摘要
【目的】利用分子生物学技术建立一种可有效转移药用野生稻有利基因的方法,为进一步大量开展利用药用野生稻有利基因提供技术基础。【方法】以植物抗性相关转录因子AP2/EREBP和bZIP家族基因保守序列为探针,采用同位素α-32P对探针进行标记,通过Southern杂交对药用野生稻TAC文库进行第一轮筛选;所获得阳性克隆用地高辛标记的探针进行第二轮筛选;最后利用PCR方法对第二轮筛选所获得的阳性克隆进行验证。通过农杆菌介导法将阳性克隆转入栽培稻品种日本晴,利用TAC载体插入片段两端的抗性标记Hpt和SacB,通过PCR扩增检测和和Southern杂交对转化植株进行鉴定。采用PEG胁迫法和生理指标变化对转基因T2植株的芽期、苗期耐旱性进行鉴定。【结果】第一轮TAC文库筛选共获得1 073个阳性克隆,其中,以AP2/EREBP的保守序列为探针筛选出606个、bZIP为探针筛选出467个。对这些阳性克隆进行第二轮筛选,从中共获得147个阳性克隆,其中,AP2/EREBP为探针筛选出95个、bZIP为探针筛选出52个。PCR验证有103个阳性克隆能够扩增出目的片段,分别是利用AP2/EREBP设计的引物筛选出63个克隆和bZIP的40个克隆,阳性克隆得率分别为66.32%和76.92%。通过农杆菌转化,以编号为52D-M16和22D-P2(与AP2/EREBP相关)以及55R-A17、49R-O14和8-A24(与bZIP相关)的克隆转化水稻日本晴均获得转化植株,转化试验结果发现外源插入片段越大则克隆转化的成苗率越低。抗性鉴定、PCR和Southern杂交结果均证明外源片段已转入受体水稻品种日本晴中。芽期抗旱性鉴定表明R12-23、R15-41和R1-8 3份材料的耐旱性优于受体日本晴,苗期鉴定显示M63-9和R12-23的耐旱性较强。综合认为,R12-23具有较好的耐旱性。【结论】通过构建药用野生稻TAC文库并结合特异克隆筛选和遗传转化方法,可以进行有利基因的转移,结合进一步的育种,有望实现药用野生稻在育种上的利用。
[Objective]Oryza officinalis Wall. has many abiotic tolerance-related genes,which are very important germplasms in rice breeding, however, it is difficult to utilize these genes in cultivated rice by interspecific hybridization due to the reproductive isolation. This study was planned to establish an efficient and quick method for transferring useful genes of O. officinalis into elite cultivated rice varieties. [Method]Positive clones were screened by southern hybridization from the TAC library of O. officinalis with the conservative sequence of AP2/EREBP and bZIP family genes as probes. The probes were labeled withα-32P during the first screening and with Digoxigenin during the second screening. PCR was used to verify the positive clones finally, which were＆amp;nbsp;introduced into cultivated rice by Agrobacterium tumefaciens-mediated transformation. The transformed plants were detected by PCR amplification with the primes designed according to the conservative sequence of Hpt and SacB that located on both sides of the fragment in TAC. Southern hybridization was also used to identify the transformation plants. PEG-6000 was used to identify the drought tolerance of T2 at germination and seedling stages.[Result]A total of 1 073 clones were developed from the TAC library with AP2/EREBP and bZIP probes that were labeled with α-P32. A total of 147 clones were obtained from the 1 073 clones with Digoxigenin labeled probes, and 95 clones detected by AP2/EREBP probe and 52 clones by bZIP probe. After PCR detection, a total of 103 clones produced PCR amplified products, among them 63 clones were detected by AP2/EREBP probe and 40 clones by bZIP probe and the percentages of positive clones were 66.32%and 76.92%, respectively. Five positive clones （49R-O14, 55R-A17, 8R-A24, 22D-P2q and 52D-M16, named by the clone’s number） were successfully introduced into Nipponbare by the Agrobacterium tumefaciens-mediated transformation. The result showed that the longer inserted fragment was more difficult to be transformed into O. sativa. The results of PCR and Southern hybridization showed that foreign fragments were transferred successfully into the genome of regenerated plantlets. Further, tests of hygromycin resistance also showed the same results. The transgenic lines showed strong tolerance to drought stress, among which R12-23, R15-41, and R1-8 were more tolerance to drought stress than Nipponbare did through detection by tolerance to drought stress at budstage;M63-9 and R12-23 were better in tolerance to drought stress than Nipponbare did through detection by tolerance to drought stress at seedingstage. It was concluded that R12-23 is high tolerance to drought stress.[Conclusion] The work demonstrated that introduction of stress-related TAC clones coupled with a transgenic validation approach is an efficient strategy to transfer agronomically important genes from O. officinalis to cultivated rice.
出处
《中国农业科学》
CAS
CSCD
北大核心
2014年第8期1445-1457,共13页
Scientia Agricultura Sinica
基金
广东省自然科学重点基金项目(10251064201000001)
国家公益性行业(农业)科研专项(201003021)
