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【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 embryonic calli of 3-hydroxy-3-methyl glutaryl coenzyme A reductase 1 (Hb HMGR1) expression vector p CAMBIA2301-PHEV2.1-Hb HMGR1 were transformed into embryogenic callus Obtained resistant transgenic material to lay the foundation for the study of Hb HMGR1 gene-specific expression of milk and improve the rubber yield. [Method] The embryogenic calli of embryogenic calli were transformed with Agrobacterium tumefaciens EHA105 expression vector p CAMBIA2301-PHEV2.1-Hb HMGR1, Sex callus for GUS staining and molecular detection. 【Result】 Five strains of Fusarium callus resistant to Agrobacterium tumefaciens were screened for 4 to 6 months, and five resistant callus lines with positive GUS detection were obtained. Among them, Nos. 2 and 11 The callus tissues were identified by PCR and amplified the same specific fragment as the positive control. The size of the target fragments of uid A, NPTII and PHEV2.1-Hb HMGR1 sequences were 829,797 and 3592 bp, respectively. The resistant calli of No.2 and No.11 were identified by reverse PCR and found that No.2 resistant callus did not amplify the target band, whereas No. 11 resistant callus obtained an about 2000 bp , Which contains a T-DNA sequence and an unknown DNA sequence, of which the unknown DNA sequence is a contiguous sequence of the genomic DNA of the rubber tree. 【Conclusion】 Transgenic callus lines containing 35S-NPTII-PHEV2.1-Hb HMGR1-35S-uid A were obtained by integrating the T-DNA of plant expression vector into the genomic DNA of resistant callus.