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铁皮石斛SEP3基因的生物信息学分析及敲除载体的构建
黄鑫,师凯辉,胡凇,田英男,冯垒,韩欣妤,孙博,王义琴,王冬梅,何玲,朱永平,和凤美
0
(云南农业大学园林园艺学院,昆明 650201)
摘要:
SEP3(SEPALLATA 3)基因是MADS-box 家族中的一员,在花器官的形成和发育过程中具有重要作用。为了解SEP3基因在铁皮石斛(Dendrobium officinale)中的成花转变机理,从铁皮石斛转录组序列中获得一个SEP3基因序列,并命名为DoSEP3。 预测其编码区长度为354 bp,预测编码117个氨基酸。 DoSEP3蛋白相对分子质量13 457.87 Da。其不稳定系数为60.53,亲水性的平均值为-0.121,是不稳定的亲水性蛋白,主要在内质网和细胞核中起作用。二级结构主要以α-螺旋为蛋白质最大量的结构元件,跨膜区分析推测DoSEP3蛋白质具有一个跨膜区。进一步的氨基酸比对显示,铁皮石斛DoSEP3蛋白与金钗石斛SEP3蛋白亲缘关系最近,可能具有类似的功能。基于生物信息学分析,利用CRISPR/cas9技术构建铁皮石斛基因敲除载体,并使用农杆菌介导法进行遗传转化,且已获得阳性原球茎,可为后续的SEP3基因表达研究奠定基础。
关键词:  铁皮石斛  SEP3基因  生物信息学分析  载体构建
DOI:10.13610/j.cnki.1672-352x.20210909.010
投稿时间:2020-10-29
基金项目:国家自然科学基金项目(31760591)资助。
Bioinformatics analysis of SEP3 gene of Dendrobium officinalee and construction of knockout vector
HUANG Xin,SHI Kaihui,HU Song,TIAN Yingnan,FENG Lei,HAN Xinyu,SUN Bo,WANG Yiqin,WANG Dongmei,HE Ling,ZHU Yongping,HE Fengmei
(College of Garden and Horticulture, Yunnan Agricultural University, Kunming 650201)
Abstract:
The SEP3 gene is a member of the MADS-box family and has an important role in the formation and development of floral organs. In order to understand the mechanism of floral transformation of the SEP3 gene in Dendrobium officinale, this gene sequence was obtained from the Dendrobium officinale sequence and named as DoSEP3. Biological information analysis indicated that the coding regions of DoSEP3 is 354 bp in length, encoding 117 amino acids. The relative molecular mass of DoSEP3 protein is 13 457.87 Da. The instability coefficient is 60.53 and the mean value of hydrophilicity is - 0.121, which is an unstable hydrophilic protein that mainly works in the endoplasmic reticulum and nucleus. In the secondary structure, α-helix is mainly the most abundant structural element of the protein, and the transmembrane region analysis presumed that the DoSEP3 has a transmembrane region. Further amino acid comparison showed that the DoSEP3 of Dendrobium officinale is closely related to SEP3 protein of Dendrobium nobile and might have similar functions. Based on the bioinformatics analysis, the CRISPR/cas9 technology was used to construct a Dendrobium officinale knockout vector, and positive protocorm was obtained through Agrobacterium tumefaciens mediated genetic transformation, which will lay a foundation for the follow-up study of the SEP3 gene expression.
Key words:  Dendrobium officinale  SEP3  bioinformatics analysis  vector construction

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