棉花是世界性重要的经济作物，是天然纤维的主要来源。棉花生殖生长过程开花、结铃直接影响棉花经济性状——棉纤维的产量和品质。随着对花发育模式研究的逐渐深入，改进后的“AE模型”相较于传统的“ABC模型”有了更广泛的适用性。然而，目前对棉花花器官发育的分子作用机理还处于不成熟阶段。发掘控制棉花花器官发育的关键基因资源，对于培育高产、优质棉花新品种具有重要意义。本实验室以棉花野生型W0为受体，以GoPGF-RNAi为载体转化棉花，在T1代发现了克隆号为182-9的花器官突变体，命名为182-9。该突变体花器官明显瓣化，花瓣和雄蕊结构异常，失去了自身独特的生理构造，均呈现类似细长叶片状态。本研究通过southern杂交、基因组重测序、转录组差异分析、q-PCR以及VIGS沉默基因等技术对182-9突变体进行了系统的分析研究，确定了T-DNA的插入位点，并对候选基因进行了克隆、表达和VIGS功能验证。此外，还对花发育中起重要作用的MADS-box基因家族进行了鉴定、分析，其中部分MADS家族基因在突变体和正常棉花花器官中差异表达，这些基因可能参与了调控花器官的形成。这些研究结果为棉花的花器官发育研究提供了理论依据，同时也为其他物种花器官发育的研究提供了一个良好的借鉴。

本论文的主要研究结果如下：

通过Southern杂交结果证明转基因突变体材料182-9中，外源基因为单拷贝插入。对转基因受体W0和突变体182-9进行高深度基因组重测序，通过基因组序列比对，预测外源DNA插入位点在棉花A11染色体的59086746-59086947区间，PCR进一步明确了A11:59086840为T-DNA插入位点。

根据棉花参考基因组TM-1（V2.1）的注释结果，在插入位点附近，存在3个候选基因GH_A11G2251、GH_A11G2252、GH_A11G2253。其中，GH_A11G2251在拟南芥中的同源基因注释为AP2-like ethylene-responsive transcription factor ANT，是一个AP2类的花功能基因；GH_A11G2252基因功能注释为Putative ribonuclease H protein，是一类转座因子基因；GH_A11G2253未有功能注释。PCR扩增结果显示三个基因在突变体182-9和受体W0之间基因组序列无差异。q-PCR结果显示， GH_A11G2251在W0和182-9中的花瓣（O2）、雄蕊（O3）、雌蕊（O4）、三个组织中表达存在明显差异；GH_A11G2252在两个材料中没有显著性差异；GH_A11G2253几乎不表达。考虑到棉花新陆早42 号相对TM-1的生长周期要短，我们采用该早熟品种为VIGS受体，沉默上述3个候选基因，观察它们的花器官表型变化。

为了进一步分析W0和182-9花器官整体的基因表达差异，我们对W0和182-9花器官转录组进行差异分析，结果显示有16343个基因在花器官中差异表达，其中8391个基因上调，7952个基因显示下调。对这些差异基因进行GO和KEGG富集分析，我们发现上调基因主要聚集在GO亚级分类的分子功能（MF，Molecular function）进程中，下调基因则主要分布在相同水平的生物学过程（BP，biological process）中。KEGG的Pathway主要富集在与光系统相关的代谢途径中。这与我们182-9出现的异常表型相一致。

已有报道证明MADS基因广泛参与了花器官的发育。我们在陆地棉基因组中鉴定出97个MADSⅠ类基因和96个MADSⅡ类基因，分别进行了系统发育进化分析，及18个组织中的表达量分析，进一步揭示了该类基因的组织特异性。之后，我们将上述两类基因分别在突变体182-9和W0花器官转录组中进行比对筛选，得到48个表达差异的花发育基因，其中包括6个MADSⅠ类基因和42个MADSⅡ类基因。

本研究结合生物信息学技术和转基因分子实验共同研究了182-9中花器官异常的可能性分子机制，挖掘出了棉花中花发育调控的相关基因，为棉花育种在花发育方面的研究提供了参考价值。

