摘要
小麦是世界上重要的粮食作物之一,为全球35%以上的人口提供每日所需的蛋白质和热量,是我国的第二大粮食作物。分离和分析小麦花药/花粉的特异性表达基因和启动子有助于深入了解小麦有性生殖过程中的基因表达调控,亦可用于雄性不育系的创建,避免水平基因转移带来的生物安全问题,具有重要的理论意义和实践应用价值。但小麦花药/花粉发育的研究却远远滞后于其他作物,如水稻、玉米等。除了少数几例小麦花药/花粉特异性基因的研究报道外,至今还未见有关小麦花药/花粉特异性启动子的相关报道。本论文对小麦花粉特异性基因TaPSG719的启动子进行了分离分析工作,具体研究结果如下:
(1)根据文献中已发表的TaPSG719基因的mRNA序列,获得了该基因1024 bp的DNA序列,经比对后发现该基因编码区域无内含子,且碱基序列完全相同。在获得TaPSG719基因部分DNA序列的基础上,采用反向PCR技术,获得了该基因起始密码子上游1,776 bp的启动子序列。用PLACE和PlantCARE数据库对分离的序列进行元件分析后发现,该序列除含有TATA-box、CAAT-box等启动子具有的一般元件外,还含有与花粉特异性表达相关的元件:AGAAA元件(10个)和GTGA元件(8个),推测其可能是花粉特异性启动子。
(2)为了验证TaPSG719启动子的调控特性,构建了含1.0 kb启动子序列的p1.0GUS载体,以及含1.7 kb该启动子序列的p1.7GFP和p1.7GUS载体。通过基因枪转化技术,将p1.7GFP载体转化小麦Bobwhite幼胚,获得了转基因植株。经GFP荧光检测,证明TaPSG719启动子可驱动GFP基因在小麦的花粉中表达。采用农杆菌转化技术,将p1.7GUS和p1.0GUS载体转化烟草,获得了转基因烟草植株。经GUS组织化学检测和GUS荧光定量分析,证实TaPSG719启动子不具有TaPSG719基因的种属特异性,其在小麦种属外的烟草中也有组织特异性启动活性,可驱动基因在花粉发育中期开始表达直至花粉发育成熟。而且1.0 kb的启动子序列就可以驱动基因的花粉特异性表达。
本文分离的小麦TaPSG719花粉特异性启动子是首次报道,该启动子的研究将有助于了解TaPSG719基因的功能,为深入分析小麦生殖发育的分子机制提供帮助,而且分离的小麦花粉特异性启动子为构建雄性不育小麦奠定了基础,具有潜在的应用价值。
Wheat is the foremost staple food crop in the world which provides both calories and proteins to over 35%of the human population.Wheat is the second most important staple crops in China.Isolation and characterization of wheat anther/pollen-specific genes and their promoters are immensely important,and has potential applications in crops biotechnology and their improvement.It would not only help us to understand gene regulation in the sexual reproduction process,but also can be used to induce sterility in the experimental plants in breeding programs,to avoid any horizontal gene transfer.But the study of wheat anther/pollen development is lagging far behind other agricultural crops, such as rice and maize.At present,only several genes involved in wheat anther/pollon development were reported.But no information about anther/pollen-specific promoter from wheat is available in the scientific literature.With the aims to get a wheat pollen-specific promoter,the upstream regulatory region of TaPSG719 gene,a wheat pollen-specific gene,was isolated and analyzed in transgenic wheat and tobacco.The main results were as follows:
(1) Based on the mRNA sequence of TaPSG719 gene reported,a 1,024 bp DNA sequence was isolated from wheat Bobwhite.Alignment of the two sequences showed that they were identical and no intron was found in the coding region of TaPSG719 gene. Based on the DNA sequence of TaPSG719 gene,a 1,776 bp promoter sequence upstream the translation start site was isolated by inverse-PCR technique.Putative functional promoter elements were analyzed by the PLACE database and the PlantCARE database. The sequences contained the basic elements,such as TATA-box and CAAT-box.Two motifs AGAAA and GTGA were detected which are known to be involved in pollen/anther-specific expression.There were ten copies of AGAAA motifs and eight copies of GTGA motifs.Many copies of AGAAA and GTGA motifs suggested that this promoter may be a pollen/anther-specific promoter.
(2) To study the regulatory characteristics of TaPSG719 promoter,two vectors containing 1.7 kb promoter sequence and one vector containing 1.0 kb promoter sequence were constructed and named p1.7GFP,p1.7GUS and p1.0GUS respectively.Transgenic wheat plants harboring p1.7GFP were obtained by particle bombardment technique.GFP fluorescence was detected in pollens of transgenic wheat.It meant that TaPSG719 promoter could start GFP gene expression in wheat pollen.Vectors p1.7GUS and p1.0GUS were transformed to tobacco respectively by Agrobacterium-mediated method and 45 transgenic plants were obtained.Histochemical GUS staining and fluorescence quantitative analysis showed that TaPSG719 promoter was a pollen-specific promoter which can drive gene expression in the middle stage of pollen development till anther mature.Although TaPSG719 gene is species-specific as it has homology with only wheat and closely related species,its promoter still has the tissue-specific activity in tobacco. And the 1.0 kb fragment upstream the coding region of TaPSG719 gene was enough to drive GUS expression exclusively and specially in pollen.
TaPSG719 promoter was anther/pollen-specific promoter first isolated from wheat. So far further detailed study of this promoter would shed light on understanding the function of TaPSG719 gene and sexual reproduction in wheat.TaPSG719 promoter drives the gene expression only in pollen grains would be useful for producing male-sterile plants in which the pollen is normal during microspore development but aborts at the late stage of pollen development.Therefore,it has potential applications in agriculture,especially in male-sterility.
引文
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