【作者】 李晓燕；

【导师】 朱睦元； 边红武；

【作者基本信息】 浙江大学 ， 遗传学， 2012， 博士

【摘要】 MicroRNA (miRNA)是一类长度在20～24个核苷酸的非编码RNA,主要在转录后水平或者翻译水平特异性调控靶基因的表达。在模式植物拟南芥中已经发现了200多个miRNA,其中与开花相关的有三个,分别为miR156, miR172和miR159,这些miRNA对植物的成花转变起到重要的调控作用。目前,针对非模式植物,特别是花卉植物中miRNA调控花发育的研究很少。本文以观赏性花卉大岩桐为材料,研究miR159和miR172对成花途径中关键靶基因的调控作用及其机理分析,探索通过操纵miRNA表达改变花卉植物花发育和花期的基因工程分子育种新策略。获得主要试验结果如下：1miR159表达对大岩桐花发育的影响GAMYB基因全序列的克隆及miR159剪切位点的验证根据GAMYB家族的序列保守性,利用5’RACE和3’RACE的方法克隆得到了大岩桐GAMYB cDNA全序列。利用RLM5’RACE方法,鉴定出SsGAMYB基因的964-985bp区域的第11位碱基是miR159的直接剪切位点,证实该基因是miR159的靶基因。在野生型大岩桐中,miR159在幼苗时期的叶片以及萼片中高表达,在雄蕊中的表达量最低；而GAMYB在雄蕊中的表达最高,而在萼片中的表达量最低。GAMYB与miR159的不同表达模式说明miR159负调控大岩桐GAMYB的表达。利用农杆菌介导法获得miR159转基因大岩桐株系本研究采用拟南芥miR159a前体的茎环序列构建组成型过表达载体35S:miR159a,同时构建抑制miR159作用的MIM159表达载体35S:MIM159.运用农杆菌介导的转基因方法,获得32株miR159a过表达株系,28株35S:MIM159转基因株系,并用RT-PCR进行了转录水平的鉴定。miR159转基因株系的表型观察及其表达检测在短日照条件下,大岩桐35S:miR159a过表达株系花期延迟37(±4.93)天,而35S:MIM159转基因株系花期提前20(±3.5)天。定量RT-PCR检测发现35S:miR159a过表达株系中miR159成熟体序列大量积累,GAMYB基因的mRNA水平降低；而在35S:MIM159株系中,miR159a的积累明显下降,GAMYB基因的mRNA水平上升。说明改变miR159的表达水平可以调控靶基因GAMYB的表达,从而提前或延迟大岩桐的开花时间。利用qRT-PCR的方法检测转基因株系中GAMYB下游基因的表达变化qRT-PCR结果显示,在35S:miR159a过表达株系的花原基中LFY基因的表达量显著下降,而在35S:MIM159株系中LFY基因的表达量显著上调.MADS-box基因(SsAG, SsAP,和SsAP3)表达水平在miR159过表达植株中上升,而在miR159抑制植株中下降。表明miR159介导的GAMYB表达调控了成花关键基因LFY的表达水平,并进一步调控下游MADS-box基因的表达。说明35S:miR159a和35S:MIM159转基因株系花期的变化很大程度上与LFY基因表达水平相关。LFY基因异常表达引起大岩桐花器官形态的变化35S:miR159a过表达株系的花形态与野生型相似,而35S:MIM159转基因株系中,高表达LFY基因的株系出现了萼片和花瓣的相互转化,即萼片花瓣化以及花瓣萼片化。qRT-PCR试验结果表明在花瓣化的萼片中SsAPs表达量明显上升；而萼片化的花瓣中SsAP,表达量显著上升,SsAP3表达量明显下降。表明萼片和花瓣的相互转化可能与第一轮和第二轮花器官中SsAP,和SsAP,异常表达相关。2miR172表达对大岩桐花发育的影响大岩桐AP2基因部分cDNA片段的克隆根据GenBank上已登录的拟南芥、番茄和葡萄的AP2类基因的同源序列设计兼并引物,利用野生型大岩桐花蕾的cDNA做模版,克隆得到SsAP2的部分片段。经核酸序列比对发现SsAP2与拟南芥,番茄以及葡萄的AP2基因具有高度同源性。miR172转基因大岩桐的获得利用农杆菌介导法成功转化大岩桐的叶片,经过4轮潮霉素抗性筛选以及基因组DNA-PCR鉴定,成功获得了35S:miR172a过表达株系24株以及抑制miR172作用的35S:MI MIM72株系21株。miR172转基因大岩桐的表型观察在长日照条件下,35S:miR172a过表达株系花期比野生型提前47(±2.16)天；35S:MIM172株系花期延迟7(士4.28)天,而且35S:MIM172株系,植株相对矮小。在35S:miR172a过表达株系中miR172的表达水平明显上升,其靶基因SsAP2的表达量明显下降；35S:MIM172株系中raiR172的积累水平受到抑制,而SsAP2的表达量明显上升,表明miR172介导调控SsAP2的表达对大岩桐成花转变具有促进作用。miR172转基因大岩桐的形态学变化在大岩桐35S:miR172a过表达株系中出现萼片花瓣化；而在35S:MIM172株系中出现花瓣比野生型卷曲。这些花器官异常的株系中,AP2的表达量均发生明显变化,表明在大岩桐中SsAP2类基因的表达异常影响花器官的正常发育。综上所述,本研究结果表明在短日照条件下,通过转基因改变miR159的表达可以有效的调控靶基因GAMYB以及下游成花关键基因LFY的表达水平,进而显著提前或延迟了大岩桐的开花时间；而在长日照条件下,通过改变miR172的表达可以调控其靶基因AP2mRNA水平,也能明显提前或延迟大岩桐的花期。说明调控开花相关miRNA的表达水平可以改变大岩桐的花发育进程。基于植物miRNA序列的高度保守性,通过转基因的方法调控开花相关miRNA表达将为观赏性花卉的花发育和花期控制提供一条分子遗传育种的新途径。更多还原

