目的：体细胞胚胎发生(Somatic embryogenesis)是棉花(Gossypium hirsutum L.)通过农
杆菌介导的遗传转化最关键的步骤之一，通常包括非愈伤组织的诱导、胚性愈伤组织的
分化、胚状体的形成以及胚状体发育成苗等过程。棉花体细胞胚胎发生存在的缺点是基
因型依赖、培养周期长、不正常胚产生效率高，严重制约了棉花基因功能的验证和转基
因育种工作。研究棉花体细胞胚胎发生的生理及分子机制可以为棉花组织培养及转基因
育种工作提供重要的理论依据和技术支撑。
方法：(1)本研究使用de novo转录组测序和iTRAQ蛋白组测序的方法，测定了新疆陆
地棉品种新陆早33号体细胞胚胎发生过程中非胚性愈伤组织、胚性愈伤组织以及胚状体
中的差异表达基因和蛋白，并对差异表达基因和蛋白进行了鉴定及表达模式分析。(2)对
差异表达基因和蛋白的相关通路进行了注释和分类，进一步明确了参与调控棉花体细胞
胚胎发生的通路。(3)利用qRT-PCR、内源含量测定以及外源添加等实验，明确相关基因、
蛋白及通路与棉花体细胞胚胎发生的关系。(4)用HPLC法，提取分离鉴定了棉花内源多
胺的种类，建立棉花组织多胺提取测定的技术体系。(5)通过分析多胺代谢途径相关生理
生化指标、基因表达、ELISA法测定相关酶活性、生理互补实验等，揭示多胺调控棉花
体细胞胚胎发生的可能机制。
结果与结论：(1)De novo转录组测序一共获得了101,670个unigene，在胚性愈伤组织
向胚状体转化过程中差异表达的基因多于非胚性愈伤组织向胚性愈伤组织转化过程中
的差异表达基因。大量的差异表达基因参与了植物激素合成及信号转导、胁迫响应反应、
ROS平衡以及多胺代谢等途径。用qRT-PCR验证了相关差异基因的真实性和准确性。内
源IAA和KT含量变化规律与其合成途径相关基因的表达模式一致。外源IAA和KT能够促
进胚性愈伤组织向胚状体的转化，不同浓度IBA的添加会对非胚性愈伤组织、胚性愈伤
组织的诱导效率有影响，外源添加多胺和过氧化氢也能在很大程度上促进胚状体的形成。
适当的胁迫条件对胚性愈伤组织的增殖以及胚状体形成都有促进作用。此外，赤霉素、
脱落酸、乙烯、油菜素内酯、水杨酸、茉莉酸及脂肪酸途径相关基因也呈现出差异表达
的规律。转录组动力学分析表明不同植物激素间的平衡调控，多胺代谢和胁迫响应过程
协同调控了棉花体细胞胚胎发生。
(2)iTRAQ蛋白组测序发现，三组样品中一共得到了5892个差异表达蛋白，这些蛋白
主要参与了催化活性、结合活性、转运活性以及结构分子活性，其中93.4%的差异蛋白
在分子功能水平参与了结构分子活性。差异表达蛋白统计分析发现，与非胚性愈伤组织
相比，胚性愈伤组织中分别有572个上调表达蛋白和452个下调表达蛋白；与胚性愈伤组
织相比，胚状体中分别有211个上调表达蛋白和647个下调表达蛋白。KEGG分析显示遗
传信息的转运、植物激素合成及信号转导、糖酵解过程、脂肪酸合成和代谢以及半乳糖
代谢等通路参与了棉花体细胞胚胎发生。通过qRT-PCR分析相关差异基因在三个时期的
表达情况进一步确定了蛋白组测序的真实性和准确性。对转录组和蛋白组的测序数据进
行关联分析发现，所测得基因序列和蛋白序列的关联系数为0.27，差异表达基因与差异
表达蛋白的关联系数高于0.6。
(3)建立了棉花组织多胺提取分离及测定的高效液相色谱法，可将腐胺、亚精胺、精
胺在 15 分钟完全分离并定量测定，线性关系良好(r>0.99)，回收率高(96.8%～103.1%)。
测定棉花体细胞胚胎发生各时期内源多胺的含量，结果显示在胚性愈伤组织以及体细胞
胚形成初期，三种类型多胺含量显著提高，腐胺的含量在胚状体中有下降趋势，而亚精
胺和精胺的含量在胚状体中没有变化。多胺合成相关基因的表达量分析发现，棉花精氨
酸合成酶 GhADC 在体细胞胚胎发生过程中的表达与多胺含量(尤其是腐胺含量)的变化
规律非常一致。此外，多胺代谢产物 H2O2 的含量在胚性愈伤组织时期也有显著的提高，
与多胺氧化酶 GhPAO1 和 GhPAO4 的表达规律一致。ELISA 酶活测定发现，多胺氧化酶
PAO 在胚性愈伤组织中表现出较高的活性，抑制 PAO 活性则表现出体细胞胚胎形成受
阻。多胺及其代谢产物 H2O2 都能够促进胚性愈伤组织的生长及胚状体的形成，同时也
能够降低多胺合成抑制剂 D-arginine 及多胺氧化酶抑制剂 1, 8-DO 的抑制效果。多胺代
谢途径另一个重要的信号分子 NO 的含量在体细胞胚胎发生过程中变化不明显，且对体
细胞胚胎发生没有显著作用。综合上面的结果，我们得出结论：多胺可能通过多胺氧化
酶调节其代谢产物过氧化氢来调控棉花体细胞胚胎发生。
 

Object: Plant regeneration via somatic embryogenesis (SE) is the key step for genetic
improvement of cotton (Gossypium hirsutum L.) through genetic engineering mediated by
Agrobacteria, and SE is also treated as a model for understanding of molecular events occurring
during plant embryo development. Somatic embryogenesis was usually considered including
twice cell reprograms involved in cellular dedifferentiation and redifferentiation. However, SE
in cotton is often hampered by recalcitrance, long culture time, and high frequency of abnormal
embryos. Low efficiency of regeneration is another major issue that limits the utilization of
Agrobacterium-mediated transformation through SE in cotton. The molecular mechanisms
underlying SE in cotton is still unclear.
