‘苹果梨’果实着色过程中PyCHI和PyF3H的克隆与表达分析Molecular cloning and expression of PyCHI and PyF3H in‘Pingguoli'during the pigmentation
杨宏霞;刘冰雁;刘振坤;鹿艳新;王雪;曲柏宏;申永杰;
摘要(Abstract):
【目的】克隆‘苹果梨’(Pyrus bretschneideri Rehd.‘Pingguoli’)Py CHI和Py F3H的全长c DNA序列,检测其在果实着色过程中的表达特性及其与花青苷积累的关系。【方法】以‘苹果梨’套袋后去袋处理的果实和未套袋的果实(CK)为试材,采用同源克隆技术克隆Py CHI和Py F3H的全长c DNA序列并进行生物信息预测分析;利用实时荧光定量PCR技术检测Py CHI和Py F3H在去袋处理与未套袋处理的不同着色时期的果实中的表达特性。【结果】Py CHI和Py F3H的全长c DNA分别为702 bp和1095 bp,分别编码233和364个氨基酸。同源性分析显示,其与沙梨、西洋梨等蔷薇科梨属植物相应基因的同源性均超过95%,相应氨基酸序列相似性均达到85%。实时荧光定量PCR分析显示,套袋果实去袋见光后Py CHI和Py F3H表达量均迅速上升,随后Py CHI表达量虽然略有下降,但在整个果实着色过程中Py CHI和Py F3H的表达量一直维持在较高水平,均显著高于对照,且与果实花青苷含量的变化规律基本一致。【结论】获得‘苹果梨’PyCHI和Py F3H的全长c DNA序列,其表达水平与果实花青苷积累关系密切,表明其在调控‘苹果梨’果实着色过程中起重要作用。
关键词(KeyWords): ‘苹果梨’;花青苷;基因克隆;表达分析
基金项目(Foundation): 国家自然科学基金(31160389)
作者(Author): 杨宏霞;刘冰雁;刘振坤;鹿艳新;王雪;曲柏宏;申永杰;
Email:
DOI: 10.13925/j.cnki.gsxb.20140419
参考文献(References):
- [1]WANG Yanling,ZHANG Yanmin,FENG Shouqian,TIAN Changping,WANG Haibo,LIU Zunchun,SONG Yang,CHEN Xuesen.Cloning,sequence analysis and expression in E.coli of Ms MYB10gene from Malus sieversii f.neidzwetzkyana[J].Scientia Agricultura Sinica,2010,43(13):2735-2743.王延玲,张艳敏,冯守千,田长平,王海波,刘遵春,宋杨,陈学森.新疆红肉苹果转录因子Ms MYB10基因的克隆、序列分析及原核表达[J].中国农业科学,2010,43(13):2735-2743.
- [2]ONO E,HOMMA Y,HORIKAWA M,KUNIKANE-D S,IMAI H,TAKAHASHI S,KAWAI Y,ISHIGURO M,FUKUI Y,NAKAYAMA T.Functional differentiation of the glycosyltransferases that contribute to the chemical diversity of bioactive flavonol glycosides in grapevines(Vitis vinifera)[J].The Plant cell,2010,22(8):2856-2871.
- [3]HUANG Chunhui,GE Cuilian,ZHANG Xiaohui,WU Han,QU Xueyan,XU Xiaobiao.Expression analysis of structural genes related to anthocyanin synthesis ina mutant of‘Hongyang’kiwifruit[J].Journal of Fruit Science,2014,31(2):169-174.黄春辉,葛翠莲,张晓慧,吴寒,曲雪艳,徐小彪.‘红阳’猕猴桃突变体果实花青苷合成相关结构基因的表达分析[J].果树学报,2014,31(2):169-174.
- [4]ZHANG Qian,YANG Jian,WANG Long,WANG Suke,LI Xiugen,ZHANG Shaoling.Advances in research of red-skinned pear coloration and anthocyanin synthesis[J].Journal of Fruit Science,2012,29(4):668-675.张茜,杨健,王龙,王苏珂,李秀根,张绍铃.红皮梨着色与花青苷合成研究进展[J].果树学报,2012,29(4):668-675.
