【作者】 李俊；

【导师】 赵洁；

【作者基本信息】 武汉大学 ， 发育生物学， 2010， 博士

【摘要】 阿拉伯半乳糖蛋白（arabinogalactan proteins, AGPs）是一类广泛分布于植物界的糖蛋白。已有研究表明,阿拉伯半乳糖蛋白在植物生长发育的不同时期起着重要作用。根据阿拉伯半乳糖蛋白的蛋白骨架特点,预测模式生物拟南芥中存在大约85个AGP基因。本文利用生物信息学、半定量及定量RT-PCR、RNAi和过表达、启动子融合及亚细胞定位载体的构建、浸花法转化拟南芥等一系列分子生物学及细胞生物学方法,对At FLA 3的表达模式、细胞亚定位及生物学功能进行了详细的研究,研究结果如下：1、AtFLA3序列的生物信息学分析表明,该基因cDNA包含一个843bp的开放阅读框,编码具有280个氨基酸的蛋白；并且AtFLA3具有4个含量丰富的氨基酸包括：脯氨酸（Pro）（10.71%）,丙氨酸（Ala）（10.71%）,丝氨酸（Ser）（10.36%）和苏氨酸（Thr）（6.79%）。其编码成熟蛋白前体具有4个不同的区域：一个N-端分泌信号,一个位于中间的、典型的、富含羟脯氨酸/脯氨酸的AGP结构域,一个类成束蛋白结构域（fasciclin-like domain）和一个C-端疏水的GPI（甘油磷酸肌醇）加锚信号。前体蛋白的第24和第25位氨基酸之间为N-端信号肽切割位点。第256位的“Ser”氨基酸为C-端甘油磷酸肌醇加尾信号的修饰位点。拟南芥基因芯片ATH1及MPSS分析表明,AtFLA3主要在花序中表达。2、定量PCR的结果表明,AtFLA3在花器官中特异表达,且该基因的表达明显受到光的调控。利用PLACE等一些网上启动子顺式元件的预测工具对AtFLA3的5’上游启动子序列进行预测,发现该启动子中含有许多真核生物顺式元件的同源序列,如与光应答、激素应答及非生物胁迫应答等相关的元件。特别是其中含有晚期花粉基因LAT52和G10的"POLLEN1LELAT52"及"GTGANTG10"的顺式调控元件,这些元件特异地在成熟花粉中起作用。我们构建了AtFLA3启动子与GUS基因融合表达载体,通过农杆菌介导的浸花法转化拟南芥植株,抗性筛选和PCR验证获得纯合的启动子与GUS融合的转基因株系。GUS染色结果显示,在AtFLA3启动子的驱动下,GUS在花药、成熟花粉及花粉管中特异表达,并且主要从第10时期的花（花期10）开始一直到第13时期的花（花期13）。花期13的柱头和引导组织中染色信号较强,但这些信号均来自于萌发的花粉管。GUS染色后的花器官半薄切片结果进一步证实AtFLA3在花粉中特异表达。3、利用基因工程手段构建eGFP （enhanced green fluorescence）报告基因分别与AtFLA3全长ORF（开放阅读框）序列和缺失C-端GPI加锚信号序列的两种融合表达载体。农杆菌介导的浸花法将eGFPm-AtFLA3和eGFPm-AtFLA3△GPI转化拟南芥。利用激光共聚焦显微技术观察这两种融合蛋白在纯合转基因幼苗胚轴细胞的亚细胞定位情况,结果表明AtFLA3定位在细胞质膜上,而且C-端GPI加锚信号序列可能影响了该蛋白在细胞质膜上的正确定位。4、对AtFLA3的T-DNA插入突变体SALK₀16582的分析表明,该突变体T-DNA的插入位于该基因的3’非编码区。通过三引物基因组PCR法鉴定纯合的SALK₀16582突变体。RNA水平鉴定表明,该突变体的T-DNA插入对编码区的表达并无影响。SALK₀16582纯合突变体与野生型植株相比,其表型无明显差异。5、利用RNAi与过量表达技术研究了AtFLA3的生物学功能。分别构建了AtFLA3的RNAi载体和过量表达载体,通过农杆菌介导的浸花法转化拟南芥植株。RNAi和过表达转基因株系与野生型植株的比较观察,发现AtFLA3的沉默和过表达均能导致角果败育。进一步的研究表明,AtFLA3的沉默导致成熟花粉中出现许多体积小且畸形花粉。FDA （fluorescin diacetate）及12-KI染色结果表明,这些异常的花粉无生活力,而且RNAi株系与野生型植株的互交实验证实,在该基因沉默株系中花粉异常是导致角果败育的主要原因。花药半薄及超薄切片的观察发现,RNAi异常花粉内壁具有缺陷,且该缺陷从单核小孢子到二核花粉过渡时期便已产生。花粉的CW （calcofluor white）染色结果表明,RNAi异常花粉中纤维素物质沉积异常,这可能引起了RNAi异常花粉内壁的缺陷,并最终导致花粉败育。由此可见,AtFLA3在花粉发育过程中起重要作用,特别是参与花粉内壁的形成。然而,在过表达植株中,成熟花粉的形态、生活力及萌发效率均正常,但花丝变短、雌蕊组织的缺陷可能是导致过表达植株败育的主要原因。更多还原

