硝态氮对组培‘嘎拉3’叶绿素合成及相关基因表达的影响
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摘要
为研究硝态氮在组培‘嘎拉3’叶绿素合成过程中的作用及其分子基础。本试验以0 mmol·L~(-1) NO_3~-的MS培养基为对照组,探讨硝态氮对组培‘嘎拉3’叶绿素和光合产物含量、叶片的解剖结构以及叶绿素合成途径关键基因MdHEMA、MdHEMD、MdCHLD、MdCHLM、MdPORA相对表达量的影响。结果表明:对照组叶片在14 d时叶缘开始变黄,21 d时由叶缘到叶脉变黄,茎基部无愈伤组织形成。对照组叶绿素含量在14 d时无明显变化,在21 d时下降幅度增大,相对于硝态氮处理,叶绿素a、叶绿素b和类胡萝卜素含量分别下降36.67%、36.84%和26.53%。对照组叶片中可溶性糖含量在21 d后显著降低且低于硝态氮处理,淀粉含量则在7 d后显著增加,高于硝态氮处理。对照组叶片解剖结构在14 d时,栅栏组织变宽与海绵组织界限不清晰,21 d后细胞变形。MdCHLD、MdHEMA和MdPORA这三个基因的相对表达量在14 d达到峰值后降低且显著低于硝态氮处理,MdCHLM在第7 d时达到峰值而MdHEMD则在第21 d达到峰值,这5个基因在缺硝态氮条件下表达趋势相似,都是先升高后降低,表明他们在叶绿素合成途径起作用。以上结果表明硝态氮通过影响叶片解剖结构和叶绿素合成途径关键酶基因的表达量来维持叶绿素含量和光合产物的相对稳定。
The effects of nitrate nitrogen on chlorophyll synthesis and its molecular basis of ‘Gala 3' in tissue culture were studied. In this study, MS medium of 0 mmol·L~(-1) NO_3~-as the control group, the effect of nitrate nitrogen on chlorophyll and photosynthetic content,leaf anatomical structure and relative expression levels of key genes Md HEMA, Md HEMD, Md CHLD, Md CHLM and Md PORA in the chlorophyll synthesis pathway of ‘Gala 3'in vitro plantlets were studied. The results showed the leaves of the control group began to turn yellow at the leaf edge at 14 d, from leaf edge to vein at 21 d, and without callus formation at the stem base. The chlorophyll content in the control group did not change significantly at 14 d, but decreased significantly at day 21, the content of chlorophyll a, chlorophyll b and carotenoid decreased by 36.67%, 36.84% and 26.53%, respectively, compared with nitrate nitrogen treatment. The content of soluble sugar decreased significantly in leaves after 21 d and was lower than that of nitrate treatment, the content of starch increased significantly after 7 d, and was higher than that of nitrate nitrogen treatment. In the control group, when the anatomical structure of the leaves was 14 d, the palisade tissue widen and the boundary between palisade tissue and sponge tissue was not clear, the cells were deformed after 21 d. The relative expression levels of the three genes Md CHLD, Md HEMA, and Md PORA were decreased significantly after the peak at 14 d and were significantly lower than those of nitrate nitrogen treatment. Md CHLM reached the peak on the 7 th day and the Md HEMD was at 21 st day. These 5 genes expressed similar tendency under the condition of no nitrogen–deficient, all of them increased first and then decreased, indicating they play the role in chlorophyll synthesis pathway. The above results indicate that nitrate nitrogen can maintains the relative stability of chlorophyll content and photosynthetic products by affecting the anatomical structure of leaves and the expression of key enzyme genes in the chlorophyll synthesis pathway.
The effects of nitrate nitrogen on chlorophyll synthesis and its molecular basis of ‘Gala 3' in tissue culture were studied. In this study, MS medium of 0 mmol·L~(-1) NO_3~-as the control group, the effect of nitrate nitrogen on chlorophyll and photosynthetic content,leaf anatomical structure and relative expression levels of key genes Md HEMA, Md HEMD, Md CHLD, Md CHLM and Md PORA in the chlorophyll synthesis pathway of ‘Gala 3'in vitro plantlets were studied. The results showed the leaves of the control group began to turn yellow at the leaf edge at 14 d, from leaf edge to vein at 21 d, and without callus formation at the stem base. The chlorophyll content in the control group did not change significantly at 14 d, but decreased significantly at day 21, the content of chlorophyll a, chlorophyll b and carotenoid decreased by 36.67%, 36.84% and 26.53%, respectively, compared with nitrate nitrogen treatment. The content of soluble sugar decreased significantly in leaves after 21 d and was lower than that of nitrate treatment, the content of starch increased significantly after 7 d, and was higher than that of nitrate nitrogen treatment. In the control group, when the anatomical structure of the leaves was 14 d, the palisade tissue widen and the boundary between palisade tissue and sponge tissue was not clear, the cells were deformed after 21 d. The relative expression levels of the three genes Md CHLD, Md HEMA, and Md PORA were decreased significantly after the peak at 14 d and were significantly lower than those of nitrate nitrogen treatment. Md CHLM reached the peak on the 7 th day and the Md HEMD was at 21 st day. These 5 genes expressed similar tendency under the condition of no nitrogen–deficient, all of them increased first and then decreased, indicating they play the role in chlorophyll synthesis pathway. The above results indicate that nitrate nitrogen can maintains the relative stability of chlorophyll content and photosynthetic products by affecting the anatomical structure of leaves and the expression of key enzyme genes in the chlorophyll synthesis pathway.
