杧果MGAPDH同源基因的克隆及其表達分析

摘 要： 甘油醛-3-磷酸脫氫酶（GAPDH）是糖代謝和能量代謝過程中的關鍵酶。根據GAPDH基因保守序列設計PCR引物，利用RT-PCR和改良RACE技術，首次從杧果中分離克隆出MGAPDH同源基因的cDNA全長序列。該序列全長為1 356 bp，開放閱讀框為1 203 bp，編碼401個氨基酸。利用分子生物學軟件對MGAPDH蛋白進行生物信息學分析，結果顯示：MGAPDH蛋白分子量為42.8 ku，等電點為8.9；該蛋白包含2個功能域，即NADB_ Rossmann superfamily和Gp_dh_C superfamily；MGAPDH蛋白不含信號肽序列和跨膜結構，是非分泌蛋白， 位于葉綠體中。氨基酸序列進化分析結果顯示，杧果MGAPDH蛋白可能與光合作用有關。半定量分析顯示，杧果MGAPDH同源基因在杧果不同組織中均表達，并且表達水平差異不明顯，說明杧果MGAPDH同源基因可作為杧果基因差異表達的內參基因。

關鍵詞： 杧果； 甘油醛-3-磷酸脫氫酶（GAPDH）； 表達分析； 序列分析

中圖分類號：Ｓ６６7．7 文獻標識碼：Ａ 文章編號：１００９－９９８０?穴２０11?雪０6－1019－０6

Molecular cloning and expression analysis of a MGAPDH homologous gene from mango

LUO Cong1， HE Xin-hua1，2*， HU Ying1， TAN Chao1， OU Shi-jin1

（1College of Agriculture， Guangxi University， Nanning， 530004； 2Guangxi Crop Genetic Improvement and Biotechnology Lab， Nanning， 530007 China）

Abstract: Glyceral-dehyde-3-phosphate dehyrogenase (GAPDH） is a key enzyme in sugar metabolism and energy metabolism. PCR primers were designed by the conserved sequences of GAPDH gene，and a full-length cDNA sequence of MGAPDH homologous gene was firstly cloned from Mango by employing RT-PCR and modified RACE technique. The full-length cDNA is 1 356 bp and contains an ORF with 1 203 bp， corresponding to a deduced protein of 401 amino acids. The results of bioinformatics analysis showed that the estimated molecular weight and isoelectric point of the putative protein were 42.8 ku and 8.9. The protein had two conserved domains， named as NADB_ Rossmann superfamily and Gp_dh_C superfamily. Bioinformatics analysis also indicated that the protein of mango MGAPDH is a non-secreting protein without any signal peptide and transmembrane domain，MGAPDH gene was located in the chloroplast. The phylogenetic analysis based on the sequence of amino acids showed that MGAPDH protein may relative to photosynthesis. Semiquantitative analysis showed that the MGAPDH homologous gene expressed in all tissues of mango， no significant differences in expression levels were founded among the tested tissues; the result indicted that MGAPDH homologous gene may be used as an internal standard for differential gene expression analysis in mango.

Key words: Mango（Mangifera indica）； Glyceral-dehyde-3-phosphate dehyrogenase (GAPDH）； Expression analysis； Sequence analysis

甘油醛-3-磷酸脫氫酶 （Glyceral-dehyde-3-phosphate dehyrogenase GAPDH） 是維持生命活動最基本的酶之一，是糖代謝和能量代謝過程中的關鍵酶，廣泛存在于所有原核和真核生物中，它是一種多功能蛋白。在哺乳動物中，它具有膜融合與膜轉運活性、微管蛋白成束活性、磷酸化活性、核RNA輸出活性、DNA修復活性和核膜融合活性等，并且它還參與細胞凋亡過程[1]。在植物中，GAPDH與糖酵解、光合作用和抗逆有關。目前報道，高等植物中存在2種不同類型的GAPDH，一類依賴于NAD+，存在于細胞質中，在糖酵解過程中起作用，是糖酵解和糖異生過程中的關鍵酶；另一類以NAPD（H）為輔酶，也可以NAD+為輔酶，存在于葉綠體，是參與卡爾文循環的關鍵酶，與植物光合作用有關[2-3]。……