【作者】 李玲；

【导师】 祝水金；

【作者基本信息】 浙江大学 ， 作物学， 2012， 博士

【摘要】 镉是毒性最强和农田受污染最普遍的重金属之一。镉在土壤-植物系统中的迁移性较为活跃,不仅严重影响作物的产量和品质,并可通过食物链富集危害人体健康。因此,土壤镉修复已成为保证农产品质量安全和持续高效的利用土地资源的一项重要科学任务。治理镉污染的土壤最有效的途径就是培育和种植非食用作物或食用器官累积量低的作物品种。这样既可以逐渐吸收消除土壤中的镉,又可以合理利用土地,保障土地持续产出。棉花作为一种重要的经济作物,主产品是棉纤维,副产品是棉籽,是一种适合用于治理土壤镉污染的作物之一本研究以转基因抗草甘膦棉种质系ZD-90、转Bt基因抗虫棉品种SGK3和陆地棉标准系TM-1为研究材料,通过二年的模拟大田试验,观察镉协迫下棉花植株在整个生育期内的生长状况,测定相关的生理和光合作用指标,并分析产量、纤维品质、种子品质以及金属离子含量及其在细胞中的分布等,探索棉花修复镉污染土壤的可能性及其耐镉性的生理生化基础。同时,以16份转基因抗虫棉棉花品种为亲本,按完全双列杂交设计配制成1套包括亲本、F1和F2的遗传材料,采用包括加性、显性和母体效应遗传模型(ADM模型),研究了棉花幼苗耐镉性的遗传特点和分子机理。主要研究结果如下：1.镉胁迫对棉花生长发育的影响2009～2010年二年不同浓度镉处理的模拟大田试验结果表明,低浓度镉处理对棉花植株生长发育具有有一定的促进作用,但高浓度镉处理对棉花植株的生长发育具有明显的抑制作用,品种间对镉胁迫的反应有显著差异。棉花植株生理生化指标测定结果表明,在低浓度镉胁迫下,棉花植株体内的抗氧化化物酶如POD、SOD、CAT和APX等迅速升高以对付镉胁迫所引起的体内活性氧的增加而免遭伤害。随着镉处理浓度的增加和胁迫时间的延长,植株体内累积的MDA量呈逐渐升高趋势,叶绿素a、叶绿素b、叶绿素a/b和叶绿素总量总体上呈逐渐降低趋势,叶片净光合速率先升高后降低,蒸腾速率和气孔导度逐渐降低,气孔限制值逐渐升高。2.镉胁迫对棉花产量及产量因素的影响参试棉花品种(系)的籽棉和皮棉产量均随着镉处理浓度的增加而显著降低。从产量构成因素来看,品种(系)间对镉处理的反应不尽一致。TM-1和SGK3的衣分均随着镉胁迫浓度的增加而降低,而ZD-90的衣分在镉处理后却反而高于对照；3个参试品种(系)的单株铃数均随着镉胁迫浓度的增加而显著降低；TM-1和ZD-90的铃重均随着镉胁迫浓度的增加而显著降低,而SGK3在浓度处理时的单铃重反而提高,中高浓度镉处理后其铃重随处理浓度增加而降低。3.镉胁迫对棉花纤维品质的影响参试品种(系)在镉处理后的纤维品质有所下降,但不同的纤维品质指标间参试品种(系)的反应不尽一致。TM-1和SGK3的纤维长度在低镉处理浓度时表现增加,在高浓度处理时表现下降,而ZD-90的纤维长度则随镉处理浓度增加而增加。3个品种(系)的纤维断裂比强度在200μM镉处理下最大,处理间差异均不显著。马克隆值均为在镉处理低浓度时增加,而高浓度处理时降低,处理间差异均不显著,其中SGK3的马克隆值高于ZD-90和TM-1。纤维伸长率在处理间差异不显著,其中ZD-90的纤维伸长率在镉处理后均低于对照。4.镉胁迫对棉花种子品质的影响镉处理后,3个棉花品种(系)的种子物理品质均有一定的下降,但不同参试品种(系)间,对镉胁迫的反应各不相同,其中TM-1的籽指、仁指和仁壳比等性状对受镉胁迫影响较大,对镉较敏感；ZD-90和SGK3二个转基因棉品种(系)的种子物理品质在处理间差异不显著,对镉胁迫反应不敏感。高浓度镉处理后,3个品种(系)种仁蛋白质含量均低于低浓度处理,但高于对照；镉处理后的油分含量与蛋白质含量变化相反。蛋白质中的氨基酸相对含量受镉处理影响较小,但参试品种间有一定的差异,其中TM-1镉处理后的半胱氨酸相对含量增加,而SGK3镉处理后的半胱氨酸含量相对降低。镉处理对棉籽油中不同脂肪酸含量的影响表现不一致,其中棕榈酸含量随镉处理浓度的升高而显著降低,亚油酸含量在不同镉处理浓度间无显著差异。3个棉花品种(系)种仁棉酚含量在镉处理后有不同程度的降低。其中ZD-90和SGK3的棉酚含量均低于TM-1。5.镉胁迫对棉花种子显微和超显微结构的影响石蜡切片结果表明,棉花种子种仁表面色素腺体密度随着镉处理浓度的升高逐渐降低,结果与棉酚含量测定结果相一致。透射电镜结果表明,镉处理浓度越高,种仁细胞受伤害越严重,表现为细胞核变得不规则,染色质的凝聚,核仁扭曲和解体；细胞膜与细胞壁分离；细胞壁变厚；细胞质浓缩,细胞器结构异常,细胞坍塌解体,对细胞造成致死性的伤害。电镜观察结合能谱分析确定镉以晶体或电子致密颗粒形式存在于蛋白质中的空泡、细胞间隙、细胞质以及附着在细胞壁上。6.镉胁迫下镉及其它金属元素在棉花体内的积累与转运不同镉浓度处理下,棉花各器官和组织镉含量测定结果表明,3个参试棉花品种(系)叶柄中累积的镉含量最高,纤维最低。不同部位累积镉能力的顺序为：纤维<种仁<棉籽壳<根系<叶片<茎秆<铃壳<叶柄。各器官对镉的转运能力、富集系数和对镉吸收能力的顺序一致,说明棉花具有较强的吸收、转运和富集重金属镉的能力,并随着土壤中的镉含量增加而增加。不同品种(系)间,镉积累与转运有明显差异,其中SGK3和ZD-90的镉累积、转运能力高于TM-1。镉与其它金属元素间相关分析表明,镉胁迫促进了纤维和棉籽壳对Mg、Mn、Cu、Zn元素的吸收和累积,影响了叶片、叶柄和铃壳对Mn、Zn元素的吸收和累积,影响了种子和茎秆对Mn、Cu元素的吸收,促进了根对Mg、Mn、Zn元素的吸收。7.棉花耐镉性的遗传分析以16份耐镉性差异明显的转基因杂交棉及其双列杂交组合为材料,采用加性、显性和母体效应遗传模型(ADM模型),对棉花耐镉相关生理性状的遗传分析。结果表明,植株幼苗干重、叶片和根系的POD活性值和MDA含量不受加性遗传效应的影响,而是受显性效应和母体效应的影响。镉处理后,幼苗干重的显性效应和母体效应均低于对照；叶片POD活性值的显性效应和母体效应均随镉处理浓度升高而增加；根系POD活性值和MDA含量的显性效应均在200μM镉处理时达到最高值。对于这些生理性状,可根据母体的总体表现利用杂种优势在处理中后期进行选择。镉处理后棉花幼苗地上部干重的遗传率高于对照,而根干重的遗传率低于对照。说明地上部干重受镉胁迫影响较小,在处理前期选择有效,而根干重受影响较大,在处理后期选择有效。8.镉胁迫对棉花基因组DNA甲基化的影响棉花基因组DNA甲基化分析研究表明,镉处理后,DNA的甲基化程度增加,未发生甲基化的比例降低,过甲基化的比例逐渐升高,表明镉胁迫可能诱导棉花基因组整体甲基化水平的提高。镉处理引起棉花叶片CCGG胞嘧啶甲基化位点数遗传频率下降和变异频率升高。由母本单独遗传的频率均低于对照,由父本单独遗传的频率均高于对照。从甲基化遗传模式来看,镉处理前主要以母本M1、H1和HM1为主,镉胁迫后主要以双亲D为主。由母本单独变异的频率以及双亲变异的频率均低于对照；由父本单独变异的频率均高于对照。从甲基化变异模式来看,以双亲变异为主,其它在镉处理前主要以母本VH1和VM1为主,镉胁迫后主要以VM2为主。更多还原

