高产脂思茅松牻牛儿基牻牛儿基焦磷酸合成酶基因的克隆及表达
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摘要
以高产脂思茅松为材料,基于转录组数据分析,采用RT-PCR方法获得思茅松牻牛儿基牻牛儿基焦磷酸合成酶(GGPS)基因的c DNA序列,Gen Bank登录号为KM382173.1.序列分析显示,思茅松GGPS基因c DNA全长1 152 bp,编码383个氨基酸,属于GGPS家族.序列比对分析显示,思茅松GGPS拥有GGPS家族特有的两个功能保守结构域FARM(DDLPSMDND)、SARM(DDILD),其与马尾松GGPS的同源性为78.07%.系统进化树分析显示,思茅松GGPS与马尾松GGPS的亲缘关系最近.荧光定量PCR分析表明,在思茅松的树皮中,GGPS基因的表达明显受到割脂物理伤害的诱导,在高产脂思茅松个体幼枝中的表达量远远高于在低产脂思茅松个体的表达量.
The present study used high-yielding-resin pine( Pinus kesiya var. langbianensis) as materials and cloned the full length of geranylgeranyl diphosphate synthase gene( GGPS) based on the transcriptome of P. kesiya by RT-PCR and named it Pk GGPS( Gen Bank ID: KM382173.1). Sequence analysis showed the open reading frame of Pk GGPS c DNA gene was 1 152 bp,encoding383 amino acids,and belonged to GGPS family. Proteinic sequence alignment revealled that PkGGPS consisted of 2 functionally conserved domains that were unique in the GGPS family,which shared 78. 07% similarity with that of Pinus massoniana. Amino acids sequences of the 2 domains were FARM( DDLPSMND) and SARM( DDILD),respectively. Phylogenetic tree analysis showed that the genetic relationship between PkGGPS and P.massoniana was the closest. The fluorescent quantitative reverse PCR proved that the expression level of Pk GGPS gene was stimulated by physical injury such as cutting the bark of P.kesiya. And the expression of PkGGPS gene in tender branch of high-yielding-resin individuals was much higher than low-yielding-resin ones.
The present study used high-yielding-resin pine( Pinus kesiya var. langbianensis) as materials and cloned the full length of geranylgeranyl diphosphate synthase gene( GGPS) based on the transcriptome of P. kesiya by RT-PCR and named it Pk GGPS( Gen Bank ID: KM382173.1). Sequence analysis showed the open reading frame of Pk GGPS c DNA gene was 1 152 bp,encoding383 amino acids,and belonged to GGPS family. Proteinic sequence alignment revealled that PkGGPS consisted of 2 functionally conserved domains that were unique in the GGPS family,which shared 78. 07% similarity with that of Pinus massoniana. Amino acids sequences of the 2 domains were FARM( DDLPSMND) and SARM( DDILD),respectively. Phylogenetic tree analysis showed that the genetic relationship between PkGGPS and P.massoniana was the closest. The fluorescent quantitative reverse PCR proved that the expression level of Pk GGPS gene was stimulated by physical injury such as cutting the bark of P.kesiya. And the expression of PkGGPS gene in tender branch of high-yielding-resin individuals was much higher than low-yielding-resin ones.
引文
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[21]李思广,付玉嫔,张快富,等.高松香思茅松无性系的选育[J].东北林业大学学报,2009,37(3):4-5.
[22]HENERY M L,MORAN G F,WALLIS I R,et al. Identification of quantitative trait loci influencing foliar concentrations of terpenes and formylated phloroglucinol compounds in Eucalyptus nitens[J]. New Phytologist,2007,176(1):82-95.
[23]INDURI B R,ELLIS D R,SLAVOV G T,et al. Identification of quantitative trait loci and candidate genes for cadmium tolerance in Populus[J]. Tree Physiology,2012,32(5):626-638.
[24]ZORRILLA-FONTANESI Y,RAMBLA J L,CABEZA A,et al. Genetic analysis of strawberry fruit aroma and identification of O-methyltransferase Fa OMT as the locus controlling natural variation in mesifurane content[J]. Plant Physiology,2012,59(2):851-870.
[25]王毅,周旭,毕玮,等.思茅松HDR基因全长c DNA克隆与序列分析[J].广西植物,2015,35(5):721-727.
[2]蒋云东,李思广,刘云彩.思茅松高产脂定向培育技术的研究进展[J].西部林业科学,2017,46(2):32-37.
[3]翁海龙,陈宏伟,段安安.思茅松高产脂优树选择[J].西北林学院学报,2010,25(3):71-74.
