Abstract：Calmodulin (CaMs) are the primary calcium-binding proteins in plants which control diverse cellular processes, including plant development and hormonal response. In this study, a genome-wide analysis conducted in Rosa roxburghii and identifified 2 RrCaM-encoding genes named RrCaM1 and RrCaM2. The RrCaM1 contains 3 introns while the RrCaM2 gene has no introns. Both of the RrCaMs contained 4 typical EF-hands. The RrCaM1 and RrCaM2 genes were located on the 3rd and 4th chromosome, repectively. The phylogenetic relationship and synteny analyses showed that RrCaM1 and RrCaM2 were clustered as 2 different subgroups. Besides, RrCaM1 and RrCaM2 shared one orthologous duplication pair with Fragaria vesca, respectively. RrCaM1 and Pyrus bretschneideri had 2 orthologous duplication pairs. CaMs genetic relationship among R. roxburghii, P. bretschneideri and Fragaria vesca was close. The results of RNA-Seq and qPCR indicated that the expression of RrCaM2 gene decreased significantly with the fruit development of R. roxburghii, while RrCaM1 was the opposite. Cis-acting element prediction and qPCR analysis showed that the promoter region of RrCaM1 contained 8 response elements such as hormone and light, while RrCaM2 lacked cis-acting elements responsive to gibberellin (GA3), low temperature and drought, which also showed completely different response patterns to salicylic acid (SA), abscisic acid (ABA), and GA3 treatments.The vitamin C (ascorbate, AsA) content and RrCaMs gene expression of R. roxburghii fruit were significantly decreased by chlorpromazine (CPZ) treatment (P<0.05). RrCaMs had a significant positive correlation with the expression of the L-galactose phosphatase (GPP) gene in AsA biosynthesis pathway and the dehydroascorbate reductase (DHAR) gene in recycling pathway. It indicates that it may be involved in the regulation of AsA accumulation in R. roxburghii fruit. This study found that the RrCaMs gene family played potentially important roles in fruit development, AsA accumulation and hormone response of R. roxburghii, and provides reference for the functional study of this gene family.
赵熳秋, 南红, 鲁敏, 安华明. 刺梨CaM基因的鉴定及其在果实中的表达特点[J]. 农业生物技术学报, 2022, 30(9): 1724-1736.
ZHAO Man-Qiu, NAN Hong, LU Min, AN Hua-Ming. Identification of CaM Genes and Its Expression Characteristics in Fruit of Rosa roxburghii. 农业生物技术学报, 2022, 30(9): 1724-1736.
[1] 安华明. 2005. 刺梨高含量AsA的积累机制及其关键酶基因的克隆与表达[D]. 博士学位论文, 浙江大学, 导师: 陈力耕, pp. 34-41. (An H M.2005. Physiological mechanism of accumulating high level l-ascorbic acid and molecular cloning and expression of its key biosynthetic enzyme in Rosa roxburghii Tratt[D]. Thesis for Ph.D., Zhejiang University, Supervisor: Chen L G, pp. 34-41.) [2] 黄艳. 2019. 桑树中钙调蛋白与类钙调蛋白基因的鉴定与功能分析[D]. 硕士学位论文, 西南大学, 导师: 何宁佳, pp. 14-44. (Huang Y.2019. Identification and functional analyses of calmodulin and calmodulin-like genes in mulberry[D]. Thesis for M.S., Southwest University, Supervisor: He N J, pp. 14-44.) [3] 李良良. 2016. 四种非生物因子对刺梨AsA相关基因表达的影响[D]. 硕士学位论文, 贵州大学, 导师: 安华明, pp. 28-34. (Li L L, 2016. Effects on AsA related gene expression of Rosa Roxburghii Tratt by four abiotic factors[D].Thesis for M.S., Guizhou University, Supervisor: An H M, pp. 28-34.) [4] 李良良, 安华明. 2016. Ca2+和 Cu2+对刺梨果实AsA代谢相关基因表达的影响[J]. 