DONG Huijie,HOU Xilin,HAN Ke,et al.Cloning and expression analysis of anthocyanins negative regulatory factor BrcLBD39 and its response to exogenous 6-BA in non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2018,41(1):49-56.[doi:10.7685/jnau.201704035]





Cloning and expression analysis of anthocyanins negative regulatory factor BrcLBD39 and its response to exogenous 6-BA in non-heading Chinese cabbage
董慧杰 侯喜林 韩克 张志硕 胡春梅
南京农业大学园艺学院/作物遗传与种质创新国家重点实验室, 江苏 南京 210095
DONG Huijie HOU Xilin HAN Ke ZHANG Zhishuo HU Chunmei
College of Horticulture/State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China
non-heading Chinese cabbageLBD39homology-based cloningsequence analysisgene expression
[目的]本文旨在探究LBD39基因在不结球白菜紫色和绿色材料中的特性及其对外源6-BA的响应,为花青苷调控机制奠定理论基础。[方法]以不结球白菜紫色自交系NJZX1-3及其绿色突变体NJZX1-0为材料,同源克隆LBD39基因的全长,应用生物信息学方法分析其核酸和蛋白序列及其进化关系,并在线预测蛋白质二级和三级结构。采用外源6-BA处理后测定叶片中花青苷含量,并通过实时荧光定量PCR技术测定了LBD39基因在外源6-BA处理后的表达水平。[结果]在2个不同材料中克隆获得的LBD39基因完全相同,序列长为876 bp,包含编码区(长为699 bp)及非编码区(长为177 bp)。其编码区序列含有1个长为696 bp的开放阅读框(ORF),编码232个氨基酸,将该基因命名为BrcLBD39。亚细胞定位预测该蛋白分布在细胞核中,蛋白相对分子质量为25.30×103,等电点为8.85。进化树分析表明,BrcLBD39蛋白序列与大白菜BraLBD39相似性为100%,其次与油菜和甘蓝关系最近,相似性分别为99%和93%。外源6-BA处理24 h以后紫色材料NJZX1-3叶片中花青苷含量均高于对照,而绿色突变体叶片基本不含花青苷;BrcLBD39基因在2个材料中具有相似的表达模式,即先下降后上升而后又下降,其中在处理后48 h时BrcLBD39基因表达量急剧增加,在紫色和绿色材料中增量分别为74.84%和49.87%。此外,BrcLBD39表达量在绿色材料中明显高于紫色材料。[结论]不结球白菜中花青苷负调控基因BrcLBD39既响应外源6-BA,又与叶片中花青苷的积累密切相关,在花青苷的生物合成过程中起着重要的调控作用。
[Objectives]The aim of the study is to investigate the characteristic of LBD39 gene responsive to 6-BA in non-heading Chinese cabbage,providing the theoretical basis of the regulation of anthocyanins. [Methods]In this experiment,the full length of LBD39 gene was cloned from two materials of non-heading Chinese cabbage,that was the purple self-line NJZX1-3 and its green mutant line NJZX1-0. The gene sequences and protein sequences were analyzed by bioinformatics,and the econdary structure and tertiary structure of BrcLBD39 protein were predicted online. The materials were treated by 6-BA,and the expression level of LBD39 was determined by quantitative real-time PCR and the total anthocyanins content was also measured. [Results]The study indicated that the sequences of the two cDNA clones were coincident completely,with a full-length of 876 bp,including the code area(699 bp) and the noncode area(177 bp). The open reading frame(ORF) of the code area of LBD39 was 696 bp in length and encoded 232 predicted amino acids,designated as BrcLBD39. Subcellular localization predicted that the protein was distributed in cell nucleus. The molecular weight of BrcLBD 39 was 25.30×103 and pI value was 8.85. The BrcLBD39 protein had 100% identities to BraLBD39,and shared the most close relationship with Brassica rapa,then Brassica napus and Brassica oleracea,with the identities of 99% and 93%,respectively. With the treatment of 6-BA,total anthocyanins content in the purple self-line NJZX1-3 increased and were higher than that of control after 24 h,however,anthocyanins were absent in the green mutant line NJZX1-0. At the same time,BrcLBD39 expressed in the two different self-lines with similar expression patterns,which declined first and then rose significantly and then fell again. The expression of BrcLBD39 increased to the maximum at 48 h,with the increment of 74.84% and 49.87% in the purple and green materials,respectively. Furthermore,the expression level of BrcLBD39 in NJZX1-0 was higher than that in NJZX1-3. [Conclusions]The negative regulatory gene BrcLBD39 was responsive to exogenous 6-BA and had close relation with the accumulation of anthocyanins in non-heading Chinese cabbage,it suggests that the gene might play a key regulatory role in anthocyanins biosynthesis.


[1] Bradshaw H D,Schemske D W. Allele substitution at a flower colour locus produces a pollinator shift in monkeyflowers[J]. Nature,2003,426(6963):176-178.
