高温胁迫下黄嘌呤脱氢酶基因超表达对水稻幼苗的保护作用

doi:10.16819/j.1001-7216.2018.7130

摘要/Abstract

摘要：

【目的】黄嘌呤脱氢酶(xanthine dehydrogenase, XDH)是嘌呤代谢的关键酶。通过分析高温胁迫对OsXDH超表达转基因水稻株系幼苗叶片生理指标的影响,探究XDH缓解水稻高温胁迫的生理机制。【方法】以OsXDH超表达转基因株系(xdh1和xdh5)及受体品种日本晴(WT)为材料,研究了高温胁迫对水稻幼苗叶片叶绿素含量、相对含水量、可溶性蛋白含量、活性氧代谢、抗氧化酶活性、XDH活性及嘌呤代谢产物尿囊素(allantoin)和尿囊酸(allantoate)含量等生理指标的影响。【结果】高温胁迫处理前,超表达株系的叶绿素含量、相对含水量、可溶性蛋白含量、活性氧代谢及抗氧化酶活性等生理指标均与野生型无显著差异;高温胁迫5 d,超表达株系的叶绿素、相对含水量、可溶性蛋白含量及抗氧化酶活性均高于野生型,而过氧化氢(H₂O₂)及丙二醛(MDA)的含量显著低于野生型;适温恢复生长5 d,野生型和超表达株系的叶绿素含量、相对含水量及可溶性蛋白含量均有所提高,超表达株系均高于野生型,H₂O₂和MDA的含量降低,超表达株系均低于野生型;受高温诱导超表达株系与野生型中XDH酶活性及尿囊素和尿囊酸的含量均提高,且在整个处理过程中超表达转基因株系均高于野生型。【结论】XDH通过调控酰脲类物质的合成,补偿自身的抗氧化能力并增强抗氧化酶系统的活性,从而有效提高水稻幼苗对高温胁迫的耐受能力。

关键词: 水稻, 黄嘌呤脱氢酶, 高温胁迫, 生理机制

Abstract:

【Objective】Xanthine dehydrogenase (XDH) is considered as a key enzyme in purine metabolism. This paper aims to reveal the physiological mechanism of XDH to relieve high temperature stress by analyzing the physiological indexes of rice seedling leaves differed in expression level of OsXDH under high temperature stress. 【Method】In this work, chlorophyll content, relative water content, soluble protein content, reactive oxygen metabolism, antioxidant enzyme activities, XDH activity, allantoin and allantoate contents were measured under high temperature stress in OsXDH over-expressed transgenic lines and wild type during seedling stage. 【Result】The results revealed that there were no significant difference between wild type and transgenic lines in all tested physiological parameters except XDH activity and ureides (allantoin and allantoate) content before high temperature stress. After high temperature stress for 5 days, compared with the transgenic lines, seedlings of wild type showed significantly decreased chlorophyll content, relative water content, soluble protein content, and antioxidant enzyme activities, but significantly increased contents of H₂O₂ and Malondialdehyde(MDA). As we know that chlorophyll content, relative water content, and soluble protein content all increased in transgenic lines and wild type after post-stress recovery, especially in transgenic lines. H₂O₂ and MDA content all decreased. For transgenic lines, these indexes were lower than those in wild type. The XDH enzyme activity and purine metabolites allantoin and allantoate contents in the wild type and transgenic lines were induced by high temperature. OsXDH over-expressed transgenic lines had significantly higher XDH enzyme activity and allantoin and allantoate contents compared with wild type throughout the process. 【Conclusion】XDH can effectively improve the tolerance of rice seedlings to high temperature stress by regulating the synthesis of ureides, compensating their antioxidant capacity and enhancing the activities of antioxidant enzymes.

Key words: rice, xanthine dehydrogenase, high temperature stress, physiological mechanism

中图分类号:

韩瑞才, 苏如奇, 万建林, 龙启樟, 曾勇军, 潘晓华, 石庆华, 吴自明. 高温胁迫下黄嘌呤脱氢酶基因超表达对水稻幼苗的保护作用[J]. 中国水稻科学, 2018, 32(4): 365-373.

Ruicai HAN, Ruqi SU, Jianlin WAN, Qizhang LONG, Yongjun ZENG, Xiaohua PAN, Qinghua SHI, Ziming WU. Protective Roles of Over-expression of OsXDH in Rice Seedlings Under High Temperature Stress[J]. Chinese Journal OF Rice Science, 2018, 32(4): 365-373.

图/表 6

图1 OsXDH超表达载体的构建、转基因植株的鉴定和目的基因表达量检测 A–超表达载体结构。LB为T-DNA左边界,Hyg为潮霉素抗性筛选基因,35S为花椰菜花叶病毒启动子,Ubi为ubiqutin基因启动子,Kpn I为酶切位点,RB为T-DNA右边界。B–T2代转基因植株PCR阳性鉴定。WT为野生型,1~16为16个随机选取的T2代转基因植株。C–转基因植株OsXDH基因的表达量。xdh1为OsXDH超表达株系1,xdh5为OsXDH超表达株系5。数据用平均数±标准差表示(n=3)。**表示在0.01水平上差异极显著(LSD)。

Fig. 1. Construction of OsXDH over-expression vector, PCR analysis of OsXDH gene in T2 transgenic plants and expression analysis of OsXDH in over-expressed lines. A, Structure of OsXDH over-expression vector. LB, Left border of T-DNA; Hyg, Hygromycin gene; 35S, 35S promoter of CaMV; Ubi, Promoter of ubiqutin; Kpn I, Restriction enzyme cutting site; RB, Right border of T-DNA. B, PCR identification of the T2 transgenic plants. WT, Wild type; 1–16, 16 randomly selected T2 transgenic plants. C, Relative expression level of OsXDH in transgenic plants. xdh1, OsXDH over-expressed transgenic line 1; xdh5, OsXDH over-expressed transgenic line 5. Values are shown as mean±SD (n=3). **, Significant difference at P<0.01 level by LSD.

