茶树CsCHLI基因的克隆及其在不同叶色白化茶树中的表达分析

doi:10.13305/j.cnki.jts.20210224.001

摘要/Abstract

摘要： 镁离子螯合酶是叶绿素合成过程中的关键酶之一,与植物叶色的白化现象密切相关。以陕茶1号、白叶1号、极白1号、黄金芽和黄金叶等5个茶树品种叶片为试验材料,分别克隆其镁离子螯合酶I亚基（Mg-chelatase I subunit,CHLI）编码基因CsCHLI全长CDS序列。多序列比对显示,白叶1号中两个碱基差异位点（G502C,C1169T）导致在AAA⁺和AAA lid结构域中有2个氨基酸残基发生改变（G168R,A390V）,黄金叶中1个碱基差异位点（G1202A）导致在AAA lid结构域中有1个氨基酸残基发生改变（R401H）。亚细胞定位结果显示,CsCHLI定位于叶绿体中,碱基或氨基酸残基的差异并不影响其亚细胞定位。RNA二级结构分析表明,碱基位点突变会影响CsCHLI的茎环结构和二级结构的稳定性。与陕茶1号相比,不同叶色白化茶树品种中总叶绿素含量分别为陕茶1号的25%~77%,CsCHLI基因表达量分别为陕茶1号的24%~46%。结果表明,茶树中CsCHLI的基因表达与叶绿素含量呈正相关。这些结果将为深入研究CsCHLI在茶树叶绿素代谢中的功能以及茶树叶色突变的作用机理提供试验证据。

关键词: 茶树, CsCHLI, 亚细胞定位, RNA二级结构, 叶绿素含量

Abstract: Magnesium chelatase is one of the key enzymes in the process of chlorophyll synthesis, which is closely related to the albino phenomenon of leaves. In this study, the leaves of tea cultivars Shaancha 1, Baiye 1, Jibai 1, Huangjinya and Haungjinye were used as materials to clone the full-length CDS sequences of CsCHLI (Mg-chelatase I subunit). Multi-sequence alignment shows that the two base differences (G502C, C1169T) were found in Baiye 1, which lead to the change of two amino acid residues (G168R, A390V) in the AAA⁺ and AAA lid domain. Meanwhile, there was an amino acid residue changed (R401H) in the AAA lid domain of Huangjinye, due to the difference of G1202A. Subcellular localization analyses illustrates that CsCHLI is localized in chloroplast and the differences of the bases or amino acid residues did not affect its subcellular localization. The RNA secondary structure analysis shows that base site mutations of CsCHLI would affect its stem-loop structure and the stability. Moreover, the chlorophyll contents in albino tea plants were 25%-77% of that in Shaancha 1, and the expressions of CsCHLI in albino tea plants were 24%-46% of that in Shaancha 1. The results show that the CsCHLI expression was positively correlated with chlorophyll content in tea plants. Furthermore, these results provided experimental evidences for the in-depth study of the chlorophyll metabolism in albino tea plants.

Key words: Camellia sinensis, CsCHLI, subcellular localization, RNA secondary structure, chlorophyll content

中图分类号:

S571.1
Q753

赵逸清, 刘政均, 张田鑫, 赵彦婷, 肖斌, 高岳芳. 茶树CsCHLI基因的克隆及其在不同叶色白化茶树中的表达分析[J]. 茶叶科学, 2021, 41(3): 327-336. doi: 10.13305/j.cnki.jts.20210224.001.

ZHAO Yiqing, LIU Zhengjun, ZHANG Tianxin, ZHAO Yanting, XIAO Bin, GAO Yuefang. Cloning of CsCHLI Gene and Its Expression Analysis in Different Albino Tea Cultivars (Camellia sinensis)[J]. Journal of Tea Science, 2021, 41(3): 327-336. doi: 10.13305/j.cnki.jts.20210224.001.

