摘要
木质素在植物体中具有运输水分、支撑植株和加强植物体免受侵害等功能,是苯丙烷代谢途径的重要产物之一。其中肉桂醇脱氢酶(cinnamyl alcohol dehydrogenase,CAD)是该途径中重要的限速酶。本研究在胡椒转录组测序的基础上,用RACE法进行克隆,对PnCAD基因全长进行生物信息学分析,并对其蛋白进行理化性质、亚细胞定位和系统进化树等分析;最后运用实时荧光定量PCR进行分析。结果表明:克隆得到CAD基因的全长cDNA,长度为1364 bp,开放阅读框(open reading frame,ORF)为1071 bp,编码356个氨基酸,预测相对分子量为3.879 kDa,等电点为6.27,属于亲水性蛋白;含有3个N-糖基化特征序列和9个磷酸化位点,可能处于细胞质内。结构域分析发现,胡椒CAD蛋白含有NAD(P)结合位点、多个催化锌和结构锌结合位点。系统进化树分析表明,胡椒CAD与细辛CAD6亲缘关系最近,胡椒和细辛的同源性最高为76%,均隶属于比较原始的双子叶植物。通过荧光定量分析发现,黄花胡椒在辣椒疫霉菌侵染下表达量升高,在8 h达到最高值,约为对照的10倍;之后在24 h时出现小幅度升高,约为对照组的6倍,之后下降。‘热引1号’胡椒的表达量在侵染8 h时达到最低,之后缓慢上升。总体来说,所有时间下黄花胡椒基因表达量均高于‘热引1号’胡椒,且差异显著。本研究结果可为今后研究胡椒抗非生物胁迫功能提供参考,为PnCAD基因的功能研究提供理论依据。
Lignin has some functions in plants,such as transporting water,supporting plants and strengthening plants against damage,etc.It is one of the important products of phenylpropane metabolism.Among them,cinnamyl alcohol dehydrogenase(CAD)is an important rate-limiting enzyme in the lignin synthesis pathway.In this experiment,on the basis of pepper transcriptome sequencing,the RACE method was used for gene cloning,bioinformatics analysis of the full-length PnCAD gene,and many analyses of its protein such as physicochemical properties,subcellular localization and phylogenetic tree.It was analyzed by qPCR.The cinnamyl alcohol dehydrogenase gene was cloned by the race method.Finally,a full-length cDNA with a length of 1364 bp was obtained,including 1071 bp open reading frame(ORF),encoding 356 amino acids.The predicted relative molecular weight was 3.879 kDa and the isoelectric point was 6.27.It belonged to hydrophilic protein.It contained three N-glycosylation characteristic sequences and nine phosphorylation sites,which were likely to be in the cytoplasm.Domain analysis showed that CAD protein contained NAD(P)binding sites,multiple catalytic zinc and structural zinc binding sites.Phylogenetic tree analysis showed that pepper CAD was closely related to Asarum CAD 6,and the highest homology was 76%,both of which belonged to primitive dicotyledons.Through fluorescence quantitative analysis,it could be found that the expression of P.flaviflorum under the infection of Phytophthora capsici increased,reached the highest value at 8 h,about 10 times that of the control,then increased slightly at 24 h,about 6 times that of the control group,and then decreased.Generally speaking,the gene expression level of P.flaviflorum at all time was higher than that of P.nigrum cv.Reyin-1 and was significantly different.The experimental data could provide reference data for the future study of the anti abiotic stress function of black pepper,and also provide a theoretical basis for the functional study of gene PnCAD.
作者
孙也乔
王珏
胡丽松
伍宝朵
郝朝运
范睿
SUN Yeqiao;WANG Jue;HU Lisong;WU Baoduo;HAO Chaoyun;FAN Rui(College of Tropical Crops,Hainan University,Haikou,Hainan 570228,China;Institute of Spices and Beverages,ChineseAcademy of Tropical Agricultural Sciences,Wanning,Hainan 571533,China;Hainan Key Laboratory of Genetics,Breeding andGermplasm Resources,Wanning,Hainan 571533,China;Hainan Key Laboratory of Genetic Improvement and Quality Control ofTropical Spice Beverage Crops,Wanning,Hainan 571533,China;Hainan SIM Soonliang Academician Workstation,Wanning,Hainan 571533,China)
出处
《热带作物学报》
CSCD
北大核心
2022年第12期2422-2430,共9页
Chinese Journal of Tropical Crops
基金
中央级公益性科研院所基本科研业务费专项(No.1630142020001)。
