白菜(Brassica campestris ssp. chinensis)BcMAX2调控腋芽生长的功能分析

doi:10.16420/j.issn.0513-353x.2022-0329

摘要/Abstract

摘要：

MAX2（MORE AXILLARY GROWTH 2）是独脚金内酯信号转导相关基因，其功能与腋芽生长有关。以白菜（Brassica campestris ssp. chinensis）分蘖菜品种‘马耳头’和普通白菜品种‘苏州青’为材料，探究MAX2对腋芽生长的影响。结果表明：从两种材料中克隆得到的BcMAX2其开放阅读框序列没有差异，‘马耳头’的BcMAX2启动子序列有2个碱基的缺失。亚细胞定位分析表明BcMAX2定位在细胞核。在拟南芥中过表达BcMAX2抑制腋芽生长，BRC1表达量升高。在‘马耳头’中沉默BcMAX2能引起BcBRC1表达量降低并促进腋芽伸长。通过对白菜cDNA文库筛选得到197个与BcMAX2互作的候选蛋白，其中多数候选互作蛋白具有结合活性和催化活性，部分候选蛋白还参与植物的信号转导和逆境响应。通过酵母双杂交和双分子荧光互补技术验证最终得到2个与BcMAX2互作的蛋白。试验表明，白菜BcMAX2可能通过促进BcBRC1的表达来负调控腋芽的生长。

关键词: 白菜, 腋芽生长, BcMAX2, BcBRC1

Abstract:

MAX2（MORE AXILLARY GROWTH 2）is a gene associated with strigolactone signal transduction. Its function is related to the growth of axillary buds. The effects of MAX2 on the growth of axillary buds were investigated by using Brassica campestris ssp. chinensis the multiple shoot branching cultivar‘Maertou’and common cultivar‘Suzhouqing’. The results showed that there were no differences in the ORF（open reading frame）sequence of BcMAX2 gene obtained from the two materials，but the BcMAX2 promoter sequence of‘Maertou’has a two-base deletion. Subcellular localization analysis indicated BcMAX2 localization in the nucleus. Overexpression of BcMAX2 in Arabidopsis increased BRC1 expression and inhibited axillary bud growth. Silencing of BcMAX2 in‘Maertou’caused the decrease of BcBRC1 expression and promoted the growth of axillary buds. Total of 197 candidate proteins interacting with BcMAX2 were obtained from the cDNA library of Brassica campestris ssp. chinensis. Most of the candidate proteins had binding and catalytic activities，and some of them were involved in signal transduction and stress response of plants. Two interacting proteins were verified by yeast two-hybrid system and bimolecular fluorescence complementation technique. The study showed that BcMAX2 may negatively regulate axillary bud growth by promoting BcBRC1 expression.

Key words: Brassica campestris ssp. chinensis, axillary bud growth, BcMAX2, BcBRC1

中图分类号:

S634.1

徐兰兰, 赵天孜, 张玮, 宗琛, 黄菲艺, 王建军, 侯喜林, 李英. 白菜(Brassica campestris ssp. chinensis)BcMAX2调控腋芽生长的功能分析[J]. 园艺学报, 2023, 50(5): 972-984.

XU Lanlan, ZHAO Tianzi, ZHANG Wei, ZONG Chen, HUANG Feiyi, WANG Jianjun, HOU Xilin, LI Ying. Function Analysis of BcMAX2 in Regulating Axillary Bud Growth in Brassica campestris ssp. chinensis[J]. Acta Horticulturae Sinica, 2023, 50(5): 972-984.