图2 高温胁迫对水稻叶片叶绿素含量、可溶性蛋白含量和相对含水量的影响 A–叶绿素含量; B–可溶性蛋白含量; C–相对含水量。BS为高温处理前,HTSFD为高温处理5 d,PSRFD为高温处理后适温恢复生长5 d。WT为野生型,xdh1为OsXDH超表达株系1,xdh5为OsXDH超表达株系5。数据用平均数±标准差表示(n=3),*表示在0.05水平上差异显著(LSD)。

Fig. 2. Effects of high temperature stress on chlorophyll content, soluble protein content and relative water content in rice leaves. A, Chlorophyll content; B, Soluble protein content; C, Relative water content. BS, Before high temperature stress; HTSFD, High temperature stress for 5 days; PSRFD, Post-stress recovery for 5 days. WT, Wild type; xdh1, OsXDH over-expressed transgenic line 1; xdh5, OsXDH over-expressed transgenic line 5. Values are shown as mean±SD (n=3). *, Significant difference at P<0.05 level by LSD.

图3 高温胁迫对水稻叶片过氧化氢含量和丙二醛含量的影响 A–过氧化氢含量; B–丙二醛含量。BS为高温处理前; HTSFD为高温处理5 d; PSRFD为适温恢复生长5 d。WT为野生型; xdh1为OsXDH超表达株系1; xdh5为OsXDH超表达株系5。数据用平均数±标准差表示(n=3)。 *和**分别表示在0.05和0.01水平上显著差异(LSD)。

Fig. 3. Effects of high temperature stress on H2O2 and Malondialdehyde(MDA) content in rice leaves. A, H2O2 content; B, MDA content. BS, Before high temperature stress; HTSFD, High temperature stress for 5 days; PSRFD, Post-stress recovery for 5 days. WT, Wild type; xdh1, OsXDH over-expressed transgenic line 1; xdh5, OsXDH over-expressed transgenic line 5. Values are shown as mean±SD (n=3). * and **, Significant difference at P<0.05 and P<0.01 levels by LSD, respectively.

图4 高温胁迫对水稻叶片超氧化物歧化酶、过氧化物酶和过氧化氢酶活性的影响 A–超氧化物歧化酶活性; B–过氧化物酶活性; C–过氧化氢酶活性。BS为高温处理前; HTSFD为高温处理5 d; PSRFD为适温恢复生长5 d。WT为野生型; xdh1为OsXDH超表达株系1; xdh5为OsXDH超表达株系5。数据用平均数±标准差表示(n=3)。 *,**分别表示在0.05和0.01水平上显著差异(LSD)。

Fig. 4. Effects of high temperature stress on activities of superoxide dismutase(SOD), peroxidase(POD) and catalase(CAT) in rice leaves. A, SOD activity; B, POD activity; C, CAT activity. BS, Before high temperature stress; HTSFD, High temperature stress for 5 days; PSRFD, Post-stress recovery for 5 days. WT, Wild type; xdh1, OsXDH over-expressed transgenic line 1; xdh5, OsXDH over-expressed transgenic line 5. Values are shown as mean±SD (n=3). * and **, Significant difference at P<0.05 and P<0.01 level by LSD, respectively.

图5 高温胁迫对水稻叶片黄嘌呤脱氢酶活性的影响 BS为高温处理前; HTSFD为高温处理5 d; PSRFD为适温恢复生长5 d。WT为野生型; xdh1为OsXDH超表达株系1; xdh5为OsXDH超表达株系5。每条通道可溶性蛋白的上样量均为50μg。以次黄嘌呤作为底物,条带亮度为XDH酶相对活性。用软件ImageJ2x分析条带亮度,显示酶活相对值。

Fig. 5. Effects of high temperature stress on xanthine dehydrogenase(XDH) activity in rice leaves. BS, Before high temperature stress; HTSFD, High temperature stress for 5 days; PSRFD, Post-stress recovery for 5 days. WT, Wild type; xdh1, OsXDH over-expressed transgenic line 1; xdh5, OsXDH over-expressed transgenic line 5. Each lane in the gel was loaded with equal content soluble protein. XDH activity was detected in gel with hypoxanthine as substrate. Numbers above the lanes indicate relative intensity obtained by scanning the formazan bands with a computing laser densitometer using ImageJ2x software.

图6 高温胁迫对水稻叶片尿囊素和尿囊酸含量的影响 A–尿囊素含量; B–尿囊酸含量。BS为高温处理前,HTSFD为高温处理5 d,PSRFD为适温恢复生长5 d。WT为野生型,xdh1为OsXDH超表达株系1,xdh5为OsXDH超表达株系5。数据用平均数±标准差表示(n=3)。*,**分别表示在0.05和0.01水平上差异显著(LSD)。

Fig. 6. Effects of high temperature stress on allantoin and allantoate contents in rice leaves. A, Allantoin content. B, Allantoate content. BS, Before high temperature stress; HTSFD, High temperature stress for 5 days; PSRFD, Post-stress recovery for 5 days. WT, Wild type; xdh1, OsXDH over-expressed transgenic line 1; xdh5, OsXDH over-expressed transgenic line 5. Values are shown as mean±SD (n=3). *, and **, Significant difference at P<0.05 and P<0.01 levels by LSD, respectively.

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