龍眼MEE70/MSI1基因DlMEE70—1a及其可變剪接體DlMEE70—1b的分離及其在體胚發(fā)生過程中的表達(dá)分析

盧秉國　練從龍　賴鐘雄

摘 要 MEE70/MSI1是擬南芥母性效應(yīng)胚胎滯育基因，在植物胚胎發(fā)生過程中具有重要作用。采用RACE技術(shù)獲得龍眼MEE70-1a（登錄號KC492117）及其可變剪接體MEE70-1b（登錄號KC492118）cDNA序列，克隆MSI1 gDNA（登錄號KC492126）序列。生物信息學(xué)分析預(yù)測DlMEE70-1a和DlMEE70-1b均為親水不穩(wěn)定的酸性蛋白，主要定位于過氧化物酶體和細(xì)胞核，各含有4和1個(gè)WD-repeat結(jié)構(gòu)域。MEE70-1a和MEE70-1b在體細(xì)胞胚胎發(fā)生過程實(shí)時(shí)熒光定量PCR分析結(jié)果表明，DlMEE70-1a和DlMEE70-1b表達(dá)趨勢以心形胚為界，除了DlMEE70-1b的不完全胚型緊實(shí)結(jié)構(gòu)（ICpEC）到球形胚（GE）階段之間，都表現(xiàn)出先降后升的趨勢；胚性愈傷組織（EC）時(shí)期兩者表達(dá)量一致且最高；心形胚（HE）時(shí)期，兩者表達(dá)量一致且最低，另DlMEE70-1b在不完全胚性緊實(shí)結(jié)構(gòu)（ICpEC）表達(dá)量與心形胚基本一致。這2個(gè)表達(dá)劑量是胚胎發(fā)生的重要保障，心形胚（HE）后，DlMEE70-1a被再次激活轉(zhuǎn)錄促進(jìn)胚胎正常發(fā)育。從DlMEE70-1a和DlMEE70-1b的理化性質(zhì)、WD重復(fù)結(jié)構(gòu)域數(shù)量、亞細(xì)胞定位以及表達(dá)量和趨勢，可以推測DlMEE70-1a需要DlMEE70-1b的協(xié)同來調(diào)控龍眼胚胎發(fā)育。

關(guān)鍵詞 龍眼；體胚發(fā)生；MEE70/MSI1；母性效應(yīng)；可變剪接；qPCR

中圖分類號 S667.2 文獻(xiàn)標(biāo)識碼 A

Gene Cloning of DlMEE70-1a and DlMEE70-1b of MEE70/MSI1

from Embryogenic Callus and Their Expression Analysis During

Somatic Embryogenesis in Dimocarpus longan Lour.

LU Bingguo， LIAN Conglong， LAI Zhongxiong*

Institute of Horticultural Biotechnology， Fujian Agriculture and Forestry University， Fuzhou， Fujian 350002， China

Abstract MEE70/MSI1， a maternal effect embryo arrest gene in Arabidopsis， plays an important role in plant embryogenesis. In the present study， the full-length cDNA sequences of DlMEE70-1a gene（Accession number： KC492117）and its alternative splicing DlMEE70-1b（Accession number： KC492118）were obtained by RT-PCR combined with RACE techniques， while the gDNA sequence of MSI1 gene（Accession number： KC492126）was also obtained. Bioinformatics analysis indicated that both of the proteins encoded by DlMEE70-1a and DlMEE70-1b were of unstable hydrophilic and acidic proteins，mainly located in peroxisomes and the nucleus and containing 4 WD-repeat domains and 1 WD-repeat domain，respectively. Real-time fluorescence quantitative PCR analysis demonstrated that， during the somatic embryogenesis， the expression levels of DlMEE70-1a and DlMEE70-1b wre bounded at the stage of the heart-shaped embryo and displayed the trend of first-decreasing-then-rising， except for DlMEE70-1b during the period of development from the incomplete compact pro-embryogenic cultures to globular embryos. High expression of DlMEE70-1a and DlMEE70-1b in the friable-embryogenic callus was important for embryogenesis，The transcription of DlMEE70-1a was activated after the heart embryo. It was interesting that DlMEE70-1a and DlMEE70-1b had the similar physical and chemical properties， the same subcellular localization and the similar expression levels and trends. Therefore， it was inferred that DlMEE70-1b was coupled with DlMEE70-1a， possessed the coordinated regulation during the embryogenesis of longan. This study provided some new clues to the elaboration of the molecular mechanism of maternal effect genes DlMEE70 in the process of somatic embryogenesis.

Key words Dimocarpus longan； Somatic embryogenesis； MEE70/MSI1；Maternal effect； Alternative splicing； qPCR

doi 10.3969/j.issn.1000-2561.2015.01.011

MEE（maternal effect embryo arrest）是Pagnussat 等[1]通過Ac/Ds轉(zhuǎn)座子系統(tǒng)篩選擬南芥雌性生殖缺陷突變體群，發(fā)現(xiàn)該突變體群自花授粉或授以野生型花粉，后代都有50%左右種子敗育，體現(xiàn)出母性效應(yīng)基因遺傳特征，遺傳分析確定MEE70和MSI1屬于同一基因。MSI1蛋白是WD40重復(fù)蛋白成員之一，一般只存在于真核生物，是保守的組蛋白綁定蛋白[2]。MSI1蛋白是染色質(zhì)排列因子CAF-1的亞基[3-5]，其還存在于RBR、CUL4-DDB1等多個(gè)染色質(zhì)修飾蛋白復(fù)合體，控制印記基因表達(dá)和關(guān)閉[6-7]。MSI1是多梳抑制蛋白復(fù)合體PRC2的亞基，在擬南芥中有3個(gè)類型PRC2，植物發(fā)育中起重要調(diào)控作用[8]。……