Effects of different phosphorus levels on proline biosynthesis and accumulation and key gene expression in sugarcane
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摘要:目的
探索不同磷肥量对甘蔗脯氨酸的合成积累影响, 以阐明磷肥对甘蔗脯氨酸合成积累及其耐旱性的作用效应.
方法以甘蔗栽培种粤糖55为研究材料, 用桶栽试验法, 在正常水分和干旱胁迫条件下, 测定4个磷肥施用水平的甘蔗植株内P5CS、δ-OAT基因表达和酶活性、以及游离脯氨酸、叶绿素含量等生理生化指标.
结果和结论无论是在正常供水条件下还是干旱胁迫下, 甘蔗P5CS、δ-OAT基因的表达及酶活性、游离脯氨酸含量均受到N、P、K配比的影响.当施用过磷酸钙为900 kg·hm-2时, 正常水分条件下, P5CS、δ-OAT酶活性均处于较低的水平, 植株游离脯氨酸含量最低; 干旱胁迫下, P5CS酶活性最高, δ-OAT酶活性亦处于较高水平, 植株游离脯氨酸含量增加到最大.基于本研究的结果, 湛江砖红壤蔗地的最佳N、P、K肥搭配方案是尿素、过磷酸钙、氯化钾的用量分别为918、900、750 kg·hm-2.相关、偏相关及通径分析的结果表明, 干旱胁迫下植株中P5CS对游离脯氨酸的合成积累贡献显著大于δ-OAT; 干旱胁迫下甘蔗合成积累游离脯氨酸的途径是以谷氨酸途径(Glu→Pro)为主, 鸟氨酸途径(Orn→Pro)为辅.
Abstract:ObjectiveDifferent phosphorus(P) fertilizer rates were applied to clarify the effect of P fertilizer on proline biosynthesis and accumulation and drought-resistance in sugarcane.
MethodSugarcane variety YT-55 was used as the plant material for barrel experiment.Under the normal and drought conditions, four P levels were designed.The physiological and biochemical indexes such as the expressions of P5CS and δ-OAT genes, enzyme activities and the contents of free proline and chlorophyll were determined.
Result and conclusionThe expressions of P5CS and δ-OAT genes, enzyme activities and the free proline content were affected by the ratios of N, P and K fertilizers under the normal and drought conditions.When the P level was 900 kg·hm-2, both P5CS and δ-OAT activities were relatively low, and the free proline content was the lowest in the plants under the normal water condition.The P5CS activity was the highest; the δ-OAT activity was also at a high level; and the free proline content was up to the maximum amount under drought stress.Based on the results, the optimum levels of N, P and K fertilizers are 918 kg·hm-2urea, 900 kg·hm-2calcium superphosphate and 750 kg·hm-2potassium chloride in Zhanjiang lateritic soil.The results of correlation, partial correlation and path analysis indicated that the P5CS was more effective than δ-OAT in free proline biosynthesis and accumulation of sugarcane under drought stress.The main pathway of free proline biosynthesis and accumulation is glutamic acid pathway (Glu→Pro) supplemented by ornithine pathway (Orn→ Pro) under drought stress.
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Keywords:
- sugarcane /
- phosphorus application rate /
- proline content /
- δ-OAT /
- P5CS /
- gene expression /
- drought stress
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图 1 不同磷肥水平脯氨酸含量的变化
图中凡是有一个相同小写字母者, 表示差异不显著(Duncan’s法, P＞0.05).
Figure 1. The changes of proline contents at different phosphorus levels
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图 2 不同磷肥水平P5CS酶活性的变化
图中凡是有一个相同小写字母者, 表示差异不显著(Duncan’s法, P＞0.05).
Figure 2. The changes of P5CS enzyme activities at different phosphorus levels
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图 3 不同磷肥水平δ-OAT酶活性的变化
图中凡是有一个相同小写字母者, 表示差异不显著Duncan’s法, P＞0.05).
Figure 3. The changes of δ-OAT enzyme activities at different phosphorus levels
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图 4 不同磷肥水平叶片叶绿素含量的变化
图中凡是有一个相同小写字母者, 表示差异不显著(Duncan’s法, P＞0.05).
Figure 4. The changes of leaf chlorophyll contents at different phosphorus levels
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图 5 叶片总RNA电泳图
泳道11 ~ 14分别为干旱胁迫下过磷酸钙用量分别为0、900、1 800、2 700 kg·hm-2的样品, 21、22分别是11、12的重复样品.
Figure 5. Electrophoresis of total RNA of leaves
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图 6 P5CS和δ-OAT的PCR产物电泳图
M:DL500 DNA Marker; 1:P5CS; 2:δ-OAT.
Figure 6. Electrophoresis of the PCR products of P5CS and δ-OAT
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图 7 干旱胁迫下甘蔗叶片中P5CS、δ-OAT基因的相对表达量
各图中柱子上凡是有一个相同小写字母者, 表示差异不显著(Duncan’s法, P＞0.05).