【Abstract】 Cadmium (Cd) is one of the most toxic heavy metals being contaminating our soils. It can be easily absorbed and accumulated by plants. The high accumulation of Cd not only inhibits the plant growth and negatively affects crop yield and quality, but also causes harm to human health through the food chain. It is therefore necessary to reduce soil Cd level to ensure the safety of agricultural products and ensure the sustainable and efficient use of the Cd contaminated soil. The most effective way of reducing soil Cd level is the breeding of Cd tolerant cultivars having no or very low Cd in their edible parts. Cotton (Gossypium hirsutum L.) is one of the important fibre crops in the world, which is widely used for fiber production. Thus it can also be used to be remediate Cd contaminated soil.In this study, we planted cotton cultivars namely ZD-90, SGK3and TM-1on Cd polluted soils in2009and2010. ZD-90is a transgenic glyphosate tolerant cotton germplasm with EPESP-G7developed by our laboratory. SGK3is an insect-resistant cultivar obtained from the Biotechnology Center of the Chinese Academy of Agricultural Sciences. TM-1is the upland cotton genetic standard line obtained from USDA, ARS, College Station, Texas, USA. A pot experiment was conducted to study the growth conditions, physiological traits and photosynthetic parameters, analyze the yield, fiber quality traits, seed quality traits, Cd accumulation and distribution, and explore the phytoremediation capability of these cultivars of Cd. Furthermore, we explored the genetic features and molecular mechanisms of the Cd resistance in cotton seedlings through the genetic models of ADM, by using sixteen transgenic insect-resistant cotton cultivars as parental with a diallel mating design. The main results were as follows:1. Effect of cadmium on the physiology and metabolism of cottonA pot experiment showed that cotton plant growth under Cd stress was dose-dependent. It is beneficial to cotton plants growth at low Cd concentrations, however, significant growth inhibition at high Cd concentrations. There are significant differences among cotton cultivars with respect to Cd stress. The physiolosical and biochemical analysis of cotton plant indicated that the activity of antioxidant enzymes such as POD, SOD, CAT and APX, etc. in plant cells greatly increased to avoid harmful effects at low Cd concentration. With the increase in Cd concentration and length of growth period, MDA content increased, chlorophyll decreased gradually, net photosynthetic rate increased at low Cd concentration and then declined. Transpiration rate and stomatal conductance decreased, stomatal limitation value increased gradually.2. Effect of cadmium on cotton yield and its componentsCotton yield significantly declined in the three cultivars under Cd stress. The mean value of lint was declined in TM-1and SGK3with an increase in Cd level, while it was increaced in ZD-90. A significant decreasing trend of bolls per