桃SERK2 的克隆及其在不同状态愈伤组织中的表达分析

doi:10.3864/j.issn.0578-1752.2019.05.010

摘要/Abstract

摘要：

【目的】分离克隆桃（Prunus persica L.）体细胞胚胎发生相关类受体蛋白激酶（somatic embryogenesis receptor-like kinases,SERKs）基因SERK2 ,检测其在不同状态桃愈伤组织中的表达差异,分析SERK2 与胚性愈伤组织发生的关系,为揭示组织培养困难树种桃胚性愈伤组织产生的分子机理奠定基础。【方法】采用同源克隆法获得PpSERK2 的cDNA全长序列,运用TMPred、DNAMAN和MEGA 5.0等生物信息学软件对序列进行分析;以‘秋蜜红’花药为外植体,接种至添加2.0 mg·L ^-1 6-BA和1.0 mg·L ^-1 2,4-D的NN69培养基诱导愈伤组织;将获得的不同状态愈伤组织制作石蜡切片并进行组织细胞学观察;通过实时荧光定量PCR（qRT-PCR）对PpSERK2 在4种不同状态愈伤组织中的表达进行分析。【结果】克隆获得‘秋蜜红’PpSERK2 的cDNA全长序列1 881 bp,编码626个氨基酸,其蛋白质理论分子量为68.99 kD,理论等电点为5.38,具有完整的SERK蛋白的保守结构域,与牡丹（Paeonia suffruticosa ）等物种的同源性为67.88%—92.71%,且与秘鲁番茄（Solanum peruvianu ）和温州蜜柑（Citrus unshiu ）的相似性最高。在与其他物种的SERK蛋白构建的系统进化树中,PpSERK2与SpSERK1、CitSERK1和PsSERK2等聚在一起,与同源性比对结果一致。以‘秋蜜红’花药为外植体诱导获得4种不同状态的愈伤组织,组织细胞学观察发现黄色疏松状（球形胚出现）、绿色紧实状（心形胚出现）和浅黄色透明状（尚未完全形成的鱼雷形胚结构出现）的3种状态的愈伤组织细胞小、排列紧密;而黄白色水渍状愈伤组织细胞体积大、排列不规则,细胞质稀且染色浅。根据形态学和组织学的鉴定结果,可知黄色疏松状、绿色紧实状和浅黄色透明状的3种状态的愈伤组织均为胚性愈伤组织,而黄白色水渍状愈伤组织为非胚性愈伤组织。实时荧光定量PCR（qRT-PCR）结果表明,PpSERK2 在‘秋蜜红’3种状态的胚性愈伤组织中的表达量显著高于非胚性愈伤组织,且在3种胚性愈伤组织中的表达量有差异,其中在黄色疏松状愈伤组织中表达量最高,绿色紧实状次之,浅黄色透明状中最低。【结论】克隆获得PpSERK2 的cDNA全长序列,其在3种状态的胚性愈伤组织中的表达量明显高于非胚性愈伤组织,且在黄色疏松状胚性愈伤组织中的表达量最高,PpSERK2 在桃体细胞胚发育的早期发挥作用。

关键词: 桃, 体细胞胚发生, SERK2, 胚性愈伤组织

Abstract:

【Objective】The somatic embryogenesis receptor-like kinase 2 (SERK2 ) gene was isolated and cloned from peach (Prunus persica L.). Here, we detected the expression profile of SERK2 in different forms of calli in peach, and analyzed the correlation between SERK2 and the regeneration of embryonic callus. This research will shed on uncovering the molecular mechanism of embryonic callus induction and regeneration in peach with difficulty on tissue culture. 【Method】The full-length cDNA sequence of PpSERK2 gene was obtained by homologous cloning. Then the sequence was analyzed by a series of bioinformatical software packages, including TMPred, DNAMAN and MEGA 5.0 etc. The callus of ‘Qiumihong’ was obtained by using anthers as explants inoculated into NN69 medium supplemented with 2.0 mg·L ^-1 6-BA and 1.0 mg·L ^-1 2,4-D. Then the different forms of calli were observed by production of paraffin section. Real-time quantitative PCR (qRT-PCR) was used to analyze the expression of PpSERK2 gene in four forms of ‘Qiumihong’ calli. 【Result】The full-length cDNA sequence of PpSERK2 gene was 1 881 bp. It encodes 626 amino acids and contains SERK conserved function domains. The theoretical isoelectric point of PpSERK2 is 5.38 and its molecular weight is 68.99 KD. The PpSERK2 has the high homology similarity from 67.88% to 92.71% at protein level, especially the highest similarity to Solanum peruvianu and Citrus unshiu . The phylogenetic analysis of SERK proteins from various plant species indicated that PpSERK2, SpSERK1, CitSERK1 and PsSERK2 were clustered together, which showing the consistent result with above protein similarity analysis. Four different forms of calli were obtained from anther of ‘Qiumihong’. The results of histocytology showed that the cells of three forms of calli, including the yellow, loose callus (global embryo), green, compact callus (heart embryo) and pale yellow, transparent callus (suspected torpedo embryo), were small and closely arranged. While the cells of yellow-white, dropsical callus was large and with irregular shapes. Based on histomorphology results, this demonstrated that the yellow, loose callus, green, compact callus and pale yellow, transparent callus were embryonic calli, while yellow-white, dropsical callus was non-embryonic callus. The results of qRT-PCR showed that transcriptional level of PpSERK2 gene in three forms of embryonic calli was remarkably higher than non-embryonic callus. Meanwhile, the transcriptional level of PpSERK2 gene was highest in the yellow, loose callus, followed by the green, compact callus, and lowest in the pale yellow and transparent callus of ‘Qiumihong’. 【Conclusion】The full-length cDNA of the PpSERK2 gene was successfully obtained. According to the results of PpSERK2 expression in four forms calli of ‘Qiumihong’, we speculated that PpSERK2 gene might play a pivotal role on the early stage during the somatic embryogenesis in peach.

Key words: peach, somatic embryogenesis, SERK2, embryonic callus

谭彬,陈谭星,韩亚萍,张亚如,郑先波,程钧,王伟,冯建灿. 桃SERK2 的克隆及其在不同状态愈伤组织中的表达分析[J]. 中国农业科学, 2019, 52(5): 882-892.

TAN Bin,CHEN TanXing,HAN YaPing,ZHANG YaRu,ZHENG XianBo,CHENG Jun,WANG Wei,FENG JianCan. Cloning and Expression Analysis of SERK2 Gene in Different Forms of Calli on Peach (Prunus persica L.)[J]. Scientia Agricultura Sinica, 2019, 52(5): 882-892.

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