园艺学报, 43(7): 1377-1382. (Li L L, An H M.2016. Effects of exogenous divalent cations Ca2+ and Cu2+ on expression of genes involved in ascorbate metabolism in Rosa roxburghii fruits[J]. Acta Horticulturae Sinica, 43(07): 1377-1382.) [5] 林素英, 梁杰, 黄志明, 等. 2012. 钙调素拮抗剂TFP对低温胁迫下枇杷幼果AsA-GSH循环的影响[J].热带作物学报, 33(11): 1980-1984. (Lin S Y, Liang J, Huang Z M, et al.2012. Effects of calmodulin antagonist TFP on AsA-GSH cycle in young loquat fruits under low temperature stress[J]. Chinese Journal of Tropical Crops, 33(11): 1980-1984.) [6] 罗充, 樊卫国, 刘进平, 等. 2004a. 钙、钙调素在刺梨果实发育过程中的含量变化研究[J]. 种子, 23(12): 6-8. (Luo C, Fan W G, Liu J P, et al.2004. Studies on changes of calcium and calmodulin contents during fruit development in Rosa roxburghii Tratt[J]. Seed, 23(12): 6-8.) [7] 罗充, 樊卫国, 刘进平, 等. 2004b. 不同钙制剂处理对刺梨果实发育的影响[J]. 贵州师范大学学报(自然科学版), 22(04): 7-11. (Luo C, Fan W G, Liu J P, et al.2004. Effects of different treatments on fruit development of Rosa roxburghii Tratt[J].Journal of Guizhou Normal University (Natural Sciences), 22(04): 7-11.) [8] 孙雅蕾, 杨曼, 安华明. 2014. 刺梨GDP-L-半乳糖磷酸酶基因的表达及其与AsA积累的关系[J]. 园艺学报, 41(6): 1175-1182. (Sun Y L,Yang M, An H M.2014. Expression of GDP-L-galactose pyrophosphatase and its relationship with ascorbate accumulation in Rosa roxburghii[J]. Acta Horticulturae Sinica, 41(6): 1175-1182.) [9] 王乐乐, 安华明. 2013. HPLC测定刺梨果实中维生素C含量方法的优化[J]. 现代食品科技, 29(2): 397-400. (Wang L L, An H M.2013. An optimized HPLC method for analyzing Vc content in Rosa roxburghii fruits[J]. Modern Food Science and Technology, 29(2): 397-400.) [10] 王雪霁. 2016. 杨树CaM及CML基因家族生物信息学分析[D]. 硕士学位论文, 中国林业科学研究院, 导师: 李潞滨, pp. 19-48. (Wang X J.2016. Bioinformatics analysis of CaM/CML gene family in Populus[D]. Thesis for M.S., Chinese Academy of Forestry, Supervisor: Li L B, pp. 19-48.) [11] 张书轩, 李良良, 鲁敏, 等. 2018. 三种植物生长调节剂对刺梨果实维生素C积累及其代谢基因表达的影响[J]. 农业生物技术学报, 26(4): 606~615. (ZHANG S X, LI L L, LU M, et al.2018. Effects of three exogenous plant growth regulators on AsA accumulation and expression of genes involved in its metabolism in Rosa roxburghii fruit[J]. Journal of Agricultural Biotechnology, 26(4): 606~615.) [12] 张雪, 杨曼, 安华明, 等. 2012. 外源Ca2+、Mg2+、Cu2+和吖啶黄素对刺梨果实维生素C合成的影响[J]. 中国农业科学, 45(6): 1144-1149. (Zhang X, Yang M, An H M, et al.2012. Effects of exogenous divalent cations Ca2+, Mg2+, and Cu2+ and acriflavine on ascorbate biosynthesis in Rosa roxburghii fruits[J]. Scientia Agricultura Sinica, 45(6): 1144-1149.) [13] Batistic O, Kudla J.2012. Analysis of calcium signaling pathways in plants[J]. Biochimica et Biophysica Acta-General Subjects, 1820(8): 1283-1293. [14] Boonburapong B, Buaboocha T.2007. Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins[J]. BMC Plant Biology, 7: 4. [15] Chen C J, Chen H, Zhang Y, et al.2020. TBtools: An integrative toolkit developed for interactive analyses of big biological data[J]. Molecular Plant, 13(8): 1194-1202. [16] Cho K M, Nguyen H T K, Kim S Y, et al.2016. CML 10, a variant of calmodulin, modulates ascorbic acid synthesis[J]. New Phytologist. 