发布日期：2019-08-15     浏览人数：352

  摘要：植物OST1在ABA信号转导、非生物逆境应答和生长发育过程中具有重要功能，但有关甜瓜OST1的研究尚未见报道。本文利用比较基因组学的方法从甜瓜基因组中鉴定出2个与拟南芥OST1直系同源的基因，并对它们的结构特征、遗传进化和顺式调控元件进行了系统分析。结果显示，这2个基因位于不同的染色体上，均含有9个外显子，编码蛋白的序列一致性为80.38%，且与黄瓜相对应的同源基因序列高度保守。二者的上游序列都含有多个应答不同激素和逆境的顺式调控元件,暗示其具有多样的生物学功能。本文为研究甜瓜OST1在逆境应答和生长发育中的功能奠定了基础。
关键词：甜瓜；OST1；特征；进化；顺式元件
Identification and characterization of two OST1 genes in melon 
Li Libin1, Liu Mingyu2, Sun Jianlei1, Wang Chongqi1, Xiao Shouhua1, Dong Yumei1§, Jiao Zigao1§
(1Vegetable and Flower Research Institute of Shandong Academy of Agricultural Sciences;Key Laboratory of Greenhouse Vegetables Biology of The Shandong Province;National Improvement Center for Vegetables, Shandong Branch,Jinan, 250100 )
2 Dongping County Seeds Limited Cooperation of Shandong Province, Dongping County of Shandong Province, 271500)
Abstract Plant OST1 gene plays important roles in ABA signaling, abiotic stress response and plant growth and development. However, the investigation of melon OST1 has not been reported at present. In this text, two novel OST1 like genes which orthologous to Arabidopsis OST1 were identified through comparative genomics method. And also their molecular features, evolution, and cis-elements were systematically analyzed. The results showed that these two melon OST1 locate on different chromosomes, and both have 9 introns. The identity of their encoded proteins is 80.38%, and they conserve with corresponding cucumber homologues. What’s more,  there are multiple cis-elements  response to  different hormones and stresses in their up-stream sequences, which suggests they may have pleiotropic roles in biological process. This text laid  a foundation for further functional dissection of them in melon abiotic stress response，growth and development.

表1 甜瓜OST1基因的命名和特征

  注：图中方框为外显子，方框之间的线条为内含子
  图1 甜瓜OST1基因的外显子-内含子结构
  2.2 甜瓜OST1基因的遗传进化和编码蛋白的基序分析
  不同物种或相同物种同一家族基因的遗传进化分析，可为研究基因的功能提供重要线索。甜瓜OST1的遗传进化分析结果表明，两个甜瓜基因CmOST1L1和CmOST1L2与黄瓜、拟南芥的OST1基因位于一个类群，分别与黄瓜的Csa003549和Csa014597直系同源，表明它们具有相似的生物学功能。编码蛋白的基序分析表明，甜瓜OST1基因编码的蛋白都含有一个激酶结构域、激酶结合位点和ATP结合区。此外，二者均都含有两个豆蔻酰化位点，三个蛋白激酶C磷酸化位点。不同的是，CmOST1L1还含有一个天冬氨酸富含区，而在CmOST1L2中没有，表明它们在编码蛋白的结构上也十分保守但又有所不同。根据结构决定功能的生物学原理，作者认为甜瓜的2个OST1基因在功能上存在分化。

