【作者】 黄杏；

【导师】 杨丽涛；

【作者基本信息】 广西大学 ， 作物栽培学与耕作学， 2012， 博士

【摘要】 甘蔗是中国乃至世界第一大糖料作物,起源于热带及亚热带地区,属喜温作物。近年来,世界范围内极端天气频发,寒害、冻害增多,这给甘蔗生产造成了巨大的损失。在生产上,甘蔗防寒抗冻的措施主要有推广应用抗寒性好的品种、改善栽培管理措施及化学调控等。脱落酸(ABA)在植物的生长发育和抗逆胁迫生长中具有重要的作用。本研究采用土培和水培两种方式种植不同抗寒性的甘蔗品种GT28和YL6,在低温胁迫前12h用ABA进行处理,研究低温胁迫下ABA提高甘蔗抗寒性的生理生化机制,并利用荧光定量PCR技术、双向电泳和质谱分析技术,探讨ABA提高甘蔗抗寒性的分子机制。主要研究结果如下：1.低温胁迫下外源ABA对甘蔗幼苗生理生化特性的影响低温胁迫改变甘蔗幼苗叶片的生理生化特性。低温胁迫后,两个甘蔗品种幼苗叶片细胞膜透性增大；丙二醛(MDA)和脯氨酸含量增加；光合相关指标净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)降低,而胞间CO2(Gi)则表现相反,浓度升高；叶绿素荧光指标最大光能转化效率(Fv/Fm)、光化学猝灭系数(qP)和PSⅡ实际量子效率(ΦPSⅡ)均降低,而初始荧光(Fo)和非光化学猝灭系数(NPQ)则升高；内源激素赤霉素(GA)和玉米素(ZR)含量下降,ABA含量升高,导致ABA/GA、ABA/IAA和ABA/ZR比值增大。低温胁迫下甘蔗幼苗脯氨酸、ABA和GA含量及ABA/GA比值存在基因型差异,抗寒性强品种脯氨酸、ABA含量和ABA/GA比值较高,GA含量较低,这是引起两个甘蔗品种不同抗寒性的重要生理基础。ABA处理后,有效缓解了低温胁迫对甘蔗的不利效应,使两个甘蔗品种幼苗叶片细胞膜、Rn、光系统Ⅱ、叶绿素含量相对稳定,降低了膜脂过氧化产物MDA和GA的含量,提高了脯氨酸、ABA含量和ABA/GA比值,从而提高两个甘蔗品种幼苗的抗寒性。2.低温胁迫下ABA及其合成抑制剂对甘蔗幼苗抗氧化酶活性的影响低温胁迫下,甘蔗幼苗抗氧化酶的活性提高。ABA处理的甘蔗幼苗ABA含量提高,抗氧化保护酶超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)、过氧化物酶(POD)、谷胱甘肽还原酶(GR)的活性提高,活性氧自由基超氧阴离子(02-)、过氧化氢(H202)及膜脂过氧化产物MDA含量下降,而ABA合成抑制剂钨酸钠处理的结果则与之相反。这说明ABA及其合成抑制剂钨酸钠处理对低温胁迫下甘蔗幼苗抗氧化保护系统具有重要的调节作用。ABA对低温胁迫下甘蔗抗寒性的增强,可能与它对抗氧化保护系统的诱导密切相关。3.低温胁迫下ABA处理对甘蔗幼苗叶片蛋白质表达的影响低温胁迫影响甘蔗幼苗叶片蛋白质表达。利用蛋白质双向电泳分析,找出两个甘蔗品种低温胁迫和ABA处理下差异蛋白点48个,质谱成功鉴定其中29个。将它们按不同功能分为7类：①参与光合作用7个,占24.1%,包括光系统Ⅱ稳定因子、叶绿体a-b结合蛋白、铁氧还蛋白-NADP-氧化还原酶、核酮糖-1,5-二磷酸羧化/加氧酶小亚基、光系统Ⅰ反应中心亚基、23kD多肽光合系统Ⅱ、叶绿体Ptr-ToxA结合蛋白；②参与抵御胁迫保护酶7个,占24.1%,包括乙二醛酶、抗坏血酸过氧化物酶、硫氧还蛋白过氧化物酶、M型硫氧还蛋白、醌还原酶、超氧化物歧化酶、谷胱甘肽硫转移酶；③参与蛋白加工6个,占20.7%,包括20S蛋白酶体、30S核糖体蛋白、60S酸性核糖体蛋白、FKBP型肽基脯氨酰顺反异构酶.BRII-KD互作蛋白、肽基脯氨酰顺反异构酶；④参与基础代谢6个,占20.7%,包括谷草转氨酶、叶绿体醛缩酶、硫胺合成酶、磷酸丙糖异构酶、果糖-1,6-二磷酸醛缩酶前体、ATP合酶δ亚基；⑤参与转录调控1个,占3.4%,为真核翻译起始因子5A；⑥参与细胞生长和分裂1个,占3.4%,为生长素结合蛋白；⑦未知功能蛋白1个,占3.4%,为假定蛋白。对其中10个编码差异蛋白质的基因mRNA的表达进行了分析,发现它们在转录水平与蛋白水平上的变化不一,相关性不高。进一步分析已鉴定蛋白的功能发现,低温胁迫通过蛋白质表达主要影响甘蔗了的光合作用和抗氧化保护系统,这也与生理参数的变化相符。ABA处理后,经过一系列蛋白的加工和折叠作用,稳定了光合系统,加强了抗氧化保护作用,从而提高植物抗寒性。4.抗寒相关基因的克隆与表达分析利用RT-PCR和RACE技术克隆了5个抗寒相关基因,获得了甘蔗抗坏血酸过氧化物酶基因cDNA全长1045bp、甘蔗谷胱甘肽-S-转移酶基因cDNA全长912bp、甘蔗ABA胁迫蛋白基因cDNA全长753bp、甘蔗铁氧还蛋白-NADP还原酶基因cDNA全长为1335bp及甘蔗脱水素基因cDNA全长578bp。成功构建了这5个基因的原核表达载体,获得了它们在大肠杆菌中的融合表达蛋白。通过荧光定量PCR分析了SoAPX、 SoGST、SoASR、SoFNR和SoDHN的mRNA在转录水平的表达,结果表明两个甘蔗品种在对抗寒基因的表达调控上存在明显差异。SoAPX基因在抗寒性强的GT28中高表达时间长于抗寒性弱的YL6；SoGSr基因在抗寒性强的GT28中启动要快于抗寒性弱的YL6;SoASR基因在抗寒性强的GT28中表达上调,并在胁迫1d时表达量最高,而在抗寒性弱的YL6中表达则下调；SoDHN基因在抗寒性强的GT28中增幅明显,最大增幅达到1200.6%,而在抗寒性弱的YL6中最大增幅仅为38.9%。可见低温胁迫下甘蔗抗寒相关基因表达的差异直接影响了基因型间的抗寒性。ABA处理后,这5个基因在两个甘蔗品种中都不同程度地被诱导,使得ABA信号的转导作用增强,甘蔗自身的抗氧化防护能力提高,膜的结构相对稳定,从而提高甘蔗抗寒性。可见,ABA对抗寒基因的诱导和调控,是其提高甘蔗抗寒性的重要分子基础。更多还原

【Abstract】 Sugarcane is the most important sugar crop in China and the world, which originated in tropical and subtropical areas and is a thermophilic crop. Extreme weather occurred frequently in worldwide that caused serious cold and/or frost damage in recent years, resulting in enormous losses in sugarcane production. The main measures to decrease the losses caused by cold and frost damage in commercial sugarcane production are selecting cold resistant sugarcane varieties, improving cultivation management and chemical regulation etc. ABA plays an important role in plant growth especially in adverse enviroment. In the present study, seedcane setts were grown in soil culture and solution culture to investigate the physiological and molecular mechanism of cold resistance enhanced by ABA application in sugarcane with strong cold resistant variety GT28and weak cold