西洋梨PcHB12基因抑制果实花青苷的合成

doi:10.16420/j.issn.0513-353x.2020-0499

摘要/Abstract

摘要：

为了解HD-ZipⅠ转录因子调控梨花青苷合成的机制,以西洋梨品种‘红茄梨’为试材,克隆了1个HD-ZipⅠ家族基因PcHB12（GenBank序列号XM_009363028）。通过系统进化树分析、qRT-PCR、酵母单杂交、电泳迁移率变动分析（Electrophoretic mobility shift assay,EMSA）和荧光素酶报告试验,探究PcHB12调控梨花青苷合成的作用。结果表明,PcHB12的开放阅读框为696 bp,编码 231个氨基酸,预测的蛋白质分子量是26.77 kD。系统进化树分析表明,PcHB12与拟南芥AtHB12蛋白序列相似度最高。‘茄梨’中PcHB12的表达量明显高于其红色芽变品种‘红茄梨’,而花青苷含量和花青苷相关基因PcMYB10.1、PcUFGT的表达量明显低于‘红茄梨’。酵母单杂交和EMSA试验发现,PcHB12结合在PcMYB10.1启动子的MBS序列。荧光素酶试验表明PcHB12负调控PcMYB10.1启动子的转录活性。因此,PcHB12可能通过负调控PcMYB10.1的表达从而抑制梨花青苷的合成。

关键词: 梨, 花青苷, PcHB12, PcMYB10.1

Abstract:

In order to understand the mechanism that the HD-ZipⅠtranscription factor regulates anthocyanin synthesis in pears,the European pear cultivar‘Red Clapp Favorite’was used as study material to clonePcHB12（GenBank accession number:XM_009363028）from the HD-ZipⅠfamily. Phylogenetic tree analysis,qRT-PCR,yeast one-hybrid assay,EMSA,and luciferase reporter assay were employed to study the regulatory function ofPcHB12on anthocyanin synthesis. The results showed that the open reading frame of PcHB12 is 696 bp,encoding 231 amino acids,and the predicted molecular weight of the protein is 26.77 kD. Phylogenetic tree analysis showed that PcHB12 has the highest similarity to the AtHB12 protein sequence of Arabidopsis thaliana. The expression level of PcHB12 in‘Clapp Favorite’is significantly higher than that in its red bud mutation cultivar‘Red Clapp Favorite’. In contrast,anthocyanin content and the expression levels of anthocyanin-related genesPcMYB10.1 and PcUFGT in ‘Clapp Favorite’were significantly lower than that in‘Red Clapp Favorite’. Yeast one-hybrid and electrophoretic mobility shift assay（EMSA）experiments found that PcHB12 binds to the MBS sequence in the PcMYB10.1 promoter. Luciferase experiments showed that PcHB12 negatively regulates transcription activity on the PcMYB10.1 promoter. Therefore,PcHB12 may negatively regulate PcMYB10.1 expression to inhibit anthocyanin synthesis in pears.

Key words: pear, anthocyanin, PcHB12, PcMYB10.1

中图分类号:

S661.2

张震, 李晨智宇, 张雅, 李玺, 王烁, 王鑫玉, 陈学森, 冯守千. 西洋梨PcHB12基因抑制果实花青苷的合成[J]. 园艺学报, 2021, 48(3): 456-464.

ZHANG Zhen, LI Chenzhiyu, ZHANG Ya, LI Xi, WANG Shuo, WANG Xinyu, CHEN Xuesen, FENG Shouqian. Inhibition of Pear Anthocyanin Synthesis by the PcHB12 Gene in European Pears[J]. Acta Horticulturae Sinica, 2021, 48(3): 456-464.

