多叶斑叶兰繁殖体系建立及基于转录组的发育调控途径功能基因研究

doi:10.13523/j.cb.20181208

中国生物工程杂志

2018, Vol. 38

Issue (12): 57-64 DOI: 10.13523/j.cb.20181208

技术与方法

多叶斑叶兰繁殖体系建立及基于转录组的发育调控途径功能基因研究

何官榕¹,何碧珠²,吴沙沙²,石京山³,陈集双^3,^**(

),兰思仁^2,^**(

)

1 福建农林大学植物保护学院福州 350002
2 福建农林大学兰科植物保护与利用国家林业与草原局重点实验室福州 350002
3 遵义医学院基础药理教育部重点实验室特色民族药学教育部国际联合实验室遵义 563000

Establishment of an Efficient Regeneration System in Goodyera foliosa and Comprehensive Analysis of Functionally Regulated Genes Involved in Developmental Regulatory Pathways Based on Transcriptome Analysis

HE Guan-rong¹,HE Bi-zhu²,WU Sha-sha²,SHI Jing-shan³,CHEN Ji-shuang^3,^**(

),LAN Si-ren^2,^**(

)

1 College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002,China
2 The Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization,Fujian Agriculture and Forestry University, Fuzhou 350002, China
3 Key Laboratory of Basic Pharmacology and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi 563000, China

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摘要：

多叶斑叶兰,是兰科斑叶兰属濒危野生植物、国家二级保护植物,具有极高的观赏和药用价值。多叶斑叶兰由于分布种群小,传播扩散能力弱,自然繁殖受到极大限制。以野生多叶斑叶兰茎段作为外植体,建立高效直接的植株再生体系。结合高通量转录组测序技术与生物信息学分析技术,深入挖掘参与多叶斑叶兰器官发育过程的功能基因。结果表明, Morel+2.0 mg/L 6-BA +0.5 mg/LKT+1.0 mg/LNAA+1g/L蛋白胨+25g/L 蔗糖+7.0 g/L Agar+1.0 g/L活性炭+30 g/L 香蕉+50 g/L土豆为最佳的芽诱导培养基;Morel+3 mg/L 6-BA+0.5mg/L NAA+0.5 mg/LKT+0.01 mg/L TDZ+2g/L蛋白胨+25g/L 蔗糖+7.0 g/L Agar+1.0 g/L活性炭30g/L 香蕉+50g/L 土豆为最佳芽增殖培养基;1/2 Morel+1.0 mg/L IBA+0.1 mg/L NAA+1 g/L+花宝2 号+25g/L 蔗糖+7.0g/L Agar +1.0 g/L活性炭+1g·L-1蛋白胨为最佳的生根培养基。转录组测序分析组装获得170688个Unigene,平均长度为584 bp, N50为833 bp。17352个Unigene比对注释到NR、Swiss-Prot、KOG、GO、KEGG数据库。野生苗与组培苗差异表达Unigene GO与KEGG功能富集分析显示, Unigene主要参与调控植物激素信号转导、植物形态发育、次生代谢过程以及能量代谢过程等生物学功能。进一步分析获得511个参与植物器官发育调控相关的转录因子。成功建立了多叶斑叶兰种质资源保存与高效离体再生体系,结合高通量转录组学技术,获得全面完整的多叶斑叶兰转录组信息特征,为后期多叶斑叶兰快速扩繁、遗传转化以及功能基因鉴定、遗传发育及其调控机制研究奠定基础。

关键词： 多叶斑叶兰; 资源保存; 植株再生; 器官发育; 转录组分析

Abstract:

Goodyera foliosa, belonged to the genus of Cymbidium, is an endangered wild and national secondary protected plant which is used as ornamental plants and for various medicinal purposes. Duo to its small distribution population and weak transmission and diffusion, the natural reproduction is greatly limited. In this study, a high efficient in vitro regeneration system was developed from stem explants of Goodyera foliosa. The functional genes involved in the morphogenesis development was deeply explored by integrating with high-throughput transcriptome sequencing and bioinformatics analysis technology. For shoot-inducing , the optical culture medium is Morel + 2.0 mg/L 6-BA + 0.5 mg/L KT+1.0 mg/L NAA + 1g/L peptone + 25g/L sucrose + 7.0 g/L Agar + 1.0 g/L active carbon + 30 g/L banana + 50 g/L potato. The optical culture medium for bud proliferation is Morel + 3 mg/L 6-BA + 0.5 mg/L NAA + 0.5 mg/L KT + 0.01 mg/L TDZ + 2g/L peptone + 25g/L sucrose + 7.0 g/L Agar + 1.0 g/L active carbon + 30g/L banana + 50g/L potato. On the rooting medium with 1/2 Morel + 1.0 mg/L IBA + 0.1 mg/L NAA + 1 g/L + Hyponex NO.2 + 25g/L sucrose + 7.0g/L Agar + 1.0 g/L active carbon + 1g/L peptone. After transcriptom sequencing and assembling, 170, 688 Unigenes were obtained. The average length and N50 length of Unigenes was 584bp and 833bp respectively. Total of 17, 352 Unigenes were completely annotated to 5 functional databases including NR, Swiss-Prot, KOG, GO and KEGG. The functional analysis of differential Unigenes was showed that hormone signal transduction, plant development, secondary metabolites and energy metabolism were significantly enriched. Moreover, 511 Unigene encoding transcription factors involved with plant organ developmental regulation were predicted. Conclusion, a comprehensive transcriptom landscape of Goodyera foliosa was described by integrating with a high efficient in vitro regeneration system and next high-throughput trancriptom sequencing. This work could provide certain reference for fast propagation, genetic transformation, functional gene mining and development mechanism research of Goodyera foliosa.

Key words: Goodyera foliosa (Lindl) Benth. Bioresource conservation Seedling regeneration Organ development Transcriptome analysis

收稿日期: 2018-11-30 出版日期: 2019-01-10

ZTFLH:

Q78

通讯作者: 陈集双,兰思仁 E-mail: biochenjs@njtech.edu.cn;lkzx@fafu.edu.cn

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引用本文:

何官榕,何碧珠,吴沙沙,石京山,陈集双,兰思仁. 多叶斑叶兰繁殖体系建立及基于转录组的发育调控途径功能基因研究[J]. 中国生物工程杂志, 2018, 38(12): 57-64.

HE Guan-rong,HE Bi-zhu,WU Sha-sha,SHI Jing-shan,CHEN Ji-shuang,LAN Si-ren. Establishment of an Efficient Regeneration System in Goodyera foliosa and Comprehensive Analysis of Functionally Regulated Genes Involved in Developmental Regulatory Pathways Based on Transcriptome Analysis. China Biotechnology, 2018, 38(12): 57-64.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181208 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I12/57

表1 6-BA含量对芽诱导的影响

表2 NAA含量对芽增殖的影响

表3 IBA的含量对生根的影响

图1 Unigene长度分析

图2 功能注释韦恩图

图3 差异Unigene GO功能富集结果

图4 差异Unigene KEGG功能富集结果

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