图/表 11

参考文献 36

[1]	Aguilar-Martínez J A, Poza-Carrión C, Cubas P. 2007. Arabidopsis BRANCHED1 acts as an integrator of branching signals within axillary buds. The Plant Cell, 19 (2):458-472. doi: 10.1105/tpc.106.048934 URL
[2]	Arite T, Umehara M, Ishikawa S, Hanada A, Maekawa M, Yamaguchi S, Kyozuka J. 2009. d14,a strigolactone-insensitive mutant of rice,shows an accelerated outgrowth of tillers. Plant and Cell Physiology, 50 (8):1416-1424. doi: 10.1093/pcp/pcp091 URL
[3]	Bennett T, Sieberer T, Willett B, Booker J, Luschnig C, Leyser O. 2006. The Arabidopsis MAX pathway controls shoot branching by regulating auxin transport. Current Biology, 16 (6):553-563. doi: 10.1016/j.cub.2006.01.058 pmid: 16546078
[4]	Bian Hai-yan, Zhong Qi-wen, Huang Si-jie, Wang Li-hui, Yang Shi-peng, Tian Jie. 2018. Analysis of gene expressions of fructan metabolism key enzymes in garlic under drought stress. Molecular Plant Breeding, 16 (20):6770-6776. (in Chinese)
	边海燕, 钟启文, 黄思杰, 王丽慧, 杨世鹏, 田洁. 2018. 干旱胁迫下大蒜果聚糖代谢关键酶基因的表达分析. 分子植物育种, 16 (20):6770-6776.
[5]	Booker J, Sieberer T, Wright W, Williamson L, Willett B, Stirnberg P, Turnbull C, Srinivasan M, Goddard P, Leyser O. 2005. MAX1 encodes a cytochrome P 450 family member that acts downstream of MAX3/4 to produce a carotenoid-derived branch-inhibiting hormone. Developmental Cell, 8 (3):443-449. doi: 10.1016/j.devcel.2005.01.009 URL
[6]	Braun N, Germain A D S, Pillot J P, Stephanie B M, Dalmais M, Antoniadi I, Li X, Alessandra M G, Signor C L, Bouteiller N, Luo D, Bendahmane A, Turnbull C, Rameau C. 2012. The pea TCP transcription factor PsBRC 1 acts downstream of strigolactones to control shoot branching. Plant Physiology, 158 (1):225-238. doi: 10.1104/pp.111.182725 pmid: 22045922
[7]	Carolien R S, Salim A B, Krol S D V, Bouwmeester H. 2013. The biology of strigolactones. Trends in Plant Science, 18 (2):72-83. doi: 10.1016/j.tplants.2012.10.003 URL
[8]	Cao Xue-wei. 2016. The formation mechanism and candidate gene identification of tillering in non-heading Chinese cabbage[Ph. D. Dissertation]. Nanjing: Nanjing Agricultural University. (in Chinese)
	曹学伟. 2016. 不结球白菜分枝性状的发生机理及其候选基因挖掘[博士论文]. 南京: 南京农业大学.
[9]	Chen X M, Shi X Y, Ai Q, Han J Y, Wang H S, Fu Q S. 2022. Transcriptomic and metabolomic analyses reveal that exogenous strigolactones alleviate the response of melon root to cadmium stress. Horticultural Plant Journal, 8 (5):637-649. doi: 10.1016/j.hpj.2022.07.001 URL
[10]	Cheng X, Ruyter-Spira C, Bouwmeester H. 2013. The interaction between strigolactones and other plant hormones in the regulation of plant development. Frontiers in Plant Science, 4:199. doi: 10.3389/fpls.2013.00199 pmid: 23785379
[11]	Cui Hong-mi. 2016. Study on the main influence factors of non-heading Chinese cabbage tillering[M. D. Dissertation]. Nanjing: Nanjing Agricultural University. (in Chinese)
	崔红米. 2016. 影响不结球白菜分枝性状的主要因素探讨[硕士论文]. 南京: 南京农业大学.
[12]	Domagalska M A, Leyser O. 2011. Signal integration in the control of shoot branching. Nature Reviews. Molecular Cell Biology, 12 (4):211-221.
