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| 蒺藜苜蓿雄性不育突变体emptyanther1与紫皮突变体purpletesta1的基因克隆与功能研究 | |
| Alternative Title | GeneCloningandFunctionalCharacterizationofMale-sterile Mutant emptyanther1and Seed Testa Mutant purple testa1in Medicago truncatula |
| 郑小玲 | |
| Thesis Advisor | 陈江华 |
| 2020 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 中国科学院西双版纳热带植物园 |
| Degree Name | 理学博士 |
| Degree Discipline | 植物学 |
| Keyword | 雄性不育，bHLH，GDSL，空花药，紫种皮 |
| Abstract | 雄性不育是植物育种和杂交制种的一个重要工具。在此基础上，利用优质基因对作物进行品种改良是农业生产的一个重要方向。豆科植物作为动物和人类主要的植物蛋白质及油脂的来源，其在国内农业生产中占据了重要地位。豆科模式植物蒺藜苜蓿（Medicago truncatula）作为优质牧草紫花苜蓿（M. sativa）的近缘种，具有二倍体基因组小、可自交、突变体资源丰富等优点。本研究我们选择蒺藜苜蓿作为研究对象，并在其Tnt1突变体库中分离到一个突变体株NF74XX。其自交后代分离出两类表型独立的突变体，即雄性不育突变体empty anther1（ean1）和紫皮突变体 purple testa1（pt1）。 雄性不育突变体主要是由孢子体型或配子体型的花药组织发育异常引起的。绒毡层作为一个为花粉发育提供营养的重要的孢子体型组织，其延迟降解会引发花粉败育，但是对于绒毡层在豆科植物的育性调控中的作用鲜有报道。此外，被子植物中已报道了许多参与绒毡层降解的bHLH蛋白，而对它们所涉及的陆生植物发育途径的进化研究也甚少被关注。在这里，我们描述了蒺藜苜蓿中的一个雄性不育突变体ean1。它表现为营养期植株生长正常，而生殖期雄性育性完全受到破坏。通过遗传学分析得出ean1为单基因隐性纯合突变体。显微镜观察发现ean1突变体表现为花粉缺失的空花药表型。我们参考拟南芥和水稻的花药发育分期，从细胞形态特征上将蒺藜苜蓿花药发育分为14期。ean1突变体在花药发育的第8期开始出现绒毡层降解缺陷，后期不能产生小孢子。且其胼胝质的合成也有缺陷。而后，通过第二代重测序技术克隆到EAN1基因，反向遗传学和遗传互补证明了EAN1基因调控蒺藜苜蓿的雄性不育表型。EAN1编码一个细胞核定位的bHLH蛋白。它在绒毡层中特异性表达，到花药发育的第7期表达量最高。EAN1与拟南芥中的同源基因具有功能保守性。进一步结合已测序的藻类、蒺藜苜蓿及其他陆生标志植物，我们重构了植物bHLH蛋白的系统进化树。结果表明，所有已报道的参与绒毡层调控的bHLH转录因子可聚类为II和III(a+c)1 两个亚家族。EAN1属于II类亚家族，蒺藜苜蓿中III(a+c)1亚家族三个成员MtAMS、MtAMSL和MtDYT1参与绒毡层和小孢子发育。EAN1能特异地与三个III(a+c)1亚家族成员形成的异源二聚体。这表明在被子植物中存在一个保守的异源二聚化机制。进一步对植物bHLH家族进行系统性的进化分析表明，II和III(a+c)1的bHLH两个亚家族保守存在于所有陆生植物，它们在陆生植物类群分化之前就已经存在。 种子休眠可分为五类，分别是生理休眠、形态休眠、形态生理休眠、物理休眠和综合休眠（物理休眠和生理休眠）。物理休眠是在种子成熟干燥过程中由于种皮或果皮不透水导致的，是植物在变化多端的自然环境下的一种自我保护机制。在自然环境下，如高温火灾干旱冰雪消融及动物消化液等诸多因素都会打破种子的物理休眠，促使种子吸水萌发。豆科许多植物都具有种子休眠特性，种皮在其中起到了关键作用。蒺藜苜蓿的种皮由外向内依次包括角质层、巨石细胞组成的表皮层、表皮层下的骨状石细胞和薄壁细胞组成的内皮层四个部分。在这里，我们描述了蒺藜苜蓿中的一个打破种子物理休眠和种皮颜色发生变化的紫皮突变体pt1。pt1可直接吸水萌发，通过遗传学分析得出它为单基因隐性纯合突变体。半薄切片结果显示pt1成熟种皮的巨石细胞结构异常。透射电镜观察发现授粉后35天的pt1种皮内巨石细胞的腔室内有电子致密物沉积。通过二代重测序克隆到了PT1基因，反向遗传学和遗传互补证明了PT1突变引起种皮物理休眠的打破和颜色改变。PT1在种皮中特异性表达。PT1编码一个高尔基体定位的乙酰脂酶/酯酶。该蛋白包含一个靠近N端的包含PMR5的N端结构域和一个紧接着的GDSL/SGNH样乙酰酯酶结构域。蛋白跨膜结构预测PT1是一个Ⅱ型一次跨膜蛋白。虽然我们对PT1的具体调控途径还不是很清楚，但pt1突变体特异地出现种皮结构变异而不影响营养期和生殖期的其它组织生长过程，这使得PT1具有潜在的农业应用价值。对于PT1深入的工作机制解析将是未来研究的重点。 随着第三代杂交体系的提出，核基因隐性纯合雄性不育系的重要性不言而喻。而不育基因在进化上的功能保守性也为作物育种研究提供了便捷。雄性不育系作为杂交育种的工具，为优质基因导入作物中培育符合人类需求的优良品种提供了一个稳定的母本。此外，作物品种的储存和繁育在生产中也是很重要的。种子物理休眠可以延长作物储存期。同时，物理休眠在基因水平上的缺失，也能便于种子批量快速的萌发。本论文的两项研究都具有潜在的农业育种应用价值。 |
| Other Abstract | Male sterility is a valuable tool in plant breeding and hybrid seed production. Based on that, it is an important direction of agricultural production to improve crop varieties by introducing high quality genes. Legumesareimportantcomponentsofsustainableagriculturalproductionof our country, providing a major source of plant protein and oil for humans and animals. As a close relative of Medicago sativa which is commonly grown as a forage crop, M. truncatula, with small diploid genome, self-pollination, abundant mutant resources, has been chosen as a legume model. In this study, we focus on characterizing a mutant strain NF74XX isolated from the Tnt1retrotransposon-tagged mutant