【作者】 胡龙兴；

【导师】 王兆龙； 黄炳茹；

【作者基本信息】 上海交通大学 ， 蔬菜学， 2010， 博士

【摘要】 干旱胁迫可造成植物在形态、生理、蛋白及基因表达等水平上的变化,是限制植物生长的主要环境因子之一。研究植物抗旱机理以及植物对干旱的适应和调节机制,对选育高效抗旱新品种,解决当前水资源短缺和生态环境恶化具有重要意义。本文首先以狗牙根为材料,对8个狗牙根基因型进行了抗旱性能评价与筛选;并以所筛选出的抗旱性差异较大的两个狗牙根品种-Tifway(抗旱)和C299(不抗旱),探讨了草坪草对干旱胁迫的光合生理反应;论文还以两个抗旱差异较大的草地早熟禾品种-Midnight(抗旱)和Brilliant(不抗旱)为材料,探讨对干旱胁迫的光合生理反应、干旱胁迫后复水过程中CTKs和KCl对草地早熟禾叶片气孔开度及光合作用的影响,并以草地早熟禾抗旱型品种为材料,通过SSH技术构建了干旱胁迫下草地早熟禾叶片消减cDNA文库,经阳性克隆测序、同源性比对、功能分类及RT-PCR分析,获得了大量与抗旱相关的候选基因片段。主要研究内容及结果如下:1.不同基因型狗牙根抗旱性能的生理评价SDS-PAGE分析表明,在轻度(6 d)到中度(10 d)干旱胁迫下,8个基因型的狗牙根叶片可溶性蛋白没有明显的变化。重度干旱胁迫(14 d)造成狗牙根叶片中可溶性蛋白降解,其中以C299和Tifeagle降解最为严重。免疫印迹检测发现,正常浇水下狗牙根叶片没有脱水素蛋白的表达。干旱胁迫6 d时,只有一条24 kDa分子量的脱水素在C299中表达。干旱胁迫第10 d时诱导表达了6条不同分子量的脱水素蛋白,其中14-、19-和24 kDa分子量的脱水素蛋白在8个狗牙根基因型中均表达,但是在C299、Tifeagle和Tifdwarf中的表达量均高于其它5个狗牙根基因型。31 kDa分子量的脱水素蛋白只在Tifway和H19中表达,其它6个基因型均没有表达。40 kDa分子量的脱水素蛋白在8个狗牙根基因型中均有表达,但是在Tifway、H19和H10中的表达量均高于其它5个基因型。以草坪外观质量(TQ)、叶片相对含水量(RWC)、相对电导率(EL)并结合可溶性蛋白图谱的变化与脱水素蛋白的表达为指标,综合评价了试验中8个狗牙根型的抗旱性能,可将试验中8个狗牙根基因型抗旱性大小排序为:(Tifway、H19)>(H10、Kan 1、Sportbermuda、Tifdwarf)>(Tifeagle、C299)。2.不同抗旱性狗牙根品种离体叶片失水率及其脱水素蛋白表达的差异抗旱品种Tifway和水分敏感品种C299叶片离体失水速度不同,Tifway的离体叶片失水率(RWL)显著低于C299。脱水至同一失水量如10%、30%、50%和65%,Tifway则需要更长的时间。叶片失水诱导了不同分子量脱水素蛋白的表达,74 kDa分子量的脱水素蛋白在不同失水量和在两个狗牙根基因型中表达量没有显著差异;14-、19-和24 kDa分子量的脱水素蛋白其表达从失水10%到65%,均随着脱水量的增加而增加,在同一失水量下,其在C299中的表达量高于Tifway。31 kDa分子量的脱水素蛋白只在Tifway中表达而不在C299中表达,且其表达量随着失水量的增加而增加。从失水量10%到65%,40 kDa分子量的脱水素蛋白其表达量随着脱水量的增加而增加,在同一失水量下,其在Tifway中的表达量显著高于C299。3.干旱胁迫下不同抗旱性狗牙根品种的光合生理差异在轻度水分胁迫下(6 d),PSII活性在C299和Tifway中均没有下降;当干旱胁迫到10 d和14 d时,C299和Tifway的Fv/Fm显著下降,且C299显著低于Tifway;干旱胁迫下光合作用显著降低,其中C299下降更快,在干旱胁迫第6、10和14 d时Tifway的Pn和gs显著高于C299。Tifway的Rubisco活性和活化率从干旱胁迫第14 d才开始下降,而C299则从干旱胁迫第10 d就开始降低。此外,SDS-PAGE蛋白图谱也显示,与光合作用相关的主要蛋白如Rubisco大亚基(49 kDa)和磷酸丙酮酸二激酶(PPDK, 98 kDa)从干旱胁迫第14 d开始在C299叶片中急剧降解,而在Tifway中没有降解。4.干旱胁迫及复水对草地早熟禾光合生理特性的影响在短期或轻度水分胁迫条件下(5 d),草地早熟禾叶片的Fv/Fm和其它的光合组分(Rubisco活性及活化率、Pnmax、CE、Vcmax、Jmax)均没有下降或有小幅降低,而Pn则伴随着gs、Tr和gm的下降而降低,并且Brilliant比Midnight下降速度快。在中度(10 d)到重度(15 d)干旱胁迫下,Rubisco活性及活化率、CE、Fv/Fm、Vcmax、Jmax等均显著下降,并且Brilliant比Midnight下降更剧烈。干旱胁迫解除后,草地早熟禾抗旱品种Midnight叶片的光合参数如Vcmax、Jmax、CE、Pnmax中均恢复到或接近对照水平,但在不抗旱品种Brilliant中均显著低于对照水平,而gs及Pn在两个品种中均未能恢复到对照水平。5.干旱胁迫后复水CTKs和KCl对草地早熟禾叶片气孔开度及光合作用的影响离体叶片脱水后复水,10μM的6-benzylaminopurine(6-BA)和50 mM的KCl溶液对诱导草地早熟禾离体叶片的气孔再开放效果最佳。草地早熟禾叶片气体交换值(Pn、gs、Tr)及气孔开度均随着土壤含水量和叶片相对含水量的下降而降低。干旱胁迫后复水,经6-BA或KCl处理过的植株其叶片气孔开度及气体交换值(Pn、gs、Tr)均显著高于未处理叶片。干旱胁迫及复水条件下,叶片气体交换值(Pn、gs、Tr)与叶片气孔孔径呈极显著线性相关。6.干旱胁迫下草地早熟禾叶片差异表达基因的分离利用SSH技术构建了干旱10 d草地早熟禾叶片消减cDNA文库。通过测序后筛选,得到了503条高质量的EST序列。序列组装拼接后分别得到了387个Uni-ESTs,其中contigs 239条和singlets 148条。通过同源性比对,获得了156条与已知功能基因同源或相似的Uni-ESTs,147条与未知功能基因同源或相似的Uni-ESTs,84条未找到同源性。经功能注释和分类后,获得了大量水分胁迫应答基因,这些基因的功能包括代谢、光合、转录、信号传导、细胞防御、细胞结构生长与分裂、运输及其它等。RT-PCR分析表明APX、MDHAR、PhGPX随干旱胁迫程度的加大而表达量上升,而RubS表达量则降低,进一步验证了消减文库的可靠性。更多还原

【Abstract】 Drought is one of the major abiotic factors limiting plant growth, which induces various changes at the morphologic, physiological, genomic and proteomic levels during adaptation to drought stress in plants. To studying the mechanisms for drought resistance and the adaptation and regulatory mechanisms to drought in plants is crucial for selecting and breeding drought-tolerant cultivars, which could be an important solution to the water shortage and the deterioration of the ecological environment.Drought tolerance of eight bermudagrass genotypes were evaluated in this study, and then two types of turfgrass, which genotypes differing in drought tolerance, warm-season turfgrass‘Tifway’(drought-tolerant),‘C299’(drought-sensitive), and