过表达兴安落叶松<i>TPP</i>基因增强拟南芥对盐胁迫的耐受能力<sup>*</sup>

doi:10.13523/j.cb.2203056

中国生物工程杂志

2022, Vol. 42

Issue (8): 21-29 DOI: 10.13523/j.cb.2203056

研究报告

过表达兴安落叶松TPP基因增强拟南芥对盐胁迫的耐受能力^*

张燕霞¹,张旭婷¹,贾永红²,雷凤燕³,王静¹,王瑞刚^1,⁴,李国婧^1,^**(

)

1.内蒙古农业大学植物逆境生理与分子生物学自治区重点实验室呼和浩特 010018
2.内蒙古农业大学职业技术学院包头 014109
3.通辽市农牧局综合保障中心通辽 028000
4.蒙树生态建设集团有限公司林木育种自治区企业重点实验室呼和浩特 011517

Overexpression of TPP from Larix gmelinii Enhanced Salt Tolerance of the Transgenic Arabidopsis thaliana

ZHANG Yan-xia¹,ZHANG Xu-ting¹,JIA Yong-hong²,LEI Feng-yan³,WANG Jing¹,WANG Rui-gang^1,⁴,LI Guo-jing^1,^**(

)

1. Inner Mongolia Key Laboratory of Plant Stress Physiology and Molecular Biology, College of Life Sciences, Inner Mongolia Agricultural University, Hohhot 010018, China
2. Vocational and Technical College, Inner Mongolia Agricultural University, Baotou 014109, China
3. Agriculture and Animal Husbandry Bureau of Ongniud Banner of Tongliao City, Tongliao 028000, China
4. Inner Mongolia Enterprise Key Laboratory of Tree Breeding, Mengshu Ecological Construction Group Co.,Ltd., Hohhot 011517, China

全文: PDF(2633 KB) HTML

摘要：

兴安落叶松(Larix gmelinii)是极为重要的造林针叶树种,具有早期速生、抗逆性强、生态效益好等特点。海藻糖参与调控干旱、寒冷、盐害等多种逆境胁迫,海藻糖-6-磷酸磷酸酶(TPP)是海藻糖合成通路的重要酶。从兴安落叶松逆境胁迫转录组中筛选到LgTPPI.1基因全长序列,克隆了其编码区(CDS),构建了重组载体并获得过表达LgTPPI.1拟南芥纯合株系。结果表明,LgTPPI.1 CDS全长1 236 bp,共编码411个氨基酸;LgTPPI.1基因的mRNA在根和茎中表达水平较低,在针叶中表达水平较高;过表达LgTPPI.1基因拟南芥在盐胁迫下海藻糖含量显著提高、脯氨酸和抗氧化酶类活性增加、胁迫响应标记基因表达上调、对盐胁迫的耐受性增强。这些结果表明,裸子植物利用与被子植物类似的海藻糖通路来耐受非生物胁迫。为后续进一步解析落叶松中海藻糖合成相关基因的功能,揭示裸子植物中针叶树对逆境的响应机制奠定基础。

关键词： 兴安落叶松; 盐胁迫; 海藻糖; TPP; 拟南芥

Abstract:

Larix gmelinii is a very important coniferous tree species for afforestation. It has the characteristics of rapid growth in early stage, strong stress resistance and good ecological benefits. Trehalose participates in the regulation of drought, cold, salt damage and other stresses. Trehalose-6-phosphate phosphatase (TPP) is an important enzyme in the trehalose synthesis pathway. In this study, the full-length sequence of LgTPPI.1 was screened from the transcriptome of L. gmelinii under stress and its coding sequence(CDS) was cloned. The recombinant vector was constructed and homozygous lines of transgenic Arabidopsis thaliana overexpressing LgTPPI.1 were obtained. The results showed that the full-length CDS of LgTPPI.1 was 1 236 bp, encoding 411 amino acids; the expression level of LgTPPI.1 was lower in roots and stems, but higher in needles. Under salt treatment, the LgTPPI.1 overexpression lines conferred stronger tolerance than the wild type A. thaliana, with elevated trehalose and proline content; increased superoxide dismutase(SOD) and catalase(CAT) activity and up-regulated expresssion of the stress-responsive marker genes. These results indicated that gymnosperm utilized similar trehalose pathway as angiosperm to telerate abiotic stress. This study provided a theoretical basis for further analysis of the function of trehalose synthetic genes and the response mechanism of conifers under stress.

