中文题名: | 基于RNA-seq解析陆地棉果枝发育的分子机制及GhSMAX1基因功能研究 |
姓名: | |
学号: | 20192306006 |
保密级别: | 公开 |
论文语种: | chi |
学科代码: | 0710 |
学科名称: | 理学 - 生物学 |
学生类型: | 博士 |
学位: | 工学博士 |
学位类型: | 学术学位 |
学位年度: | 2024 |
学校: | 石河子大学 |
院系: | |
专业: | |
研究方向: | 植物分子育种 |
第一导师姓名: | |
第一导师单位: | |
第二导师姓名: | |
完成日期: | 2024-06-30 |
答辩日期: | 2024-07-06 |
外文题名: | Molecular mechanisms of fruiting branch development in upland cotton based on RNA-seq analysis and functional study of the GhSMAX1 gene |
中文关键词: | |
外文关键词: |   ; Fruit branch ; Plant hormones ; GhSMAX1 ; Plant architecture ; GhSMXL |
中文摘要: |
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棉花是全球重要的纤维和油料作物,也是我国国计民生的重要战略物资和棉纺织工业的关键原料。新疆是中国最大的棉花生产基地,其种植面积、单产、总产、商品调出量连续29年居全国第一,形成了“中国棉花看新疆”的格局。但是,新疆棉花种植面临着水资源短缺、土壤退化等生产环境恶化的问题,同时棉花品种“多乱杂”现象突出,机采棉的质量较差,导致新疆棉花的整体质量和经济效益不高,阻碍了棉花产业的发展。研发抗逆、早熟和紧凑型棉花株型品种是新疆棉花育种的重要目标之一。棉花植株的高度、分枝、成熟时间和生长习性等性状决定着其物种特异性,也影响着棉花栽培适应性、管理模式、产量和收获效率。因此,挖掘控制果枝发育的关键基因,解析棉花果枝发育的分子调控机制,为棉花育种提供科学依据,具有重要的理论和现实意义。本研究通过重离子辐射诱变陆地棉品种新陆早62号(XLZ62)获得不同果枝类型的棉花种质,在此基础上对一个长果枝突变棉花种质和XLZ62果枝3个发育时期的组织进行转录组测序(RNA-seq)分析,探索长短果枝基因表达差异,挖掘控制果枝发育的关键基因,并解析这些基因在果枝发育中的功能,旨在为高产和机械化收获棉花品种的培育提供理论指导和基因资源。本论文的主要研究内容和结果如下: 1. 重离子辐射诱变XLZ62创制陆地棉新种质 本研究利用重离子束诱变技术对XLZ62种子进行处理,经田间连续种植多代获得了107份在分枝数目与长度、株高和叶片大小等相关的突变体棉花材料。通过调查统计XLZ62和不同突变体在株高、棉铃数目、第一果枝高度、第四果枝长度、叶片数量和果枝夹角等农艺性状,进一步筛选到7份具有较大表型变异的突变体种质,包括:III式型果枝(M41)、营养枝生长旺盛型(M55);叶枝生长旺盛的丛生铃(M38);叶枝旺盛的塔型(M49)、果枝长且分枝少(M71);矮杆(M50)等。其中,M41果枝长度和数目显著增加,棉铃数增多,与XLZ62的果枝相比具有明显差异。本研究以XLZ62和M41为主要研究材料,开展果枝发育过程中的RNA-seq研究。 2. 长短果枝陆地棉果枝发育3个时期的RNA-seq分析 分别取样XLZ62和M41第四果枝的初始腋芽(S1)、腋芽伸长形成果枝第一节间时期的顶芽(S2)、第二节间时期的顶芽(S3)组织。其中,XLZ62的3个发育时期分别命名为XS1、XS2、XS3,M41的3个发育时期分别命名为MS1、MS2、MS3,每个样品三次独立重复,共18个样本进行RNA-seq分析。文库测序获得了总计937,872,376条clean reads,总序列量达到144.63 Gb。在6个组织中总共识别出45,203个表达基因,其中12,420个基因在所有组织中均有表达。XLZ62中有36,708个表达基因,14,741个在3个发育时期均有表达。M41中有36,534个表达基因,14,901个在3个发育时期均有表达。比较XLZ62和M41在三个发育时期的差异表达基因(Differentially expressed gene, DEG),XLZ62在XS2 vs XS1中的DEGs最多,上调724个,下调1378个;共有的DEGs为39个;M41在MS3 vs MS2的DEGs最多,上调1576个,下调1437个;共有的DEGs为64个。XLZ62和M41两品种间比较显示,MS3 vs XS3的DEGs最多,上调871个,下调1810个;3个发育时期共有的DEGs为410个。GO富集分析发现,这410个DEGs主要涉及蛋白磷酸化、跨膜运输和谷胱甘肽代谢过程;KEGG分析显示主要富集在内分泌抵抗、mRNA监控和植物激素信号通路。其中,植物激素信号通路AUXs、CKs、SLs在调控棉花果枝发育中起关键作用,特别是GhSMAX1基因在M41中差异明显。因此,我们对棉花SMXL基因家族成员进行了全基因组鉴定和分子特征分析。 3. 棉花SMXL基因家族成员的全基因组鉴定、分子进化和表达特征分析 拟南芥SUPPRESSOR OF MAX2 1 (SMAX1)-like (SMXL)蛋白SMXL6, SMXL7, SMXL8和水稻D53作为SLs信号通路中重要的靶蛋白促进分枝。SMXL基因家族在棉花中还未开展系统的鉴定和功能研究。本研究首先对绿藻门、苔藓门和被子植物中已经测序的21个物种的SMXL基因家族成员开展全基因组鉴定,并探讨了这些基因在植物中的系统进化史。从陆地棉(Gossypium hirsutum)、海岛棉(G. barbadense)、亚洲棉(G. arboreum)、雷蒙德氏棉(G. raimondii)和草棉(G. herbaceum)中分别鉴定出18、18、9、9和9个共63个SMXL基因成员,分为4个进化分枝。氨基酸比对表明SMXL蛋白具有一个保守的Clp-N domains、P-loop NTPase和EAR基序。不同组织中的表达(qRT-PCR)分析表明,GhSMAX1-1在叶中高表达,GhSMAX1-2、GhSMXL6/7-1/7-2在茎中高表达。GR24处理棉花幼苗48 h后,发现GhSMXL6/7-1/7-2响应GR24上调表达。在胁迫条件4 ℃、37 ℃、0.4 mol/L NaCl以及200 g/L PEG6000处理24 h后,GhSMAX1-1/2的表达量在4 ℃和37 ℃胁迫下上调。GhSMXL6的表达量在盐胁迫后下调。在干旱条件下,除GhSMAX1-2以外的所有基因的表达量均上调。蛋白共表达网络分析发现GhSMXL6、GhSMXL7-1和GhSMXL7-2主要与促进生长发育和诱导芽分化的蛋白互作,如bZIP、MYB和AP2/ERF等转录因子。 4. GhSMXL基因家族成员的功能分析 利用烟草脆裂病毒(Tobacco rattle virus, TRV)诱导的基因沉默(Virus-induced gene silence, VIGS)实验表明GhSMAX1-1/1-2以及GhSMXL6/7-1/7-2沉默后均抑制腋芽的发育,并且这5个GhSMXL基因的沉默均导致植株矮化,其中GhSMAX1基因沉默的株高降低最明显。此外,GhSMXL6和GhSMXL7-1/2沉默后叶片还表现出黄化和萎蔫的表型。另外,沉默一个负调控因子BRC1抑制子的编码基因GhTIE1促进了顶芽和侧芽的生长。拟南芥原生质体的亚细胞定位分析显示,GhSMAX1蛋白定位在细胞核中,GhD14蛋白定位在细胞质和细胞核中。酵母双杂交(Y2H)显示GhSMAX1和GhD14蛋白并不互作。在模式植物拟南芥(Col-0)中过表达GhSMAX1基因,发现转基因拟南芥的茎分枝数量和株高均显著高于Col-0,并且幼苗的下胚轴明显伸长。这些结果初步表明GhSMXL基因促进棉花分枝发育,GhSMAX1基因在控制株高和腋芽发育方面起着重要的作用。VIGS植株TRV:GhSMAX1和对照TRV:00植株顶芽的RNA-seq分析。发现GhSMAX1基因沉默后抑制了大量AUXs信号通路相关基因的表达,例如ARF9、LAX5和GH3.6;而促进了ETHs信号通路相关基因的表达,例如EIN3、ERF1B、ERF.C.3、ARF、ARR-B和AP2-EREBP等。 总之,本研究通过重离子束诱变获得长果枝突变体M41,并利用RNA-seq技术分析了果枝发育的调控网络。发现了一个编码SLs信号转导靶蛋白的GhSMAX1基因,并对棉花SMXL基因家族进行了生物信息学和表达特征分析。5个GhSMXL基因的沉默均抑制了棉花果枝发育和植株高度。其中,过量表达GhSMAX1基因促进拟南芥植株分枝和株高。本研究为优化棉花株型结构,提高棉花机械化收获和生产效率提供了理论基础和优质基因资源。 |
