李森;程宵婧;侯非凡;公菲菲;刘娟;杜崴;亢秀萍;邢国明;

• 1:山西农业大学园艺学院/山西省设施园艺工程技术中心
• 2:大同黄花产业发展研究院

摘要(Abstract)：

为了拓宽对黄花菜基因功能研究的技术体系,本研究以黄花菜‘大同黄花’为试验材料,基于转录组数据库检索和PCR技术克隆得到了黄花菜HcPDS基因完整编码序列,序列全长1710 bp,编码570个氨基酸。该基因编码的蛋白质具有PDS基因家族典型的Phytoene desat结构域,分子量为63.719 72 kD,等电点为7.53,是亲水不稳定性蛋白。通过序列比对,发现HcPDS基因与水仙、芦笋等植物中的PDS基因具有较高的同源性。通过构建黄花菜病毒诱导的基因沉默(VIGS)体系,黄花菜幼苗被侵染后,接种pTRV2-HcPDS的植株新叶出现不完全的光漂白现象,叶片中的HcPDS基因表达量相比接种TRV2空载的阴性对照和空白对照植株分别下降了73.33%和71.72%。空白对照和阴性对照植株叶片则无光漂白现象出现,且空白对照和阴性对照的HcPDS基因表达量相比无显著差异。所构建的黄花菜VIGS体系可有效沉默HcPDS基因,为黄花菜功能基因的鉴定与分析奠定了技术基础。

关键词(KeyWords)： 黄花菜;基因克隆;病毒诱导的基因沉默;表达鉴定

Abstract:

Keywords:

基金项目(Foundation): 山西省高等学校科技创新项目(2019L0375);; 山西省农业重点研发计划(重点)项目(201703D211001-04-05;201903D211011-01);; 山西农业大学青年拔尖创新人才支持计划(BJRC201601)

作者(Author): 李森;程宵婧;侯非凡;公菲菲;刘娟;杜崴;亢秀萍;邢国明;

Email:

DOI: 10.13320/j.cnki.jauh.2021.0004

参考文献(References)：

• [1]席海秀,李洪艳,佟少明.植物八氢番茄红素脱氢酶(PDS)基因保守结构域模式与系统进化分析[J].植物生理学报,2013,49(12):1407-1412.
• [2]牛义岭,姜秀明,许向阳.番茄GRAS转录因子家族基因SIGLD1的克隆及VIGS载体构建[J].基因组学与应用生物学,2016,35(8):2155-2160.
• [3]Zhou X,Huang C.Virus-induced gene silencing using begomovirus satellite molecules[J].Methods in Molecular Biology,2012,894(5):57-67.
• [4]Kandoth P K,Heinz R,Yeckel G,et al.A virusinduced gene silencing method to study soybean cyst nematode parasitism in Glycine max[J].BMC Research Notes,2013,6(7):255.
• [5]Pflieger S,Mms R,Blanchet S,et al.VIGS technology:An attractive tool for functional genomics studies in legumes[J].Functional Plant Biology,2013,40(12):2-7.
• [6]Sun Z,Gantt C E.Differential expression of two isopentenyl pyrophosphate isomerases and enhanced carotenoid accumulation in a unicellular chlorophyte[J].Proc Natl Acad,USA,1998,95(19):14585.
• [7]Bennypaul H S,Mutti J S,Rustgi S,et al.Virusinduced gene silencing (VIGS) of genes expressed in root,leaf,and meiotic tissues of wheat[J].Functional&Integrative Genomics,2012,12(1):143-156.
• [8]Zhong X,Yuan X,Wu Z,et al.Virus-induced gene silencing for comparative functional studies in Gladiolus hybridus[J].Plant Cell Reports,2014,33(2):301-312.
• [9]Stefanie W,Andrea S,Stefan G,et al.Highly Efficient Virus-induced Gene Silencing (VIGS) in California Poppy (Eschscholzia californica):An Evaluation of VIGSas a Strategy to Obtain Functional Data from Nonmodel Plants.Annals of Botany,2007,100(3):641-649.
• [10]徐华.百合VIGS体系的建立及LhDTX 35基因的克隆和功能研究[D].北京：中国农业科学院,2018.
• [11]侯非凡.黄花菜秋水仙碱合成相关基因筛选及遗传图谱构建[D].太谷：山西农业大学,2017.
• [12]公菲菲,李森,杜崴,高阳,侯非凡,亢秀萍.黄花菜生物钟基因HcLHY的克隆及时空表达分析[J].河北农业大学学报,2020,43(3):37-44.
• [13]Saitou N,Nei M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4(4),406-425.
• [14]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J].Methods (San Diego,Calif.),2001,25(4):402-408.
• [15]杨善朝.SPSS统计软件应用基础[M].南宁：广西师范大学出版社,2001.
• [16]Li S,Li K,Cao D,et al.Genome-wide analysis of tomato NF-Y factors and their role in fruit ripening[J].BMC Genomics,2016,17(1):36.
• [17]Nelson D R,Schuler M A,Paquette S M,et al.Comparative genomics of rice and Arabidopsis.Analysis of 727 cytochrome P 450 genes and pseudogenes from a monocot and a dicot[J].Plant Physiology,2004,135(2):756-772.
• [18]Li C,Yoshikawa N.Virus-induced gene silencing of Ngene in tobacco by apple latent spherical virus vectors[J].Methods in Molecular Biology,2015,1236:229-240.
• [19]Bilichak A,Kovalchuk I.Increasing a stable transformation efficiency of Arabidopsis by manipulating the endogenous gene expression using virus-induced gene silencing[J].Methods in Molecular Biology,2017,1456:225-236.