[13]	Ferrero M, Pagliarani C, Novák O, Ferrandino A, Cardinale F, Visentin I. 2018. Exogenous strigolactone interacts with abscisic acid-mediated accumulation of anthocyanins in grapevine berries. J Exp Bot, 69:2391-2401. doi: 10.1093/jxb/ery033 pmid: 29401281
[14]	Gomez-Roldan V, Fermas S, Brewer P B, Puech-Pagès V, Dun E A, Pillot J P, Letisse F, Matusova R, Danoun S, Portais J C. 2008. Strigolactone inhibition of shoot branching. Nature, 455 (7210):189-194. doi: 10.1038/nature07271
[15]	Hamiaux C, Drummond R S M, Janssen B J, Ledger S E, Cooney J M, Newcomb R D, Snowden K C. 2012. DAD2 is an α/β hydrolase likely to be involved in the perception of the plant branching hormone,strigolactone. Current Biology, 22 (21):2032-2036. doi: 10.1016/j.cub.2012.08.007 pmid: 22959345
[16]	Hou Xilin, Li Ying, Huang Feiyi. 2020. Advances in molecular biology of main characters and breeding technology in non-heading Chinese cabbage (Brassica campestris ssp. chinensis). Acta Horticulturae Sinica, 47 (9):1663-1677. (in Chinese)
	侯喜林, 李英, 黄菲艺. 2020. 不结球白菜(Brassica campestris ssp. chinensis)主要性状及育种技术的分子生物学研究新进展. 园艺学报, 47 (9):1663-1677.
[17]	Hu J, Ji Y Y, Hu X T, Sun S Y, Wang X L. 2020. BES1 functions as the co-regulator of D53-like SMXLs to inhibit BRC1 expression in strigolactone-regulated shoot branching in Arabidopsis. Plant Communications, 1 (3):30-41.
[18]	Jiang L, Liu X, Xiong G S, Liu H H, Chen F L, Wang L, Meng X B, Liu G F, Hong Y, Yuan Y D, Yi W, Zhao L H, Ma H L, He Y Z, Wu H S, Melcher K, Qian Q, Xu H E, Wang Y H, Li J Y. 2013. DWARF 53 acts as a repressor of strigolactone signalling in rice. Nature, 504 (7480):401-405. doi: 10.1038/nature12870
[19]	Johnson X, Brcich T, Dun E A, Goussot M, Haurogné K, Beveridge C A, Rameau C. 2006. Branching genes are conserved across species. Genes controlling a novel signal in pea are coregulated by other long-distance signals. Plant Physiology, 142 (3):1014-1026. doi: 10.1104/pp.106.087676 pmid: 16980559
[20]	Li Ju. 2015. Expression analysis of TCP family transcription factor genes BcBRC1 and tillering traits genetic analysis in non-heading Chinese cabbage[M. D. Dissertation]. Nanjing: Nanjing Agricultural University. (in Chinese)
	李菊. 2015. 不结球白菜TCP家族转录因子基因BcBRC1的表达分析及分枝性状遗传分析[硕士论文]. 南京: 南京农业大学.
[21]	Li Z D, Li Y, Liu T K, Zhang C W, Xiao D, Hou X L. 2022. Non-heading Chinese cabbage database:an open-access platform for the genomics of Brassica campestris(syn. Brassica rapa)ssp. chinensis. Plants, 11 (8):101596181.
[22]	Seale M, Bennett T, Leyser O. 2017. BRC1 expression regulates bud activation potential but is not necessary or sufficient for bud growth inhibition in Arabidopsis. Development, 144 (9):1661-1673.
[23]	Shen R X, Ma X L, Wang H Y. 2020. SMXL6/7/8 :Dual-function transcriptional repressors of strigolactone signaling. Molecular Plant, 13 (9):1244-1246. doi: 10.1016/j.molp.2020.08.002 URL
[24]	Snowden K C, Simkin A J, Janssen B J, Templeton K R, Loucas H M, Simons J L, Karunairetnam S, Gleave A P, Klee C H J. 2005. The DECREASED APICAL DOMINANCE1/Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE8 gene affects branch production and plays a role in leaf senescence,root growth,and flower development. The Plant cell, 17 (3):746-759. doi: 10.1105/tpc.104.027714 URL