Key words: Larix gmelinii Salt stresses Trehalose TPP Arabidopsis thaliana

收稿日期: 2022-03-28 出版日期: 2022-09-07

ZTFLH:

Q812

基金资助: * 内蒙古自治区科技计划(2019GG007);国家转基因生物新品种培育科技重大专项(2018ZX08020-003);内蒙古自治区高等学校创新团队发展计划(NMGIRT2222)

通讯作者: 李国婧 E-mail: liguojing@imau.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	张燕霞
	张旭婷
	贾永红
	雷凤燕
	王静
	王瑞刚
	李国婧

引用本文:

张燕霞,张旭婷,贾永红,雷凤燕,王静,王瑞刚,李国婧. 过表达兴安落叶松TPP基因增强拟南芥对盐胁迫的耐受能力^*[J]. 中国生物工程杂志, 2022, 42(8): 21-29.

ZHANG Yan-xia,ZHANG Xu-ting,JIA Yong-hong,LEI Feng-yan,WANG Jing,WANG Rui-gang,LI Guo-jing. Overexpression of TPP from Larix gmelinii Enhanced Salt Tolerance of the Transgenic Arabidopsis thaliana. China Biotechnology, 2022, 42(8): 21-29.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2203056 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I8/21

表1 本研究所用到的引物列表

图1 LgTPPI.1基因的克隆产物及表达载体酶切验证电泳图

图2 LgTPPI.1基因及其编码的蛋白质序列

图3 LgTPPI.1与拟南芥同源蛋白的系统进化分析

图4 LgTPPI.1基因在兴安落叶松不同组织的表达模式分析

图5 35S∷HA-LgTPPI.1转基因拟南芥纯合株系中LgTPPI.1的表达量检测

图6 拟南芥过表达LgTPPI.1纯合株系和野生型对盐胁迫的耐受性比较

图7 盐胁迫下过表达LgTPPI.1基因株系和野生型拟南芥生理指标变化

图8 过表达LgTPPI.1基因株系和野生型拟南芥中胁迫响应基因表达量检测

图9 盐胁迫处理前后过表达LgTPPI.1基因拟南芥纯合株系和野生型拟南芥中海藻糖含量比较

[1]	Schiraldi C, di Lernia I, de Rosa M. Trehalose production: exploiting novel approaches. Trends in Biotechnology, 2002, 20(10): 420-425. pmid: 12220904
[2]	Kaplan F, Kopka J, Haskell D W, et al. Exploring the temperature-stress metabolome of Arabidopsis. Plant Physiology, 2004, 136(4): 4159-4168. pmid: 15557093
[3]	Pramanik M H R, Imai R. Functional identification of a trehalose 6-phosphate phosphatase gene that is involved in transient induction of trehalose biosynthesis during chilling stress in rice. Plant Molecular Biology, 2005, 58(6): 751-762. doi: 10.1007/s11103-005-7404-4 pmid: 16240171
[4]	Han B Y, Fu L L, Zhang D, et al. Inter species and intra species analysis of trehalose contents and the biosynthesis pathway gene family reveals crucial roles of trehalose in osmotic-stress tolerance in cassava. International Journal of Molecular Sciences, 2016, 17(7): 1077. doi: 10.3390/ijms17071077
[5]	Iturriaga G, Cushman M A F, Cushman J C. An EST catalogue from the resurrection plant Selaginella lepidophylla reveals abiotic stress-adaptive genes. Plant Science, 2006, 170(6): 1173-1184. doi: 10.1016/j.plantsci.2006.02.004
[6]	Ambastha V, Tiwari B S. Cellular water and anhydrobiosis in plants. Journal of Plant Growth Regulation, 2015, 34(3): 665-671. doi: 10.1007/s00344-015-9497-6
[7]	Ge L F, Chao D Y, Shi M, et al. Overexpression of the trehalose-6-phosphate phosphatase gene OsTPP1 confers stress tolerance in rice and results in the activation of stress responsive genes. Planta, 2008, 228(1): 191-201. doi: 10.1007/s00425-008-0729-x
[8]	冯小雨. 小麦TaTPP7基因调控抗旱及耐盐机制研究. 杨陵: 西北农林科技大学, 2021.
	Feng X Y. Regulation of drought and salt tolerance by TaTPP7 gene in wheat. Yangling: Northwest Agriculture & Forestry University, 2021.
[9]	Acosta-Pérez P, Camacho-Zamora B D, Espinoza-Sánchez E A, et al. Characterization of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase genes and analysis of its differential expression in maize (Zea mays) seedlings under drought stress. Plants (Basel, Switzerland), 2020, 9(3): 315.