图2 甜瓜、黄瓜OST1基因与拟南芥SnRK2的进化分析
表 2甜瓜OST1的结构域和蛋白基序分析

  注：表中数字代表基序或者结构域的数目，-代表没有。
  2.3甜瓜OST1的顺式调控元件分析和功能预测
  基因上游序列中顺式调控元件的分析对于揭示基因的功能具有重要意义。分析发现，甜瓜2个OST1的上游序列都含有多个应答激素和逆境的顺式调控元件，但类型和数目不尽一致。CmOST1L1分别含有一个ABA应答元件，一个生长素应答元件、一个茉莉酸甲酯应答元件，一个水杨酸应答元件，三个热胁迫应答元件、三个病原物应答元件；而CmOST1L2含有两个茉莉酸甲酯应答元件、一个热胁迫和干旱应答元件、三个病原物应答元件。这说明第一它们在激素和逆境应答方面存在一定差异，第二它们不仅能够应答非生物逆境胁迫，而且可能在生物逆境应答中也具有一定功能。这与拟南芥OST1研究的结果相一致[25,26]。根据顺式调控元件分析的结果，推测甜瓜的2个OST1基因在功能上存在分化，CmOST1L1可应答ABA、生长素和茉莉酸甲酯信号，响应热胁迫应答和生物逆境；而CmOST1L2可响应干旱、热胁迫信号及病原物和茉莉酸甲酯信号。
表3甜瓜OST1的顺式调控元件