[2] Yamasaki H,Sakihama Y,Ikehara N. Flavonoid-peroxidase reaction as a detoxification mechanism of plant cells against H2O2[J]. Plant Physiology,1997,115(4):1405.
[3] Steyn W J,Wand S J E,Holcroft D M,et al. Anthocyanins in vegetative tissues:a proposed unified function in photoprotection[J]. New Phytologist,2002,155(3):349-361.
[4] Koes R,Verweij W,Quattrocchio F. Flavonoids:a colorful model for the regulation and evolution of biochemical pathways[J]. Trends in Plant Science,2005,10(5):236-242.
[5] Zhu H F,Fitzsimmons K,Khandelwal A,et al. CPC,a single-repeat R3 MYB,is a negative regulator of anthocyanin biosynthesis in Arabidopsis[J]. Molecular Plant,2009,2(4):790-802.
[6] Rubin G,Tohge T,Matsuda F,et al. Members of the LBD family of transcription factors repress anthocyanin synthesis and affect additional nitrogen responses in Arabidopsis[J]. The Plant Cell,2009,21(11):3567-3584.
[7] Guo N,Cheng F,Wu J,et al. Anthocyanin biosynthetic genes in Brassica rapa[J]. BMC Genomics,2014,15:426.
[8] 王小青,韩键,文杨,等. 呈色机制不同的桃叶片花色素苷积累及合成相关基因表达的季节性差异[J]. 南京农业大学学报,2016,39(6):924-931. DOI:10.7685/njau.201602027. Wang X Q,Han J,Wen Y,et al. Seasonal difference in the expression pattern of genes related to anthocyanin accumulation and biosynthesis in leaves of peach with different coloration modes[J]. Journal of Nanjing Agricultural University,2016,39(6):924-931(in Chinese with English abstract).
[9] Longo L,Vasapollo G. Extraction and identification of anthocyanins from Smilax aspera L. berries[J]. Food Chemistry,2004,94(2):226-231.
[10] 李妍,王雪花,陈忠文,等. 不结球白菜抗坏血酸合成基因BcGME的同源克隆及胁迫下的表达分析[J]. 南京农业大学学报,2016,39(2):205-212. DOI:10.7685/jnau.201607026. Li Y,Wang X H,Chen Z W,et al. Homologous cloning and expression analysis of ascorbic acid biosynthesis gene BcGME under stress from non-heading Chinese cabbage[J]. Journal of Nanjing Agricultural University,2016,39(2):205-212(in Chinese with English abstract).
[11] 谭国飞,王枫,贾晓玲,等. 芹菜甘露醇脱氢酶基因的分离与表达分析[J]. 园艺学报,2013,40(11):2189-2198. Tan G F,Wang F,Jia X L,et al. Isolation and expression of mannitol dehydrogenase gene in celery[J]. Acta Horticulturae Sinica,2013,40(11):2189-2198(in Chinese with English abstract).
[12] Shuai B,Reynaga-Pena C G,Springer P S. The lateral organ boundaries gene defines a novel,plant-specific gene family[J]. Plant Physiology,2002,129(2):747-761.
[13] Husbands A,Bell E M,Shuai B,et al. LATERAL ORGAN BOUNDARIES defines a new family of DNA-binding transcription factors and can interact with specific bHLH proteins[J]. Nucleic Acids Research,2007,35(19):6663-6671.
[14] Okushima Y,Fukaki H,Onoda M,et al. ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis[J]. The Plant Cell,2007,19(1):118-30.
[15] Chiu L W,Zhou X,Burke S,et al. The purple cauliflower arises from activation of a MYB transcription factor[J]. Plant Physiology,2010,154(3):1470-1480.
[16] Hayashi K,Matsumoto S,Tsukazaki H,et al. Mapping of a novel locus regulating anthocyanin pigmentation in Brassica rapa[J]. Breeding Science,2010,60(1):76-80.
[17] 刘瑾,汪维红,张德双,等. 控制白菜叶片紫色的pur基因初步定位[J]. 华北农学报,2013,28(1):49-53. Liu J,Wang W H,Zhang D S,et al. Primary mapping of pur,a gene controlling purple leaf color in Brassica rapa[J]. Acta Agriculturae Boreali-Sinica,2013,28(1):49-53(in Chinese with English abstract).
[18] 王海竹,徐启江,闫海芳. 光照、糖和激素对花青素合成调控的综述[J]. 江西农业学报,2016,28(9):35-41. Wang H Z,Xu Q J,Yan H F. Summary of regulation of anthocyanin synthesis by light,sugar and hormone[J]. Acta Agriculturae Jiangxi,2016,28(9):35-41(in Chinese with English abstract).
[19] 鲁绪才. 6-BA调控叶片衰老的研究进展[J]. 安徽农业科学,2013,41(9):3761-3762. Lu X C. Research progress of 6-BA on leaf senescence regulation[J]. Journal of Anhui Agricultural Sciences,2013,41(9):3761-3762(in Chinese with English abstract).