[10]	丁泽红, 吴春来, 颜彦, 等. 木薯海藻糖-6-磷酸酯酶MeTPP6基因克隆及其表达分析. 江苏农业科学, 2019, 47(6): 31-35.
	Ding Z H, Wu C L, Yan Y, et al. Cloning and expression analysis of trehalose-6-phosphate phosphatase gene MeTPP6 in cassava. Jiangsu Agricultural Sciences, 2019, 47(6): 31-35.
[11]	赵天济. 柠条锦鸡儿海藻糖-6-磷酸磷酸酯酶基因的克隆及特性分析. 呼和浩特: 内蒙古大学, 2016.
	Zhao T J. Isolation and characterization of trehalose-6-phosphate phosphatase gene from Caragana korshinskii. Hohhot: Inner Mongolia University, 2016.
[12]	吕程佳, 李芳, 王梦荷, 等. 茶树TPP基因家族的鉴定及其对低温胁迫的响应. 南京农业大学学报, 2022, 45(1): 47-55.
	Lü C J, Li F, Wang M H, et al. Identification and response of TPP gene family to low temperature stress in tea plant. Journal of Nanjing Agricultural University, 2022, 45(1): 47-55.
[13]	李辉, 李德芳, 邓勇, 等. 红麻海藻糖生物合成关键酶基因HcTPPJ的克隆及响应逆境的表达分析. 作物学报, 2020, 46(12): 1914-1922. doi: 10.3724/SP.J.1006.2020.04006
	Li H, Li D F, Deng Y, et al. Cloning of the key enzyme gene HcTPPJ in trehalose biosynthesis of kenaf and its expression in response to abiotic stress in kenaf. Acta Agronomica Sinica, 2020, 46(12): 1914-1922. doi: 10.3724/SP.J.1006.2020.04006
[14]	马常耕, 孙晓梅. 我国落叶松遗传改良现状及发展方向. 世界林业研究, 2008, 21(3): 58-63.
	Ma C G, Sun X M. Larch genetic improvement and its future development in China. World Forestry Research, 2008, 21(3): 58-63.
[15]	白晓明. 长白落叶松LoCAT基因的克隆及功能分析. 哈尔滨: 东北林业大学, 2019.
	Bai X M. Cloning and function analysis of LoCAT gene from Larix olgensis. Harbin: Northeast Forestry University, 2019.
[16]	朱虹. TPPB的克隆及抗旱功能分析. 福州: 福建农林大学, 2019.
	Zhu H. Cloning and drought resistance function analysis of plant trehalose synthesis pathway gene TPPB. Fuzhou: Fujian Agriculture and Forestry University, 2019.
[17]	Krasensky J, Broyart C, Rabanal F A, et al. The redox-sensitive chloroplast trehalose-6-phosphate phosphatase AtTPPD regulates salt stress tolerance. Antioxidants & Redox Signaling, 2014, 21(9): 1289-1304.
[18]	Lin Q F, Yang J, Wang Q L, et al. Overexpression of the trehalose-6-phosphate phosphatase family gene AtTPPF improves the drought tolerance of Arabidopsis thaliana. BMC Plant Biology, 2019, 19(1): 381. doi: 10.1186/s12870-019-1986-5
[19]	Lin Q F, Wang S, Dao Y H, et al. Arabidopsis thaliana trehalose-6-phosphate phosphatase gene TPPI enhances drought tolerance by regulating stomatal apertures. Journal of Experimental Botany, 2020, 71(14): 4285-4297. doi: 10.1093/jxb/eraa173
[20]	Nuccio M L, Wu J, Mowers R, et al. Expression of trehalose-6-phosphate phosphatase in maize ears improves yield in well-watered and drought conditions. Nature Biotechnology, 2015, 33(8): 862-869. doi: 10.1038/nbt.3277
[21]	Jiang D G, Chen W T, Gao J E, et al. Overexpression of the trehalose-6-phosphate phosphatase OsTPP3 increases drought tolerance in rice. Plant Biotechnology Reports, 2019, 13(3): 285-292. doi: 10.1007/s11816-019-00541-4
[22]	王佳丽, 黄贤金, 钟太洋, 等. 盐碱地可持续利用研究综述. 地理学报, 2011, 66(5): 673-684.
	Wang J L, Huang X J, Zhong T Y, et al. Review on sustainable utilization of salt-affected land. Acta Geographica Sinica, 2011, 66(5): 673-684.

