青花菜Ogu不育胞质分子鉴定和序列分析

Molecular identification of Ogu cytoplasmic male sterile and sequence analysis in broccoli(Brassica oleracea var. italica)

雄性不育是农作物利用杂种优势、进行轮回选择和群体改良的重要手段,在农作物生产中具有巨大的利用价值。该研究为了鉴定青花菜细胞质雄性不育材料的不育胞质类型,以期今后为青花菜种质资源的收集、利用及分子标记辅助育种提供新的不育标记。根据Gen Bank中orf138基因保守序列设计特异引物,对20个青花菜种质资源基因组DNA进行PCR扩增。结果表明:特异引物P1/P2在12个青花菜雄性不育基因型中均扩增出392 bp的片段,在8个可育基因型中未扩增出条带,与田间育性鉴定结果相符。获得青花菜Ogu胞质雄性不育的特异基因orf138序列,Gen Bank中的登录号为HQ149728;用Blastn在Gen Bank中进行同源性比对分析,发现12个不育材料的特异片段与已报道的萝卜Ogu CMS所具有的Ogu orf138基因(Genbank登录号:Z18896.1)同源度高达100%。序列同源比对发现orf138基因存在变异位点。研究结果可为青花菜雄性不育细胞质的分子鉴定、进一步阐明胞质雄性不育败育机理,以及指导青花菜新型不育系的创建和杂种优势高效利用提供理论依据。

Aroma is an important character of tea,and terpenoids,with preferable fragrance and relatively high boiling point,are substantial substances contributing to tea aroma,which not only determine the aroma quality of tea,but also provide a classification basis of scent types.In plants,sesquiterpenes and dolichol and so forth are synthesize via the cytoplasmic mevalonate-dependent pathway.The enzyme HMG-CoA reductase （HMGR）catalyzes the conversion of 3-hydroxy-3-methylglutaryl coenzyme A （HMG-CoA）to mevalonate,which is the committed step in the synthesis of isoprenoids via the MVA pathway.To help understand the molecular and genetic mechanisms underlying terpenoid synthesis of tea,a full-length cDNA encoding HMGR was cloned from tea （Camellia sinensis （L.）O.Kuntze）by using the RACE-PCR technique （designated as CsHMGR 1）.It comprised 1 979 bp,with a 1 722 bp intact open read-ing frame encoding a 573-amino-acid protein.The deduced protein showed 80% to 82% similarities to homologs from rubber tree （Hevea brasiliensis ）,common camptotheca fruit （Camptotheca acuminate ）,ginseng （Panax ginseng ）, litchis （Litchi chinensis ）,American ginseng （Panax quinquefolius ）,rooted salvia （Salvia miltiorrhiza ）,Momordica grosvenori （Siraltia grosvenorii ）,and longan （Dimocarpus longan）.The phylogenetic tree,constructed with the cat-alytic domalned of CsHMGR1 and homologs from other species,indicated that CsHMGR1 belonged to the eukaryotic class I HMGR family.CsHMGR1 consisted of two transmembrane domalns,implying that it may be localized to en-doplasmic reticulum （ER）similarly to other eukaryotic homologs.It also contalned two HMG-CoA binding sites,two NADP（H）-binding sites,four conserved catalytic active residues and a phosphorylation site,indicating that phospho-rylation/dephosphorylation is likely a crucial mode of regulation of its biochemical activity.Tissue expression analysis indicated that CsHMGR 1 was expressed comparatively in the leaf buds of C .sinensis cv.Dayelong and in both leaf buds and floral buds of the mother plants.The regulation of expression and physiological activity of CsHMGR1 are likely to impact greatly on tea quality,and CsHMGR1 may provide a basis of the quality evaluation and breeding of tea given that its function is further resolved.