cool-season turfgrass‘Midnight’(drought-tolerant),‘Brilliant’(drought-sensitive) were used to investigate photosynthetic response to drought stress. The effects of foliar application of CTKs and KCl on stomatal aperture and photosynthesis after re-watering were examined in Kentucky bluegrass drought-sensitive cultivars. Finally, a subtracted cDNA library was constructed using the approaches of suppression subtractive hybridization (SSH) in leaves of Kentucky bluegrass. A large number of candidate genes which related to drought tolerance were obtained after colony sequencing, BlastX, functional classification and RT-PCR analysis. The main contents and results of this study are as follows:1. Evaluation of drought tolerance in different genotypes of bermudagrassSDS-PAGE analysis demonstrated that no obvious changes in soluble protein profiles occurred in eight bermudagrass genotypes under mild to moderate drought stress. Severe drought stress led to degradation in soluble proteins in eight bermudagrass genotypes, with more significant degradation in‘C299’and‘Tifeagle’than the other genotypes. No dehydrin polypepides was detected in fully hydrated leaves of well-watered plants. Immunoblot analysis with anti-dehydrin polyclonal antibodies detected one dehydrin polypeptide (24 kDa) in‘C299’but not in other cultivars at 6 d of drought. At 10 d of drought stress, six dehydrin polypeptides of 74, 40, 31, 24, 19, and 14 kDa were detected in eight genotypes. The 74-, 19-, and 14-kDa polypeptides accumulated in all genotypes, and no genotypic differences were observed in the level of expression. The 40-kDa polypeptides had lower expression level in‘C299’,‘Tifdwarf’, and‘Tifeagle’. The31-kDa dehydrin polypeptide was detected only in‘H19’and‘Tifway’, but was not expressed in the other six genotypes. The 24-kDa polypeptides expressed in all genotypes, but the intensity in‘Tifdwarf’,‘Tifeagle’, and‘C299’were higher than in the other genotypes. The drought-resistance of eight bermudagrass genotypes were evaluated base on the TQ, RWC, EL, changes of soluble proteins, and the levels of dehydrin expression, the order were as follows: (‘Tifway’,‘H19’) > (‘H10’,‘Kan 1’,‘Sportbermuda’,‘Tifdwarf’) > (‘Tifeagle’,‘C299’).2. Variations in relative water loss rate and expression levels of dehydrin in detached leaves of bermudagrass genotypes differing in drought toleranceDrought-tolerant‘Tifway’had a slower RWL than drought-sensitive‘C299’during 7 h of dehydration period. The immunoblot analysis indicated that dehydration induced the accumulation of 74-, 40-, 31, 24-, 19-, and 14 kDa dehydrin polypeptides across the RWL range in both genotypes. The intensity of 74-kDa dehydrin polypeptides was did not change with increasing RWL from 10% to 65% in both‘Tifway’and‘C299’. The intensity of 24-, 19-, and 14-kDa dehydrins increased with RWL from 10% to 65% for both genotypes, but‘C299’had higher expression level than‘Tifway’at the same water deficit. The accumulation of 31-kDa dehydrin was detected in‘Tifway’, but not in‘C299’during dehydration. As RWL increased from 10% to 65%, the intensity of 31-kDa dehydrin increased in‘Tifway’. With the increase in RWL, the intensity of 40-kDa dehydrin proteins was elevated. The accumulation of 40- kDa dehydrin polypeptides in‘Tifway’was more pronounced than in‘C299’at the same level of water deficit (RWL from 10% to 65%).3. Variations in photosynthetic physiology for two bermudagrass genotypes differing in drought-tolerance under drought stressNo reduction was observed for PSII activity in both‘C299’and‘Tifway’under mild drought stress (6 d), while significant decrease was observed for Fv/Fm in both genotypes at 10 and 14 d of drought stress, but to a greater extent for‘C299’. Drought stress led to a significant decrease in photosynthesis, with more dramatic decline in‘C299’than in‘Tifway’.