  注: ABＲE,ABA 应答顺式元件； AuxRR-core为生长素应答元件；Box-W1为真菌激发子应答元件；CGTCA- motif,莉酸甲酯应答元件；HSE,热胁迫应答元件；MBS,为干旱胁迫应答元件；TC-rich repeats 为防卫和逆境应答元件；TCA-element,水杨酸应答元件；W box 为防卫应答元件；－代表没有。
  3结论和讨论
  本研究在甜瓜基因组中鉴定出2个与拟南芥OST1基因直系同源的基因CmOST1L1和CmOST1L2，二者位于甜瓜的不同染色体上，且均含有9个外显子，与黄瓜OST1研究的结果一致，并与黄瓜的2个OST1基因在编码区序列和结构上高度保守，但二者在基因上游的顺式调控元件上明显不同，在编码蛋白的结构上也有所差异。因此，推测这2个基因具有相似的生物学功能但又有所分化。拟南芥OST1的研究表明，它不仅在ABA信号应答和渗透胁迫应答方面具有重要功能，而且还能调节蔗糖代谢和促进植株生长。此外，拟南芥的OST1基因在免疫反应过程中也具有重要功能。因而有关这类基因的研究对于揭示植物逆境应答基因调控网络具有重要意义，而且它们在提高农作物产量和抗逆方面具有重要的应用价值。笔者认为甜瓜的OST1基因在逆境应答和生长发育过程中也具有重要的生物学功能。至于它们在不同生物学过程中的具体功能如何，表达调控的机制如何，与拟南芥及其它物种的OST1在功能上有何异同，需要结合分子和细胞生物学的手段以及转基因研究进行深入探讨。
参考文献
[1] Fujii H, Verslues P E, Zhu J K. Arabidopsis decuple mutant reveals the importance of SnRK2 kinases in osmotic stress responses in vivo[J]. Proc Natl Acad Sci U S A. 2011, 108(4):1717-1722.
[2] Kulik A, Wawer I, Krzywińska E, et al. SnRK2 protein kinases—key regulators of plant response to abiotic stresses[J]. OMICS. 2011, 15(12): 859-872.
[3] Hirayama T and Umezawa T. The PP2C-SnRK2 complex: the central regulator of an abscisic acid signaling pathway[J]. Plant Signaling & Behavior, 2010, 5(2): 160-163.
[4]Soon F F, Ng L M, Zhou X E, et al. Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases[J]. Science, 2012, 335(6064):85-88. 
[5] Sun L, Wang Y P, Chen P, et al.Transcriptional regulation of SlPYL, SlPP2C, and SlSnRK2 gene families encoding ABA signal core components during tomato fruit development and drought stress[J]. J Exp Bot. 2011, 62(15):5659-5669. 
[6] Li  L B, Zhang Y R, Liu K C, et al. Identification and bioinformatics analysis of SnRK2 and CIPK family genes in sorghum[J]. Agricultural Sciences in China, 2010, 9(1), 19-30.
[7] Huai J，Wang M，He J，et al．Cloning and characterization of the SnRK2 gene family from Zea mays[J]． Plant Cell Report，2008，27: 1861-1868.
[8] Kobayashi Y，Yamamoto S，Minami H，et al．Differential activation of rice sucrose nonfermenting 1-related protein kinase2 family by hyperosmotic stress and abscisic acid[J]．Plant Cell，2004，16: 1163-1177.
[9] Acharya B R, Jeon B W, Zhang W, et al. Open Stomata 1 (OST1) is limiting in abscisic acid responses of Arabidopsis guard cells[J]. New Phytol. 2013, 200(4):1049-1063.
[10] Vlad F, Rubio S, Rodrigues A, et al. Protein phosphatases 2C regulate the activation of the Snf1-related kinase OST1 by abscisic acid in Arabidopsis. Plant Cell. 2009, 21(10):3170-3184.
[11] Yoshida R, Umezawa T, Mizoguchi T, et al. The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis[J]. J Biol Chem. 2006, 281(8):5310-5318.
[12] Fujita Y, Yoshida T, Yamaguchi-Shinozaki K. Pivotal role of the AREB/ABF-SnRK2 pathway in ABRE-mediated transcription in response to osmoticstress in plants[J]. Physiol Plant. 2013, 147(1):15-27.
[13] Boudsocq M, Droillard M J, Barbier-Brygoo H, et al. Different phosphorylation mechanisms are involved in the activation of sucrose non-fermenting 1 related protein kinases 2 by osmotic stresses and abscisic acid[J]. Plant Mol Biol. 2007, 63(4):491-503.
[14] Vlad F, Droillard M J, Valot B, et al. Phospho-site mapping, genetic and in planta activation studies reveal key aspects of the different phosphorylation mechanisms involved in activation of SnRK2s[J]. Plant J. 2010,63(5):778-790. 
[15] Wang P, Du Y, Hou Y J, et al. Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1[J]. Proc Natl Acad Sci U S A. 2015, 112(2):613-618. 
[16] Fujii H, Zhu J K. Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress[J]. Proc Natl Acad Sci U S A. 2009, 106(20):8380-8385.
[17] Nakashima K, Fujita Y, Kanamori N, et al. Three Arabidopsis SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3 involved in ABA-Signaling are essential for the control of seed development and dormancy. Plant and Cell Physiology, 2009, 50(7):1345-1363
[18] Zheng Z, Xu X, Crosley R A,. The protein kinase SnRK2.6 mediates the regulation of sucrose metabolism and plant growth in Arabidopsis[J]. Plant Physiol. 2010, 153(1):99-113.
[19] Xue S, Hu H, Ries A, et al. Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO2 signal transduction in guard cell[J]. EMBO J. 2011, 30(8):1645-1658.
[20] Mogami J, Fujita Y, Yoshida T, et al. Two distinct families of protein kinases are required for plant growth under high external Mg2+ concentrations in Arabidopsis[J]. Plant Physiology, 2015, 167(3):1039-1057.
[21] Ding Y L, Li H, Zhang X Y, et al. OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis[J]. Developmental Cell, 2015, 32(3):278-289. 
[22] Wang M, Yuan F, Hao H, et al. BolOST1, an ortholog of open stomata 1 with alternative splicing products in Brassica oleracea, positively modulates drought responses in plants[J]. Biochem Biophys Res Commun. 2013, 442(3-4):214-220.
[23] Han Y, Dang R, Li J, et al. Sucrose nonfermenting1-related protein kinase2.6, an ortholog of open stomata1, is a negative regulator of strawberry fruit development and ripening[J]. Plant Physiol. 2015, 167(3):915-930
[24] 李利斌，曹齐卫，张允楠，等. 黄瓜全基因组SnRK2 基因的鉴定和序列特征分析[J]. 核农学报2014，28(5): 800-807. 
[25]Melotto M, Underwood W, Koczan J, et al. Plant stomata function in innate immunity against bacterial invasion[J]. Cell, 2006, 126(5): 969-980.
[26] Montillet J L, Leonhardt N, Mondy S, et al. An abscisic acid-independent oxylipin pathway controls stomatal closure and immune defense in Arabidopsis[J]. PLoS Biol, 2013, 11(3):e1001513.