[25]	Stirnberg P, Furner I J, Leyseret H M O. 2007. MAX2 participates in an SCF complex which acts locally at the node to suppress shoot branching. The Plant Journal, 50 (1):80-94. doi: 10.1111/j.1365-313X.2007.03032.x URL
[26]	Stirnberg P, van de Sande K, Leyser H M. 2002. MAX1 and MAX2 control shoot lateral branching in Arabidopsis. Development, 129 (5):1131-1141. doi: 10.1242/dev.129.5.1131 pmid: 11874909
[27]	Soundappan I, Bennett T, Morffy N, Liang Y, Nelson D C. 2015. SMAX1-LIKE/D 53 family members enable distinct MAX2-dependent responses to strigolactones and karrikins in Arabidopsis. The Plant Cell, 27 (11):3143-3159. doi: 10.1105/tpc.15.00562 pmid: 26546447
[28]	Sun W J, Ji X L, Song L Q, Wang X F, You C X, Hao Y J. 2021. Functional identification of MdSMXL8.2,the homologous gene of strigolactones pathway repressor protein gene in Malus × domestica. Horticultural Plant Journal, 7 (4):275-285. doi: 10.1016/j.hpj.2021.01.001 URL
[29]	Umehara M, Hanada A, Yoshida S, Akiyama K, Arite T, Noriko T K, Magome H, Kamiya Y, Shirasu K, Yoneyama K, Kyozuka J, Yamaguchi S. 2008. Inhibition of shoot branching by new terpenoid plant hormones. Nature, 455 (7210):195-200. doi: 10.1038/nature07272
[30]	Wang L, Wang B, Jiang L, Liu X, Li J Y. 2015. Strigolactone signaling in Arabidopsis regulates shoot development by targeting D53-Like SMXL repressor proteins for ubiquitination and degradation. The Plant cell, 27 (11):3128-3142. doi: 10.1105/tpc.15.00605 pmid: 26546446
[31]	Wang L, Wang B, Yu H, Guo H Y, Lin T, Kou L Q, Wang A Q, Shao N, Ma H Y, Xiong G S, Li X Q, Yang J, Chu J F, Li J Y. 2020. Transcriptional regulation of strigolactone signalling in Arabidopsis. Nature, 583 (7815):277-281. doi: 10.1038/s41586-020-2382-x
[32]	Wang H W, Chen W X, Eggert K, Charnikhova T, Bouwmeester H, Schweizer P, Hajirezaei M R, Seiler C, Sreenivasulu N, Wirén N V, Kuhlmann M. 2018. Abscisic acid influences tillering by modulation of strigolactones in barley. Journal of Experimental Botany, 69 (16):3883-3898. doi: 10.1093/jxb/ery200 pmid: 29982677
[33]	Waters M T, Brewer P B, Bussel J D, Smith S M, Beveridge C A. 2012. The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones. Plant Physiology, 159 (3):1073-1085. doi: 10.1104/pp.112.196253 pmid: 22623516
[34]	Yang Xue-dong, Dai Wei, Zhang Chang-wei, Wang Jin-yan, Kong Min, Hou Xi-lin. 2012. The technology system establishment of VIGS in non-heading Chinese cabbage. Acta Horticulturae Sinica, 39 (11):2168-2174. (in Chinese)
	杨学东, 戴薇, 张昌伟, 王金彦, 孔敏, 侯喜林. 2012. 白菜病毒诱导基因沉默技术体系的建立. 园艺学报, 39 (11):2168-2174.
[35]	Zhang Wei, Guo Ming-liang, Huang Fei-yi, Long Yan, Hou Xi-lin, Li Ying. 2021. Homologous cloning and functional analysis of the tillering regulation gene BcMAX1 in non-heading Chinese cabbage. Journal of Nanjing Agricultural University, 44 (2):241-248. (in Chinese)
	张玮, 郭明亮, 黄菲艺, 龙言, 侯喜林, 李英. 2021. 不结球白菜分枝调控基因BcMAX1的同源克隆及功能分析. 南京农业大学学报, 44 (2):241-248.
[36]	Zou J H, Zhang S Y, Zhang W P, Li G, Chen Z X, Zhai W X, Zhao X F, Pan X B, Xie Q, Zhu L H. 2006. The rice HIGH‐TILLERING DWARF 1 encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds. The Plant Journal, 48 (5):687-698. doi: 10.1111/tpj.2006.48.issue-5 URL

