摘要
本研究以冰草属植物中的4个品种——蒙古冰草新品系(A.mongolicumKeng)、航道冰草(A.cristatumcv.Fairway)、诺丹冰草(A.desertorumcv.Nordan)和一个种间杂种冰草——蒙农杂种冰草(A.cristatum×A.desertorumcv.Hycrest-Mengnong)为材料,分别以幼穗为外植体建立了冰草组织培养再生体系。在此基础上,以调控脯氨酸生物合成最后一步的关键酶的突变体基因P5CS为目标基因,bar基因为筛选标记基因,用基因枪法轰击幼穗诱导的愈伤组织,获得转基因植株。利用PCR方法从拟南芥(Arabidopsisthaliana)基因组DNA中分离得到了CBF4及其启动子,并对CBF4启动子进行了序列分析和初步的功能分析。结果表明:1.冰草幼穗的取样时期为孕穗期,长度介于1.0～3.0cm的幼穗为最适宜的外植体;诱导愈伤组织的最适培养基为改良MS+2,4-D2.0mg/L,诱导率83.5%;分化培养基为MS+KT0.2mg/L,分化率74.5%;生根培养基为1/2MS,生根率100%。2.以幼穗诱导的愈伤组织为受体,采用基因枪轰击法转化冰草获得转基因植株;PCR和Southern检测表明外源基因P5CS已整合到冰草属植物基因组DNA中;RT-PCR检测表明目的基因已在冰草转基因植株的转录水平表达;P5CS基因的遗传转化率为0.09%～0.11%。3.采用PCR方法从拟南芥中分离了CBF4基因及其启动子序列。
This paper tells how the wheatgrass tissue culture and regeneration system with the immature inflorescence as explant was established using four species, namely: A. mongolicum Keng, A.cristatum cv. Fairway, A.desertorum cv. Nordan, and A.cristatum×A.desertorum cv. Hycrest-Mengnong as materials. Based on the established regeneration system, the P5CS gene, which regulates the last step of key enzymes mutant gene of proline synthesis in a plant, was transformed into wheatgrass with phosphinothricin acetyltransferase (bar) conferring herbicide resistance as selecting gene. The transformation was conducted through microprojectile bombardment of callus derived from immature inflorescence.The CBF4 gene and its promoter had been isolated from Arabidopsis thaliana by PCR. The sequence analysis and function assay were conducted.The primary results and progress are summarized as follows:1. The inflorescence of a length of 1.0 to 3.0 cm is optimal to be used as explant to induce the callus. The feasible medium for callus induction from immature inflorescence was the improved MS+2,4-D (2.0 mg/L). The induction frequency was above 83.5%. The differentiation medium was MS+KT (0.2 mg/L), with a differentiation ratio of 74.5%. The rooting medium was 1/2 MS with 100% rooting ratio.2. The transgenic plants were obtained by microprojectile bombardment of callus induced from immature inflorescence,the results of PCR and Southern analysis displayed that the exogenous P5CS gene had integrated into the genome of transgenic wheatgrass,and the assay of RT-PCR showed that the transgenic P5CS had expressed at a transcript level. The transgenic frequencies of P5CS gene were 0.09%～0.11% in 2003.3. The CBF4 gene and its promoter were isolated from Arabidopsis thaliana by PCR. The analysis of CBF4 promoter sequence with bioinformatics software indicated that it could be one of the inducible promoters by outside environmental stresses. The GUS expression cassette driven by CBF4 promoter was constructed and transformed into tabacco(Wisconsin 3
出处
《草地学报》
CAS
CSCD
2005年第2期172-173,共2页
Acta Agrestia Sinica
基金
国家转基因植物与产业化研究专项(J00-B-001-13)和(J2002-B-008)资助项目
