【作者】 王舟；

【导师】 刘建秀；

【作者基本信息】 南京农业大学 ， 植物学， 2010， 博士

【摘要】 结缕草(Zoysia japonica)是目前正在国内外广泛应用的主要暖季型草坪草之一,具有弹性好、耐践踏、养护投入低等优良坪用特性,是绿化、足球场草坪和高尔夫球场草坪的主要建群种。由于我国结缕草相关研究工作起步较晚,其功能基因的发掘和分子育种有待进一步深入研究。为了揭示草坪草抗逆分子机制,获得具有自主知识产权的优良抗逆基因,本文在筛选抗寒性强的结缕草品种的基础上,克隆其抗寒相关转录因子ZjDREB1基因,并对其表达模式和功能进行了分析。主要研究内容和结果如下：1.以美国引进的并在国内外草坪生产上普遍应用的结缕草三个优良品种——Meyer、Palisades和兰引3号为试验材料,通过叶片电解质外渗法,利用半致死温度(LT50)为评价指标,鉴定各品种的抗寒性。结果表明,三种结缕草品种的半致死温度(LT50)分别为Meyer的-10.3℃、Palisades的-8.9℃和兰引3号的-7.2℃。LT50值由低到高,推断出抗寒性由强到弱依次为Meyer> Palisades>兰引3号。2.根据结缕草近缘植物DREB转录因子的AP2/EREBP保守结构域序列,通过RT-PCR和RACE的方法从冷诱导的抗寒结缕草品种Meyer中扩增到了一个新的DREB同源基因,命名为ZjDREB1 (GenBank登录号：GQ848096)。该基因的开放阅读框(ORF)为774 bp,编码257个氨基酸,分子量和等电点(pI)分别为28.85 kDa和5.53。序列分析表明,ZjDREB1推测的蛋白具备DREB类转录因子的三个典型特征:AP2/EREBP保守DNA结合域、碱性核定位信号(NLS)和酸性激活区。根据氨基酸序列相似性构建的系统进化树显示,结缕草ZjDREB1与狗牙根BeDREB1、BeDREB2同源性最高,达84%,属于DREB基因家族的A-2亚群。进一步分离克隆了ZjDREB1的基因组DNA序列(GenBank登录号为GQ864011),分析发现其由2个外显子和1个内含子组成。这是首个从结缕草中分离得到的DREB类转录因子基因。3.为在研究ZjDREB1基因的表达模式时提供分子内参,采用RT-PCR和RACE技术扩增出1560 bp的结缕草肌动蛋白基因全长cDNA序列。序列分析表明,该基因的开放阅读框(ORF)为1134 bp,编码377个氨基酸,5,非编码区117 bp,3,非编码区309 bp,分子量和等电点(pI)分别为41.72 kDa和5.23。所得序列与GenBank中收录的其它植物肌动蛋白核苷酸序列的一致性均在85%以上,氨基酸序列的一致性高达97%以上。将其命名为ZjACT, GenBank登录号为GU290545。根据高等植物肌动蛋白相似性构建的系统进化树显示,结缕草肌动蛋白ZjACT与大麦Hvactin和圆锥小麦TtACT-1肌动蛋白之间的亲缘关系最为密切,在进化中分化时间最为接近。进一步分离克隆了结缕草Actin基因的基因组DNA序列(GenBank登录号GU290546),分析发现其由4个外显子和3个内含子组成。本研究有助于揭示植物Actin基因家族的进化历史,为研究植物Actin基因家族功能和进化上的多样性奠定理论基础,同时也为开展草坪草和牧草Actin基因的功能分析和利用研究提供参考。4.以克隆得到的结缕草肌动蛋白基因ZjACT为内参,利用实时定量PCR方法分析了三个结缕草品种ZjDREB1基因在冷胁迫处理下的表达差异。结果表明,室温下ZjDREB1基本没有表达。4℃处理1 h后,该基因开始被诱导,并在持续的冷胁迫下表达量快速增加,处理6 h后,表达量达峰值。抗寒性强的品种ZjDREB1基因的表达量始终高于抗寒性弱的品种。随着温度的降低, ZjDREB1的表达水平也随之增高,0℃时的表达量要大于8℃的。进一步的序列结构分析表明,三个结缕草品种ZjDREB1基因的cDNA序列、基因组DNA序列和氨基酸序列都存在差异,推测是导致品种间表达模式显著变化的可能原因之一。对不同品种间基因表达差异与冷耐受性的相关性研究,为揭示结缕草抗寒分子机制提供了依据。5.以已克隆得到的结缕草ZjDREB1全长cDNA为模板,用引入了BamHⅠ和HindⅢ酶切位点的引物,通过PCR方法获得该基因的编码框全长,并将其构建到原核表达载体pET-30a(+)上。重组载体pET-ZjDREB1转化大肠杆菌BL21(DE3),获得含ZjDREB1基因的重组工程菌,进而分析它在冷胁迫下可能具有的功能。经IPTG诱导表达,SDS-PAGE检测获得了分子量约为36 kDa的融合蛋白,大小与预期一致。在大肠杆菌的抗寒性试验中,与对照菌相比,表达ZjDREB1融合蛋白的重组菌在低温条件下表现出显著提高的细胞活力,提示ZjDREB1蛋白可能具有提高大肠杆菌(很可能还包括其他生物)对低温的抗性。由此推断,ZjDREB1是潜在的可用于改良植物对环境胁迫耐受性的候选基因。6.将结缕草ZjDREB1基因导入植物表达载体pCAMBIA1301中,构建了重组表达载体pCAM-ZjDREB1,转入农杆菌LBA4404,并通过浸花法转化模式植物拟南芥。T0代植株经潮霉素抗性筛选得到10株抗性苗。利用PCR和半定量RT-PCR技术再对抗性苗进行分子鉴定,最终获得5株阳性植株。结果表明,结缕草ZjDREB1基因已整合到拟南芥基因组中,并在转录水平上表达。以电解质渗漏法检测了植株的抗寒性,结果显示过表达ZjDREB1基因的拟南芥转基因植株(LT50=-8.6℃)的抗寒能力较野生型对照植株(LT50=-5.5℃)有明显的提高。这些结果说明ZjDREB1基因具有在草坪草和牧草抗逆基因工程改良中应用的潜力。7.为了改良重要的多年生暖季型草坪草假俭草(Eremochloa ophiuroides)的抗逆性,以茎段侧芽来源的胚性愈伤组织为受体材料,通过农杆菌介导法将结缕草耐逆相关转录因子ZjDREB1基因导入假俭草优良种质E126中。分别就转化体系中适宜的筛选剂浓度、抑菌素浓度、菌液浓度、侵染时间以及共培养时间等对转化效率的影响进行了研究,建立了高效的假俭草遗传转化和再生体系。各种影响转化效率因素的优化试验表明,愈伤筛选和再生苗筛选的最佳潮霉素浓度均为30 mg/L,头孢霉素作为抑菌素的最佳浓度为400 mg/L,转化时菌液浓度OD600为0.1-0.3、侵染30 min、共培养3 d为最优转化条件,在侵染和共培养期间加入100μM乙酰丁香酮可提高转化频率。经PCR、半定量RI-PCR和叶片的潮霉素抗性检测,获得了5株阳性转基因植株。抗寒性鉴定表明,过表达ZjDREB1基因的假俭草转基因植株的半致死温度(LT50=-4.4℃)显著低于野生型对照植株(LT5o=-1.9℃)。初步证明,ZjDREB1基因已整合到假俭草基因组中,可以作为后期转基因假俭草植株抗性研究及培育转基因假俭草新抗性品种的材料。8.为进一步研究结缕草胁迫响应信号转导途径中除DREB以外的其他关键基因(如COR、ICE、AREB/ABF、MYC/MYB、bZIP等),揭示结缕草抗逆分子机制,建立结缕草功能基因组学研究基础平台,构建了首个结缕草低温和干旱诱导的标准cDNA文库。以经低温、干旱处理的结缕草为材料,取其叶片提取总RNA并分离纯化mRNA,反转录合成双链cDNA后,采用Gateway(?)技术构建结缕草低温和干旱诱导的标准cDNA文库。原始文库滴度1.76×106pfu/ml,库容7.04×106pfu,重组率90%,插入片段平均长度大于1 kb。文库质量优良,可能包含大量新基因,不仅为结缕草基因组资源提供了材料,也为后期进行大规模EST测序、发掘新抗逆相关基因、制作基因芯片等研究奠定了基础。更多还原

