| 摘要: |
| 以罗汉果授粉后50 d和70 d果实为材料,利用抑制消减杂交技术构建了罗汉果果实在不同生育时期皂苷生物合成相关基因的差减cDNA文库。在正向差减文库(70 d为tester,50 d果实为driver)中随机挑选了641个cDNA阳性克隆测序,得到622条有效序列,重组率在96%以上。外源片段的长度分布在101～934 bp之间,平均长度约500 bp。BLASTN结果显示:201个ESTs序列没有同源序列,可能是新基因; 另外421个ESTs序列在核酸数据库中存在同源序列,其序列和已知功能(主要是能量代谢、次生代谢、转录因子、衰老及抗病性等)的蛋白有高度相似性。构建的cDNA文库符合SSH文库的质量标准,能满足下一步的实验要求。该SSH-cDNA文库为研究罗汉果差异表达基因、探讨三萜皂苷生物合成的分子机理、提高罗汉果甜苷V的质量和产量奠定了基础。 |
| 关键词: 罗汉果 甜苷V 抑制消减杂交 三萜皂苷 EST |
| DOI：10.3969/j.issn.1000-3142.2011.05.020 |
| 分类号:Q541 |
| Fund project: |
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| Construction of SSH-cDNA library from different developmental stages of Siraitia grosvenorii |
|
TANG Qi1, QIU De-You2, MA Xiao-Jun1,3*,
MO Chang-Ming3, FU Wei1
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1.Institute of Medicinal Plant, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193,
China;2.The Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China;3.Guangxi Branch
Institute, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Nanning 530023, China
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| Abstract: |
| In order to reveal the differentially-expressed genes involved in triterpenoid saponin biosynthesis of Siraitia grosvenorii,50 DAF(days after flowering)and 70 DAF fruits from different developmental stages were employed to construct subtractive cDNA library by suppression subtractive hybridization(SSH)in this study. Total 641 positive clones were selected randomly and sequenced from the forward-subtracted cDNA library(70 DAF as the tester and 50 DAF as the driver),and 622 high quality sequences were obtained. The rate of the recombination fraction was above 96%. The distribution of inserted fragments ranged from 101 bp and 934 bp,and the average fragment size was 500 bp. Followed by BLASTN and BLASTX for sequences dates,201 ESTs showed no significant matches to any other sequences in NCBI nucleotide sequence database,they were probably novel genes. The other 421 ESTs carried with remarkable identity to proteins with known function in the database,which fell into several functional categories including energy and secondary metabolism,transcription factors,ripening,senescence and pathogen-resistance. Further results indicated that the cDNA library quality were conformed to SSH library standard and could be used in further studies,also would provide reference for studying the differentially expressed genes,exploring molecular mechanism of triterpenoid saponin biosynthesis,and increasing productivity and quality of mogrosides in S.grosvenorii. |
| Key words: Siraitia grosvenorii mogrosides V suppression subtractive hybridization triterpenoid saponin Expressed sequence tags |