[20] Nakamura N,Nakanae H,Maekawa S. Effects of light and kinetin on anthocyanin accumulation in the petals of Rosa hybrida,Hort cv. Ehigasa.[J]. Zeitschrift Für Pflanzenphysiologie,1980,98(3):263-270.
[21] Wade H K,Sohal A K,Jenkins G I. Arabidopsis ICX1 is a negative regulator of several pathways regulating flavonoid biosynthesis genes[J]. Plant Physiology,2003,131(2):707-715.
[22] Das P K,Shin D H,Choi S B,et al. Cytokinins enhance sugar-induced anthocyanin biosynthesis in Arabidopsis[J]. Molecules and Cells,2012,34(1):93-101.
[23] Das P K,Shin D H,Choi S B,et al. Sugar-hormone cross-talk in anthocyanin biosynthesis[J]. Molecules and Cells,2012,34(6):501-507.
[24] Zhou L L,Shi M Z,Xie D Y. Regulation of anthocyanin biosynthesis by nitrogen in TTG1-GL3/TT8-PAP1-programmed red cells of Arabidopsis thaliana[J]. Planta,2012,236(3):825-837.
[25] Albert N W,Davies K M,Lewis D H,et al. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots[J]. The Plant Cell,2014,26(3):962-980.
[26] 郭宁. 白菜花青苷和黄酮醇苷自然变异及遗传机制研究[D]. 北京:中国农业科学院,2014. Guo N. Natural variation and genetic mechanism of anthocyanin and flavonol glycosides metabolism in Brassica rapa[D]. Beijing:Chinese Academy of Agricultural Sciences,2014(in Chinese with English abstract).


[1]张爱芬,王立,侯喜林,等.不结球白菜S 位点受体激酶基因片段的克隆与表达分析[J].南京农业大学学报,2011,34(3):25.[doi:10.7685/j.issn.1000-2030.2011.03.005]
 ZHANG Ai-fen,WANG Li,HOU Xi-lin,et al.Cloning and expression analysis of SRK gene fragment in non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2011,34(1):25.[doi:10.7685/j.issn.1000-2030.2011.03.005]
 HUANG Jian-feng,XU Xiao-meng,SHEN Qi-rong,et al.Physiological mechanisms for the difference of nitrate accumulation in two cultivars of non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2011,34(1):74.[doi:10.7685/j.issn.1000-2030.2011.01.014]
 CHEN Yi-bo,HOU Xi-lin,CHEN Xiao-feng.Studies on heat tolerance mechanism of non-heading Chinese cabbage(Brassica campestris ssp.chinensis)[J].Journal of Nanjing Agricultural University,2010,33(1):27.[doi:10.7685/j.issn.1000-2030.2010.01.006]
 ZHENG Jia-qiu,HOU Xi-lin,ZHU hong-fang.Induction of heat shock protein synthesis and chilling tolerance in Brassica campestris ssp.chinensis[J].Journal of Nanjing Agricultural University,2010,33(1):30.[doi:10.7685/j.issn.1000-2030.2010.02.006]
 BAN Qing-yu,GENG Jian-feng,HOU Xi-lin,et al.QTL mapping for proline and soluble protein content of leaves in non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2010,33(1):35.[doi:10.7685/j.issn.1000-2030.2010.02.007]
 HAO Hui-nan,WANG Qian,HOU Xi-lin,et al.Segregation analysis of the main agronomic characters of non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2010,33(1):8.[doi:10.7685/j.issn.1000-2030.2010.04.002]
 RONG Zi-long,HOU Xi-lin,SHI Gong-jun,et al.Cloning and expression analysis of late bolting BcFLC1 gene from Brassica campestris ssp.chinensis[J].Journal of Nanjing Agricultural University,2010,33(1):23.[doi:10.7685/j.issn.1000-2030.2010.06.005]
 SHEN Shan-na,HOU Xi-lin.Changes of protective substances and enzymes in non-heading Chinese cabbage after infection by downy mildew[J].Journal of Nanjing Agricultural University,2009,32(1):23.[doi:10.7685/j.issn.1000-2030.2009.01.005]
 CHENG Yan,BAN Qing-yu,WANG Qian,et al.Isolated microspore culture and ploidy identification of microspore-derived plants in Brassica campestris ssp.chinensis[J].Journal of Nanjing Agricultural University,2009,32(1):25.[doi:10.7685/j.issn.1000-2030.2009.02.006]
 LIU Lin,HOU Xi-lin,WANG Li-ying,et al.Changes of four protective enzyme activities and relationships to resistance in non-heading Chinese cabbage after infection of Turnip mosaic virus[J].Journal of Nanjing Agricultural University,2009,32(1):14.[doi:10.7685/j.issn.1000-2030.2009.03.003]


更新日期/Last Update: 1900-01-01