[1]	赵晓艳,陈允妲,章雅倩,吴晓玉,王飞,陈金印. **Myxococcus sp.V11海藻糖合酶TreS II分子改造 ^***[J]. 中国生物工程杂志, 2020, 40(3): 79-87.
[2]	封金云,宿玲恰,吴敬. 多酶复配合成海藻糖及其分离提取的研究 ^*[J]. 中国生物工程杂志, 2019, 39(7): 65-70.
[3]	杜立,宿玲恰,吴敬. 提高源自Bacillus circulans 251的β-CGTase对麦芽糖亲和性及其在生产海藻糖中的应用 ^*[J]. 中国生物工程杂志, 2019, 39(5): 96-104.
[4]	杨飞芸,杨天瑞,刘坤,崔爽,王瑞刚,李国婧. 异源表达CiRS基因拟南芥的黄酮代谢及抑菌能力研究 ^*[J]. 中国生物工程杂志, 2019, 39(11): 22-30.
[5]	刘翠翠, 胡梦蝶, 王志, 代俊, 姚娟, 李沛, 李志军, 陈雄, 李欣. 鲁氏酵母胞内海藻糖积累过程的代谢特征分析[J]. 中国生物工程杂志, 2017, 37(9): 41-47.
[6]	王曦光, 王娟, 张琳. 拟南芥蛋白质丰度与基因翻译效率关联分析[J]. 中国生物工程杂志, 2017, 37(2): 40-47.
[7]	杨飞芸,武燕燕,崔爽,张秀娟,王瑞刚,李国婧. 异源表达CiRS基因通过生成白藜芦醇增强拟南芥的抗氧化能力 ^*[J]. 中国生物工程杂志, 2017, 37(12): 27-33.
[8]	赵一瑾, 王腾飞, 汪俊卿, 王瑞明. 以CotC为分子载体在枯草芽孢杆菌表面展示海藻糖合酶[J]. 中国生物工程杂志, 2017, 37(1): 71-80.
[9]	李梦悦, 王腾飞, 汪俊卿, 赵一瑾, 程成, 王瑞明. 海藻糖合酶在毕赤酵母表面的展示[J]. 中国生物工程杂志, 2016, 36(2): 73-80.
[10]	于秀敏, 岳文冉, 张燕娜, 杨飞芸, 王瑞刚, 李国婧, 杨杞. *异源表达CkLEA1基因增强了拟南芥对非生物胁迫的耐受性*[J]. 中国生物工程杂志, 2016, 36(10): 28-34.
[11]	孙瑞芬, 张艳芳, 郭树春, 于海峰, 李素萍, 乔慧蕾, 聂惠, 安玉麟. *向日葵ACC氧化酶基因(HaACO1)的克隆及表达分析*[J]. 中国生物工程杂志, 2015, 35(9): 21-27.
[12]	聂利珍, 于肖夏, 李国婧, 孙杰, 姜超, 于卓. *Rd29A启动子驱动AtCDPK1基因转化马铃薯的研究*[J]. 中国生物工程杂志, 2015, 35(11): 13-22.
[13]	艾君, 姜潮, 刘敏, 王晓艳, 田海山, 李校堃. 拟南芥双油体蛋白融合表达KGF-2及其生物学活性研究[J]. 中国生物工程杂志, 2015, 35(1): 21-26.
[14]	孙瑞芬, 张艳芳, 郭树春, 于海峰, 李素萍, 乔慧蕾, 聂惠, 安玉麟. 向日葵盐胁迫相关基因的cDNA-AFLP差异表达[J]. 中国生物工程杂志, 2015, 35(1): 34-40.
[15]	于欣鑫, 高晋君, 李勇, 李晶. flg22诱导的拟南芥转录组分析及芥子油苷代谢途径的变化[J]. 中国生物工程杂志, 2014, 34(5): 30-38.

Viewed

Full text

Abstract

Cited

Shared

Discussed