外文摘要: |
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Cotton is a globally important fiber and oil crop, as well as a critical strategic resource for China’s national economy and people’s livelihood and a key raw material for the cotton textile industry. Xinjiang is the largest cotton production base in China, leading the country for 29 consecutive years in planting area, unit yield, total yield, and commercial output, thereby establishing the pattern of “Chinese cotton depends on Xinjiang”. However, Xinjiang’s cotton cultivation faces issues such as water resource scarcity and soil degradation, which deteriorate the production environment. Additionally, the prominence of mixed and disordered cotton varieties and the poor quality of machine-picked cotton result in low overall quality and economic efficiency of Xinjiang cotton, hindering the development of the cotton industry. Developing stress-resistant, early maturing, and compact cotton plant varieties is one of the crucial goals of Xinjiang cotton breeding. The agronomic traits such as plant height, branching, maturity time, and growth habits determine cotton species specificity and affect their adaptability to cultivation, management mode, yield, and harvesting efficiency. Therefore, exploring key genes controlling fruit branch development and analyzing the molecular regulatory mechanisms of cotton fruit branch development to provide a scientific basis for cotton breeding have substantial theoretical and practical significance. In this study, we obtained different fruit branch types of cotton germplasm by heavy ion radiation mutagenesis of the upland cotton variety Xinluzao 62 (XLZ62). Transcriptome sequencing (RNA-seq) analysis was conducted on tissues of a long fruit branch mutant cotton germplasm and XLZ62 fruit branches at three developmental stages to explore the gene expression differences between long and short fruit branches, identify key genes controlling fruit branch development, and analyze the functions of these genes in fruit branch development, aiming to provide theoretical guidance and genetic resources for the breeding of high-yield and mechanized cotton varieties. The main research contents and results of this study are as follows: 1. Creation of new upland cotton germplasm by heavy ion radiation mutagenesis of XLZ62 The heavy ion beam mutagenesis technology was utilized to treat XLZ62 cotton seeds, yielding 107 mutant lines with variations in branching number and length, plant height, and leaf size after multiple generations of continuous field planting. Comprehensive investigations and statistical analyses of agronomic traits, such as plant height, number of cotton bolls, height of the first fruit branch, length of the fourth fruit branch, number of leaves, and angles of fruit branches, were conducted on XLZ62 and its mutants. As a result, seven mutant germplasms exhibiting significant phenotypic variations were identified, including: type III fruit branch (M41), vigorous vegetative branch growth type (M55); cluster bolls with vigorous vegetative branches (M38); tower type with vigorous leaf branch growth (M49), long fruit branch with few branches (M71); dwarf type (M50). Among them, M41 had significantly increased fruit branch length and number of cotton bolls, showing obvious differences compared to XLZ62 fruit branches. This study used XLZ62 and M41 as the main research materials to conduct RNA-seq studies during the fruit branch development process. 