农业生物技术学报2013，Vol.21Issue(8)：911-919,9.DOI:10.3969/j.issn.1674-7968.2013.08.004
大白菜BrTCP24基因的克隆与功能分析
Cloning and Functional Analysis of BrTCP24 Gene in Chinese Cabbage (Brassica rapa L.ssp.pekinensis)
摘要
Abstract
It is very important to isolate and characterize the genes responsible for negative control of the leaf heading growth of Chinese cabbage,which can be help to speed up breeding progress of the small heading Chinese cabbage varieties that meet the current market demands.Here,a full-length TCP cDNA named BrTCP24,which belongs to the second subgroups of TCP domain family,was isolated from leaves of Chinese cabbage inbred line Fushanbaotou (Brassica rapa L.ssp.pekinensis).The full-length cDNA of BrTCP24 consisted of 1 221 nucleotides,and was predicted to code a 406-amino acid polypeptide.In addition,there were no introns in BrTCP24 gene.The phylogenetic analysis about BrTCP24 and TCP families in Arabidopsis was carried out using the software of MEGA4.0.The result indicated that BrTCP24 gene and Arabidopsis AtTCP3 gene would belong to the same branch,which suggested they had closely genetic relationship.The alignment of predicted amino acid sequences of BrTCP24 and Arabidopsis AtTCP3 indicated that there was 55.15％ identity between them.Additionally,these two proteins contained conserved TCP domain and had 91.53％ identity in this domain.These results suggested that BrTCP24 and Arabidopsis AtTCP3 would have similar biological functions.The semiquantitive RT-PCR indicated that BrTCP24 gene was expressed in roots,dwarf stems,rosette leaves,folding leaves,flowers,siliques and bud flowers examined in Chinese cabbage.Among them,rosette leaves had the highest mRNA level,followed by roots,folding leaves,flowers,siliques and bud flowers,while dwarf stems had the lowest mRNA level.Interestingly,the expression level of BrTCP24 didn't effect by 5 μmol NAA treatment in 12 h period.To test the function of BrTCP24,we then engineered Arabidopsis plants that would over-express BrTCP24 ectopically,driven by CaMV 35S promoter,and obtained 17 transgenic lines by Kanamycin and PCR screening.Using RT-PCR method,we randomly detected 5 transgenic lines and found all of them could express the BrTCP24 gene.To detect the transgenic copy numbers,the Realtime quantitative PCR method was employed.Only one copy ofBrTCP24 gene was found to be inserted in all transgenic lines examined.Compared to the wild type plants,35S::BrTCP24 transgenic plants had shorter hypocotyls and smaller leaves.Furthermore,the transcripts levels of several organ size-associated genes,including aintegumenta(ANT),Arabidopsis thaliana expansin 10(AtEXP10),Arabidopsis thaliana growth regulating factor 5(AtGRF5),Arabidopsis thaliana GRF-interacting factor 1 (AtGIF1) and cyclin D3;1 (CycD3;1)which regulated either cell proliferation or cell growth were down-regulated in 35S::BrTCP24 transgenic plants.Taken together,the Chinese cabbage BrTCP24 gene can be involved in the negative control of organ size by inhibiting cell growth.关键词
大白菜/BrTCP24基因/器官大小Key words
Chinese cabbage/BrTCP24 gene/Organ size引用本文复制引用
王凤德,谭婷婷,张一卉,李景娟,李化银,李利斌,刘立锋,高建伟..大白菜BrTCP24基因的克隆与功能分析[J].农业生物技术学报,2013,21(8):911-919,9.基金项目
国家自然科学基金项目(31101553),国家高技术研究发展计划(863)项目(2012AA100103009)资助,山东省优秀中青年科学家科研奖励基金项目(BS2010SW027)和山东省良种工程项目(2011lzgcshucaizy （31101553）
2012lzgcshucaizy) （）
