日本落叶松内源GUS基因鉴定及其酶活性分析  

Identification of Endogenous GUS Gene and Its Enzyme Activity Analysis in Larix kaempferi(Lamb.)Carr.

作　　者：叶查龙 张陈谊 杨玲[2] 孙晓梅[1] 李万峰[1] YE Zha-long;ZHANG Chen-yi;YANG Ling;SUN Xiao-mei;LI Wan-feng(State Key Laboratory of Tree Genetics and Breeding,Key Laboratory of Tree Breeding and Cultivation of the National Forestry and Grassland Administration,Research Institute of Forestry,Chinese Academy of Forestry,Beijing 100091,China;State Key Laboratory of Tree Genetics and Breeding,School of Forestry,Northeast Forestry University,Harbin 150040,Heilongjiang,China)

机构地区：[1]林木遗传育种全国重点实验室国家林业和草原局林木培育重点实验室中国林业科学研究院林业研究所,北京100091 [2]林木遗传育种全国重点实验室东北林业大学林学院,黑龙江哈尔滨150040

出　　处：《林业科学研究》2024年第6期86-92,共7页Forest Research

基　　金：科技创新2030-重大项目(2023ZD04070,2022ZD04016)。

摘　　要：[目的]研究日本落叶松内源β-葡糖苷酸酶(GUS)基因及其酶活性,分析GUS作为报告基因的可靠性。[方法]利用pCAMBIA1301载体上的GUS基因序列在日本落叶松参考基因组中进行比对,鉴定并克隆出日本落叶松GUS(LaGUS)基因。在NCBI利用blastp查找其他物种中LaGUS的同源序列并构建进化树。利用已有的转录组数据和实时荧光定量PCR分析LaGUS基因在日本落叶松中的表达模式。利用组织化学的方法,以X-gluc作为GUS酶反应底物,检测日本落叶松体细胞胚胎发生过程中内源GUS的酶活性。[结果]在日本落叶松中鉴定到一个LaGUS基因,CDS长3435 bp,编码1144个氨基酸。25种植物中具有与LaGUS基因相似的序列。LaGUS基因的表达量在活动期高于休眠期,在体胚苗中高于在胚性愈伤组织和体细胞胚胎中。组织化学染色表明,日本落叶松胚性愈伤组织、体细胞胚胎和体胚苗在室温下染色6 d时均观察到蓝色,但体胚苗的染色程度高于胚性愈伤组织和体细胞胚胎。[结论]日本落叶松具有内源GUS活性,在基因工程中利用GUS作为报告基因可能存在一定干扰。[Objective]The endogenous enzyme activity ofβ-glucuronidase(GUS)in Larix kaempferi(Lamb.)Carr.was detected,and the feasibility of exogenous GUS as a reporter gene in genetic engineering was analyzed.[Method]GUS protein sequences from pCAMBIA1301 vector was used to blast the reference genome of L.kaempferi,and then GUS gene in L.kaempferi(LaGUS)was identified and cloned.Expression pattern of LaGUS was analyzed using the published transcriptome data and quantitative reverse transcription PCR.Enzyme activity of endogenous GUS during L.kaempferi embryogenesis was examined with histochemical method with X-gluc as the substrate of enzyme reaction.[Result]The endogenous GUS gene,LaGUS was identified in L.kaempferi with a coding sequence of 3435 bp,encoding 1144 amino acids.It was found that sequences similar to LaGUS existed in at least 25 plant species.The expression level of LaGUS was higher in the active stage than in the dormant stage of L.kaempferi,and higher in somatic seedlings than in embryogenic callus and somatic embryo.Histochemical analysis showed that blue color was observed in embryogenic callus,somatic embryo and somatic seedlings after staining at room temperature for six days,and the degree of staining in somatic seedlings was higher than that in embryogenic callus and somatic embryo.[Conclusion]L.kaempferi has endogenous GUS activity,and using GUS as a reporter gene in genetic engineering may have certain interference.

关 键 词：日本落叶松 内源GUS 体细胞胚胎发生 报告基因 

分 类 号：S722.3[农业科学—林木遗传育种]