砷胁迫下两种凤尾厥属植物细胞分裂素的形态、分解和作用

发布时间：2018-08-03 17:03

【摘要】：细胞分裂素在植物生长发育过程中起着重要作用。为研究砷胁迫下细胞分裂素(CTK)在凤尾蕨属两种不同砷富集能力植物中的合成、分解及作用,首先,确立细胞分裂素的检测方法,建立对不同形态的细胞分裂素提纯、分离及测定的方法；其次,通过室内水培实验,研究As(V)对砷超富集植物大叶井口边草(Pteris cretica var. nervosa)和非砷超富集植物剑叶凤尾蕨(Pteris ensiformis)叶片内源CTK存在形态的影响,以及在土培(0,50,100,200 mg·kg-1)条件下,比较研究两种植物CTK含量与细胞分裂素氧化酶(CKX)的活性变化的关系；最后,在5mg·L-1砷胁迫下,通过外源叶片喷施一定浓度梯度(0,5,20,40 mg·L-1)的激动素(6-KT),测定两种凤尾蕨属植物地上部叶片对砷富集随时间动态(1、4、7、10、13d)的变化,并观测细胞分裂素对植物叶片细胞超微结构的影响。主要研究结果如下：1.用高效液相色谱同时测定植物内源结合态和游离态细胞分裂素方法的建立。样品以含有少量2,6-二叔丁基-4-甲基苯酚改良的Bieleski溶剂提取,提取液经聚乙烯吡咯烷酮(PVPP)柱、二乙基氨基乙基交联葡聚糖凝胶(DEAE)柱和Sep-Pak C18小柱进行固相萃取净化。净化后的样品采用Agilent Zorbax SB-C18反相色谱柱(5μm×4.6mm×250mm),甲醇-乙腈-1%乙酸作为流动相梯度洗脱分离,流速为0.6mL/min,紫外检测波长为269 nm,柱温45℃。该条件下,异戊烯基腺嘌呤(iP)异戊烯基腺嘌呤核苷(iPA)、7-p-葡糖基-N6-(异戊二烯基)腺嘌呤(iP7G)、反式玉米素(Z)、反式玉米素核苷(ZR)和反式玉米素-7-葡糖苷(Z7G)6种细胞分裂素能达到基线分离,各组分的检测限在1.72-5.59 ng之间,加标回收率在92.9%-104.2%范围内。2.水培实验结果表明,在一定浓度梯度(0,2,5,10 mg·L-1)砷胁迫下,大叶井口边草(P. cretica var. nervosa)和剑叶凤尾蕨(P. ensiformis)生长受到不同的影响,大叶井口边草地上部鲜重在不同浓度As(V)胁迫下无显著差异,剑叶凤尾蕨在10mg· L-1 As(V)处理时显著低于对照组,仅为对照组的77%。大叶井口边草叶片内源ZR7G、ZR、iP7G、iPA含量在10 mg·L-1 As(Ⅴ)处理时显著高于对照组,而Z和iP与对照组相比无显著差异。剑叶凤尾蕨叶片内源ZR含量在10 mg·L-1As(Ⅴ)仅为对照组的36.38%,Z、iP、iPA、ZR7G和iP7G与对照组相比则无显著差异。大叶井口边草叶片结合态细胞分裂素在10mg·L-1 As(Ⅴ)胁迫下显著高于对照组,是对照组的2.48倍；剑叶凤尾蕨在10mg·L-1 As(Ⅴ)胁迫下是对照组的1.7倍。低浓度砷胁迫能提高两种植物CTKs的含量,但高浓度胁迫对剑叶凤尾蕨CTKs含量起抑制作用,对大叶井口边草则无显著影响。3.大叶井口边草叶片中CTKs总量与CKX活性呈负相关；剑叶凤尾蕨CTKs总量与CKX活性随着胁迫增强无显著变化。此外,大叶井口边草游离态Z含量与其CKX活性呈显著负相关,而剑叶凤尾蕨游离态Z含量与CKX活性呈极显著正相关。4.外源添加不同浓度6-KT对超富集植物大叶井口边草和非超富集植物剑叶凤尾蕨的生长均有促进作用,增加植物生物量,提高植物活力指数。在喷施6-KT浓度为5 mg·L-1时对大叶井口边草的从根部向地上部转运砷有显著促进作用,同时增加叶绿素a/b的值；同样地,6-KT促进剑叶凤尾蕨吸收砷,但叶绿素a/b的值随着6-KT浓度增加而降低,可能是促进砷的吸收但剑叶凤尾蕨体内没有相应的解毒机制而产生毒害,降低光合作用。外源6-KT能增加大叶井口边草叶绿体中淀粉粒的存储,增强细胞活性；6-KT对剑叶凤尾蕨超微结构有一定的修复功能,在40 mg·L-1处理时剑叶凤尾蕨富集的砷量比5 mg·L-1处理多,但植物细胞更加完整。因此,外源喷施6-KT能促进植物提取砷并维持其良好生长。
[Abstract]:Cytokinin plays an important role in the process of plant growth and development. In order to study the synthesis, decomposition and effect of cytokinin (CTK) in two different arsenic enrichment plants of the genus pteropteris under arsenic stress, first, establish a method for detection of cytokinin, and establish a method for purification, separation and determination of different types of cytokinin. Secondly, the effects of As (V) on the morphology of endogenous CTK in the leaves of the arsenic super enriched plant (Pteris cretica var. nervosa) and the non arsenic hyperaccumulator (Pteris ensiformis) were studied by the laboratory hydroponic experiment. The content of the two plants and the cell fractions were compared under the condition of the soil culture (0,50100200 mg kg-1). The relationship between the changes in the activity of CKX, and at the end of 5mg. L-1 arsenic stress, by spraying a certain concentration gradient (0,5,20,40 mg. L-1) by exogenous leaves (6-KT) to determine the variation of arsenic concentration with time dynamics (1,4,7,10,13d) in the upper leaves of two species of pteropteris, and the observation of cytokinin to plant leaf cells. The main results are as follows: 1. the method of simultaneous determination of endogenous and free cytokinin by high performance liquid chromatography was established. The sample was extracted by Bieleski solvent containing a small amount of 2,6- di-tert butyl -4- methyl phenol, and the extraction solution was mixed with polyvinylpyrrolidone (PVPP) column and two ethyl amino ethyl ester. DEAE column and Sep-Pak C18 column were purified by solid phase extraction. The purified samples were purified by Agilent Zorbax SB-C18 reversed phase chromatography column (5 mu m x 4.6mm x 250mm), methanol acetonitrile -1% acetic acid was used as a mobile phase gradient elution