引物名称	引物序列（5′-3′）	用途
Primer name	Primer sequence	Usage
BcMAX2	F：ATGGCTTCCACCACTCTCTGC	ORF扩增
BcMAX2	R：TCAGTCAATGATGATGCGGCT	Complete ORF amplification
BcMAX2-P	F：TTCAACCGAATCAATCCACTATTCT	启动子扩增
BcMAX2-P	R：CTCCAAGATTTGCTCTCATGGCTTCCAC	Complete promoter amplification
pRI101-BcMAX2	F：TTCTTCACTGTTGATACATATGATGGCTTCCACCACTCTCTGC	载体构建
pRI101-BcMAX2	R：TCGCCCTTGCTCACCATGGATCCTCAGTCAATGATGATGCGGCT	Construct of vector
RT-BcMAX2	F：AGGAGCTTGTGCTTGACGTT	RT-PCR
RT-BcMAX2	R：AAGACAGCGACAACAACCCT
Actin1	F：GGAGCTGAGAGATTCCGTTG	白菜内参基因
Actin1	R：GAACCACCACTGAGGACGAT	Internal reference gene in Brassica
Actin2	F：TTGACAATTGATGCAAACAATGACG	拟南芥内参基因
Actin2	R：CCATTGCTTAATTCCACGGACAAAC	Internal reference genein Arabidopsis
BcMAX2-BD	F：GGAATTCCATATGATGGCTTCCACCACTCTCTGC	诱饵载体构建
BcMAX2-BD	R：GGAATTCGTCAATGATGATGCGGCT	Construct of bait vector
BcNF-YC4-AD	F：CATATGGCCATGGAGGCCAGTGAATTCATGGACAACAACAACCAGCA	载体构建
BcNF-YC4-AD	R：ATCTGCAGCTCGAGCTCGATGGATCCGGAAGCAAGCTCATTACTAGT	Construct of vector
BcCEBiP-AD	F：CATATGGCCATGGAGGCCAGTGAATTCATGGAAACTTCCCGTTTTACCC	载体构建
BcCEBiP-AD	R：ATCTGCAGCTCGAGCTCGATGGATCCGGGAGAAGGCATGCACAGAAC	Construct of vector
BcBOLA-AD	F：CATATGGCCATGGAGGCCAGTGAATTCATGGAGGAGACAGATCGTTCA GATCGTTC	载体构建 Construct of vector
BcBOLA-AD	R：ATCTGCAGCTCGAGCTCGATGGATCCGGGATGGGAATGTCTTGATGGG
AD	F：CTATTCGATGATGAAGATACCCCACCAAACCC	cDNA插入片段扩增
AD	R：GTGAACTTGCGGGGTTTTTCAGTATCTACGATT	Amplification of cDNA fragments
pCAMBIA1300-35S -NYFP-BcNF-YC4	F：GAATTCATGGACAACAACAACCAGCA	BiFC载体构建
pCAMBIA1300-35S -NYFP-BcNF-YC4	R：GTCGACAAGCAAGCTCATTACTAGT	Construct of vector BiFC
pCAMBIA1300-35S -NYFP-BcCEBiP	F：GGATCCATGGAAACTTCCCGTTTTACCC	BiFC载体构建
pCAMBIA1300-35S -NYFP-BcCEBiP	R：ACTAGTGAGAAGGCATGCACAGAAC	Construct of vector BiFC
pCAMBIA1300-35S -NYFP-BcBOLA	F：GGATCCATGGAGGAGACAGATCGTTC	BiFC载体构建
pCAMBIA1300-35S -NYFP-BcBOLA	R：ACTAGTGATGGGAATGTCTTGATGGG	Construct of vector BiFC
pCAMBIA1300-35S -CYFP-BcMAX2	F：GAATTCATGGCTTCCACCACTCTCTGC	BiFC载体构建
pCAMBIA1300-35S -CYFP-BcMAX2	R：GTCGACGGTCAATGATGATGCGGCT	Construct of vector BiFC