209(2): 664-678. [17] Dai C, Lee Y, Lee I C, et al.2018. Calmodulin 1 regulates senescence and ABA response in Arabidopsis[J]. Frontiers in Plant Science, 9: 803. [18] DeFalco T A, Bender K W, Snedden W A.2010. Breaking the code: Ca2+ sensors in plant signalling[J]. Biochemical Journal ,425(1): 27-40. [19] Ding X C, Zhang L P, Hao Y W, et al.2018. Genome-wide identification and expression analyses of the calmodulin and calmodulin-like proteins reveal their involvement in stress response and fruit ripening in papaya[J]. Postharvest Biology and Technology, 143: 13-27. [20] Dowdle J, Ishikawa T, Gatzek S, et al.2007. Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability[J]. The Plant Journal, 52(4): 673-689. [21] Gasteiger E, Hoogland C, Gattiker A, et al.2003. ExPASy: The proteomics server for in-depth protein knowledge and analysis[J]. Nucleic Acids Research, 31(13): 3784-3788. [22] Ififigeneia M, Angelos K K.2017. Genetic control of ascorbic acid biosynthesis and recycling in horticultural crops[J]. Frontiers in Chemistry, 5(50): 1-8. [23] Imai T, Karita S, Shiratori, G, et al.1998. L-galactono-gamma-lactone dehydrogenase from sweet potato: Purifification and cDNA sequence analysis[J]. Plant and Cell Physiology, 39(12): 1350-1358. [24] Kanchiswamy C N, Mohanta T K, Capuzzo A, et al.2013. Differential expression of CPKs and cytosolic Ca2+ variation in resistant and susceptible apple cultivars (Malus × domestica) in response to the pathogen Erwinia amylovora and mechanical wounding[J]. BMC Genomics, 14:760. [25] Kohagen M, Lepsik M, Jungwirth P.2014. Calcium binding to calmodulin by molecular dynamics with effective polarization[J]. Journal of Physical Chemistry Letters, 5(22): 3964-3969. [26] Laing W A, Bulley S, Wright M, et al.2004. A highly specifific L-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis[J]. Proceedings of the National Academy of Sciences of the USA, 101(48): 16976-16981. [27] Letunic I, Bork P.2016. Interactive tree of life (iTOL) v3: An online tool for the display and annotation of phylogenetic and other tree[J]. Nucleic Acids Research, 44(W1): W242-W245. [28] Li C L, Meng D, Zhang J H, et al.2019. Genome-wide identification and expression analysis of calmodulin and calmodulin-like genes in apple (Malus×domestica)[J]. Plant Physiology and Biochemistry, 139: 600-612. [29] Li M J, Ma F W, Guo C M, et al.2010. Ascorbic acid formation and profiling of genes expressed in its synthesis and recycling in apple leaves of different ages[J]. Plant Physiology and Biochemistry, 48(4): 216-224. [30] Liu Y, Yang T Y, Lin Z k, et al.2019. A WRKY transcription factor PbrWRKY53 from Pyrus betulaefolia is involved in drought tolerance and AsA accumulation[J]. Plant Biotechnology Journal, 17(9): 1770-1787. [31] Lu M, Ma W T, Liu Y Q, et al.2020. Transcriptome analysis reveals candidate lignin-related genes and transcription factors in Rosa roxburghii during fruit ripening[J]. Plant Molecular Biology Reporter, 38(2): 331-342. [32] Luan S, Kudla J, Rodrõ Â, et al.2002. Calmodulins and calcineurin B-like proteins: Calcium sensors for specific signal response coupling in plants[J]. The Plant Cell, 14(Suppl): S389-S400. [33] McCormack E, Braam J.2003. Calmodulins and related potential calcium sensors of Arabidopsis[J]. The