mechanisms of TLP family genes.     

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Nudix基因家族的全基因组鉴定及表达分析

[目的]Nudix是一类能够催化各种核苷二磷酸衍生物水解的酶,具有维持遗传物质稳定,响应逆境胁迫等生物学功能.本研究旨在对陆地棉Nudix基因进行全基因组分析,为深入研究Nudix基因家族参与棉纤维生长发育调控机制提供参考.[方法]通过生物信息学方法对陆地棉基因组(ZJU_v2.1)的Nudix基因进行全基因组鉴定,并...     

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...     

陆地棉三个WRKY基因的克隆及表达分析

WRKY转录因子可调控下游抗逆基因的表达,进而在植物生长发育以及对外界环境的反应中起着重要的调控作用,目前对棉花WRKY家族基因的研究比较少。本研究利用电子克隆的方法,从陆地棉品种山农圣杂3号中克隆得到了3个具有完整开放阅读框的棉花WRKY基因,聚类分析表明它们同属于WRKY家族中的第Ⅱ类。利用RT-PCR结果表明:250mmol/LNaCl盐胁迫和20%PEG6000干旱胁迫下同时诱导GhWRKY4的基因表达,GhWRKY5仅受干旱胁迫下的诱导,而GhWRKY6对这两种逆境胁迫都没有变化。     

棉花抗黄萎病相关基因的序列和表达分析

黄萎病是棉花生产的主要病害,克隆抗病基因是培育棉花抗黄萎病品种的关键。本文根据前期的定位结果,结合棉花基因组测序信息,提取定位区段内基因组序列,预测获得63个基因。Gene Ontology分析表明63个基因参加多种生物进程,其中6个基因参与植物抗逆进程。根据Gene Ontology分析和前人研究结果,选择15个基因进行序列分析。启动子序列分析表明启动子区域包含各种抗逆的调控元件,其中6个基因包含了W-box元件。对海7124和苏棉8号棉花幼苗进行黄萎病菌处理,取病菌处理后不同时期的根,选择14个基因进行表达分析,结果表明在黄萎病菌处理后,有8个基因表达出现变化,其中在海7124和苏棉8号之间表达差异最大的基因是跨膜蛋白(Transmembrane protein 214-a isoform 1)、细胞色素P450(Cytochrome p450)和Udp-糖基转移酶UGT89A2(Udp-glycosyl transferase 89a2-like)基因。本研究为抗病基因克隆提供了候选基因。     

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。     

Proteomics Analysis of Gossypium barbadense Leaves with Resistance to Verticillium dahliae under Infection Stress

[Objective] The purpose of this study is to analyze the resistance mechanism of island cotton under Verticillium wilt stress and to find possible resistance genes. [Method] The cotton resistance mechanism under the stress of Verticillium dahliae was studied by protein two-dimensional electrophoresis and mass spectrometry at the protein level. [Result] In the leaves, eleven proteins were down-regulated and 15 proteins were up-regulated after 2 hours of inoculation with Verticillium dahliae. The down-regulated proteins were mainly related to photosynthesis and carbon assimilation, and then we deduce that Verticillium dahliae is mainly broken cotton photosynthetic system to cause cotton susceptibility. The up-regulated proteins were mainly related to photosynthesis and benzoquinone reductase, beta-D-galactosidase, 14-3-3f protein and other disease-resistant protein. [Conclusion] It is speculated that the defense mechanism of island cotton on Verticillium wilt may occur at two levels: one is passive defense, it is manifested that the isoproteins of the down-regulated proteins such as chloroplast II AB binding protein and ribulose diphosphate carboxylase are highly expressed to maintain the stability of the island cotton photosynthetic system, the histone and 14-3-3f protein are highly expressed to keep the cell stability; the second is active defense, it is manifested that the high expressed β-D-galactosidase and benzoquinone reductase may be involved in the resistance of island cotton to Verticillium wilt.     