Method: RNA-Sequencing and isobaric tags for relative and absolute quantitation(iTRAQ)
based comparative proteome was used to analyze the differentially expressed genes and proteins
during somatic embryogenesis of cotton cultivar Xinluzao 33 from non-embryogenic callus
(NEC), embryogenic callus (EC) and somatic embryos (SEs). The genes differentially
expressed (DEGs) and differentially expressed proteins (DEPs) amongst NEC, EC and SEs
were identified, annotated and classified in GO, COG, NR, SWISS and KEGG. qRT-PCR
confirmed the sequencing data. Endogenous levels of plant hormones, polyamines, ROS, H2O2
and NO were determinated, the effect of certain chemicals and pathways were investigated.
Complementary physiological experiments and ELISA assay were conducted for confirming
the mechanisms of polyamines on cotton somatic embryogenesis.
Results:(1)A total of 101, 670 unigenes were de novo assembled. More DEGs were found
between SEs and EC than between EC and NEC. A significant number of DEGs were related
to hormone homeostasis, stress and ROS responses, and metabolism of polyamines.
Quantitative real-time PCR analysis further confirmed the authenticity and accuracy of the
transcriptome expression results. To confirm the expression dynamics of selected DEGs
involved in various pathways, experiments were set up to investigate the effects of hormones
(Indole-3-acetic acid, IAA; Kinetin, KT), polyamines, H2O2 and stresses on SE. Our results
showed that exogenous application of IAA and KT positively regulated the development of EC
and SEs, and that PAs and H2O2 promoted the conversion of EC into SEs. Furthermore, we
found that low and moderate stress is beneficial for proliferation of EC. Together, our global
analysis of transcriptomic dynamics reveals that hormone homeostasis, polyamines, and stress
response synergistically regulating SE in cotton.
(2)In the proteome profile, 5892 proteins were identified and almost of them classified
into catalytic activity, binding activity, transporter activity and structural molecular activity
covered 93.4% in molecular function level. Distribution analysis of differentially expressed
proteins (DEPs) suggested 572 and 211 proteins were up-regulated, 452 and 647 proteins were
down-regulated during EC and SE differentiation respectively. KEGG (Kyoto Encyclopedia of
Genes and Genomes) analysis indicated genetics information transmission, plant hormone
transduction, glycolysis, fatty acid biosynthesis and metabolism, galactose metabolism were
involved in somatic embryogenesis. Quantitative real-time PCR analysis further confirmed the
authenticity and accuracy of the proteomic expression results. Proteome profile analysis
facilitated identification of interesting genes and provided insights into the molecular
mechanisms underlying somatic embryogenesis in Gossypium hirsutum L.
(3)HPLC method for determination of cotton cultures polyamine was established through
relevant and recovery analysis. Putrescine (Put), spermidine (Spd) and spermine (Spm)
significantly increased from the EC stage to the early phase of embryo differentiation. The
levels of Put then decreased until the somatic embryo stage whereas Spd and Spm remained
nearly the same. The expression profiles of GhADC genes were consistent with changes in Put
during cotton SE. The H2O2 concentrations began to increase significantly at the EC stage,
during which time both GhPAO1 and GhPAO4 expressions were highest and PAO activity was
significantly increased. Exogenous Put, Spd, Spm and H2O2 not only enhanced embryogenic
callus growth and embryo formation, but also alleviated the effects of D-arginine and 1, 8-
diamino-octane, which are inhibitors of polyamine synthesis and PAO activity. Overall, the
results suggest that both PAs and their metabolic product H2O2 are essential for the conversion
of EC into somatic embryos in cotton.

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