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本研究利用巴西橡胶树(Hevea brasiliensis)这一重要产胶植物的基因组文库进行了简单重复序列(SSRs),也即微卫星序列的分离鉴定。对来自无性系GT1的小插入基因组文库进行SSRs(AC/TG及CT/GA基序)筛选鉴定共获得154个克隆。采用载体通用引物及重复序列进行PCR扩增发现,其中114个为阳性克隆。限制性酶切分析结果显示,这些阳性克隆中有6个为重复拷贝,故予以剔除。之后,对剩余108个克隆中的50个进行了序列分析。此外,试验中对来自优良无性系RRII105的大插入基因组文库也进行了筛选。所获24个阳性克隆的测序结果也证实了橡胶基因组中微卫星序列的存在。经过最终的序列分析,本研究共鉴定出67个具有不同特点的简单及复合型微卫星序列,其中59个来自GT1,其余8个来自RRII105。所检测到的重复基序包括二核苷酸(TG/AC,AG/TC,TA/AT),三核苷酸(AAG,AGG,ATT),四核苷酸(GAAA,AAGG,ATCC,TAAA,AAAT),以及一个五核苷酸(GAAAT)。与其它作物的报道结果类似,在橡胶基因组中也观察到大量的CT/GA重复。
In this study, simple repetitive sequences (SSRs), ie microsatellite sequences, were isolated and identified using the genomic library of Hevea brasiliensis, a major rubber-producing plant. A total of 154 clones were screened for SSRs (AC / TG and CT / GA motif) screening of small insertional genomic libraries from clonal GT1. Using vector universal primers and repeated sequences of PCR amplification found that 114 were positive clones. Restriction analysis showed that 6 of these positive clones were duplicates, so they were deleted. After that, 50 of the remaining 108 clones were sequenced. In addition, large insertion genomic libraries from elite clones RRII105 were also screened in the assay. Sequencing of the 24 positive clones obtained also confirmed the presence of microsatellite sequences in the rubber genome. After the final sequence analysis, we identified 67 simple and complex microsatellite sequences with different characteristics, of which 59 were from GT1 and the remaining 8 were from RRII105. The repetitive motifs detected included dinucleotides (TG / AC, AG / TC, TA / AT), trinucleotides (AAG, AGG, ATT), tetranucleotides (GAAA, AAGG, ATCC, TAAA , AAAT), and a pentanucleotide (GAAAT). Similar to the reported results of other crops, a large number of CT / GA repeats were also observed in the rubber genome.