橡胶树乳管特异性启动子连接HbHMGR1基因的易碎胚性愈伤组织转化

Transformation of friable embryogenic callus with HbHMGR1 gene connected with laticifer specific promoter in Hevea brasiliensis

【目的】以3-羟基-3-甲基戊二酸单酰辅酶A还原酶1基因（Hb HMGR1）乳管表达载体p CAMBIA2301-PHEV2.1-Hb HMGR1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Hb HMGR1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p CAMBIA2301-PHEV2.1-Hb HMGR1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,uid A、NPTII和PHEV2.1-Hb HMGR1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序列是橡胶树品种热研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-Hb HMGR1-35S-uid A的转基因愈伤组织系。

【Objective】The laticifer expression vector p CAMBIA2301-PHEV2.1-Hb HMGR1 containing 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 gene（Hb HMGR1） 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 Hb HMGR1 gene in laticifer and increasing rubber yield. 【Method】The expression vector p CAMBIA2301-PHEV2.1-Hb HMGR1 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 kanamycinresistant 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 uid A, NPTII and PHEV2.1-Hb HMGR1 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-Hb HMGR1-35S-uid A.