Figure 7. Relative expressions of P5CS and δ-OAT genes in sugarcane leaves under drought stress
表 1 正常水分下生理指标间的简单和偏相关分析1)
Table 1 Simple and partial correlation analyses of physiological parameters at the normal water level
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表 2 干旱胁迫下个生理指标间的简单和偏相关性分析
Table 2 Simple and partial correlation analyses of physiological parameters under drought stress
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表 3 正常水分下X1、X2和X3对Y的通径分析1)
Table 3 The path analyses of X1, X2 and X3 to Y at the normal water level
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表 4 干旱胁迫下X1、X2和X3对Y的通径分析1)
Table 4 The path analyses of X1, X2 and X3 to Y under drought stress
下载: 导出CSV
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[1] 轻工业部甘蔗糖业科学研究所, 广东省农业科学院.中国甘蔗栽培学[M].北京:中国农业出版社, 1985:225-228. [2] 焦蓉, 刘好宝, 刘贯山, 等.论脯氨酸积累与植物抗渗透胁迫[J].中国农学通报, 2011, 27(7):216-221. http://d.old.wanfangdata.com.cn/Periodical/zgnxtb200204025 [3] HU C A, DELAUNEY A J, VERMA D P S.A bifunctional enzyme (△1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants [J].Proc Natl Acad Sci USA, 1992, 89 (19):93549358. doi: 10.1073/pnas.89.19.9354
[4] DELAUNEY A J, HU C A, KISHORP B K, et al.Cloning of ornithine-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis[J].J Biol Chem, 1993, 268(25):1867318678.
[5] SZABADOS L, SAVOUR A.Proline:A multifunctional amino acid[J].Trends Plant Sci, 2009, 15(2):89-97. http://d.old.wanfangdata.com.cn/NSTLQK/NSTL_QKJJ022499954/
[6] LEHMANN S, FUNK D, SZABADOS L.Proline metabolism and transport in plant development[J ].Amino Acids, 2010, 39(4):949-962. doi: 10.1007/s00726-010-0525-3
[7] ROOSENS N H C J, THU T T, ISKANDAR H M, et al.Isolation of ornithine-δ- aminotransferase cDNA and effect of salt stress on its expression in Arabidopsis[J].Plant Physiol, 1998, 117(1):263-271. doi: 10.1104/pp.117.1.263
[8] 钱大文, 周鸿凯, 江大可, 等.NaCl胁迫下海马齿(Sesuvium portulacastrum L.)植株内游离脯氨酸的合成积累途径[J].中国野生植物资源, 2013, 32(3):35-39. doi: 10.3969/j.issn.1006-9690.2013.03.010 [9] 黄诚梅, 毕黎明, 杨丽涛, 等.聚乙二醇胁迫对甘蔗伸长期间叶中脯氨酸积累及其代谢关键酶活性的影响[J].植物生理学通讯, 2007, 43(1):77-80. http://d.old.wanfangdata.com.cn/Periodical/zwslxtx200701016 [10] 杨俊兴, 张彤, 吴冬秀.磷素营养对植物抗旱性的影响[J].广东微量元素科学, 2003, 10(12):13-19. doi: 10.3969/j.issn.1006-446X.2003.12.002 [11] 李唯.植物生理学[M].北京:高等教育出版社, 2012:46-47. [12] 舒玉. 磷肥对草地早熟禾和黑麦草抗旱性的影响[D]. 哈尔滨: 东北林业大学, 2008. [13] 赵海超, 龚学臣, 抗艳红, 等.不同施磷量对马铃薯苗期抗旱性的影响[J].安徽农业科学, 2012, 40(6):33143316. doi: 10.3969/j.issn.0517-6611.2012.06.038 [14] XU L, HUANG H R, YANG L T.Combined application of NPK on yield quality of sugarcane applied through SSDI [J].Sugar Tech, 2010, 12(2):104-107. doi: 10.1007/s12355-010-0021-9
[15] 刘永贤, 曾维宾, 熊柳梅, 等不同磷肥用量对桂中蔗区甘蔗生长性状与产量的影响[J].南方农业学报, 2012, 43(5):634-636. doi: 10.3969/j:issn.2095-1191.2012.05.634 [16] 许树宁, 方锋学, 唐仕云, 等.不同施肥量对甘蔗产量和蔗糖分的影响[J].现代农业科技, 2012(19):9-10. doi: 10.3969/j.issn.1007-5739.2012.19.001 [17] 陈如凯, 张木清, 陆裔波.干旱胁迫对甘蔗生理影响的研究[J].甘蔗, 1995, 2(1):1-6. http://d.old.wanfangdata.com.cn/Periodical/gxnykx201208008 [18] 杨建波, 诸葛少军, 黎海涛, 等.干旱胁迫对甘蔗生长生理的影响及品种抗旱性评价[J].南方农业学报, 2012, 43(8):1114-1120. doi: 10.3969/j:issn.2095-1191.2012.08.1114 [19] BATES L S, WALDREN R P, TEARE I D.Rapid determination of free proline for water-stress studies[J].Plant Soil, 1973, 39(1):205-207. doi: 10.1007/BF00018060