2. RNA-seq analysis of upland cotton fruit branch development at three stages of long and short fruit branches Tissues were sampled from the initial axillary bud (S1), the top bud at the first internode formation stage of the axillary bud elongating to form the fruit branch (S2), and the top bud at the second internode stage (S3) of the fourth fruit branch of XLZ62 and M41. The three developmental stages of XLZ62 were named XS1, XS2, XS3, and those of M41 were named MS1, MS2, MS3, respectively. RNA-seq analysis was performed on a total of 18 samples, with three independent replicates for each sample. Library sequencing yielded a total of 937,872,376 clean reads, with a total sequence amount of 144.63 Gb. A total of 45,203 expressed genes were identified across the six tissues, of which 12,420 genes were expressed in all tissues. In XLZ62, 36,708 genes were expressed, and 14,741 were expressed in all three developmental stages. In M41, 36,534 genes were expressed, and 14,901 were expressed in all three developmental stages. Comparing differentially expressed genes (DEGs) between XLZ62 and M41 at three developmental stages, the most DEGs in XLZ62 were in XS2 vs XS1, with 724 upregulated and 1378 downregulated genes; the shared DEGs were 39. In M41, the most DEGs were in MS3 vs MS2, with 1576 upregulated and 1437 downregulated genes; the shared DEGs were 64. Comparison between the two varieties showed the most DEGs in MS3 vs XS3, with 871 upregulated and 1810 downregulated genes; the shared DEGs in three developmental stages were 410. GO enrichment analysis revealed that these 410 DEGs mainly involved protein phosphorylation, transmembrane transport, and glutathione metabolic processes; KEGG analysis showed they were mainly enriched in endocrine resistance, mRNA surveillance, and plant hormone signal transduction pathways. Among them, AUXs, CKs, and SLs in the plant hormone signal transduction pathway played key roles in regulating cotton fruit branch development, especially the GhSMAX1 gene, which was significantly different in M41. Therefore, we conducted genome-wide identification and molecular characterization analysis of the cotton SMXL gene family members. 3. Genome-wide identification, molecular evolution, and expression characteristics analysis of cotton SMXL gene family members Arabidopsis SUPPRESSOR OF MAX2 1 (SMAX1)-like (SMXL) proteins SMXL6, SMXL7, SMXL8, and rice D53 are important target proteins in the SLs signal pathway promoting branching. The SMXL gene family has not yet been systematically identified and functionally studied in cotton. This study first conducted genome-wide identification of SMXL gene family members in 21 sequenced species from Chlorophyta, Bryophyta, and Angiosperms, and explored the phylogenetic evolutionary history of these genes in plants. A total of 63 SMXL gene members were identified from Gossypium hirsutum (18), G. barbadense (18), G. arboreum (9), G. raimondii (9), and G. herbaceum (9), which are classified into four evolutionary clades. Amino acid alignment indicated