引文
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[10]李文庆,张民,束怀瑞.氮素在果树上的生理作用[J].山东农业大学学报:自然科学版,2002,33(1):96-100
[11]曲文章,蔡伯岩,荡妙真,等.氮素水平对甜菜主要营养的影响[J].中国甜菜糖业,1999(2):1-6
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[15] Fallahi E, Possingham JV. Productivity, postharvest physiology and soil nitrate movement as influenced by nitrogen application to ‘Delicious’ apple[J]. Acta Hortic, 2000,512:149-157
[16] Makino A, Shimada T, Takumi S, et al. Does decrease in Ribulose–1,5–bisphosphate carboxylase by antisense rbcs lead to a higher N–use efficiency of photosynthesis under conditions of saturating CO2 and light in rice plant?[J]. Plant physiol, 1997,114(2):483-491
[17]李彩,彭良志,党江波,等.不同施氮水平纽荷尔脐橙叶绿素含量的季节变化[J].中国南方果树,2012,41(2):14-18
[18]田娟,张崇玉,熊永琴,等.输液肥对柑橘叶片叶绿素变化的影响[J].贵州农业科学,2008,36(4):143-145
[19]周学伍,黄辉北,黄永.柑橘叶片叶绿素含量的年周期变化及与N含量的关系[J].西南农业大学学报,1985(4):34-37
[20]赵世杰,史国安,董新纯.植物生理学实验指导[M].北京:中国农业科学技术出版社,2002:55-57,83-88
[21]曾小鲁.实用生物学制片技术[M].北京:高等教育出版社,1987:177-178
[22] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative pcr and the 2-??ct method[J]. Methods, 2001,25(4):402-408
[23]胡美君,郭延平,沈允钢,等.柑橘属光合作用的环境调节[J].应用生态学报,2006,17(3):537-540
[24]李林锋.氮磷钾配方施肥对鸦胆子幼苗光合特性的影响[J].江西农业大学学报,2010,32(6):1136-1141
[25] Boussadia O, Steppe K, Zgallai H, et al. Effects of nitrogen defficiency on leaf photosynthesis, carbohydrate status and biomass production and biomass production in two olive cultivars ‘Meski’and ‘Koroneiki’[J]. Sci Horti, 2010,123(3):336-342
[26] Slewinski TL, Braun DM. Current perspectives on the regulation of whole–plant carbohydrate partitioning[J]. Plant Sci,2010,178(4):341-349
[27] Knoblauch M, Peters WS. Münch, morphology, microfluidics–our structural problem with the phloem[J]. Plant Cell and Environ, 2010,33(9):1439-1452
[28]张桂茹,杜维广,满为群,等.不同光合特性大豆叶的比较解剖研究[J].植物学通报,2002,19(2):208-214
[29]王亚菲.施氮量对棉花生长发育和叶片微观结构的影响[D].保定:河北农业大学,2015:25-30
[30] Morales F, Grasa R, Abadía A, et al. Iron chlorosis paradox in fruit trees[J]. J Plant Nutr, 1998,21(4):815-825
[31] Armbruster U, Pesaresi P, Pribil M, et al. Update on chloroplast research:new tools, new topics, and new trends[J]. Mol Plant, 2011,4(1):1-16
[2] Kumar AM, Caankovszki G, Soll D. A second and differentially expressed glutamyl–t RNA reductase gene from Arabidopsis thaliana[J]. Plant Mol Biol, 1996,30(3):419-426
[3] Adhikari ND, Froehlich JE, Strand DD, et al. GUN4–porphyrin complexes bind the CNH/GUN5 subunit of Mg–chelatase and promote chlorophyll biosynthesis in Arabidopsis[J]. Plant Cell, 2011,23(4):1449-1467
[4]李清.大豆叶片黄化基因的克隆与功能分析[D].北京:中国科学院大学,2016:20-24
[5]张修德.苹果叶绿素合成酶关键基因MdHEMA1基因克隆和功能验证[D].北京:中国农业科学院.2016:19-21
[6]张宝懿,刘诗诗,崔继哲.镁原卟啉Ⅸ甲基转移酶研究进展[J].核农学报,2017,31(6):1086-1091
[7] Meskauskiene R, Nater M, Goslings D, et al. FLU:A negative regulator of chlorophyll biosynthesis in Arabidopsis thalian A[J]. Proc Natl Acad Sci, 2001,98(22):826-831