‘Tifway’maintained significantly higher Pn, gs than‘C299’at 6, 10 and 14 d of drought treatment.Rubisco activity in‘C299’declined during drought stress to below the well-watered control level at 10 d of treatment, but the decline was not detected until 14 d in‘Tifway’. Analysis using SDS-PAGE demonstrated that no obvious changes in Rubisco (49-kDa large subunit) and PPDK (98-kDa) abundance occurred during drought stress compared with the well-watered control (0 d) in‘Tifway’. Great change was observed in‘C299’for Rubisco and PPDK abundance.4. Effects of drought stress and re-watering on photosynthetic components in Kentucky bluegrassThere was no decline or some reductions in leaf Fv/Fm and other photosynthetic components (Rubisco activity and activation state, Pnmax, CE, Vcmax, Jmax) under short-term or mild drought stress (5 d) in Kentucky bluegrass. Net photosynthetic rate declined with the decreasing of gs, Tr and gm in both‘Midnight’and‘Brilliant’, but to a great extent for ‘Brilliant’. Significant decrease was observed in Rubisco activity, activation state, Pnmax, CE, Vcmax, Jmax, in both cultivars under moderate to severe drought stress, with more dramatic decline in‘Brilliant’than‘Midnight’.Photosynthetic parameters such as Vcmax, Jmax, CE, Pnmax were recovered to or close to the well-watered control level in drought-tolerant‘Midnight’after water deficit was alleviated, whereas significantly lower than the well-watered control level for drought-sensitive‘Brilliant’. Leaf Pn and gs were still not recovered to the control level after re-watering in both cultivars.5. Effects of CTKs and KCl on stomatal aperture and photosynthesis after re-watering10μM of 6-BA and 50 mM KC1 was sufficient for stomatal re-opening in Kentucky bluegrass after rehydration. Leaf Pn, gs, Tr, and stomatal aperture of Kentucky bluegrass decreased with the decreasing of RWC and SWC under drought stress. Leaf Pn, gs, Tr, and stomatal aperture in 6-BA- or KCl-treated plants significantly higher than untreated plants after re-watering. The decrease in the size of stomatal aperture was highly correlated to the decline in Pn, gs, and Tr under drought stress and during re-watering.6. Isolation of differentially expressed genes in leaves of Kentucky bluegrassA subtracted cDNA library was constructed using SSH approaches, which consist of 503 high quality EST sequences after sequencing. 387 Uni-ESTs were obtained after assembling, which consist of 239 contigs and 148 singlets. BlastX alignment results demonstrated that 156 fragments were homologous with function-known genes, and 147 fragments were homologous with function-unknown genes. The rest 84 ESTs with no hit in published database may represent novel genes. The 156 fragments matched to function-known proteins were further classified into 9 categories, which functions are related to metabolism, protein destination, photosynthesis, transcription, signaling and transduction, cellular defense, cell structure, growth and division, transport, and others. RT-PCR analysis indicated that the expression level of APX, MDHAR, PhGPX increased, whereas the RubS decreased with the progression of drought stress.更多还原