plant was found in the three cultivars. As compared to control plants, boll weight increased to the highest at200μM Cd level and then declined significantly at600μM Cd level for SGK3, while a continuously decreasing trend was found for ZD-90and TM-1.3. Effect of cadmium on cotton fiber quality traitsFiber quality traits of the three cotton cultivars decreased to a little under Cd stress. The main difference exsised between fiber quality and cultivars. It was beneficial to fiber length at low Cd concentrations, which was inhibited at high Cd concentrations in TM-1and SGK3, but increased in ZD-90. Fiber strength was found increasing to the highest at200μM Cd level and there was no significant difference in the exposed cultivars. Micronaire was acceptable at low Cd concentrations, and showed a decline at high Cd concentrations. Higher micronaire was observed in SGK.3than that in ZD-90and TM-1. No significant difference was found for fiber elongation at all Cd levels in the three cultivars. It was lower at all Cd treatments than control.4. Effect of cadmium on cotton seed qualityThe physical quality traits of cottonseeds showed a little decrease in the three cotton cultivars under Cd stress, but cultivar specific response was different. TM-1was sensitive to Cd as a marked decrease was found in seed index, kernel index and kernel/hull. ZD-90and SGK3was not sensitive to Cd because there were no significant differences in seed physical traits. Seed protein content at higher Cd level was lower in comparison with lower Cd level, but was still higher than control. The change of oil content is opposite to that of protein content. Relative content of amino acids was less influenced by Cd stress, but there were some differences among cultivars. Cys content was increased in TM-1and decreased in SGK3. It was inconsistent with different fatty acids content under Cd stress. Palmitic acid conent in the seed decreased significantly with increasing Cd supply. There were not significant changes in the content of linoleic acid in seed under the various treatments compared to controls. A decreasing trend was observed for gossypol content in the three cultivars. Gossypol content in ZD-90and SGK3was lower than that in TM-1.5. Effect of cadmium on microstructure and ultra-microstructure of cotton seedsParaffin showed that the pigment gland density on surface of seeds was decreased with the increase in Cd concentration, which was consistant with the trend of gossypol content in cotton seeds. TEM results showed that the damage to the seed cells became more and more prominent with increasing concentration of cadmium as compared to control. The main alterations were such as plasmolysis, disintegration of nucleus, shrinkage of cytoplasm, thickening and constriction of cell wall, abnormal structures of organelle, cell collapse and disintegration. TEM observation and ED AX analysis confirmed that Cd was existed in the form of rings and crystals as well as electron dense granules, distributed