【作者】 马金霞；

【导师】 敖光明；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 高等植物雄性生殖过程发生在雄蕊内。花粉作为雄配子体,包含了与雌配子体结合完成受精过程所需要的全部遗传信息,在植物的整个生殖周期中发挥着重要作用。发育过程中发生的一系列事件是有时空顺序性的,而且特化细胞类型及其功能的建立及保持需要特定的几组基因的转录活化。植物雄蕊中花药内雄配子体发育的起始与成熟是由一系列基因所控制,在花粉母细胞减数分裂和形成小孢子期与后期形成雄配子过程中均有一系列基因调节。正是由于花粉的这一重要作用,使其成为生物化学和分子生物学领域研究基本规律性问题的首选对象。利用分子生物学手段对植物花粉发育过程中特异表达的基因的功能进行研究,对于我们从分子水平了解植物花粉的发育过程具有重要的意义。 本研究小组通过差异筛选法,并结合冷噬菌斑筛选,从玉米成熟花粉cDNA文库中克隆到一个玉米花粉特异表达的cDNA,采用5’RACE,3’RACE技术及重叠PCR获得了zm401cDNA的全长（1149bp）。Northern杂交表明zm401是一个玉米花粉特异表达的基因。采用生物学软件对zm401cDNA的全长序列和结构进行分析,发现zm401符合非编码RNA基因的特点,属于类似于mRNA的非编码基因这一类。为了进一步验证全长zm401能否编码小肽,本文对其全长cDNA进行了体外翻译。虽然没有翻译出小肽,但是还不能肯定地说全长zm401不编码。全长zm401究竟是以RNA还是小肽的形式起作用,还在进一步的研究中。 本文将全长zm401的抑制表达载体和ZM13启动子驱动下的超表达载体通过基因枪法导入玉米愈伤,经分子检测共得到16株Southern阳性转基因玉米,其中10株为转入抑制表达表达载体的转基因玉米,6株为转入超表达载体的转基因玉米。通过对转基因玉米雄穗表型变化进行分析发现,插入抑制表达载体的转基因玉米的雄穗外稃不张开,花粉不能散出;转超表达载体转基因玉米,雄穗顶端的小穗发育不良,不能长成正常大小,干瘪、枯死。对转基因玉米花药的组织切片显微结构进行观察,发现转抑制表达载体和ZM13启动子驱动的转超表达载体的转基因玉米小孢子空瘪、变形,无内容物,绒毡层与非转基因玉米相比出现退化延迟现象,花粉形态异常。通过对转基因玉米花药形态的观察及花粉活力的测定,表明全长zm401的转基因玉米花药出现退化或皱缩现象,花粉经I₂-KI染色后,明显可见淀粉积累减少,花粉形态异常且大小不一。在T1代转基因玉米中,同样可见花药退化或皱缩现象,花粉黑染率较低。从而证明全长zm401基因在小孢子和花药壁发育中具有重要的作用。为了进一步研究全长zm401的功能,我们将其全长cDNA转入烟草。全长zm401在烟草中异位表达时转基因烟草也表现出发育延迟,绒毡层退化延迟,小孢子减数分裂不同步的现象,表明全长zm401的确在小孢子和绒毡层发育中有重要作用。更多还原

【Abstract】 Male reproductive processes in flowering plants take place in the stamen. The male gametophyte (pollen grain) of flowering plants is the producer and the carrier of gametes. In a single unit each grain contains all the genetic information required to unite with the female gamete at fertilization and to form a new sporophyte. The developmental events leading to anther formation and pollen release are exquisitely timed and choreographed. Transcriptional activation of specific gene sets is required for the reestablishment and maintenance of differentiated cell types and functions during anther development. The begaining and mature of male gametophyte of flowering plants are controlled by a series of genes in the male stamen. Genes regular meiosis of pollen mother cells, microspore forming stage and the forming process of the male gametophyte. Because of crucial function in the plant’s reproductive cycle, pollen is the object of biochemical and molecular biological research. Identifying genes that are responsible for pollen development is very important.In our previous study, a cDNA library to poly (A) RNA isolated from mature pollen of zea mays was constructed. Differential screening and Cold-plaque screening obtained one cDNA fragment; designated zm401 (Zea mays), full length of zm401 cDNA was generated by 5’ and 3’ RACE method. zm401 cDNA was 1149 bp in length. zm401 was specifically or preferentially expressed in mature pollen. The analysis results by biologic software suggest that zm401 was one of a growing number of non-coding mRNA-like RNA transcripts that exert their cellular functions directly as RNA. We performed in vitro translation to further study if zm401 could be translated in vitro. The result that zm401 cDNA could not be translated confirmed the conclusion that zm401 could be a member of a new category of non-coding RNA. But we could not know if zm401 can be translated in vivo, or execute its function by RNA form. The exact functions of zm401 remain to be elucidated, and further analysis of zm401 may provide a way to elucidate the mechanism of anther development.To gain more information about the function of zm401 gene, plant expression vectors with zm401 RNAi construct and sense zm401gene promoted by ZM13 promoter were constructed. Maize embryogenic callus were transformed by microprojectile bombardment with these plant expression vectors. After selecting for hygromycin resistance, 10 plants with zm401 RNAi construct and 6 plants with sense zm401 respectively were obtained by genomic PCR, southern blotting analysis. The aberrant phenotypes were observed in the anther of the transgenic plants with zm401 RNAi construct and sense zm401 gene. The tassels of transgenic plants with zm401 RNAi construct could not open when the tassels of control plants did, and the top tassels of transgenic plants with sense zm401gene were dysplasia, shriveled and withered. Histological differences were observable for transgenic plants. Comparing the non-transformant with transgenic plant, microspores in the non-transforming were rounded normally; the shape of most of the pollen grains of transgenic plants became distorted. The degradation of the tapetum was delayed for transgenic plants. Most of the pollen grains in the transgenic plants were distored and were not stained with the solution of I2-KI, indicating the absence ofaccumulated starch. In contrast, most of the pollen grains in the non-transformant were rounded and were deeply stained by solution of I2-KI. To further study the function of zm401, we transformed it into tobacco. All transgenic tobacco plants expressing zm401 showed various levels of sterility, ranging from abortive flower development to male sterility. Further analyses on anther development of transgenic plants indicated multiple abnormalities of anther development at late stages. These abnormalities include lagged degradation of the tapetum and connective tissue, failed deposition of fibrous bands in endothecium cells, and aborted pollen grain development. All these results strongly suggest that zm401 has更多还原