砷脅迫下兩種鳳尾厥屬植物細(xì)胞分裂素的形態(tài)、分解和作用
發(fā)布時(shí)間：2018-08-03 17:03
【摘要】：細(xì)胞分裂素在植物生長發(fā)育過程中起著重要作用。為研究砷脅迫下細(xì)胞分裂素(CTK)在鳳尾蕨屬兩種不同砷富集能力植物中的合成、分解及作用,首先,確立細(xì)胞分裂素的檢測方法,建立對(duì)不同形態(tài)的細(xì)胞分裂素提純、分離及測定的方法；其次,通過室內(nèi)水培實(shí)驗(yàn),研究As(V)對(duì)砷超富集植物大葉井口邊草(Pteris cretica var. nervosa)和非砷超富集植物劍葉鳳尾蕨(Pteris ensiformis)葉片內(nèi)源CTK存在形態(tài)的影響,以及在土培(0,50,100,200 mg·kg-1)條件下,比較研究兩種植物CTK含量與細(xì)胞分裂素氧化酶(CKX)的活性變化的關(guān)系；最后,在5mg·L-1砷脅迫下,通過外源葉片噴施一定濃度梯度(0,5,20,40 mg·L-1)的激動(dòng)素(6-KT),測定兩種鳳尾蕨屬植物地上部葉片對(duì)砷富集隨時(shí)間動(dòng)態(tài)(1、4、7、10、13d)的變化,并觀測細(xì)胞分裂素對(duì)植物葉片細(xì)胞超微結(jié)構(gòu)的影響。主要研究結(jié)果如下：1.用高效液相色譜同時(shí)測定植物內(nèi)源結(jié)合態(tài)和游離態(tài)細(xì)胞分裂素方法的建立。樣品以含有少量2,6-二叔丁基-4-甲基苯酚改良的Bieleski溶劑提取,提取液經(jīng)聚乙烯吡咯烷酮(PVPP)柱、二乙基氨基乙基交聯(lián)葡聚糖凝膠(DEAE)柱和Sep-Pak C18小柱進(jìn)行固相萃取凈化。凈化后的樣品采用Agilent Zorbax SB-C18反相色譜柱(5μm×4.6mm×250mm),甲醇-乙腈-1%乙酸作為流動(dòng)相梯度洗脫分離,流速為0.6mL/min,紫外檢測波長為269 nm,柱溫45℃。該條件下,異戊烯基腺嘌呤(iP)異戊烯基腺嘌呤核苷(iPA)、7-p-葡糖基-N6-(異戊二烯基)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種細(xì)胞分裂素能達(dá)到基線分離,各組分的檢測限在1.72-5.59 ng之間,加標(biāo)回收率在92.9%-104.2%范圍內(nèi)。2.水培實(shí)驗(yàn)結(jié)果表明,在一定濃度梯度(0,2,5,10 mg·L-1)砷脅迫下,大葉井口邊草(P. cretica var. nervosa)和劍葉鳳尾蕨(P. ensiformis)生長受到不同的影響,大葉井口邊草地上部鮮重在不同濃度As(V)脅迫下無顯著差異,劍葉鳳尾蕨在10mg· L-1 As(V)處理時(shí)顯著低于對(duì)照組,僅為對(duì)照組的77%。大葉井口邊草葉片內(nèi)源ZR7G、ZR、iP7G、iPA含量在10 mg·L-1 As(Ⅴ)處理時(shí)顯著高于對(duì)照組,而Z和iP與對(duì)照組相比無顯著差異。劍葉鳳尾蕨葉片內(nèi)源ZR含量在10 mg·L-1As(Ⅴ)僅為對(duì)照組的36.38%,Z、iP、iPA、ZR7G和iP7G與對(duì)照組相比則無顯著差異。大葉井口邊草葉片結(jié)合態(tài)細(xì)胞分裂素在10mg·L-1 As(Ⅴ)脅迫下顯著高于對(duì)照組,是對(duì)照組的2.48倍；劍葉鳳尾蕨在10mg·L-1 As(Ⅴ)脅迫下是對(duì)照組的1.7倍。低濃度砷脅迫能提高兩種植物CTKs的含量,但高濃度脅迫對(duì)劍葉鳳尾蕨CTKs含量起抑制作用,對(duì)大葉井口邊草則無顯著影響。3.大葉井口邊草葉片中CTKs總量與CKX活性呈負(fù)相關(guān)；劍葉鳳尾蕨CTKs總量與CKX活性隨著脅迫增強(qiáng)無顯著變化。此外,大葉井口邊草游離態(tài)Z含量與其CKX活性呈顯著負(fù)相關(guān),而劍葉鳳尾蕨游離態(tài)Z含量與CKX活性呈極顯著正相關(guān)。4.外源添加不同濃度6-KT對(duì)超富集植物大葉井口邊草和非超富集植物劍葉鳳尾蕨的生長均有促進(jìn)作用,增加植物生物量,提高植物活力指數(shù)。在噴施6-KT濃度為5 mg·L-1時(shí)對(duì)大葉井口邊草的從根部向地上部轉(zhuǎn)運(yùn)砷有顯著促進(jìn)作用,同時(shí)增加葉綠素a/b的值；同樣地,6-KT促進(jìn)劍葉鳳尾蕨吸收砷,但葉綠素a/b的值隨著6-KT濃度增加而降低,可能是促進(jìn)砷的吸收但劍葉鳳尾蕨體內(nèi)沒有相應(yīng)的解毒機(jī)制而產(chǎn)生毒害,降低光合作用。外源6-KT能增加大葉井口邊草葉綠體中淀粉粒的存儲(chǔ),增強(qiáng)細(xì)胞活性；6-KT對(duì)劍葉鳳尾蕨超微結(jié)構(gòu)有一定的修復(fù)功能,在40 mg·L-1處理時(shí)劍葉鳳尾蕨富集的砷量比5 mg·L-1處理多,但植物細(xì)胞更加完整。因此,外源噴施6-KT能促進(jìn)植物提取砷并維持其良好生長。
[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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：X173
本文編號(hào)：2162402
[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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：X173
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