图/表 6

表1 引物序列列表

Table 1 The list of primer sequences

用途 Purpose	引物名称 Primer name	序列（5′-3′） Sequence
荧光实时定量 qRT-PCR	PcHB12-F	GCTTGTTGGAGTCTATATTCG
	PcHB12-R	TCTGTAGTCTTGCTCTATCTG
	PcMYB10.1-F	GAAACAGGTCTAAACAGATGCAG
	PcMYB10.1-R	GAGGTCTTATGACGGATGTG
	PcUFGT-F	GTCAGCCGTTTGGGCA
	PcUFGT-R	GAGTGTAAACCAGGTCGTTG
	PcActin-F	TGGGCTTTGCTCCTCTTAC
	PcActin-R	CCTTCGTGCTCATCTTACC
电泳迁移率变动分析 Electrophoretic mobility shift assay	PcHB12（pET32a）-F	GGATTCATGGACAGGAGGAAAGAGGAGT
电泳迁移率变动分析 Electrophoretic mobility shift assay	PcHB12（pET32a）-R	GTCGACTCAGGTCCAGAAATTTAACCAGTG
荧光素酶报告基因试验 Luciferase reporter assay	PcHB12（pHBT-AvrRpm1-HA）-F	GGATCCATGGACAGGAGGAAAGAGGAGT
	PcHB12 (pHBT-AvrRpm1-HA)-R	AGGCCTTCAGGTCCAGAAATTTAACCAGTG
	proPcMYB10.1（pFRK1-LUC）-F	GGATCCGTTCCACTACATCCCATAGGTCAT
	proPcMYB10.1（pFRK1-LUC）-R	CCATGGGGTGCCACTT TCCATCTCC
酵母单杂交试验 Yeast one-hybrid assay	PcHB12（PGADT7）-F	CATATGATGGACAGGAGGAAAGAGGAGT
	PcHB12（PGADT7）-R	GGATTCTCAGGTCCAGAAATTTAACCAGTG
	proPcMYB10.1（PHIS-2）-F	GGATCCGTTCCACTACATCCCATAGGTCAT
	proPcMYB10.1（PHIS2）-R	CCATGGGGTGCCACTTTCCATCTCC

图1 ‘茄梨’和‘红茄梨’幼果果实表型（A）、花青苷含量（B）及PcHB12与花青苷相关基因的相对表达量（C）

Fig. 1 Phenotype（A）,anthocyanin content（B）,relative expressions of PcHB12 and other anthocyanin-related genes（C）in young fruit of‘Clapp Favorite'and‘Red Clapp Favorite’pears **P < 0.01.

图2 ‘红茄梨’PcHB12基因RT-PCR产物

Fig. 2 RT-PCR product of PcHB12 in‘Red Clapp Favorite’

图3 不同物种HD-Zip蛋白系统进化树分析节点处数字代表亲缘关系的远近。

Fig. 3 Phylogenetic tree analysis of HD-Zips from different species The numbers at node represent the distance of the kinship.

图4 PcHB12与PcMYB10.1启动子结合 A:酵母单杂试验验证PcHB12可以结合PcMYB10.1启动子;B:EMSA试验验证PcHB12可以结合PcMYB10.1启动子的MBS位点。“+”代表添加探针或转录因子;“-”代表不添加探针或转录因子;“× 25”“× 50”“× 100”分别代表添加探针的25倍、50倍和100倍。

Fig. 4 PcHB12 binds to the promoter of PcMYB10.1 A:PcHB12 bound with PcMYB10.1 promoter through yeast one-hybrid assay;B:PcHB12 bound with MBS motif of PcMYB10.1 promoter through EMSA assay. The “+”and “-”indicate the presence and absence of the probe and protein. The “× 25”“× 50”and“× 100”indicat 25-fold,50-fold and 100-fold excess of probe.

图5 相对荧光素酶活性

Fig. 5 Relative LUC activity **P< 0.01.