参考文献

[1] Satou M, Enoki H, Oikawa A, et al Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins[J]. Plant Molecular Biology, 2014, 85(4/5): 411-428.
[2] Zeng X, Tang R, Guo H, et al.A naturally occurring conditional albino mutant in rice caused by defects in the plastid-localized OsABCI8 transporter[J]. Plant Molecular Biology, 2017, 94(1/2): 137-148.
[3] Sawers R J, Viney J, Farmer P R, et al.The maize Oil yellow1 (Oy1) gene encodes the I subunit of magnesium chelatase[J]. Plant Molecular Biology , 2006, 60(1): 95-106.
[4] Gao M, Hu L, Li Y, et al.The chlorophyll-deficient golden leaf mutation in cucumber is due to a single nucleotide substitution in CsChlI for magnesium chelatase I subunit[J]. Theoretical and Applied Genetics, 2016, 129(10): 1961-1973.
[5] Liu M, Wang Y, Nie Z, et al.Double mutation of two homologous genes YL1 and YL2 results in a leaf yellowing phenotype in soybean [Glycine max (L.) Merr][J]. Plant Molecular Biology, 2020, 103: 527-543. doi: 10.1007/s11103-020-01008-9.
[6] 卢翠, 沈程文. 茶树白化变异研究进展[J]. 茶叶科学, 2016, 36(5): 445-451.
Lu C, Shen C W.Research progress of albino tea plant (Camellia sinensis (L.) O. Kuntze)[J]. Journal of Tea Science, 2016, 36(5): 445-451.
[7] Cheng S, Fu X, Liao Y, et al.Differential accumulation of specialized metabolite L-theanine in green and albino-induced yellow tea (Camellia sinensis) leaves[J]. Food Chemistry, 2019, 276: 93-100.
[8] Lu M, Han J, Zhu B, et al.Significantly increased amino acid accumulation in a novel albino branch of the tea plant (Camellia sinensis)[J]. Planta, 2019, 249(2): 363-376.
[9] Farmer D A, Brindley A A, Hitchcock A, et al.The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase[J]. Biochemical Journal, 2019, 476(13): 1875-1887.
[10] Sawicki A, Zhou S, Kwiatkowski K, et al.1-N-histidine phosphorylation of ChlD by the AAA+ ChlI2 stimulates magnesium chelatase activity in chlorophyll synthesis[J]. Biochemical Journal, 2017, 474(12): 2095-2105.
[11] Huang Y, Li H.Arabidopsis CHLI2 can substitute for CHLI1[J]. Plant Physiology, 2009, 150(2): 636-645.
[12] Wang C, Zhang L, Li Y, et al.Single nucleotide mutagenesis of the TaCHLI gene suppressed chlorophyll and fatty acid biosynthesis in common wheat seedlings[J]. Frontiers in Plant Science, 2020, 11: 97. doi: 10.3389/fpls.2020.00097.
[13] Du H, Qi M, Cui X, et al.Proteomic and functional analysis of soybean chlorophyll-deficient mutant cd1 and the underlying gene encoding the CHLI subunit of Mg-chelatase[J]. Molecular Breeding, 2018, 38(6): 71. doi: 10.1007/s11032-018-0819-9.
[14] Zhang H, Liu L, Cai M, et al.A point mutation of magnesium chelatase OsCHLI gene dampens the interaction between CHLI and CHLD subunits in rice[J]. Plant Molecular Biology Reporter, 2015, 33(6): 1975-1987.
[15] Liu X, Yu W, Wang G, et al.Comparative proteomic and physiological analysis reveals the variation mechanisms of leaf coloration and carbon fixation in a xantha mutant of Ginkgo biloba L[J]. International Journal of Molecular Sciences, 2016, 17(11): 1794. doi: 10.3389/fpls.2020.00097.
[16] 郭俊红, 王伟东, 谷星, 等. 茶树WRKY转录因子基因CsWRKY57的克隆及表达分析[J]. 茶叶科学, 2017, 37(4): 411-419.
Guo J H, Wang W D, Gu X, et al.Cloning and expression analysis of WRKY transcription factor gene CsWRKY57 in tea plant (Camellia sinensis)[J]. Journal of Tea Science, 2017, 37(4): 411-419.
[17] Xia E H, Li F D, Tong W et al. Tea Plant Information Archive: a comprehensive genomics and bioinformatics platform for tea plant[J]. Plant Biotechnology Journal, 2019, 17(10): 1938-1953.
[18] 向芬, 李维, 刘红艳,等. 茶树叶绿素测定方法的比较研究[J]. 茶叶通讯, 2016, 43(4):37-40.
Xiang F, Li W, Liu H Y, et al.Comparisonon methods of chlorophyll extraction in Camellia sinensis[J]. Journal of Tea Communication, 2016, 43(4):37-40.
[19] Wellburn A R, Lichtenthaler H.Formulae and program to determine total carotenoids and chlorophylls a and b of leaf extracts in different solvents[M]//Advances in Photosynthesis Researchr. Dordrecht: Springer Link, 1984: 9-12.
[20] Hao X, Horvath D P, Chao W S, et al.Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)[J]. International Journal of Molecular Sciences, 2014, 15(12): 22155-22172.
[21] Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-CT method[J]. Methods, 2001, 25(4): 402-408.
[22] Zhang D, Chang E, Yu X, et al.Molecular characterization of magnesium chelatase in soybean [Glycine max (L.) Merr.][J]. Frontiers in Plant Science, 2018, 9: 720. doi: 10.3389/fpls.2018.00720.
[23] 吴全金. ‘白鸡冠’茶树响应光调控的基因差异及理化特征分析[D]. 福州: 福建农林大学, 2015.
Wu Q J.Gene differential analysis and physicochemical characteristics of Camellia sinensis cv. Baijiguan in response to light [D]. Fuzhou: Fujian Agriculture and Forestry University, 2015.
[24] Li Q, Fang C, Duan Z, et al.Functional conservation and divergence of GmCHLI genes in polyploid soybean[J]. The Plant Journal, 2016, 88(4): 584-596.
[25] Brzezowski P, Sharifi M N, Dent R M, et al.Mg chelatase in chlorophyll synthesis and retrograde signaling in Chlamydomonas reinhardtii: CHLI2 cannot substitute for CHLI1[J]. Journal of Experimental Botany, 2016, 67(13): 3925-3938.