【Abstract】 Arabinogalactan-proteins （AGPs） are glycoproteins that widely distributed in plant kingdom. And AGPs have been implicated in a variety of plant development processes including sexual plant reproduction. According to AGPs structure characteristics, there are existing approximately 85 AGPs genes in Arabidopsis annotated genome. In present paper, bioinformatical analysis, semi quantitative and quantitative PCR, RNA interference, overexpression, promoter fusion, subcellular localization and floral-dip transformation of Arabidopsis combine with other molecular and cytological methods were employed to investigate the biological functions of AtFLA3 in Arabidopsis. Main results were described as follows:1. The result of bioinformatic analysis indicated that AtFLA3 has an 843-bp-long open reading frame encoding 280 amino acids. And the most abundant residues of the deduced protein are Pro （10.71%）, Ala （10.71%）, Ser （10.36%） and Thr （6.79%）. And it has four distinct regions including:an N-terminal signal sequence for secretion, a central hydrooxyproline/proline rich AGP domain, a fasciclin-like domain and a C-terminal hydrophobic sequence considered as a GPI addition signal. The position between 24 and 25 amino acids has the max cleavage site probability for N-Signal peptide cleavage. The potential C-terminal GPI-Modification site is the 256 amino acid "S". The results of Arabidopsis Affymetrix GeneChip ATH1 and MPSS indicated AtFLA3 mainly expressed in inflorescence.2. The results of quantitative PCR showed that AtFLA3 is specifically expressed in floral organs and regulated by light. Some web tools for prediction cis-acting regulatory DNA elements were employed to analysis the CRE sequence in the promoter region of AtFLA3. The promoter of AtFLA3 contains many CREs homolog to eukaryotes such as:light responsive elements, hormone responsive elements, abiotic stress responsive elements and so on. Especially, there are existing three "POLLEN1LELAT52" and one "GTGANTG10" elements which were discovered in late pollen genes LAt52 and G1O respectively. The pAtFLA3::GUS fusion vector was constructed and transformed into Arabidopsis via floral-dip method. And then the homozygous pAtFLA3::GUS transgenic lines were obtained after resistant selection and PCR confirmation. GUS staining results indicated that AtFLA3 specifically expressed in anthers, mature pollens and pollen tubes, while no signal can be detected in other parts of the flowers. And this specific expression started at floral stage 10 and reached maximum at flower stage 13. At floral stage 13, GUS signal was seen in the stigma and transmitting tract and these singals might come from pollen tubes rather than female organs. Semi-thin section of the GUS stained floral organs further indicated AtFLA3 specifically expressed in pollen grains.3. We constructed AtFLA3 and eGFP reporter gene fusion vector eGFPm-AtFLA3 and eGFPm-AtFLA3AGPI respectively, then transformed into Arabidopsis via floral-dip method. Hypocotyl cells of the homozygous transgenic seedlings were used for study the subcellular localization pattern under confocal microscopy. The results indicated that AtFLA3 exhibits cell membrane localization, and GPI-anchoring signal sequence may affect its localization.4. Analysis of SALK₀16582 mutant showed that the T-DNA region insert into the 3’untranslated region of AtFLA3. Homozygous mutants of SALK₀16582 were testified by PCR using genomic DNA as template. Further investigation indicated that no reduction of AtFLA3 expression can be detected in homozygous lines. Meanwhile, no obvious differences can be observed compared with wild-type plants.5. RNA interference and overexpression methods were utilized to reveal the biological function of AtFLA3 in Arabidopsis. So we constructed AtFLA3 RNAi and overexpression vectors respectively, and then transformed into Arabidopsis. Phenotypical observation showed that both RNAi and overexpression plants were sterility. The RNAi transgenic lines had many small, shrunken, wrinkled pollen grains. Malformed pollen grains of RNAi plants showed no viability seen by FDA and I2-KI staining. The results of genetic crosses further confirmed the pollen defection was the only cause for sterility in RNAi plants. Further observations indicated that the intine layer of aborted pollens in RNAi plants is not well defined, and this abnormality occurs during the transition from uninucleate stage microspore to bicellular stage pollen. Calcofluor white staining showed that abnormal cellulose distribution in aborted pollen, which may incur the defection in pollen intine layer and finally lead to pollen sterility in RNAi plants. To sum up, AtFLA3 plays important role in microspore development, especially in pollen intine formation.However, the OE pollens were as normal as wild-type pollens. The defection of stamen filaments elongation and female organs were the two main reasons for sterility in OE plants.更多还原