in intercellular space, cytoplasm and cell wall.6. Accumulation and transportation of Cd and other metal elements in cotton plant under Cd stressThe results of Cd accumulation in cotton plant under Cd stress showed that petiole was the highest part in Cd accumulation, and fiber was the lowest. The ability of uptake and accumulation in different parts of the three varieties with the rise of Cd level was in order:fiber <seed<seed shell<root<leaf<shoot<boll shell<petiole. The translocation ability, bioconcentration factor and absorbency showed the same order in different organs and increaced with Cd concentration. It indicated that cotton has higher potential for absorbing and removing Cd from moderately contaminated soils and can be used for the phytoremediation of polluted soils. There was significant difference on Cd accumulation and transportation among cultivars, it was higher in ZD-90and SGK.3than that in TM-1. Correlation analysis showed that the uptake and accumulation of Mg, Mn, Cu, Zn in fiber and seed shell, and Mg, Mn, Zn in root, Mn, Zn in leaf, petiole and boll shell, and Mn, Cu in seed and shoot were greatly influenced by Cd stress.7. Genetic features analysis of the resistance to Cd of cotton seedlingsGenetic analysis on physiological traits showed that there were not genetic effects on plant dry weight, POD activity and MDA content in leaf and root, but the dominant effects and maternal effects. For the dominant effects and maternal effects, it was lower of seedling dry weight under Cd stress than that in control and increased with Cd levels of leaf POD activity. The dominant effects of root POD activity and MDA content were increasing to the highest at200p.M Cd level. For these physiological charecters, it could be selected according to maternal effects and using heterosis at longer treatment time. The heritability of shoot dry weight was higher than control in Cd treatments, while was lower in root dry weight. It showed that less influence was found on shoot dry weight and much influence on root dry weight under Cd stress.8. Effect of cadmium on cotton genomic DNA methylationMASP analysis showed that Cd may induce the changes of genomic methylation levels because of the rise in DNA methylation level and the reduction of unmethylation under Cd stress. The genetic frequency of CCGG cytosine methylation loci decreased and variation frequency increased under Cd stress. The frequency inherited from the female parent alone was lower while thet inherited from the male parent alone was higher than the control. According to the inheritance of cytosine methylation patterns, M1, Hland HM1patterns of the female parent served as the main patterns at control, D pattern of parents played a major role. The variation frequency from the female and both parents was lower than that of the control, but was higher than the control from the male parent. According to the variation of cytosine methylation patterns, the D pattern of parents played a major role.更多还原