resistant variety YL6using real-time PCR,2-DE and MS. The main results were as follows.1. Effects of exogenous ABA on physiological and biochemical of sugacane settlings under cold stressLow temperature caused significant changes in the physiological and biochemical characteristics in leaves of sugarcane plants. The results showed that, under the cold stress condition, the plasma membrane permeability and contents of MDA and proline increased; for photosynthesis related parameters, Pn, Gs, Tr decreased while Ci increased; for fluorescent parameters, Fv/Fm, qP and ΦPSII increased, but Fo and NPQ decreased; for endogenous hormones, GA and ZR content decreased while ABA content increased, and the ratios of ABA/GA, ABA/IAA, ABA/ZR increased. Genotypic differences in response to the contents of proline, ABA and GA, and the ratio of ABA/GA exist between strong cold resistant variety GT28and weak cold resistant variety YL6under cold stress. The contents of proline and ABA and the ratio of ABA/GA are higher and the content of GA is lower in the strong clod resistant variety, which is the vital physiological basis that caused two sugarcane varieties with different cold resistance. It is concluded that the ABA application effectively alleviated the adverse effect of low temperature on the plant growth, which kept the relative stability of cell membrane, Pn, PS Ⅱ and chlorophyll, decreased the contents of MDA and GA, and increased the contents of proline and ABA and the ratio of ABA/GA, thereby increased the cold resistance of sugarcane settlings.2. Effect of ABA and its biosynthesis inhibitor on the activities of antioxidant enzymes in sugarcane settling under cold stressThe activities of antioxidant enzymes were increased under cold stress. After ABA application, the content of ABA and the activities of SOD, APX, CAT, POD and GR increased in sugarcane settlings, while the contents of O2-, H2O2and MDA decreased. After tungstate (biosynthesis inhibitor of ABA) application, the result was contray. It is concluded that ABA and its biosynthesis inhibitor have an important regulatory role to the antioxidant protection system in sugarcane settling under cold stress. The cold resistance in sugarcane enhanced by ABA application under cold stress may be associated with the induction of antioxidant protection system.3. Effects of exogenous ABA on protein expression in sugacane settlings under cold stressThe protein expression was changed in settling leaves under cold stress. The results of2-D gel electrophoresis analysis showed that48differential protein spots were detected under cold stress and cold stress plus ABA application conditions in two sugarcane varieties, and29of them were successfully identified through mass spectrometry. The29proteins were involved into seven categories. Seven proteins participate in photosynthesis, accounted for24.1%, including photosystem Ⅱ stability/assembly factor, chlorophyll a-b binding protein, ferredoxin-NADP-reductase, ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, photosystem I reaction center subunit Ⅱ,23kD polypeptide of photosystem II and chloroplast-localized Ptr ToxA-binding protein;7proteins participate in defense responses, accounted for24.1%, including glyoxylase, ascorbate peroxidase, thioredoxin peroxidase, thioredoxin M-type, quinone reductase, super-oxide dismutase and glutathione