【作者】 马红珍；

【导师】 郭天财； 康国章；

【作者基本信息】 河南农业大学 ， 作物栽培学与耕作学， 2013， 博士

【摘要】 本文采用cDNA末端快速扩增法（Rapid-amplification of cDNA ends, RACE），在小麦中首次克隆出BTF3基因（命名为TaBTF3）的序列全长，并采用VIGS（Virus-inducedgene silencing, VIGS）技术对其进行基因功能鉴定。主要研究结果如下：1.采用RACE-PCR技术从小麦中首次克隆到TaBTF3基因，其cDNA序列全长882bp，最大ORF序列为534bp，该基因编码一个由177个氨基酸组成的蛋白，预测分子量为20KD。基因组DNA序列全长1742bp，包含5个外显子、4个内含子，内含子和外显子的剪切位点严格符合GT/AC规律。实时定量PCR检测表明，TaBTF3在小麦根、叶、叶鞘、旗叶和幼穗中均有不同程度的表达，在旗叶中表达量最高，且受PEG、冷（4℃）、NaCl、MeJA、ABA和SA诱导上调表达。蛋白亚细胞定位结果表明，TaBTF3蛋白主要定位于细胞核与细胞质。Southern杂交结果显示，TaBTF3是多拷贝基因，以多拷贝的形式存在。2.选用大麦条纹花叶病毒（BSMV）载体，在豫麦34中成功沉默报告基因TaPDS（小麦八氢番茄红素脱氢酶基因），TaPDS-VIGS小麦植株叶片呈现明显的光漂白表型。GFP蛋白荧光检测结果表明，在BSMV-GFP植株的叶片与根系中均可以检测到较强的GFP荧光信号，BSMV诱导的基因沉默是系统性沉默，并不仅仅局限于接种叶片。高效稳定的小麦VIGS技术体系的成功建立，为小麦TaBTF3基因功能分析验证提供了技术平台。3.成功获得TaBTF3-VIGS植株与BSMV-GFP对照植株，并对BSMV-VIGS植株进行鉴定、生理指标检测。沉默TaBTF3基因导致叶绿素含量降低、MDA与H2O2含量显著升高。通过荧光定量PCR检测线粒体、叶绿体编码基因的表达量变化，结果表明，与BSMV-GFP对照植株相比，沉默TaBTF3基因导致线粒体、叶绿体编码基因的表达量显著下降。进一步采用石蜡切片技术观察TaBTF3-VIGS植株叶肉细胞的变化，结果显示，与对照相比，TaBTF3-VIGS植株的叶肉细胞发育异常、体积变小、排列松散、无序、胞间隙较大。由此表明，TaBTF3可能与线粒体、叶绿体和叶肉细胞的生长发育有关。4.通过荧光定量PCR检测小麦逆境相关基因的表达量，并对TaBTF3-VIGS植株与BSMV-GFP对照植株进行胁迫耐受性检测。结果表明，在正常生长条件下，沉默TaBTF3基因导致逆境相关基因的表达被抑制。逆境胁迫处理结果显示，TaBTF3-VIGS植株的相对电导率与失水率显著高于对照，而相对含水量与游离脯氨酸含量则明显降低。沉默TaBTF3降低了小麦植株对干旱和冻害胁迫的耐受性。这些结果表明，TaBTF3参与了植物逆境胁迫耐受性过程。更多还原

【Abstract】 In this paper, the full length cDNA sequences of wheat BTF3gene (named TaBTF3)was first cloned by using the RACE (Rapid-amplification of cDNA ends, RACE)method, and the VIGS (Virus-induced gene silencing, VIGS) method was used toidentify the functions of TaBTF3gene.The main results were as follows:1. The first TaBTF3gene was cloned from wheat by RACE-PCR method, thefull-length cDNA sequence of the TaBTF3gene was882bp with an open readingframe (ORF) of534bp, and the predicted TaBTF3protein has177amino acids with acalculated molecular mass of20KD. The genomic DNA sequence of TaBTF3was1742bp, containing five exons and four introns, with all splicing sites complying withthe GT-AC. The Real-time fluorescent quantitative PCR analysis showed that theexpression of TaBTF3was constitutive, and the higher level of the transcript wasfound in booting flag leaves, and the expression levels of TaBTF3increasedsignificantly under PEG, cold and NaCl stress conditions, or treated by ABA, MeJAand SA. The subcellular localization of TaBTF3protein was examined by usingpolyethylene glycol (PEG)-mediated Arabidopsis protoplasts transformation method,gene copy number of TaBTF3was tested by using southern blotting. Results showedthat TaBTF3is a multi-copy gene and might be predominantly localized in thenucleus and cytoplasm.2. The stable and efficient VIGS system with barley strip mosaic virus (BSMV)vector was established in our laboratory. We successfully silenced the wheat PDSgene (VIGS reporter gene-TaPDS) by using this BSMV derived vector，which resultsin significantly photobleaching phenotype. The strong GFP fluorescence was detectedin both leaves and roots of the BSMV-GFP wheat plants, thus, the spreading ofBSMV is systematic, not only limited to the inoculation sites. The established VIGSsystem will be beneficial to the gene function analysis of TaBTF3in wheat for thenext step.3. The TaBTF3-VIGS and BSMV-GFP transgenic wheat plants were successfullyobtained by using the VIGS method, the identification and physiological parameterstest of the BSMV-VIGS plants were carried out. Results showed that silencing ofTaBTF3leads to a significant decrease in the chlorophyll content, but increased inMDA and H2O2contents. The transcript levels of chloroplast-and mitochondrial-encoded genes were examined by using the real-time quantitative PCRmethod. To further investigate the structural changes of the wheat mesophyll cells ofTaBTF3-VIGS plants, paraffin slices were prepared and observed under lightmicroscopy. Compared with the BSMV-GFP control plants, the expression levels ofthe chloroplast-and mitochondrial-encoded genes were significantly inhibited, andthe significant differences in the mesophyll cell structure were also observed, the cellswere smaller in size, irregularly shaped, disorderly and loosely arranged, and hadenlarged intercellular spaces. These results indicated that TaBTF3may involve in thegrowth and development of chloroplast, mitochondria and mesophyll cell in wheat.4. The transcript accumulation of stress-related genes were examined by using thereal-time quantitative PCR method， and the abiotic stresses tolerance ofTaBTF3-VIGS plants and BSMV-GFP control plants were examined. Results showedthat the transcript accumulation of stress-related genes was significantly reducedunder normal growth conditions. TaBTF3-silenced wheat plants exhibited markedlyhigher relative electrical conductivity and water loss rate, lower content of the freeproline and relative water content under stress conditions compared to theBSMV-GFP plants. Silencing of TaBTF3results in impaired tolerance to freeze anddrought stresses, the results above implied that TaBTF3may involve in the process ofabiotic stresses tolerance.更多还原