[20] KAVI KISHOR P B, HONG Z L, MIAO G H, et al.Overexpression of Δ1-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants[J].Plant Physiol, 1995, 108 (4):1387-1394. doi: 10.1104/pp.108.4.1387
[21] 黄诚梅. 甘蔗脯氨酸积累与△1-吡咯啉-5-羧酸合成酶(ScP5CS)基因克隆及转化研究[D]. 南宁: 广西大学, 2007: 17-18. [22] KIM H R, RHO H W, PARK J W, et al.Assay of ornithine aminotransferase with ninhydrin[J].Analytical Biochem, 1994, 223(2):205-207. http://d.old.wanfangdata.com.cn/NSTLQK/NSTL_QKJJ0216957306/
[23] 李合生.植物生理生化实验原理和技术[M].北京:高等教育出版社, 2000:164-165. [24] LIVAK K J, SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods, 2001, 25(4):402-408. doi: 10.1006/meth.2001.1262
[25] 阙友雄, 许莉萍, 徐景升, 等.甘蔗基因表达定量PCR分析中内参基因的选择[J].热带作物学报, 2009, 30 (3):274-278. doi: 10.3969/j.issn.1000-2561.2009.03.006 [26] IGARASHI Y, YOSHIBA Y, TAKESHITA T.Molecular cloning and characterization of a cDNA encoding proline transporter in rice[J].Plant Cell Physiol, 2000, 41(6):750-756. doi: 10.1093/pcp/41.6.750
[27] BRUGIEREA N, DUBOISB F, LIMAMIA A M.Glutamine synthetase in the phloem plays a major role in controlling proline production[J].Plant Cell, 1999, 11(10):19952012. doi: 10.1105/tpc.11.10.1995
[28] 王庭璋, 胡望雄, 徐建红, 等.植物基因组上游开放阅读框的挖掘方法及其翻译调控[J].中国农业科学, 2013, 46(16):3314-3323. doi: 10.3864/j.issn.0578-1752.2013.16.002 [29] 刘丽, 甘志军, 王宪泽.植物氮代谢硝酸还原酶水平调控机制的研究进展[J].西北植物学报, 2004, 24 (7):1355-1361. doi: 10.3321/j.issn:1000-4025.2004.07.038 [30] 谭彩霞, 郭静, 陈静, 等.小麦籽粒淀粉合成酶基因表达与酶活性特征的研究[J].麦类作物学报, 2009, 29 (1):24-30. http://d.old.wanfangdata.com.cn/Periodical/mlzwxb200901005 [31] VOCTBERG G S, SHARP R E.Growth of the maize primary root at low water potcntia:3:Role of increased proline deposition in osmotic adjustment[J].Plant Physiol, 1991, 96(4):1125-1130. doi: 10.1104/pp.96.4.1125
[32] SCHWACKE R, GRALLATH S, BRCITKREUZ K E, et al.LeProTl, a transproter for proline, glycine betaine and γ-amino butyric acid in tomato pollen[J].The Plant Cell, 1999, 11(3):377-391.
[33] ROSA P, ROSARIO A, JUAN M R.Evaluation of the role of genes encoding for Δ1-pyrroline-5-carboxylate synthetase (P5CS) during drought stress in arbuscular mycorrhizal Glycine max and Lactuca sativa plants[J].Physiol Mol Plant P, 2004, 65(4):211-221. doi: 10.1016/j.pmpp.2005.02.003
[34] 王丽媛, 丁国华, 黎莉.脯氨酸代谢的研究进展[J].哈尔滨师范大学自然科学学报, 2010, 26(2):84-89. doi: 10.3969/j.issn.1000-5617.2010.02.024 [35] 曹芳, 魏永胜.渗透胁迫下硝酸钾对烟草脯氨酸代谢途径的影响[J].西北农业学报, 2010, 19 (9):144148. doi: 10.3969/j.issn.1004-1389.2010.09.029 [36] 姜淑欣, 刘党校, 庞红喜, 等.PEG胁迫及复水对不同抗旱性小麦幼苗脯氨酸代谢关键酶活性的影响[J].西北植物学报, 2014, 34(8):1581-1587. http://d.old.wanfangdata.com.cn/Periodical/xbzwxb201408012 [37] CHOUDHARY N L, SAIRAM R K, TYAGI A.Expression of D1-pyrroline-5-carboxylate synthetase gene during drought in rice(Oryza sativa L.)[J].Indian J Biochem Bio, 2005, 42(6):366-370. https://www.ncbi.nlm.nih.gov/pubmed/22226812
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