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在优化花培诱导培养基成分的基础上,通过延长花药愈伤组织在诱导培养基上培养的时间和降低共培养过程中农杆菌浓度等,成功地建立了以花药愈伤组织为受体的农杆菌转化体系.并将白叶枯菌抗性基因Xa21作为该体系的模式基因导入到多个粳稻品种的花药愈伤组织中,共得到了145个独立的转基因株系.分子检测和田间抗性检测发现其中的140个株系的基因组含有外源的Xa21基因,包括单倍体45株、二倍体87株和混倍体植株8株.根据后代性状分离比和染色体的FISH分析可以确定,在87个二倍体植株中有15株为纯合的加倍单倍体转基因植株;再加上田间自然加倍和由秋水仙碱处理单倍体所获得的28株加倍单倍体,共得到了43个纯合的加倍单倍体转基因株系.还将该转化体系用于反向遗传学的研究.一个在植物体内能转录产生双链RNA(double strand RNA,dsRNA)发夹结构的质粒pZ2RNAi被导入到水稻单倍体基因组中,该dsRNA的靶目标是水稻的OsMADS2基因的转录物,结果表明,在转pZ2RNAi的单倍体水稻中,靶基因OsMADS2的表达被特异地抑制,并导致花器官形态的明显改变.因此,单倍体转化体系与RNAi技术相结合有可能成为在水稻和其他植物中功能基因组研究的一个新途径.
On the basis of optimizing the components of culture medium induced by anther culture, the anther callus was successfully established by prolonging the culture time of anther callus on induction medium and decreasing the Agrobacterium concentration in the process of co-culture Bacillus transformation system, and the Xanthomonas oryzae resistance gene Xa21 was introduced into the anther callus of several japonica rice varieties as a model gene of the system, and 145 independent transgenic lines were obtained. Molecular detection and field resistance The results showed that Xa21 gene was exogenous in 140 lines including 45 haploid, 87 diploid and 8 diploid plants.According to offspring trait segregation ratio and chromosome FISH analysis, Fifteen out of 87 diploid plants were homozygous doubled haploid transgenic plants; together with 28 doubling haploids that were naturally doubled in the field and haploid obtained with colchicine, a total of 43 homozygous doubled haploid transgenic lines.The transformation system was also used for reverse genetics.A plasmid pZ2RNAi that can transcribe double hairpin RNA (dsRNA) hairpin structure in plants Guided To the haploid genome of rice, the target of the dsRNA is the transcript of the OsMADS2 gene in rice. The results show that the expression of the target gene OsMADS2 is specifically inhibited in the haploid rice transformed with pZ2 RNAi and the flower organ morphology Therefore, the combination of haploid transformation system and RNAi technology may become a new way to study the functional genomics in rice and other plants.