[4]许林红,蒋云东,付玉嫔,等.思茅松高产脂半同胞家系选育[J].西北林学院学报,2014,29(3):109-112.
[5]翁海龙,贾红亮,陈宏伟,等.思茅松高产脂优树产脂量相关因子分析[J].东北林业大学学报,2008,36(11):69-70.
[6]李思广,蒋云东,李明.思茅松树脂道数量与产脂力回归关系分析[J].福建林业科技,2007,34(1):59-62.
[7]VRANOVE,COMAN D,GRUISSEM W. Network analysis of the MVA and MEP pathways for isoprenoid synthesis[J]. Annual Review of Plant Biology,2013,64(1):665-700.
[8]马靓,丁鹏,杨广笑,等.植物类萜生物合成途径及关键酶的研究进展[J].生物技术通报,2006(S1):22-30.
[9]VANDERMOTEN S,HAUBRUGE,CUSSON M. New insights into short-chain prenyltransferases:structural features,evolutionary history and potential for selective inhibition[J]. Cellular and Molecular Life Sciences,2009,66(23):3 685-3 695.
[10]IGNEA C,IOANNOU E,GEORGANTEA P,et al. Reconstructing the chemical diversity of labdane-type diterpene biosynthesis in yeast[J]. Metabolic Engineering,2015,28:91-103.
[11]LIU Q,ZHOU Z,WEI Y,et al. Genome-wide identification of differentially expressed genes associated with the high yielding of oleoresin in secondary xylem of masson pine(Pinus massoniana Lamb.)by transcriptomic analysis[J]. PLoS ONE,2015,10(7):e132 624.
[12]肖明贵,余龙江,刘智,等.曼地亚红豆杉植株中GGPP合成酶的克隆与分析[J].中国生物工程杂志,2004,24(2):45-50.
[13]ZHANG M,SU P,ZHOU Y J,et al. Identification of geranylgeranyl diphosphate synthase genes from Tripterygium wilfordii[J]. Plant Cell Reports,2015,34(12):2 179-2 188.
[14]LIANG M H,LIANG Y J,JIN H H,et al. Characterization and functional identification of a gene encoding geranylgeranyl diphosphate synthase from Dunaliella bardawil[J]. Journal of Agricultural&Food Chemistry,2015,63(35):7 805-7 812.
[15]陈博雯,覃子海,王鹏良,等.马尾松GGPPS基因克隆及序列分析[J].西部林业科学,2016,45(2):1-6.
[16]方洁,李春芳,马春雷,等.茶树GGPS基因家族的克隆、生物信息学和表达分析[J].茶叶科学,2017,37(2):130-138.
[17]王毅,周旭,毕玮,等.思茅松1-脱氧-D-木酮糖-5-磷酸合酶(DXS)基因的克隆及功能分析[J].林业科学研究,2015,28(6):833-838.
[18] OHNUMA S,HIROOKA K,OHTO C,et al. Conversion from archaeal geranylgeranyl diphosphate synthase to farnesyl diphosphate synthase[J]. Journal of Biological Chemistry,1997,272(8):5 192-5 198.
[19]杨章旗.马尾松不同年龄产脂量及松香组分分析[J].林业科学,2014,50(6):147-151.
[20]ZHANG S,JIANG J,LUAN Q. Genetic and correlation analysis of oleoresin chemical components in slash pine[J]. Genetics&Molecular Research,2016,15(3):1-12.
[21]李思广,付玉嫔,张快富,等.高松香思茅松无性系的选育[J].东北林业大学学报,2009,37(3):4-5.
[22]HENERY M L,MORAN G F,WALLIS I R,et al. Identification of quantitative trait loci influencing foliar concentrations of terpenes and formylated phloroglucinol compounds in Eucalyptus nitens[J]. New Phytologist,2007,176(1):82-95.
[23]INDURI B R,ELLIS D R,SLAVOV G T,et al. Identification of quantitative trait loci and candidate genes for cadmium tolerance in Populus[J]. Tree Physiology,2012,32(5):626-638.
[24]ZORRILLA-FONTANESI Y,RAMBLA J L,CABEZA A,et al. Genetic analysis of strawberry fruit aroma and identification of O-methyltransferase Fa OMT as the locus controlling natural variation in mesifurane content[J]. Plant Physiology,2012,59(2):851-870.
[25]王毅,周旭,毕玮,等.思茅松HDR基因全长c DNA克隆与序列分析[J].广西植物,2015,35(5):721-727.