collection of M.truncatula. Two mutant alleles with different phenotypes were found in the population of the inbred offspring of NF74XX, namely, male-sterile mutant empty anther1(ean1) and seed coat mutantpurple testa1(pt1).Male-sterile mutants are commonly caused due to abnormal development of either the sporophytic or gametophytic anther tissues. Tapetum, a key nutritive layer of the sporophytic tissue, nourishes the developingpollengrains, and delayed degenerationof tapetumwould induce pollen abortion. However, the regulatory functions of tapetum in legume fertility have rarely been reported. Moreover, numerous bHLH proteins have beenreported toinvolvein thedegeneration of the tapetum in angiosperms, butrelatively little attention has been given to the evolution of the involved developmental pathways across land plants. Here, we characterized a M.truncatula male-sterile mutant, ean1,which has no visible defect in vegetative organs but showed completely male sterility in reproductive stage. Genetic analysis showed that recessive ean1mutant was caused by loss-of-function of a single gene. Microscopic observations revealed that ean1mutant presented the appearance of empty anthers. Referring to the anther development of Arabidopsis thaliana and Oryza sativa, we divided the M. truncatula anther development into 14 stages based on the morphological and cellular features. The ean1mutant was defective in tapetum degeneration at stage8, which led to the microspore abortion during later stages. In addition, callosesynthesis was affected in ean1mutant. Subsequently, we cloned theEAN1 gene by re-sequencing, and characterizedEAN1mutation causes the male sterility by reverse genetics and genetic complementation.EAN1encodes a nucleus-localized bHLH transcription factor, which is strongly expressed in tapetum with most highly expressed level detected in stage 7of the anther. The EAN1isfunctionally conserved with its homologous genes in A.thaliana. The phylogenetic tree of plant bHLH proteins was reconstructed by collecting bHLH sequences from algae, M. truncatulaand other representative land plants. The results showed that all the reported bHLH transcription factors involved in the regulation of tapetum degeneration could be grouped into two subfamilies, namely, II and III(a+c)1. EAN1belongs to the subfamily II, and three subfamilies III(a+c)1members in M. truncatula,MtAMS, MtAMSL and MtDYT1, also participate in tapetum and microspore development. EAN1 specifically forms heterodimers with the three subfamily III(a+c)1 members, which suggests there is a conserved heterodimerization mechanism in angiosperms. Further analysis shows these two subfamilies are conserved in land plants, and they have existed before the differentiation of groups of land plant.There are five classes of seed dormancy: physiological dormancy, morphological dormancy, morphophysiological dormancy, physical dormancy and combinational dormancy (physiological dormancy and physical dormancy). Physical dormancy is caused by the present of a water-impermeable seed or fruit coat during maturing and drying of the seed. It