New Phytologist, 159(3): 585-598. [34] McCormack E, Tsai Y C, Braam J.2005. Handling calcium signaling: Arabidopsis CaMs and CMLs[J]. Trends in Plant Science, 10(8): 383-389. [35] Mohanta T K, Kumar P, Bae H H.2017. Genomics and evolutionary aspect of calcium signaling event in calmodulin and calmodulin-like proteins in plants[J]. BMC Plant Biology, 17: 38. [36] Mohanta T K, Mohanta N, Mohanta Y, et al.2015. Genome-wide identification of calcineurin B-Like (CBL) gene family of plants reveals novel conserved motifs and evolutionary aspects in calcium signaling events[J]. BMC Plant Biology, 15: 189. [37] Qin A, Shi Q, Yu X.2011. Ascorbic acid contents in transgenic potato plants overexpressing two dehydroascorbate reductase genes[J]. Molecular Biology Reports, 38(3): 1557-1566. [38] Shi J Y, Du X.2020. Identifcation characterization and expression analysis of calmodulin and calmodulin-like proteins in Solanum pennellii[J]. Scientific Reports, 10(1): 7474. [39] Smirnoff N, Wheeler G L.2000. Ascorbic acid in plants: Biosynthesis and function[J]. Critical Reviews in Biochemistry and Molecular Biology, 35(4): 291-314. [40] Snedden W A, Fromm H.2001. Calmodulin as a versatile calcium signal transducer in plants[J]. The New Phytologist, 151(1): 35-66. [41] Sun Q G, Yu S H, Guo Z F.2020. Calmodulin-like (CML) gene family in Medicago truncatula: Genome-wide identifification, characterization and expression analysis[J]. International Journal of Molecular Sciences, 21(19): 7142. [42] Tang J, Lin J, Li X, et al.2017. Characterization and expression profiling analysis of calmodulin genes in response to salt and osmotic stresses in pear (Pyrus bretschneideri Rehd.) and in comparison with Arabidopsis[J]. BioMed Research International, 2017: 7904162. [43] Vandelle E, Vannozzi A, Wong D, et al.2018. Identification characterization and expression analysis of calmodulin and calmodulin-like genes in grapevine (Vitis vinifera) reveal likely roles in stress responses[J]. Plant Physiology and Biochemistry, 129: 221-237. [44] Wang Y P, Tang H, Debarry J D, et al.2012. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity[J] .Nucleic Acids Research, 40(7): e49. [45] Wheeler G L, Jones M A, Smirnoff N.1998. The biosynthetic pathway of vitamin C in higher plants[J]. Nature, 393(6683): 365-369. [46] Wu X M, Qiao Z, Liu H P, et al.2017. CML20, an Arabidopsis calmodulin-like protein, negatively regulates guard cell aba signaling and drought stress tolerance[J]. Frontiers in Plant Science, 8: 824. [47] Xing C H, Liu Y, Zhao L Y, et al.2019. A novel MYB transcription factor regulates ascorbic acid synthesis and affects cold tolerance[J]. Plant Cell & Environment, 42(3): 832-845. [48] Yang T B, Peng H, Bauchan G R.2014. Functional analysis of tomato calmodulin gene family during fruit development and ripening[J]. Horticulture Research, 1(1): 14057-14066. [49] Zhang K, Yue D Y, Wei W, et al.2016. Characterization and functional analysis of calmodulin and calmodulin-like genes in Fragaria vesca[J]. Frontiers in Plant Science, 7: 1820. [50] Zhu X Y, Dunand C, Snedden W, et al.2015. CaM and CML emergence in the green lineage[J]. Trends in Plant Science, 20(8): 483-489.