引物名称	引物序列（5′-3′）	用途
Primer name	Primer sequence	Usage
BcMAX2	F：ATGGCTTCCACCACTCTCTGC	ORF扩增
BcMAX2	R：TCAGTCAATGATGATGCGGCT	Complete ORF amplification
BcMAX2-P	F：TTCAACCGAATCAATCCACTATTCT	启动子扩增
BcMAX2-P	R：CTCCAAGATTTGCTCTCATGGCTTCCAC	Complete promoter amplification
pRI101-BcMAX2	F：TTCTTCACTGTTGATACATATGATGGCTTCCACCACTCTCTGC	载体构建
pRI101-BcMAX2	R：TCGCCCTTGCTCACCATGGATCCTCAGTCAATGATGATGCGGCT	Construct of vector
RT-BcMAX2	F：AGGAGCTTGTGCTTGACGTT	RT-PCR
RT-BcMAX2	R：AAGACAGCGACAACAACCCT
Actin1	F：GGAGCTGAGAGATTCCGTTG	白菜内参基因
Actin1	R：GAACCACCACTGAGGACGAT	Internal reference gene in Brassica
Actin2	F：TTGACAATTGATGCAAACAATGACG	拟南芥内参基因
Actin2	R：CCATTGCTTAATTCCACGGACAAAC	Internal reference genein Arabidopsis
BcMAX2-BD	F：GGAATTCCATATGATGGCTTCCACCACTCTCTGC	诱饵载体构建
BcMAX2-BD	R：GGAATTCGTCAATGATGATGCGGCT	Construct of bait vector
BcNF-YC4-AD	F：CATATGGCCATGGAGGCCAGTGAATTCATGGACAACAACAACCAGCA	载体构建
BcNF-YC4-AD	R：ATCTGCAGCTCGAGCTCGATGGATCCGGAAGCAAGCTCATTACTAGT	Construct of vector
BcCEBiP-AD	F：CATATGGCCATGGAGGCCAGTGAATTCATGGAAACTTCCCGTTTTACCC	载体构建
BcCEBiP-AD	R：ATCTGCAGCTCGAGCTCGATGGATCCGGGAGAAGGCATGCACAGAAC	Construct of vector
BcBOLA-AD	F：CATATGGCCATGGAGGCCAGTGAATTCATGGAGGAGACAGATCGTTCA GATCGTTC	载体构建 Construct of vector
BcBOLA-AD	R：ATCTGCAGCTCGAGCTCGATGGATCCGGGATGGGAATGTCTTGATGGG
AD	F：CTATTCGATGATGAAGATACCCCACCAAACCC	cDNA插入片段扩增
AD	R：GTGAACTTGCGGGGTTTTTCAGTATCTACGATT	Amplification of cDNA fragments
pCAMBIA1300-35S -NYFP-BcNF-YC4	F：GAATTCATGGACAACAACAACCAGCA	BiFC载体构建
pCAMBIA1300-35S -NYFP-BcNF-YC4	R：GTCGACAAGCAAGCTCATTACTAGT	Construct of vector BiFC
pCAMBIA1300-35S -NYFP-BcCEBiP	F：GGATCCATGGAAACTTCCCGTTTTACCC	BiFC载体构建
pCAMBIA1300-35S -NYFP-BcCEBiP	R：ACTAGTGAGAAGGCATGCACAGAAC	Construct of vector BiFC
pCAMBIA1300-35S -NYFP-BcBOLA	F：GGATCCATGGAGGAGACAGATCGTTC	BiFC载体构建
pCAMBIA1300-35S -NYFP-BcBOLA	R：ACTAGTGATGGGAATGTCTTGATGGG	Construct of vector BiFC
pCAMBIA1300-35S -CYFP-BcMAX2	F：GAATTCATGGCTTCCACCACTCTCTGC	BiFC载体构建
pCAMBIA1300-35S -CYFP-BcMAX2	R：GTCGACGGTCAATGATGATGCGGCT	Construct of vector BiFC