【Abstract】 Zoysiagrass (Zoysia japonica) is one of the major warm-season grasses widely utilized both at home and abroad, with excellent attributes of a very dense turf by means of slow growing underground rhizomes and rapid developing above-ground stolons, well adapted for lawns and golf turfs in the transitional and warm climatic regions, besides requiring minimal maintenance inputs. As the studies regarding zoysiagrass just got off to a late start in China, novel functional gene discovery and molecular breeding remain to be further researched. In order to reveal the molecular mechanisms involved in stress tolerance of turfgrass and obtain favorable genes of proprietary intellectual property, we isolated the ZjDREBl gene encoding a DRE-binding transcription factor from a cold-tolerant zoysiagrass cultivar on the basis of freeze tolerance evaluation, and analyzed its expression pattern as well as function. The main results are given as follows:1. Low temperature is one of the primary limiting factors for distribution and widespread use of released zoysiagrass(Zoysia spp.) cultivars in the transition zones and temperate regions. Three cultivars of zoysiagrass used in this study (Meyer, Palisades and Lanyin 3) were all introduced from the United States, and in common use both at home and abroad. Low-temperature tolerance was evaluated by measuring leaf electrolyte leakage (EL). The temperature at which 50% of the plants were killed (LT50) was determined. Our results showed that the lethal temperature (LT50) of the three zoysiagrass accessions predicted by EL was respectively -10.3℃in Meyer,-8.9℃in Palisades and -7.2℃in Lanyin 3. According to LT50 values, it was concluded that the freeze tolerance of the three cultivars ranked as:Meyer> Palisades> Lanyin 3.2. Dehydration-responsive element binding (DREB) proteins, specifically interacting with the DRE cis-acting element, have been identified as a group of important transcription factors that regulate the expression of many stress-inducible genes in plants. In this work, a novel DREB ortholog, designated as ZjDREBl (GenBank accession no.:GQ848096), from the cold-tolerant zoysiagrass cultivar Meyer was isolated using RT-PCR and RACE methods. It contained an open reading frame (ORF) of 774 bp encoding 257 amino acid residues. The predicted molecular mass of the deduced protein was 28.85 kDa and theoretical isoelectric point (pI) was 5.53. The deduced protein sequence featured a conserved AP2/EREBP DNA-binding domain, and comprised of a nuclear localization signal (NLS) and an acidic activation region, showing the typical characteristics of the DREB gene family. The phylogenetic tree constructed on the basis of amino acid sequences suggested that ZjDREBl from zoysiagrass and BeDREBl,2 from bermuda grass shared the highest 84% identity, and ZjDREB1 was classified into the A-2 group of DREB proteins. A comparison of the cDNA and its genomic counterpart (GenBank GU290546) demonstrated that the gene consisted of 2 exons and 1 intron. To our knowledge, this is the first report on isolation of DREB-like genes in zoysiagrass.3. In order to be used as an internal control in subsequent expression analysis of ZjDREB1, a 1560-bp full-length cDNA of actin was cloned from the leaves of zoysiagrass using RT-PCR and RACE methods. The sequence analysis revealed that it contained an ORF of 1134 bp encoding a protein composed of 377 amino acids, a 5’-UTR of 157 bp and a 3’-UTR of 495 bp. The predicted molecular mass of the deduced protein was 41.72 kDa and theoretical isoelectric point (pI) was 5.23. Homologous alignment showed that it shared over 85% of nucleotide identities and 97% of amino acid identities with actins from other plants in GenBank. The full-length cDNA was designated as