百子蓮體細胞發育受體激酶APSERK1（Somatic Embryogenesis Receptor Kinase1）基因全長克隆及表達分析

張琰 石玉波

摘 要：根據前期百子蓮（Agapanthus praecox ssp.orientalis）轉錄組測序分析的結果，獲得了1個與胚性能力相關的關鍵基因Somatic Embryogenesis Receptor Kinase1（SERK1）同源性較高的核心片段。采用cDNA末端快速擴增（rapid amplification of cDNA ends，RACE）方法得到了百子蓮SERK基因cDNA全長序列，命名為ApSERK1。序列分析表明，百子蓮ApSERK1基因開放閱讀框（ORF）為1800bp，編碼1個含有600個氨基酸的蛋白質。氨基酸同源性比對發現，百子蓮ApSERK1蛋白序列與水稻、小蘭嶼蝴蝶蘭、鐵皮石斛、椰子樹和菠蘿等植物蛋白序列的相似性可達到90%以上。實時熒光定量qRT-PCR結果表明，ApSERK1基因有一定時空表達差異，在百子蓮的種子、小花梗中表達含量最高，且隨著外源生長素濃度的增加，胚性愈傷組織的胚性能力呈現下降趨勢。推測ApSERK1基因是衡量植物細胞胚性能力的重要指標。

關鍵詞：百子蓮；體細胞發育受體激酶；基因克隆；表達分析

中圖分類號 S682.32 文獻標識碼 A 文章編號 1007-7731（2018）21-0036-05

Molecular Cloning and Expression Analysis of Somatic Embryogenesis Receptor Kinase1 in Agapanthus praecox

Zhang Yan et al.

（1Shanghai Vocational Technical College of Agriculture and Forestry，Shanghai 201600，China）

Abstract：A core fragment highly homologous with the key gene of embryo ability pathway， Somatic Embryogenesis Receptor Kinase1（SERK1 was obtained on the basis of preliminary Agapanthus praecox ssp. orientalis transcriptome sequencingresults. The full length sequence of cDNA， the SERK1 gene of A. praecox was gained by the RACE method， which was named ApSERK1 . The results of sequence analysis showed that the full length of cDNA was 1800bp， which was composed of 600 encoded amino acids.The amino acid homology comparison found that the ApSERK1 protein sequence of baizilian could be more than 90% similar to the plant protein sequences of rice， xiaolangyu butterfly orchid， dendrobium candidum， coconut tree and pineapple.The results of real-time quantitative PCR showed that ApSERK1 gene had a certain temporal and spatial expression difference， with the highest expression content in the seed and floret stalk， and the germinal capacity of embryonal callus decreased with the increase of exogenous auxin concentration.It is speculated that ApSERK1 gene is an important indicator of the embryonic ability of plant cells.

Key words：Agapanthus praecox ssp. orientalis；Somatic cell Receptor kinase；Gene cloning；Expression analysis

基于德國植物生理學家Haberlandt（1902）提出的細胞全能性（Cell Totipotency）理論[1]，植物體細胞胚胎發生（Somatic Embryogenesis，SE）是指在離體培養條件下，單倍體或雙倍體的體細胞向類似合子胚的體細胞胚胎（Somatic Embryos）發生方式轉變，并發育成新個體的形態發生過程[2]。自從1958 年英國生物學家F.C.Steward和J.Reinert分別以胡蘿卜的根系作為外植體成功誘導出體細胞再生植株以來[3]，目前在裸子植物、雙子葉植物、單子葉植物中已有300多種植物利用其莖段、下胚軸、子葉、葉片、葉柄、果肉、花芽、根、合子胚、胚乳等作為外植體建立了SE 技術體系[4]。體細胞發育受體激酶 SERK（Somatic Embryogenesis Recepotor Kinase）基因無疑是檢驗植物體細胞體系建立的1個關鍵性基因。1997年Schmidt等首先從胡蘿卜懸浮胚性細胞中檢測出DcSERK基因的差異表達，并將其作為胚性能力檢驗的首要分子標記物[5]。……