参考文献 39

[1]	An J P, Wang X F, Zhang X W, Xu H F, Bi S Q, You C X, Hao Y J. 2020. An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation. Plant Biotechnology Journal, 18 (2):337-353. doi: 10.1111/pbi.v18.2 URL
[2]	An X H, Tian Y, Chen C Q, Liu X J, Liu D D, Xie X B, Cheng C G, Cong P H, Hao Y J. 2015. MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples. Plant and Cell Physiology, 56 (4):650-662. doi: 10.1093/pcp/pcu205 URL
[3]	An X H, Tian Y, Chen K Q, Wang X F, Hao Y J. 2012. The apple WD 40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation. Journal of Plant Physiology, 169 (7):710-717. doi: 10.1016/j.jplph.2012.01.015 URL
[4]	Bi Siqi, An Jianping, Wang Xiaofei, Hao Yujin, Rui Lin, Li Tong, Han Yuepeng, You Chunxiang. 2019. Ethylene response factor MdERF 3 promotes anthocyanin and proanthocyanidin accumulation in apple. Acta Horticulturae Sinica, 46 (12):2277-2285. (in Chinese)
	毕思琦, 安建平, 王小非, 郝玉金, 芮麟, 李彤, 韩月彭, 由春香. 2019. 苹果乙烯响应因子MdERF3促进花青苷和原花青苷积累. 园艺学报, 46 (12):2277-2285.
[5]	Butelli E, Titta L, Giorgio M, Mock H, Matros A, Peterek S, Schijlen E, Hall R D, Bovy A, Luo J, Martin C. 2008. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nature Biotechnology, 26:1301-1308. doi: 10.1038/nbt.1506 URL
[6]	Espley R V, Hellens R P, Putterill J, Stevenson D E, Kutty-Amma S, Allan A C. 2007. Red colouration in apple fruit is due to the activity of the MYB transcription factor,MdMYB10. Plant Journal, 49 (3):414-427. doi: 10.1111/tpj.2007.49.issue-3 URL
[7]	Fang H C, Dong Y H, Yue X X, Hu J F, Jiang S H, Xu H F, WangY C, Su M Y, Zhang J, Zhang Z Y, Wang N, Chen X S. 2019. The B-box zinc finger protein MdBBX 20 integrates anthocyanin accumulation in response to ultraviolet radiation and low temperature. Plant Cell and Environment, 42 (7):2090-2104. doi: 10.1111/pce.v42.7 URL
[8]	Feng S Q, Sun S S, Chen X L, Wu S J, Wang D Y, Chen X S. 2015. PyMYB10 and PyMYB10.1 interact with bHLH to enhance anthocyanin accumulation in pears. PLoS ONE,10 (11): https://doi.org/10.1371/journal.pone.0142112.
[9]	Feng S Q, Wang Y L, Yang S, Xu Y T, Chen X S. 2010. Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10. Planta, 232 (1):245-255. doi: 10.1007/s00425-010-1170-5 URL
[10]	Feng S Q, Xu Y C, Yang L, Sun S S, Wang D Y, Chen X S. 2015. Genome-wide identification and characterization of R2R3-MYB transcription factors in pear. Scientia Horticulturae, 197:176-182. doi: 10.1016/j.scienta.2015.09.033 URL
[11]	Fu Z Z, Shang H Q, Jiang H, Gao J, Dong X Y, Wang H J, Li Y M, Wang L M, Zhang J, Shu Q Y, Chao Y C, Xu M L, Wang R, Wang L S, Zhang H C. 2020. Systematic identification of the light-quality responding anthocyanin synthesis-related transcripts in petunia petals. Horticultural Plant Journal, 6 (6):428-438. doi: 10.1016/j.hpj.2020.11.006 URL
[12]	Javelle M, Vernoud V, Rogowsky P M, Ingram G C. 2011. Epidermis:the formation and functions of a fundamental plant tissue. New Phytologist, 189:17-39. doi: 10.1111/nph.2010.189.issue-1 URL
[13]	Jiang S H, Chen M, He N B, Chen X L, Wang N, Sun Q G, Zhang T L, Xu H F, Fang H C, Wang Y C, Zhang Z Y, Wu S J, Chen X S. 2019. MdGSTF6,activated by MdMYB1,plays an essential role in anthocyanin accumulation in apple. Horticulture Research, 6 (1): https://doi.org/10.1038/s41438-019-0118-6.
[14]	Jiang Y H, Liu C H, Yan D, Wen X H, Liu Y L, Wang H J, Dai J Y, Zhang Y J, Liu Y F, Zhou B, Ren X L. 2017. MdHB1down-regulation activates anthocyanin biosynthesis in the white-fleshed apple cultivar‘Granny Smith’. Journal of Experimental Botany, 68 (5):1055-1069. doi: 10.1093/jxb/erx029 URL