S-transferase;6proteins participate in protein processing, accounted for20.7%, including20S proteasome,3OS ribosomal protein,60S acidic ribosomal protein, FKBP type peptidyl-prolyl cis-trans isomerase, BRII-KD interacting protein and peptidyl-prolyl cis-trans isomerase;6proteins participate in metabolism, accounted for20.7%, including aspartate aminotransferase, chloroplastic aldolase, thiamine biosynthesis, triosephosphate isomerase, fructose1,6-bisphosphate aldolase precursor and ATP synthase delta chain;1protein participates in transcription, accounted for3.4%, which is eukaryotic translation initiation factor5A;1protein participates in cell growth an division, accounted for3.4%, which is an auxin-binding protein; and1protein is unclassified, accounted for3.4%, which is a hypothetical protein. Gene expression analysis of10differential proteins done by quantitative real time PCR showed that the mRNA level was not correlated well with the protein level. It was found that cold stress mainly affected photosynthesis and antioxidant protection system through changes in protein expressions in sugarcane, which is consistent with result of physiological analysis. After ABA treatment, stabilized the photo synthetic system and strengthened the role of antioxidant protection through a series of protein processing and folding, thereby increased the cold resistance of sugarcane settlings.4. Cloning and expression analysis of cold resisitance genesFive cold resistance genes were cloned by RT-PCR and RACE. A full length of SoAPX gene was obtained and the cDNA was1045bp. A full length of SoGST gene was obtained and the cDNA was912bp. A full length of SoASR gene was obtained and the cDNA was753bp. A full length of SoFNR gene was obtained and the cDNA was1335bp. A full length of SoDHN gene was obtained and the cDNA was578bp. All the genes were expressed in Prokaryon and transformed into E. coli and expressed successfully. The mRNA expressions of SoAPX, SoGST, SoASR, SoFNR and SoDHN were analyzed at the transcriptional level. The results showed that, low temperature caused significant differences in gene expression and regulation of cold resistance in two sugarcane plants. SoAPX gene is highly expressed in strong cold resistant variety GT28longer than weak cold resistant variety YL6, SoGST gene is started in stong cold resistant variety GT28faster than weak cold resistant variety YL6, SoASR gene is up-regulated in stong cold resistant variety GT28and the highest expression level in1day after cold stress, but it is down-regulated in weak resistant variety YL6, SoDHN gene significantly increased in stong cold resistant variety GT28and the highest increase reaches by1200.6%, while the highest increase in weak resistant variety YL6is only38.9%. It indicated that, the differential expression of sugarcane cold-related gene is directly impacted in the cold resistant genotypes. After ABA pretreatment, the expressions of the five genes were induced in different extent, which refleted that the role of ABA signal transdution was enhanced, the capacity of antioxidant defense was increased, the structure of membrane was relatively stable, thereby increasing the cold resistance of sugarcane settlings. The induction and regulation of cold resistance related genes by ABA application is an important molecular basis to improve the cold resistance in sugarcane.更多还原