太子参醛氧化酶基因克隆及表达分析
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摘要
目的:获取太子参醛氧化酶(abscisic acid aldehyde oxidase,AAO)基因。方法:基于AAO基因的同源性和太子参转录组数据库,以块根为材料,利用3'-RACE技术和PCR技术克隆获取AAO基因序列,通过qRT-PCR技术检测该基因在太子参茎、叶、须根,不同生长时期及经外源脱落酸及其抑制剂处理后的块根中表达情况。结果:克隆获得太子参Ph AAO基因序列长6 773 bp,含完整开放阅读框(4 053 bp),由10个外显子和9个内含子组成,氨基酸结构预测其含有钼-黄素酶家族基因特有的结构域。qRT-PCR分析显示Ph AAO基因在太子参叶、须根中显著表达,在块根形成的关键时期6月中旬表达量最高;脱落酸和氟啶酮处理后PhAAO基因表达上调,其中氟啶酮处理组较为明显。结论:获得太子参Ph AAO基因序列,并进一步研究该基因的表达情况,为后续研究其功能特点,探讨太子参脱落酸生物合成分子机制奠定理论基础。
Objective: To obtain nucleotide sequence information of abscisic acid aldehyde oxidase( AAO) gene. Methods: The AAO gene was obtained by cloning with 3'-RACE technique and PCR technique. The expression in the stems,leaves,fibrous roots,roots at different growth stages and the roots treated with exogenous abscisic acid and fluridone of Pseudostellaria heterophylla was detected by qRT-PCR. Results: The results showed that the sequence of Ph AAO gene was 6 773 bp included a complete ORF( 4 053 bp) and 10 exons and 9 introns,which containing molybdenum-lysin family-specific domai by bioinformatics analysis. qRT-PCR analysis showed that Ph AAO gene had higher expression level in leaves and tuberous root and in mid-June which was the critical period than other tissues and growth periods of Pseudostellaria heterophylla. Compared with the control group,the Ph AAO gene showed up-regulated expression in Pseudostellaria heterophylla root of abscisic acid and fluridone treatment group,especially the fluridone treatment group. Conclusion: The study obtains Ph AAO gene of Pseudostellaria heterophylla. In addition,the expression of the gene is analyzed. The research is great significance for the further investigation of the Ph AAO gene functional identification and provides a theoretical basis for the further analysis on the abscisic acid biosynthesis of Pseudostellaria heterophylla.
Objective: To obtain nucleotide sequence information of abscisic acid aldehyde oxidase( AAO) gene. Methods: The AAO gene was obtained by cloning with 3'-RACE technique and PCR technique. The expression in the stems,leaves,fibrous roots,roots at different growth stages and the roots treated with exogenous abscisic acid and fluridone of Pseudostellaria heterophylla was detected by qRT-PCR. Results: The results showed that the sequence of Ph AAO gene was 6 773 bp included a complete ORF( 4 053 bp) and 10 exons and 9 introns,which containing molybdenum-lysin family-specific domai by bioinformatics analysis. qRT-PCR analysis showed that Ph AAO gene had higher expression level in leaves and tuberous root and in mid-June which was the critical period than other tissues and growth periods of Pseudostellaria heterophylla. Compared with the control group,the Ph AAO gene showed up-regulated expression in Pseudostellaria heterophylla root of abscisic acid and fluridone treatment group,especially the fluridone treatment group. Conclusion: The study obtains Ph AAO gene of Pseudostellaria heterophylla. In addition,the expression of the gene is analyzed. The research is great significance for the further investigation of the Ph AAO gene functional identification and provides a theoretical basis for the further analysis on the abscisic acid biosynthesis of Pseudostellaria heterophylla.
引文
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[6]龙登凯,李军,周涛,等.太子参类胡萝卜素双加氧裂解酶家族4成员的克隆及表达特性分析[J].中国中药杂志,2016,41(13):2404-2410.
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[9]丁铃,江维克,周涛,等.太子参肌动蛋白基因Ph ACT2的全长c DNA克隆与生物信息学分析[J].分子植物育种,2017,(2):460-467.
[10]Seo M,Koiw Ai H,Akaba S,et al.Abscisic aldehyde oxidase in leaves of Arabidopsis thaliana[J].Plant Journal,2000,23(4):481-488.
[11]朱攀攀,刘长英,赵爱春,等.桑树脱落酸生物合成相关基因的鉴定及转录表达分析[J].中国农业科学,2015,48(5):1011-1022.
[12]Zhu JK.Salt and drought stress signal transductionin plants[J].Annual Review of Plant Biology,2002,53:247-273.
[13]肖承鸿,周涛,江维克,等.贵州太子参生物量与次生代谢物积累的动态变化分析[J].中国药学杂志,2013,48(16):1346-1351.
[2]Milborrow BV.The pathway of biosynthesis of abscisic acid in vascular plant:a review of the present state of knowledge of ABA biosynthesis[J].Journal of Experimental Botany,2001,52:1145-1164.
[3]陶均,李玲.高等植物脱落酸生物合成的酶调控[J].植物学报,2002,19(6):675-683.
[4]王庆美,张立明,王振林.甘薯内源激素变化与块根形成膨大的关系[J].中国农业科学,2005,38(12):2414-2420.
[5]郑伟,周涛,李军,等.太子参玉米黄质环氧化酶基因的克隆与表达分析[J].中国中药杂志,2017,42(4):669-674.
[6]龙登凯,李军,周涛,等.太子参类胡萝卜素双加氧裂解酶家族4成员的克隆及表达特性分析[J].中国中药杂志,2016,41(13):2404-2410.
[7]Li J,Zheng W,Long D,et al.De novo sequencing and assembly analysis of the Pseudostellaria heterophylla transcriptome[J].PLo S ONE,2016,11(10):1-19.
[8]Ren H,Gao Z,Chen L,et al.Dynamic analysis of ABA accumulation in relation to the rate of ABA catabolism in maize tissues under water deficit[J].Journal of Experimental Botany,2007,58(2):211-221.
[9]丁铃,江维克,周涛,等.太子参肌动蛋白基因Ph ACT2的全长c DNA克隆与生物信息学分析[J].分子植物育种,2017,(2):460-467.
[10]Seo M,Koiw Ai H,Akaba S,et al.Abscisic aldehyde oxidase in leaves of Arabidopsis thaliana[J].Plant Journal,2000,23(4):481-488.
[11]朱攀攀,刘长英,赵爱春,等.桑树脱落酸生物合成相关基因的鉴定及转录表达分析[J].中国农业科学,2015,48(5):1011-1022.
[12]Zhu JK.Salt and drought stress signal transductionin plants[J].Annual Review of Plant Biology,2002,53:247-273.
[13]肖承鸿,周涛,江维克,等.贵州太子参生物量与次生代谢物积累的动态变化分析[J].中国药学杂志,2013,48(16):1346-1351.