李哲　贺永国　黄绵佳　曾宪海

摘要：【目的】以3-羟基-3-甲基戊二酸单酰辅酶A还原酶1基因（HbHMGR1）乳管表达载体pCAMBIA2301- PHEV2.1-HbHMGR1转化橡胶树易碎胚性愈伤组织，获得抗性转基因材料，为研究HbHMGR1基因的乳管特异性表达及提高橡胶产量打下基础。【方法】用根癌农杆菌EHA105介导表达载体pCAMBIA2301-PHEV2.1-HbHMGR1转化橡胶树品种热研8-79花药易碎胚性愈伤组织，经卡那霉素筛选数月后，对抗性愈伤组织进行GUS染色和分子检测。【结果】经过对根癌农杆菌侵染的易碎愈伤组织进行4～6个月筛选，得到5个GUS检测呈阳性的抗性愈伤组织系；取其中的2号和11号抗性愈伤组织系进行PCR鉴定，均能扩增出与阳性对照相同的特异片段，uidA、NPTII和PHEV2.1-

HbHMGR1序列的目的片段大小分别为829、797和3592 bp。2号和11号抗性愈伤组织系经反向PCR鉴定，发现2号抗性愈伤组织系未扩增出目的条带，而11号抗性愈伤组织系扩增获得一条约2000 bp的条带，包含T-DNA序列和一段未知的DNA序列，其中未知DNA序列是橡膠树品种热研8-79基因组的一段连续序列。【结论】将植物表达载体的T-DNA整合到抗性愈伤组织系基因组DNA中，可获得一个含35S-NPTII-PHEV2.1-HbHMGR1-35S-uidA的转基因愈伤组织系。

关键词： 橡胶树；HbHMGR1基因；乳管特异性启动子PHEV2.1；易碎愈傷组织；遗传转化

中图分类号： S794.1 文献标志码：A 文章编号：2095-1191（2016）05-0597-07

Abstract：【Objective】The laticifer expression vector pCAMBIA2301-PHEV2.1-HbHMGR1 containing 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 gene（HbHMGR1） was constructed successfully， and transform friable embryogenic callus of rubber tree clone to obtain transgenic materials， in order to provide basis for studying specific expression of exogenous HbHMGR1 gene in laticifer and increasing rubber yield. 【Method】The expression vector pCAMBIA2301-PHEV2.1-HbHMGR1 were transfered into friable embryogenic callus of elite rubber tree clone Reyan 8-79 by mediation of Agrobacterium tumefaciens strain EHA105. Friable calluses were cultivated in subculture medium supplemented with 75 mg/L kanamycin for months. Then the kanamycin-resistant calluses were detected by GUS staining. 【Result】A total of 5 kanamycin-resistant friable embryogenic callus lines were obtained after screening of 4-6 months， and GUS detection showed that， they were positive. In addition， PCR identification results showed that， the specific DNA fragments could be amplified from resistant callus lines 2 and 11 as same as positive control， the target fragments of uidA， NPTII and PHEV2.1-HbHMGR1 were 829， 797 and 3592 bp in length， respectively. The inverse PCR（IPCR） identification results showed that， the positive fragments could not be amplified from line 2， while one band with the length of 2000 bp was amplified from the line 11， which contained T-DNA sequence and an unknown DNA sequence. Then this unknown DNA sequence was identified as a segment of continuous sequence of genome of rubber tree clone Reyan 8-79 by using genome database of rubber tree. 【Conclusion】The T-DNA region of plant expression vector is integrated into genome of resistant callus line， so as to obtain one transgenic callus line with 35S-NPTII-PHEV2.1-HbHMGR1-35S-uidA.

Key words： Hevea brasiliensis； HbHMGR1 gene； laticifer specific promoter PHEV2.1； friable callus； genetic transformation

0 引言

【研究意义】橡胶树是重要的热带经济作物，其产出的天然橡胶是重要战略物资（张志平等，2014）。近年来，随着国民经济和国防事业的快速发展，我国天然橡胶的自给率逐年下降，进口依赖度不断攀升（王少明，2011）。然而，橡胶树适种区域有限，通过扩大种植面积增加橡胶产量的潜力极小，因此开展橡胶树品种改良、提高橡胶单产的研究迫在眉睫。……