that SMXL proteins have a conserved Clp-N domain, P-loop NTPase, and EAR motif. Expression analysis (qRT-PCR) in different tissues showed that GhSMAX1-1 was highly expressed in leaves, and GhSMAX1-2, GhSMXL6/7-1/7-2 were highly expressed in stems. After 48 h of GR24 treatment of cotton seedlings, GhSMXL6/7-1/7-2 were upregulated in response to GR24. Under stress conditions of 4 °C, 37 °C, 0.4 mol/L NaCl, and 200 g/L PEG6000 treatments for 24 h, respectively. The expression levels of GhSMAX1-1 and GhSMAX1-2 were upregulated at both 4 °C and 37 °C treatments. In contrast, GhSMXL6 was notably downregulated under 0.4 mol/L NaCl stress. Furthermore, under 37 °C conditions, all examined genes, except GhSMAX1-2, exhibited upregulation. Protein co-expression network analysis found that GhSMXL6, GhSMXL7-1, and GhSMXL7-2 mainly interacted with proteins promoting growth and inducing bud differentiation, such as transcription factors bZIP, MYB, and AP2/ERF. 4. Functional analysis of GhSMXL gene members Tobacco rattle virus (TRV)-induced gene silencing (Virus-induced gene silence, VIGS) experiments showed that silencing GhSMAX1-1/1-2 and GhSMXL6/7-1/7-2 inhibited axillary bud development and silencing these five GhSMXL genes led to plant dwarfing, with the most significant reduction in plant height observed in GhSMAX1 gene silencing. In addition, silencing GhSMXL6 and GhSMXL7-1/2 resulted in leaf chlorosis and wilting phenotypes. Moreover, silencing GhTIE1, encoding a negative regulator BRC1 inhibitor, promoted the growth of apical and lateral buds. Overexpressing the GhSMAX1 gene in Arabidopsis (Col-0) significantly increased the number of stem branches and plant height in transgenic Arabidopsis compared to Col-0, and the hypocotyls of seedlings were significantly elongated. These results preliminarily indicated that GhSMXL genes promote cotton branching development, and the GhSMAX1 gene plays an important role in controlling plant height and axillary bud development. Subcellular localization analysis of Arabidopsis protoplasts showed that GhD14 protein was localized in the cytoplasm and nucleus, while GhSMAX1 protein was localized in the nucleus. Yeast two-hybrid (Y2H) showed that GhSMAX1 and GhD14 proteins did not interact. RNA-seq analysis of apical buds from TRV:GhSMAX1 and control TRV:00 plants demonstrated that silencing the GhSMAX1 gene inhibited the expression of multiple genes within the AUXs signaling pathway, including ARF9, LAX5, and GH3.6. Conversely, it upregulated genes associated with the ETHs signaling pathway, such as EIN3, ERF1B, ERF. C. 3, ARF, ARR-B, and AP2-EREBP. In conclusion, this study obtained a long fruit branch mutant M41 through heavy ion beam mutagenesis and analyzed the regulatory network of fruit branch development using RNA-seq technology. A GhSMAX1 gene encoding an SLs signal transduction target protein was discovered, and bioinformatics and expression characteristic analyses of the cotton SMXL gene family were performed. Silencing five GhSMXL genes inhibited cotton fruit branch development and plant height. Overexpression of the GhSMAX1 gene promoted plant branching and height in Arabidopsis. This study provides a theoretical foundation and high-quality genetic resources for optimizing cotton plant architecture and improving cotton mechanized harvesting and production efficiency. |
参考文献: |
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中图分类号: | Q78 |
开放日期: | 2024-07-11 |