杉木涩籽形成过程的花青素还原酶基因表达
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摘要
为探究杉木涩籽形成过程中,花青素还原酶(ANR)基因在类黄酮生物合成途径中的分子调控机制,以种子为试验材料,通过RT-PCR结合RACE的方法对杉木ANR基因进行全长克隆,运用q PCR技术分析不同涩籽率的杉木家系中ANR基因在种子不同发育时期的表达模式。结果显示,克隆到的ANR基因全长c DNA序列为1 357 bp,具有一个1 056 bp的开放阅读框,编码351个氨基酸。保守域预测显示ANR蛋白属于SDR超家族,具有一个FR_SDR_e特异性结构域。q PCR结果发现,在高涩籽率杉木中,90、100 d的表达量一直显著高于中、低涩籽率杉木;而在中、低涩籽率杉木中,ANR基因的表达模式仅仅是种子发育80 d时较高,之后明显下调。表明杉木涩籽率可能与其ANR基因表达相关。
To explore the role of anthocyanin reductase( ANR) gene in flavonoid biosynthesis pathway during the formation of sterile seeds of Chinese fir,the full-length ANR gene was cloned by RT-PCR combined with RACE,and the ANR gene expression in different development stages of seeds was analyzed by q PCR. The results showed that the full-length c DNA sequence of the cloned ANR gene was 1 357 bp,it contains a 1 056 bp open reading frame and encodes 351 amino acids. Conservative domain prediction indicated that ANR protein belongs to SDR superfamily and has a FR_SDR_e specific domain. q PCR results showed that ANR gene expression in family with high sterile seed rate was significantly higher at 90 and 100 d of seed development than the families with middle and low sterile seed rate. However,the ANR gene expression in families with middle and low sterile seed rate was only higher at 80 d of seed development,and then decreased. The results showed that the sterile seed rate of Chinese fir might be related to ANR gene expression.
To explore the role of anthocyanin reductase( ANR) gene in flavonoid biosynthesis pathway during the formation of sterile seeds of Chinese fir,the full-length ANR gene was cloned by RT-PCR combined with RACE,and the ANR gene expression in different development stages of seeds was analyzed by q PCR. The results showed that the full-length c DNA sequence of the cloned ANR gene was 1 357 bp,it contains a 1 056 bp open reading frame and encodes 351 amino acids. Conservative domain prediction indicated that ANR protein belongs to SDR superfamily and has a FR_SDR_e specific domain. q PCR results showed that ANR gene expression in family with high sterile seed rate was significantly higher at 90 and 100 d of seed development than the families with middle and low sterile seed rate. However,the ANR gene expression in families with middle and low sterile seed rate was only higher at 80 d of seed development,and then decreased. The results showed that the sterile seed rate of Chinese fir might be related to ANR gene expression.
引文
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[17]KUMAR V,YADAV S K. Overexpression of Cs DFR and Cs ANR enhanced flavonoids accumulation and antioxidant potential of roots in tobacco[J]. Plant Root,2013,7:65-76.
[18]MO Y,NAGEL C,TAYLOR L P. Biochemical complementation of chalcone synthase mutants defines a role for flavonols in functional pollen[J]. Proceedings of the National Academy of Sciences of the United States of America,1992,89(15):7 213-7 217.
[19]YLSTRA B,TOURAEV A,MORENO R M B,et al. Flavonols stimulate development,germination,and tube growth of tobacco pollen[J]. Plant Physiology,1992,100(2):902-907.
[20]MAHAJAN M,AHUJA P S,YADAV S K. Post-transcriptional silencing of flavonol synthase mRNA in tobacco leads to fruits with arrested seed set[J]. PLoS One,2011,6(12):e28 315.
[21]乔小燕,马春雷,陈亮.植物类黄酮生物合成途径及重要基因的调控[J].天然产物研究与开发,2009,21(2):354-360,207.
[22]ZHU Y,WANG H Y,PENG Q Z,et al. Functional characterization of an anthocyanidin reductase gene from the fibers of upland cotton(Gossypium hirsutum)[J]. Planta,2015,241(5):1 075-1 089.
[23]BAUDRY A,HEIM M A,DUBREUCQ B,et al. TT2,TT8,and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana[J]. The Plant Journal,2004,39(3):366-380.
[24]戴鹏辉,任锐,曹福祥,等.珙桐MYB转录因子Di MYB1基因的克隆及表达分析[J].植物生理学报,2016,52(8):1 255-1 262.
[2]管康林.杉木种子空瘪涩粒成因的综合分析[J].浙江林学院学报,1997,14(1):88-93.
