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水稻Oryza sativa是世界上重要的禾本科Gramineae粮食作物之一。据估计,到2030年,禾本科作物的产量至少需要提高40%才能满足人口增长所带来的粮食需求[1]。生长发育良好的水稻根系不仅使植株适应不利的生长环境,而且还能提高植株对水分和营养的利用率[2],从而保证结实率和产量。水稻的种子根只在植株发育初期起作用,随后的整个生育期主要通过发育更为完善和发达的侧根和冠根维持生长发育。因此,发掘和鉴定调控水稻侧根和根冠生长发育的蛋白对水稻根系遗传发育及分子调控机制研究显得尤为重要。目前,在水稻根系发育研究中已获得了一些突变体表型及其调控侧根和冠根发育的关键基因,这些基因的功能缺失突变能特异地影响根系的生长发育和形态建成[3-5]。水稻侧根通过中柱鞘和内皮层细胞不断分裂而形成[6]。已有研究表明:真核生物中含锌指结构域蛋白能特异结合DNA,RNA和蛋白质,从而参与调控真核生物细胞的发育和凋亡过程及相关信号传导途径[7-8]。拟南芥Arabidopsis thaliana C1-1i亚族中GIS,GIS2,ZFP8和ZFP5蛋白受赤霉素和细胞分裂素诱导,参与调控表皮毛的生长发育及相关信号传导途径[9-12];而C1-2i亚族中的Zat6被证实参与调控种子根的生长发育和磷酸盐的吸收[13]。最近,水稻中已鉴定出189个含C2H2型锌指结构蛋白,其中至少26个蛋白响应不同的生物胁迫和非生物胁迫[14]。这些研究结果预示着含锌指结构域的蛋白在水稻生长发育过程中起着重要的调节作用。在前期研究中,我们已鉴定到1个水稻调控侧根生长发育的C2HC型锌指结构蛋白。本研究拟利用酵母双杂交技术筛选鉴定与该蛋白互作的调控蛋白,以期为水稻根系生长发育分子机制的解析奠定工作基础。
Screening and identification of a protein for controlling lateral root development in Oryza sativa
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摘要: CCHC型锌指结构蛋白OsZFP参与调控水稻Oryza sativa侧根的生长发育,但其相关互作蛋白及调控机制未知。以水稻‘日本晴’‘Nipponbare’为试验材料,克隆了OsZFP基因,利用EcoRⅠ和SalⅠ酶切位点构建酵母双杂交钓饵表达载体pGBKT7+OsZFP,验证该表达载体对酵母菌株Y2H无毒性及报告基因自激活现象;采用酵母双杂交技术,从已构建的水稻cDNA文库中筛选到1个阳性互作蛋白,经美国国家生物技术信息中心数据库(NCBI)同源性比对,鉴定为含有T-complex polypeptide 1(TCP-1)结构域的分子伴侣蛋白第7个亚基(Os06g0687700),命名为chaperonin containing TCP-1 eta subunit(CCT-eta),进而通过酵母一对一回复验证该互作蛋白。基于CCT-eta亚基与OsZFP蛋白互作,推测分子伴侣蛋白亚基CCT-eta可能参与调控水稻侧根的生长发育。Abstract: OsZFP, a CCHC-type zinc finger protein, has reportedly participated in lateral root development in rice. To determine the molecular mechanism and its interacting proteins in a regulated network, the OsZFP gene from Oryza sativa. 'Nipponbare' was cloned, and then a yeast bait vector, pGBKT7 + OsZFP, was constructed by EcoRⅠ and SalⅠ for cDNA library screening of rice. Results showed no toxicity or autoactivation with transformation of the Y2H strain containing the pGBKT7 + OsZFP bait vector. In a yeast two-hybrid assay, a positive interacted protein was obtained and confirmed from the cDNA library. Sequencing and homologous alignment from the National Center for Biotechnology Information (NCBI) showed that the interacting protein (named chaperonin containing a T-complex polypeptide 1 (TCP-1) eta subunit, CCT-eta) was the eta subunit of the chaperone which contained a TCP-1 domain. Based on the interaction between OsZFP and the CCT-eta subunit, it was speculated that the CCT-eta subunit of the chaperone protein may be involved in controlling lateral root development in rice.
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Key words:
- botany /
- Oryza sativa /
- regulated protein /
- vector construction /
- yeast two-hybrid /
- protein interaction
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链接本文:
https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2017.06.008