陆地棉PPO基因全基因组鉴定及对黄萎病菌的响应分析

【目的】多酚氧化酶(Polyphenol oxidases,PPO)广泛参与植物抵抗病虫害等生物逆境胁迫的过程。本研究旨在研究棉花中的PPO基因及其表达模式,验证多酚氧化酶与棉花抗黄萎病的关系。【方法】分析了黄萎病菌V991侵染下异源四倍体陆地棉TM-1根系中PPO酶活力变化,并利用TM-1的基因组数据库,鉴定相关基因,并进行生物信息学和表达分析。【结果】共筛选到13个候选GhPPO基因。这些基因都不含内含子,所编码的蛋白包含2个铜离子结合位点和PPO的保守序列(酪氨酸酶、DWL和KFDV保守结构域)。进化分析显示,陆地棉PPO基因的种内相似性大于种间相似性,并且相互之间形成了多对重复基因。GhPPO的表达量差异较大,少数基因具有组织特异表达的特性,多数基因在棉花不同组织中表达量都很低。实时荧光定量聚合酶链式反应分析结果表明:GhPPO6D在棉花根系中表达量较高,且在黄萎病菌V991侵染后上调。【结论】GhPPO6D可能参与了棉花与黄萎病菌的互作过程,与V991侵染后棉花根系PPO酶活力上升相关。     

棉花RAV基因家族的全基因组分析

在二倍体棉花D5基因组(Gossypium raimondii Ulb.)数据库中鉴定出10个RAV基因,分布于4、5、8、9、13号染色体;在二倍体棉花A2基因组(Gossypium arboreum L.)数据库中鉴定出10个RAV基因,与棉花D5基因组的R AV成员的数量和序列具有一一对应的同源关系,推测棉花A、D组的祖先种中可能存在10个RAV基因。对植物R AV蛋白序列做系统发育分析,将R AV成员分为4个组;发现棉花R AV基因可能参与了棉属所特有的基因组多倍化事件的证据。对N CBI中陆地棉(Gssypium hirsutum L.)EST、Unigene数据库做比对统计,得到陆地棉不同组织中R AV基因表达情况;对陆地棉受黄萎病菌胁迫后的荧光定量检测,发现棉花RAV基因与棉花响应黄萎病菌的胁迫相关。     

Integrated Evaluation and the Physiological and Biochemical Responses of Semi-Wild Cotton under Complex Salt-Alkali Stress

[Objective] The genetic diversity of semi-wild cotton was abundant, so more new elite resistant genes could be found and applied to improve the resistance of cotton. The aim of this study was to primarily study on the regulation of response to salt-alkali stress, explore the method of integrated evaluation and characterize the resistance of semi-wild cotton. [Method] Four semi-wild and two cultivated Gossypium hirsutum accessions were evaluated for phonotypical, physiological and biochemical traits under alkali-salt versus normal conditions in hydroponic solutions. The alkali-salt tolerance was determined based on principal component analysis, cluster analysis, correlation analysis, grey rational analysis and analysis of variance. [Result] On the basis of the overall results, the alkali-salt stress tolerance of the six accessions was ranked as: Marie-galante85 > Latifolium32 > CRI16 > CRI12 > Latifolium40 > Latifolium130. Roots were found to be the most important responsive systems to complex alkali-salt stress. At seedling growth stage, the active scavenging system played a crucial role in response to alkali-salt stress. In addition, the salt tolerant and salt sensitive accessions showed different response tends towards leaf peroxidase, root ascorbate peroxidase and root catalase within 48 h, suggesting the accessions have different levels of response to salt stress. [Conclusion]Our study identified the most alkali-salt tolerant accessions and provided basic concept of complex alkali-salt tolerance mechanism within cotton accessions. And, our study provided a simple and rapid, highly accurate and precise method for evaluating salt resistance of cotton, and proved that the balance of ROS system play an important role in response to salt-alkali stress. Hence, mining of salt tolerant genes from these accessions may facilitate the development of novel salt tolerant variety.     

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.