is a self-protection mechanism for plants to adapt the varied natural environment. In nature, many factors, including high temperatures, fire, drying, freezing/thawing, and digestive tracts of animals, render the seed coat permeable to water. Physical dormancy is widespread among legume species, and the seed coat plays key roles in this process. The seed coat structure of M. truncatulaconsists of four parts, which are the outer cuticle layer, followed by an epidermal layer of macrosclereids, a subepidermal layer of osteosclereids and internal parenchyma cells. Here, we characterized a pt1mutant with altered seed coat color in M. truncatula. Seeds of pt1could directly imbibe water to germination, indicating that the physical dormancy of pt1is broken. Genetic analysis showedthat pt1mutant was controlled by a single recessive gene. Semi-thin sections revealed an abnormal structure of macrosclereids in mature seed coat of the pt1mutant. Transmission electron microscopy analysis displayed electron dense deposition in the lumen of macrosclereids of the pt1mutantat 35 days after pollination. We clonedthePT1geneby re-sequencing, and confirmedPT1mutation brokes the physical dormancy and affects the testa color by reverse genetics and genetic complementation. PT1is specifically expressed in the seed coat, and it encodes a Golgi-locolized acetyllipase/esteraseand contains a PMR5 N terminal domain and a GDSL/SGNH-like acyl esterase domain. PT1is a typical type IIone-spantransmembrane protein resulting from the prediction of transmembrane helices. Although the specific regulatory pathway of PT1is not clear, pt1mutants show seed coat structure variation without other defect during the vegetative and reproductive growth,making PT1potentially valuable for agricultural applications. In-depth analysis of the working mechanism of PT1will be the key point in the future research. Recessive nuclear male-sterile lines and evolutionarily conserved function of the male-sterile genes play a key role in the “third-generation” hybrid system and crop breeding. As a tool for crop breeding, recessive nuclear male-sterile lines could provide a stable female parent source for hybrid seed production. In addition, the storage and breeding of crop varieties are important in agricultural production. The physical dormancy of seeds can prolong the storage timeof crops, and the deletion of physical dormancy at the gene level can facilitate the rapid germination of seeds in batches. Both studies above mentioned have potential values forcrop breeding. Key words: Male-sterility,bHLH,GDSL,empty anther1,purple testa1 |
| Pages | 135 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | https://ir.xtbg.ac.cn/handle/353005/11704 |
| Collection | 西双版纳热带植物园毕业生学位论文 |
| Affiliation | 1.中国科学院大学; 2.中国科学院西双版纳热带植物园 |
| Recommended Citation GB/T 7714 | 郑小玲. 蒺藜苜蓿雄性不育突变体emptyanther1与紫皮突变体purpletesta1的基因克隆与功能研究[D]. 中国科学院西双版纳热带植物园. 中国科学院大学,2020. |
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| 郑小玲.pdf（8075KB） | 学位论文 | 开放获取 | CC BY-NC-SA | Application Full Text | ||
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