[8] Goslings D, Meskauskiene R, Kim C, et al. Concurrent interactions of heme and FLU with Glu t RNA reductase(HEMA1), the target of metabolic feedback inhibition of tetrapyrrole biosynthesis, in dark–and lightgrown Arabidopsis plants[J]. Plant J,2010,40(6):957-967
[9] Xu GH, Fan XR, Miller AJ. Plant nitrogen assimilation and use efficiency[J]. Annu Rev Plant Biol, 2012,63(1):153-182
[10]李文庆,张民,束怀瑞.氮素在果树上的生理作用[J].山东农业大学学报:自然科学版,2002,33(1):96-100
[11]曲文章,蔡伯岩,荡妙真,等.氮素水平对甜菜主要营养的影响[J].中国甜菜糖业,1999(2):1-6
[12] Dejong TM, Day KR, Johnson RS. Partitioning of leaf nitrogen with respect to within canopy light exposure and nitrogen availability in peach[J]. Trees, 1989(3):89-95
[13] Stassen PJC, Terblanche JH, Strydom DK. The effect of time and rate of nitrogen application on development and composition of peach trees[J]. Agroplantae,1981(13):55-61
[14] Nii N, Kato M. Starch accumulation and photosynthesis on leaves of young peach trees grown under different levels of nitrogen application[J]. J Jpn Soc Hortic Sci, 1993,62(3):547-554
[15] Fallahi E, Possingham JV. Productivity, postharvest physiology and soil nitrate movement as influenced by nitrogen application to ‘Delicious’ apple[J]. Acta Hortic, 2000,512:149-157
[16] Makino A, Shimada T, Takumi S, et al. Does decrease in Ribulose–1,5–bisphosphate carboxylase by antisense rbcs lead to a higher N–use efficiency of photosynthesis under conditions of saturating CO2 and light in rice plant?[J]. Plant physiol, 1997,114(2):483-491
[17]李彩,彭良志,党江波,等.不同施氮水平纽荷尔脐橙叶绿素含量的季节变化[J].中国南方果树,2012,41(2):14-18
[18]田娟,张崇玉,熊永琴,等.输液肥对柑橘叶片叶绿素变化的影响[J].贵州农业科学,2008,36(4):143-145
[19]周学伍,黄辉北,黄永.柑橘叶片叶绿素含量的年周期变化及与N含量的关系[J].西南农业大学学报,1985(4):34-37
[20]赵世杰,史国安,董新纯.植物生理学实验指导[M].北京:中国农业科学技术出版社,2002:55-57,83-88
[21]曾小鲁.实用生物学制片技术[M].北京:高等教育出版社,1987:177-178
[22] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative pcr and the 2-??ct method[J]. Methods, 2001,25(4):402-408
[23]胡美君,郭延平,沈允钢,等.柑橘属光合作用的环境调节[J].应用生态学报,2006,17(3):537-540
[24]李林锋.氮磷钾配方施肥对鸦胆子幼苗光合特性的影响[J].江西农业大学学报,2010,32(6):1136-1141
[25] Boussadia O, Steppe K, Zgallai H, et al. Effects of nitrogen defficiency on leaf photosynthesis, carbohydrate status and biomass production and biomass production in two olive cultivars ‘Meski’and ‘Koroneiki’[J]. Sci Horti, 2010,123(3):336-342
[26] Slewinski TL, Braun DM. Current perspectives on the regulation of whole–plant carbohydrate partitioning[J]. Plant Sci,2010,178(4):341-349
[27] Knoblauch M, Peters WS. Münch, morphology, microfluidics–our structural problem with the phloem[J]. Plant Cell and Environ, 2010,33(9):1439-1452
[28]张桂茹,杜维广,满为群,等.不同光合特性大豆叶的比较解剖研究[J].植物学通报,2002,19(2):208-214
[29]王亚菲.施氮量对棉花生长发育和叶片微观结构的影响[D].保定:河北农业大学,2015:25-30
[30] Morales F, Grasa R, Abadía A, et al. Iron chlorosis paradox in fruit trees[J]. J Plant Nutr, 1998,21(4):815-825
[31] Armbruster U, Pesaresi P, Pribil M, et al. Update on chloroplast research:new tools, new topics, and new trends[J]. Mol Plant, 2011,4(1):1-16
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