ZjACT with its accession number GU290545. The phylogenetic tree constructed on the basis of amino acid sequences suggested that the relationship of ZjACT from zoysiagrass was most intimate with Hvactin from barley and TtACT-1 from poulard wheat, and they might have the same differential time in evolution. A comparison of the cDNA and its genomic counterpart (GenBank accession no. GU290546) demonstrated that the gene consisted of 4 exons and 3 introns. These results provided clues for studies on evolution of Actin gene family, and laid the foundation of researches into its functional and evolutionary diversity, as well as served as references for functional analysis and utilization of Actin gene from turf and forage grasses.4. Using Actin gene ZjACT as the internal control, expression profiling of ZjDREB1 genes from three zoysiagrass cultivars in response to cold was quantitatively analyzed by Real Time PCR. The results showed that there was no expression of ZjDREBl in room temperature. Its transcripts started to rise after 1 h at 4℃, rapidly and strongly up-regulated by chilling treatment, then reached a maximum level after 6 h of exposure to low temperature. The expression level of ZjDREB1 in the cold-tolerant cultivar was higher than that of the cold-susceptible one. A decreasing positive temperature resulted in higher accumulation of ZjDREBl transcripts with a dramatic increase below 8℃and peaking at 0℃. Further sequence analysis on ZjDREB1 genes from the three cultivars showed differences among cDNA sequence, genomic DNA sequence and the deduced amino acid sequence, inferring one of the possible reasons why their expression patterns differed significantly. Studies on the correlation of the function of the differentially expressed genes and the cold tolerance in different cultivars may provide some new insights into the molecular mechanism of zoysiagrass in response to cold stress.5. Based on the full cDNA sequence, two primers were designed with the flanking restriction sites of BamHⅠand HindⅢ. Using the zoysiagrass cDNA isolated previously as template, the complete coding sequence for the ZjDREB1 ORF was PCR amplified, and inserted into the prokaryotic expression vector pET-30a(+). Recombinant pET-ZjDREB1 vector was then introduced into Escherichia coli BL21(DE3) host strain to analyze its possible function under cold stress. A fusion protein about 36 kDa was expressed in E.coli cells harboring pET-ZjDREB1 after the induction of IPTG by SDS-PAGE analysis. Compared with control cells, those recombinant ones expressing ZjDREB1 fusion protein showed significantly improved cell viability at low temperature, implying that the protein may play an important role in resistance of E. coli (and probably other organisms) to low temperatures. It is concluded that ZjDREB1 is suggested to be potentially useful for improving plant tolerance to environmental stresses.6. The ZjDREB1 gene isolated from zoysiagrass was inserted into the plant expression vector pCAMBIA1301 to construct the recombinant pCAM-ZjDREB1 plasmid. It was introduced into Agrobacterium tumefaciens LBA4404 host strain, and then transformed into wild type Arabidopsis thaliana through floral-dip method. A total of 10 resistant plants were obtained in T0 generation via hygromycin screening. Among them 5 positive seedlings were further verified by PCR and semi-quantitative RT-PCR analysis. The results indicated that exogenous ZjDREB1 gene had been integrated into the Arabidopsis genome and expressed at the