【Abstract】 MicroRNAs are endogenous20～24nt noncoding RNAs that regulate their target genes via post-transcriptional repression or complementary sequences degradation. In Arabidopsis, about200miRNA have been identified. Among numerous miRNAs, only three families are involved in flowering time control:miR156, miR172and miR159, which play crucial roles in flowering pathway. Current knowledge about miRNA regulation of flowering time control in non-model plant, especially in ornament plants is still largely unknown. The aim of this study is to investigate whether genetic modification of miR159and miR172expression can offer an effective approach for regulation of flowering characteristics in gloxinia.1Function of miR159in flower developmentIdentification and sequence analysis of SsGAMYB. To obtain the full length of GAMYB in gloxinia, the fragments with conserved MYB domain were cloned by rapid amplification of cDNA ends (RACE). A putative complementary site of miR159a existed in the region (964～985bp) and the cleavage site was detect by RNA ligase-mediated5’rapid amplification (RLM5’-RACE). In gloxinia, mature miR159a highly accumulated in young leaves and sepals, and lowly expressed in stamens. SsGAMYB was highly expressed in stamens and carpals, while its expression level was almost undetectable in sepals. Thus, the different expression patterns revealed that miR159negatively regulated SsGAMYB in gloxinia.Transgenic gloxinia was generated using Agrobacterium-mediated transformation. We used the AtmiR159a precursor to overexpress miR159a based on the conserved function in plants. In addition, we carried out target mimicry to repress the activity of the endogenous miR159a. The transgenic plantlets were detected by reverse transcription polymerase chain reaction (RT-PCR) with gene-specific primers. Distinct bands of expected size were obtained from thirty-two plantlets of AtmiR159a overexpressing35S:miR159a transgenic lines and twenty-eight transgenic plants of MIM159overexpressing35S:MIM159lines. Altered flowering time in transgenic gloxinia. Compared with wild-type gloxinia, under short-day conditions, overexpression of miR159delayed the timing of flowering to37(±4.93) days whereas suppression of miR159accelerated flowering to20(±3.5) days. Mature miR159a level was significantly increased in35S:miR159a lines and decreased in35S:MIM159lines. While a low level of SsGAMYB was observed in35S:miR159a lines, whereas the SsGAMYB level was significantly elevated in35S:MIM159lines. These results revealed that the alteration of miR159can regulate the mRNA level of SsGAMYB and effect flowering time in transgenic lines.Expression of flowering genes downstream SsGAMYB in transgenic gloxinia. Quantitative RT-PCR analysis of flower buds showed that overexpression of miR159resulted in a significant decline in SsLFY transcript level in35S:miR159a lines, while suppression of miR159caused an increase in SsLFY in35S:MIM159lines under SD conditions. Meanwhile, the mRNA levels of SsAG, SsAP1and SsAP3were decreased dramatically in35S:miR159a lines but increased in35S:MIM159lines. These results revealed that alteration of miR159in gloxinia can affect the expression level of LFY and regulate the flowering time.Effects of altered LFY expression on floral organ development. Phenotype observation indicated that the floral morphology of35S:miR159a appeared relatively normal compared to the wild-type. Nonetheless, abnormal morphology appeared in a few of35S:MIM159lines (3of28lines). In the flower bud stage, sepals were converted partly into petals in the35S:MIM159lines, disturbing the normal development of sepals. Quantitative RT-PCR analysis showed that AP3expression level was significantly higher in abnormal sepals, while API transcript level was notablely higher and AP3was drastically lower in abnormal petals compared to wild-type flowers. It revealed that the conversion of petals and sepals might be caused by disturbed expression levels of API and AP3between the first and second whorls.2Function of miR172in flower developmentIdentification partial cDNA of SsAP2. Based on the conserved domain of AP2, degenerate primers are designed according to homologous genes in Arabidopsis, tomato and grape to amplify partial cDNA from gloxinia. Phylogenetic analysis showed that the polypeptide of SsAP2shared significant similarity with those of Arabidopsis, tomato and grape.Generation of transgenic gloxinia lines that overexpress or suppress miR172a. Transgenic gloxinia was generated using Agrobacterium-mediated transformation. After four fortnights of in vivo culture, twenty-four plantlets of35S:miR172a transgenic lines and twenty-one transgenic plants of35S:MIM172were obtained.Phenotype of transgenic gloxinias. Compared with wild-type gloxinia, under long-day conditions, overexpression of miR172a accelerated flowering to47(±2.16) days whereas suppression of miR172delayed the timing of flowering to7(±4.28) days. Mature miR172level was significantly increased and SsAP2was dramatically lower in35S:miR172a lines. While a low level of mature miR172and an elevated level of SsAP2were observed in35S.MIM172lines. These results revealed that miR172mediated cleavage of SsAP2can effect flowering time in transgenic lines.Morphological changes in transgenic gloxinias. Nonetheless, abnormal morphology appeared in a few of35S:miR172a lines (2of241ines). In the flower bud stage, sepals were converted partly into petals in the35S:miR172a lines and petals were rolled downward in35S:MIM172lines with a reduced plant height. It suggested that alteration of SsAP2activity disrupted the formation of floral organs.Our results suggested that regulation of GAMYB and flowering genes downstream by miR159is a functionally effective pathway in triggering flowering under short days. While manipulating expression level of miR172completely affected the expression of SsAP2and controlled the flowering time. Thus, manipulation of miRNAs involved in flowering time provides an applicable approach for commercial ornamental plants breeding.更多还原