Cotton is a worldwide cash crop and a major source of natural fiber. Flowering and boll-setting during cotton reproductive growth directly affected the economic characters of cotton —— The yield and quality of cotton. With the further research on flower development pattern, the improved “AE” model has more extensive applicability than the traditional “ABC “model. However, the molecular mechanism of cotton organ development is still in the immature stage. It is very important to explore the key gene resources which control the development of cotton organs for breeding new cotton varieties with high yield and high quality. Four floral organ mutants, named 182-9, were found in the T1 generation of cotton transformed with the wild type W0 as the receptor and the GoPGF-RNAi as the vector. The mutant had obvious petalization of flower organs, abnormal structure of petals and Stamens, and lost its own unique physiological structure, which were similar to elongated leaves. In this study, T-DNA insertion sites were identified by southern blot analysis, genomic resequencing, transcriptome differential analysis, q-PCR and VIGS silencing gene analysis, the candidate gene was cloned, expressed and the VIGS function was verified. The MADS-box gene family, which plays an important role in flower development, was identified and analyzed. Some of the MADS genes were differentially expressed in mutant and normal cotton organs, these genes may be involved in regulating flower organ formation. These results provide a theoretical basis for the research of cotton floral organ development, and also provide a good reference for the research of other species floral organ development.

The main findings of this paper are as follows:

Southern Blot analysis showed that the foreign gene was inserted by single copy in the transgenic mutant 182-9．The high-depth genome resequencing of the transgenic receptor W0 and the mutant 182-9 was carried out. By comparing the genomic sequences, the insertion sites of exogenous DNA were predicted to be in the 59086746-59086947 region of the Cotton A11 chromosome, PCR further identified A11:59086840 as the T-DNA insertion site.

According to the Annotation of Reference Genome TM-1(V2.1) , there are three candidate genes GH_A11G2251、GH_A11G2252、GH_A11G2253 near the insertion site. The homologous gene of GH_A11G2251 in Arabidopsis is annotated as AP2-like ethylene-responsive transcription factor (AP2) , which is a functional gene of AP2．The function of GH_A11G2252 gene is annotated as Putative ribonuclease H protein, which is a type of transposable factor gene. No functional comments on GH_A11G2253. The results of PCR amplification showed that there was no difference in the genome sequence between the mutant 182-9 and the receptor W0. The results of q-PCR showed that there were significant differences in the expression of GH_A11G2251 in petal（O2）, pistil（O3） and stamen（O4） in W0 and 182-9, no significant difference in GH_A11G2252 in the two materials, and GH_A11G2253 was not expressed. Considering that the growth cycle of Cotton Xinluzao 42 is shorter than that of TM-1, we adopted this early maturing cultivar as VIGS receptor to silence the three candidate genes mentioned above and observe their floral organ phenotypic changes.

In order to further analyze the differences of gene expression between W0 and 182-9 floral organs, we analyzed the transcriptome differences between W0 and 182-9 floral organs. The results showed that 16343 genes were differentially expressed in floral organs, among which 7952 genes were up-regulated, 8,391 genes down-regulated. In the enrichment analysis of GO and KEGG, we found that the up-regulated genes were mainly clustered in the Molecular function (MF) process of GO subclassification, while the down-regulated genes were mainly distributed in the same level of biological process (BP). KEGG Pathway is mainly concentrated in the Pathway related to photosystem. It's consistent with the abnormal phenotype we found in 182-9.

It has been reported that MADS gene is involved in the development of flower organs. We identified 97 in upland cotton genome by MADSⅠgenes and 96 by MADSⅡclass genes, phylogenetic evolution is analyzed respectively, and 18 expression analysis in the organization, and further reveals the tissue specificity of this gene. After that, we compared the two genes in the mutant 182-9 and W0 floral organ transcriptome, and got 48 differentially expressed floral developmental genes, including 6 MADSⅠgenes and 42 MADSⅡgenes.

In this study, the molecular mechanism of 182-9 floral organ abnormalities was investigated by combining bioinformatics technique and transgenic molecular experiments, and the genes involved in the regulation of floral development in cotton were discovered, it provides reference value for the research of cotton breeding in flower development.

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