[15]	Kobayashi S, Goto-Yamamoto N, Hirochika H. 2004. Retrotransposon-induced mutations in grape skin color. Science, 304 (5673):982. doi: 10.1126/science.1095011 URL
[16]	Kubo H, Peeters A J M, Aarts M G M, Pereira A, Koornneef M. 1999. ANTHOCYANINLESS2,a homeobox gene affecting anthocyanin distribution and root development in Arabidopsis. Plant Cell, 11:1217-1226. pmid: 10402424
[17]	Liu R H, Liu J R, Chen B Q. 2005. Apples prevent mammary tumors in rats. Journal of Agricultural and Food Chemistry, 53:2341-2343. doi: 10.1021/jf058010c URL
[18]	Liu W J, Wang Y C, Yu L, Jiang H Y, Guo Z W, Xu H F, Jiang S H, Fang H C, Zhang J, Su M Y, Zhang Z, Chen X L, Chen X S, Wang N. 2019. MdWRKY11 participates in anthocyanin accumulation in red-fleshed apples by affecting MYB transcription factors and the photoresponse factor MdHY5. Journal of Agricultural and Food Chemistry, 67 (32):8783-8793. doi: 10.1021/acs.jafc.9b02920 URL
[19]	Liu Xiao-fen, Li Fang, Yin Xue-ren, Xu Chang-jie, Chen Kun-song. 2013. Recent advances in the transcriptional regulation of anthocyanin biosynthesis. Acta Horticulturae Sinica, 40 (11):2295-2306. (in Chinese)
	刘晓芬, 李方, 殷学仁, 徐昌杰, 陈昆松. 2013. 花青苷生物合成转录调控研究进展. 园艺学报, 40 (11):2295-2306.
[20]	Lu P T, Zhang C Q, Liu J T, Liu X W, Jiang G M, Jiang X Q, Khan M A, Wang L S, Hong B, Gao J P. 2014. RhHB1mediates the antagonism of gibberellins to ABA and ethylene during rose( Rosa hybrida)petal senescence. Plant Journal, 78 (4):578-590. doi: 10.1111/tpj.12494 URL
[21]	Manavella P A, Arce A L, Dezar C A, Bitton F, Renou J P, Crespi M, Chan R L. 2006. Crosstalk between ethylene and drought signaling pathways is mediated by the sunflower Hahb-4 transcription factor. Plant Journal, 48:125-137. pmid: 16972869
[22]	Meng J X, Gao Y, Han M L, Liu P Y, Yang C, Shen T, Li H H. 2020. In vitro anthocyanin induction and metabolite analysis in Malus spectabilis leaves under low nitrogen conditions. Horticultural Plant Journal, 6 (5):284-292. doi: 10.1016/j.hpj.2020.06.004 URL
[23]	Miao Z Q, Zhao P X, Mao J L, Yu L H, Yuan Y, Tang H, Liu Z B, Xiang C B. 2018. HOMEOBOXPROTEIN52 mediates the crosstalk between ethylene and auxin signaling during primary root elongation by modulating auxin transport-related gene expression. Plant Cell, 30:2761-2778. doi: 10.1105/tpc.18.00584 URL
[24]	Ré D A, Capella M, Bonaventure G, Chan R L. 2014. Arabidopsis AtHB7 and AtHB12 evolved divergently to fine tune processes associated with growth and responses to water stress. BMC Plant Biology, 14 (1):150. doi: 10.1186/1471-2229-14-150 URL
[25]	Romani F, Ribone P A, Capella M, Miguel V N, Chan R L. 2016. A matter of quantity:common features in the drought response of transgenic plants overexpressing HD-Zip I transcription factors. Plant Science, 251:139-154. doi: 10.1016/j.plantsci.2016.03.004 URL
[26]	Rossi A, Serraino I, Dugo P, Paola R D, Mondello L, Genovese T, Morabito D, Dugo G, Sautebin L, Caputi A P, Cuzzocrea S. 2003. Protective effects of anthocyanins from blackberry in a rat model of acute lung inflammation. Free Radical Research Communications, 37:891-900.
[27]	Sessa G, Morelli G, Ruberti I. 1993. The Athb-1 and - 2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities. The EMBO Journal, 12 (9):3507-3517. doi: 10.1002/embj.1993.12.issue-9 URL