separation, the flow velocity was 0.6mL/min, UV detection wavelength was 269 nm, and the column temperature was 45. Methotrexate (iP) isoamyl adenine nucleoside (iPA), 7-p- glucosyl -N6- (iP7G), trans zein (Z), trans zein nucleoside (ZR) and trans zein -7- glucoside (Z7G) can be separated from 6 kinds of cytokinins. The detection limit of each component is between 1.72-5.59 ng, and the recovery rate is in the range of 92.9%-104.2%. The results showed that under a certain concentration gradient (0,2,5,10 mg. L-1) arsenic stress, the growth of P. cretica var. nervosa and P. ensiformis (P. ensiformis) had different effects. There was no significant difference in the fresh weight of the upper leaf edge of the well mouth under the stress of As (V) at different concentrations. The contents of ZR7G, ZR, iP7G, iPA in the leaves of 77%. Da leaves of the control group were significantly higher than those of the control group, while Z and iP were significantly higher than those of the control group, while Z and iP were not significantly different from those of the control group. The endogenous ZR content of the leaves of the leaf pteropteris was only 36.38% of the 10 mg. There was no significant difference between the group and the control group. The leaf combined cytokinin of the leaf blade of the well mouth was significantly higher than that of the control group under the stress of 10mg. L-1 As (V), 2.48 times that of the control group, and 1.7 times as much as the control group under the stress of 10mg. L-1 As (V). The low concentration of arsenic stress could raise the content of two plant CTKs, but the high concentration stress was on the sword. The CTKs content of Ye Fengwei fern had no significant effect on the leaf edge grass of the big leaf well mouth. The total amount of CTKs and the activity of CKX were negatively correlated with the.3. in the leaves of the well mouth. The total CTKs and CKX activity of the pteropteris were not significantly changed with the stress. In addition, the free Z content in the leaf edge of the big leaf wellhead was negatively correlated with the activity of CKX, but the blade of the blade was negatively correlated with the activity of CKX. The content of free Z in pteropteris pteropteris has a very significant positive correlation with the activity of CKX..4. exogenous adding different concentrations of 6-KT can promote the growth of the hyperenriched plant leaf edge grass and the non hyperaccumulator pteropteris pteroptera, increasing the plant biomass and improving the plant vitality index. In the application of the spraying 6-KT concentration of 5 mg. L-1 to the leaf edge grass of the large leaf well mouth The roots transport arsenic to the ground significantly and increase the value of chlorophyll a/b; similarly, 6-KT promotes the absorption of arsenic in the pteropteris pteropteris, but the value of chlorophyll a/b decreases with the increase of the concentration of 6-KT, which may promote the absorption of arsenic, but there is no corresponding detoxification mechanism in the pteropteris pteropteris. 6-KT can increase the storage of starch granules in the chloroplast of the leaf of the well mouth and enhance the cell activity. 6-KT has a certain repair function for the ultrastructure of Ye Fengwei fern, and the arsenic content of the pteropteris fern is more than 5 mg. L-1 when treated with 40 mg. L-1, but the plant cells are more complete. Therefore, exogenous spraying 6-KT can promote the extraction of arsenic and vitamins in plants. Hold it well.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X173

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