陆地棉PLDα基因生物信息学分析及CRISPR/Cas9载体的构建  

Bioinformatics Analysis of Upland Cotton PLDαGene and Construction of CRISPR/Cas9 Vector

作　　者：王永兴 蓝师磊 程文翰 WANG Yongxing;LAN Shilei;CHENG Wenhan(College of Biological Engineering,Jingchu University of Technology,Jingmen 448000,China;Hubei Engineering Research Center for Specialty Flowers Biological Breeding,Jingmen 448000,China)

机构地区：[1]荆楚理工学院生物工程学院,湖北荆门448000 [2]湖北省特色花卉生物育种工程研究中心,湖北荆门448000

出　　处：《荆楚理工学院学报》2025年第2期1-9,共9页Journal of Jingchu University of Technology

基　　金：湖北省自然科学基金计划项目(2023AFB931);湖北省教育厅科学研究计划资助项目(Q20224301);荆楚理工学院校级科研项目(YY202206);湖北省大学生创新训练项目(202211336008)。

摘　　要：PLDα属于磷脂酶D(phospholipose D,PLD)的一种,在植物生长发育和各种胁迫的信号转导过程中发挥着关键作用,本文以陆地棉作为研究材料,通过生物信息学方法对PLD基因进行了分析,检测了在陆地棉下胚轴不同组培时期PLDα的表达量,构建了拟南芥和陆地棉PLDα的CRISPR/Cas9载体,以期通过表达调控促进陆地棉的体细胞胚胎再生过程。试验通过对已报导陆地棉PLDα氨基酸序列比对,获取陆地棉中可能存在的PLD基因序列,选取可信度高的基因,利用q-PCR技术对陆地棉PLDα基因在陆地棉体细胞胚胎再生不同阶段的表达量进行检测,通过构建表达框并与CRISPR/Cas9载体连接,获取PLDα基因敲除载体。结果表明陆地棉中可能存在的PLD基因家族成员有34个,根据陆地棉下胚轴在组培过程中,四个时间点上PLDα1的表达量分析发现,在组培初期愈伤组织较少时其表达量较高,随着组培时间延长愈伤组织逐渐增多时表达量逐渐下降。通过载体构建分别得到了携有拟南芥PLDα1与陆地棉PLDα1的CRISPR/Cas9敲除载体的农杆菌,对缩短棉花组培进程有重要意义。PLDα belongs to phospholipase D(PLD)family,and plays a key role in the process of plant growth and development,and signal transduction under various stresses.In this paper,upland cotton was used as the research material,and the PLD gene was analyzed by bioinformatics methods.The expression of PLDα in the hypocotyls of upland cotton at different tissue culture stages was examined,and the CRISPR/Cas9 vectors of PLDα in Arabidopsis thaliana and upland cotton were constructed,in order to promote the somatic embryo regeneration process of upland cotton through expression regulation.In the experiment,by aligning the reported PLDα amino acid sequences of upland cotton,the potential PLD gene sequences in the upland cotton were obtained,and the genes with high reliability were selected.The expression levels of the PLDα gene in upland cotton were analyzed by q-PCR during different stages of somatic embryogenesis,and the PLDα gene knockout vector was obtained by constructing the expression cassette and connecting it with the CRISPR/Cas9 vector.The results showed that there were 34 potential PLD gene family members in upland cotton.According to the expression analysis of PLDα1 at four time points in the hypocotyl of upland cotton during the tissue culture process,it was found that the expression level was high in the early stage of tissue culture when there were fewer callus,and the expression level decreased gradually with the extension of tissue culture time as the callus gradually increased.Agrobacterium carrying the CRISPR/Cas9 knockout vectors for Arabidopsis thaliana PLDα1 and upland cotton PLDα1 were obtained through vector construction,which is of great significance for shortening the cotton tissue culture process.

关 键 词：PLDα 陆地棉 生物信息学分析 CRISPR/Cas9 

分 类 号：Q37[生物学—遗传学]