transcription level. Low-temperature tolerance was evaluated by measuring leaf electrolyte leakage. Our results demonstrated that transgenic plants overexpressing ZjDREB1 gene (LT50=-8.6℃) had a significantly greater cold tolerance than that of wild-type control plants (LT50=-5.5℃). It implied a promising future of the applications of ZjDREB1 gene in genetic engineering for stress tolerance improvement of turf and forage grasses.7. In order to improve stress tolerance of centipedegrass(Eremochloa ophiuroides), an important perennial warm-season grass, Agrobacterium tumefaciens strain LBA4404 harboring the plasmid pCAMBIA1301 containing stress tolerance-related transcription factor ZjDREB1 gene from zoysiagrass was used to transform axillary bud-derived embryogenic calluses of the good selection ’E126’. So as to systematically optimize the conditions for centipedegrass transformation, several factors known to influence Agrobacterium-mediated DNA transfer were examined, including concentration of selective agent and bacteriostat, bacterial culture OD600, duration of infection and co-cultivation. Consequently, an efficient and reproducible transformation system for the production of transgenic centipedegrass plants was established. It was found that the efficiency of transformation highly relied on the following optimal conditions:30 mg/L hygromycin for selection,400 mg/L cefotaxime for bacteriostasis, bacterial culture OD600 of 0.1-0.3, infection for 30 min and co-cultivation for 3 d. Furthermore, the inclusion of 100 mM acetosyringone in both the infection and co-cultivation media led to an increase in transformation frequency. Thus, the hygromycin-selected plants were obtained and 5 putative transgenic plants were confirmed by PCR, semi-quantitative RT-PCR analysis and test of leaf-dip in hygromycin, preliminarily indicating the integration of exogenous ZjDREB1 gene into the genome of centipedegrass. The low-temperature tolerance assessment demonstrated that LT50 of transgenic plants overexpressing ZjDREB1 gene (-4.4℃) was significantly lower than that of wild-type control plants (-1.9℃). It suggested that the positive plants could be used for the subsequent studies on their stress tolerance and cultivation of new varieties of transgenic centipedegrass with improved resistance.8. For a better understanding of orther key genes from zoysiagrass in signal transduction pathways under stress conditions (e.g. COR, ICE, AREBIABF, MYC/MYB, bZIP), exploring molecular mechanism involved in stress response of zoysiagrass, and developing a functional genomics platform, the first cold- and drought-induced cDNA library of zoysiagrass was constructed. Plants of zoysiagrass were subjected to cold and drought stress respectively. Total RNA was extracted from their leaves. Then mRNA was isolated and purified, reverse transcribed into double-stranded cDNA. The directional cDNA library enriched for full-length sequences was constructed using Gateway(?) technology. The results indicated that the titer of the original library was 1.76×106 pfu/ml, the capacity was 7.04×106 pfu, and the average insert size was larger than 1 kb with the recombination efficiency of 90%. It is suggested that the cDNA library was successfully established in high quality, most likely containing lots of novel genes. Providing resources for zoysiagrass genomics, the library can be an effective tool for further studies on high-throughput EST sequencing, new stress-responsive genes screening, gene chips preparing etc.更多还原