[28]	Song Yang, Liu Hongdi, Wang Haibo, Zhang Hongjun, Liu Fengzhi. 2019. Molecular cloning and functional characterization of anthocyanin synthesis related genes VcTTG1 of blueberry. Acta Horticulturae Sinica, 46 (7):1270-1278. (in Chinese)
	宋杨, 刘红弟, 王海波, 张红军, 刘凤之. 2019. 越橘花青苷合成相关基因 VcTTG1的克隆与功能鉴定. 园艺学报, 46 (7):1270-1278.
[29]	Sun Q G, Jiang S H, Zhang T L, Xu H F, Fang H C, Zhang J, Su M Y, Wang Y C, Zhang Z Y, Wang N, Chen X S. 2019. Apple NAC transcription factor MdNAC52 regulates biosynthesis of anthocyanin and proanthocyanidin through MdMYB9 and MdMYB11. Plant Science,289: https://doi.org/10.1016/j.plantsci.2019.110286.
[30]	Tsuda T, Horio F, Uchida K, Aoki H, Osawa T. 2003. Dietary cyaniding 3- O-beta-D-glucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice. Journal of Nutrition, 133:2125-2130. doi: 10.1093/jn/133.7.2125 URL
[31]	Wang N, Xu H F, Jiang S H, Zhang Z Y, Lu N L, Qiu H R, Qu C Z, Wang Y C, Wu S J, Chen X S. 2017. MYB12 and MYB22play essential roles in proanthocyanidin and flavonol synthesis in red-fleshed apple(Malus sieversii f. niedzwetzkyana). Plant Journal, 90:276-292. doi: 10.1111/tpj.2017.90.issue-2 URL
[32]	Wang Y C, Wang N, Xu H F, Jiang S H, Fang H C, Su M Y, Zhang Z Y, Zhang T L, Chen X S. 2018. Auxin regulates anthocyanin biosynthesis through the aux/IAA-ARF signaling pathway in apple. Horticulture Research, 5 (1):59. doi: 10.1038/s41438-018-0068-4 URL
[33]	Xie X B, Li S, Zhang R F, Zhao J, Chen Y C, Zhao Q, Yao Y X, You C X, Zhang X S, Hao Y J. 2012. The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples. Plant Cell and Environment, 35 (11):1884-1897. doi: 10.1111/j.1365-3040.2012.02523.x URL
[34]	Yang Guanxian, Xu Haifeng, Zhang Jing, Wang Nan, Fang Hongcheng, Jiang Shenghui, Wang Yicheng, Su Mengyu, Chen Xuesen. 2019. Functional identification of apple anthocyanin regulatory gene MdMYB111. Acta Horticulturae Sinica, 46 (5):832-840. (in Chinese)
	杨官显, 许海峰, 张静, 王楠, 房鸿成, 姜生辉, 王意程, 苏梦雨, 陈学森. 2019. 苹果花青苷调控基因 MdMYB111的功能鉴定. 园艺学报, 46 (5):832-840.
[35]	Zhang C M, Hao Y J. 2020. Advances in genomic,transcriptomic,and metabolomic analyses of fruit quality in fruit crops. Horticultural Plant Journal, 6 (6):361-371. doi: 10.1016/j.hpj.2020.11.001 URL
[36]	Zhang F, Zuo K J, Zhang J Q, Liu X, Zhang L D, Sun X F, Tang K X. 2010. An L 1 box binding protein,GbML1,interacts with GbMYB25 to control cotton fibre development. Journal of Experimental Botany, 61 (13):3599-3613. doi: 10.1093/jxb/erq173 pmid: 20667961
[37]	Zhang J, Xu H F, Wang N, Jiang S H, Fang H C, Zhang Z Y, Yang G X, Wang Y C, Su M Y, Xu L, Chen X S. 2018. The ethylene response factor MdERF1B regulates anthocyanin and proanthocyanidin biosynthesis in apple. Plant Molecular Biology, 98:205-218. doi: 10.1007/s11103-018-0770-5 pmid: 30182194
[38]	Zhang Jing, Ci Zhijuan, Xu Haifeng, Jiang Shenghui, Fang Hongcheng, Wang Yicheng, Zhang Zongying, Yang Guanxian, Chen Xensen. 2019. Cloning and functional characterization of an ethylene response factor MdERF1B-like in apple. Acta Horticulturae Sinica, 46 (6):1033-1044. (in Chinese)
	张静, 慈志娟, 许海峰, 姜生辉, 房鸿成, 王意程, 张宗营, 杨官显, 陈学森. 2019. 苹果乙烯响应因子MdERF1B-like的克隆与功能鉴定. 园艺学报, 46 (6):1033-1044.
[39]	Zhang Z, Tian C P, Zhang Y, Li C Z Y, Li X, Yu Q, Wang S, Wang X Y, Chen X S, Feng S Q. 2020. Transcriptomic and metabolomic analysis provides insights into anthocyanin and procyanidin accumulation in pear. BMC Plant Biology, https://doi.org/10.1186/s12870-020-02344-0.