[3]黄烺增,谢永强,齐清琳,等.杉木种子涩籽胚败育过程及其生理生化指标[J].福建林学院学报,1993,13(4):401-406.
[4]黄烺增.杉木种子涩籽胚败育与过氧化物酶同工酶活性及脂质过氧化作用的关系[J].西北植物学报,1996,16(5):83-88.
[5]林小琴,马志慧,何宗明,等.杉木种子多酚提取工艺优化[J].森林与环境学报,2017,37(1):47-53.
[6]XIE D Y,SHARMA S B,PAIVA N L,et al. Role of anthocyanidin reductase,encoded by BANYULS in plant flavonoid biosynthesis[J]. Science,2003,299(5 605):396-399.
[7]DEBEAUJON I,NESI N,PEREZ P,et al. Proanthocyanidin-accumulating cells in Arabidopsis testa:regulation of differentiation and role in seed development[J]. The Plant Cell,2003,15(11):2 514-2 531.
[8]李军,梁燕梅,赵爱春,等.桑树花青素还原酶基因Ma ANR的克隆和表达分析[J].蚕业科学,2016,42(4):570-575.
[9]DEVIC M,GUILLEMINOT J,DEBEAUJON I,et al. The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development[J]. The Plant Journal,1999,19(4):387-398.
[10]THIRUGNANASAMBANTHAM K,MURALIDARAN S,MANDAL A K A. Molecular cloning,computational and expression analysis of anthocyanidin reductase in Tea(Camellia sinensis)[J]. Applied Biochemistry and Biotechnology,2014,174(1):130-145.
[11] OPPERMANN U,FILLING C,HULT M,et al. Short-chain dehydrogenases/reductases(SDR):the 2002 update[J].Chemico-Biological Interactions,2003,143/144:247-253.
[12]SHEN G A,PANG Y Z,WU W S,et al. Isolation and characterization of a putative anthocyanidin reductase gene from Ginkgo biloba[J]. Journal of Plant Physiology,2006,163(2):224-227.
[13]ALBERT S,DELSENY M,DEVIC M. BANYULS,a novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat[J]. The Plant Journal,1997,11(2):289-299.
[14]姜春丽,苏新堯,许亚春,等.光照对药食同源植物苦荞麦种子中原花青素积累关键酶Ft ANR,Ft LAR基因表达的影响[J].中国中药杂志,2018,43(3):469-477.
[15] LIU Y,SHI Z,MAXIMOVA S,et al. Proanthocyanidin synthesis in Theobroma cacao:genes encoding anthocyanidin synthase,anthocyanidin reductase,and leucoanthocyanidin reductase[J]. BMC Plant Biology,2013,13:202.
[16]LEPINIEC L,DEBEAUJON I,ROUTABOUL J M,et al. Genetics and biochemistry of seed flavonoids[J]. Annual Review of Plant Biology,2006,57:405-430.
[17]KUMAR V,YADAV S K. Overexpression of Cs DFR and Cs ANR enhanced flavonoids accumulation and antioxidant potential of roots in tobacco[J]. Plant Root,2013,7:65-76.
[18]MO Y,NAGEL C,TAYLOR L P. Biochemical complementation of chalcone synthase mutants defines a role for flavonols in functional pollen[J]. Proceedings of the National Academy of Sciences of the United States of America,1992,89(15):7 213-7 217.
[19]YLSTRA B,TOURAEV A,MORENO R M B,et al. Flavonols stimulate development,germination,and tube growth of tobacco pollen[J]. Plant Physiology,1992,100(2):902-907.
[20]MAHAJAN M,AHUJA P S,YADAV S K. Post-transcriptional silencing of flavonol synthase mRNA in tobacco leads to fruits with arrested seed set[J]. PLoS One,2011,6(12):e28 315.
[21]乔小燕,马春雷,陈亮.植物类黄酮生物合成途径及重要基因的调控[J].天然产物研究与开发,2009,21(2):354-360,207.
[22]ZHU Y,WANG H Y,PENG Q Z,et al. Functional characterization of an anthocyanidin reductase gene from the fibers of upland cotton(Gossypium hirsutum)[J]. Planta,2015,241(5):1 075-1 089.
[23]BAUDRY A,HEIM M A,DUBREUCQ B,et al. TT2,TT8,and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana[J]. The Plant Journal,2004,39(3):366-380.
[24]戴鹏辉,任锐,曹福祥,等.珙桐MYB转录因子Di MYB1基因的克隆及表达分析[J].植物生理学报,2016,52(8):1 255-1 262.