水稻籽粒低镉蛋白LCD互作蛋白的筛选与鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2020-0510

摘要/Abstract

摘要：

水稻低镉基因（Low cadmium,LCD）参与水稻籽粒镉（Cd）的积累,筛选与LCD互作的蛋白有利于揭示LCD介导的籽粒Cd积累调控的分子机制。以粳稻日本晴为试验材料构建酵母双杂交膜系统和核系统cDNA文库,以LCD为诱饵蛋白,分别进行膜系统和核系统筛选,获得29个与LCD互作的候选蛋白,并对这些基因进行gene ontology（GO）注释分析。进一步通过一对一互作验证,获得21个互作蛋白,包括膜系统蛋白16个,核系统蛋白5个,其中,6个蛋白与物质转运相关,5个蛋白与植物耐逆相关,4个蛋白与植物发育响应相关,6个蛋白与细胞生理代谢相关。

关键词: 水稻, LCD, 低镉, 酵母双杂交, 互作蛋白

Abstract:

Low cadmium（LCD）gene is involved in grain Cd accumulation of rice. To screen LCD-interacting proteins is beneficial for revealing the molecular mechanism of LCD-mediated grain Cd accumulation and regulation. Using Oryza sativa ssp. japonica cv. Nipponbare as experimental material,the cDNA libraries of yeast two hybrid membrane system and nuclear system were constructed,and LCD was used as bait protein to screen the membrane system and nuclear system respectively. A total of 29 genes were obtained,and the gene ontology（GO）analyses were conducted to annotate their molecular function. Further,these genes were cloned and one-to-one interaction verification with LCD was performed. Finally,a total of 21 LCD-interacting proteins including 16 from membrane system and 5 from nuclear system were identified. Among them,6 proteins were related to the tolerance and transport of metal ions,5 were related to the stress responses,4 were related to plant development responses,and the rest 6 were related to cellular and physiological metabolisms.

Key words: rice, LCD, low cadmium, yeast two-hybrid（Y2H）, interacting protein

郝小花, 戴佳利, 暨文劲, 黄丹, 李东屏, 田连福. 水稻籽粒低镉蛋白LCD互作蛋白的筛选与鉴定[J]. 生物技术通报, 2020, 36(11): 21-29.

HAO Xiao-hua, DAI Jia-li, JI Wen-jin, HUANG Dan, LI Dong-ping, TIAN Lian-fu. Screening and Identification of LCD-interacting Proteins in Rice[J]. Biotechnology Bulletin, 2020, 36(11): 21-29.

图/表 12

参考文献 44

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引物名称	引物序列（5'-3'）	酶切位点
pBT3-N-F	GGCCATTACGGCCATGGTGGAAACCACAGTT	Sfi I
pBT3-N-R	GGCCGAGGCGGCCTTTTTTACAAGGCCACTT	Sfi I
pGBKT7-F	GGAATTCATGGTGGAAACCACAGTTGATGTT	EcoR I
pGBKT7-R	CGGGATCCGTTTTTTACAAGGCCACTTCCAG	BamH I

引物名称	引物序列（5'-3'）	酶切位点
pBT3-N-F	GGCCATTACGGCCATGGTGGAAACCACAGTT	Sfi I
pBT3-N-R	GGCCGAGGCGGCCTTTTTTACAAGGCCACTT	Sfi I
pGBKT7-F	GGAATTCATGGTGGAAACCACAGTTGATGTT	EcoR I
pGBKT7-R	CGGGATCCGTTTTTTACAAGGCCACTTCCAG	BamH I

引物名称	引物序列（5'-3'）
pPR3-N-F	GTCGAAAATTCAAGACAAG
pPR3-N-R	AAGCGTGACATAACTAATTA
pGADT7-F	GGAGTACCCATACGACGTACC
pGADT7-R	TATCTACGATTCATCTGCAGC

引物名称	引物序列（5'-3'）
pPR3-N-F	GTCGAAAATTCAAGACAAG
pPR3-N-R	AAGCGTGACATAACTAATTA
pGADT7-F	GGAGTACCCATACGACGTACC
pGADT7-R	TATCTACGATTCATCTGCAGC

组合	质粒1	质粒2	目的	缺陷培养基	生长状态
1	pBT3-N-LCD	-	自激活检测	SD-Leu SD-Lue-Trp	√ ×
2	pBT3-N-LCD	pOst-Nub1	阳性对照	SD-Lue-Trp SD-Lue-Trp-His SD-Lue-Trp-His-Ade	√ √ √
3	pBT3-N-LCD	pPR3-N	阴性对照	SD-Lue-Trp SD-Lue-Trp-His SD-Lue-Trp-His-Ade	√ ×/少量 ×