菠萝AcHMGR基因的克隆与表达分析

Cloning and Expression Analysis of AcHMGR Gene in Pineapple

以菠萝(Ananas comosus L.cv.Comte de Paris)果实为材料,采用RT-PCR结合RACE方法得到3-羟基-3-甲基戊二酸单酰辅酶A还原酶基因(命名为AcHMGR)的c DNA及基因组DNA全长。AcHMGR的c DNA全长2 407 bp,其开放读码框长度为1 740 bp,编码579个氨基酸;其基因组DNA全长为4 115 bp,从起始密码子到终止密码子的长度为3 764 bp,含有4个外显子和3个内含子。荧光定量PCR结果表明,对照的菠萝果实中,AcHMGR基因表达量在花后0 d最高,从花后30 d开始,其表达量急剧下降,之后维持在一个较低水平直到果实成熟。经20 mg/L N-(2-氯-4-吡啶基)-N'-苯基脲(CPPU)处理后,显著提高了AcHMGR基因在花后30 d果实中的相对表达量,为同期对照的1.8倍;但其他时期的相对表达量与对照相比无明显差异。研究结果表明,AcHMGR基因在菠萝果实早期发育过程中表达量较高,CPPU处理提高了AcHMGR基因的相对表达量并显著提高果实的重量,说明CPPU处理后AcHMGR基因可能在促进果实重量的增加中起着重要作用。

In this study, one gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase（ HMGR）（designated as AcHMGR） was cloned from pineapple（Ananas comosus L. cv. Comte de Paris） fruit using RT-PCR and RACE. The full-lengths c DNA of AcHMGR was 2 407 bp, and the open reading frames was 1 740 bp, which encoding a putative protein of 579 amino acids. The genomic DNA sequence of AcHMGR was 4 115 bp, with the length of 3 764 bp, from the start codon to the terminator codon, which contained 4 exons and 3 introns.Phylogenetic tree analysis indicated that AcHMGR belonged to plant HMGR groups. The result of quantitative real-time PCR showed that the relative expressions of AcHMGR was the highest at 0 days after flowering（DAF）,then it sharply decreased after 30 DAF until maturation. After 20 mg/L N-（2-chloro-4-pyridyl）-N＇-phenylurea（CPPU） treatment, the relative expressions of AcHMGR in fruit was significantly up-regulated at 30 DAF（1.8-fold compared with the control）, but the expressions had no obviously difference compared with the control at other development stages of pineapple fruit. The results showed that AcHMGR was highly expressed at the early development of pineapple fruit, and the expression was up-regulated by CPPU treatment. On the other hand,CPPU treatment significantly increased fruit weight, which meaning AcHMGR might play an important role during the process of CPPU increased pineapple fruit weight.