CHI Xiufeng,JIANG Xinqiang,LI Yanshuo,et al.Cloning and Construction of VIGS Expression Vector of LtXTH5 in Lilium tsingtauense[J].Northern Horticulture,2020,44(15):74-81.[doi:10.11937/bfyy.20193398]
青岛百合LtXTH5的克隆及VIGS表达载体构建
- Title:
- Cloning and Construction of VIGS Expression Vector of LtXTH5 in Lilium tsingtauense
- Keywords:
- Lilium tsingtauense; LtXTH5; cloning; sequence analysis; silence vector
- 文献标志码:
- A
- 摘要:
- 以青岛百合(Lilium tsingtauense)的花被片作为试材，采用了RT-PCR的方法从青岛百合中分离到LtXTH5基因，并进行生物信息学分析和病毒诱导沉默表达载体的构建，研究了花发育基因LtXTH5的基本理化性质和载体构建方法，以期为后期LtXTH5生物学功能的验证奠定基础。结果表明：LtXTH5基因（登录号MN245449）的开放阅读框包含882 bp，编码293个氨基酸；生物信息学分析预测LtXTH5蛋白相对分子量为33.8 kDa，分子式为C1536H2283N407O436S12，理论等电点（pI）为7.03，脂肪指数68.23，不稳定指数为31.68，LtXTH5是亲水性蛋白，具有信号肽；基序分析发现不同植物XTH中的保守基序数目不同，LtXTH5含有9个基序；序列比对结果表明LtXTH5氨基酸序列含有DEIDFEFLG催化活性位点和GH16_XET结构域，是典型的XTH家族成员；系统进化树分析表明LtXTH5与拟南芥AtXTH5亲缘关系最近，属于I/II亚家族，并且具有转糖基酶或水解酶活性。利用LtXTH5的5′端537 bp片段和pTRV2载体质粒成功构建了沉默载体（TRV2-LtXTH5），并将TRV2-LtXTH5载体成功转至农杆菌GV3101中，下一步拟用已转入TRV2-LtXTH5载体的农杆菌菌液对烟草进行侵染，进一步探究LtXTH5基因的功能。
- Abstract:
- Using the tepals of Lilium tsingtaense as the test material，LtXTH5 gene was isolated from Lilium tsingtaense by RT-PCR，and the bioinformatics analysis and the construction of virus induced silencing expression vector were carried out.The basic physical and chemical properties and vector construction method of the flower development gene LtXTH5 were studied in order to provide the basis for the verification and identification of biological functions of LtXTH5 in the later stage.The results showed that the open reading frame of LtXTH5 gene (MN245449) contained 882 bp and encoded 293 amino acids.Bioinformatics analysis predicted that the relative molecular weight of LtXTH5 was 33.8 kDa，the molecular formula was C1536H2283N407O436S12，the theoretical isoelectric point (pI) was 7.03，the fat index was 68.23，and the instability index was 31.68.LtXTH5 was a hydrophilic protein with signal peptide.Motif analysis of XTHs from different plants showed that the number of conserved motif was different among different plants.LtXTH5 contains nine motifs.Sequence alignment indicated LtXTH5 was a typical member of XTH family，which include DEIDFEFLG catalytic active sites and GH16_XET domain.Phylogenetic analysis showed that LtXTH5 was closely related to AtXTH5 in Arabidopsis thaliana，belonging to I/II subfamily，and had transglycosylase (XET) or hydrolase (XEH) activities.Using the 5′ end 537 bp fragment of LtXTH5 and pTRV2 vector plasmid，a silencing vector (TRV2-LtXTH5) was successfully constructed，and TRV2-LtXTH5 vector was successfully transferred to agrobacterium GV3101.In the next step，we plan to use the agrobacterium liquid which had been transferred into TRV2-LtXTH5 vector to infect tobacco and further explore the function of LtXTH5 gene.
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备注/Memo
第一作者简介：池秀凤（1995-），女，硕士研究生，研究方向为园林植物种质创新与利用。E-mail：970366420@qq.com.责任作者：刘庆华（1962-），男，博士，教授，现主要从事园林植物种质创新与利用等研究工作。E-mail：lqh6205@163.com.基金项目：山东省林业科技创新资助项目（LYCX06-2018-34）；青岛农业大学博士基金资助项目（6631113340）；青岛农业大学研究生创新计划资助项目（QYC201827）；青岛农业大学大学生创新创业资助项目（2019-172）。收稿日期：2019-09-05