- [5]SUN Bailing,QU Baihong,LI Wei,WANG Xu.Cloning and expression analysis of gene fragments of related fruit coloring enzymes in Pingguoli[J].Journal of Jilin Agricultural University,2012,34(4):423-427,442.孙百灵,曲柏宏,李伟,王旭.‘苹果梨’果实着色相关酶基因片段克隆及表达分析[J].吉林农业大学学报,2012,34(4):423-427,442.
- [6]WANG Xu,QU Baihong,LIU Zhenkun.Cloning and constructing the plant expression vector of phenylalanine ammonia lyase in Pingguoli in Yanbian[J].Northern Horticulture,2013(7):106-112.王旭,曲柏宏,刘振坤.延边‘苹果梨’苯丙氨酸解氨酶基因克隆及植物表达载体的构建[J].北方园艺,2013(7):106-112.
- [7]FENG Shouqian,CHEN Xuesen,ZHANG Chunyu,LIU Xiaojing,LIU Zunchun,WANG Haibo,WANG Yanling,ZHOU Cha-ohua.A study of the relationship between anthocyanin biosynthesis and related enzymes activity in Pyrus pyrifolia‘Mantianhong’and its bud sport‘sAoguan’[J].Scientia Agricultura Sinica,2008,41(10):3184-3190.冯守千,陈学森,张春雨,刘晓静,刘遵春,王海波,王延玲,周朝华.砂梨品种‘满天红’及其芽变品系‘奥冠’花青苷合成与相关酶活性研究[J].中国农业科学,2008,41(10):3184-3190.
- [8]YANG Hongli,WANG Yanhang,JIANG Zhengwang,HUANG Hongwen.Construction of c DNA library of‘Hongyang’kiwifruit and analysis of F3H expression[J].Hereditas(Beijing),2009,31(12):1265-1272.杨红丽,王彦昌,姜正旺,黄宏文.‘红阳’猕猴桃c DNA文库构建及F3H基因的表达初探[J].遗传,2009,31(12):1265-1272.
- [9]FENG F,LI M,MA F,CHENG L.Phenylpropanoid metabolites and expression of key genes involved in anthocyanin biosynthesis in the shaded peel of apple fruit in response to sun exposure[J].Plant Physiology and Biochemistry,2013,69:54-61.
- [10]NIU Shanshan,XU Changjie,ZHANG Wangshu,ZHANG Bo,LI Xian,WANG Kuilin,FERGUSON I B,ALLAN A C,CHEN Kunsong.Coordinated regulation of anthocyanin biosynthesis in Chinese bayberry(Myrica rubra)fruit by a R2R3 MYB transcription factors[J].Planta,2010,231(4):887-899.
- [11]KIM S H,LEE J R,HONG S T,YOO Y K,AN G,KIM S R.Molecular cloning and analysis of anthocyanin biosynthesis genes preferentially expressed in apple skins[J].Plant Science,2003,165:403-413.
- [12]HONDA C,KOTODA N,WADA M,KONDO S,KOBYASHI S,SOEJIMA J,ZHANG Zilian.TSUDA T,MORIGUCHI T.Anthocyanin biosynthetic genes are coordinately expressed during red coloration in apple skin[J].Plant Physiology and Biochemistry,2002,40(11):955-962.
- [13]ZHANG Xueying,ZHANG Shanglong,LUO Jun,YE Zhengwen,LI Shicheng.Advances in research on fruit anthoyanin synthesis[J].Journal of Fruit Science,2004,21(5):456-460.张学英,张上隆,骆军,叶正文,李世诚.果实花色素苷合成研究进展[J].果树学报,2004,21(5):456-460.
- [14]LIN W K,MICHELETTI D,PALMER J,VOLZ R,LOZANO L,ESPLEY R,HELLENS R P,CHAGNèD,ROWAN D D,TROGGIO M,IGLESIAS I,ALLAN A C.High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex[J].Plant,Cell&Environment,2011,34(7):1176-1190.
- [15]ZHANG X,ALLAN A C,YI Q,CHEN L,LI K,SHU Q,SU J.Differential gene expression analysis of Yunnan red pear,Pyrus Pyrifolia during fruit skin coloration[J].Plant Molecular Biology Reporter,2010,29(2):305-314.